Threshold color control system

• US 5,036,000 A
• Filed: 07/20/1987
• Issued: 07/30/1991
• Est. Priority Date: 12/16/1986
• Status: Expired due to Term

• Current Assignee: Orasure Technologies Incorporated
• Original Assignee: Enzymatics Incorporated (Qiagen NV)
• Inventors: Palmer, John L., Timmerman, Marsha W.
• Primary Examiner(s): Kepplinger, Esther L.
• Assistant Examiner(s): Scheiner, Toni R.
• Application Number:
  US07/075,817
• Time in Patent Office: 1,471 Days
• Field of Search: 435/25, 435/26, 435/175, 435/176, 435/177, 435/178, 435/179, 435/805, 435/810, 435/10, 435/174, 435/14, 435/288, 435/182, 422/56
• 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: G01N 33/521 Single-layer analytical elements
• CPC Class Codes: Y10S 436/904 Oxidation - reduction indicators

Abstract

A system for quantitative colorimetric analysis of biological fluids or organic compounds, including NAD(P)H, or a substrate of an enzyme which reacts with the formation or consumption 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 gives a digital reading of the organic material the concentration which is sought to be determined; the concentration of NAD(P)H is determined by a color change or color "signal" when the NAD(P)H is above a threshold concentration and by the absence of a color signal when the concentration of NAD(P)H is below the threshold concentration. The system includes a chromogen, an electron-accepting reactant which, until exhausted, prevents a visible color change due to accumulation of reduced chromogen, and a catalyst. 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.

Claims

1. A colorimetric assay system for measurement of the concentration of NAD(P)H, NAD(P), or an analyte in a sample, which analyte reacts to form or consume NAD(P)H, wherein the assay system comprises:

a chromogen which is capable of accepting electrons from NAD(P)H and which produces a color change upon reduction by NAD(P)H,

a non-chromogenic competing reactant capable of accepting electrons from NAD(P)H, in an amount sufficient to increase the range of concentrations that can be measured by the color change of the chromogen when the non-chromogenic competing reactant is reduced, wherein the non-chromogenic competing reactant prevents a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P)H is exceeded,

and an electron carrier catalyst capable of transferring electrons from NAD(P)H to the chromogen,

wherein the change in color caused by the 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 assay system of claim 1 wherein the non-chromogenic competing reactint is capable of accepting electrons directly from NAD(P)H, from reduced chromogen, or from both NAD(P)H and reduced chromogen.

3. The assay system of claim 1 wherein the non-chromogenic competing reactant is capable of oxidizing the reduced chromogen.

4. The assay system of claim 2 wherein the reactant accepts electrons directly from NAD(P)H and reacts preferentially with the NAD(P)H.

5. The assay system of claim 2 wherein the non-chromogenic competing reactant accepts electrons from both the reduced chromogen and the NAD(P)H.

6. The assay system of claim 1 wherein the non-chromogenic competing reactant is a weak oxidizing agent which accepts electrons more readily than NAD(P).

7. The assay system of claim 6 wherein the non-chromogenic competing reactant is selected from the group consisting of triethylenediamine chloride salt of Fe(III), Fe(III) citrate complex, Fe(III) EDTA complex, Fe(III) sorbitol complex, hexamino cobalt (III) chloride, potassium hexcacyanocobaltate (III) and sodium hexanitrocobaltate (III).

8. The assay system of claim 6 wherein the non-chromogenic competing reactant is selected from the group consisting of sodium potassium periodate, paranitrobenzyl disulfide, 2,2'"'"'-dithiobis-(pyride-N-oxide), and 2,2'"'"'-dithiobis(4-tert-butyl-1-isopropyl imidazole).

9. The assay system of claim 6 wherein the non-chromogenic competing reactant is a water soluble alkali metal salt of ferricyanide.

10. The assay system of claim 6 wherein the non-chromogenic competing reactant is a weak oxidizing agent selected from the group consisting of trichloro-melanine, hydrogen peroxide, and an organic peroxide.

11. The assay system of claim 9 wherein the salt is a sodium or potassium salt.

12. The assay system of claim 10 wherein the organic peroxide is selected from the group consisting of a lower alkyl peroxide and benzoyl perioxide.

