Methods for DNA conjugation onto solid phase including related optical biodiscs and disc drive systems
First Claim
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1. A method of evaluating a solid phase for use in a dual bead assay, the method comprising the steps of:
- selecting a test solid phase;
binding a probe to the test solid phase in the presence or absence of a cross-linking agent;
determining a total amount of probe bound to the test solid phase in the presence or absence of a cross-linking agent;
determining a percentage of probe bound covalently to the solid phase;
determining an amount of probe bound to the solid phase non-covalently; and
calculating a percentage of probe bound covalently to the solid phase, wherein if no less than a pre-determined minimum threshold of the probes is bound covalently, the solid phase is suitable for use in a dual bead assay.
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Abstract
The invention provides for methods of conjugating biochemical probes onto a solid phase for use in biomedical assays as implemented in conjunction with an optical bio-disc system. The method includes determining the suitability of a test solid phase for purposes of use in a dual bead assay, selecting a test solid phase, conjugating a probe to the test solid phase in the presence or absence of a cross-linking agent, and determining the total amount of probe bound to the test solid phase in the presence or absence of a cross-linking agent. The method is further employed to determine the percentage of probe bound covalently or non-covalently to the solid phase and calculating the percentage of probe bound covalently thereby selecting the solid phase with the highest conjugation efficiency. The invention is further directed at methods for determining whether a target agent is present in a biological sample. A bio-disc for performing a dual bead assay according to these methods is also provided.
101 Citations
115 Claims
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1. A method of evaluating a solid phase for use in a dual bead assay, the method comprising the steps of:
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selecting a test solid phase;
binding a probe to the test solid phase in the presence or absence of a cross-linking agent;
determining a total amount of probe bound to the test solid phase in the presence or absence of a cross-linking agent;
determining a percentage of probe bound covalently to the solid phase;
determining an amount of probe bound to the solid phase non-covalently; and
calculating a percentage of probe bound covalently to the solid phase, wherein if no less than a pre-determined minimum threshold of the probes is bound covalently, the solid phase is suitable for use in a dual bead assay. - View Dependent Claims (2, 3, 4, 7, 13, 14, 15)
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- 5. The method according to claim I wherein said probe is nucleic acid.
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16. A method for DNA conjugation onto a solid phase for determining the suitability of a test solid phase for use in a dual bead assay, said method comprising the steps of:
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selecting a test solid phase;
conjugating a probe onto the test solid phase;
washing the solid phase employing a conjugate dilution buffer;
heat treating the solid to thereby remove the non-covalently bound probes, and;
calculating the percentage of probes bound covalently to the solid phase.
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29. An optical bio-disc, comprising:
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a substrate having a tracking groove formed therein;
a reflective layer formed on at least a portion of said substrate so that an incident beam of electromagnetic energy may track along said groove;
an active layer associated with said substrate; and
a capture agent having an affinity for said active layer so that said capture agent is immobilized by said active layer so that a percentage of said capture agent bound covalently to said active layer may be calculated.
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51. An optical bio-disc, comprising:
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a substrate having encoded information associated therewith, said encoded information being readable by a disc drive assembly to control rotation of the disc;
a target zone associated with said substrate, said target zone disposed at a predetermined location relative to said substrate;
an active layer associated with said target zone; and
a plurality of capture agents attached to said active layer so that when said substrate is rotated, said capture agents remain attached to said active layer to thereby maintain a number of said capture agents within said target zone so that when a dual bead complex including covalently bound probes is introduced into said target zone, said capture agent sequesters said dual bead complex therein to thereby allow detection of captured dual bead complex. - View Dependent Claims (52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79)
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67. A method of preparing a dual bead assay for use in an optical bio-disc, said method comprising the steps of:
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providing a mixture of capture beads that have transport probes covalently bound thereto;
suspending said mixture of capture beads in a hybridization solution;
adding to said mixture a target agent that hybridizes with said transport probes;
adding to said mixture reporter beads including covalently bound signal probes attached thereto;
allowing said signal probes to hybridize with said target agent to thereby form a dual bead complex including at least one capture bead and one reporter bead;
separating said dual bead complex from unbound reporter beads;
removing from said mixture said unbound reporter beads; and
loading said mixture including said dual bead complex into an optical bio-disc for analysis.
