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. An analysis method comprising:
encapsulating cells into a plurality of droplets to create at least one droplet containing a single cell, wherein the droplet includes one or more binding agents, wherein the binding agents provide an analyte identifier for a target analyte and an identifier specific for the droplet;
lysing the cell within the droplet to release the target analyte;
binding the one or more binding agents to the target analyte; and
detecting the target analyte identifier, thereby detecting the target analyte.
2. The method of claim 1 , wherein the identifier specific for the droplet is detected to associate the detection of the target analyte with the droplet.
3. The method of claim 1 , wherein the target analyte is a protein or a nucleic acid.
4. The method of claim 1 , wherein encapsulating the cells into the plurality of droplets includes diluting the concentration of single cells in a collection to a level where less than about one cell is present for every 10 droplets formed.
5. The method of claim 1 , wherein the lysing step involves exposing the cell to lysis reagents that release the cellular nucleic acids, proteins, and other components from the cell.
6. The method of claim 1 , wherein the detecting step provides for analysis of molecules released from single cells lysed inside droplets, wherein the molecules include cytoplasmic or nuclear proteins.
7. The method of claim 1 , wherein the one or more binding agents include a first barcoded binder that includes the analyte identifier.
8. The method of claim 7 , wherein the analyte identifier comprises an oligonucleotide sequence that constitutes a unique identifier or barcode.
9. The method of claim 7 , wherein the first barcoded binder is a nucleic acid with a barcode sequence attached thereto.
10. The method of claim 9 , wherein the barcode sequence is designed to include no homopolymer repeats.
11. The method of claim 1 , wherein the lysing step includes one selected from the group consisting of: (1) co-flowing a lysis buffer in a laminar flow alongside the incoming cell stream in the flow path just before the droplet-forming microfluidic nozzle; (2) introducing a lysis buffer within droplet library reagents; (3) use of a temperature or other inducible protease or lysis reagent; (4) mechanical abrasion inside droplets traveling through microfluidic turns and constrictions; and (5) laser-induced lysis.
12. The method of claim 1 , wherein one or more of the plurality of droplets include multiple sets of binding pairs for multi-plex target analysis.
13. The method of claim 1 , wherein the droplet further contains a sample identifier and all individual analyte molecules from the cell are labeled with the sample identifier.
14. The method of claim 13 , wherein the sample identifier combines with the analyte identifier so all analytes can be traced to a sample.
15. The method of claim 1 , wherein the detecting step comprises nucleic acid sequencing.
16. A droplet library comprising:
a plurality of droplets, wherein at least one droplet comprises, in an aqueous phase fluid,
a single cell,
one or more binding agents that provide an analyte identifier for a target analyte and an identifier specific for the droplet, and
lysis reagents to lyse the cell.
17. The library of claim 16 , further comprising a number of library elements, each element comprising a single cell encapsulated in a droplet, that are pooled together in a single collection.
18. The library of claim 16 , wherein the one or more binding agents include a first barcoded binder that includes the analyte identifier.
19. The library of claim 18 , wherein the analyte identifier comprises an oligonucleotide sequence that constitutes a unique identifier or barcode.
20. The library of claim 19 , wherein the first barcoded binder is a nucleic acid with a barcode sequence attached thereto.