Massively parallel on-chip coalescence of microemulsions

Patent metadata

Field	Value
Publication number	US-12539516-B2
Application number	US-202017135689-A
Country	US
Kind code	B2
Filing date	Dec 28, 2020
Priority date	Mar 18, 2015
Publication date	Feb 3, 2026
Grant date	Feb 3, 2026

Abstract

Embodiments disclosed herein are directed to microfluidic devices that allow for scalable on-chip screening of combinatorial libraries and methods of use thereof. Droplets comprising individual molecular species to be screened are loaded onto the microfluidic device. The droplets are labeled by methods known in the art, including but not limited to barcoding, such that the molecular species in each droplet can be uniquely identified. The device randomly sorts the droplets into individual microwells of an array of microwells designed to hold a certain number of individual droplets in order to derive combinations of the various molecular species. The paired droplets are then merged in parallel to form merged droplets in each microwell, thereby avoiding issues associated with single stream merging. Each microwell is then scanned, e.g., using microscopy, such as high content imaging microscopy, to detect the optical labels, thereby identifying the combination of molecular species in each microwell.

First claim

What is claimed is:

1 . A method for combinatorial screening of molecular species comprising:
generating at least one set of droplets, each droplet comprising one or more molecular species; randomly distributing droplets from the at least one set of droplets onto a microfluidic device comprising an array of microwells defined in a top layer above a single flow channel, the microwells sized to capture at least two droplets, wherein each microwell is configured to capture between two and ten droplets and the microwell configuration is in the shape of two or more interconnected hemispheres, by flowing the at least one set of droplets through the flow channel in a carrier oil, such that the droplets are able to rise out of the carrier oil via buoyancy and into available microwell spaces; and merging the droplets captured in each microwell to form a single merged droplet comprising the combination of the one or more molecular species from each individual droplet captured in the microwell.

2 . The method of claim 1 , wherein the microfluidic device comprises at least one droplet input for receiving one or more sets of droplets.

3 . The method of claim 1 , wherein the droplets of the at least one set of droplets further comprise an optical barcode.

4 . The method of claim 3 , wherein the optical barcode comprises an optically detectable label that identifies the one or more molecular species in a given droplet.

5 . The method of claim 4 , wherein the optically detectable label is a fluorophore, and wherein optionally the fluorophore is bound to or encapsulated within a bead, or wherein the optically detectable label is an object of a different size, shape, color, or combination thereof.

6 . The method of claim 5 , wherein the optical barcode comprises a sub-set of fluorescent beads from a set of at least 37 distinct fluorescent beads or the sub-set comprises at least 2, at least 3, at least 4, at least 5, or at least 6 distinct fluorescent beads.

7 . The method of claim 3 , further comprising identifying the one or more molecular species in the merged droplets by imaging each microwell to detect the optical barcode, eluting the merged droplets off the microfluidic device and identifying the merged droplets by the optical barcode, or both.

8 . The method of claim 7 , wherein identifying the merged droplets by the optical barcode comprises sequentially hybridizing labeled probes, each probe hybridizing to a specific oligonucleotide sequence out of a set of possible oligonucleotide sequences at each position in the optical barcode.

9 . The method of claim 7 , wherein identifying the merged droplets by the optical barcode comprises generating an amplicon of the optical barcode and sequencing the optical barcode.

10 . The method of claim 7 , wherein the droplets of the at least one set of droplets further comprise one or more reporter agents.

11 . The method of claim 10 , wherein;
a) the one or more molecular species is a nucleic acid or a cell, and the one or more reporter agents are barcoded primers designed to amplify a target sequence from the nucleic acid or cell; or b) the one or more agents is a peptide or protein, and the one or more reporter agents is a barcoded molecule specific for the peptide or protein.

12 . The method of claim 11 , wherein the barcoded molecule specific for the peptide or protein is a barcoded antibody or antibody fragment.

13 . The method of claim 10 , further comprising;
eluting the merged droplets off the microfluidic device; and sorting the merged droplet contents by the one or more reporter agents.

14 . The method of claim 1 , wherein the droplets of the at least one set of droplets further comprise an optical barcode and a reporter agent.

15 . The method of claim 14 , further comprising:
eluting the merged droplets from the device; sorting the merged droplets by the optical barcode; and detecting the reporter agent.

16 . The method of claim 13 , wherein eluting the merged droplets comprises inverting the device such that the merged droplets float into an elution solution in the flow channel.

17 . The method of claim 1 , further comprising washing out surfactant from the merged droplets to prevent redistribution of droplet contents to another microwell.

18 . The method of claim 17 , wherein washing out the surfactant comprises flowing through the flow channel a solution comprising a compound that sequesters surfactant.

19 . The method of claim 18 , wherein the compound that sequesters surfactant is perfluorooctanol.

20 . The method of claim 1 , further comprising introducing an additional molecular species to the merged droplets by flowing the additional molecular species through the flow channel such that the additional molecular species is delivered to each microwell.

21 . The method of claim 1 , wherein the droplets are merged by electrocoalescence, thermal coalescence, acoustic coalescence, or changes in surfactant concentration.

22 . The method of claim 1 , wherein the at least one set of droplets comprises at least two sets of droplets.

23 . The method of claim 22 , wherein the at least two sets of droplets comprises a first set of droplets and a second set of droplets,
wherein the first set of droplets comprises an antibiotic from a set of antibiotics and wherein the second set of droplets comprises an antibiotic adjuvant from a set of antibiotic adjuvants and one or more bacteria, wherein at least a subset of the merged droplets comprises an antibiotic from the set of antibiotics and an adjuvant from a set of antibiotic adjuvants.

24 . The method of claim 23 , wherein the first and second set of droplets further comprise an optical barcode.

25 . The method of claim 24 , wherein the optical barcode comprises an optically detectable label that identifies the one or more molecular species in a given droplet.

26 . The method of claim 25 , wherein the optically detectable label is a fluorophore or an object of a different size, shape, color, or combination thereof.

27 . The method of claim 25 , wherein the optical barcode comprises a sub-set of fluorescent beads from a set of at least 37 distinct fluorescent beads.

28 . The method of claim 24 , further comprising determining an efficacy of the antibiotic and antibiotic adjuvant combination in each microwell by imaging each microwell to obtain a bacterial phenotype, the optical barcode identifying the combination of antibiotic and antibiotic adjuvant.

29 . A method for combinatorial screening of molecular species comprising:
generating at least one set of droplets, each droplet comprising one or more molecular species; randomly distributing droplets from the at least one set of droplets onto a microfluidic device comprising an array of microwells defined in a top layer above a single flow channel, the microwells configured to capture between two and ten droplets from the at least one set of droplets based on buoyancy and the microwell configuration being in the shape of two or more interconnected hemispheres; flowing the at least one set of droplets through the flow channel in a carrier oil, such that the droplets are able to rise out of the carrier oil via buoyancy and into available microwell spaces; and merging the droplets captured in each microwell to form a single merged droplet compri

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology

Inventors

• Blainey Paul
• Kulesa Anthony
• Kehe Jared

Classifications

• G01N2333/195

  from bacteria · CPC title

• C40B60/08

  Integrated apparatus specially adapted for both creating and screening libraries · CPC title

• C40B50/10

  involving encoding steps · CPC title

• B01L2400/0469

  Buoyancy · CPC title

• B01L2400/046

  Chemical or electrochemical formation of bubbles · CPC title