Multi-parameter assays including analysis discs and methods relating thereto

• US 20020151043A1
• Filed: 04/11/2002
• Published: 10/17/2002
• Est. Priority Date: 04/11/2001
• Status: Active Grant

• Current Assignee: Vindur Technologies, Inc.
• Original Assignee: Nagaoka & Co., Ltd.
• Inventors: Gordon, John Francis
• Granted Patent: US 7,033,747 B2
• Time in Patent Office: Days
• US Class Current: 435/287.2
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0694 Creating chemical gradients in a fluid
• CPC Class Codes: B01L 2300/0803 Disc shape
• CPC Class Codes: B01L 2300/0883 Serpentine channels
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• 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: G01N 21/07 Centrifugal type cuvettes G01N21/09 takes precedence
• CPC Class Codes: G01N 21/64 Fluorescence; Phosphorescence
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/76 Chemiluminescence; Bioluminescence
• CPC Class Codes: G01N 21/77 by observing the effect on a chemical indicator
• CPC Class Codes: G01N 33/523 the element being adapted for a specific analyte
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 35/00069 whereby the sample substrate is of the bio-disk type, i.e. having the format of an optical disk

Abstract

Methods and apparatus for testing a sample for target molecules or chemicals. The apparatus includes a rotatable optical disc having a reaction chamber and having at least two groups of beads or micro-particles, the different group of beads having at least two different densities, sizes, shapes, and/or color, and each bead in a group having different probes attached thereto. A sample is added to the reaction chamber and the disc is rotated. The reaction chamber has a density gradient medium that causes beads of different densities to remain at different radial locations, depending on the density of the beads. The beads are then inspected by directing a beam of electromagnetic radiation onto the disc. The beam may be reflected from or transmitted through the disc. The amount, or presence, or absence of targets is determined by analyzing the signal returned from the beam. Related methods of performing assays and making the disc apparatus are provided.

Claims

1. A disc system for conducting an assay on a sample, comprising;

a rotatable disc with a chamber for receiving the sample;

a medium in the chamber for forming a density gradient; and

a plurality of beads including at least two different groups of beads, each of the beads in each group having a common physical characteristic and having attached thereto at least one capture probe with affinity to a specific target.

2. The system according to claim 1 wherein said common physical characteristic of the beads of one group is different from the physical characteristic associated with the other groups.

3. The system according to claim 2 wherein said physical characteristic is density such that when the disc is rotated, the beads from the different groups move radially within the density gradient to different radial locations within the chamber.

4. The system according to claim 2 wherein said physical characteristic is bead size.

5. The system according to claim 2 wherein said physical characteristic is color.

6. The system according to claim 2 wherein said physical characteristic is a predetermined fluorescent characteristic.

7. The system of claim 2 wherein the rotatable disc includes an upper cover disc, a base, and a body disposed between the cover and the base, the body defining the chamber.

8. The system of claim 2 wherein the chamber has two sub-chambers with a controllable barrier therebetween, said controllable barrier being controlled by centripetal forces.

9. The system of claim 2 wherein the chamber has two sub-chambers with a controllable barrier therebetween, said controllable barrier being controlled by electrical or mechanical means.

10. The system of claim 2 wherein the capture probe is selected from the group comprising antigens, antibodies, DNA, RNA, lectins, protein receptors, ligands, biotin, and streptavidin.

11. The system of claim 2 wherein the density gradient in the medium is formed by application of centripetal force when the disc is rotated.

12. The system of claim 2 further comprising a detection assembly including a radiation source and a detector for detecting radiation modulated by the sample.

13. The system of claim 12 wherein the radiation source includes a laser.

15. The method according to claim 14 wherein the beads of one group have a different physical characteristic as that of the other groups.

16. The method according to claim 15 wherein said physical characteristic is density such that when the disc is rotated, the beads from the different groups move radially within the density gradient to different radial locations within the chamber.

17. The method according to claim 15 wherein said physical characteristic is bead size.

18. The method according to claim 15 wherein said physical characteristic is color.

19. The method according to claim 15 wherein said physical characteristic is a pre-determined fluorescent characteristic.

20. The method according to claim 15 wherein the chamber includes a reaction chamber for receiving the sample and a separation chamber separated by a barrier.

21. The method of claim 20 including means for controlling rotation of the disc to maintain a speed sufficient to cause the beads to move from the reaction chamber through the barrier into the separation chamber.

