Ultra-sensitive detection of molecules or particles using beads or other capture objects

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.

What is claimed: 
     
         1 - 141 . (canceled) 
     
     
         142 . A method for determining a measure of the concentration of analyte molecules or particles in a fluid sample, comprising:
 (1) preparing a plurality of capture objects that each are associated with either at least one analyte molecule or particle of the fluid sample or are free of any analyte molecules or particles of the fluid sample;   (2) individually isolating at least a portion of the capture objects subjected to the preparing step from each other;   (3) addressing at least a portion of the capture objects individually isolated in the isolating step and determining a measure indicative of the number or fraction of the individually isolated capture objects associated with at least one analyte molecule or particle of the fluid sample; and   (4) determining a measure of the concentration of analyte molecules or particles in the fluid sample based at least in part on the measure determined in step (3)   
     
     
         143 . The method of  claim 142 , wherein the addressing step is performed using optical techniques. 
     
     
         144 . The method of  claim 143 , wherein the optical techniques are capable of substantially simultaneously addressing at least a portion of the capture objects individually isolated in the isolating step. 
     
     
         145 . The method of  claim 142 , wherein the addressing step comprises determining the number of the individually isolated capture objects associated with at least one analyte molecule or particle of the fluid sample. 
     
     
         146 . The method of  claim 145 , wherein the addressing step further comprises determining a measure indicative of a ratio of the number of the individually isolated capture objects associated with at least one analyte molecule or particle of the fluid sample to the number of the individually isolated capture objects not associated with an analyte molecule or particle of the fluid sample. 
     
     
         147 . The method of  claim 145 , wherein the step of determining a measure of the concentration of analyte molecules or particles in the fluid sample is based at least in part on a Poisson distribution analysis involving the number of the individually isolated capture objects associated with at least one analyte molecule or particle of the fluid sample. 
     
     
         148 . The method of  claim 142 , wherein the individually addressing step comprises determining the fraction of the individually isolated capture objects associated with at least one analyte molecule or particle of the fluid sample. 
     
     
         149 . The method of  claim 142 , wherein the capture objects comprise or are each contained within a liquid droplet, and the step of individually isolating the at least a portion of the capture objects comprises suspending the liquid droplets in a fluid that is immiscible with the liquid droplets. 
     
     
         150 . The method of  claim 149 , wherein the capture objects are beads. 
     
     
         151 . 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;   detecting at each addressed location the presence or absence of a capture object and whether, if present, the capture object is associated with any of analyte molecules or particles; and   determining a measure of the concentration of analyte molecules or particles in the fluid sample based at least in part by determining a measure indicative of the number or fraction of the capture objects subjected to the addressing step determined to be associated with at least one analyte molecule or particle.   
     
     
         152 . The method of  claim 151 , wherein the step of determining a measure of the concentration of analyte molecules or particles in the fluid sample comprises determining the number of the capture objects subjected to the addressing step determined to be associated with at least one analyte molecule or particle. 
     
     
         153 . The method of  claim 152 , wherein the step of determining a measure of the concentration of analyte molecules or particles in the fluid sample comprises determining the ratio of the number of capture objects subjected to the addressing step determined to be associated with at least one analyte molecule or particle of the fluid sample to the number of capture objects subjected to the addressing step determined not to be associated with an analyte molecule or particle of the fluid sample. 
     
     
         154 . The method of  claim 152 , wherein the step of determining a measure of the concentration of analyte molecules or particles in the fluid sample is based at least in part on a Poisson distribution analysis involving the number of capture objects subjected to the addressing step determined to be associated with at least one analyte molecule or particle of the fluid sample. 
     
     
         155 . The method of  claim 151 , wherein the step of determining a measure of the concentration of analyte molecules or particles in the fluid sample comprises determining the fraction of the capture objects subjected to the addressing step determined to be associated with at least one analyte molecule or particle. 
     
     
         156 . A system for determining a measure of the concentration of analyte molecules or particles in a fluid sample, comprising:
 at least one detector configured to address individual locations of an assay substrate, wherein the at least one detector is able to detect:
 at least one signal indicative of the presence or absence of a capture object at each location addressed, and 
 at least one signal indicative of the presence or absence of an analyte molecule or particle immobilized with respect to a capture object at each location addressed; and 
   at least one computer comprising a processor programmed and configured to determine:
 (a) from the at least one signal indicative of the presence or absence of a capture object, a measure indicative of the total number of the individual locations containing a capture object or indicative of the number of the individual locations containing a capture object not associated with an analyte molecule, and 
 (b) from the at least one signal indicative of the presence or absence of an analyte molecule or particle, a measure indicative of the number of the individual locations containing at least one analyte molecule or particle, 
 wherein the at least one computer comprises a processor programmed and configured to determine a measure indicative of the concentration of analyte molecules or particles in the fluid sample based at least in part on (a) and (b). 
   
     
     
         157 . An article, comprising:
 a plurality of beads having an average diameter between about 0.1 micrometer and about 10 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,   wherein at least some of the reaction vessels of the plurality of reaction vessels contain a bead of the plurality of beads.   
     
     
         158 . A kit, comprising:
 a plurality of beads having an average diameter between about 0.1 micrometer and about 10 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.   
     
     
         159 . A kit for at least one assay for detecting at least one analyte molecule or particle, comprising:
 beads having an average diameter between about 0.1 micrometer and about 10 micrometers;   wherein the beads have a binding surface having affinity for the at least one type of analyte molecule or particle; and   wherein a total number of the beads having a binding surface having affinity for the at least one type of analyte molecule or particle provided per assay is between about 10,000 and about 1,000,000.   
     
     
         160 . A kit for at least one assay for detecting at least one analyte molecule or particle, comprising:
 beads having an average diameter between about 0.1 micrometer and about 10 micrometers; and   capture components having affinity for the at least one type of analyte molecule or particle, wherein the capture components are associated with or associable with the beads;   wherein a total number of the beads provided per assay is between about 10,000 and about 1,000,000.   
     
     
         161 . A kit for an assay for detecting at least one analyte molecule or particle, comprising:
 beads having an average diameter between about 0.1 micrometer and about 10 micrometers, wherein the beads are able to produce or be made to produce a detectable signal;   capture components having affinity for the at least one type of analyte molecule or particle, wherein the capture components are associated with or associable with the beads;   binding ligands comprising beta-galactosidase; and   resorufin-beta-D-galactopyranoside molecules.