Methods and compositions for performing molecular separations

• US 6,440,284 B1
• Filed: 12/17/1999
• Issued: 08/27/2002
• Est. Priority Date: 12/17/1997
• Status: Expired due to Term

• Current Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Caliper Holdings Incorporated
• Inventors: Dubrow, Robert S.
• Primary Examiner(s): Warden, Jill
• Assistant Examiner(s): STARSIAK, JOHN S
• Application Number:
  US09/466,106
• Time in Patent Office: 984 Days
• Field of Search: 204/450, 204/451, 204/454, 204/455, 204/600, 204/601, 204/605, 427/105, 427/106, 427/107, 435/287.2, 422/68.1, 436/86, 436/89, 210/198.2
• US Class Current: 204/455
• CPC Class Codes: G01N 27/44747 Composition of gel or of carrier mixture
• CPC Class Codes: G01N 27/44752 Controlling the zeta potential, e.g. by wall coatings
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027

Abstract

The present invention provides methods of electrophoretically separating macromolecular species, as well as compositions and systems useful in carrying out such methods. Specifically, the methods of the present invention comprise providing a substrate that has at least a first capillary channel disposed therein. The surface of the channel has a first surface charge associated therewith, and is filled with a water soluble surface adsorbing polymer solution that bears a net charge that is the same as the charge on the capillary surface.

Claims

1. A method of manufacturing a microfabricated channel system, the method comprising:

providing a device comprising at least one microchannel; and

disposing a polymer in the at least one microchannel, the polymer comprising a net charge, the net charge being of the same type as at least one surface of the microchannel.

2. A method of claim 1, wherein the device comprises at least two microchannels.

3. A method of claim 2, wherein the polymer has a net charge of between about 0.01% and about 1%.

4. The method of claim 1, wherein the polymer has a molecular weight between about 1 Kd and about 6,000 Kd.

5. The method of claim 1, wherein the polymer is a water soluble polymer.

6. The method of claim 1, wherein the polymer is a water soluble surface adsorbing polymer.

7. The method of claim 1,wherein the polymer is a water soluble silica adsorbing polymer.

8. The method of claim 1, wherein the polymer is a water soluble surface adsorbing acrylic polymer.

9. The method of claim 1, wherein the polymer is a water soluble surface adsorbing polydimethylacrylamide polymer-co-acrylic acid polymer.

10. The method of claim 1, wherein the polymer is a water soluble surface adsorbing dimethylacrylamide polymer incorporating a selected percentage of charged acrylic acid monomers.

11. The method of claim 1, wherein the polymer is a water soluble surface adsorbing polymer which polymer is an acrylic polymer selected from a polyacrylamide, a polymethylacrylamide, and a polydimethylacrylamide.

12. The method of claim 1, wherein the polymer is a water soluble surface adsorbing polymer formed from a carboxylic acid monomer.

13. The method of claim 1, wherein the polymer is a water soluble surface adsorbing polymer formed from a monomer selected from:

acrylic acid, bisacrylamidoacetic acid, 4,4-Bis(4-hydroxyphenyl)pentanoic acid, 3-butene-1,2,3-tricarboxylic acid, 2-carboxyethylacrylate, itaconic acid, methacrylic acid, and 4-vinylbenzoic acid.

14. The method of claim 1, wherein the polymer is a water soluble surface adsorbing polymer formed from a sulfonic acid or phosphoric acid monomer.

15. The method of claim 1, wherein the polymer is a water soluble surface adsorbing polymer formed from a sulfonic acid or phosphoric acid monomer selected from 2-acrylamido-2-methyl-1-propanesulfonic acid, 2-methyl-2-propene-1-sulfonic acid, 2-propene-1-sulfonic acid, 4-styrenesulfonic acid, 2-sulfoethyl methacrylate, 3-sulfopropyldimethyl-3-methacrylamidopropylammonium inner salt, 3-sulfopropyl methacrylate, vinylsulfonic acid, Bis(2-methacryloxyethyl)phosphate, and monoacryloxyethyl phosphate.

