HIGH SENSITIVITY DETERMINATION OF THE CONCENTRATION OF ANALYTE MOLECULES OR PARTICLES IN A FLUID SAMPLE
First Claim
1. A method of detecting analyte molecules or particles in a fluid sample containing or suspected of containing analyte molecules or particles, comprising:
- partitioning the fluid sample across an array comprising a plurality of reaction vessels, so that at least some of the reaction vessels contain no analyte molecules or particles and at least some of the reaction vessels contain at least one analyte molecule or particle;
immobilizing at least one binding ligand with respect to an analyte molecule or particle within each reaction vessel containing at least one analyte molecule or particle;
exposing the at least one binding ligand to a liquid in which is solubilized or suspended precursor labeling agent molecules, wherein exposure of the precursor labeling agent molecules to the at least one binding ligand converts at least some of the precursor labeling agent molecules into a labeling agent molecules which are insoluble in the liquid and/or which become immobilized within the reaction vessel; and
determining from detecting the presence of the labeling agent molecules the number of reaction vessels which contain an analyte molecule or particle.
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Accused Products
Abstract
The present invention relates to methods, systems, and kits for detecting, quantifying and/or analyzing a fluid sample comprising molecules or particles at low concentration. In certain embodiments, the methods for detection and/or quantifying analyte molecules in a sample comprise capturing a plurality of analyte molecules on a substrate (e.g., an array comprising a plurality of reaction vessels). The substrate may then be exposed to additional reaction components such as at least one binding ligand. The substrate may additionally be exposed to a precursor labeling agent molecule, wherein the precursor labeling agent molecule, in some cases, is converted to a labeling agent molecule, which may be detected, either directly or indirectly, which determination may be related to the presence of and/or may be employed to quantify the analyte molecules. Although the various aspects of the present invention may use a number of different assay formats, in one embodiment, the assays are conducted in a plurality of reaction vessels defined, at least in part, by the distal ends of fiber optic strands.
148 Citations
114 Claims
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1. A method of detecting analyte molecules or particles in a fluid sample containing or suspected of containing analyte molecules or particles, comprising:
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partitioning the fluid sample across an array comprising a plurality of reaction vessels, so that at least some of the reaction vessels contain no analyte molecules or particles and at least some of the reaction vessels contain at least one analyte molecule or particle; immobilizing at least one binding ligand with respect to an analyte molecule or particle within each reaction vessel containing at least one analyte molecule or particle; exposing the at least one binding ligand to a liquid in which is solubilized or suspended precursor labeling agent molecules, wherein exposure of the precursor labeling agent molecules to the at least one binding ligand converts at least some of the precursor labeling agent molecules into a labeling agent molecules which are insoluble in the liquid and/or which become immobilized within the reaction vessel; and determining from detecting the presence of the labeling agent molecules the number of reaction vessels which contain an analyte molecule or particle. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17-41. -41. (canceled)
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42. A system for detecting analyte molecules or particles, comprising;
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an array comprising a plurality of reaction vessels, wherein at least some of the reaction vessels contain no analyte molecules or particles and at least some of the reaction vessels contain at least one analyte molecule or particle; at least one binding ligand immobilized with respect an analyte molecule or particle within each reaction vessel containing an analyte molecule or particle; and precursor labeling agent molecules solubilized or suspended in a liquid contained within the reaction vessels, wherein the precursor labeling agent molecules are able to convert upon exposure to a binding ligand to labeling agent molecules that are insoluble within the liquid and/or that become immobilized within reaction vessels containing a binding ligand.
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43-75. -75. (canceled)
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76. A method of detecting analyte molecules or particles in a fluid sample containing or suspected of containing analyte molecules or particles, comprising:
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providing a fluid sample containing or suspected of containing analyte molecules or particles; immobilizing at least one binding ligand with respect to at least some of the analyte molecules or particles; exposing the at least one binding ligand to a liquid in which is solubilized or suspended precursor labeling agent molecules, wherein exposing the precursor labeling agent molecules to the at least one binding ligand converts at least some of the precursor labeling agent molecules into labeling agent molecules which are insoluble in the liquid and/or which become immobilized within the reaction vessel; and determining a measure of the concentration of the analyte molecules or particles in the fluid sample based on the detection of labeling agent molecules wherein the true concentration of the analyte molecules or particles in the fluid sample is less than about 100×
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15 molar, and wherein the measure of the concentration determined in the determining act differs from the true concentration by no greater than 10%.
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77-80. -80. (canceled)
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81. A kit for detecting analyte molecules or particles comprising:
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an array comprising a plurality of reaction vessels, each reaction vessel having a volume not exceeding about 100 femtoliters and each reaction vessel containing at least one analyte capture component immobilized or able to become immobilized within the reaction vessels having binding specificity for the analyte molecules or particles; at least one binding ligand having binding specificity for the analyte molecules or particles; and precursor labeling agent molecules able to be solubilized or suspended in a liquid, wherein the precursor labeling agent molecules are able to convert upon exposure to the at least one binding ligand to labeling agent molecules that are insoluble within the liquid and/or that become immobilized within the reaction vessels.
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82-112. -112. (canceled)
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113. A method of detecting analyte molecules or particles in a fluid sample containing or suspected of containing analyte molecules or particles, comprising:
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partitioning the fluid sample across an array comprising a plurality of reaction vessels, so that at least some of the reaction vessels contain no analyte molecules or particles and at least some of the reaction vessels contain at least one analyte molecule or particle; immobilizing at least one binding ligand comprising a binding site with respect to an analyte molecule or particle within each reaction vessel containing at least one analyte molecule or particle; applying an enzymatic component to the array and capturing the enzymatic component with the binding site; contacting the enzymatic component with precursor labeling agent molecules, wherein the precursor labeling agent molecules are converted to labeling agent molecules upon contact with enzymatic components; detecting the labeling agent molecules; and determining from the detection of the labeling agent molecules the number of reaction vessels which contain an analyte molecule or particle.
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114-146. -146. (canceled)
Specification