Multi-purpose optical analysis disc for conducting assays and related methods for attaching capture agents

• US 20050003459A1
• Filed: 01/21/2003
• Published: 01/06/2005
• Est. Priority Date: 01/30/2002
• Status: Abandoned Application

• Current Assignee: Vindur Technologies, Inc.
• Original Assignee: Vindur Technologies, Inc.
• Inventors: Krutzik, Siegfried Richard
• Application Number:
  US10/348,049
• Publication Number:
  US 20050003459A1
• Time in Patent Office: Days
• US Class Current: 435/7.900
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 33/553 Metal or metal coated
• 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

The present invention relates to optical bio-disc systems and related test methods and to immobilizing receptor molecules on optical bio-discs. When a sample is injected into a fluidic circuit, the target agent binds to a capture agent or probe bound in a capture zone. A signal is generated from tags attached to a reporter probe that has specific affinity to the target agent. The assays and methods of the present invention are implemented on a bio-disc. The bio-disc includes a flow channel having capture zones in fluid communication with a mixing chamber and a peripheral waste reservoir. The bio-disc is implemented on an optical disc that has information encoding format such as a CD. A bio-disc drive assembly is employed to rotate the disc, read and process any encoded information, and analyze the samples in the flow channel of the bio-disc.

Claims

1. An optical bio-disc, comprising:

a substantially circular substrate having a center and an outer edge;

a metal layer associated with the substrate;

a target zone disposed between the center and the outer edge; and

at least one capture agent that binds to the metal layer such that the capture agent is immobilized on the metal layer within the target zone to thereby form a capture zone.

2. The optical bio-disc according to claim 1 wherein the metal layer is selected from the group comprising gold, aluminum, silver, nickel, and reflective metal alloys.

3. The optical bio-disc according to claim 1 wherein the substrate includes encoded information associated therewith, the encoded information being readable by a disc drive assembly to control rotation of the bio-disc.

4. The optical bio-disc according to claim 1 further comprising a circumferential peripheral waste reservoir formed adjacent to said outer edge of said substrate.

5. The optical bio-disc according to claim 4 further comprising a flow channel in fluid communication with said capture zone and said peripheral reservoir and an input site in fluid communication with the flow channel.

12. A method of using the optical bio-disc according to claim 1, said method of using comprising:

providing a sample containing an analyte of interest onto the capture zone to thereby place the analyte and capture agent in close proximity to each other;

incubating the sample at a pre-determined time and temperature to allow sufficient binding of the analyte to the capture agent;

washing the flow channel to remove excess sample;

providing a plurality of signal agents having specific affinity to the analyte bound on the capture agent in the capture zone, each of said plurality of signal agents having conjugated thereto a reporter;

washing the flow channel to remove excess signal agents; and

scanning a beam of electromagnetic radiation through the capture zone to determine the presence and amount of said reporters.

13. The method according to claim 12 wherein said capture agent is an antibody.

14. The method according to claim 12 wherein said signal agent is an antibody.

15. The method according to claim 12 wherein said reporter is a bead.

16. The method according to claim 15 wherein said bead is fluorescent labeled.

17. The method according to claim 16 wherein said bead is selected from the group comprising 0.02 um, 0.2 um, 0.5 um, 1 um, 2 um and 6 um polystyrene bead.

18. The method according to claim 12 wherein said reporter is an enzyme.

19. The method according to claim 18 further comprising the step of providing an enzyme substrate to the capture zones, wherein an enzyme substrate reaction occurs when the enzyme is present in the capture zone to thereby produce a detectable product.

20. The method according to claim 19 further comprising the step of washing the flow channel to remove excess substrate.

21. The method according to claim 20 further comprising the step of scanning a beam of electromagnetic radiation through the capture zone to determine the presence and amount of enzyme products.

6. An optical bio-disc, comprising:

a substantially circular substrate having a center and an outer edge;

a metal layer associated with the substrate;

a target zone disposed between the center and the outer edge;

an active layer formed on the surface of said metal layer; and

at least one capture agent that binds to said active layer such that the capture agent is immobilized on said active layer within the target zone to thereby form a capture zone, wherein the active layer is formulated to immobilize a pellet formed by an enzyme reaction.

7. The optical bio-disc according to claim 6 wherein the metal layer is selected from the group comprising aluminum, gold, silver, nickel, and reflective metal alloys.

