Nanopores in zero mode waveguides

• US 9,267,917 B2
• Filed: 11/05/2012
• Issued: 02/23/2016
• Est. Priority Date: 11/04/2011
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA), Northeastern University
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Wanunu, Meni, Korlach, Jonas, Foquet, Mathieu, Turner, Stephen
• Primary Examiner(s): Bhat, Narayan
• Application Number:
  US13/669,186
• Publication Number:
  US 20130240356A1
• Time in Patent Office: 1,205 Days
• Field of Search: 435/6.1, 435/283.1, 435/287.2, 435/288.4, 435/288.7, 536/23.1, 422/68.1, 422/82.07, 422/82.11, 385/129, 204/451
• US Class Current: 1/1
• CPC Class Codes: G01N 27/447 using electrophoresis
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• 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: G02B 6/00 Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings

Abstract

Methods, devices, substrates, and systems are disclosed involving arrays of zero-mode waveguides having nanopores extending through the bases that form the bottoms of the zero-mode-waveguides. Electric fields across the nanopores are used to attach single biomolecules such as polymerase enzymes within each zero-mode-waveguide. Electric fields across the nanopores can also be used for the active loading of nucleic acid templates into enzymes attached within the zero mode waveguides.

Claims

1. An array of zero mode waveguides, each zero mode waveguide comprising an aperture having walls and a base, the base comprising a thin membrane, the array comprising a top solution above the zero mode waveguides extending into each zero mode waveguide, and a bottom solution below the thin membranes comprising the bases of the zero mode waveguides, wherein each zero mode waveguide further comprises a nanopore extending through the thin membrane to the solution below the zero mode waveguide.

2. The array of claim 1 wherein each aperture extends through an opaque cladding layer to a base layer comprising the thin membrane.

3. The array of claim 2 wherein the opaque cladding layer comprises a metal.

4. The array of claim 2 wherein the opaque cladding layer comprises aluminum.

5. The array of claim 2 wherein the thin membrane comprises silicon nitride.

6. The array of claim 1 wherein the apertures have a cross sectional dimension of between about 20 nm and 300 nm.

7. The array of claim 1 wherein the nanopores have a cross sectional dimension between about 2 nm and 10 nm.

8. The array of claim 1 wherein the thin membrane has a thickness from 5 nm to 30 nm.

9. A method for isolating a single biomolecule within a zero mode waveguide comprising:

providing an array of claim 1;

exposing the bottom of the zero mode waveguide to the bottom solution, wherein the bottom solution comprises attachment molecules, the attachment molecules having a threading portion which can extend through the nanopore, and a blocking portion which does not pass through the nanopore, wherein the threading portion comprises a binding moiety;

providing an electric field across the array that drives the threading portion of a single attachment molecule into the nanopore of a plurality of zero mode waveguides;

adding biomolecules to the top solution, the biomolecules comprising a group capable of reaction with the binding moiety on an attachment molecule;

allowing the biomolecules to react with the binding moiety whereby a single biomolecule is isolated within a single zero mode waveguide of a plurality of the zero mode waveguides.

10. The method of claim 9 wherein the blocking portions of the attachment molecules comprise a protein, a bead, or a nanoparticle.

11. The method of claim 9 wherein the biomolecule is connected to an avidin or a streptavidin.

12. The method of claim 9 wherein threading portion of the attachment molecules comprise a nucleic acid.

13. The method of claim 12 wherein the nucleic acid comprises a DNA.

14. The method of claim 9 wherein the binding moiety on the threading portion comprises a biotin.

15. The method of claim 9 wherein the group on the biomolecule capable of reacting with the binding moiety comprises an avidin or a streptavidin.

16. The method of claim 9 wherein the biomolecule comprises a polymerase enzyme.

17. A method for isolating single polymerase enzymes within a zero mode waveguides comprising:

providing an array of claim 1;

exposing the bottom of the zero mode waveguide to the bottom solution, wherein the bottom solution comprises attachment molecules, the attachment molecules having a threading portion which can extend through the nanopore, and a blocking portion which does not pass through the nanopore, wherein the threading portion comprises a binding moiety;

providing an electric field across the array that drives the threading portion of a single attachment molecule into the nanopore of a plurality of zero mode waveguides;

adding polymerase enzymes, each comprising a group capable of reaction with the binding moiety to the top solution; and

allowing the polymerase enzymes to react with the binding moiety whereby a single polymerases enzyme is isolated in a single zero mode waveguide of a plurality of the zero mode waveguides.

18. The method of claim 17 further comprising adding nucleic acid templates to the top solution and providing an electric field across the array to drive the nucleic acid templates into the zero mode waveguides.