16. The method of claim 15, wherein the negatively charged monomer units are selected from:

acrylic acid, bisacrylamidoacetic acid, 4,4-Bis(4-hydroxyphenyl)pentanoic acid, 3-butene-1,2,3-tricarboxylic acid, 2-carboxyethylacrylate, itaconic acid, methacrylic acid, 4-vinylbenzoic acid, sulfonic acid, 2-acrylamido2-methyl-1-propanesulfonic acid, 2-methyl-2-propene-1-sulfonic acid, 2-propene-1-sulfonic acid, 4-styrenesulfonic acid, 2-sulfoethyl methacrylate, 3-sulfopropyldimethyl-3-methacrylamidopropylammonium inner salt, 3-sulfopropyl methacrylate, vinylsulfonic acid, Bis(2-methacryloxyethyl)phosphate, and monoacryloxyethyl phosphate.

17. The method of claim 1, wherein the surface of the microchannel comprise a first surface which is negatively charged and wherein monomer subunits of the polymer comprise negatively charged monomer subunits.

18. The method of claim 1, the method further comprising filling the microchannel with a water soluble hydrophilic polymer solution.

19. The method of claim 1, wherein the microchannel comprises a region of positive charge and the polymer is formed from positively charged monomer units.

20. The method of claim 1, wherein the microchannel comprises a region of positive charge and the polymer is formed from positively charged monomer units, which monomer units are quaternary amine monomers.

21. The method of claim 1, wherein the microchannel comprises a region of positive charge and the polymer is formed from positively charged monomer units, which monomer units are quaternary amine monomers selected from 2-acryloxyethyltrimethylammonium chloride, diallyldimethylammonium chloride, 2-methacryloxyethyltrimethylammonium chloride, and 3-methacryloxy-2-hydroxypropyltrimethylammonium chloride.

22. The method of claim 1, wherein the polymer is provided in an aqueous solution at a concentration between about 0.01% and 30% (w/v).

23. The method of claim 1, wherein the polymer is in a solution which has a solution viscosity, as used within the microchannel, of from about 2 to about 1000 centipoise.

24. The method of claim 1, wherein the polymer is in a solution which has a solution viscosity as measured at 20°

C. using an Ubberholde viscometer of about 5.5 to about 60.5 centipoise.

25. The method of claim 1, wherein the polymer is present in a solution which further comprises a buffer for controlling pH or conductivity, or wherein the solution further comprises an additional polymer.

26. The method of claim 1, wherein the microchannel further comprises a neutral polymer.

27. The method of claim 1, wherein the microchannel further comprises a neutral polymer, which neutral polymer enhances sieving of an analyte in the microchannel.

28. The method of claim 1, wherein the polymer is formed by 2-Methyl-1-propanol polymerization of polydimethylacrylamide.

29. The method of claim 1, wherein the polymer is formed by 2-Methyl-1-propanol polymerization of polydimethylacrylamide/acrylic acid.

30. The method of claim 1, wherein the polymer is formed by aqueous polymerization of medium molecular weight polydimethylacrylamide/acrylic acid.

31. The method of claim 1, wherein the polymer is formed by aqueous polymerization of high molecular weight polydimethylacrylamide/acrylic acid.

32. The method of claim 1, wherein the polymer is disposed in the microchannel by placing one end of the channel into contact with a solution comprising the polymer and allowing the polymer to wick into the channel.

33. The method of claim 1, wherein the polymer is disposed in the microchannel by driving the polymer into the channel using vacuum or pressure.

34. The method of claim 1, wherein the polymer is disposed in the microchannel by placing the microchannel in contact with a polymer solution comprising the polymer at a reservoir comprising the polymer solution disposed at the end of the channel, and applying a slight pressure to force the polymer into the microchannel.

35. The method of claim 1, further comprising adding DNA to the microchannel.

36. The method of claim 35, wherein the planar microfabricated structure is provided by fabricating a channel network into the surface of a planar substrate and overlaying a second substrate on the surface of the first substrate to cover and seal the channels, thereby providing the microscale capillary channels.

37. The method of claim 1, wherein the device is a planar microfabricated structure comprising a plurality of microscale capillary channels fabricated therein.

38. The method of claim 1, wherein the microchannel has a charged surface capable of supporting an electroosmotic mobility (μ

EO) of at least about 1×

10<sup>−

5</sup> cm²V<sup>−

1</sup>s<sup>−

1</sup>, for a buffer when said buffer is in contact with said microchannel, which buffer comprises from about 1 mM to about 100 mM sodium borate at a pH of from about 6 to about 9.