13. The assay system of claim 1 wherein the catalyst is diaphorase and non-chromogenic competing reactant is not a substrate for the diaphorase.

14. The assay system of claim 1 wherein the catalyst is an organic polycyclic compound.

15. The assay system of claim 14 wherein the catalyst is an organic polycyclic compound selected from the group consisting of medola blue, phenazonium methosulfate, N-methyl-phenazonium methosulfate, methylene blue and anthraquinones substituted with a member selected from the group consisting of methyl-, chloro-, bromo-, amino-, hydroxy-, nitro-, methoxy and sulfonic acid.

16. The assay system of claim 1 which further comprises a metal salt which enhances the catalytic activity of the catalyst.

17. The assay system of claim 16 wherein the catalyst is an organic ligand complex of the metal salt.

18. The assay system of claim 7 wherein the catalyst is ferrocene.

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

20. The assay system of claim 1 wherein the concentration of an analyte which reacts to form or consume NAD(P)H is measured.

21. The assay system of claim 20 wherein the analyte is selected form the group consisting of glucose, alcohol, lactic acid, glycerol, ketones and beta-hydroxybutyrate.

22. The assay system of claim 21 wherein the analyte is generated in situ, and wherein the assay system further comprises an NAD(P)-dependent dehydrogenase capable of oxidizing the analyte.

23. The assay system of claim 1 wherein said assay system comprises a series of defined areas, and wherein each area contains the non-chromogenic competing reactant in different preselected concentrations, wherein each different preselected concentration is capable of preventing a visible color change due to accumulation of reduced chromogen until a different predetermined threshold amount of NAD(P)H is exceeded, and wherein a visible color change is indicative of an amount of NAD(P)H above the predetermined threshold and the absence of a visible color change is indicative of an amount of NAD(P)H below the predetermined threshold.

24. The assay system of claim 23 wherein the concentration of an analyte which reacts to form or consume NAD(P)H is measured.

25. The assay system of claim 23, wherein the assay system further comprises an NAD(P)-dependent dehydrogenase capable of oxidizing the analyte.

26. The assay system of claim 23 wherein the analyte is selected from the group consisting of glucose, alcohol, lactic acid, glycerol, ketones and beta-hydroxybutyrate.

27. The assay system of claim 26 wherein the analyte is generated in situ.

28. The assay system of claim 23 wherein the catalyst is an organic polycyclic compound, the chromogen is a tetrazolium salt and the non-chromogenic competing reactant is a weak oxidizing agent which accepts electrons more readily than NAD(P).

29. The assay system of claim 28 wherein the non-chromogenic competing reagent is selected from the group consisting of triethyldiamine salt of Fe(III), Fe(III) EDTA complex, Fe(III) sorbitol complex, hexamino cobalt (III) chloride, potassium hexacyanocobaltate (III) and sodium hexanitrocobaltate (III).

30. The assay system of claim 28 wherein the non-chromogenic competing reactant is selected from the group consisting of sodium potassium periodate, paranitrobenzyl disulfide and 2,2'"'"'-diathiobis (4-tert-butyl-1-isopropyl imidazole).

31. The assay system of claim 28 wherein the non-chromogenic competing reactant is a water-soluble alkali metal salt of ferricyanide.

32. The assay system of claim 31 wherein the salt is a sodium or potassium salt.

33. The assay system of claim 28 wherein the non-chromogenic competing reactant is a weak oxidizing agent selected from the group consisting of trichloro-melanine, hydrogen perioxide and organic peroxides.

34. The assay system of claim 33 wherein the organic peroxide is selected from the group consisting of a lower alkyl peroxide and benzoyl peroxide.

35. The assay system of claim 1 wherein the non-chromogenic competing reactant is capable of reoxidizing dye formed upon the reduction of the chromogen.

36. The assay system of claim 1 wherein the sample is urine, the analyte is uric acid, and the concentration of uric acid is determined.

37. The assay system of claim 1 wherein the sample is saliva, the analyte is alcohol, and the concentration of alcohol is determined.

38. The assay system of claim 1 wherein the sample is blood, the analyte is cholesterol, and the concentration of cholesterol is determined.