22. The method according to claim 15 wherein the labeled tag is a bead.

23. The method according to claim 15 wherein the labeled tag is selected from the group comprised of fluorescent labeled antibodies, oligonucleotides, lectins, protein receptors, biotin, and streptavidin.

24. The method of either claim 22 or 23 including the further step of optically analyzing the chamber for the presence of beads, tags, labels, and binding partner complexes.

25. The method of claim 24 wherein said optically analyzing step includes directing a beam of radiation to the chamber and detecting a beam modulated by the sample.

27. The method according to claim 26 wherein the micro-particles of one group have a different physical characteristic as compared to the other groups.

28. The method according to claim 27 wherein said physical characteristic is density such that when the disc is rotated, the micro-particles from the different groups move radially within the density gradient to different radial locations within the chamber.

29. The method according to claim 27 wherein said physical characteristic is micro-particle size.

30. The method according to claim 27 wherein said physical characteristic is color.

31. The method according to claim 27 wherein said physical characteristic is a pre-determined fluorescent characteristic.

32. The method according to claim 27 wherein said physical characteristic is a pre-determined phosphorescent characteristic.

33. The method according to claim 27 wherein said physical characteristic is a pre-determined chemiluminescent characteristic.

34. The method according to claim 27 further comprising the step of introducing a labeled reagent into the reaction chamber prior to rotating the disc.

35. The method according to claim 34 wherein said labeled reagent may be mixed with the test sample, prior to introducing the mixture into the reaction chamber.

37. The device according to claim 36 wherein the rotatable disc is adapted to rotate about its central aperture which is adapted to releasably engage with a rotatable shaft.

38. The device according to claim 36 wherein the rotatable disc includes a plurality of radially extending reaction chambers and separation chambers.

39. The device according to claim 36 wherein the density gradient forming medium is a gel provided by Pharmacia Biotech under the trademark Percoll.

40. The device according to claim 39 wherein the density gradient may be preformed in the gel, or generated by the application of a centripetal force.

41. The device according to claim 40 wherein the density gradient is formed and maintained by rotating the disc about its central axis.

42. The device according to claim 36 wherein the density gradient medium is transmissive to light.

14. A method for conducting an assay, said method comprising the steps of:

introducing at least one sample into a chamber of a disc, each chamber containing a density gradient medium;

introducing at least two different groups of beads, each of the beads in each group having a common physical characteristic and having attached thereto at least one capture probe with affinity to at least one target in the sample;

allowing interaction between the sample and the beads to thereby promote capture of any target present in the sample;

adding a labeled tag into the reaction chamber, each of said labeled tags having affinity to any target present in the sample;

allowing the labeled tag to bind to the target that is bound to any of the beads; and

rotating the disc to cause beads to move radially outwardly into the density gradient medium.

26. A method for conducting an assay, said method comprising the steps of:

providing a rotatable disc which defines at least one reaction chamber and at least one separation chamber, said reaction chamber being coupled to said separation chamber by controllable barrier means, said separation chamber having a density gradient forming medium;

introducing a plurality of micro-particles including at least two different groups into said reaction chamber, each group of micro-particles having a common physical characteristic and being adapted to receive specific target agents;

introducing at least one test sample into said reaction chamber;

allowing any target agent present in the sample to bind to the micro-particles;

rotating the disc to provide a centripetal force;

controlling the controllable barrier means between said reaction and separation chambers, so that the plurality of micro-particles can move from the reaction chamber to the separation chamber and into the density gradient medium under the centripetal force to separate the micro-particles according to their physical characteristic; and

analyzing the contents of the separation chamber by optical means.

36. A device for conducting an assay, said device comprising:

a rotatable disc which defines at least one reaction chamber and at least one separation chamber, said reaction chamber being coupled to said separation chamber by controllable barrier means; and

a density gradient forming medium located in the separation chamber so that in use, a plurality of particles having different densities may be separated along the density gradient of the medium.

43. A method for conducting a cellular assay, said method comprising the steps of:

providing a rotatable disc which defines at least one reaction chamber and at least one separation chamber, said reaction chamber being coupled to said separation chamber by controllable barrier means, and said separation chamber having a density gradient forming medium;

introducing at least two groups of beads into said reaction chamber, each of the beads in each group having a common physical characteristic and having attached thereto at least one capture probe with affinity to a specific cell surface marker;

introducing at least one sample of cells under test into said reaction chamber;

allowing any cell surface marker on any target cell present in the sample to bind to a respective capture probe on the beads thereby forming a bead-cell binding partner complex;

rotating the disc to provide a centripetal force;

controlling the controllable barrier means between said reaction and separation chambers, so that the plurality of beads and any bead-cell binding partner complex can move from the reaction chamber to the separation chamber and into the density gradient medium under the centripetal force to separate the beads and the binding partner complexes; and

analyzing the contents of the separation chamber by optical means for the presence, location, and amount of bead-cell binding complexes.

