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1. (WO2017136751) MULTI-STAGE, MULTIPLEXED TARGET ISOLATION AND PROCESSING FROM HETEROGENEOUS POPULATIONS
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WHAT IS CLAIMED IS:

1. A method for isolating cells, comprising:

providing a plurality of beads in a chamber, said beads capable of binding a cell-specific binding marker so as to attach to a specific cell-type;

providing cells to the first chamber, the cells including cells of the specific cell-type, such that ceils of the specific cell-type bind to specific beads of the plurality of beads; and retaining the cells of the specific cell-type bound to the specific beads in the chamber while removing cells not of the specific cell-type from the chamber.

2. The method of claim 1, further comprising releasing the cells of the specific cell-type from the specific beads.

3. The method of claim 1, wherein the ceils of the specific cell-type are retained in the chamber by filtration,

4. The method of claim 1, wherein the cells of the specific cell-type are retained in the chamber by electrophoresis.

5. The method of claim 1, wherein the ceils of the specific cell-type are retained in the chamber by dielectrophoresis.

6. The method of claim 1, wherein the cells of the specific cell-type are retained in the chamber by electro-osmotic flow.

7. The method of claim 1, wherein the cells of the specific cell-type are retained in the chamber by radiation pressure.

8. The method of claim 7, wherein the radiation pressure is photon pressure.

9. The method of claim 1, wherein the ceils of the specific cell-type are retained in the chamber by surface immobilization.

10. The method of claim 1, wherein the specific beads are magnetized and the cells of the specific cell-type are retained in the chamber by applying a magnetic field.

11. The method of claim 1, wherein the specific beads are magnetized and the cells of the specific cell-type are retained in the chamber by applying acoustic waves.

12. The method of claim 1, wherein the specific beads are magnetized and the cells of the specific cell-type are retained in the chamber by applying ultrasonic waves.

13. The method of claim 1, wherein the specific beads are magnetized and the ceils of the specific cell-type are retained in the chamber by applying surface acoustic waves.

14. A method for isolating cells, comprising:

providing a plurality of magnetic beads in a first chamber, said magnetic beads capable of binding a cell-specific binding marker so as to attach to a specific cell-type;

providing cells to the first chamber, the cells including cells of the specific cell-type, such that ceils of the specific cell-type bind to magnetic beads of the plurality of magnetic beads; applying a magnetic field to the first chamber and moving the magnetic field in a predetermined directions to transfer the magnetic beads to a second chamber, the second chamber in fluid com muni cation with the first chamber;

blocking fluid communication between the second chamber and the first chamber; washing the first chamber,

unblocking fluid communication between the second chamber and the first chamber; removing the magnetic beads, including the magnetic beads bound to the cells of the specific cell-type, from the second chamber by applying the magnetic field to the second chamber in a second predetermined direction;

releasing the cells of the specific cell-type from the magnetic beads,

15. The method of claim 14, wherein releasing the ceils comprises eluting.

16. The method of claim 14, wherein the chamber comprises a volume of about 10 nl to about 10 μl.

17. The method of claim 14, wherein said step of applying a magnetic field comprises activating a stationary magnet.

18. The method of claim 7, wherein said step of applying a magnetic field comprises bringing a permanent magnet into proximity of the first chamber.

19. The method of claim 18, wherein the permanent magnet comprises a Rare Earth magnet,

20. The method of claim 18, wherein the permanent magnet comprises a Neodynium magnet.

21. The method of claim 14, wherein said step of applying a magnetic field comprises bringing an electro-magnet into proximity of the first chamber.

22. The method of claim 14, wherein the removing the magnetic beads from the second chamber comprises returning the magnetic beads to the first chamber, and further comprising blocking fluid communication between the second chamber and the first chamber; washing the second chamber; and

unblocking fluid communication between the second chamber and the first chamber, prior to releasing the ceils of the specific cell-type from the magnetic beads.