8. The optical bio-disc according to claim 6 wherein the substrate includes encoded information associated therewith, the encoded information being readable by a disc drive assembly to control rotation of the bio-disc.

9. The optical bio-disc according to claim 6 further comprising a circumferential peripheral waste reservoir formed near the outer edge of said substrate.

10. The optical bio-disc according to claim 6 further comprising an enzyme, wherein the enzyme, when exposed to an enzyme substrate, produces a signal detectable by an incident beam of electromagnetic radiation.

11. The optical bio-disc according to claim 6 further comprising a flow channel in fluid communication said the capture zone and said peripheral reservoir, and an input site in fluid communication with said flow channel.

22. A method of making an optical assay disc for performing a binding assay, said method of making comprising:

providing a substantially circular rotatable substrate having a center, an outer edge, and a metal layer associated thereto;

depositing a plurality of capture agents onto said metal layer, thereby forming a capture zone;

incubating said capture agents on said metal layer to thereby facilitate binding of one or more capture agents onto the metal layer;

washing the capture zones to remove excess capture agents;

attaching a cap portion to said metal layer using an adhesive member with fluidic channels formed therein thereby forming fluidic circuits, said fluidic channels aligned such that the capture zones are incorporated in said fluidic circuits;

blocking unoccupied sites within said fluidic circuit with a blocking agent;

incubating said blocking agent in said fluidic circuit to thereby facilitate binding of the blocking agent onto the unoccupied sites within said fluidic circuit;

aspirating said blocking agent out of said fluidic circuit; and

washing said fluidic circuit to remove residual blocking agent from said fluidic circuit.

23. The method of claim 22, further comprising the step of depositing an affinity agent onto said metal layer prior to depositing said capture agent.

24. The method of claim 23, wherein the affinity agent is selected from the group comprising streptavidin, Neutravidin, avidin, biotin, biotin-BSA, biotin-PEG, and functionalized derivatives thereof.

25. The method of claim 22, further comprising the step of forming a circumferential peripheral waste reservoir proximal said outer edge of said substrate.

26. The method of claim 22, wherein the step of washing involves rotating for a sufficient period of time at a sufficient speed so that non-immobilized capture agents are moved away from the capture zones.

27. The method of claim 22, wherein the capture agent is a primary capture antibody.

28. The method of claim 22, wherein the capture agent is a secondary capture antibody.

29. The method of claim 27, wherein the capture antibody is independently selected from the group comprising IgG, biotinylated-IgG, anti-HCG antibody, and anti-myoglobin antibody.

30. The method of claim 28, further comprising the step of depositing a primary capture antibody onto said secondary capture antibody, wherein said secondary capture antibody is bound to said metal layer in the capture zone during the depositing of capture agents step.

31. The method of claim 30, further comprising the steps of incubating said substrate and washing said substrate following depositing said primary capture antibody on said secondary capture antibody.

32. The method of claim 31, wherein the step of incubating involves incubating for a sufficient period of time, at a sufficient temperature to allow immobilization of said primary capture antibody onto secondary capture antibody.

33. The method of claim 22, further including the step of blocking non-capture areas within the capture zone with blocking agents to thereby prevent non-specific binding of analytes, signal probes, and reporters onto non-capture areas.

34. The optical bio-disc as made in conjunction with the method recited in claim 22.

35. An optical bio-disc for performing an immunochemical assay, said disc comprising:

a substantially circular rotatable substrate having a center and an outer edge;

a metal layer disposed over said substrate;

a cap portion integrally attached to the metal layer by an adhesive member, the adhesive member having one or more portions removed, thereby forming one or more channels defined there between; and

one or more capture agents immobilized on the metal layer, the capture agents defining discrete capture zones within the one or more channels.

36. The disc according to claim 35, wherein the capture agents are immobilized by a cross-linking system.

37. The disc according to claim 35, wherein the capture agents are immobilized by the metal layer.

38. The disc according to claim 36, wherein the capture agents are antibodies having a selective affinity for analytes of interest.

39. The disc of claim 38, wherein the capture agents are selected from the group comprising antibodies for HCG and myoglobin.

40. The disc according to claim 36, wherein the capture agents are antibodies having a selective affinity for primary antibodies, said primary antibodies having a selective affinity for HCG.

