Massively parallel on-chip coalescence of microemulsions

Patent metadata

Field	Value
Publication number	US-2018071738-A1
Application number	US-201615559381-A
Country	US
Kind code	A1
Filing date	Mar 18, 2016
Priority date	Mar 18, 2015
Publication date	Mar 15, 2018
Grant date	—

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

1 . A microfluidic device comprising:
at least one droplet input for receiving one or more sets of droplets, each set of droplets comprising one or more agents; and an array of microwells for receiving one or more droplets from the one or more sets of droplets.

2 . The microfluidic device of claim 1 , further comprising;
at least one flow channel from which droplets from each droplet set are randomly distributed into the array of microwells such that droplets from each droplet set are randomly co-located in individual microwells of the array; a loading mechanism, wherein the loading mechanism comprises a bottom clamp on which the array of microwells is placed, one or more removable spacers that when inserted define a flow channel beneath the array of microwells, a top clamp, and one or more connectors for securing the top clamp to the bottom clamp such that the array of microwells is secured against the spacer when inserted or the bottom clamp when the spacers are removed; or both the at least one flow channel and the loading mechanism.

3 . (canceled)

4 . The microfluidic device of claim 2 , wherein the droplets rise or sink via buoyancy from the flow channel into empty microwell spaces.

5 . The microfluidic device of claim 1 , wherein the microwells are sized to capture at least two droplets, at least three droplets, at least three droplets, at least four droplets, at least five droplets, or at least six droplets.

6 . (canceled)

7 . The microfluidic device of any one of claims 1 to 6 , wherein each microwell is sized to capture droplets of a same size or each microwell is sized to capture droplet of a different size.

8 . (canceled)

9 . The microfluidic device of claim 1 , wherein the microwells comprise a single linear shape sized to hold at least two droplets, the microwells comprise a single circular shape that is sized to hold at least two droplets, or the microwells comprise a set of interconnected hemispheres sized to hold at least two droplets.

10 . (canceled)

11 . (canceled)

12 . The microfluidic device of claim 9 , wherein the interconnected hemispheres are arranged in a linear fashion, or wherein the interconnected hemispheres are arranged in a radial fashion.

13 . (canceled)

14 . The microfluidic device of claim 1 , wherein the device comprises a single flow channel.

15 . The microfluidic device of claim 2 , wherein the flow channel is approximately 10 mm to approximately 50 mm in width; or approximately 25 mm to approximately 100 mm in length; or approximately 100 μm to 500 μm in depth/height.

16 . The microfluidic device of claim 1 , further comprising a coalescing mechanism for merging the two droplets in a microwell space to form a single droplet.

17 . The microfluidic device of claim 16 , wherein the coalescing mechanism coalesces the droplets by electrocoalescence, thermal coalescence, acoustic coalescence, changes in surfactant concentration, or a combination thereof.

18 . A system comprising:
microfluidic device of claim 1 ; and an automated high content imaging device to image each microwell.

19 . 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 sets of droplets onto a microfluidic device comprising an array of microwells, the microwells sized to capture at least two droplets, by flowing the at least one set of droplets through at least one flow channel located beneath the microwells; 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.

20 . The method of claim 19 , wherein the microfluidic device comprising at least one droplet input for receiving one or more sets of droplets, each set od droplets comprising one or more agents; and an array of microwells for receiving the one or more droplets from the one or more set of droplets.

21 . The method of claim 19 , wherein the droplets of at least one set of the at least two sets of droplets further comprises an optical barcode.

22 . The method of claim 19 , wherein the optical barcode comprises an optically detectable label that identifies the one or more molecular species in a given droplet.

23 . The method of claim 22 , 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 lable is an object of a different size, shape, color, or combination thereof.

24 . (canceled)

25 . The method of claim 24 , 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.

26 . (canceled)

27 . (canceled)

28 . The method of claim 21 , further comprising identifying the one or more agents in the merged droplets by imaging each microwell to detect the optically detectable label, eluting the merged droplets off the microfluidic device and identifying the merged droplets by the optical barcodes, or both.

29 . (canceled)

30 . The method of claim 28 , wherein identifying the merged droplets by the optical barcodes 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.

31 . The method of claim 28 , wherein identifying the merged droplets by the optical barcode comprises generating an amplicon of the optical barcode and sequencing the optical barcode.

32 . The method of claim 28 , wherein the droplets of at least one set of the at least two sets of droplets further comprise one or more reporter agents.

33 . The method of claim 32 , wherein;
a) the one or more agents 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.

34 . (canceled)

35 . The method of claim 33 , wherein the barcoded molecule specific for the peptide or protein is a barcoded antibody or antibody fragment.

36 . The method of claim 32 , further comprising;
eluting the merged droplets off the microfluidic device; and sorting the merged droplet contents by the one or more reporter agents.

37 . The method of claim 19 , wherein the droplets of at least one set of the at least two sets of droplets further comprises an optical barcode and a reporter agent.

38 . The method of claim 37 , further comprising:
eluting the merged droplets from the device; sorting the merged droplets by optical barcode; and detecting the detection agent.

39 . The method of claim 29 , wherein eluting the merged droplets comprises inverting the device such that the merged droplets float into

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology

Inventors

• Blainey Paul
• Kulesa Anthony
• Kehe Jared

Classifications

• B01L3/502715

  characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces · CPC title

• B01J2219/00418

  using pressure · CPC title

• B01J2219/00317

  Microwell devices, i.e. having large numbers of wells · CPC title

• B01J2219/00547

  Bar codes · CPC title

• B01L2200/0689

  Sealing · CPC title