Integrated portable biological detection system

• US 6,403,367 B1
• Filed: 12/22/1999
• Issued: 06/11/2002
• Est. Priority Date: 07/07/1994
• Status: Expired due to Fees

• Current Assignee: Gamida for Life B.V.
• Original Assignee: Nanogen, Inc.
• Inventors: Diver, Jonathan, Willoughby, David, Sheldon, Ed, Jalali, Shila, OʼConnell, James P., Wu, Lei, Cheng, Jing, Heller, Michael J., Smolko, Dan
• Primary Examiner(s): Redding, David A.
• Application Number:
  US09/470,448
• Time in Patent Office: 902 Days
• Field of Search: 204/547, 204/643, 204/545, 204/403, 204/409, 435/287.1, 435/287.2, 435/288.5, 422/66, 422/50, 422/81, 422/82.01, 422/129
• US Class Current: 435/287.1
• 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 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• 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: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00686 Automatic
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00713 Electrochemical synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00853 Employing electrode arrangements
• CPC Class Codes: B01J 2219/00914 by dielectrophoresis
• CPC Class Codes: B01L 2200/0631 Purification arrangements, e.g. solid phase extraction [SPE]
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• 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 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 2300/087 : Multiple sequential chambers
• CPC Class Codes: B01L 2300/0877 : Flow chambers
• CPC Class Codes: B01L 2400/0415 : electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0421 : electrophoretic flow
• CPC Class Codes: B01L 2400/0424 : Dielectrophoretic forces
• CPC Class Codes: B01L 2400/0496 : Travelling waves, e.g. in combination with electrical or acoustic forces
• CPC Class Codes: B01L 2400/0644 : rotary valves
• 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 3/502753 : 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: B01L 3/502761 : specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 7/52 : with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• 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: B82Y 10/00 : Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 5/00 : Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• 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: C07K 1/045 : using devices to improve synthesis, e.g. reactors, special vessels
• CPC Class Codes: C12N 13/00 : Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• 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 1/6825 : Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6832 : Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/107 : incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2531/101 : Linear amplification, i.e. non exponential
• CPC Class Codes: C12Q 2531/113 : PCR
• CPC Class Codes: C12Q 2531/119 : Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2565/515 : characterised by the interaction between or sequential use of two or more arrays
• CPC Class Codes: C12Q 2565/607 : being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 27/44743 : Introducing samples
• CPC Class Codes: G11C 13/0014 : comprising cells based on organic memory material
• CPC Class Codes: G11C 13/0019 : comprising bio-molecules
• CPC Class Codes: G11C 13/04 : using optical elements ; using other beam accessed elements, e.g. electron or ion beam
• CPC Class Codes: G11C 19/00 : Digital stores in which the information is moved stepwise, e.g. shift registers
• CPC Class Codes: H01L 25/50 : Multistep manufacturing processes of assemblies consisting of devices, each device being of a type provided for in group H01L27/00 or H01L29/00 H01L21/50 takes precedence
• CPC Class Codes: H01L 2924/00 : Indexing scheme for arrangements or methods for connecting or disconnecting semiconductor or solid-state bodies as covered by H01L24/00
• CPC Class Codes: H01L 2924/0002 : Not covered by any one of groups H01L24/00, H01L24/00 and H01L2224/00

Abstract

We have performed separation of bacterial and cancer cells from peripheral human blood in microfabricated electronic chips by dielectrophoresis. The isolated cells were examined by staining the nuclei with fluorescent dye followed by laser induced fluorescence imaging. We have also released DNA and RNA from the isolated cells electronically and detected specific marker sequences by DNA amplification followed by electronic hybridization to immobilized capture probes. Efforts towards the construction of a “laboratory-on-a-chip” system are presented which involves the selection of DNA probes, dyes, reagents and prototyping of the fully integrated portable instrument.

Claims

1. An integrated system for the analysis of eukaroytic and/or prokaryotic cells in a biological sample comprising:

a. an electronic system for cell separation, cell lysis, sample preparation, and sample analysis further comprising;

at least one input and at least one output port connected to a flow cell, a plurality of individually addressable electrodes positioned within said flow cell wherein each electrode is capable of being electrically biased separately from the other electrodes, and a heating element connected to said flow cell, said flow cell adapted to receive said sample via the input, said electrodes being operatively coupled to a power source adapted to electronically disrupt cell membranes within said sample, said electrodes further comprising an active matrix array adapted to bind to predetermined molecules of interest within said sample for analysis of said molecules;

b. a detector operatively positioned to detect said molecules bound to said active matrix array by a detectable signal; and

c. a power source.