39. The assay system of claim 1, wherein the concentration of an analyte which reacts to form NAD(P)H is determined and the assay system further comprises an NAD(P)-dependent dehydrogenase capable of oxidizing the analyte.

40. A diagnostic colorimetric device for determination of the concentration of NAD(P)H, NAD(P), or an analyte which reacts to form or consume NAD(P)H, wherein the device comprises:

a physical support means,

a chromogen which is capable of accepting electrons from NAD(P)H and which produces a color change upon reduction by NAD(P)H,

a non-chromogenic competing reactant which is capable of accepting electrons from NAD(P)H, in an amount sufficient to prevent a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P)H is exceeded,

an electron-carrier catalyst capable of transferring electrons from NAD(P)H to the chromogen,

a coenzyme selected from the group consisting of NAD(P) and NAD(P)H,

wherein a visible color change is indicative of an amount of NAD(P)H above the predetermined threshold and the absence of a visible color change is indicative of an amount of NAD(P)H below the predetermined threshold.

41. The device of claim 40 wherein the coenzyme is NAD(P).

42. The device of claim 40 wherein the physical support means is a water-absorbing material.

43. The device of claim 42 wherein the water-absorbing material is selected from the group consisting of chromogenic pater, gelatin and a synthetic resin.

44. The device of claim 40 wherein the physical support means is a synthetic resin selected from the group consisting of polyethylene, polypropylene, polyalkyleneacetate and polycarbonate.

45. The device of claim 42 wherein the water-absorbing material is a multilayer dry gel inside a controlled volume capillary.

46. The device of claim 45 wherein the gel is selected from the group consisting of gelatin, agarose, agar, polyvinyl alcohol, polyvinyl pyrolidone, alginate, carrageenan, dextran, xanthan gum and mixtures thereof.

47. The device of claim 42 wherein the physical support means are in a form selected from the group consisting of sheets, rods, webs, filters and strips.

48. The device of claim 42 wherein the water absorbing material is a metal oxide.

49. The device of claim 48 wherein the water absorbing metal oxide is a controlled pore alumina catalytic ring.

50. The device of claim 42 wherein the water-absorbing material is a hybrid ceramic/polymer.

51. The device of claim 50 wherein the hybrid ceramic/polymer material is a polyvinyl chloride sheet that contains embedded silica particles.

52. The device of claim 42 wherein the physical support means is selected from the group consisting of glass, cellulose, wood, metal and textiles.

53. The device of claim 42 wherein the physical support means is a controlled-pore polycarbonate membrane.

54. The device of claim 52 wherein the concentration of non-chromogenic competing reactant to NAD(P)H at the threshold is either 1 to 1 or 2 to 1.

55. The device of claim 40 wherein the non-chromogenic competing reactant is a weak oxidizing agent which accepts electrons more readily than NAD(P).

56. The device of claim 55 wherein the catalyst is an organic polycyclic compound, and the chromogen is a tetrazolium salt.

57. The device of claim 40 which further comprises a metal salt capable of enhancing the catalytic activity of the catalyst.

58. The device of claim 40 wherein the concentration of an analyte which reacts to form or consume NAD(P)H is measured.

59. The device of claim 58 which further comprises an NAD(P)-dependent dehydrogenase capable of oxidizing the analyte.

60. The device of claim 59 wherein the analyte is selected from the group consisting of alcohol, lactic acid, glucose, glycerol, ketones and beta-hydroxybutyrate.

61. The device of claim 60 wherein the analyte is generated in situ.

62. The device of claim 58 wherein the analyte is generated in situ from triglyceride.

63. The device of claim 59 wherein the analyte is generated in situ.

64. The device of claim 40 wherein the non-chromogenic competing reactant is capable of reoxidizing dye formed upon the reduction of the chromogen.

82. The device of claim 40 wherein the catalyst is diaphorase and the non-chromogenic competing reactant is not a substrate for the diaphorase.