44. The method according to claim 43 wherein the beads of one group have a different physical characteristic as the other groups.

45. The method according to claim 44 wherein said physical characteristic is density such that when the disc is rotated, the beads and bead-cell binding partner complexes from the different groups move radially within the density gradient to different radial locations within the chamber according to density.

46. The method according to claim 44 wherein said physical characteristic is bead size.

47. The method according to claim 44 wherein said physical characteristic is color.

48. The method according to claim 44 wherein said physical characteristic is a pre-determined fluorescent characteristic.

49. The method according to claim 44 wherein said physical characteristic is a pre-determined phosphorescent characteristic.

50. The method according to claim 44 wherein said physical characteristic is a pre-determined chemiluminescent characteristic.

51. The method according to claim 44 wherein said target cell is a white blood cell.

52. The method according to claim 51 wherein said white blood cell is selected from the group consisting of lymphocytes, monocytes, basophils, eosinophils, and neutrophils.

53. The method according to claim 52 wherein said lymphocyte is either a T-cell, B-cell, or natural killer cell.

54. The method according to claim 53 wherein said T-cell is either a helper cell or a suppressor cell.

55. The method according to any one of claims 44, 51, 52, 53, or 54 wherein said surface marker is a cluster designation marker.

56. The method according to claim 55 wherein said cluster designation marker is selected from the group comprising CD2, CD3, CD4, CD5, CD6, CD7, CD8, and CD14.

58. The method according to claim 57 wherein the micro-particles of one group have a different physical characteristic as compared to the other groups.

59. The method according to claim 58 wherein said physical characteristic is density such that when the disc is rotated, the micro-particles from the different groups move radially within the density gradient to different radial locations within the chamber according to density.

60. The method according to claim 58 wherein said physical characteristic is micro-particle size.

61. The method according to claim 58 wherein said physical characteristic is color.

62. The method according to claim 58 wherein said physical characteristic is micro-particle shape.

63. The method according to claim 58 wherein said physical characteristic is a pre-determined fluorescent characteristic.

64. The method according to claim 58 wherein said physical characteristic is a pre-determined phosphorescent characteristic.

65. The method according to claim 58 wherein said physical characteristic is a pre-determined chemiluminescent characteristic.

105. A method of using the analysis disc made according to claim 59, said method of using comprising the steps of:

adding at least one test sample into the reaction chamber containing said plurality of micro-particles;

allowing any target present in the test sample to form a complex with the micro-particles;

adding a plurality of labeled tags into the reaction chamber including at least two groups, each of the tags in each group having a common label and having affinity to one target in the test sample;

allowing any labeled tag to bind to its respective target bound to any of the micro-particles; and

rotating the disc to provide a centrifugal force to generate a density gradient in the density forming medium and move the micro-particles in the reaction chamber through the controllable barrier into the separation chamber to separate the micro-particles according to sedimentation rate or elution time.

106. The method according to claim 105 further comprising the steps of:

directing an incident beam of electromagnetic radiation on a fixed point within the separation chamber; and

detecting the micro-particles or the labeled tags while the particles flow through the separation chamber to determine sedimentation rate or elution time of the micro-particles through the medium and to determine the presence and amount of any target bound to the micro-particles.

107. The method according to claim 106 further comprising the step of scanning the incident beam over the entire separation chamber to determine the presence of micro-particles and labeled tags that may remain at their isopycnic points.

113. The method according to either claim 105 or 111 wherein said common label is a fluorescent label.

114. The method according to either claim 105 or 111 wherein said common label is color.

115. The method according to either claim 105 or 111 wherein said labeled tag is a fluorescent bead.

116. The method according to either claim 105 or 111 wherein said labeled tag is an infrared bead.

117. The method according to either claim 105 or 111 wherein said labeled tag is a fluorescent labeled antibody.

57. A method of making an analysis disc to test for the presence of at least one target agent in at least one sample, the method comprising the steps of:

providing a base having a center and an outer edge;

encoding information on an information layer associated with the base, the encoded information being readable by a disc drive assembly to control rotation of the disc;

providing a body defining a radially extending analysis chamber, said analysis chamber including a reaction chamber and at least one separation chamber, said reaction chamber being coupled to said separation chamber by controllable barrier means;

depositing a density gradient forming medium into said separation chamber;

depositing a plurality of micro-particles into the reaction chamber including at least two different groups of micro-particles, each of the micro-particles in each group having a common physical characteristic and having attached thereto a plurality of capture probes; and

providing a cover disc including an aperture, said aperture being linked to the reaction chamber.

