Nanopore sequencers

• US 11,567,060 B2
• Filed: 06/19/2018
• Issued: 01/31/2023
• Est. Priority Date: 06/20/2017
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Boyanov, Boyan, Akolkar, Rohan N., Fisher, Jeffrey S., Mandell, Jeffrey G., Qiang, Liangliang, Barnard, Steven M.
• Primary Examiner(s): Ball, J. Christopher
• Application Number:
  US16/624,567
• Publication Number:
  US 20200132664A1
• Time in Patent Office: 1,687 Days
• Field of Search: None
• CPC Class Codes: C12N 1/06 Lysis of microorganisms
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• 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

Example nanopore sequencers include a cis well, a trans well, and a nanopore fluidically connecting the cis and trans wells. In one example sequencer, a modified electrolyte (including an electrolyte and a cation complexing agent) is present in the cis well, or the trans well, or in the cis and the trans wells. In another example sequencer, a gel state polyelectrolyte is present in the cis well, or the trans well, or in the cis and the trans wells.

Claims

1. A nanopore sequencer, comprising:

a cis well;

a trans well;

a nanopore fluidically connecting the cis well and the trans well; and

a modified electrolyte in the cis well, or the trans well, or the cis and trans wells, the modified electrolyte including an electrolyte and a cation complexing agent;

wherein;

the modified electrolyte is in the cis well, the nanopore sequencer further comprises a non-modified electrolyte in the trans well, and the non-modified electrolyte includes the electrolyte without the cation complexing agent;

or

the modified electrolyte is in the trans well, the nanopore sequencer further comprises the non-modified electrolyte in the cis well, and the non-modified electrolyte includes the electrolyte without the cation complexing agent.

2. The nanopore sequencer as defined in claim 1, further comprising a plurality of the trans wells defined in a substrate, each of which is fluidically connected to the cis well by a respective nanopore, wherein a density of the plurality of trans wells ranges from about 10 trans wells per mm² of the substrate to about 1,000,000 trans wells per mm² of the substrate.

3. The nanopore sequencer as defined in claim 1, wherein the modified electrolyte includes greater than 0 mM to about 500 mM of the cation complexing agent.

4. A nanopore sequencer, comprising:

a cis well;

a trans well;

a nanopore fluidically connecting the cis well and the trans well; and

a modified electrolyte in the cis well, or the trans well, or the cis and trans wells, the modified electrolyte including an electrolyte and a cation complexing agent;

wherein;

the electrolyte includes a potassium cation and an associated anion, a sodium cation and the associated anion, or combinations thereof; and

the cation complexing agent is selected from the group consisting of a crown ether, a calixarene, valinomycin, and derivatives thereof.

5. The nanopore sequencer as defined in claim 4, further comprising a plurality of the trans wells defined in a substrate, each of which is fluidically connected to the cis well by a respective nanopore, wherein a density of the plurality of trans wells ranges from about 10 trans wells per mm² of the substrate to about 1,000,000 trans wells per mm² of the substrate.

6. The nanopore sequencer as defined in claim 4, wherein the modified electrolyte includes greater than 0 mM to about 500 mM of the cation complexing agent.

7. A method of using a nanopore sequencer, the nanopore sequencer comprising:

a cis well;

a trans well;

a nanopore fluidically connecting the cis well and the trans well; and

a modified electrolyte in the cis well, or the trans well, or the cis and trans wells, the modified electrolyte including an electrolyte and a cation complexing agent;

wherein the method comprises;

controlling depletion of a redox reagent of the electrolyte in the nanopore sequencer by applying a voltage bias ranging from about −

1 V to about 1 V between the cis well of the nanopore sequencer and the trans well of the nanopore sequencer.

8. The method as defined in claim 7, further comprising providing the nanopore sequencer with:

the modified electrolyte in the cis well, and the electrolyte, without the cation complexing agent, in the trans well;

or

the modified electrolyte in the trans well, and the electrolyte, without the cation complexing agent, in the cis well.