65. A diagnostic colorimetric device for determination of the concentration of NAD(P)H, NAD(P), or an analyte which reacts to form or consume NAD(P)H, wherein the device comprises:

a physical support means,

a chromogen which is capable of accepting electrons from NAD(P)H and which produces a color change upon reduction by NAD(P)H,

a non-chromogenic competing reactant which is capable of accepting electrons from NAD(P)H, in an amount sufficient to prevent a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P)H is exceeded,

an electron-carrier catalyst capable of transferring electrons from NAD(P)H to the chromogen,

wherein the physical support means has a series of defined areas, each area containing a different pre-selected concentration of the non-chromogenic competing reactant and wherein each pre-selected concentration of non-chromogenic competing reactant is capable of preventing a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P)H is exceeded.

66. The device of claim 65 wherein the non-chromogenic competing reactant is a weak oxidizing agent which accepts electrons more readily than NAD(P).

67. The device of claim 65 wherein the concentration of an analyte which reacts to form or consume NAD(P)H is measured.

68. The device of claim 67 which further comprises an NAD(P)-dependent dehydrogenase capable of oxidizing the analyte.

69. The device of claim 68 wherein the analyte is selected from the group consisting of glucose, alcohol, lactic acid, glycerol, ketones and beta-hydroxybutyrate.

70. The device of claim 68 wherein the analyte is generated in situ.

71. The device of claim 65 wherein the non-chromogenic competing reactant is capable of reoxidizing dye formed upon the reduction of the chromogen.

83. The device of claim 65 wherein the catalyst is diaphorase and the non-chromogenic competing reactant is not a substrate for the diaphorase.

72. A diagnostic colorimetric device for determination of the concentration of NAD(P)H, NAD(P), or an analyte which reacts to form or consume NAD(P)H, wherein the device comprises:

a physical support means,

a chromogen which is capable of accepting electrons from NAD(P)H and which produces a color change upon reduction by NAD(P)H,

a non-chromogenic competing reactant which is capable of accepting electrons from NAD(P)H, in an amount sufficient to prevent a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P)H is exceeded,

an electron-carrier catalyst capable of transferring electrons from NAD(P)H to the chromogen,

NAD(P), and

an NAD(P)-dependent dehydrogenase capable of oxidizing the analyte to produce NAD(P)H in an amount proportional to the analyte,

wherein the physical support means has a series of defined areas, each area containing a different pre-selected concentration of the non-chromogenic competing reactant and wherein each pre-selected concentration of non-chromogenic competing reactant is capable of preventing a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P)H is exceeded.

73. The device of claim 72, wherein the non-chromogenic competing reactant is capable of reoxidizing dye formed upon the reduction of the chromogen.

84. The device of claim 72 wherein the catalyst is diaphorase and the non-chromogenic competing reactant is not a substrate for the diaphorase.

74. A digital colorimetric method for determining the amount of an organic analyte in a sample, wherein the method comprises:

(a) contacting the sample with an assay system comprising an NAD(P)-dependent dehydrogenase capable of oxidizing the analyte to produce NAD(P)H in an amount proportional to the analyte, NAD(P), a chromogen capable of producing a color change upon reduction by NAD(P)H in the presence of an electron-carrier catalyst, an electron-carrier catalyst, and a non-chromogenic competing reactant capable of accepting electrons from NAD(P)H, wherein the non-chromogenic competing reactant is present in an amount sufficient to prevent a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P) is exceeded, and

(b) determining the amount of NAD(P)H as an indirect determination of the analyte, wherein a visible color change is indicative of an amount of NAD(P)H above the predetermined threshold an the absence of a visible color change is indicative of an amount of NAD(P)H below the predetermined threshold.

75. The method of claim 74 wherein the analyte is selected from the group consisting of glucose, alcohol, lactic acid, glycerol, ketones and beta-hydroxybutyrate.

76. The method of claim 74 wherein the non-chromogenic competing reactant is capable of reoxidizing dye formed upon the reduction of the chromogen.

77. The method of claim 74 wherein the analyte is generated in situ.

78. The method of claim 74 wherein the sample is not diluted prior to adding the sample to the assay system.

79. The method of claim 74 wherein the sample is urine and the analyte is uric acid.

80. The method of claim 74 wherein the sample is saliva and the analyte is alcohol.

81. The method of claim 74 wherein the sample is blood and the analyte is cholesterol.

85. The method of claim 75 wherein the catalyst is diaphorase and the non-chromogenic competing reactant is not a substrate for the diaphorase.