66. A method of using the disc made according to any one of the claims 57, 58, 59, 60, 61,62, 63, 64, or 65, said method of using the disc including the steps of:

adding at least one sample into the reaction chamber through said aperture;

allowing any target present in the sample to bind to their respective capture probes attached to the micro-particles of the different groups;

adding a labeled tag into the reaction chamber, said labeled tag having affinity to the targets present in the solution;

allowing the labeled tags to bind to any target that is bound to any of the micro-particles; and

rotating said analysis disc so that the plurality of micro-particles move from the reaction chamber through the controllable barrier to the separation chamber and into the density gradient medium under a centripetal force to separate the micro-particles according to their physical characteristics.

67. The method according to claim 66 wherein the capture probe is selected from the group comprising antigens, antibodies, DNA, RNA, lectins, protein receptors, ligands, biotin, and streptavidin.

68. The method according to claim 66 wherein the target is selected from the group comprising antigens, antibodies, DNA, RNA, lectins, protein receptors, ligands, biotin, and streptavidin.

69. The method according to claim 66 wherein the target is a cell.

70. The method according to claim 66 wherein the target is a cell surface marker.

71. The method according to claim 66 wherein the target is streptavidinated.

72. The method according to claim 66 wherein the target is biotinylated.

73. The method according to claim 66 wherein the labeled tag is a reporter micro-particle having attached thereto a reporter probe that has affinity to a different portion of the target so when the target is present, a bead micro-particle partner complex forms from the binding of the capture probe and the reporter probe to the same target.

74. The method according to claim 73 wherein the reporter micro-particle is fluorescent.

75. The method according to claim 73 wherein said reporter micro-particle is streptavidinated.

76. The method according to claim 73 wherein said reporter micro-particle is biotinylated.

77. The method according to claim 73 further comprising the steps of:

detecting the presence of the target by scanning a beam of electromagnetic energy from a disc drive assembly over the separation chamber; and

analyzing returned electromagnetic energy to determine the location and amount of capture micro-particles, reporter micro-particles, labeled tags, micro-particle to micro-particle complexes, and micro-particle-cell complexes.

79. The device according to claim 78 including a density gradient forming medium located in the separation chamber so that in use, a plurality of particles having different densities may be separated along the density gradient of the medium.

80. The device according to claim 79 wherein the rotatable disc is adapted to rotate about its central aperture which is adapted to releasably engage with a rotatable shaft.

81. The device according to claim 79 wherein the rotatable disc includes a plurality of radially extending reaction chambers and separation chambers.

82. The device according to claim 79 wherein the density gradient forming medium is a gel provided by Pharmacia Biotech under the trademark Percoll.

83. The device according to claim 82 wherein the density gradient may be preformed in the gel, or generated by the application of a centripetal force.

84. The device according to claim 83 wherein the density gradient is formed and maintained by rotating the disc about its central axis.

85. The device according to claim 79 wherein the density gradient medium is transparent to light.

78. A device for conducting an assay, the device comprising:

a rotatable disc including an upper cover disc, a base, and a body disposed between the cover and the base, the body defining an analysis chamber;

each of said analysis chamber comprising at least one reaction chamber and at least one separation chamber, said reaction chamber being in fluid communication with said separation chamber.

86. A method of making an analysis disc to test for the presence of at least one target agent in at least one sample, said method comprising the steps of:

providing a base having a center and an outer edge;

encoding information on an information layer associated with the base, the encoded information being readable by a disc drive assembly to control rotation of the disc;

providing a body defining a radially extending analysis chamber, said analysis chamber including a reaction chamber and at least one separation chamber, said reaction chamber being in fluid communication with said separation chamber;

depositing a density gradient forming medium into said separation chamber;

depositing a plurality of micro-particles into the reaction chamber including at least two different groups of micro-particles, each of the micro-particles in each group having a common physical characteristic and having attached thereto a plurality of capture probes; and

providing a cover disc including an aperture that is linked to the reaction chamber.

87. The method according to claim 86 wherein the micro-particles of one group have a different physical characteristic as the other groups.

