Chemical functionalization of solid-state nanopores and nanopore arrays and applications thereof

• US 10,101,315 B2
• Filed: 01/20/2017
• Issued: 10/16/2018
• Est. Priority Date: 05/08/2007
• Status: Active Grant

• Current Assignee: Trustees of Boston University
• Original Assignee: Trustees of Boston University
• Inventors: Meller, Amit, Wanunu, Meni
• Primary Examiner(s): Hixson, Christopher Adam
• Application Number:
  US15/411,782
• Publication Number:
  US 20170198346A1
• Time in Patent Office: 634 Days
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68
• CPC Class Codes: Y10T 428/2982 Particulate matter [e.g., sphere, flake, etc.]
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Chemical functionalization of solid-state nanopores and nanopore arrays and applications thereof. Nanopores are extremely sensitive single-molecule sensors. Recently, electron beams have been used to fabricate synthetic nanopores in thin solid-state membranes with sub-nanometer resolution. A new class of chemically modified nanopore sensors are provided with two approaches for monolayer coating of nanopores by: (1) self-assembly from solution, in which nanopores −10 nm diameter can be reproductibly coated, and (2) self-assembly under voltage-driven electrolyte flow, in which 5 nm nanopores may be coated. Applications of chemically modified nanopore are provided including: the detection of biopolymers such as DNA and RNA; immobilizing enzymes or other proteins for detection or for generating chemical gradients; and localized pH sensing.

Claims

1. A method for characterizing nucleotide sequences of mixtures of DNA molecules comprising:

providing an optically-addressable nanopore array comprising a solid-state electrically insulating membrane having a plurality of apertures each aperture having a surface with an organic monolayer coating and a protein nanopore immobilized therein by the organic monolayer coating;

receiving fluorophore-labeled DNA molecules through the nanopores;

optically probing the nanopore array; and

detecting variations in fluorescence from the fluorophore-labeled DNA molecules at each nanopore, the variations in fluorescence being indicative of a nucleotide sequence of a fluorophore-labeled DNA molecule as it translocates through a nanopore.

2. The method of claim 1 wherein said organic monolayer coating comprises at least one epoxy, methoxyethylene glycol, amine, carboxylic acid or aldehyde.

3. The method of claim 1 wherein said organic monolayer coating comprises a methoxyethylene glycol-terminated silane monolayer.

4. The method of claim 1 wherein said protein nanopore is an α

-hemolysin or a mutant thereof.

5. The method of claim 1 wherein said nanopores of said optically-addressable nanopore array are spaced so that fluorescence detected from adjacent nanopores is spatially resolved.

6. The method of claim 5 wherein said optically-addressable nanopore array has at least a 500 nm spacing between adjacent nanopores.

7. The method of claim 1 wherein said step of detecting includes collecting said fluorescence with a microscope.

8. The method of claim 1 wherein said DNA molecules are single stranded DNA molecules.

9. The method of claim 1 wherein said insulating solid-state membrane separates a cis chamber from a trans chamber.

10. The method of claim 1 wherein said fluorescence is generated by fluorescence resonance energy transfer (FRET).

26. The method of claim 1 wherein said organic monolayer coating includes a reactive monolayer coating forming covalent bonds with a surface of said membrane.

11. A method for characterizing a mixture of DNA molecules comprising:

providing an optically-addressable nanopore array comprising a solid-state electrically insulating membrane that has a plurality of apertures and that separates a cis chamber from a trans chamber, wherein each aperture has a surface with an organic monolayer coating and a protein nanopore immobilized therein by the organic monolayer coating;

translocating fluorophore-labeled single-stranded DNAs of the DNA molecules through nanopores of the array from the cis chamber to the trans chamber;

optically probing the nanopore array;

detecting with optical sensors variations in fluorescence from the fluorophore-labeled single stranded DNAs in the proximity to the membrane at each nanopore, wherein the variations in fluorescence are indicative of sequences of the DNA molecules.

12. The method of claim 11 wherein said organic monolayer coating comprises at least one epoxy, methoxyethylene glycol, amine, carboxylic acid or aldehyde.

13. The method of claim 11 wherein said organic monolayer coating comprises a methoxyethylene glycol-terminated silane monolayer.

14. The method of claim 11 wherein said protein nanopore is an α

-hemolysin or a mutant thereof.

27. The method of claim 11 wherein said organic monolayer coating includes a reactive monolayer coating forming covalent bonds with a surface of said membrane.

15. A method for characterizing analytes comprising:

providing an optically-addressable nanopore array comprising a solid-state electrically insulating membrane having a plurality of apertures and separating a cis chamber from a trans chamber, wherein each aperture has a surface with an organic monolayer coating and a protein nanopore immobilized therein by the organic monolayer coating;

receiving a mixture of fluorophore-labeled DNAs from the cis chamber to the trans chamber through the nanopores;

optically probing the nanopore array; and

detecting variations in fluorescence from the fluorophore-labeled DNA as a function of time in proximity to the membrane at each nanopore on a trans side of the nanopore array, the variations in fluorescence as a function of time being indicative of sequences of the fluorophore-labeled DNAs as they translocate through the nanopores.

16. The method of claim 15 wherein said organic monolayer coating comprises at least one epoxy, methoxyethylene glycol, amine, carboxylic acid or aldehyde.

17. The method of claim 15 wherein said organic monolayer coating comprises a methoxyethylene glycol-terminated silane monolayer.

18. The method of claim 15 wherein said protein nanopore is an α

-hemolysin or a mutant thereof.

28. The method of claim 15 wherein said organic monolayer coating includes a reactive monolayer coating forming covalent bonds with a surface of said membrane.

19. A method for characterizing local chemical environments to determine nucleotide sequences, the method comprising the steps of:

providing an optically-addressable nanopore array comprising a solid-state electrically insulating membrane having a plurality of apertures, each aperture having a surface with an organic monolayer coating and a protein nanopore immobilized therein by the organic monolayer coating;

receiving environmentally sensitive reagents from a cis chamber in a trans chamber through the nanopores wherein the environmentally sensitive reagents comprise a mixture of fluorophore-labeled single-stranded DNAs;

optically probing the nanopore array as environmentally sensitive reagents translocate through the nanopores; and

detecting variations in fluorescence from the fluorophore-labeled DNA as a function of time from the environmentally sensitive reagents in proximity to the membrane at the nanopores, wherein the variations in fluorescence at each nanopore is indicative of a nucleotide sequence of a fluorophore-labeled single-stranded DNA.

20. The method of claim 19 wherein said organic monolayer coating comprises at least one epoxy, methoxyethylene glycol, amine, carboxylic acid or aldehyde.

21. The method of claim 19 wherein said protein nanopore is an α

-hemolysin or a mutant thereof.

22. The method of claim 19 wherein said solid-state insulating membrane separates a cis chamber from a trans chamber and wherein said environmentally sensitive reagents are charged and are translocated from the cis chamber to the trans chamber under an electrical field across said insulating solid-state membrane.

23. The method of claim 19 wherein said nanopores of said optically-addressable nanopore array are spaced so that fluorescence detected from adjacent nanopores is spatially resolved.

24. The method of claim 19 wherein said step of detecting includes collecting said fluorescence with a microscope.

29. The method of claim 19 wherein said organic monolayer coating includes a reactive monolayer coating forming covalent bonds with a surface of said solid-state electrically insulating membrane.

25. The method of 19 wherein said organic monolayer coating comprises a methoxyethylene glycol-terminated silane monolayer.