NANOPORES IN ZERO MODE WAVEGUIDES

• US 20130240356A1
• Filed: 11/05/2012
• Published: 09/19/2013
• 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
• Granted Patent: US 9,267,917 B2
• Time in Patent Office: Days
• US Class Current: 204/451
• 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, and each zero mode waveguide having a nanopore extending through its base.

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

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

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

5. The array of claim 2 wherein the base layer 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.

8-10. -10. (canceled)

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

providing an array of zero mode waveguides on a substrate, each zero mode waveguide having a base, each zero mode waveguide having a nanopore extending through its base;

exposing the bottom of the zero mode waveguide to a solution comprising 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 pore, wherein the threading portion comprises a binding moiety;

providing an electric field across the array that drives the threading portion of the attachment molecules into the nanopores;

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

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

12. The method of claim 11 wherein the blocking portions of the attachment molecules comprise a protein, a bead, or a nanoparticle.

13. The method of claim 11 wherein the biomolecule is connected to avidin or streptavidin.

14. The method of claim 11 wherein threading portion of the attachment molecules comprise a nucleic acid.

15. The method of claim 14 wherein the nucleic acid comprises DNA.

16. The method of claim 11 wherein the binding moiety on the threading portion comprises biotin.

17. The method of claim 11 wherein the group on the biomolecule capable of reacting with the binding moiety comprises avidin or streptavidin.

18. The method of claim 11 wherein the biomolecule comprises a polymerase enzyme.

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

providing an array of zero mode waveguides on a substrate, each zero mode waveguide having a nanopore through its base;

exposing the bottom of the zero mode waveguide to a solution comprising 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 pore, wherein the threading portion comprises an binding moiety;

providing an electric field across the array that drives the threading portion of the attachment molecules into the nanopores;

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

allowing the polymerase enzymes to react with the binding moieties whereby a single biomolecule is isolated in a plurality of the zero mode waveguides.

20. The method of claim 19 further comprising adding nucleic acid templates to the top of the array and providing an electric field across the array to drive the nucleic acid templates into the zero mode waveguides.

21. The method of claim 20 wherein a salt gradient is formed in order to enhance driving of the biomolecules into the zero mode waveguides.

22-26. -26. (canceled)