High speed molecular sensing with nanopores

• US 10,421,995 B2
• Filed: 10/23/2014
• Issued: 09/24/2019
• Est. Priority Date: 10/23/2013
• Status: Active Grant

• Current Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Original Assignee: Genia Technologies, Inc. (Roche Holding AG)
• Inventors: Davis, Randall
• Primary Examiner(s): Leavitt, Maria G
• Application Number:
  US14/521,469
• Publication Number:
  US 20150111779A1
• Time in Patent Office: 1,797 Days
• Field of Search: None
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• 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: H03B 1/00 Details

Abstract

Described herein are methods and devices for capturing and determining the identity of molecules using nanopores. The molecules can be counted, sorted and/or binned rapidly in a parallel manner using a large number of nanopores (e.g., 132,000 nanopores reading 180 million molecules in 1 hour). This fast capture and reading of a molecule can be used to capture probe molecules or other molecules that have been generated to represent an original, hard to detect molecule or portions of an original molecule. Precise counting of sample molecules or surrogates for sample molecules can occur. The methods and devices described herein can, among other things, replace flow cytometers and other counting instruments (e.g., while providing increased precision and throughput relative to a flow cytometer). In some cases, the devices and methods capture and hold particular molecules or surrogates of molecules in the nanopores and then eject them into clean solution to perform a capture, sorting, and binning function similar to flow cytometers.

Claims

1. A method for molecular counting and/or sorting, comprising:

a. providing a nanopore array comprising multiple nanopores in a lipid bilayer membrane and multiple nanopore detectors, each of said nanopore detectors comprising a top electrode and a bottom conductive electrode, wherein the top electrode is in contact with a conductive solution and the bottom conductive electrode is positioned adjacent to an individual nanopore, wherein the bottom conductive electrode is adapted to detect a current passing through the individual nanopore, and wherein the individual nanopore is individually addressable by the adjacent bottom conductive electrode;

b. providing a plurality of oligonucleotide markers wherein each of the oligonucleotide markers comprise oligonucleotides of between 10 and 100 nucleotides, wherein at least two of the oligonucleotides of between 10 and 100 nucleotides hybridize with a nucleic acid sample to produce a combined probe, wherein a first oligonucleotide of the at least two of the oligonucleotides has a label molecule attached to it and a second oligonucleotide has a sequence of bases that provides a unique current level in the nanopore for each of the oligonucleotide markers, wherein the second oligonucleotide hybridizes to an oligonucleotide that is attached to a bead for capture and isolation of the combined probe,

c. capturing and identifying at least one oligonucleotide marker within the plurality of oligonucleotide markers with at least one nanopore and at least one nanopore detector within the nanopore array at a rate of at least about one oligonucleotide marker per second per at least one nanopore, wherein the at least one oligonucleotide marker is identified based on a current that flows through the individual nanopore and an applied voltage of between 40 to 200 mV at which the oligonucleotide marker is released from the nanopore, and

d. sorting the plurality of oligonucleotide markers into selected oligonucleotide markers and unselected oligonucleotide markers, wherein the selected oligonucleotide markers are captured, identified, and held in nanopores within the nanopore array, and wherein the unselected oligonucleotide markers are captured, identified, and released from nanopores within the nanopore array; and

further wherein the selected oligonucleotide markers are released as a group when the percentage of selected oligonucleotide markers that are captured is at least 50%.

2. The method of claim 1, wherein the nanopore array is configured to have a plurality of regions configured to perform the method on different samples.

3. The method of claim 1, wherein each oligonucleotide marker within the plurality of oligonucleotide markers are generated by:

a. hybridizing the first oligonucleotide to a first region of the nucleic acid sample;

b. hybridizing the second oligonucleotide to a second region of the nucleic acid sample, wherein the first region of the nucleic acid sample is adjacent to the second region of the nucleic acid sample, thereby positioning the second oligonucleotide adjacent to the first oligonucleotide;

c. ligating the first oligonucleotide to the second oligonucleotide to produce the combined probe; and

d. hybridizing the second oligonucleotide to the oligonucleotide that is attached to the bead for capture and isolation of the combined probe, thereby producing each oligonucleotide marker within the plurality of the oligonucleotide markers.

4. The method of claim 1, further comprising determining copy number variation of a nucleic acid sequence in the nucleic acid sample.

5. The method of claim 1, further comprising quantifying relative RNA expression levels in the nucleic acid sample.

6. The method of claim 1, further comprising performing an ELISA assay on the first oligonucleotide label molecule.

7. The method of claim 1, wherein the first oligonucleotide comprises biotin.

8. The method of claim 1, wherein the bead is magnetic.

9. The method of claim 1, further comprising concentrating the markers adjacent or in proximity to the nanopore array with a magnetic field.