METHOD AND DEVICE FOR DETECTING THE PRESENCE OF A SINGLE TARGET NUCLEIC ACID IN A SAMPLE

• US 20080171382A1
• Filed: 08/13/2007
• Published: 07/17/2008
• Est. Priority Date: 04/17/1997
• Status: Abandoned Application

• Current Assignee: United States Department Of Health And Human Services (Government of the United States of America), Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Cytonix, The United States of America As Represented By The Secretary of Agriculture
• Inventors: Silver, Jonathan E., BROWN, James F.
• Application Number:
  US11/837,564
• Publication Number:
  US 20080171382A1
• Time in Patent Office: Days
• US Class Current: 435/287.2
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/06 Fluid handling related problems
• CPC Class Codes: B01L 2200/0642 Filling fluids into wells by specific techniques
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: B01L 3/50851 specially adapted for heating or cooling samples laboratory heating apparatus B01L7/00; incubators C12M
• CPC Class Codes: B01L 3/5088 confining liquids at a location by surface tension, e.g. virtual wells on plates, wires B01L3/50857 takes precedence
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 : Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: Y10S 436/805 : Optical property
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

A microfluidic device comprising a first sample chamber and a first sample portion positioned in the first sample chamber. If the first sample portion contains a single molecule of a target nucleic acid, the first sample portion would attain a detectable concentration of the target nucleic acid after a single round of amplification. Also, a microfluidic device comprising a first sample chamber and at least one amplification targeting reagent positioned in the first sample chamber. If a sample portion which comprises at least a single molecule of a target nucleic acid is positioned in the first sample chamber, the sample portion would attain a detectable concentration of the target nucleic acid after a single round of amplification.

Claims

1. A microfluidic device comprising:

a first sample chamber; and

a first sample portion, said first sample portion being positioned in said first sample chamber, whereby if said first sample portion contains at least a single molecule of a target nucleic acid, said first sample portion would attain a detectable concentration of said target nucleic acid within a portion of said first sample chamber after a single round of amplification.

2. A microfluidic device as recited in claim 1, wherein if said first sample portion contains at least a single molecule of said target nucleic acid, said first sample portion would attain a detectable concentration of said target nucleic acid within a portion of said first sample chamber after a single amplification step.

3. A microfluidic device as recited in claim 1, wherein said first sample chamber further comprises constituents for enabling amplification of a target nucleic acid.

4. A microfluidic device as recited in claim 1, wherein said single round of amplification comprises at least one thermocycle step.

5. A microfluidic device as recited in claim 1, wherein said single round of amplification comprises at least one homogeneous amplification step.

6. A microfluidic device as recited in claim 1, wherein said first sample chamber has a volume of about 1 picoliter or less.

7. A microfluidic device as recited in claim 1, wherein said first sample chamber has a volume in the range of from about 1 picoliter to about 1 microliter.

8. A microfluidic device as recited in claim 1, wherein said first sample chamber has a volume of about 10 picoliters or less.

9. A microfluidic device as recited in claim 1, wherein said first sample chamber has a volume of about 100 picoliters.

10. A microfluidic device as recited in claim 1, wherein said first sample chamber has a volume of about 1 nanoliter.

11. A microfluidic device as recited in claim 1, wherein said first sample chamber has a volume of 10 nanoliters or less.

12. A microfluidic device as recited in claim 1, wherein said first sample chamber has a volume of about 10 nanoliters.

13. A microfluidic device as recited in claim 1, wherein said first sample chamber has at least one dimension of 2 mm or less.

14. A microfluidic device as recited in claim 1, wherein said first sample chamber has at least one dimension of 1 mm or less.

15. A microfluidic device as recited in claim 1, wherein said first sample chamber has at least one dimension of 100 microns or less.

16. A microfluidic device as recited in claim 1, wherein said first sample chamber has at least one dimension of 20 microns or less.

17. A microfluidic device as recited in claim 1, wherein said first sample chamber has at least one dimension of a few microns or less.

18. A microfluidic device as recited in claim 1, wherein said first sample portion has a volume of about 1 picoliter or less.

19. A microfluidic device as recited in claim 1, wherein said first sample portion has a volume in the range of from about 1 picoliter to about 1 microliter.

20. A microfluidic device as recited in claim 1, wherein said first sample portion has a volume of about 10 picoliters or less.

21. A microfluidic device as recited in claim 1, wherein said first sample portion has a volume which is nanoliter-sized.

22. A microfluidic device as recited in claim 1, wherein said first sample portion has a volume of about 1 nanoliter or less.

23. A microfluidic device as recited in claim 1, wherein said first sample portion has a volume of about 10 nanoliters or less.

24. A microfluidic device as recited in claim 1, wherein said first sample portion has a volume of about 100 nanoliters or less.