23. The method of claim 22, wherein releasing the ceils comprises eluting.

24. The method of claim 22, wherein the chamber comprises a volume of about 10 nl to about 10 μl.

25. The method of claim 22, wherein said step of applying a magnetic field comprises bringing a permanent magnet into proximity of the first chamber.

26. The method of claim 25, wherein the permanent magnet comprises a Rare Earth magnet.

27. The method of claim 25, wherein the permanent magnet comprises a Neodynium magnet.

28. The method of claim 22, wherein said step of applying a magnetic field comprises bringing an electro-magnet into proximity of the first chamber.

29. A method for isolating cells, comprising:

providing a plurality of magnetic beads in a first chamber, said magnetic beads capable of binding to a target cell;

providing cells to the first chamber, the cells including target cells, such that the target cells bind to magnetic beads of the plurality of magnetic beads;

a) applying a moveable magnetic field to transfer the magnetic beads between the first chamber and a second chamber;

b) blocking fluid communication between the second chamber and the first chamber; c) washing one of the first chamber and the second chamber to remove non-target cells; d) unblocking fluid communication between the second chamber and the first chamber; e) repeating steps (a)-(d) a number of times;

eluting the target cells from the magnetic beads.

30. The method of claim 29, wherein the number of times that steps (a)-(d) are repeated is predetermined.

31. The method of claim 29, wherein at least a portion of the first chamber or a portion of the second chamber is transparent.

32. The method of claim 29, wherein the number of times that steps (a)-(d) are repeated is adaptively determined based on observations of ceils or beads in the chamber.

33. The method of claim 29, wherein the number of times that steps (a)-(d) are repeated is adaptively determined based on analysis of waste cells or beads resulting from washing.

34. The method of claim 29, wherein releasing the cells comprises eluting.

35. The method of claim 29, wherein the chamber comprises a volume of about 10 nl to about 10 μl.

36. The method of claim 29, wherein said step of applying a magnetic field comprises bringing a permanent magnet into proximity of the first chamber.

37. The method of claim 36, wherein the permanent magnet comprises a Rare Earth magnet.

38. The method of claim 37, wherein the permanent magnet comprises a Neodynium magnet.

39. The method of claim 29, wherein said step of applying a magnetic field comprises bringing an electro-magnet into proximity of the first chamber.

40. A method for isolating target substrates,, comprising:

(a) providing a plurality of magnetic beads to a plurality of respective first chambers, each respective plurality of magnetic beads provided to each first chamber containing only magnetic beads capable of binding to a respective target substrate such that each respective mixing chamber includes magnetic beads capable of binding to only one target substrate type,

(b) providing a sample comprising multiple target substrate types to a plurality of respective first chambers,

(c) applying a moveable magnetic field to transfer the magnetic beads, including any substrates bound thereto, between the first chambers and a second chambers;

(d) blocking fluid communication between the second chambers and the first chambers;

(e) washing select ones of the first chambers and the second chambers to remove non-bound substrates;

(f) unblocking fluid communication between the second chambers and the first chambers;

(g) repeating steps (c)-(f) a predetermined number of times;

(h) releasing the respective target substrates from the magnetic beads.

41. The method of claim 40, wherein the plurality of first chambers comprises 2 - 1000 first chambers.

42. The method of claim 40, wherein releasing the respective target substrates comprises eluting.

43. The method of claim 40, wherein the chamber comprises a volume of about 10 nl to about 10 μl.

44. The method of claim 40, wherein said step of applying a magnetic field comprises bringing a permanent magnet into proximity of the first chambers.

45. The method of claim 44, wherein the permanent magnet comprises a Rare Earth magnet,

46. The method of claim 45, wherein the permanent magnet comprises a Neodynium magnet.

47. The method of claim 40, wherein said step of applying a magnetic field comprises bringing an electro-magnet into proximity of the first chamber,

48. The method of claim 40, wherein said step of applying a magnetic field comprises activating a stationary magnet.

49. The method of claim 48, wherein the stationary magnet is an electromagnet.

50. The method of claim 40, wherein the target substrates include at least one of a molecule, cell, DNA, DNA fragment, RNA, and RNA fragment.

