MOLECULAR DIAGNOSTIC ASSAY DEVICE AND METHOD OF USE

• US 20150010904A1
• Filed: 09/24/2014
• Published: 01/08/2015
• Est. Priority Date: 09/16/2011
• Status: Active Grant

• Current Assignee: Credo Biomedical Pte Ltd.
• Original Assignee: Credo Biomedical Pte Ltd.
• Inventors: Wong, Winston Jr., Kao, Stephen Chang-Chi
• Granted Patent: US 10,598,656 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: G01N 2021/752 Devices comprising reaction zones
• CPC Class Codes: G01N 2021/757 using immobilised reagents
• CPC Class Codes: G01N 21/80 Indicating pH value
• CPC Class Codes: G01N 2333/918 Carboxylic ester hydrolases (3.1.1)
• CPC Class Codes: G01N 2458/15 Non-radioactive isotope labels, e.g. for detection by mass spectrometry
• CPC Class Codes: G01N 33/54386 Analytical elements
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Abstract

Lateral flow devices and methods of use for a molecular diagnostic assay are provided. The method is suitable for detection or monitoring of targets, including biological, chemical, and material targets that exist in very low concentrations in biological samples. The methods and devices of the present application are amenable to power source-free point of care testing.

Claims

1. A method of detecting an analyte in a test sample, the method comprising;

1) providing a lateral flow assay device that comprises a chromatographic medium that includes;

(a) a sample loading zone located upstream of a detection zone;

(b) a reporting carrier zone located between the sample loading zone and a detection zone, wherein said reporting carrier zone comprises a reporting carrier capable of forming a complex with the analyte said reporting carrier comprising a carrier and one or more proficient enzyme cassettes; and

(c) a detection zone, wherein the detection zone comprises a capture component for the analyte and an indicator;

ii) Contacting the sample application zone with the test sample, wherein the test sample travels through the reporting carrier zone along the chromatographic medium from the sample loading zone to the detection zone and beyond the detection zone;

iii) adding a substrate to the detection zone wherein the substrate undergoes a reaction in the presence of proficient enzyme analyte containing reporting carrier; and

iv) generating a response of the indicator within the detection zone that corresponds to the presence or absence of the analyte in the test sample.

2. The method of claim 1, wherein the lateral flow device further comprises a control zone down stream of the detection zone and the control zone comprises a capture component for the reporting carrier and an indicator;

adding a substrate to the control zone wherein the substrate undergoes a reaction in the presence of proficient enzyme containing reporting carrier; and

generating a response of the indicator within the control zone that corresponds to the presence or absence of the reporting carrier.

3. The method of claim 1 further comprising maintaining a reaction temperature.

4. The method of claim 1, wherein the analyte is a nucleic acid.

5. The method of claim 1, wherein the proficient enzyme conjugate comprises an antibody.

6. The method of claim 1, wherein the proficient enzyme conjugate comprises a nucleic acid.

7. The method of claim 1, wherein the proficient enzyme conjugate comprises a proficient enzyme selected from the group consisting of:

urease, phosphocholine phosphatase, betagalactosidase, xylose reductase, shikimate dehydrogenase, malate dehydrogenase, carboxylesterase, neopullulanase, subtilisin, 4-phytase, acetylcholinesterase, laccase, bacterial leucyl aminopeptidase, tripeptidyl-peptidase I, coagulation factor VIIa, trypsin, beta-fructofuranosidase.

8. The method of claim 3, where the reaction temperature is maintained to about 4°

C., to about 95°

C., or higher while still maintaining enzyme activity.

9. The method of claim 7, wherein the antibody interacts non-covalently with a proficient enzyme to form the proficient enzyme conjugate.

10. The method of claim 8, wherein the nucleic acid is covalently attached to a proficient enzyme to form the proficient enzyme conjugate.

11. The method of claim 7, wherein the antibody is covalently attached to a proficient enzyme to form the proficient enzyme conjugate.

12. The method of claim 1, further providing one or more inactive pro-enzymes in the detection zone.

13. The method of claim 1, wherein the activity of the proficient enzyme conjugate is detected by monitoring the effect of the proficient enzyme assisted reaction.

18. The method of claim 1, wherein the activity of the proficient enzyme conjugate is detected by colorimetric change.

19. The method of claim 1, wherein the activity of the proficient enzyme conjugate is detected by fluorescence emission.

20. The method of claim 1, wherein the activity of the proficient enzyme conjugate is detected by electrochemical methods.

21. The method of claim 18, wherein the colorimetric change is due to silver ion reduction.

22. The method of claim 1, wherein the activity of the proficient enzyme conjugate is detected by precipitation of a soluble component.

26. The method of claim 1 further comprising adding a pre-reporter carrier to the test sample prior to contacting the sample application zone with the test sample.

27. The method of claim 1 further comprising an absorptive pad at the distal end of the chromatographic medium.

28. The method of claim 1, wherein the substrate is allyl hexanoate and proficient enzyme is carboxylesterase.

17. The method of claim 17, wherein the pH change is determined using a pH sensitive cellulose acetate coupled dye.

14. The method of claim 15, wherein the effect of the proficient enzyme assisted reaction is proton release.

15. The method of claim 16, wherein the proton release produces a pH change.

16. The method of claim 17, wherein the pH change is determined using a pH sensitive potassium I-hydroxy-4-[1-(2-hydroxyethylsulphonyl)phenylazo]-naphthalene-2-sulphonate indicator polymer that is immobilized on the membrane.

