Apparatus and methods for active biological sample preparation

• US 6,129,828 A
• Filed: 09/06/1996
• Issued: 10/10/2000
• Est. Priority Date: 09/06/1996
• Status: Expired due to Fees

• Current Assignee: Nanogen, Inc.
• Original Assignee: Nanogen, Inc.
• Inventors: Jackson, Thomas R., Heller, Michael J., Swanson, Paul D., Scott, Bradley S., Sheldon, Edward L. III
• Primary Examiner(s): Warden, Jill
• Assistant Examiner(s): STARSIAK, JOHN S
• Application Number:
  US08/709,358
• Time in Patent Office: 1,495 Days
• Field of Search: 204/518, 204/519, 204/543, 204/544, 204/627, 204/628, 204/615, 204/648, 204/465, 204/553, 435/287.2, 435/306.1, 536/25.4, 536/25.41, 536/25.42
• US Class Current: 204/518
• CPC Class Codes: B01D 57/02 by electrophoresis treatment of water, waste water, sewage or sludge by electrophoresis C02F1/469; electrophoretic production of compounds or non-metals C25B7/00; investigating or analysing materials by using electrophoresis G01N27/26
• CPC Class Codes: B01J 19/0093 Microreactors, e.g. miniaturised or microfabricated reactors laboratory containers with capillary fluid transport in microfabricated channels or chambers B01L3/5027
• CPC Class Codes: B01L 2200/0631 Purification arrangements, e.g. solid phase extraction [SPE]
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0424 Dielectrophoretic forces
• CPC Class Codes: B01L 2400/0644 rotary valves
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B03C 5/026 using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]
• CPC Class Codes: B03C 5/028 using travelling electric fields, i.e. travelling wave dielectrophoresis [TWD]
• CPC Class Codes: C12N 13/00 Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: G01N 27/44743 Introducing samples
• CPC Class Codes: H01L 29/6656 using multiple spacer layers, e.g. multiple sidewall spacers

Abstract

Systems and methods for the electronic sample preparation of biological materials utilize the differential charge-to-mass ratio and/or the differential affinity of sample constituents to separation materials for sample preparation. An integrated system is provided for performing some or all of the processes of: receipt of biological materials, cell selection, sample purification, sample concentration, buffer exchange, complexity reduction and/or diagnosis and analysis. In one embodiment, one or more sample chambers adapted to receive a buffer solution are formed adjacent to a spacer region which may include a trap or other affinity material, electrophoretic motion of the materials to be prepared being effected through operation of electrodes. In another aspect of this invention, a transporter or dipstick serves to collect and permit transport of materials, such as nucleic acids, most preferably DNA and/or RNA. In one embodiment, a membrane or trap is held in a frame which is adapted to mate with a channel formed in the spacer region. In another aspect of this invention, an electrophoretic system for biological sample preparation is operated in a manner so as to utilize the differential charge-to-mass ratio so as to control the migration of materials within the solution. In one aspect, bunching of selected materials is achieved by operation of two electrodes in a manner so as to reduce the spatial dispersion of those materials. In another aspect of this invention, a vertically disposed sample preparation unit includes an upper reservoir including and a collection chamber. A sample is preferably pre-prepared and densified, applied to the conductive polymer, electrophoresed so as to move nucleic acids into the conductive polymer and move undesired material away from the conductive polymer. Integrated systems are described in which cell separation, purification, complexity reduction and diagnosis may be performed together. In the preferred embodiment, cell separation and sample purification are performed in a first region, the steps of denaturation, complexity reduction and diagnosis being performed in a second region.