25. A microfluidic device as recited in claim 1, wherein said first sample portion has a volume of about 1 microliter or less.

26. A microfluidic device as recited in claim 1, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by about 2 mm or less.

27. A microfluidic device as recited in claim 1, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by about 1 mm or less.

28. A microfluidic device as recited in claim 1, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by about 500 microns or less.

29. A microfluidic device as recited in claim 1, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by about 100 microns or less.

30. A microfluidic device as recited in claim 1, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by about 20 microns or less.

31. A microfluidic device as recited in claim 1, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by a few microns or less.

32. A microfluidic device as recited in claim 1, wherein said first sample chamber contains at least one amplification targeting reagent.

33. A microfluidic device as recited in claim 1, wherein said microfluidic device comprises a plurality of sample chambers.

34. A microfluidic device as recited in claim 33, wherein at least said first sample chamber contains at least a first amplification targeting reagent, and at least a second sample chamber contains at least a second amplification targeting reagent, said first amplification targeting reagent differing from said second amplification targeting reagent.

35. A microfluidic device as recited in claim 33, wherein each of said sample chambers has a volume of about 1 picoliter or less.

36. A microfluidic device as recited in claim 33, wherein each of said sample chambers has a volume in the range of from about 1 picoliter to about 1 microliter.

37. A microfluidic device as recited in claim 33, wherein each of said sample chambers has a volume of about 10 picoliters or less.

38. A microfluidic device as recited in claim 33, wherein each of said sample chambers has a volume of about 100 picoliters.

39. A microfluidic device as recited in claim 33, wherein each of said sample chambers has a volume of about 1 nanoliter.

40. A microfluidic device as recited in claim 33, wherein each of said sample chambers has a volume of 10 nanoliters or less.

41. A microfluidic device as recited in claim 33, wherein each of said sample chambers has a volume of about 10 nanoliters.

42. A microfluidic device as recited in claim 33, wherein each of said sample chambers contains at least one amplification targeting reagent.

43. A microfluidic device as recited in claim 1 wherein said microfluidic device comprises from about 10,000 to over 100,000 sample chambers.

44. A microfluidic device as recited in claim 1, wherein said first sample chamber has at least one dimension of 500 microns or less.

45. A microfluidic device as recited in claim 44, wherein said first sample chamber has at least one dimension of 100 microns or less.

46. A microfluidic device as recited in claim 1, wherein said first sample chamber has opposing barriers separated by 500 microns or less.

47. A microfluidic device as recited in claim 46, wherein said first sample chamber has opposing barriers separated by 100 microns or less.

48. A microfluidic device as recited in claim 1, wherein said microfluidic device further comprises at least one cured displacing fluid which seals said first sample portion within said first sample chamber.

49. A microfluidic device as recited in claim 1, wherein said microfluidic device further comprises at least one cured adhesive which seals said first sample portion within said first sample chamber.

50. A microfluidic device as recited in claim 1, wherein said microfluidic device further comprises a sealing device adjacent to said first sample chamber.

51. A microfluidic device as recited in claim 50, wherein said sealing device is selected from the group consisting of microscope slide coverslips, tapes, films, silicon films, silicon devices, and devices comprising an array of reactants.

52. A microfluidic device as recited in claim 1, wherein said first sample chamber comprises a first microcapillary device.

53. A microfluidic device as recited in claim 52, wherein said microfluidic device comprises a plurality of microcapillary devices.

54. A microfluidic device comprising:

a first sample chamber; and

at least one amplification targeting reagent positioned in said first sample chamber,

whereby if a sample portion which comprises at least a single molecule of a target nucleic acid is positioned in said first sample chamber, said first sample portion would attain a detectable concentration of said target nucleic acid within a portion of said first sample chamber after a single round of amplification.

55. A microfluidic device as recited in claim 54, wherein if said first sample portion contains at least a single molecule of said target nucleic acid, said first sample portion would attain a detectable concentration of said target nucleic acid within a portion of said first sample chamber after a single amplification step.

56. A microfluidic device as recited in claim 54, wherein said first sample chamber further comprises constituents for enabling amplification of a target nucleic acid.

57. A microfluidic device as recited in claim 54, wherein said single round of amplification comprises at least one thermocycle step.

58. A microfluidic device as recited in claim 54, wherein said single round of amplification comprises at least one homogeneous amplification step.

59. A microfluidic device as recited in claim 54, wherein said first sample chamber has a volume of about 1 picoliter or less.

60. A microfluidic device as recited in claim 54, wherein said first sample chamber has a volume in the range of from about 1 picoliter to about 1 microliter.

61. A microfluidic device as recited in claim 54, wherein said first sample chamber has a volume of about 10 picoliters or less.

62. A microfluidic device as recited in claim 54, wherein said first sample chamber has a volume of about 100 picoliters.

