Nanopore based sequencer

• US 9,593,372 B2
• Filed: 12/19/2014
• Issued: 03/14/2017
• Est. Priority Date: 10/06/2008
• Status: Active Grant

• Current Assignee: Arizona Board of Regents (University of Arizona)
• Original Assignee: Arizona Board of Regents (University of Arizona)
• Inventors: Lindsay, Stuart, Zhang, Peiming
• Primary Examiner(s): NOGUEROLA, ALEXANDER STEPHAN
• Application Number:
  US14/577,715
• Publication Number:
  US 20150211059A1
• Time in Patent Office: 816 Days
• US Class Current: 1/1
• CPC Class Codes: C07D 235/24 with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• 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

Abstract

The present invention is directed to systems, devices and methods for identifying biopolymers, such as strands of DNA, as they pass through a constriction such as a carbon nanotube nanopore. More particularly, the invention is directed to such systems, devices and methods in which a newly translocated portion of the biopolymer forms a temporary electrical circuit between the nanotube nanopore and a second electrode, which may also be a nanotube. Further, the invention is directed to such systems, devices and methods in which the constriction is provided with a functionalized unit which, together with a newly translocated portion of the biopolymer, forms a temporary electrical circuit that can be used to characterize that portion of the biopolymer.

Claims

1. A method of sequencing a target molecule, the method comprising:

providing an apparatus including;

a substrate;

a first electrode passing through at least a portion of the substrate, the first electrode including a nanopore and configured to permit the translocation of a target molecule therethrough, the first electrode including a first recognition element connected to an end thereof;

a second electrode supported by the substrate and disposed in a fixed relationship with respect to said first electrode, the second electrode forming a first gap with the first electrode, the second electrode including a second recognition element connected to an end thereof; and

an electrical circuit configured to detect, upon passage of the target molecule past said gap, an electrical current between the first electrode and the second electrode; and

a first chamber and a second chamber, the first chamber being located on a first side of the device and the second chamber being located on a second side of the device;

introducing a target molecule into the second chamber;

electrophoresing the target molecule so that it translocates through the nanopore; and

detecting an electrical current passing through the first electrode, the first recognition element, the target molecule, the second recognition element, and the second electrode.

2. The method of claim 1, wherein the first and second recognition elements are the same.

3. The method of claim 1, each of the first recognition element and the second recognition element configured to recognize a plurality of target molecules, the first recognition element and the second recognition element further configured to form a set of hydrogen bonds with each target molecule.

4. The method of claim 1, wherein at least one of the first recognition element and the second recognition element includes thiobenzoic acid.

5. The method of claim 1, wherein the target molecule is a DNA molecule.

6. The method of claim 1, the detecting the electrical current including detecting switching between two levels of the electrical current.

7. The method of claim 1, further comprising identifying, based on the electrical current, the target molecule.

8. The method of claim 1, further comprising identifying, based on the time-varying magnitude of the electrical current, the target molecule.

9. The method of claim 1, wherein at least one of the first recognition element and the second recognition element includes 4-(mercaptomethyl)-1H-imidazole-2-carboxamide.

10. The method of claim 1, wherein at least one of the first recognition element and the second recognition element includes 5-(2-aminoethyl)-1H-imidazole-2-carboxamide.

11. The method of claim 1, wherein the universal base reader includes thiobenzoic acid.