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 a plurality of types of analyte molecules or particles, said plurality including at least a first type of analyte molecules or particles and a second type of analyte molecules or particles in a fluid sample, comprising:
- providing a substrate comprising a plurality of locations, at least a portion of which locations contain a first type of capture object and at least a portion of which locations contain a second type of capture object, wherein with respect to the total number of capture objects present on the substrate, the ratio of capture objects comprising at least one analyte molecule or particle to capture objects 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 first type of capture object or the presence or absence of a second type of capture object and whether, if present, the first type of capture object and the second type of capture object is associated with any of the first type of analyte molecules or particles or any of the second type of analyte molecules or particles, respectively;
determining a measure of the concentration of the first type of analyte molecules or particles in the fluid sample at least in part by determining the fraction of the first type of capture objects subjected to the addressing step determined to be associated with at least one analyte molecule or particle of the first type; and
determining a measure of the concentration of the second type of analyte molecules or particles in the fluid sample at least in part by determining the fraction of the second type of capture objects subjected to the addressing step determined to be associated with at least one analyte molecule or particle of the second type.
1 Assignment
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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
21 Claims
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1. A method for determining a measure of the concentration of a plurality of types of analyte molecules or particles, said plurality including at least a first type of analyte molecules or particles and a second type 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 first type of capture object and at least a portion of which locations contain a second type of capture object, wherein with respect to the total number of capture objects present on the substrate, the ratio of capture objects comprising at least one analyte molecule or particle to capture objects 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 first type of capture object or the presence or absence of a second type of capture object and whether, if present, the first type of capture object and the second type of capture object is associated with any of the first type of analyte molecules or particles or any of the second type of analyte molecules or particles, respectively; determining a measure of the concentration of the first type of analyte molecules or particles in the fluid sample at least in part by determining the fraction of the first type of capture objects subjected to the addressing step determined to be associated with at least one analyte molecule or particle of the first type; and determining a measure of the concentration of the second type of analyte molecules or particles in the fluid sample at least in part by determining the fraction of the second type of capture objects subjected to the addressing step determined to be associated with at least one analyte molecule or particle of the second type. - View Dependent Claims (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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2. A method for determining a measure of the concentration of a plurality of types of analyte molecules or particles, said plurality including at least a first type of analyte molecules or particles and a second type of analyte molecules and 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 first type of capture object and at least a portion of which locations contain a second type of capture object, wherein with respect to the total number of capture objects present on the substrate, the ratio of capture objects comprising at least one analyte molecule or particle associated with a binding ligand to capture objects 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 the first type of capture object or the presence of absence of a second type of capture object and whether, if present, the first type of capture object and the second type of capture object is associated with any of the first type of analyte molecules or particles or any of the second type of analyte molecules or particles, respectively; and determining a measure of the concentration of the first type of analyte molecules or particles in the fluid sample at least in part by determining the fraction of the first type of capture objects subjected to the addressing step determined to be associated with at least one analyte molecule or particle of the first type; and determining a measure of the concentration of the second type of analyte molecules or particles in the fluid sample at least in part by determining the fraction of the second type of capture objects subjected to the addressing step determined to be associated with at least one analyte molecule or particle of the second type.
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3. A method for determining a measure of the concentration of a plurality of types of analyte molecules or particles, said plurality including at least a first type of analyte molecules or particles and a second type of analyte molecules and particles in a fluid sample, comprising:
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immobilizing a plurality of the first type of analyte molecules or particles and a plurality of a second type of analyte molecules and particles with respect to a plurality of first type of capture objects and a plurality of a second type of capture objects, respectively; spatially segregating at least a portion of the plurality of first type of capture objects and the plurality of second type of capture objects into a plurality of separate locations; addressing at least some of the plurality of locations and determining the number of locations containing a first type of capture object and the number of locations containing a second type of capture object; further determining the number of said locations containing a first type of capture object and a first type of analyte molecule or particle and the number of said locations containing a second type of capture object and a second type of analyte molecule or particle; determining a measure of the concentration of the first type of analyte molecules or particles in the fluid sample based at least in part on the ratio of the number of locations containing a first type of capture object and a first type of analyte molecule and particle, to the number of locations containing a first type of capture object; and determining a measure of the concentration of the second type of analyte molecules or particles in the fluid sample based at least in part on the ratio of the number of locations containing a second type of capture object and a second type of analyte molecule and particle, to the number of locations containing a second type of capture object.
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4. A method for determining a measure of the concentration of a plurality of types of analyte molecules or particles, said plurality including at least a first type of analyte molecules or particles in a fluid sample and a second type of analyte molecules or particles, comprising:
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immobilizing a plurality of the first type of analyte molecules or particles and a plurality of a second type of analyte molecules or particles with respect to a plurality of a first type of capture objects and a plurality of second type of capture objects, respectively; spatially segregating at least a portion of the plurality of first type of capture objects and the plurality of second type of capture objects into a plurality of separate locations; addressing at least some of the plurality of locations and determining the number of locations containing a first type of capture object and the number of locations containing a second type of capture object; further determining the number of said locations containing a first type of capture object and a first type of analyte molecule or particle and the number of locations containing a second type of capture object and a second type of analyte molecule or particle; determining a measure of the concentration of first type of analyte molecules or particles in the fluid sample based at least in part on the ratio of the number of locations containing a first type of capture object and a first type of analyte molecule and particle, to the number of locations containing a first type of capture object but not containing any first type of analyte molecules or particles; and determining a measure of the concentration of second type of analyte molecules or particles in the fluid sample based at least in part on the ratio of the number of locations containing a second type of capture object and a first type of analyte molecule and particle, to the number of locations containing a second type of capture object but not containing any second type of analyte molecules or particles.
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5. 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 at least one 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 fraction of said capture objects associated with at least one 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 fraction of capture objects subjected to the addressing step determined to be associated with at least one analyte molecule or particle.
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6. A method for determining a measure of the concentration of a plurality of types of analyte molecules or particles, said plurality including at least a first type of analyte molecules or particles and a second type of analyte molecules or particles in a fluid sample, comprising:
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providing a plurality of a first type of capture objects and a plurality of a second type of capture objects, wherein the first type of capture objects are each associated with either at least one first type of analyte molecule or particle or are free of any analyte molecules or particles, and the second type of capture objects are each associated with either at least one second type of 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 fraction of said first type of capture objects associated with at least first type of one analyte molecule or particle and the fraction of said second type of capture objects associated with at least one second type of analyte molecule or particle; determining a measure of the concentration of first type of analyte molecules or particles in the fluid sample based at least in part on the fraction of first type of capture objects subjected to the addressing step determined to be associated with at least one first type of analyte molecule or particle; and determining a measure of the concentration of second type of analyte molecules or particles in the fluid sample based at least in part on the fraction of second type of capture objects subjected to the addressing step determined to be associated with at least one second type of analyte molecule or particle.
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Specification