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 screening method comprising:
 (a) providing at least one cell and a scaffold in a compartment, wherein the scaffold comprises:
 (i) an effector bound to the scaffold by a cleavable linker; and 
 (ii) a barcode corresponding to and for identifying the effector; 
   (b) cleaving the cleavable linker to release the effector from the scaffold, thereby generating a released effector in the compartment;   (c) detecting a signal from the compartment, wherein the signal results from an interaction between the released effector and the at least one cell;   (d) identifying the compartment as a positive compartment or a negative compartment based on the signal; and,   (e) performing at least one of:
 (i) sorting the positive compartment, sorting contents of the positive compartment, sorting the scaffold of the positive compartment, sorting the barcode of the positive compartment, physically separating the positive compartment from the negative compartment in space, or any combination thereof, based on the identifying of (d); 
 (ii) measuring a property of the barcode in the positive compartment to elucidate the structure of the effector in the positive compartment, based on the identifying of (d); or 
 (iii) adding a secondary barcode to tag the barcode in the positive compartment, based on the identifying of (d). 
   
     
     
         22 . The method of  claim 21 , wherein the barcode is a nucleic acid barcode. 
     
     
         23 . The method of  claim 21 , wherein the barcode comprises a property or subunit unique to the effector. 
     
     
         24 . The method of  claim 21 , wherein the barcode comprises a property or subunit unique to the scaffold. 
     
     
         25 . The method of  claim 21 , wherein the barcode comprises at least one barcode subunit, the effector comprises at least one effector subunit, and the at least one barcode subunit corresponds to and identifies the effector or the at least one effector subunit. 
     
     
         26 . The method of  claim 21 , wherein the barcode is covalently bound to the scaffold. 
     
     
         27 . The method of  claim 21 , wherein the effector is selected from the group consisting of: a peptide, a protein, an enzyme, a small molecule, and a nucleic acid. 
     
     
         28 . The method of  claim 25 , wherein at least two of the at least one effector subunit are covalently attached to one another. 
     
     
         29 . The method of  claim 21 , wherein the scaffold comprises at least one of: a solid support, a bead, a fiber, a nanofibrous scaffold, a molecular cage, a dendrimer, and a multi-valent molecular assembly. 
     
     
         30 . The method of  claim 21 , wherein the scaffold comprises a polymer bead comprising an internal section and an outer surface, and wherein the internal section comprises a plurality of reactive groups and the bead surface comprises a surface modification rendering the bead surface orthogonally protected to the reactive groups. 
     
     
         31 . The method of  claim 21 , wherein the scaffold is at least 1 micrometer (μm) in diameter. 
     
     
         32 . The method of  claim 21 , wherein the identifying in (d) further comprises using a computer, using an image processing device, using a computer program or an algorithm, defining a threshold for the signal, or any combination thereof. 
     
     
         33 . The method of  claim 32 , further comprising providing or obtaining a system comprising a detector, the computer, the image processing device, the algorithm, or a combination thereof. 
     
     
         34 . The method of  claim 21 , wherein the signal results from an activity-based assay. 
     
     
         35 . The method of  claim 21 , wherein the cleavable linker is a photocleavable linker, configured to be cleaved upon exposure to light. 
     
     
         36 . The method of  claim 21 , wherein the cleaving releases a pre-determined quantity or dose of the effector into the compartment. 
     
     
         37 . The method of  claim 21 , wherein the barcode comprises a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a peptide nucleic acid, or a peptide. 
     
     
         38 . The method of  claim 22 , further comprising providing a reagent in the compartment, wherein the reagent is configured to cleave, nick, prime, or amplify the nucleic acid barcode, or any combination thereof. 
     
     
         39 . The method of  claim 38 , wherein the reagent comprises a nuclease, a restriction enzyme, a primer, a polymerase, a deoxyribonucleoside triphosphate (dNTP), a polymerase chain reaction (PCR) mix, or any combination thereof. 
     
     
         40 . The method of  claim 22 , further comprising amplifying or sequencing the nucleic acid barcode. 
     
     
         41 . The method of  claim 21 , further comprising providing a plurality of different scaffolds in a plurality of compartments, wherein a subset of the plurality of compartments each comprises at least one cell and at least one scaffold, and wherein the concentrations of the released effectors in the subset of the plurality of compartments differ by no more than 2-fold. 
     
     
         42 . The method of  claim 41 , wherein the plurality of different scaffolds each comprise a unique effector, and wherein the plurality of different scaffolds comprise at least 10,000 unique effectors. 
     
     
         43 . The method of  claim 42 , wherein each unique effector is screened at least 2 times.