41. The disc according to claim 40, wherein the capture agents are anti-mouse antibodies produced in goats.

42. The disc according to claim 40, wherein said primary antibodies having a selective affinity for HCG.

43. An optical disc and drive system for performing a binding assay, the system comprising:

an optical assay disc comprising;

a substrate, said substrate having a circumferential peripheral reservoir formed therein;

a metal layer disposed over the substrate;

a cap portion integrally attached to the metal layer by an adhesive member, the adhesive member having one or more portions removed, thereby forming one or more channels defined there between; and

one or more capture agents immobilized on the metal layer, the capture agents defining discrete capture zones within the one or more channels; and

an optical disc drive comprising;

a light source for directing light to said assay disc at said capture zones;

a detector for detecting light reflected from or transmitted through the disc at the capture zones and providing a signal; and

a processor for using the signal to count items in the sample bound to the capture agents.

44. The system of claim 43, wherein the detector is on the same side of the disc as the light source for detecting light reflected from the capture zones.

45. The system of claim 43, wherein the detector is on the opposite side of the disc as the light source for detecting light transmitted through the capture zones.

46. The disc of claim 43, wherein the processor includes image recognition software for detecting and imaging beads, colloidal particles, fluospheres, and enzyme precipitates.

135. The system according to claim 43 wherein said detector is a fluorescence detector.

47. An optical bio-disc for performing an immunochemical assay, said disc comprising:

a substantially circular rotatable substrate having a center and an outer edge, said substrate having formed therein a circumferential peripheral reservoir proximal to said outer edge;

a metal layer disposed over said substrate;

a cap portion integrally attached to the metal layer by an adhesive member, the adhesive member having one or more portions removed, thereby forming one or more channels defined there between; and

one or more capture antibodies immobilized on the metal layer, the capture antibodies defining discrete capture zones within the one or more channels.

48. The optical bio-disc according to claim 47 further comprising one or more absorber pads in said peripheral reservoir.

49. An optical bio-disc for performing an immunochemical assay, said disc comprising:

a substantially circular rotatable substrate having a center and an outer edge, said substrate having formed therein an outer circumferential peripheral reservoir proximal to the outer edge;

a metal layer disposed over said substrate;

a cap portion integrally attached to the metal layer by an adhesive member, the adhesive member having one or more portions removed, thereby forming one or more flow channels defined there between; and

one or more capture antibodies immobilized on the metal layer, the capture antibodies defining discrete capture zones within the one or more channels.

50. The optical bio-disc according to claim 49 further comprising an inner peripheral reservoir formed adjacent and in fluid communication with said outer reservoir.

51. The optical bio-disc according to claim 49 further comprising one or more mixing wells formed in said substrate in pre determined locations between said center and outer edges within said flow channels.

52. The optical bio-disc according to claim 49 further comprising absorber pads located within said outer peripheral reservoir.

53. The optical bio-disc of claim 50 further comprising arc shaped lands separating said outer and inner reservoirs, said lands having pass through ports to thereby place said reservoirs in fluid communication with each other.

54. An optical bio-disc, comprising:

a rotatable substrate having a center and an outer edge;

an outer reservoir formed in said substrate proximal to said outer edge;

an inner reservoir formed in said substrate proximal to and in fluid communication with said outer reservoir;

a channel layer positioned adjacent said substrate, said channel layer having at least one fluidic channel formed therein which is in fluid communication with said inner reservoir, thereby forming a fluidic circuit; and

a cap portion positioned adjacent said channel layer.

55. The optical bio-disc according to claim 54 further comprising an absorber pad located in said outer reservoir.

56. The optical bio-disc according to claim 55 further comprising an inlet port formed in said cap portion, said inlet port located in a predetermined location in said fluidic channel to thereby allow liquid to flow from the inlet port through the length of the fluidic channel and into the inner and outer reservoirs.

57. The optical bio-disc according to claim 54 further comprising a vent port formed in said cap portion, said vent port located in a predetermined location in said outer reservoir to thereby allow venting of air inside said fluidic circuit to prevent air blockage within the fluidic circuit.

58. The optical bio-disc of claim 54 further comprising a first reflective metal layer disposed over said substrate.

59. The optical bio-disc of claim 58 further comprising target zones formed on said first metal layer, said target zones formed within said fluidic channel.