2. An integrated system of claim 1 wherein the sample preparation further includes the step of cell separation.

3. An integrated system of claim 2 wherein the cell separation is performed electronically.

4. An integrated system of claim 1 further comprising an illumination source operatively positioned to provide radiation to the active matrix array.

5. An integrated system of claim 4 wherein the illumination source is a laser.

6. An integrated system of claim 5 wherein the laser is a diode laser.

7. An integrated system of claim 5 wherein the system includes a beam splitter.

8. An integrated system of claim 7 wherein the beam splitter is positioned to receive the illumination and reflect at least a portion thereof to the active matrix array, and to pass at least a portion of the radiation from the active matrix array.

9. An integrated system of claim 1 wherein said plurality of individually addressable electrodes comprising the active matrix array are used as a unit for each of said cell separation, cell lysis, and sample analysis.

10. An integrated system of claim 1 wherein said plurality of individually addressable electrodes comprising the active matrix array are used independent of one another in the formation of subsets of electrodes for cell separation, cell lysis, and sample analysis.

11. An integrated system of claim 1 wherein the sample preparation includes further reaction steps after said electronic lysis.

12. An integrated system of claim 11 wherein the further reaction includes enzymatic reaction.

13. An integrated system of claim 11 wherein the further reaction includes chemical reaction.

14. An integrated system of claim 11 wherein the further reaction includes protease reactions.

15. An integrated system of claim 14 wherein the protease reaction includes protease-K reaction.

16. An integrated system of claim 1 wherein the further reaction includes an amplification reaction.

17. An integrated system of claim 16 wherein the amplification includes SDA.

18. An integrated system of claim 16 wherein the amplification includes PCR.

19. An integrated system of claim 16 wherein the amplification includes linear amplification.

20. An integrated system of claim 16 wherein the amplification includes exponential amplification.

21. An integrated system of claim 1 wherein the power source provides a time varying signal to at least certain of the electrodes.

22. An integrated system of claim 21 wherein the time varying signal includes an AC component.

23. An integrated system of claim 22 wherein the AC component is sinusoidal.

24. An integrated system of claim 21 wherein the time varying signal includes an offset signal.

25. An integrated system of claim 24 wherein the offset is a DC offset.

26. An integrated system of claim 1 further including a control system.

27. An integrated system of claim 26 wherein the control system is a computer.

28. An integrated system of claim 27 wherein the computer is a personal computer.

29. An integrated system of claim 28 wherein the personal computer is a notebook computer.

30. An integrated system of claim 1 wherein the flow cell of the electronic system includes at least one port in addition to the input and the output ports.

31. An integrated system of claim 30 wherein the flow cell of the electronic system includes at least two ports in addition to the input and the output ports.

32. An integrated system of claim 31 wherein the ports are at substantially 90 degrees to another port.

33. An integrated system of claim 30 wherein at least two of the ports are in opposition to one another.

34. An integrated system of claim 1 wherein the detector is a CCD detector.

35. An integrated system of claim 1 wherein the system is a closed system.

36. An integrated system of claim 1 wherein the system is a self contained system.

37. An integrated system of claim 1 wherein the system further includes a fluidic system.

38. An integrated system of claim 37 wherein the fluidic system includes a pump.

39. An integrated system of claim 37 wherein the fluidic system includes a plurality of valves.

40. An integrated system of claim 1 wherein the sample analysis includes binding of molecules to the array selected from the group consisting of hybridization and protein-ligand binding interactions.

41. An integrated system of claim 40 wherein the hybridization is DNA-DNA hybridization.

42. An integrated system of claim 40 wherein the hybridization is DNA/RNA hybridization.

43. An integrated system of claim 40 wherein the hybridization is pNA/nucleic acid hybridization.

44. An integrated system of claim 40 wherein the protein-ligand binding is selected from the group consisting of receptor-ligand, antigen-antibody, and enzyme-substrate binding.

45. An integrated system for the analysis of a biological sample containing eukaryotic and/or prokaryotic cells, comprising:

a. a first electronic system for sample preparation, said system further comprising;

at least one input port, a first flow cell connected to the input, a plurality of electrodes positioned within said flow cell wherein each electrode is capable of being electrically biased separately from the other electrodes, and at least one output port, said flow cell adapted to receive said sample via the input, said electrodes being operatively coupled to a power source adapted to electronically disrupt cell membranes within said sample;

b. a second electronic system for sample analysis, said system further comprising;

at least one input port, a second flow cell connected to the input, a plurality of electrodes positioned within said second flow cell wherein each electrode is capable of being electrically biased separately from the other electrodes, said electrodes comprising an active matrix array adapted to bind to predetermined molecules of interest within said sample for analysis of said molecules, and at least one output port, said input of the second electronic system being coupled to at least one output port of the first electronic system;

c. a detector operatively positioned to detect said molecules bound to said active matrix array by a detectable signal; and

d. a power source.