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80. A method of preparing a dual bead assay for use in an optical bio-disc, said method comprising the steps of:
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providing a mixture of capture beads having transport probes covalently attached thereto;
suspending said mixture of capture beads in a hybridization solution;
adding to said mixture a target agent that hybridizes with said transport probes;
allowing said transport probes to hybridize with said target agent to thereby form a hybridized partial complex including at least one capture bead;
separating within said mixture said hybridized partial complex from unbound target agents;
adding to said mixture reporter beads including signal probes covalently attached thereto;
allowing said signal probes to hybridize with said target agent to thereby form a dual bead complex including at least one capture bead and one reporter bead;
separating said dual bead complex from unbound reporter beads;
removing from said mixture said unbound reporter beads; and
loading said mixture including said dual bead complex into an optical bio-disc for analysis. - View Dependent Claims (81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92)
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93. A method of testing for the presence of a target-DNA in a DNA sample by use of an optical bio-disc, said method comprising the steps of:
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preparing a DNA sample to be tested for the presence of a target-DNA;
preparing a plurality of reporter beads each having covalently attached thereto a plurality of strands of signal-DNA and an anchor agent, the target-DNA and the signal-DNA being complementary;
preparing a plurality of capture beads each having covalently attached thereto a plurality of transport-DNA, the target-DNA and transport-DNA being complimentary;
mixing said DNA sample, said plurality of reporter beads, and said plurality of capture beads to thereby form a test sample, the transport-DNA and the signal-DNA being non-complimentary;
allowing hybridization between said signal-DNA, any target-DNA, and transport-DNA existing in the DNA sample to thereby form a dual bead complex including at least one capture bead and one reporter bead;
removing from the test sample reporter beads and capture beads that are not associated with the dual bead complex;
depositing said test sample in a flow channel of an optical bio-disc which is in fluid communication with a target zone, the target zone including a plurality of capture agents each including an amino group that attaches to an active layer to immobilize the capture agents within the target zone;
allowing any anchor agent to bind with the capture agents so that reporter beads associated with the dual bead complex are maintained within the target zone; and
detecting any dual bead complexes in the target zone to thereby determine whether target-DNA is present in the DNA sample. - View Dependent Claims (97, 108)
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94. A method of testing for the presence of a target-DNA in a test sample by use of an optical bio-disc, said method comprising the steps of:
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preparing a test sample to be tested for the presence of a target-DNA;
preparing a plurality of reporter beads each having covalently attached thereto a plurality of strands of signal-DNA, the target-DNA and the signal-DNA being complementary;
preparing a plurality of capture beads each having covalently attached thereto a plurality of transport-DNA and an anchor agent, the target-DNA and transport-DNA being complimentary;
depositing a plurality of capture beads and reporter beads in a mixing chamber, each of said reporter beads and said capture beads including signal-DNA and transport-DNA, respectively, being non-complimentary to each other;
depositing said test sample in the mixing chamber of an optical bio-disc which is linked to a target zone by a connecting flow channel allowing any target-DNA existing in the test sample to bind to the signal-DNA and the transport-DNA on the reporter and the capture bead, respectively, to thereby form a dual bead complex;
rotating the optical bio-disc to cause the dual bead complex to move from the mixing chamber through the flow channel and into the target zone, the target zone including a plurality of capture agents each including an amino group that attaches to an active layer to immobilize the capture agents within the target zone, said capture agent having affinity for the anchor agent;
allowing any anchor agent to bind with the capture agent so that capture beads associated with dual bead complex are maintained within the capture zone;
removing from the target zone reporter beads that are free of any dual bead complex; and
detecting any dual bead complex in the target zone to thereby determine whether target-DNA is present in the test sample.