25. The method of claim 25, wherein the pH sensitive polymer is selected from the group consisting of methyl acrylic acid, methyl methacrylate, methacrylic acid 2-(dimethylamino) ethyl ester, and N-hydroxymethyl acrylamide.

23. The method of claim 24, wherein the soluble component is a protein or pH sensitive polymer.

24. The method of claim 25, wherein the protein is BSA.

29. A lateral flow assay device for detecting the presence of an analyte within a test sample, the lateral flow assay device comprising:

a chromatographic medium that includes;

a sample loading zone located upstream of a detection zone;

a reporting carrier zone located between the sample loading zone and a detection zone, wherein said reporting carrier zone comprises a reporting carrier capable of forming a complex with the analyte said reporting carrier comprising a carrier and one or more proficient enzyme cassettes; and

a detection zone, wherein the detection zone comprises a capture component for the analyte and an indicator, wherein the indicator detects a reaction of a substrate in the presence of a proficient enzyme thereby detecting the presence of the analyte.

31. The lateral flow assay device of claim 31 wherein the proficient enzyme conjugate comprises an antibody.

30. The lateral flow assay device of claim 31 further comprising a control zone, wherein the control zone comprises a capture component for the reporting carrier and an indicator for detecting a reaction of a substrate in the presence of a proficient enzyme;

wherein the product of the enzyme reporting carrier complex and substrate is detected thereby detecting the presence of the reporting carrier.

32. The lateral flow assay device of claim 31 wherein the proficient enzyme conjugate comprises a nucleic acid.

33. The lateral flow assay device of claim 31 wherein the proficient enzyme conjugate comprises a proficient enzyme selected from the group consisting of:

urease, phosphocholine phosphatase, betagalactosidase, xylose reductase, shikimate dehydrogenase, malate dehydrogenase, carboxylesterase, neopullulanase, subtilisin, 4-phytase, acetylcholinesterase, laccase, bacterial leucyl aminopeptidase, tripeptidyl-peptidase 1, coagulation factor VIIa, trypsin, beta-fructofuranosidase.

34. The lateral flow assay device of claim 31 wherein the antibody interacts non-covalently with a proficient enzyme to form the proficient enzyme conjugate.

35. The lateral flow assay device of claim 31 wherein the nucleic acid is covalently attached to a proficient enzyme to form the proficient enzyme conjugate.

36. The lateral flow assay device of claim 31 further providing one or more inactive pro-enzymes in the detection zone.

37. The lateral flow assay device of claim 31, wherein the activity of the proficient enzyme conjugate is detected by monitoring the effect of the proficient enzyme assisted reaction.

38. The lateral flow assay device of claim 31, wherein the indicator is a pH sensitive indicator.

39. The lateral flow assay device of claim 31, wherein the indicator is a pH sensitive indicator is Potassium I-hydroxy-4-[1-(2-hydroxyethylsulphonyl)phenylazo]-naphthalene-2-sulphonate.

40. The lateral flow assay device of claim 31, wherein the indicator is a pH sensitive indicator is a pH sensitive cellulose acetate coupled dye.

41. The lateral flow assay device of claim 31, wherein the reporting carrier zone comprises a conjugate pad.

42. The lateral flow assay device of claim 31, wherein the sample loading zone comprises a sample loading pad.

43. The lateral flow assay device of claim 31, further comprising a rigid or flexible backing material.

44. The lateral flow assay device of claim 31, wherein the antibody is associated with the proficient enzyme by noncovalent interactions.

45. The lateral flow assay device of claim 31, wherein the antibody or nucleic acid is covalently attached to the proficient enzyme.

46. The lateral flow assay device of claim 31, further comprising a source of one or more inactive pro-enzymes.

47. The lateral flow assay device of claim 31, wherein the detection zone detects a product of the proficient enzyme by detection of a pH change.

48. The lateral flow assay device of claim 31, wherein the pH change is determined using a pH sensitive hydrogel.

49. The lateral flow assay device of claim 31, wherein the detection zone detects the product of the proficient enzyme by calorimetric change.

50. The lateral flow assay device of claim 31, wherein the detection zone detects the product of the proficient enzyme by fluorescence emission.

51. The lateral flow assay device of claim 31, wherein the detection zone detects the product of the proficient enzyme by electrochemical methods.

52. The lateral flow assay device of claim 51, wherein the colorimetric change is due to silver ion reduction.

53. The lateral flow assay device of claim 31, the detection zone detects the product of the proficient enzyme by precipitation of a soluble component.

54. The lateral flow assay device of claim 31, wherein the soluble component is a protein or pH sensitive polymer.

57. The lateral flow assay device of claim 31 further comprising an absorptive pad at the distal end of the chromatographic medium.

58. The lateral flow assay device of claim 31, wherein the substrate is allyl hexanoate and proficient enzyme is carboxylesterase.