46. An integrated system of claim 45 wherein the sample preparation further includes the step of cell separation.

47. An integrated system of claim 46 wherein the cell separation is performed electronically.

48. An integrated system of claim 45 further comprising an illumination source operatively positioned to provide radiation to the active matrix array.

49. An integrated system of claim 48 wherein the illumination source is a laser.

50. An integrated system of claim 49 wherein the laser is a diode laser.

51. An integrated system of claim 48 wherein the system includes a beam splitter.

52. An integrated system of claim 51 wherein the beam splitter is positioned to receive the illumination and reflect at least a portion thereof to the active matrix array, and to pass at least a portion of the radiation from the active matrix array.

53. An integrated system of claim 45 wherein said plurality of individually addressable electrodes of said first electronic system are used as a unit for each of said cell separation and cell lysis.

54. An integrated system of claim 45 wherein said plurality of individually addressable electrodes of said second electronic system are used as a unit for said analysis.

55. An integrated system of claim 45 wherein said plurality of individually addressable electrodes of said first electronic system are used independent of one another in the formation of subsets of electrodes for cell separation and cell lysis.

56. An integrated system of claim 45 wherein said plurality of individually addressable electrodes of said second electronic system are used independent of one another in the formation of subsets of electrodes for said analysis.

57. An integrated system of claim 45 wherein the sample preparation includes further reaction steps after said electronic lysis.

58. An integrated system of claim 57 wherein the further reaction includes enzymatic reaction.

59. An integrated system of claim 57 wherein the further reaction includes chemical reaction.

60. An integrated system of claim 57 wherein the further reaction includes protease reactions.

61. An integrated system of claim 60 wherein the protease reaction includes protease-K reaction.

62. An integrated system of claim 57 wherein the further reaction includes an amplification reaction.

63. An integrated system of claim 62 wherein the amplification includes SDA.

64. An integrated system of claim 62 wherein the amplification includes PCR.

65. An integrated system of claim 62 wherein the amplification includes linear amplification.

66. An integrated system of claim 62 wherein the amplification includes exponential amplification.

67. An integrated system of claim 45 wherein the power source provides a time varying signal to at least certain of the electrodes.

68. An integrated system of claim 67 wherein the time varying signal includes an AC component.

69. An integrated system of claim 68 wherein the AC component is sinusoidal.

70. An integrated system of claim 67 wherein the time varying signal includes an offset signal.

71. An integrated system of claim 70 wherein the offset is a DC offset.

72. An integrated system of claim 45 further including a control system.

73. An integrated system of claim 72 wherein the control system is a computer.

74. An integrated system of claim 73 wherein the computer is a personal computer.

75. An integrated system of claim 74 wherein the personal computer is a notebook computer.

76. An integrated system of claim 45 wherein the first flow cell of the first electronic system includes at least one port in addition to the input and the output ports.

77. An integrated system of claim 76 wherein the first flow cell of the first electronic system includes at least two ports in addition to the input and the output ports.

78. An integrated system of claim 77 wherein the ports are at substantially 90 degrees to another port.

79. An integrated system of claim 76 wherein at least two of the ports are in opposition to one another.

80. An integrated system of claim 45 further including at least one heater element.

81. An integrated system of claim 80 wherein a heater is disposed on the first electronic system.

82. An integrated system of claim 80 wherein a heater is disposed on the second electronic system.

83. An integrated system of claim 80 wherein the heater element is in thermal contact with either of the first or second electronic systems.

84. An integrated system of claim 45 wherein the detector is a CCD detector.

85. An integrated system of claim 45 wherein the system is a closed system.

86. An integrated system of claim 45 wherein the system is a self contained system.

87. An integrated system of claim 45 wherein the system further includes a fluidic system.

88. An integrated system of claim 87 wherein the fluidic system includes a pump.

89. An integrated system of claim 87 wherein the fluidic system includes a plurality of valves.

90. An integrated system of claim 45 wherein the sample analysis includes binding of molecules to the array selected from the group consisting of hybridization and protein-ligand binding interactions.

91. An integrated system of claim 90 wherein the hybridization is DNA-DNA hybridization.

92. An integrated system of claim 90 wherein the hybridization is DNA/RNA hybridization.

93. An integrated system of claim 90 wherein the hybridization is pNA/nucleic acid hybridization.

94. An integrated of claim 90 wherein the protein-ligand binding is selected from the group consisting of receptor-ligand, antigen-antibody, and enzyme-substrate binding.