39. The method of claim 1, wherein the microchannel has an internal cross-sectional dimension of between about 1 μ

m and about 500 μ

m.

40. The method of claim 1, wherein the at least one microchannel comprises a material selected from:

silica, polydimethylsiloxanes (PDMS), polymethylmethacrylate (PMMA), polyurethane, polyvinylchloride (PVC), polystyrene, polysulfone, polycarbonate, polytetrafluoroethylene.

41. A microfabricated channel system made by the method of claim 1.

42. A device, comprising:

at least one microchannel, the microchannel comprising a polymer disposed therein, the polymer comprising a net charge, the net charge being of the same type as at least one surface of the microchannel.

43. The device of claim 42, comprising at least two intersecting microchannels.

44. The device of claim 43, wherein the polymer has a net charge of between about 0.01% and about 1%.

45. The device of claim 42, wherein the polymer has a molecular weight between about 1 Kd and about 6,000 Kd.

46. The device of claim 42, wherein the polymer is a water soluble polymer.

47. The device of claim 42, wherein the polymer is a water soluble surface adsorbing polymer.

48. The device of claim 42, wherein the polymer is a water soluble silica adsorbing polymer.

49. The device of claim 42, wherein the polymer is a water soluble surface adsorbing acrylic polymer.

50. The device of claim 42, wherein the polymer is a water soluble surface adsorbing polydimethylacrylamide polymer-co-acrylic acid polymer.

51. The device of claim 42, wherein the polymer is a water soluble surface adsorbing dimethylacrylamide polymer incorporating a selected percentage of charged acrylic acid monomers.

52. The device of claim 42, wherein the polymer is a water soluble surface adsorbing acrylic polymer selected from a polyacrylamide, a polymethylacrylamide, and a polydimethylacrylamide.

53. The device of claim 47, wherein the polymer is a water soluble surface adsorbing polymer formed from a carboxylic acid monomer.

54. The device of claim 42, wherein the polymer is a water soluble surface adsorbing polymer is formed from a monomer selected from:

acrylic acid, bisacrylamidoacetic acid, 4,4-Bis(4-hydroxyphenyl)pentanoic acid, 3-butene-1,2,3-tricarboxylic acid, 2-carboxyethylacrylate, itaconic acid, methacrylic acid, and 4-vinylbenzoic acid.

55. The device of claim 42, wherein the polymer is a water soluble surface adsorbing polymer formed from a sulfonic acid or phosphoric acid monomer.

56. The device of claim 42, wherein the polymer is a water soluble surface adsorbing polymer formed from a sulfonic acid or phosphoric acid monomer selected from 2-acrylamido-2-methyl-1-propanesulfonic acid, 2-methyl-2-propene-1-sulfonic acid, 2-propene- 1-sulfonic acid, 4-styrenesulfonic acid, 2-sulfoethyl methacrylate, 3-sulfopropyldimethyl-3-methacrylamidopropylammonium inner salt, 3-sulfopropyl methacrylate, vinylsulfonic acid, Bis(2-methacryloxyethyl)phosphate, and monoacryloxyethyl phosphate.

57. The device of claim 56, wherein the negatively charged monomer units are selected from:

acrylic acid, bisacrylamidoacetic acid, 4,4-Bis(4-hydroxyphenyl)pentanoic acid, 3-butene-1,2,3-tricarboxylic acid, 2-carboxyethylacrylate, itaconic acid, methacrylic acid, 4-vinylbenzoic acid, sulfonic acid, 2-acrylamido-2-methyl-1-propanesulfonic acid, 2-methyl-2-propene-1-sulfonic acid, 2-propene-1-sulfonic acid, 4-styrenesulfonic acid, 2-sulfoethyl methacrylate, 3-sulfopropyldimethyl-3-methacrylamidopropylammonium inner salt, 3-sulfopropyl methacrylate, vinylsulfonic acid, Bis(2-methacryloxyethyl)phosphate, and monoacryloxyethyl phosphate.

58. The device of claim 42, wherein the surface of the microchannel comprises a first surface which is negatively charged and monomer subunits of the polymer comprise negatively charged monomer subunits.

59. The device of claim 42, wherein the microchannel is filled with a water soluble hydrophilic polymer solution.

60. The device of claim 42, wherein the microchannel comprises a region of positive charge and the polymer is formed from positively charged monomer units.