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95. A method of testing for the presence of a target-RNA in a test sample by use of an optical bio-disc, said method comprising the steps of:
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preparing a test sample to be tested for the presence of a target-RNA;
preparing a plurality of reporter beads each having covalently attached thereto a plurality of strands of signal-DNA, the target-RNA and the signal-DNA being complementary;
preparing a plurality of capture beads each having covalently attached thereto a plurality of transport-DNA and an anchor agent, the target-RNA and transport-DNA being complimentary;
depositing a plurality of capture beads and reporter beads in a mixing chamber, each of said reporter beads and capture beads including the signal-DNA and the transport-DNA, respectively, being non-complimentary to each other;
depositing said test sample in the mixing chamber of an optical bio-disc which is linked to a target zone by a connecting flow channel allowing any target-RNA existing in the test sample to hybridize with the signal-DNA and the transport-DNA on the reporter and the capture bead, respectively, to thereby form a dual bead complex;
rotating the optical bio-disc to cause the dual bead complex to move from the mixing chamber through the flow channel and into the target zone, the target zone including a plurality of capture agents each including an amino group that attaches to an active layer to immobilize the capture agents within the target zone, said capture agent and said anchor agent having affinity to each other;
allowing any anchor agent to bind with the capture agent so that capture beads associated with dual bead complex are maintained within the capture zone;
removing from the target zone reporter beads that are free of any dual bead complex; and
detecting any dual bead complex in the target zone to thereby determine whether target-RNA is present in the test sample.
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96. A method of testing for the presence of a target-antigen in a test sample by use of an optical bio-disc, said method comprising the steps of:
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preparing a test sample to be tested for the presence of a target-antigen;
preparing a plurality of reporter beads each having covalently attached thereto a plurality of signal-antibody, the signal-antibody having an affinity to epitopes on the target-antigen;
preparing a plurality of capture beads each having covalently attached thereto a plurality of transport-antibody and an anchor agent, the transport-antibody having affinity to epitopes on the target-antigen;
depositing the capture beads and the reporter beads in a mixing chamber, each of said reporter beads and capture beads including the signal-antibody and the transport-antibody, respectively, having no affinity to each other;
depositing said test sample in the mixing chamber of an optical bio-disc which is linked to a target zone by a connecting flow channel allowing any target-antigen existing in the test sample to bind to the signal-antibody and the transport-antibody on the reporter and the capture bead, respectively, to thereby form a dual bead complex;
rotating the optical bio-disc to cause the dual bead complex to move from the mixing chamber through the flow channel and into the target zone, the target zone including a plurality of capture agents each including an amino group that attaches to an active layer to immobilize the capture agents within the target zone;
allowing any anchor agent to bind with the capture agent so that capture beads associated with dual bead complex are maintained within the capture zone;
removing from the target zone reporter beads that are free of any dual bead complex; and
detecting any dual bead complex in the target zone to thereby determine whether target-antigen is present in the test sample.
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98. A method of making an optical bio-disc for testing for the presence of a target-DNA in a DNA sample, said method comprising the steps of:
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providing a substrate having a center and an outer edge;
encoding information on an information layer associated with the substrate, said encoded information being readable by a disc drive assembly to control rotation of the disc;
forming a target zone in association with said substrate, said target zone disposed at a predetermined location relative to said center of said substrate;
applying an active layer in said target zone;
depositing within said target zone, a plurality of strands of capture-DNA each including an amino group that covalently attaches to said active layer to immobilize said strands of capture-DNA within said target zone;
forming a flow channel in fluid communication with said target zone;
forming a mixing chamber in fluid communication with the flow channel;
depositing a plurality of reporter beads in the mixing chamber, each of said reporters including a signal-DNA that has an affinity for the target-DNA;
depositing a plurality of capture beads in the mixing chamber, each of said capture bead including a transport-DNA that hybridizes with a portion of the target-DNA and is complementary to said capture-DNA, the transport-DNA and signal-DNA being non-complimentary; and
designating an input site associated with the mixing chamber, the input site implemented to receive a DNA sample to be tested for the presence of any target-DNA, so that when the DNA sample is deposited in the mixing chamber hybridization occurs between the signal-DNA, the target-DNA, and the transport-DNA to thereby form a dual bead complex including at least one reporter bead and one capture bead, so that when the disc is rotated, the dual bead complex move into the target zone and hybridization occurs between the anchor-DNA and the capture-DNA to thereby place the dual bead complex in the target zone.