60. The optical bio-disc of claim 59 further comprising a second reflective metal layer disposed over said cap portion.

61. The optical bio-disc of claim 54 further comprising a semi-reflective metal layer disposed over said substrate.

62. The optical bio-disc according to claim 54 wherein said substrate includes encoded information associated therewith, the encoded information being readable by a disc drive assembly to control rotation of the bio-disc, incubation time, incubation temperature, and specific steps of a binding assay.

63. The optical bio-disc according to claim 54 wherein the fluidic channel is radially directed.

64. The optical bio-disc according to claim 63 wherein the radially directed fluidic channel is in fluid communication with said inner reservoir.

65. The optical bio-disc according to claim 59 further comprising one or more capture antibodies immobilized within said target zone, the capture antibodies defining discrete capture zones within the one or more target zones in the at least one fluidic channel.

66. The optical bio-disc according to claim 61 further comprising one or more capture antibodies immobilized in pre-determined location on said semi-reflective layer, the capture antibodies defining discrete capture zones within the at least one fluidic channel.

67. The optical bio-disc according to claim 65 wherein said discrete capture zones are arranged in a micro-array format.

68. The optical bio-disc according to claim 54 wherein said outer and inner reservoirs are circumferential and arranged in an annular format proximal each other.

69. The optical bio-disc according to claim 68 wherein said outer and inner reservoirs are separated from each other by arcuate lands.

70. The optical bio-disc according to claim 69 wherein said arcuate lands include pass through ports to thereby place said outer and inner reservoirs in fluid communication.

71. The optical bio-disc according to claim 54 further comprising a mixing well formed in said substrate in fluid communication with said at least one fluidic channel.

126. The optical bio-disc according to claims 70 wherein said outer and inner reservoirs are large enough to contain waste solutions from multi-step assay procedures involving multiple spin wash steps.

72. An optical bio-disc, comprising:

a rotatable substrate;

a cap portion having a center and an outer edge;

an outer reservoir formed in said cap portion proximal to said outer edge;

an inner reservoir formed in said cap portion proximal to and in fluid communication with said outer reservoir; and

a channel layer positioned between said substrate and said cap, said channel layer having at least one fluidic channel formed therein which is in fluid communication with said inner reservoir thereby forming a fluidic circuit.

73. The optical bio-disc according to claim 72 further comprising an absorber pad located in said outer reservoir.

74. The optical bio-disc according to claim 73 further comprising an inlet port formed in said cap portion, said inlet port located in a predetermined location in said at least one fluidic channel to thereby allow liquid to flow from the inlet port through the length of the fluidic channel and into the inner and outer reservoirs.

75. The optical bio-disc according to claim 72 further comprising a vent port formed in said cap portion, said vent port located in a predetermined location in said outer reservoir to thereby allow venting of air inside said fluidic circuit to prevent air blockage within the fluidic circuit.

76. The optical bio-disc of claim 72 further comprising a first reflective metal layer disposed over said substrate.

77. The optical bio-disc of claim 76 further comprising target zones formed on said metal layer.

78. The optical bio-disc of claim 77 further comprising a second reflective metal layer disposed over said cap portion.

79. The optical bio-disc of claim 72 further comprising a semi-reflective metal layer disposed over said substrate.

80. The optical bio-disc according to claim 72 wherein said substrate includes encoded information associated therewith, the encoded information being readable by a disc drive assembly to control rotation of the bio-disc and to control the assay.

81. The optical bio-disc according to claim 72 wherein the at least one fluidic channel is radially directed.

82. The optical bio-disc according to claim 81 wherein the radially directed fluidic channel is in fluid communication with said inner reservoir.

83. The optical bio-disc according to claim 77 further comprising one or more capture antibodies immobilized within said target zone, the capture antibodies defining discrete capture zones within the one or more target zones in the at least one fluidic channel.

84. The optical bio-disc according to claim 79 further comprising one or more capture antibodies immobilized in pre-determined location on said semi-reflective layer, the capture antibodies defining discrete capture zones within the at least one fluidic channel.

85. The optical bio-disc according to claim 83 wherein said discrete capture zones are arranged in a micro-array format.

86. The optical bio-disc according to claim 72 wherein said outer and inner reservoirs are circumferential and arranged in an annular format proximal each other.

