Analysis Devices, Kits, and Related Methods for Digital Quantification of Nucleic Acids and Other Analytes

• US 20120264132A1
• Filed: 04/05/2012
• Published: 10/18/2012
• Est. Priority Date: 03/24/2009
• Status: Active Grant

• Current Assignee: California Institute of Technology, University of Chicago
• Original Assignee: University of Chicago
• Inventors: Ismagilov, Rustem F., Shen, Feng, Kreutz, Jason E., Sun, Bing, Du, Wenbin
• Granted Patent: US 9,447,461 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: B01F 33/304 the mixing being performed in a mixing chamber where the products are brought into contact
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0896 Nanoscaled
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502738 characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

Provided are devices and methods for effecting processing of samples, including essentially isothermal amplification of nucleic acids, at multiple reaction locations in a single device. In some embodiments, the disclosed devices and methods provide for effecting parallel sample processing in several hundred locations on a single device.

Claims

1. A method of amplifying a nucleic acid molecule, comprising:

contacting (a) a sample comprising at least one nucleic acid molecule disposed at a plurality of first areas, with (b) at least one component of an amplification reagent disposed in a plurality of second areas,

the contacting being effected by placing the first and second areas into direct fluid communication with one another; and

the contacting comprises effecting relative motion between a substrate comprising the first area with a substrate comprising the second area; and

exposing the area having the at least one nucleic acid molecule to conditions effective for amplification of the at least one nucleic acid molecule.

2. The method of claim 1, wherein the amplification is essentially isothermal

3. The method of claim 1, wherein at least two of the plurality of first areas differ from one another in volume, wherein at least two of the plurality of second areas differ from one another in volume, wherein at least one first area differs in volume from at least one second area, or any combination thereof.

4. The method of claim 1, wherein the amplification is multiplexed amplification.

5. The motion of claim 1, wherein the relative motion comprises rotation, linear translation, or both.

6. The method of claim 1, wherein the relative motion is effected manually.

7. The method of claim 1 wherein the relative motion is effected under control of a programmed controller.

8. The method of claim 1 wherein placing the first and second areas into direct fluid communication with one another comprises removing a barrier between the first and second areas.

9. The method of claim 1, further comprising estimating the level of nucleic acid present in the sample.

10. The method of claim 1, wherein at least 10 first areas are placed into direct fluid communication with at least 10 second areas essentially simultaneously.

11. The method of claim 1, wherein at least 50 first areas are placed into direct fluid communication with at least 50 second areas essentially simultaneously.

12. The method of claim 1, wherein at least 500 first areas are placed into direct fluid communication with at least 500 second areas essentially simultaneously.

13. The method of claim 1, wherein at least one of the first and second areas has a volume of from 0.1 nL to about 1000 nL.

14. The method of claim 1, wherein the area having the at least one nucleic acid molecule is estimated to contain one molecule of nucleic acid.

15. The method of claim 1, further comprising detecting amplified nucleic acid.

16. The method of claim 1, further comprising correlating an estimated level of the at least one nucleic acid in the sample to a disease state in the source of the sample.

17. The method of claim 1, further comprising isolating the at least one nucleic acid.

18. The method of claim 17, further comprising disposing the at least one nucleic acid at the first area.

19. The method of claim 1, further comprising introducing the sample at the first area, introducing the amplification reagent at the second area, or both.

20. The method of claim 19, wherein introducing the sample at the first area comprises exerting the sample through a conduit in fluid communication with the first area.

21. The method of claim 20, wherein the conduit is formed in a substrate that comprises the first area.

22. The method of claim 19, wherein introducing the sample at the second area comprises exerting the sample through a conduit in fluid communication with the second area.

23. The method of claim 22, wherein the conduit is formed in a substrate that comprises the second area.

24. The method of claim 1, further comprising distributing the sample between the first area and a first control area.

25. The method of claim 24, wherein sample at the first control area remains uncontacted with the at least one amplification reagent.

26. The method of claim 1, further comprising distributing the at least one amplification reagent between the second area and a second control area.

27. The method of claim 26, wherein the reagent at the second control area remains uncontacted with the sample.

28. The method of claim 2, wherein the essentially isothermal amplification comprises fewer than 10 changes in temperature.

29. The method of claim 1, further comprising amplification product into direct fluid communication with a third area.

30. A device, comprising:

a first substrate having a first population of areas,

at least one area of the first population of areas having at least one satellite area disposed proximate to the at least one area, the at least one satellite area being adapted to retain material from the at least one area;

a second substrate having a second plurality of area formed therein,

the first and second substrates being engagable with one another such that relative motion between the first and second substrates places at least some of the first population of areas in register with at least some of the second population of areas so as to place the first and second areas into fluid communication with one another.