Claims

1. An apparatus for active biological sample preparation adapted to separate desired materials from undesired materials comprising:

a first sample chamber, adapted to receive a buffer solution,

a second sample chamber, adapted to receive a buffer solution,

a spacer compartment disposed between the first sample chamber and the second sample chamber, the spacer compartment providing a trap having a differential effect on the desired materials versus undesired materials,

a first electrode adapted for electrical contact with the buffer solution when located within the first sample chamber, and

a second electrode adapted for electrical contact with the buffer solution when in the second sample chamber, characterized by having

a protective layer between the first sample chamber and the first electrode, thereby forming a first electrode chamber within the first sample chamber, wherein the protective layer is a membrane.

2. The apparatus for active biological sample preparation of claim 1 wherein the trap is hydrophobic.

3. The apparatus of claim 1 for active biological sample preparation wherein the trap is a protein trap.

4. The apparatus of claim 3 wherein the protein trap is polyvinylidene diflouride (PVDF).

5. The apparatus of claim 3 wherein the protein trap is nitrocellulose.

6. The apparatus of claim 1 for active biological sample preparation wherein the trap is a DNA trap.

7. The apparatus of claim 1 further comprising a third electrode disposed proximal to the trap.

8. The apparatus of claim 7 for active biological sample preparation wherein the third electrode and trap comprise a metal-coated filter material.

9. The apparatus of claim 1 further comprising a third electrode formed integral with the trap.

10. The apparatus of claim 1 for active biological sample preparation further comprising a control system operatively coupled to the electrodes.

11. The apparatus of claim 1 for active biological sample preparation wherein the spacer compartment has a length, a width, and a depth defining a volume.

12. The apparatus of claim 11 for active biological sample preparation wherein the volume of the spacer compartment is less than 50% of a volume of the first sample chamber.

13. The apparatus of claim 12 for active biological sample preparation wherein the volume of the spacer compartment is 30% or less of the volume of the first sample chamber.

14. The apparatus of claim 13 for active biological sample preparation wherein the volume of the spacer compartment is 25% or less of the volume of the first sample chamber.

15. The apparatus of claim 1 for active biological sample preparation wherein the length of the sample chamber is 20% or less of the distance between the first electrode and the second electrode.

16. The apparatus of claim 15 for active biological sample preparation wherein the length of the sample chamber is 10% or less of the distance between the first electrode and the second electrode.

17. The apparatus of claim 16 for active biological sample preparation wherein the length of the sample chamber is 5% or less of the distance between the first electrode and the second electrode.

18. The apparatus of claim 1 for active biological sample preparation wherein the length of the sample chamber is 5 nm or less.

19. The apparatus of claim 18 for active biological sample preparation wherein the length of the sample chamber is 4 mm or less.

20. The apparatus of claim 19 for active biological sample preparation wherein the length of the sample chamber is from approximately 1 to approximately 2 mm.

21. The apparatus of claim 1 for active biological sample preparation wherein the membrane is an ultrafiltration membrane.

22. The apparatus of claim 1 for active biological sample preparation wherein the membrane is a cellulose acetate membrane.

23. The apparatus of claim 1 wherein the first electrode chamber has a volume equal to at least 10 times a volume of at least one of the first or second sample chambers.

24. The apparatus of claim 1 for active biological sample preparation wherein the first sample chamber is disposed vertically above the spacer compartment.

25. A method for active biological sample preparation of a sample comprising a collection of materials including desired materials and undesired materials having differential charge-to-mass ratios, wherein

the desired material comprises specific cells and the undesired material comprises a remainder of the sample,

the separation being achieved in an apparatus as defined in claim 1 comprising the steps of:

providing the sample to the first sample chamber of the apparatus,

electrophoresing the sample within the apparatus to affect net differential migration between the desired material and the undesired material, whereby the desired material is located within the trap and the undesired material is in the second chamber,

removing the desired material from the, apparatus, whereby relatively purified desired materials are prepared.

