SANDWICH ASSAYS IN DROPLETS
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.
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.