Devices and systems for molecular barcoding of nucleic acid targets in single cells

Patent metadata

Field	Value
Publication number	US-2016289669-A1
Application number	US-201615004618-A
Country	US
Kind code	A1
Filing date	Jan 22, 2016
Priority date	Jan 22, 2015
Publication date	Oct 6, 2016
Grant date	—

Abstract

Disclosed herein are devices and systems comprising a) a substrate comprising at least 100 microwells and a plurality of beads, wherein a plurality of the at least 100 microwells each contain a single bead, and wherein the ratio of the average diameter of the microwells to the diameter of the beads ranges from about 1.2 to about 1.8; b) a flow cell in fluid communication with the substrate; and c) at least one inlet port and at least one outlet port, wherein the at least one inlet port and at least one outlet port are capable of directing a flow of a fluid through the flow cell, thereby contacting the microwells with the fluid.

First claim

1 . (canceled)

2 . A device comprising:
a substrate comprising at least 100 microwells, wherein each microwell has a volume ranging from about 1,000 μm 3 to about 786,000 μm 3 , and wherein a surface of the at least 100 microwells is coated with a surface coating to improve wettability; and a flow cell in fluid communication with the substrate.

3 . (canceled)

4 . (canceled)

5 . (canceled)

6 . (canceled)

7 . The device of claim 2 , further comprising at least one inlet port and at least one outlet port, wherein the at least one inlet port and the at least one outlet port are in fluid communication with the flow cell via fluid channels having axes that form an angle relative to the plane of the substrate, and wherein the at least one inlet port and the at least one outlet port are capable of directing a flow of a fluid through the flow cell, thereby contacting the microwells with the fluid.

8 . (canceled)

9 . The device of claim 2 , wherein the substrate comprises from 1,000 to 5,000,000 microwells.

10 . (canceled)

11 . The device of claim 2 , wherein each microwell has a volume ranging from about 21,000 μm 3 to about 170,000 μm 3 .

12 . (canceled)

13 . The device of claim 2 , wherein the microwells have a non-circular cross section in the plane of the substrate, and wherein the non-circular cross section in the plane of the substrate is square or hexagonal.

14 . The device of claim 2 , wherein the aspect ratio of average diameter to depth for the at least 100 microwells ranges from about 0.1 to 2.

15 . (canceled)

16 . The device of claim 2 , wherein the dimension of each microwell allows each microwell to contain at most one bead.

17 . The device of claim 2 , wherein the ratio of the average diameter of the microwells to the diameter of the beads ranges from about 1.2 to about 1.8.

18 . The device of claim 2 , wherein the side walls of the microwells have a positive draft angle of about 1 to 15 degrees.

19 . (canceled)

20 . The device of claim 2 , wherein the substrate further comprises surface features that surround each microwell or straddle the surface between microwells of the at least 100 microwells.

21 . The device of claim 20 , wherein the substrate further comprises surface features that surround each microwell or straddle the surface between microwells of the at least 100 microwells, wherein the surface features are selected from the group consisting of rounded, domed, ridged, and peaked surface features, or any combination thereof.

22 . The device of claim 2 , wherein the percentage of the at least 100 microwells that contain a single bead is at least about 10%.

23 . (canceled)

24 . The device of claim 2 , wherein the percentage of the at least 100 microwells that contain a single cell is between about 0.01% and about 15%.

25 . (canceled)

26 . The device of claim 2 , wherein a surface of the at least 100 microwells is coated with polyethylene glycol (PEG), poly-Hema, pluronic acid F68, pluronic acid F108, polysorbate 20, silicon dioxide (SiO2), or any combination thereof.

27 . The device of claim 2 , wherein a surface of the at least 100 microwells comprises a plasma-treated surface.

28 . (canceled)

29 . (canceled)

30 . (canceled)

31 . (canceled)

32 . (canceled)

33 . The device of claim 2 , wherein the substrate is fabricated from a material selected from the group consisting of silicon, fused-silica, glass, a polymer, a metal, an elastomer, polydimethylsiloxane, agarose, and a hydrogel, or any combination thereof.

34 . (canceled)

35 . (canceled)

36 . The device of claim 2 , wherein the flow cell is fabricated from a material selected from the group consisting of silicon, fused-silica, glass, polydimethylsiloxane (PDMS; elastomer), polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), polyimide, cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET), epoxy resin, and metal, or any combination of these materials.

37 . (canceled)

38 . (canceled)

39 . (canceled)

40 . The device of claim 2 , further comprising a pipette tip interface for loading or removing samples, assay reagents, bead suspensions, or waste from the device.

41 . The device of claim 40 , wherein the pipette tip interface comprises a conical feature that mates to a pipette tip to form a fluid connection with the inlet port or outlet port.

42 . The device of claim 41 , wherein the conical feature is comprised of a compliant material that forms a substantially leak-proof seal with the pipette tip.

43 . (canceled)

44 . The device of claim 41 , further comprising a valve that prevents fluid flow within the device unless a pipette tip is inserted into the conical feature of the pipette tip interface.

45 . The device of claim 2 , wherein each single bead of a plurality of beads contained within the at least 100 microwells comprises a plurality of tethered stochastic labels capable of attaching to a target nucleic acid molecule in a stochastic manner, wherein each stochastic label in the plurality of tethered stochastic labels comprises a cell label that is identical for all of the stochastic labels attached to that bead, but is different for stochastic labels attached to different beads, wherein the plurality of tethered stochastic labels attached to a single bead further comprises a diverse set of molecular labels, and wherein each stochastic label in the plurality of tethered stochastic labels further comprises a target nucleic acid molecule binding region.

46 .- 51 . (canceled)

52 . A system comprising:
a device comprising:
a substrate comprising at least 100 microwells, wherein each microwell has a volume ranging from about 1,000 μm 3 to about 786,000 μm 3 , and wherein a surface of the at least 100 microwells is coated with a surface coating to improve wettability;
a flow cell in fluid communication with the substrate; and
at least one inlet port and at least one outlet port, wherein the at least one inlet port and at least one outlet port are capable of directing a flow of a fluid through the flow cell, thereby contacting the microwells with the fluid; and
a flow controller; wherein the flow controller is configured to control the delivery of fluids.

53 . (canceled)

54 . The system of claim 52 , further comprising fluids wherein the fluids comprise cell samples, bead suspensions, assay reagents, or any combination thereof.

55 . (canceled)

56 . The system of claim 54 , wherein cell samples and bead suspensions are dispensed or injected directly into the device by the user.

57 . The system of claim 54 , wherein beads and assay reagents other than cell samples are preloaded in the device.

58 . The system of claim 52 , wherein the flow controller is configured to intersperse fluid injections into the flow ce

Assignees

• Becton Dickinson Co

Inventors

• Fan Christina
• Fodor Stephen P A
• Fu Glenn
• Facer Geoff
• Stern David
• Lai Janice
• Chaney Ari
• Spuhler Phillip
• Li Sixing
• Huang Xiaohua Chen
• Metzler Kimberly R
• Walczak Elisabeth Marie

Classifications

• B01L3/502746

  characterised by the means for controlling flow resistance, e.g. flow controllers, baffles or throttle valves · CPC title

• B01L2300/0654

  Lenses; Optical fibres · CPC title

• B01L3/502738

  characterised by integrated valves (throttle valves in microfluidic sample containers B01L3/502746) · CPC title

• B01L2300/0819

  Microarrays; Biochips · CPC title

• B01L2300/12

  Specific details about materials · CPC title