95. An integrated system for the analysis of a biological sample containing eukaroytic and/or prokaryotic cells, comprising:

a. a first nonelectronic system for sample preparation, said system further comprising;

at least one input port, a first flow cell connected to the input, a heater element, and at least one output port, said flow cell adapted to receive said sample via the input, said heater being operatively coupled to a power source adapted to heat said flow cell to a temperature sufficient to induce disruption of cell membranes within said sample;

b. a second electronic system for sample analysis, said system further comprising;

at least one input port, a second flow cell connected to the input, a plurality of electrodes positioned within said second flow cell wherein each electrode is capable of being electrically biased separately from the other electrodes, said electrodes comprising an active matrix array adapted to bind to predetermined molecules of interest within said sample for analysis of said molecules, and at least one output port, said input of the second electronic system being coupled to at least one output port of the first nonelectronic system;

c. a detector operatively positioned to detect said molecules bound to said active matrix array by a detectable signal; and

d. a power source.

96. An integrated system of claim 95 further comprising an illumination source operatively positioned to provide radiation to the active matrix array.

97. An integrated system of claim 96 wherein the illumination source is a laser.

98. An integrated system of claim 97 wherein the laser is a diode laser.

99. An integrated system of claim 96 wherein the system includes a beam splitter.

100. An integrated system of claim 99 wherein the beam splitter is positioned to receive the illumination and reflect at least a portion thereof to the active matrix array, and to pass at least a portion of the radiation from the active matrix array.

101. An integrated system of claim 95 wherein said plurality of individually addressable electrodes of said electronic system are used as a unit for said analysis.

102. An integrated system of claim 95 wherein said plurality of individually addressable electrodes of said electronic system are used independent of one another in the formation of subsets of electrodes for said analysis.

103. An integrated system of claim 95 wherein the sample preparation includes further reaction steps after said heater induced lysis.

104. An integrated system of claim 103 wherein the further reaction includes enzymatic reaction.

105. An integrated system of claim 103 wherein the further reaction includes chemical reaction.

106. An integrated system of claim 103 wherein the further reaction includes protease reactions.

107. An integrated system of claim 106 wherein the protease reaction includes protease-K reaction.

108. An integrated system of claim 103 wherein the further reaction includes an amplification reaction.

109. An integrated system of claim 108 wherein the amplification includes SDA.

110. An integrated system of claim 108 wherein the amplification includes PCR.

111. An integrated system of claim 108 wherein the amplification includes linear amplification.

112. An integrated system of claim 108 wherein the amplification includes exponential amplification.

113. An integrated system of claim 95 wherein the power source provides a time varying signal to at least certain of the electrodes.

114. An integrated system of claim 113 wherein the time varying signal includes an AC component.

115. An integrated system of claim 114 wherein the AC component is sinusoidal.

116. An integrated system of claim 113 wherein the time varying signal includes an offset signal.

117. An integrated system of claim 116 wherein the offset is a DC offset.

118. An integrated system of claim 95 further including a control system.

119. An integrated system of claim 118 wherein the control system is a computer.

120. An integrated system of claim 119 wherein the computer is a personal computer.

121. An integrated system of claim 120 wherein the personal computer is a notebook computer.

122. An integrated system of claim 95 wherein the first flow cell of the nonelectronic system includes at least one port in addition to the input and the output ports.

123. An integrated system of claim 122 wherein the first flow cell of the nonelectronic system includes at least two ports in addition to the input and the output ports.

124. An integrated system of claim 123 wherein the ports are at substantially 90 degrees to another port.

125. An integrated system of claim 122 wherein at least two of the ports are in opposition to one another.

126. An integrated system of claim 95 further including at least two heater elements.

127. An integrated system of claim 126 wherein a heater is disposed on the electronic system.

128. An integrated system of claim 126 wherein a heater element is in thermal contact with either of the nonelectronic or electronic systems.

129. An integrated system of claim 95 wherein the detector is a CCD detector.

130. An integrated system of claim 95 wherein the system is a closed system.

131. An integrated system of claim 95 wherein the system is a self contained system.

132. An integrated system of claim 95 wherein the system further includes a fluidic system.

133. An integrated system of claim 132 wherein the fluidic system includes a pump.

134. An integrated system of claim 132 wherein the fluidic system includes a plurality of valves.

135. An integrated system of claim 95 wherein the sample analysis includes binding of molecules to the array selected from the group consisting of hybridization and protein-ligand binding interactions.

136. An integrated system of claim 135 wherein the hybridization is DNA-DNA hybridization.

137. An integrated system of claim 135 wherein the hybridization is DNA/RNA hybridization.

138. An integrated system of claim 135 wherein the hybridization is pNA/nucleic acid hybridization.

139. An integrated of claim 135 wherein the protein-ligand binding is selected from the group consisting of receptor-ligand, antigen-antibody, and enzyme-substrate binding.