26. The method of claim 25 wherein the desired material is eluted from the trap.

27. A method for arrive biological sample preparation of a sample comprising a collection of materials including desired materials and undesired materials having differential charge-to-mass ratios wherein

the desired material comprises at least one of DNA and RNA and the undesired material comprises protein,

the separation being achieved in an apparatus as defined in claim 1 comprising the steps of:

providing the sample to the first sample chamber of the apparatus,

electrophoresing the sample within the apparatus to affect net differential migration between the desired material and the undesired material, whereby undesired material is located within the trap and the desired material is in the second chamber,

removing the desired material from the apparatus, whereby relatively purified desired materials are prepared.

28. The apparatus for biological sample preparation of claim 1 wherein the trap is a RNA trap.

29. The apparatus for biological sample preparation of claim 1 wherein the trap is a dipstick.

30. A method for active biological same preparation of a sample comprising a collection of materials including desired materials and undesired materials having differential charge-to-mass ratios, the separation being achieved in an apparatus as defined in any of claims 1-19 and 21-24 comprising the steps of:

providing the sample materials to the first sample chamber of the apparatus,

electrophoresing the sample within the apparatus to affect net differential migration between the desired material and the undesired material whereby one of the desired or undesired materials is located within the trap and the other material is in the second sample chamber,

removing the desired material from the apparatus, whereby relatively purified desired materials are prepared.

31. The method of claim 3 wherein after at least a portion of the desired material has contacted the trap, a potential is applied to the undesired materials in at least one of the first or second sample chambers so as to repel them from the trap.

32. The method of claim 30 for active biological sample preparation wherein the sample is first electrophoretically transported through the first sample chamber prior to contacting the trap.

33. The method of claim 32 for active biological sample preparation wherein the desired material is located within the trap.

34. The method of claim 33 for active biological sample preparation comprising the additional step of removing the desired material and trap from the apparatus.

35. The method of claim 33 for active biological sample preparation comprising the additional step of removing the desired material from the trap.

36. The method of claim 35 for active biological sample preparation comprising the additional step of removing the desired material from the trap to one of the first or second sample chambers.

37. The method of claim 36 for active biological sample preparation wherein the desired material is removed from the trap to the first sample chamber.

38. The method of claim 36 for active biological sample preparation wherein the desired material is removed from the trap to the second sample chamber.

39. The method of claim 32 for active biological sample preparation wherein the undesired material is located within the trap.

40. The method of claim 39 for active biological sample preparation comprising the additional step of removing the desired material from the system by contact with solution in one of the first or second sample chambers.

41. The method of claim 39 for active biological sample preparation wherein the desired materials are electrophoresed through the trap prior to the undesired materials being trapped in the trap.

42. The method of claim 30 for active biological sample preparation wherein the sample is initially placed in the first sample chamber immediately adjacent the trap.

43. The method of claim 42 for active biological sample preparation wherein the desired material is retained in the trap and the undesired material is electrophoresed through the trap into the second sample chamber.

44. The method of claim 43 for active biological sample preparation wherein the desired material is eluted from the trap after the undesired materials are electrophoresed through the trap.

45. The method of claim 44 for active biological sample preparation wherein the desired materials are eluted into the second sample chamber.

46. The method of claim 30 for active biological sample preparation wherein the sample is subject to a proteinase step prior to providing the sample materials to the first sample chamber of the device.

47. The method of claim 30 for active biological sample preparation wherein the sample is subject to a densification step prior to electrophoretic transport of charged materials.

48. The method of claim 47 for active biological sample preparation which further comprises the step of subjecting the sample to a proteinase prior to subjecting the sample to the densification step.

49. The method of claim 30 for active biological sample preparation wherein the sample is subject to a proteinase prior to electrophoretic transport of charged materials.

50. A device for purification of DNA from a sample including proteinaceous material having a charge-to-mass ratio which is greater than that of the DNA, the device comprising:

an upper reservoir adapted for receipt of a buffer solution, and for receipt of a sample solution containing DNA and proteinaceous material,

a collection chamber, disposed downstream of the upper reservoir, adapted for receipt of a buffer solution and for receipt of the DNA, characterized by having a membrane defining at least a portion of the collection chamber,

a polymer, disposed upstream and adjacent to the collection chamber, wherein

the polymer is in a fixed position relative to the collection chamber,

electrodes adapted for electrical contact with the buffer solution to provide electrophoretic movement of the DNA and proteinaceous material through the polymer and collection chamber, wherein

the electrodes are disposed in a direction which is parallel to the membrane, and

the distance between the electrodes and the membrane is equal to or greater than the height of the collection chamber.