31. The device of claim 30, wherein the at least one satellite area is adapted to retain material not retained by a first and second area placed into fluid communication with one another.

32. The device of claim 30, wherein at least one of the first or second substrates has a thickness in the range of from about 10 micrometers to about 5000 micrometers.

33. The device of claim 30, wherein at least one of the first or second populations of areas comprises an area having a volume in the range of from about 1 pL to about 1 microL.

34. The device of claim 30, wherein the relative motion gives rise to at least one reaction region defined by an area of the first population of areas in fluid communication with an area of the second population of areas, the at least one reaction region having a volume in the range of from about 1 pL to about 1 microL.

35. The device of claim 30, wherein the first population of areas comprises two or more areas that differ from one another in volume and the second population of areas comprises two or more areas that differ from one another in volume.

36. The device of claim 30, wherein at least at least one satellite area is disposed proximate to at least one reaction region.

37. The device of claim 30, wherein the relative motion gives rise to at least about 10 reaction regions.

38. The device of claim 30, further comprising a supply of a reagent adapted to participate in nucleic acid amplification.

39. The device of claim 38, wherein the reagent is packaged together with the first and second substrates.

40. A method of effecting amplification of at least one nucleic acid target molecule, comprising:

contacting

(1) a sample material disposed in a plurality of first areas,

the sample material comprising a nucleic acid target, and

at least one of the first areas containing one molecule of the nucleic acid target, with

(2) a reactant material disposed in a plurality of second areas,

the contacting being effected by pairwise placement of at least some of the first areas and at least some of the second areas into direct fluid communication with one another,

the contacting effecting amplification of at least one nucleic acid target molecule.

41. The method of claim 40 wherein the sample material comprises a reagent.

42. The method of claim 41, wherein the material comprises an amplification reagent.

43. The method of claim 40, wherein the amplification is essentially isothermal

44. The method of claim 40, further comprising estimating the level of the at least one nucleic acid target.

45. The method of claim 40, wherein the reactant material comprises an amplification reagent.

46. The method of claim 45, further comprising exposing an area containing having the at least one nucleic acid molecule to conditions effective for amplification of the at least one nucleic acid target so as to give rise to an amplification product.

47. The method of claim 46, wherein the amplification is essentially isothermal.

48. The method of claim 46, further comprising estimating the level of the at least one nucleic acid target.

49. The method of claim 40, wherein the pairwise placement is effected by relative motion of the first and second areas.

50. The method of claim 49, wherein the pairwise placement places at least 10 first areas into direct fluid communication with at least 10 second areas.

51. The method of claim 50, wherein the pairwise placement places at least 50 first areas into direct fluid communication with at least 50 second areas.

52. The method of claim 49, wherein the relative motion is effected manually.

53. The method of claim 49, wherein the relative motion is effected by a controller.

54. A method, comprising:

dispersing a first sample that comprises at least one molecule of interest among a plurality of first areas, at least one of the first areas containing a single molecule of interest;

dispersing a reactant material into a plurality of second areas; and

effecting pairwise placement of at least some of the plurality of first areas into direct fluid communication with at least some of the plurality of second areas so as to contact reactant material with the first sample.

55. The method of claim 54, wherein (a) the first sample is dispersed among the plurality of the first areas at essentially the same time among the plurality of first areas, (b) the reactant material is dispersed among the plurality of second areas at essentially the same time among the plurality of second areas, (c) the pairwise placement of the at least some of the plurality of first areas into direct fluid communication with at least some of the plurality of second areas occurs at essentially simultaneously the same time among the majority of any pairs formed by first and second areas.

56. The method of claim 54, wherein the first sample comprises a reagent, and wherein the method further comprises effecting a reaction between the reagent and at least one molecule of interest.

57. The method of claim 56, wherein the reaction comprises nucleic acid amplification.

58. The method of claim 57, wherein the amplification is essentially isothermal.

59. The method of claim 57, further comprising recovering a product of the nucleic acid amplification.

60. The method of claim 56, wherein the pairwise placement is effected by relative motion of the first and second areas.

61. The method of claim 54, further comprising effecting a reaction between the contacted reactant material and the first sample.

62. The method of claim 61, wherein the reactant material comprises an amplification reagent.

63. The method of claim 61, wherein the reaction comprises nucleic acid amplification of the at least one molecule of interest.

64. The method of claim 63, wherein the nucleic acid amplification is essentially isothermal.

65. The method of claim 64, further comprising recovering a product of the nucleic acid amplification.

66. The method of claim 54, wherein the pairwise placement places at least 10 first areas into direct fluid communication with at least 10 second areas.

67. The method of claim 66, wherein the pairwise placement places at least 50 first areas into direct fluid communication with at least 50 second areas.

68. The method of claim 54, wherein the pairwise placement is effected by relative motion of the first and second areas.