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

• US 9,903,820 B2
• Filed: 07/31/2015
• Issued: 02/27/2018
• 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): XU, XIAOYUN
• Application Number:
  US14/815,727
• Publication Number:
  US 20160103067A1
• Time in Patent Office: 942 Days
• Field of Search: None
• 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 reproducibly ceased, and (2) self-assembly under voltage driven electrolyte flow, in which 5 nm nanopore 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 determining nucleotide sequences of polynucleotides, the method comprising the steps of:

providing an optically-addressable nanopore array comprising a solid-state insulating membrane having a plurality of nanopores, wherein the optically-addressable nanopore array separates a cis chamber from a trans chamber and wherein the nanopores are spaced so that fluorescence from adjacent nanopores is spatially resolved;

translocating fluorophore-labeled polynucleotides through the nanopores from the cis chamber to the trans chamber; and

optically probing the nanopore array wherein an optical sensor of a plurality of optical sensors selectively addresses each nanopore in the array to detect fluorescent emission variations as a function of time at each nanopore in the array, and wherein each of said fluorescent emission variations from every nucleotide as a function of time from a nanopore is indicative of a nucleotide sequence of a polynucleotide translocating therethrough.

2. The method of claim 1 wherein each of said nanopores comprises a solid-state nanopore through said membrane and a protein nanopore immobilized therein.

3. The method of claim 2 wherein said protein nanopore is immobilized by an organic chemical coating on said surface.

4. The method of claim 3 wherein said protein nanopore is an α

-hemolysin or a mutant thereof.

5. The method of claim 1 wherein said optically-addressable nanopore array has at least a 500 nm spacing between adjacent nanopores.

6. The method of claim 1 wherein said step of optically probing includes collecting said fluorescent emission variations with a microscope.

7. The method of claim 1 wherein said polynucleotide is single stranded DNA.

8. The method of claim 1 wherein said fluorescent emission variations are generated by fluorescence resonance energy transfer (FRET).

9. The method of claim 1 wherein said fluorophore-labeled polynucleotides are charged and are translocated from said cis chamber to said trans chamber by an electric field across said solid-state insulating membrane.