61. The device of claim 42, wherein the microchannel comprises a region of positive charge and the polymer is formed from positively charged monomer units, which monomer units are quaternary amine monomers.

62. The device of claim 42, wherein the microchannel comprises a region of positive charge and the polymer is formed from positively charged monomer units, which monomer units ate quaternary amine monomers selected from 2-acryloxyethyltrimethylammonium chloride, diallyldimethylammonium chloride, 2-methacryloxyethyltrimethylammonium chloride, and 3-methacryloxy-2-hydroxypropyltrimethylammonium chloride.

63. The device of claim 42, wherein the polymer is in an aqueous solution at a concentration between about 0.01% and 30% (w/v).

64. The device of claim 42, wherein the polymer is in a solution which has a solution viscosity, within the microchannel, of from about 2 to about 1000 centipoise.

65. The device of claim 42, wherein the polymer is in a solution which has a solution viscosity, as measured at 20°

C. using an Ubberholde viscometer, of about 5.5 to about 60.5 centipoise.

66. The device of claim 42, wherein the polymer is in a solution which further comprises a buffer for controlling pH or conductivity, or wherein the solution comprises one or more additional polymers.

67. The device of claim 42, wherein the microchannel further comprises a neutral polymer.

68. The device of claim 42, wherein the microchannel further comprises a neutral polymer, which neutral polymer enhances sieving of an analyte in the microchannel.

69. The device of claim 42, wherein the polymer is formed by 2-Methyl-1-propanol polymerization of polydimethylacrylamide.

70. The device of claim 42, wherein the polymer is formed by 2-Methyl-1-propanol polymerization of polydimethylacrylamide/acrylic acid.

71. The device of claim 42, wherein the polymer is formed by aqueous polymerization of medium molecular weight polydimethylacrylamide/acrylic acid.

72. The device of claim 42, wherein the polymer is formed by aqueous polymerization of high molecular weight polydimethylacrylamide/acrylic acid.

73. The device of claim 42, wherein the polymer is disposed in the microchannel by placing one end of the channel into contact with a solution comprising the polymer and allowing the polymer to wick into the channel.

74. The device of claim 42, wherein the polymer is disposed in the microchannel by driving the polymer into the channel using vacuum or pressure.

75. The device of claim 42, wherein the polymer is disposed in the microchannel by placing the microchannel in contact with a polymer solution comprising the polymer at a reservoir comprising the polymer solution disposed at the end of the channel, and applying a slight pressure to force the polymer into the microchannel.

76. The device of claim 42, further comprising DNA in the microchannel.

77. The device of claim 76, wherein the planar microfabricated structure is provided by fabricating a channel network into the surface of a planar substrate and overlaying a second substrate on the surface of the first substrate to cover and seal the channels, thereby providing the microscale capillary channels.

78. The device of claim 42, wherein the device comprises a planar microfabricated structure comprising a plurality of microscale capillary channels fabricated therein, wherein the at least one microchannel is one of the plurality of microscale capillary channels.

79. The device of claim 42, wherein the microchannel has a charged surface capable of supporting an electroosmotic mobility (μ

EO) of at least about 1×

10<sup>−

5</sup> cm²V<sup>−

1</sup>s<sup>−

1</sup>, for a buffer when that buffer is in contact with those walls, which buffer comprises from about 1 mM to about 100 mM sodium borate at a pH of from about 6 to about 9.

80. The device of claim 42, wherein the microchannel has an internal cross-sectional dimension of between about 1 μ

m and about 500 μ

m.

81. The device of claim 42, wherein the at least one microchannel comprises a material selected from:

silica, polydimethylsiloxanes (PDMS), polymethylmethacrylate (PMMA), polyurethane, polyvinylchloride (PVC), polystyrene, polysulfone, polycarbonate, polytetrafluoroethylene.

82. The device of claim 42, further comprising an electrode in fluidic contact with the polymer in the microscale channel.

83. The device of claim 42, further comprising a plurality of electrodes in fluidic contact with the polymer in the microscale channel, which electrodes during operation of the device, apply an electric field along the microscale channel, thereby providing for electrophoresis of analytes through the microscale channel.

84. The device of claim 42, further comprising at least one well or reservoir in fluidic contact with the microscale channel, which well or reservoir is coupled to a pressure source, which pressure source directs flow of the polymer into the channel.