87. The optical bio-disc according to claim 86 wherein said outer and inner reservoirs are separated from each other by arcuate lands.

88. The optical bio-disc according to claim 87 wherein said arcuate lands include pass through ports to thereby place said outer and inner reservoirs in fluid communication.

89. The optical bio-disc according to claim 72 further comprising a mixing well formed in said substrate in fluid communication with said at least one fluidic channel.

127. The optical bio disc according to claim 73 further comprising drying agents deposited in said absorber pads, said drying agents keeps reagents deposited in the bio-disc free from moisture to thereby preserve functional activity of said reagents during disc storage and increase disc shelflife.

128. The optical bio-disc according to claim 82 wherein said radially directed fluidic channel that is in fluid communication with said inner reservoir allows the utilization of sub-micron reporters.

90. An optical bio-disc, comprising:

a rotatable substrate having a center and an outer edge;

a reservoir formed in said substrate proximal to said outer edge;

a channel layer positioned adjacent said substrate, said channel layer having at least one fluidic channel formed therein which is in fluid communication with said reservoir thereby forming a fluidic circuit; and

a cap portion positioned adjacent said channel layer.

92. The optical bio-disc according to claim 90 wherein said reservoir is separated into at least two uni-radial fluidly independent members.

93. The optical bio-disc according to claim 92 further comprising absorber pads in said fluidly independent reservoirs.

94. The optical bio-disc according to claim 93 further comprising an inlet port formed in said cap portion, said inlet port located in a predetermined location in said at least one fluidic channel to thereby allow liquid to flow from the inlet port through the length of the fluidic channel and into said reservoir.

136. The optical bio disc according to claim 93 further comprising drying agents deposited in said absorber pads, said drying agents keeps reagents deposited in the bio-disc free from moisture to thereby preserve functional activity of said reagents during disc storage and increase disc shelflife.

91. An optical bio-disc, comprising:

a rotatable substrate;

a cap portion having a center and an outer edge;

a reservoir formed in said cap portion proximal to said outer edge; and

a channel layer positioned between said substrate and said cap, said channel layer having at least one fluidic channel formed therein which is in fluid communication with said reservoir thereby forming a fluidic circuit.

95. The optical bio-disc according to 94 further comprising a vent port formed in said cap portion, said vent port located in a predetermined location in each of said independent reservoirs to thereby allow venting of air inside said fluidic circuit to prevent air blockage within the fluidic circuit.

96. The optical bio-disc according to claim 95 further comprising one or more capture antibodies immobilized in pre-determined location on said substrate, the capture antibodies defining discrete capture zones within the at least one fluidic circuit.

97. An optical bio-disc, comprising:

a rotatable substrate;

a cap portion having a center and an outer edge;

an outer reservoir formed in said cap portion proximal to said outer edge;

an inner reservoir formed in said cap portion proximal to said outer reservoir; and

a channel layer positioned between said substrate and said cap, said channel layer having at least one fluidic channel formed therein which is in fluid communication with said inner reservoir.

98. The optical bio-disc according to claim 97 wherein said outer reservoir is separated into uni-radial fluidly independent members.

99. The optical bio-disc according to claim 97 wherein said inner reservoir is separated into uni-radial fluidly independent members.

100. A method of making an optical bio-disc, said method of making comprising:

providing a substantially circular substrate having a center, an outer edge, and a metal layer associated thereto;

forming a circumferential peripheral waste reservoir, said waste reservoir located proximal said outer edge of said substrate;

depositing one or more capture agents onto said metal layer, thereby forming a capture zone;

incubating said one or more capture agents on said metal layer to thereby facilitate binding of the capture agents onto the metal layer;

washing the capture zone to remove unbound capture agents; and

attaching a cap portion to said metal layer using an adhesive member having flow channels formed therein thereby forming fluidic circuits, said cap portion having at least one inlet and vent port formed therein.

101. The method of claim 100 further comprising the step of forming one or more mixing wells located between said substrate center and said waste reservoir.

102. The method of claim 100 further comprising the step of placing one or more absorber pads into said waste reservoir.

103. The optical bio-disc made according claim 102 wherein said inlet port, flow channel, and reservoirs are in fluid communication with each other, comprising said fluidic circuit, such that when sample is added into the inlet port, said sample moves into said flow channel, and when the disc is rotated, said sample moves from the flow channel into said inner then outer reservoir, and into said absorber pads.