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99. A method of making an optical bio-disc for determining the presence of a target-DNA in a test sample, said method comprising the steps of:
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providing a substrate having a center and an outer edge;
encoding information on an information layer associated with the substrate, the encoded information being readable by a disc drive assembly to control rotation of the disc;
forming a target zone in association with the substrate, the target zone disposed at a predetermined location relative to the center of the substrate;
applying an active layer in the target zone;
depositing within the target zone, a plurality of strands of capture-DNA each including an amino group that covalently attaches to the active layer to immobilize the strands of capture-DNA within the target zone; and
forming a flow channel in fluid communication with the target zone. - View Dependent Claims (100, 101, 102)
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103. A method of making an optical bio-disc for determining the presence of a target-antigen in a test sample, said method comprising the steps of:
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providing a substrate having a center;
encoding information on an information layer associated with the substrate, the encoded information being readable by a disc drive assembly to control rotation of the disc;
forming a target zone in association with the substrate, the target zone disposed at a predetermined location relative to the center of the substrate;
depositing an active layer in the target zone;
depositing in the target zone, a plurality of capture agents each including an amino group that covalently attaches to the active layer to immobilize the capture agents within the target zone; and
forming a flow channel in fluid communication with the target zone, the flow channel implemented to receive a test sample including target-antigen. - View Dependent Claims (104, 105, 106, 115)
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107. A method of making an optical bio-disc to test for the presence of a target agent in a test sample, the method comprising the steps of:
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providing a substrate having a center and an outer edge;
encoding information on an information layer associated with the substrate, the encoded information being readable by a disc drive assembly to control rotation of the disc;
forming a target zone in association with the substrate, the target zone disposed at a predetermined location relative to the center of the substrate;
depositing an active layer in the target zone;
depositing a plurality of capture agents in the target zone, each capture agent including an amino group that covalently attaches to the active layer to immobilize the capture agent within the target zone;
forming a flow channel in fluid communication with the target zone;
forming a mixing chamber in fluid communication with the flow channel;
depositing a plurality of reporter beads in the mixing chamber, each of the reporter beads having covalently attached thereto a plurality of signal probes, each of the signal probe having affinity to the target agent; and
depositing a plurality of capture beads in the mixing chamber, each of the capture beads having covalently attached thereto a plurality of transport probes and an anchor agent, each of the transport probe having affinity to the target agent, the transport probes and signal probes having no affinity toward each other, and the capture agents and the anchor agents having specific affinity to each other.
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109. An optical bio-disc, comprising:
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a substrate having a center and an outer edge, said substrate forming a distal layer of the bio-disc, said substrate having a top surface and a bottom surface relative to an interrogation beam of electromagnetic energy directed from a disc drive;
a reflective layer formed on the bottom surface of said substrate;
an active layer associated with said substrate and said reflective layer; and
a strand of capture DNA including an amino group which has an affinity for said active layer so that said amino group covalently attaches to said active layer to immobilize said strand of DNA in a target zone disposed between said center and said outer edge. - View Dependent Claims (110, 111, 112, 113, 114)
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Specification
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Current AssigneeNagaoka & Co., Ltd.
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Original AssigneeNagaoka & Co., Ltd.
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InventorsPhan, Brigitte Chau, Lam, Amethyst Hoang, Virtanen, Jorma Antero, Yeung, KaYuen
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Application NumberUS10/086,941Publication NumberTime in Patent OfficeDaysField of SearchUS Class Current435/6CPC Class CodesC12Q 1/68 involving nucleic acidsC12Q 1/6825 Nucleic acid detection invo...C12Q 2537/125 Sandwich assay formatC12Q 2563/116 electrical properties of nu...C12Q 2563/149 Particles, e.g. beadsG01N 33/53 Immunoassay; Biospecific bi...G01N 33/531 Production of immunochemica...G01N 33/542 with steric inhibition or s...G01N 33/543 with an insoluble carrier f...
