Single-molecule capacitive nucleic acid sequencing with nanoscale electrode pairs

• US 9,868,987 B2
• Filed: 10/21/2015
• Issued: 01/16/2018
• Est. Priority Date: 05/06/2013
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Turner, Stephen
• Primary Examiner(s): Forman, Betty J
• Application Number:
  US14/918,957
• Publication Number:
  US 20160083789A1
• Time in Patent Office: 818 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2563/157 Nanotubes or nanorods
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: G01N 27/227 Sensors changing capacitance upon adsorption or absorption of fluid components, e.g. electrolyte-insulator-semiconductor sensors, MOS capacitors G01N27/225 takes precedence
• CPC Class Codes: G01N 27/4145 specially adapted for biomolecules, e.g. gate electrode with immobilised receptors
• CPC Class Codes: G01N 27/4146 involving nanosized elements, e.g. nanotubes, nanowires

Abstract

Sequencing methods, devices, and systems are described. Arrays of nanoscale electronic elements comprising two electrodes separated by an insulating layer are used to provide sequence information about a template nucleic acid in a polymerase-template complex bound proximate to the insulating region. A sequencing reaction mixture comprising nucleotide analogs having impedance labels is introduced to the array of nanoscale electronic elements under conditions of polymerase mediated nucleic acid synthesis. The time sequence of incorporation of nucleotide analogs is determined by identifying the types of labels of the nucleotide analogs that are incorporated into the growing strand using measured impedance.

Claims

1. A method for single molecule nucleic acid sequencing comprising:

providing a substrate comprising an array of nanoscale capacitive devices, each capacitive device comprising two nanoscale electrodes separated by an insulating region, wherein a single polymerase enzyme complex comprising a single polymerase enzyme and a template nucleic acid is attached to the insulating region;

exposing the substrate to a plurality of types of nucleotide analogs, each type comprising a different capacitive label attached to the phosphate portion of the nucleotide analog under conditions whereby polymerase mediated nucleic acid synthesis occurs, resulting in cleavage of the capacitive label and the growth of a nascent nucleic acid strand;

applying a voltage across the two nanoscale electrodes in each device, whereby when a nucleotide analog resides in the active site of the enzyme, the capacitive label on the nucleotide analog produces a measurable change in the capacitance measured at the nanoscale electrodes, such change in capacitance occurring before the cleavage of the capacitive label;

measuring the capacitance at the nanoscale electrodes over time, whereby the capacitance over time indicates an incorporation event and identifies the type of nucleotide analog by its capacitive label; and

using the measured capacitance at the electrodes over time to determine a sequence of the template nucleic acid.

2. The method of claim 1 wherein the voltage applied across the nanoscale electrodes is an AC voltage.

3. The method of claim 2 wherein the AC voltage at the nanoscale electrodes is brought to different frequency levels while a nucleotide analog is within the active site of the enzyme.

4. The method of claim 1 wherein the measured over time includes the measurement of current oscillation color.

5. The method of claim 1 wherein the measured capacitance over time comprises a measurement of conductance over time across the nanoscale electrodes.

6. The method of claim 1 wherein the substrate is exposed to four types of nucleotide analogs corresponding to A, G, C, T, or A, G, C, U, each of the four types of nucleotide analogs having a different capacitive label.

7. The method of claim 1 wherein the nanoscale electrodes are co-planar with the insulating region.

8. The method of claim 1 wherein the two nanoscale electrodes are disposed on the surface such that each comprises a wall, and the polymerase enzyme complex is attached to the insulating region between the walls.

9. The method of claim 1 wherein the two nanoscale electrodes are disposed vertically with respect to the substrate.

10. The method of claim 1 wherein at least one of the capacitive labels comprises a charged label.

11. The method of claim 1 wherein at least one of the capacitive labels comprises a negatively charged label.

12. The method of claim 1 wherein at least one of the capacitive labels comprises a nanoparticle.

13. The method of claim 12 wherein the nanoparticle comprises a metal, an oxide, or a polymer.

14. The method of claim 1 wherein the substrate comprises 100,000 to 10 million capacitive devices.

15. The method of claim 1 wherein each capacitive device further comprises a reference electrode.

16. The method of claim 1 wherein each capacitive device further comprises a control electrode.

17. The method of claim 1 wherein each capacitive device further comprises a reference electrode and a control electrode.

18. The method of claim 1 wherein the time constant for charging the capacitive devices is less than 1 microsecond.

19. The method of claim 1 wherein the time constant for charging the capacitive devices is less than 100 microseconds.