113. A method of using the optical bio-disc made according to claim 100, said method of using comprising:

providing a sample containing an analyte of interest into the flow channel and onto the capture zone to thereby place the analyte and capture agent in close proximity to each other;

incubating the sample at a pre-determined time and temperature to allow sufficient binding of the analyte to the capture agent;

spinning the disc at a first pre-determined speed to remove unbound sample, said sample moving from said flow channel into said waste reservoir during spinning;

providing a solution containing plurality of signal agents having specific affinity to the analyte bound on the capture agent in the capture zone, said signal agent having conjugated thereto a reporter;

spinning the disc at a second pre-determined speed to remove excess solution of signal agents, said solution moving from said flow channel into said waste reservoir during spinning; and

scanning a beam of electromagnetic radiation through the capture zone to determine the presence and amount of said reporters.

114. The method of claim 113 wherein said sample and solution of signal agents are absorbed into said absorber pads in said waste reservoir during the spin steps.

115. The method according to claim 113 wherein said capture agent is an antibody.

116. The method according to claim 113 wherein said signal agent is a tagged antibody.

117. The method according to claim 113 wherein said reporter is a bead.

118. The method according to claim 117 wherein said bead is selected from the group comprising beads labeled with fluorophores, and chromophores.

119. The method according to claim 118 wherein said bead is selected from the group comprising 0.02 um, 0.2 um, 0.5 um, 1 um, 2 um, and 6 um polystyrene bead.

120. The method according to claim 113 wherein said reporter is an enzyme.

121. The method according to claim 120 further comprising the step of providing an enzyme substrate into the flow channels and onto the capture zone.

122. The method according to claim 121 further comprising the step of spinning the disc to remove excess enzyme substrate from the flow channel.

123. The method according to claim 122 further comprising the step of scanning a beam of electromagnetic radiation through the capture zone to determine the presence and amount of enzyme products.

125. A method of using the optical bio-disc made according to claim 100 said method of using comprising:

mixing a sample containing analytes of interest with a suspension containing plurality of signal agents having specific affinity to the analytes, each of said signal agents having conjugated thereto a reporter;

incubating the sample and signal agent suspension at a pre-determined time and temperature to allow sufficient binding of the analytes to the signal agents to thereby form analyte-signal agent complexes;

introducing said complexes into the flow channel and onto the capture zone to thereby place said complexes and capture agents in close proximity to each other;

incubating the complexes at a pre-determined time and temperature to allow sufficient binding of the analyte-signal agent complexes to the capture agents;

spinning the disc at a pre-determined speed to remove the suspension containing unbound complexes, said suspension moving from said flow channel into said waste reservoir during spinning; and

scanning a beam of electromagnetic radiation through the capture zone to determine the presence and amount of said reporters.

132. The method of making an optical bio-disc according to claim 100 said method of making further comprising:

blocking unoccupied sites within said fluidic circuit with a blocking agent;

incubating said blocking agent in said fluidic circuit to thereby facilitate binding of the blocking agent onto the unoccupied sites within said fluidic circuit;

aspirating said blocking agent out of said fluidic circuit; and

washing said fluidic circuit to remove residual blocking agent out of said fluidic circuit.

133. The method of claim 119 wherein said bead carries different chemical functionalities.

134. The method of claim 133 wherein said chemical functionalities are selected from the group comprising carboxyl, amino, aldehyde, and hydrazine functional groups.

104. A method of making an optical bio-disc, said method of making comprising:

providing a substantially circular substrate having a center, an outer edge, and a metal layer associated thereto;

forming an outer circumferential peripheral waste reservoir proximal said outer edge of said substrate;

forming an inner circumferential reservoir proximal said outer waste reservoir of said substrate, said outer and inner reservoirs separated by a raised land;

forming pass through ports in said raised land to thereby place said outer and inner reservoirs in fluid communication;

depositing one or more capture agents onto said metal layer, thereby forming a capture zone;

incubating said one or more capture agents on said metal layer to thereby facilitate binding of the capture agents onto the metal layer;

washing the capture zone to remove unbound capture agents; and

attaching a cap portion to said metal layer using an adhesive member having flow channels formed therein thereby forming fluidic circuits, said cap portion having at least one inlet and vent port formed therein.

