ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES USING BEADS OR OTHER CAPTURE OBJECTS
First Claim
1. A method for determining a measure of the concentration of analyte molecules or particles in a fluid sample, comprising:
- exposing a plurality of capture objects that each include a binding surface having affinity for at least one type of analyte molecule or particle, to a solution containing or suspected of containing the at least one type of analyte molecules or particles;
immobilizing analyte molecules or particles with respect to the plurality of capture objects such that at least some of the capture objects associate with at least one analyte molecule or particle and a statistically significant fraction of the capture objects do not associate with any analyte molecule or particle;
spatially segregating at least a portion of the capture objects subjected to the immobilizing step into a plurality of separate locations;
addressing at least a portion of the plurality of locations subjected to the spatially segregating step and determining the number of said locations containing an analyte molecule or particle; and
determining a measure of the concentration of analyte molecules or particles in the fluid sample based at least in part on the number of locations determined to contain an analyte molecule or particle.
1 Assignment
0 Petitions
Accused Products
Abstract
The present invention relates to systems and methods for detecting analyte molecules or particles in a fluid sample and in some cases, determining a measure of the concentration of the molecules or particles in the fluid sample. Methods of the present invention may comprise immobilizing a plurality of analyte molecules or particles with respect to a plurality of capture objects. At least a portion of the plurality of capture objects may be spatially separated into a plurality of locations. A measure of the concentration of analyte molecules in a fluid sample may be determined, at least in part, on the number of reaction vessels comprising an analyte molecule immobilized with respect to a capture object. In some cases, the assay may additionally comprise steps including binding ligands, precursor labeling agents, and/or enzymatic components.
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Citations
132 Claims
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1. A method for determining a measure of the concentration of analyte molecules or particles in a fluid sample, comprising:
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exposing a plurality of capture objects that each include a binding surface having affinity for at least one type of analyte molecule or particle, to a solution containing or suspected of containing the at least one type of analyte molecules or particles; immobilizing analyte molecules or particles with respect to the plurality of capture objects such that at least some of the capture objects associate with at least one analyte molecule or particle and a statistically significant fraction of the capture objects do not associate with any analyte molecule or particle; spatially segregating at least a portion of the capture objects subjected to the immobilizing step into a plurality of separate locations; addressing at least a portion of the plurality of locations subjected to the spatially segregating step and determining the number of said locations containing an analyte molecule or particle; and determining a measure of the concentration of analyte molecules or particles in the fluid sample based at least in part on the number of locations determined to contain an analyte molecule or particle. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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2. A method for determining a measure of the concentration of analyte molecules or particles in a fluid sample, comprising:
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exposing a plurality of capture objects that each include a binding surface having affinity for at least one type of analyte molecule or particle, to a solution containing or suspected of containing the at least one type of analyte molecules or particles to form capture objects comprising at least one immobilized analyte molecule or particle; mixing the capture objects prepared in the exposing step to a plurality of binding ligands such that at least some of the capture objects associate with a single binding ligand and a statistically significant fraction of the capture objects do not associate with any binding ligand; spatially segregating at least a portion of the capture objects subjected to the mixing step into a plurality of locations; addressing at least a portion of the plurality of locations subjected to the spatially segregating step and determining the number of locations containing a binding ligand; and determining a measure of the concentration of analyte molecules or particles in the fluid sample based at least in part on the number of locations determined to contain a binding ligand.
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14-39. -39. (canceled)
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40. A method for determining a measure of the concentration of analyte molecules or particles in a fluid sample, comprising:
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providing a substrate comprising a plurality of locations, at least a portion of which locations contain a bead, wherein with respect to the total number of beads present on the substrate, the ratio of beads comprising at least one analyte molecule or particle to beads comprising no analyte molecules or particles is between about 8;
1 and about 1;
10,000,000;addressing at least a portion of the plurality of locations, wherein during the addressing step at least two of the plurality of locations is addressed at least partially concurrently; detecting at each addressed location the presence or absence of a bead and whether, if present, the bead comprises any analyte molecules or particles; and determining a measure of the concentration of analyte molecules or particles in the fluid sample at least in part by determining the number of locations addressed containing a bead comprising at least one analyte molecule or particle.
