Semi-permeable arrays for analyzing biological systems and methods of using same

Patent metadata

Field	Value
Publication number	US-12071663-B2
Application number	US-201716070505-A
Country	US
Kind code	B2
Filing date	Jan 17, 2017
Priority date	Jan 15, 2016
Publication date	Aug 27, 2024
Grant date	Aug 27, 2024

Abstract

The present application provides a method of assembling a container for one or multiple parallel steps of biochemical analysis on one or more cells comprising performing molecular bonding of a porous membrane on an apical or basal surface of an array having a plurality of wells, wherein the molecular bonding substantially isolates each well from adjacent wells.

First claim

What is claimed is:

1. A method for high-throughput parallel single cell biochemical analysis in a container, the method comprising providing an array container, the array container comprising:
an array having a plurality of wells, the array comprising:
(a) a first functionalized surface of each well, wherein the first functionalized surface comprises an affinity resin; and
(b) a second functionalized surface of a top surface of the array, wherein the top surface is in planar configuration with the openings of the wells, and wherein the second functionalized surface provides accessible ionic functional groups, and

a porous membrane configured to be positioned on the top surface and over the openings of the wells, wherein the porous membrane and the top surface of the array can cooperate by establishing molecular bonding so as to substantially isolate each well, or a subset of wells, from adjacent wells, and wherein isolation of each well or subset of wells comprises confining cells, macromolecules, or a combination thereof, inside the wells by sealing the loaded wells with the porous membrane, wherein the porous membrane pore size selectively isolates the cells and the macromolecules while allowing flow of liquid and/or molecules of a certain smaller size through the porous membrane.

2. The method of claim 1 , wherein the array comprises at least one additional functionalized surface of each well, wherein the at least one additional functionalized surface provides for multiplexing reactions.

3. The method of claim 1 , wherein the array comprises an array material comprising polydimethylsiloxane, polycarbonate, polystyrene, polymethyl-methacrylate, polyvinylidene difluoride, polyvinylchloride, polypropylene, cyclic olefin co-polymer, a glass, silicon, or polystyrene modified with dextran; and/or wherein the array comprises an array material doped with an ionic functional group anywhere from 1% to 30% (by molar basis) providing access for further modification via molecular bonding or providing charged moieties at surfaces.

4. The method of claim 1 , wherein the top surface comprises an array material functionalized to an organosilane and/or provides free alcohols upon treatment; wherein the top surface comprises organosiloxane functional groups conjugated to polydimethylsiloxane; or wherein the top surface comprises functional groups conjugated to cyclic olefin co-polymer using aryl diazonium salts.

5. The method of claim 4 , wherein the free alcohols are reacted with an amino-silane to create an amine functionalized surface.

6. The method of claim 1 , wherein the ionic functional group is a positively charged functional group.

7. The method of claim 1 , wherein the affinity resin comprises a negatively charged surface, wherein the negatively charged surface repels mRNA, and wherein the negatively charged surface enables mRNA to be captured by poly(dT) beads.

8. The method of claim 1 , wherein the porous membrane is an ion exchange membrane and/or an ultrafiltration membrane, and wherein the ultrafiltration membrane comprises a weak cationic exchange surface or a polymeric surface functionalized with chitosan.

9. An array container for multiple parallel single-cell biochemical analysis, comprising
an array having a plurality of wells, wherein said array has a functionalized top surface in a planar configuration with the openings of the wells, and
a porous membrane configured to be positioned on the top surface of the array and over the openings of the wells,
wherein the porous membrane and the functionalized top surface of the array can cooperate by establishing molecular bonding so as to substantially isolate each well, or a subset of wells, from adjacent wells, wherein isolation of each well or subset of wells comprises confining cells, macromolecules, or a combination thereof, inside the wells by sealing the loaded wells with the porous membrane, wherein the porous membrane pore size selectively isolates the cells and the macromolecules, while allowing flow of liquid and/or molecules of a certain smaller size through the porous membrane.

10. The array container of claim 9 , wherein the array container comprises a plurality of Unique Molecular Identifiers (UMIs).

11. The array container of claim 9 , wherein molecular bonding comprises one or more of covalent bonding, ion-ion bonding, dipole-dipole bonding, ion-dipole bonding, hydrogen bonding, and van der Waals bonding.

12. The array container of claim 9 , wherein the molecular bonding is effective when the container is immersed in an aqueous solution.

13. The array container of claim 9 , wherein the plurality of wells comprises about at least 1,000 or at least 10,000 or at least about 100,000 or at least 200,000 or at least 500,000 or up to 1,000,000 wells or about 50,000 to 1,000,000 wells.

14. The array container of claim 9 , wherein the wells are micro-sized wells, nano-sized wells, or pico-sized wells; wherein the array has about 80,000 50 μm-wells, or 250,000 30 μm-wells, or 1,000,000 15 μm-wells; wherein the wells have a size less than or equal to about 50 microns on each side; and/or wherein the distance between adjacent wells is about 15 or 30 or 40 or 50 microns.

15. The array container of claim 9 , wherein the well volume is equal or less than a microliter.

16. The array container of claim 9 , wherein the molecular bonding between the porous membrane and the top surface of the array is reversible.

17. The array container of claim 9 , wherein the top surface of the array bears a charge of a first polarity; the porous membrane bears a charge of a second polarity, the second polarity being opposite the first polarity and is hydrated; wherein the charged porous membrane is on the charged top surface of the array.

18. The array container of claim 17 , wherein the first polarity is positive and the second polarity is negative.

19. The array container of claim 9 , wherein the top surface comprises organosiloxane functional groups.

20. The array container of claim 9 , wherein the array comprises polydimethylsiloxane (PDMS), polycarbonate (PC), polystyrene (PS), polymethyl-methacrylate (PMMA), PVDF, polyvinylchloride (PVC), polypropylene (PP), cyclic olefin co-polymer (COC), a glass, or is silicon.

21. The array container of claim 17 , wherein a charge of a first polarity is induced on the top surface of the array by: plasma treating the array; and coating the top surface with an organosiloxane functional group.

22. The array container of claim 21 , further comprising treating the top surface of the array with a polysaccharide.

23. The array container of claim 9 , wherein the porous membrane is functionalized by a reactive functional group.

24. The array container of claim 23 , wherein the top surface of the array is functionalized by a reactive functional group.

25. The array container of claim 23 , wherein the top surface of the array is functionalized with an amine, an amine silane, a thiosilane, a methacrylate silane, a poly(allylamine), poly(lysine), BSA, epoxide silane, or chitosan.

26. The array container of claim 9 , wherein part or all of the inside surface of the wells of the array is functionalized with a different molecule than the top surface of the array.

27. The array container of claim 9 , wherein part or all of the inside surface of the wells is functionalized to activate loaded cells, capture secreted products, to

Assignees

• Massachusetts Inst Technology

Inventors

• Gierahn Todd M
• Love J Christopher
• Hughes Travis K
• Wadsworth Ii Marc H
• Shalek Alexander K
• Carroll Shaina

Classifications

• B01J2219/00572

  Chemical means · CPC title

• B01J19/0046

  Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays (synthesis methods per se C40B50/00) · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q2565/514

  characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array · CPC title

• C12Q2563/179

  the label being a nucleic acid · CPC title