63. A microfluidic device as recited in claim 54, wherein said first sample chamber has a volume of about 1 nanoliter.

64. A microfluidic device as recited in claim 54, wherein said first sample chamber has a volume of 10 nanoliters or less.

65. A microfluidic device as recited in claim 54, wherein said first sample chamber has a volume of about 10 nanoliters.

66. A microfluidic device as recited in claim 54, wherein said first sample chamber has at least one dimension of 2 mm or less.

67. A microfluidic device as recited in claim 54, wherein said first sample chamber has at least one dimension of 1 mm or less.

68. A microfluidic device as recited in claim 54, wherein said first sample chamber has at least one dimension of 100 microns or less.

69. A microfluidic device as recited in claim 54, wherein said first sample chamber has at least one dimension of 20 microns or less.

70. A microfluidic device as recited in claim 54, wherein said first sample chamber has at least one dimension of a few microns or less.

71. A microfluidic device as recited in claim 54, wherein said first sample chamber has at least one dimension of 500 microns or less.

72. A microfluidic device as recited in claim 71, wherein said first sample chamber has at least one dimension of 100 microns or less.

73. A microfluidic device as recited in claim 54, wherein said first sample chamber has opposing barriers separated by 500 microns or less.

74. A microfluidic device as recited in claim 73, wherein said first sample chamber has opposing barriers separated by 100 microns or less.

75. A microfluidic device as recited in claim 54, wherein said microfluidic device comprises a plurality of sample chambers.

76. A microfluidic device as recited in claim 75, wherein each of said sample chambers has a volume of about 1 picoliter or less.

77. A microfluidic device as recited in claim 75, wherein each of said sample chambers has a volume in the range of from about 1 picoliter to about 1 microliter.

78. A microfluidic device as recited in claim 75, wherein each of said sample chambers has a volume of about 10 picoliters or less.

79. A microfluidic device as recited in claim 75, wherein each of said sample chambers has a volume of about 100 picoliters.

80. A microfluidic device as recited in claim 75, wherein each of said sample chambers has a volume of about 1 nanoliter.

81. A microfluidic device as recited in claim 75, wherein each of said sample chambers has a volume of 10 nanoliters or less.

82. A microfluidic device as recited in claim 75, wherein each of said sample chambers has a volume of about 10 nanoliters.

83. A microfluidic device as recited in claim 75, wherein each of said sample chambers contains at least one amplification targeting reagent.

84. A microfluidic device as recited in claim 75, wherein at least said first sample chamber contains at least a first amplification targeting reagent, and at least a second sample chamber contains at least a second amplification targeting reagent, said first amplification targeting reagent differing from said second amplification targeting reagent.

85. A microfluidic device as recited in claim 84, wherein said first sample chamber further contains at least a first sample portion.

86. A microfluidic device as recited in claim 54, wherein said first sample chamber further contains at least a first sample portion.

87. A microfluidic device as recited in claim 86, wherein said first sample portion has a volume of about 1 picoliter or less.

88. A microfluidic device as recited in claim 86, wherein said first sample portion has a volume in the range of from about 1 picoliter to about 1 microliter.

89. A microfluidic device as recited in claim 86, wherein said first sample portion has a volume of about 10 picoliters or less.

90. A microfluidic device as recited in claim 86, wherein said first sample portion has a volume which is nanoliter-sized.

91. A microfluidic device as recited in claim 86, wherein said first sample portion has a volume of about 1 nanoliter or less.

92. A microfluidic device as recited in claim 86, wherein said first sample portion has a volume of about 10 nanoliters or less.

93. A microfluidic device as recited in claim 86, wherein said first sample portion has a volume of about 100 nanoliters or less.

94. A microfluidic device as recited in claim 86, wherein said first sample portion has a volume of about 1 microliter or less.

95. A microfluidic device as recited in claim 86, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by about 2 mm or less.

96. A microfluidic device as recited in claim 86, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by about 1 mm or less.

97. A microfluidic device as recited in claim 86, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by about 500 microns or less.

98. A microfluidic device as recited in claim 86, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by about 100 microns or less.

99. A microfluidic device as recited in claim 86, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by about 20 microns or less.

100. A microfluidic device as recited in claim 86, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by a few microns or less.

101. A microfluidic device as recited in claim 86, wherein said microfluidic device further comprises at least one cured displacing fluid which seals said first sample portion within said first sample chamber.

102. A microfluidic device as recited in claim 86, wherein said microfluidic device further comprises at least one cured adhesive which seals said first sample portion within said first sample chamber.

103. A microfluidic device as recited in claim 54 wherein said microfluidic device comprises from about 10,000 to over 100,000 sample chambers.

104. A microfluidic device as recited in claim 54, wherein said first sample chamber comprises a first microcapillary device.

105. A microfluidic device as recited in claim 104, wherein said microfluidic device comprises a plurality of microcapillary devices.