Methods for determining the concentration of an analyte in solution

Abstract

Disclosed is a method for measuring the concentration of an analyte or analytes in a solution. Although the methods can be conducted using a number of different assay formats, in one embodiment, the assays are conducted in reaction vessels defined, at least in part, by the distal ends of fiber optic strands.

1 . A method of determining the concentration of an analyte in a fluid sample to be tested, the method comprising the steps of:
 (a) partitioning at least a portion of the analyte molecules in the fluid sample across a plurality of reaction vessels so that a statistically significant fraction of the reaction vessels contain analyte and a statistically significant fraction of the reaction vessels contain no analyte;   (b) determining the presence or absence of the analyte in each reaction vessel to identify the number of reaction vessels that contain analyte and/or to identify the number of reaction vessels that contain no analyte; and   (c) determining the concentration of the analyte in the fluid sample from the number of reaction vessels that contain the analyte.   
     
     
         2 - 11 . (canceled) 
     
     
         12 . A method of determining the concentration of an analyte in a fluid sample to be tested, the method comprising the steps of:
 (a) exposing the fluid sample to a plurality of reaction vessels under conditions so that analyte is captured in a statistically significant fraction of the reaction vessels, wherein each reaction vessel comprises a microwell and each reaction vessel defines a binding surface that has a capture component immobilized thereon;   (b) determining the presence or absence of the analyte in each reaction vessel so as to identify the number of reaction vessels that contain captured analyte and/or the number of reaction vessels that do not contain captured analyte; and   (c) determining the concentration of the analyte in the fluid sample to be tested from the number of reaction vessels that contain and/or do not contain the analyte.   
     
     
         13 - 16 . (canceled) 
     
     
         17 . A method of determining the concentration of an analyte in a fluid sample to be tested, the method comprising the steps of:
 (a) partitioning at least a portion of the analyte molecules in the fluid sample into a plurality of reaction vessels, so that, for substantially all of the reaction vessels, each reaction vessel contains either no analyte or a single molecule of the analyte;   (b) determining the presence or absence of the analyte in each reaction vessel to provide a number of the reaction vessels that contain the analyte; and   (c) determining the concentration of the analyte in the fluid sample from the number of reaction vessels that contain the analyte.   
     
     
         18 - 20 . (canceled) 
     
     
         21 . A method of determining the concentration of an analyte in a fluid sample to be tested, the method comprising the steps of:
 (a) partitioning the sample to be tested into a plurality of second, smaller fluid samples of equal volume so that a statistically significant fraction of the second, smaller fluid samples contain either a single molecule of the analyte or no analyte;   (b) determining the presence or absence of the analyte in each of the second samples so as to identify the number of second samples that contain the analyte; and   (c) determining the concentration of the analyte in the sample to be tested from the number of second samples that contain the analyte.   
     
     
         22 - 35 . (canceled) 
     
     
         36 . A method of detecting analyte molecules or particles in a fluid sample, comprising:
 partitioning a fluid sample containing or suspected of containing analyte molecules or particles 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 within each reaction vessel containing at least one analyte molecule or particle, a first binding ligand comprising a label;   applying an enzyme to the array and capturing the enzyme with the label;   contacting the enzyme with an enzymatic substrate, wherein the enzymatic substrate produces a detectable product upon contact with the enzyme;   detecting the detectable product; and   determining from detecting the presence of the detectable product the number of reaction vessels which contain an analyte molecule or particle.   
     
     
         37 . A method of detecting analyte molecules or particles in a fluid, comprising:
 partitioning a fluid containing or suspected of containing analyte molecules or particles 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;   exposing the reaction vessels to an enzymatic substrate, wherein the enzymatic substrate produces a detectable product in reaction vessels containing at least one analyte molecule or particle;   detecting the detectable product; and   determining from detecting the presence of the detectable product the number of reaction vessels which contain at least one analyte molecule or particle.   
     
     
         38 . A method of detecting analyte molecules or particles in a fluid, comprising:
 immobilizing with respect to each analyte molecules or particles, an enzyme molecule;   partitioning the enzyme molecules across an array comprising a plurality of reaction vessels, so that at least some of the reaction vessels contain no enzyme molecules and at least some of the reaction vessels contain at least one enzyme molecule;   exposing the reaction vessels to an enzymatic substrate, wherein the enzymatic substrate produces a detectable product in reaction vessels containing at least one enzyme molecule; and   determining from detecting the presence of the detectable product the number of reaction vessels which contain at least one enzyme molecule.   
     
     
         39 . The method of  claim 1 , wherein the analyte molecules are exposed to a plurality of secondary binding ligands after the analyte molecules have been partitioned across the plurality of reaction vessels, wherein at least one secondary binding ligand becomes associated with each analyte molecule. 
     
     
         40 . The method of  claim 39 , wherein the presence or absence of the analyte in each reaction vessel is determined by determining the presence or absence of a secondary binding ligand in each reaction vessel. 
     
     
         41 . The method of  claim 40 , wherein the number of reaction vessels that contain or do not contain the analyte is determined by determining the number of reaction vessels that contain or do not contain the secondary binding ligand. 
     
     
         42 . The method of  claim 1 , wherein at least one wash step is performed. 
     
     
         43 . The method of  claim 1 , wherein the reaction vessels are sealed during at least step (b). 
     
     
         44 . The method of  claim 1 , wherein each reaction vessel is exposed to a plurality of reaction components, wherein at least one of a chromogenic, fluorogenic, or chemiluminescent product is produced upon exposure of plurality of reaction components to the secondary binding ligand. 
     
     
         45 . The method of  claim 44 , wherein the presence or absence of the analyte in each reaction vessel is determined by determining the presence or absence of at least one chromogenic, fluorogenic, or chemiluminescent product in each reaction vessel. 
     
     
         46 . The method of  claim 45 , wherein the concentration of the analyte in the fluid sample is determined from determining the number of reaction vessels that contain or do not contain at least one chromogenic, fluorogenic, or chemiluminescent product. 
     
     
         47 . The method of  claim 1 , wherein the partitioning of at least a portion of the analyte molecules in the fluid sample across a plurality of reaction vessels comprises partitioning the fluid sample across a plurality of wells of an array comprising the plurality of wells. 
     
     
         48 . The method of  claim 1 , wherein at least one analyte of a first type and at least one analyte of a second type are partitioned across the plurality of reaction vessels. 
     
     
         49 . The method of  claim 48 , wherein the reaction vessels comprises at least one capture component per reaction vessel, wherein the capture component is able to immobilize at least one analyte molecule. 
     
     
         50 . The method of  claim 49 , wherein the first analyte or the second analyte is able to be immobilized with respect to a first capture component. 
     
     
         51 . The method of  claim 50 , wherein the reaction vessels comprise at least one capture component of a first type per reaction vessel and at least one capture component of a second type per reaction vessel.