High Speed Molecular Sensing with Nanopores

• US 20150111779A1
• Filed: 10/23/2014
• Published: 04/23/2015
• 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
• Granted Patent: US 10,421,995 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• 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 an array of nanopores, wherein an individual nanopore of said array is individually addressable by an adjacent sensing electrode;

b. providing a plurality of markers that each comprise nucleotides, wherein at least two of the nucleotides hybridize with a nucleic acid sample, and wherein the markers are capable of being captured by the individual nanopore and identified using the sensing electrode; and

c. capturing and identifying the markers with the array of nanopores at a rate of at least about 1 marker per second per nanopore.

2. The method of claim 1, further comprising releasing the captured markers from the nanopore.

3. The method of claim 1, wherein the plurality of markers comprise markers to be sorted, wherein the markers to be sorted are captured, identified and held in the nanopores, and wherein markers other than the markers to be sorted are captured, identified and released from the nanopores.

4. The method of claim 3, wherein the markers to be sorted are released as a group and collected.

5. The method of claim 3, wherein the markers to be sorted are released as a group when the ratio of the number of markers to be sorted divided by a remaining number of markers that are captured and identified by the nanopores increases above a threshold.

6. The method of claim 1, wherein the array of nanopores is configured to have a plurality of regions capable of performing the method on different samples.

7. The method of claim 1, wherein the markers are identified based on a current that flows through the individual nanopore and/or a voltage at which the marker leaves the nanopore.

8. The method of claim 1, wherein the markers each comprise a single stranded nucleic acid molecule attached to a bead.

9. The method of claim 1, wherein the markers are generated by:

a. hybridizing a first probe to a nucleic acid sample;

b. hybridizing a second probe to the nucleic acid sample adjacent to the first probe;

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

d. capturing the combined probe with a bead attached to an oligonucleotide, wherein the oligonucleotide hybridizes with the combined probe.

10. The method of claim 9, further comprising determining copy number variation of a nucleic acid sequence in the nucleic acid sample.

11. The method of claim 9, further comprising quantifying relative RNA expression levels in the nucleic acid sample.

12. The method of claim 9, further comprising performing an ELISA assay on the nucleic acid sample.

13. The method of claim 9, wherein the first probe comprises biotin.

14. The method of claim 9, wherein the bead is magnetic.

15. The method of claim 14, further comprising concentrating the markers adjacent or in proximity to the array of nanopores with a magnetic field.

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

a. providing an array of nanopores, wherein an individual nanopore of said array is individually addressable by an adjacent sensing electrode operated in non-faradaic mode;

b. providing a plurality of markers capable of being captured by the individual nanopore and identified using the sensing electrode; and

c. capturing and identifying the markers with the array of nanopores at a rate of at least about 1 marker per second per nanopore.

17. A method for sequencing, counting, and/or sorting molecules, comprising:

a. providing an array of nanopores, wherein an individual nanopore of said array is individually addressable by an adjacent sensing electrode operated in non-faradic mode or faradaic mode;

b. providing a plurality of magnetically attractable beads each coupled to a molecule among a plurality of molecules to be sequenced, counted and/or sorted using the array of nanopores;

c. concentrating the magnetically attractable beads in the vicinity of the array of nanopores with a magnet; and

d. sequencing, counting and/or sorting the molecules with the array of nanopores.

18. The method of claim 17, wherein the magnetically attractable beads comprise metal.

19. The method of claim 17, wherein the magnetically attractable beads comprise a permanent magnetic material.

20. The method of claim 17, wherein the concentration of the magnetically attractable beads near the array of nanopores is increased by at least 100-fold by said concentrating.