88. The method according to claim 87 wherein said physical characteristic is buoyant density such that when the disc is rotated, the micro-particles from the different groups move radially within the density gradient to different radial locations within the chamber according to buoyant density.

89. The method according to claim 87 wherein said physical characteristic is micro-particle size.

90. The method according to claim 87 wherein said physical characteristic is color.

91. The method according to claim 87 wherein said physical characteristic is shape.

92. The method according to claim 87 wherein said physical characteristic is a pre-determined fluorescent characteristic.

93. The method according to claim 87 wherein said physical characteristic is a pre-determined phosphorescent characteristic.

94. The method according to claim 87 wherein said physical characteristic is a pre-determined chemiluminescent characteristic.

108. A method of using the analysis disc made according to any one of the claims 86, 87, 88, 89, 90, 91, 92, 93, or 94, said method of using comprising the steps of:

adding at least one test sample into the reaction chamber containing said plurality of micro-particles;

allowing any target present in the test sample to form a complex with the micro-particles;

adding a plurality of labeled tags into the reaction chamber including at least two groups, each of the tags in each group having a common label and having affinity to one target in the test sample;

allowing any labeled tag to bind to its respective target bound to any of the micro-particles; and

rotating the disc to provide a centrifugal force to generate a density gradient in the density forming medium and move the micro-particles in the reaction chamber into the separation chamber to separate the micro-particles according to sedimentation rate or elution time.

109. The method according to claim 108 further comprising the steps of:

directing an incident beam of electromagnetic radiation on a fixed point within the separation chamber; and

detecting the micro-particles or the labeled tags while the particles flow through the separation chamber to determine sedimentation rate or elution time of the micro-particles through the medium and to determine the presence and amount of any target bound to the micro-particles.

110. The method according to claim 109 further comprising the step of scanning the incident beam over the entire separation chamber to determine the presence of micro-particles and labeled tags that may remain at their isopycnic points.

111. A method of using the analysis disc made according to claim 88, said method of using comprising steps of:

depositing at least one test sample into the reaction chamber;

allowing any target present in the test sample to form a complex with the micro-particles in the reaction chamber;

adding a plurality of labeled tags into the reaction chamber including at least two groups, each of the tags in each group having a common label and having affinity to one target in the test sample;

allowing any labeled tag to bind to its respective target bound to any of the micro-particles; and

rotating the disc to provide a centrifugal force to generate a density gradient in the density forming medium and move the micro-particles in the reaction chamber into the separation chamber to separate the micro-particles according to buoyant density.

112. The method according to claim 111 further comprising the step of scanning the incident beam over the entire separation chamber to determine the presence of micro-particles that may remain at their isopycnic points.

95. A method of making an analysis disc to test for the presence of at least one target agent in at least one sample, said method comprising the steps of:

providing a base having a center and an outer edge;

encoding information on an information layer associated with the base, the encoded information being readable by a disc drive assembly to control rotation of the disc;

providing a body defining a radially extending analysis chamber, said analysis chamber including a reaction chamber and at least one separation chamber, said reaction chamber being in fluid communication with said separation chamber; and

providing a cover disc including an aperture that is linked to the reaction chamber.

96. A method of using the analysis disc made according to claim 95, said method of using comprising the steps of:

preparing a plurality of micro-particles including at least two different groups of micro-particles, each of the micro-particles in each group having a common physical characteristic and having attached thereto a plurality of capture probes;

mixing the plurality of micro-particles, at least one test sample, and a density gradient forming medium to form an assay solution;

allowing any target present in the test sample to form a complex with the micro-particles;

depositing the assay solution into the analysis chamber; and

rotating the disc to provide a centrifugal force to generate a density gradient in the density forming medium.

97. The method according to claim 96 wherein the micro-particles of one group have a different physical characteristic as the other groups.

98. The method according to claim 97 wherein said physical characteristic is buoyant density such that when the disc is rotated, the micro-particles from the different groups move radially within the density gradient to different radial locations within the chamber according to buoyant density.

99. The method according to claim 97 wherein said physical characteristic is micro-particle size.

100. The method according to claim 97 wherein said physical characteristic is color.

101. The method according to claim 97 wherein said physical characteristic is shape.

102. The method according to claim 97 wherein said physical characteristic is a pre-determined fluorescent characteristic.

103. The method according to claim 97 wherein said physical characteristic is a pre-determined phosphorescent characteristic.

104. The method according to claim 97 wherein said physical characteristic is a pre-determined chemiluminescent characteristic.