51. The device of claim 50 for purification of DNA wherein the membrane is an ultrafiltration membrane.

52. The device claim 50 for purification of DNA wherein the membrane comprises a molecular weight cut-off membrane.

53. The device of claim 50 for purification of DNA wherein the molecular weight cut-off membrane retains the DNA within the collection chamber.

54. The device of claim 50 for purification of DNA wherein the collection chamber has a volume which is smaller than a volume of the polymer.

55. The device of claim 54 wherein the collection chamber volume is less than or equal to half the volume of the polymer.

56. The device of claim 55 for purification of DNA wherein the volume of the collection chamber is equal to or less than 1/3 the volume of the polymer.

57. The device of claim 50 for purification of DNA further comprising a membrane between the polymer and the collection chamber.

58. The device of claim 57 for purification of DNA wherein the membrane has a pore size which permits transport of both the DNA and proteinaceous materials through the membrane.

59. The device of claim 50 for purification of DNA further comprising a lower reservoir adapted to receive a buffer solution and disposed beneath the collection chamber.

60. The device of claim 50 wherein the polymer is a molecular sieve.

61. The device of claim 60 wherein the molecular sieve is acrylamide.

62. The device of claim 50 further comprising a buffer.

63. The device of claim 62 wherein the buffer is histidine.

64. The device of claim 50 wherein the polymer has a thickness in the direction of migration of substantially 10 to 20 mm.

65. A method for purification of DNA from a sample mixture including DNA and other charged materials, including certain materials having a higher charge-to-mass ratio than the DNA, in a device as defined in any of claims 50 to 64, the method comprising the steps of:

placing the sample mixture in the upstream buffer chamber above the polymer,

activating the electrodes so as to electrophorese the sample mixture into the polymer for a time sufficient to cause the undesired material to substantially move through the polymer, and

eluting the DNA into the collection chamber.

66. The method of claim 65 for purification of DNA from a sample mixture which further comprises the step of densification of the sample mixture prior to electrophoresing the sample mixture.

67. The method of claim 66 for purification of DNA from a sample mixture wherein the densification step includes the addition of sucrose.

68. The method of claim 62 for purification of DNA from a sample mixture which comprises the further step of lysing of cells prior to the placement of the sample mixture in the upstream buffer chamber.

69. The method of claim 68 for purification of DNA from a sample mixture which comprises the further step of shearing the sample prior to placement of the sample mixture in the upstream buffer chamber.

70. The method of claim 65 for purification of DNA from a sample mixture which comprises the further step of subjecting the sample to a proteinase prior to electrophoresing the sample mixture.

71. The method of claim 70 for purification of DNA from a sample mixture wherein the sample mixture is subject to an RNAse step prior to said proteinase step.

72. The method of claim 71 wherein the proteinase is proteinase K.

73. The method of claim 65 for purification of DNA from a sample mixture which comprises the further step of changing the buffer solution prior to the step of eluting the DNA into the collection chamber.

74. The method of claim 65 for purification of DNA from a sample mixture which comprises the further step of adding a membrane to the collection chamber prior to eluting the DNA into the collection chamber.

75. The method of claim 65 for purification of DNA from a sample mixture which comprises the further step of extracting the DNA from the collection chamber.

76. The method of claim 75 for purification of DNA from a sample mixture wherein the DNA is extracted from the collection chamber by piercing of the collection chamber.

77. The method of claim 75 for purification of DNA from a sample mixture wherein the DNA is extracted from the collection chamber through a valve.