Methods and systems for microfluidic screening

Abstract

Provided are methods and systems useful for screening large libraries of effector molecules. Such methods and systems are particularly useful in microfluidic systems and devices. The methods and systems provided herein utilize encoded effectors to screen large libraries of effectors.

1 .- 20 . (canceled) 
     
     
         21 . A method comprising:
 (a) providing or obtaining a microfluidic device comprising a droplet formation region connected to a flow path;   (b) providing or obtaining an effector library comprising a plurality of different effectors, wherein an effector of the plurality of different effectors is bound to a scaffold of a plurality of scaffolds via a cleavable linker and is releasable from the scaffold upon cleavage of the cleavable linker, and wherein the scaffold further comprises an encoding corresponding to and for identifying a structure of the effector;   (c) generating a plurality of droplets in the droplet formation region and encapsulating the effector library in the plurality of droplets, wherein each droplet of a subset of the plurality of droplets comprises at least one scaffold of the plurality of scaffolds;   (d) incubating the plurality of droplets in the flow path for an incubation time, wherein the incubation time is at least 14 minutes and a dispersion ratio of incubation times of the plurality of droplets is less than 4%;   (e) measuring a signal indicative of an activity of the effector from the plurality of droplets; and   (f) performing at least one of:
 (i) sorting a droplet, contents of a droplet, a scaffold, an encoding, or any combination thereof, based on the signal; 
 (ii) measuring a property of an encoding to identify a structure of the effector in a droplet, based on the signal; or 
 (iii) adding a barcode to tag an encoding in the droplet, based on the signal. 
   
     
     
         22 . The method of  claim 21 , wherein the dispersion ratio is at most 2.5%. 
     
     
         23 . The method of  claim 21 , wherein the dispersion ratio is at most 3%. 
     
     
         24 . The method of  claim 21 , wherein the microfluidic device generates the plurality of droplets at a frequency of at least 70 Hz. 
     
     
         25 . The method of  claim 21 , wherein the effector library comprises at least 100,000 different effectors. 
     
     
         26 . The method of  claim 21 , wherein the encoding is a nucleic acid encoding comprising a nucleic acid sequence unique to the effector bound to the scaffold. 
     
     
         27 . The method of  claim 26 , wherein both the nucleic acid encoding and the effector are covalently bound to the scaffold prior to encapsulation in a droplet. 
     
     
         28 . The method of  claim 26 , wherein measuring the property of the encoding comprises sequencing the encoding. 
     
     
         29 . The method of  claim 21 , wherein the encoding is unique to the identity of the effector. 
     
     
         30 . The method of  claim 21 , wherein the encoding further corresponds to and identifies the scaffold. 
     
     
         31 . The method of  claim 21 , wherein the encoding comprises a plurality of subunits of the encoding and the effector comprises a plurality of subunits of the effector, and one or more of the plurality of subunits of the encoding each correspond to and identify the effector or one or more of the plurality of subunits of the effector. 
     
     
         32 . The method of  claim 21 , wherein the cleavable linker is a photocleavable linker releasable by light. 
     
     
         33 . The method of  claim 21 , wherein upon cleavage of the cleavable linker, a pre-determined amount or dose of the effector is released into the droplet. 
     
     
         34 . The method of  claim 21 , wherein the activity is measured in an activity-based assay, and wherein the droplet further comprises an assay reagent. 
     
     
         35 . The method of  claim 34 , wherein the microfluidic device comprises a first inlet for accepting the assay reagent and a second inlet for accepting the effector library, wherein the assay reagent comprises a first fluid, wherein the effector library is suspended in a second fluid different from the first fluid, and wherein the second fluid flows through the second inlet 
     
     
         36 . The method of  claim 21 , wherein the microfluidic device further comprises a detection region and a sorting region for generating the signal and sorting the droplets based on the signal. 
     
     
         37 . The method of  claim 21 , wherein the signal comprises electromagnetic radiation, thermal radiation, fluorescence, luminescence, or any combination thereof. 
     
     
         38 . The method of  claim 21 , wherein the encoding comprises deoxyribonucleic acid, ribonucleic acid, a peptide, or a peptide nucleic acid. 
     
     
         39 . The method of  claim 21 , wherein the scaffold is selected from the group consisting of: a solid support, a bead, a fiber, a nanofibrous scaffold, a molecular cage, a dendrimer, and a multi-valent molecular assembly. 
     
     
         40 . The method of  claim 21 , wherein the scaffold is at least 1 micrometer (μm) in diameter. 
     
     
         41 . The method of  claim 21 , wherein the plurality of droplets are aqueous droplets surrounded by an immiscible carrier fluorinated oil.