56. The lateral flow assay device of claim 56, wherein the pH sensitive polymer is selected from the group consisting of methyl acrylic acid, methyl methacrylate, methacrylic acid 2-(dimethylamino) ethyl ester, and N-hydroxymethyl acrylamide.

55. The lateral flow assay device of claim 56, wherein the protein is BSA.

59. A lateral flow assay kit for detecting the presence of an analyte within a test sample, the lateral flow assay device comprising:

a porous membrane comprising;

a sample loading zone;

a reporting carrier zone down stream of the loading zone, wherein said reporting carrier zone comprises a reporting carrier capable of forming a complex with the analyte;

a detection zone down stream of the reporting carrier zone, wherein the detection zone comprises a capture component and an indicator; and

a substrate for the proficient enzyme;

wherein the substrate is applied to the detection zone after the test sample has been allowed to flow through the lateral flow device and the product of the enzyme and substrate is detected.

61. The lateral flow assay kit of claim 61 wherein the proficient enzyme conjugate comprises an antibody.

60. The lateral flow assay kit of claim 61 further comprising a control zone, wherein the control zone comprises a capture component for the reporting carrier and an indicator for detecting a reaction of a substrate in the presence of a proficient enzyme;

wherein the product of the enzyme reporting carrier complex and substrate is detected thereby detecting the presence of the reporting carrier.

62. The lateral flow assay kit of claim 61 wherein the proficient enzyme conjugate comprises a nucleic acid.

63. The lateral flow assay kit of claim 61 wherein the proficient enzyme conjugate comprises a proficient enzyme selected from the group consisting of urease, phosphocholine phosphatase, betagalactosidase, xylose reductase, shikimate dehydrogenase, malate dehydrogenase, carboxylesterase, neopullulanase, subtilisin, 4-phytase, acetylcholinesterase, laccase, bacterial leucyl aminopeptidase, tripeptidyl-peptidase I, coagulation factor VIIa, trypsin, beta-fructofuranosidase.

64. The lateral flow assay kit of claim 61 wherein the antibody interacts non-covalently with a proficient enzyme to form the proficient enzyme conjugate.

65. The lateral flow assay kit of claim 61 wherein the nucleic acid is covalently attached to a proficient enzyme to form the proficient enzyme conjugate.

66. The lateral flow assay kit of claim 61 further providing one or more inactive pro-enzymes in the detection zone.

67. The lateral flow assay kit of claim 61, wherein the activity of the proficient enzyme conjugate is detected by monitoring the effect of the proficient enzyme assisted reaction.

68. The lateral flow assay kit of claim 61, wherein the indicator is a pH sensitive indicator.

71. The lateral flow assay kit of claim 61, wherein the reporting carrier zone comprises a conjugate pad.

72. The lateral flow assay kit of claim 61, wherein the sample loading zone comprises a sample loading pad.

73. The lateral flow assay kit of claim 61, further comprising a rigid or flexible backing material.

74. The lateral flow assay kit of claim 61, wherein the antibody is associated with the proficient enzyme by noncovalent interactions.

75. The lateral flow assay kit of claim 61, wherein the antibody or nucleic acid is covalently attached to the proficient enzyme.

76. The lateral flow assay kit of claim 61, further comprising a source of one or more inactive pro-enzymes.

77. The lateral flow assay kit of claim 61, wherein the detection zone detects a product of the proficient enzyme by detection of a pH change.

78. The lateral flow assay kit of claim 79, wherein the pH change is determined using a pH sensitive hydrogel.

79. The lateral flow assay kit of claim 61, wherein the detection zone detects the product of the proficient enzyme by colorimetric change.

80. The lateral flow assay kit of claim 61, wherein the detection zone detects the product of the proficient enzyme by fluorescence emission.

81. The lateral flow assay kit of claim 61, wherein the detection zone detects the product of the proficient enzyme by electrochemical methods.

82. The lateral flow assay kit of claim 81, wherein the colorimetric change is due to silver ion reduction.

83. The lateral flow assay device of claim 61, the detection zone detects the product of the proficient enzyme by precipitation of a soluble component.

84. The lateral flow assay device of claim 83, wherein the soluble component is a protein or pH sensitive polymer.

85. The lateral flow assay device of claim 84, wherein the protein is BSA.

86. The lateral flow assay device of claim 84, wherein the pH sensitive polymer is selected from the group consisting of methyl acrylic acid, methyl methacrylate, methacrylic acid 2-(dimethylamino) ethyl ester, and N-hydroxymethyl acrylamide.

87. The lateral flow assay device of claim 60 further comprising an absorptive pad at the distal end of the chromatographic medium.

88. The lateral flow assay device of claim 60, wherein the substrate is allyl hexanoate and proficient enzyme is carboxylesterase.

70. The lateral flow assay kit of claim 70, wherein the indicator is a pH sensitive cellulose acetate coupled dye.

69. The lateral flow assay kit of claim 70, wherein the indicator is a pH sensitive indicator is Potassium I-hydroxy-4-[1-(2-hydroxyethylsulphonyl)phenylazo]-naphthalene-2-sulphonate.