51. A method for mixing substrates in on a microfluidic platform, comprising:

inserting a plurality of a first target substrate in a chamber;

inserting a plurality of second target substrate in the chamber;

inserting a plurality of magnetic beads in the chamber;

applying a magnetic field environment to the chamber; and

changing the magnetic field environment to move the beads in a first direction.

52. A method for mixing substrates in on a microfluidic platform, comprising:

inserting a plurality of a first target substrate in a chamber;

inserting a plurality of second target substrate in the chamber;

inserting a plurality of magnetic beads in the chamber;

applying a moveable magnetic field to the chamber;

moving the magnetic field in a first direction,

moving the magnetic field in a second direction different from the first direction; and removing the magnetic beads from the chamber.

53. The method of claim 52, wherein the chamber comprises a volume of about 10 nl to about 10 μl.

54. The method of claim 52, wherein said step of applying a magnetic field comprises activating a stationary magnet.

55. The method of claim 52, wherein the stationary magnet is an electromagnet.

56. The method of claim 52, wherein said step of applying a moveable magnetic field comprises bringing a permanent magnet into proximity of the chamber.

57. The method of claim 56, wherein the permanent magnet comprises a Rare Earth magnet,

58. The method of claim 57, wherein the permanent magnet comprises a Neodynium magnet.

59. The method of claim 52, wherein said step of applying a moveable magnetic field comprises bringing an electro-magnet into proximity of the first chamber.

60. The method of claim 52, wherein the first target substrate includes at least one of a molecule, cell, DNA, DNA fragment, RNA, and RNA fragment.

61. The method of claim 52, wherein the second target substrate includes at least one of a molecule, cell, DNA, DNA fragment, RNA, and RNA fragment.

62. A substrate-isolation platform, comprising:

a microfluidic chip, comprising a plurality of processing units, each processing unit comprising: an inlet port, a plurality of first chambers connected to the inlet port by a fluid channel, the fluid channel comprising a plurality of valves, a plurality of second chambers, each of the second chambers connected to a respective first chamber by a fluid channel, each fluid channel including a controllable blocking valve, and a plurality of respective outlet ports, each outlet port in fluid communication with a respective one of said second chambers and each outlet port including a blocking valve, and

a magnet adjacent the microfluidic chip, wherein relative positioning of the magnet and the microfluidic chip is variable.;

a valve control capable of actuating certain ones of the controllable blocking valves in response to a control signal.

63. The platform of claim 62, wherein at least one of the first chambers and the second chambers are ring chambers.

64. The platform of claim 62, wherein the microfluidic chip comprises a volume of about 10 nl to about 10 μl.

65. The platform of claim 62, wherein the microfluidic chip comprises at least two layers,

66. The platform of claim 65, wherein at least one layer comprises high thermal conductivity.

67. The platform of claim 65, wherein at least one layer comprises a quart layer.

68. The platform of claim 65, wherein at least one layer comprises a silica layer.

69. The platform of claim 62, wherein the magnet produces a dynamic magnetic field.

70. The platform of claim 62, wherein the magnet is an electromagnet.

71. The platform of claim 62, wherein magnet is a permanent magnet.

72. The platform of claim 71, wherein the magnet comprises a Rare Earth magnet,

73. The platform of claim 72, wherein the magnet comprises a Neodynium magnet.

74. The platform of claim 62, wherein each microchip comprises multiple processing units.

75. The platform of claim 74, wherein each microchip comprises 2-1000 processing units.

76. The platform of claim 74, wherein at least a portion of the first chamber or a portion of the second chamber is transparent.

