Sandwich assays in droplets

Abstract

The invention generally relates to performing sandwich assays in droplets. In certain embodiments, the invention provides methods for detecting a target analyte that involve forming a compartmentalized portion of fluid including a portion of a sample suspected of containing a target analyte and a sample identifier, a first binding agent having a target identifier, and a second binding agent specific to the target analyte under conditions that produce a complex of the first and second binding agents with the target analyte, separating the complexes, and detecting the complexes, thereby detecting the target analyte.

What is claimed is: 
     
         1 . A method for identifying a target analyte, the method comprising:
 forming one or more compartmentalized portions of fluid, each comprising a target analyte, a first binding agent comprising a target identifier, and a second binding agent specific to the target analyte under conditions that produce a complex of the first and second binding agents with the target analyte;   separating the complexes; and   detecting the target identifier, thereby detecting the target analyte.   
     
     
         2 . The method according to  claim 1 , further comprising the step of counting the target identifier. 
     
     
         3 . The method according to  claim 1 , wherein each of the one or more compartmentalized portions comprises a different target identifier. 
     
     
         4 . The method according to  claim 1 , wherein the compartmentalized portion is a droplet. 
     
     
         5 . The method according to  claim 4 , wherein the droplet is in a microchannel. 
     
     
         6 . The method according to  claim 4 , wherein the droplet is surrounded by an immiscible carrier fluid. 
     
     
         7 . The method according to  claim 6 , wherein the carrier fluid is oil. 
     
     
         8 . The method according to  claim 1 , wherein the forming step comprises merging a droplet comprising either the first and/or the second binding agents with a droplet comprising the target analyte. 
     
     
         9 . The method according to  claim 1 , wherein the forming step comprises flowing a droplet comprising either the first and/or the second binding agents such that it interacts with a fluid stream comprising the target analyte to cause a portion of the fluid stream to integrate with the droplet. 
     
     
         10 . The method according to  claim 1 , wherein the compartmentalized portion further comprises a competitive binding inhibitor. 
     
     
         11 . The method according to  claim 1 , wherein the target identifier is releasably attached to the first binding agent. 
     
     
         12 . The method according to  claim 1 , wherein a portion of the second binding agent is functionalized for immobilization to a support. 
     
     
         13 . The method of  claim 12 , wherein the support is a solid support. 
     
     
         14 . The method of  claim 12 , wherein the portion of the second binding agent is a terminal portion of the second binding agent. 
     
     
         15 . The method according to  claim 1 , wherein the separating step comprises:
 immobilizing the complexes to a support via the functionalized portion of the second binding agent; and   releasing contents from the compartmentalized portion.   
     
     
         16 . The method of  claim 11 , further comprising the step of separating target identifiers not in a complex from target identifiers in a complex. 
     
     
         17 . The method according to  claim 1 , wherein the separating step comprises:
 releasing the contents from the compartmentalized portion;   immobilizing the complexes to the solid support via the functionalized portion of the second binding agent; and   washing the released contents to remove unbound sample components.   
     
     
         18 . The method according to  claim 1 , wherein the target analyte is derived from a sample and wherein the compartmentalized portion further comprises an identifier of the sample. 
     
     
         19 . The method of  claim 18 , wherein the identifier of the sample is capable of complexing with the target identifier. 
     
     
         20 . The method according to  claim 1 , wherein the target identifier is a barcode oligonucleotide.