105. The method of claim 104 further including a step of forming one or more mixing wells located between said substrate center and said waste reservoir.

106. The method according to claim 105 further comprising the step of depositing a plurality of signal agents into said mixing wells, each of said signal agents having attached thereto a reporter.

107. The method of claim 106 wherein said reporter is detectable using an optical disc drive.

108. The method of claim 106 wherein said reporter is a bead.

109. The method of claim 108 wherein said bead is a fluorescent bead.

110. The method of claim 109 wherein said fluorescent bead is detected using a fluorescent type optical disc reader.

111. The method according to claim 105 further including a step of placing one or more absorber pads into said waste reservoir.

112. The optical bio-disc made according to claim 111 wherein said inlet port, fluidic channel, and reservoirs are in fluid communication with each other, thereby forming a fluidic circuit, such that when sample is added to the inlet port, said sample moves into said flow channel, and when the disc is rotated, said sample moves from the flow channel into said inner then outer reservoir, and into said absorber pads.

124. A method of using the optical bio-disc made according to the claim 106, said method of using comprising:

providing a sample containing analytes of interest into said inlet port to said flow channel, and into said mixing wells;

incubating the sample in the mixing wells for a sufficient time to allow binding of the analytes of interest to the signal agents to thereby form a suspension of analyte-signal agent complexes;

spinning the disc to move said suspension from said mixing well into the capture zone within the fluidic channel to thereby place the analyte complexes and capture agents in close proximity to each other;

incubating the sample at a pre-determined time and temperature to allow sufficient binding of the analyte-signal agent complexes to the capture agent;

spinning the disc to remove unbound complexes in said suspension, said suspension moving from said flow channel into said waste reservoir during spinning; and

scanning a beam of electromagnetic radiation through the capture zone to determine the presence and amount of said reporters.

129. The method according to claim 108 wherein said reporter bead is a nanosphere.

130. The method according to claim 129 wherein said nanosphere is selected from the group comprising colloidal particles between 4 to 50 nm in diameter.

131. The method according to claim 129 wherein said nanosphere is labeled with a tag selected from the group comprising fluophores and luminophores.

137. A method of attaching a capture agent having a reactive amino group onto a carboxy modified polystyrene substrate, said method comprising the steps of:

activating the carboxy terminal end of said carboxy modified polystyrene using N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylamino)propyl carbodiimide thereby generating a N-hydroxysuccinimide ester active group; and

attaching said capture agent having an amino group onto the carbonyl of the N-hydroxysuccinimide ester.

148. The method according to claim 137 wherein said capture agent is selected from the group comprising antibodies, receptor molecules, antigens, oligonucleotides, and ligands.

149. An optical bio-disc having capture agents attached thereto, said capture agents attached to said bio-disc according to the methods of claim 137.

150. An optical bio-disc utilized according to the methods recited in claim 137.

138. A method of attaching a capture agent onto an amino modified polystyrene substrate, said method comprising the steps of:

activating said amino modified polystyrene by attaching glutaraldehyde onto the amino terminal end of said amino modified polystyrene in the presence of sodium cyanoborohydride; and

attaching said capture agent onto the aldehyde activated amino modified polystyrene.

139. A method of attaching a capture agent having a maleimide group attached thereto onto an amino modified polystyrene substrate, said method comprising the steps of:

binding a s-acetylthioacetic acid-N-hydroxysuccinimide onto the amino end of said amino modified polystyrene;

removing a protective acetyl group from the sulfur end of the s-acetylthioacetic acid-N-hydroxysuccinimide using hydroxylamine thereby generating a sulfhydryl activated amino modified polystyrene; and

attaching said capture agent having a maleimide group attached thereto onto said sulfhydryl activated amino modified polystyrene.

140. A method of attaching a sulfhydryl derivatized capture agent onto an amino modified polystyrene substrate, said method comprising the steps of:

binding a gamma-maleimidobutyric acid-N-hydroxysuccinimide onto the amino end of said amino modified polystyrene; and

attaching said sulfhydryl derivatized capture agent onto a double bond of the introduced heterocyclic group of the gamma-maleimidobutyric acid.