9. The method as defined in claim 7, further comprising:

incorporating the cation complexing agent into the electrolyte to form the modified electrolyte; and

introducing the modified electrolyte to the cis well, or the trans well, or the cis and trans wells.

10. The method as defined in claim 7, wherein:

the electrolyte includes a potassium cation and an associated anion, a sodium cation and the associated anion, or combinations thereof; and

the cation complexing agent is selected from the group consisting of a crown ether, a calixarene, valinomycin, and derivatives thereof.

11. A nanopore sequencer, comprising:

a cis well;

a trans well;

a nanopore fluidically connecting the cis well and the trans well; and

a polyelectrolyte in a gel state in the cis well, or the trans well, or the cis and trans wells.

12. The nanopore sequencer as defined in claim 11, wherein:

the polyelectrolyte is in the cis well, and the nanopore sequencer further comprises an electrolyte in the trans well;

or

the polyelectrolyte is in the trans well, and the nanopore sequencer further comprises an electrolyte in the cis well.

13. The nanopore sequencer as defined in claim 12, wherein:

the polyelectrolyte is selected from the group consisting of polydiallyldimethylammonium chloride, an ionic form of polyethyleneimine, an ionic form of linear polyethyleneimine, poly(allylamine hydrochloride), and an ionic form of poly(4-vinylpyridine); and

the electrolyte includes a potassium cation and an associated anion, a sodium anion and the associated anion, or combinations thereof.

14. The nanopore sequencer as defined in claim 11, further comprising a plurality of the trans wells defined in a substrate, each of which is fluidically connected to the cis well by a respective nanopore, and wherein a density of the plurality of trans wells ranges from about 10 trans wells per mm² of the substrate to about 1,000,000 trans wells per mm² of the substrate.

15. The nanopore sequencer as defined in claim 11, wherein:

the polyelectrolyte is present in both the cis and trans wells; and

the polyelectrolyte is selected from the group consisting of polydiallyldimethylammonium chloride, an ionic form of polyethyleneimine, an ionic form of linear polyethyleneimine, poly(allylamine hydrochloride), and an ionic form of poly(4-vinylpyridine).

16. A method of using the nanopore sequencer of claim 11, the method comprising:

controlling depletion of a redox reagent of the polyelectrolyte in the nanopore sequencer by applying a voltage bias ranging from about −

1 V to about 1 V between the cis well of the nanopore sequencer and the trans well of the nanopore sequencer.

17. A nanopore sequencer, comprising:

a cis well;

a trans well;

a nanopore fluidically connecting the cis well and the trans well;

a cis well electrode structure associated with the cis well, the cis well electrode structure including a first base electrode and a redox solid immobilized to the first base electrode;

a trans well electrode structure associated with the trans well, the trans well electrode structure including a second base electrode and the redox solid immobilized to the second base electrode; and

an electrolyte including a cation to be consumed or released in response to respective redox reactions at the cis well electrode structure and the trans well electrode structure.

18. The nanopore sequencer as defined in claim 17, wherein the redox solid is selected from the group consisting of tetracyanoquinodimethane (TCNQ), Prussian blue, and polypyrrole.

19. The nanopore sequencer as defined in claim 17, wherein the first and second base electrodes are independently selected from the group consisting of graphite, platinum, gold, silver, copper, carbon fiber, diamond, and palladium.

20. A method of using the nanopore sequencer of claim 17, the method comprising:

controlling depletion of a redox reagent of the electrolyte in the nanopore sequencer by;

applying a first voltage to the cis well electrode structure, thereby initiating a cathodic reaction that consumes a cation of the electrolyte in the nanopore sequencer; and

applying a second voltage to the trans well electrode structure, thereby initiating an anodic reaction that releases the cation of the electrolyte.

21. The method as defined in claim 20, wherein the redox solid is selected from the group consisting of tetracyanoquinodimethane (TCNQ), Prussian blue, and polypyrrole.