77. The platform of claim 74, wherein the first chambers comprise a plurality of sub-chambers.

78. The platform of claim 77, wherein comprising a sufficient number of said sub-chambers to enable mixing of at least 10 components in pre-defined ratios.

79. The platform of claim 74, wherein the second chambers comprise a plurality of sub-chambers.

80. The platform of claim 77, wherein comprising a sufficient number of said sub-chambers to enable mixing of at least 10 components in pre-defined ratios.

81 . A method of integrated subtype purification and RNA- Seq on the platform of claim 62.

82. A method for isolating a target substrate, comprising;

providing a plurality of first magnetic beads in a first chamber, said first magnetic beads capable of binding a cell-specific marker so as to attach a specific cell-type;

providing cells to the first chamber, the cells including cells of the specific cell-type, such that cells of the specific cell -type bind to the first magnetic beads;

applying a magnetic field to the first chamber and moving the magnetic field in a first predetermined direction to transfer the first magnetic beads to a second chamber, the second chamber in fluid communication with the first chamber;

blocking fluid communication between the second chamber and the first chamber; washing the first chamber;

unblocking fluid communication between the second chamber and the first chamber; releasing the cells from the first magnetic beads;

removing the first magnetic beads from the second chamber by applying a magnetic field from the second chamber in a second predetermined direction;

blocking fluid communication between the second chamber and the first chamber; lysing the cells;

capturing target substrates from the lysed cells using second magnetic beads; applying a magnetic field to the second chamber and moving the magnetic field in a third predetermined direction to transfer the target substrates captured by the second magnetic beads to the first chamber;

mixing the second magnetic beads and the captured target substrates with mRNA-seq reagents;

cycling through a range of temperatures to create a PCR product; and

applying DNA-binding beads to the PCR product to clean up DNA.

83. The method of claim 82, wherein the second magnetic beads are oligo (dT) beads.

84. The method of claim 82, wherein the second magnetic beads are Solid Phase Reversible Immobilization beads.

85. The method of claim 82, wherein the DNA-binding beads are charge switch silica beads.

86. The method of claim 82, wherein the DNA-binding beads are solid phase reversible immobilization (SPRI) beads.

87. The method of claim 82, wherein the DNA-binding beads are dsDNA antibodies.

88. The method of claim 82, wherein the target substrates are RNA.

89. The method of claim 82, wherein the cycling ranges from about 5 cycles to about 100 cycles.

90. The method of claim 82, wherein the cycling ranges from about 10 cycles to about 90 cycles.

91. The method of claim 82, wherein the cycling ranges from about 5 cycles to about 90 cycles.

92. The method of claim 82, wherein the cycling ranges from about 10 cycles to about 80 cycles,

93. The method of claim 82, wherein the cycling ranges from about 10 cycles to about 70 cycles.

94. The method of claim 82, wherein the cycling ranges from about 10 cycles to about 60 cycles.

95. The method of claim 82, wherein the cycling ranges from about 10 cycles to about 50 cycles.

96. The method of claim 82, wherein the cycling ranges from about 10 cycles to about 40 cycles.

97. The method of claim 82, wherein the cycling ranges from about 10 cycles to about 30 cycles.

98. A method of integrated subtype purification and RNA- Seq on a platform comprising a microfluidic chip, comprising a plurality of processing units, each processing unit comprising: an inlet port, a plurality of first chambers connected to the inlet port by a fluid channel, the fluid channel comprising a plurality of valves, a plurality of second chambers, each of the second chambers connected to a respective first chamber by a fluid channel, each fluid channel including a controllable blocking valve, and a plurality of respective outlet ports, each outlet port in fluid communication with a respective one of said second chambers and each outlet port including a blocking valve; and a movable magnet adjacent the microfluidic chip, the method comprising:

providing a plurality of first magnetic beads in a first chamber, said first magnetic beads capable of binding a cell-specific marker so as to attach a specific cell-type;