141. A method of attaching a sulfhydryl derivatized capture agent onto a polystyrene substrate containing a functional maleimide active group, said method comprising the step of attaching said sulfhydryl derivatized capture agent onto the maleimide active group.

142. A method of immobilizing a capture agent having an amino group onto a gold surface, said method comprising the steps of:

attaching a mercaptoundecanoic acid molecule onto said gold surface through the thiol terminal group of the mercaptoundecanoic acid;

activating the carboxy terminal end of said mercaptoundecanoic acid molecule using N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylamino)propyl carbodiimide thereby generating a N-hydroxysuccinimide active group; and

attaching said capture agent having an amino group onto the carbonyl group of the N-hydroxysuccinimide ester.

143. A method of immobilizing a capture agent having a reactive amino group onto a gold surface comprising the steps of:

attaching a mercaptoundecanoic acid molecule onto said gold surface through the thiol terminal group of the mercaptoundecanoic acid;

activating the carboxy terminal end of said mercaptoundecanoic acid molecule using N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylamino)propyl carbodiimide thereby generating a N-hydroxysuccinimide ester;

attaching amino groups of bovine serum albumin onto the carbonyl group of the N-hydroxysuccinimide ester;

conjugating a dextran aldehyde crosslinker onto said bovine serum albumin in the presence of sodium cyanoborohydride, said dextran aldehyde crosslinker having high binding capacity for substances with reactive amino groups; and

attaching said capture agent having a reactive amino group onto the said dextran aldehyde crosslinker.

144. A method of immobilizing a capture agent having a reactive amino group onto a gold surface, said method comprising the steps of:

attaching a mercaptoundecanoic acid molecule onto said gold surface through the thiol terminal group of the mercaptoundecanoic acid;

activating the carboxy terminal end of said mercaptoundecanoic acid molecule using N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylamino)propyl carbodiimide thereby generating a first N-hydroxysuccinimide ester active group;

attaching one or more carboxyl groups of bovine serum albumin onto said first N-hydroxysuccinimide ester active group;

activating remaining free carboxyl groups of said bovine serum albumin using N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylamino)propyl carbodiimide thereby generating a second N-hydroxysuccinimide ester active group; and

attaching said capture agent having a reactive amino group onto the carbon of said second N-hydroxysuccinimide ester active group.

145. A method of immobilizing a capture agent having a reactive amino group onto a gold surface, said method comprising the steps of:

attaching a mercaptoundecanoic acid molecule onto said gold surface through the thiol terminal group of the mercaptoundecanoic acid;

activating the carboxy terminal end of said mercaptoundecanoic acid molecule using N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylamino)propyl carbodiimide thereby generating a N-hydroxysuccinimide ester active group;

attaching amino groups of poly-L-lysine onto the carbon of the N-hydroxysuccinimide ester active group;

conjugating a dextran aldehyde crosslinker onto said poly-L-lysine in the presence of sodium cyanoborohydride, said dextran aldehyde crosslinker having high binding capacity for substances with amino groups; and

attaching said capture agent having a reactive amino group onto the said dextran aldehyde crosslinker.

146. A method of immobilizing a streptavidinated capture agent onto a gold surface, said method comprising the steps of:

attaching a mercaptoundecanoic acid molecule onto said gold surface through the thiol terminal group of the mercaptoundecanoic acid;

activating the carboxy terminal end of said mercaptoundecanoic acid molecule using N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylamino)propyl carbodiimide thereby generating a N-hydroxysuccinimide ester active group;

attaching amino groups of a biotinylated bovine serum albumin molecule onto the carbonyl group of the N-hydroxysuccinimide ester active group; and

attaching said streptavidinated capture agent onto said biotinylated bovine serum albumin.

147. A method of immobilizing a biotinylated capture agent onto a gold surface, said method comprising the steps of:

attaching a mercaptoundecanoic acid molecule onto said gold surface through the thiol terminal group of the mercaptoundecanoic acid;

activating the carboxy terminal end of said mercaptoundecanoic acid molecule using N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylamino)propyl carbodiimide thereby generating a N-hydroxysuccinimide ester active group;

attaching amino groups of a streptavidin molecule onto the carbonyl group of the N-hydroxysuccinimide ester active group; and

attaching said biotinylated capture agent onto said streptavidin molecule.

151. (CANCELED)

152. (CANCELED)

153. (CANCELED)