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41. A method for determining a measure of the concentration of analyte molecules or particles in a fluid sample, comprising:
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providing a substrate comprising a plurality of locations, at least a portion of which contain a bead, wherein with respect to the total number of beads present on the substrate, the ratio of beads comprising at least one analyte molecule or particle associated with a binding ligand to beads comprising no analyte molecules or particles associated with a binding ligand is between about 8;
1 and about 1;
10,000,000;addressing at least a portion of the plurality of locations, wherein during the addressing step at least two of the plurality of locations is addressed at least partially concurrently; detecting at each addressed location the presence or absence of a bead and whether, if present, the bead comprises any analyte molecules or particles associated with a binding ligand; and determining a measure of the concentration of analyte molecules or particles in the fluid sample at least in part by determining the number of locations addressed containing a bead comprising at least one analyte molecule or particle associated with a binding ligand.
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42-70. -70. (canceled)
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71. An article or kit, comprising:
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a plurality of beads having an average diameter between about 0.1 micrometer and about 100 micrometers; and a substrate comprising a plurality of reaction vessels, wherein the average depth of the reaction vessels is between about 1.0 times and about 1.5 times the average diameter of the beads and the average diameter of the reactions vessels is between about 1.0 times and about 1.9 times the average diameter of the beads.
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72-84. -84. (canceled)
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85. A method for determining a measure of the concentration of analyte molecules or particles in a fluid sample, comprising:
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exposing a plurality of capture objects that each include a binding surface having affinity for at least one type of analyte molecule or particle, to a solution containing or suspected of containing the at least one type of analyte molecules or particles, wherein at least some of the capture objects become associated with at least one analyte molecule or particle;
mixing the plurality of capture objects prepared in the exposing step to a plurality of binding ligands comprising an enzymatic component such that a statistically significant fraction of the capture objects associated with at least one analyte molecule or particle associate with a single binding ligand;spatially segregating at least a portion of the capture objects subjected to the mixing step into a plurality of separate locations; and determining a measure of the concentration of analyte molecules or particles in the fluid sample based at least in part by addressing at least a portion of the plurality of locations subjected to the spatially segregating step to determine the presence of the enzymatic component or a product of a reaction involving the enzymatic component.
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86-101. -101. (canceled)
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102. A method for determining a measure of the concentration of analyte molecules or particles in a fluid sample, comprising:
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immobilizing a plurality of analyte molecules or particles with respect to a plurality of beads; spatially segregating at least a portion of the plurality of beads into a plurality of separate locations; addressing at least some of the plurality of locations and determining the number of locations containing a bead; further determining the number of said locations containing a bead and an analyte molecule or particle; and determining a measure of the concentration of analyte molecules or particles in the fluid sample based at least in part on the ratio of the number of locations containing a bead and an analyte molecule and particle, to the number of locations containing a bead.
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103. A method for determining a measure of the concentration of analyte molecules or particles in a fluid sample, comprising:
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immobilizing a plurality of analyte molecules or particles with respect to a plurality of beads; spatially segregating at least a portion of the plurality of beads into a plurality of separate locations; addressing at least some of the plurality of locations and determining the number of locations containing a bead; further determining the number of said locations containing a bead and an analyte molecule or particle; and determining a measure of the concentration of analyte molecules or particles in the fluid sample based at least in part on the ratio of the number of locations containing a bead and an analyte molecule and particle, to the number of locations containing a bead but not containing any analyte molecules or particles.
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104-130. -130. (canceled)
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131. A method for determining a measure of the concentration of analyte molecules or particles in a fluid sample, comprising:
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providing a plurality of capture objects that each are associated with either a single analyte molecule or particle or are free of any analyte molecules or particles; individually addressing at least a portion of the capture objects and determining the number of said capture objects associated with an analyte molecule or particle; and determining a measure of the concentration of analyte molecules or particles in the fluid sample based at least in part on the number of capture objects subjected to the addressing step determined to be associated with an analyte molecule or particle.
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132-141. -141. (canceled)
Specification