providing cells to the first chamber, the cells including cells of the specific cell-type, such that ceils of the specific ceil-type bind to the first magnetic beads;

applying a magnetic field to the first chamber and moving the magnetic field in a first predetermined direction to transfer the first magnetic beads to a second chamber, the second chamber in fluid communication with the first chamber;

blocking fluid communication between the second chamber and the first chamber; washing the first chamber;

unblocking fluid communication between the second chamber and the first chamber; releasing the cells from the first magnetic beads;

removing the first magnetic beads from the second chamber by applying a magnetic field from the second chamber in a second predetermined direction;

blocking fluid communication between the second chamber and the first chamber; lysing the cells in the second chamber;

capturing target substrates from the lysed cells using dynamically altered magnetic beads; applying a magnetic field to the second chamber and moving the magnetic field in a third predetermined direction to transfer the target substrates captured by the dynamically altered magnetic beads to the first chamber,

mixing the dynamically altered magnetic beads and the captured target substrates with mRNA-seq reagents;

cycling through a range of temperatures to create a PGR product; and

applying DNA-binding beads to the PGR product to clean up DNA.

99. The method of claim 89, wherein the second magnetic beads are oligo (dT) beads.

100. The method of claim 89, wherein the second magnetic beads are Solid Phase Reversible Immobilization beads.

101. The method of claim 89, wherein the DNA-binding beads are charge switch silica beads.

102. The method of claim 89, wherein the DNA-binding beads are solid phase reversible immobilization (SPRI) beads.

103. The method of claim 89, wherein the DNA-binding beads are dsDNA antibodies.

104. A kit comprising an integrated subtype purification and RNA- Seq on a platform comprising a microfluidic chip, comprising a plurality of processing units, each processing unit comprising: an inlet port, a plurality of first chambers connected to the inlet port by a fluid channel, the fluid channel comprising a plurality of valves, a plurality of second chambers, each of the second chambers connected to a respective first chamber by a fluid channel, each fluid channel including a controllable blocking valve, and a plurality of respective outlet ports, each outlet port in fluid communication with a respective one of said second chambers and each outlet port including a blocking valve; and a movable magnet adjacent the microfluidic chip, the method comprising:

providing a plurality of first magnetic beads in a first chamber, said first magnetic beads capable of binding a cell-specific marker so as to attach a specific cell-type;

providing cells to the first chamber, the cells including cells of the specific cell-type, such that cells of the specific cell-type bind to the first magnetic beads;

applying a magnetic field to the first chamber and moving the magnetic field in a first predetemiined direction to transfer the first magnetic beads to a second chamber, the second chamber in fluid com muni cation with the first chamber;

blocking fluid communication between the second chamber and the first chamber; washing the first chamber;

unblocking fluid communication between the second chamber and the first chamber; releasing the cells from the first magnetic beads,

removing the first magnetic beads from the second chamber by applying a magnetic field from the second chamber in a second predetermined direction;

blocking fluid communication between the second chamber and the first chamber; lysing the cells in the second chamber;

capturing target substrates from the lysed cells using dynamically altered magnetic beads; applying a magnetic field to the second chamber and moving the magnetic field in a third predetermined direction to transfer the target substrates captured by the dynamically altered magnetic beads to the first chamber;

mixing the dynamically altered magnetic beads and the captured target substrates with mRNA-seq reagents;

cycling through a range of temperatures to create a PCR product; and

applying DNA-binding beads to the PCR product to clean up DNA.

105. The kit according to claim 104, further comprising magnetic beads in a first chamber comprises the first magnetic beads conjugated to an antibody wherein the first magnetic beads are capable of binding a cell-specific marker so as to attach a specific cell-type.

106. The kit according to claim 105, wherein the antibody and cell-specific marker are cleaved.

107. The kit according to claim 105, wherein the antibody and cell-specific marker are enzymatically cleaved.