# US 11,029,306 B2 · Nanopore-based sequencing using voltage mode with hybrid mode stimuli

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- Number: US 11,029,306 B2
- Document ID: US11029306B2
- Status: Granted
- Kind: B2
- Application number: 16/297,428
- Filed: 2019-03-08
- Published: 2019-07-04
- Granted: 2021-06-08
- Earliest claimed: 2016-09-15
- Assignees: Roche Sequencing Solutions, Inc.
- Applicants: Roche Sequencing Solutions, Inc.
- Inventors: Roger J. A. Chen; Hui Tian; J. William Maney, Jr.
- Web page: https://patentgrep.com/patent/US11029306B2

## CPC

- 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)}
- C12Q 1/6869: Methods for sequencing
- G01N 27/44713: {Particularly adapted electric power supply}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A liquid voltage is applied to a first side of a lipid bilayer. The liquid voltage comprises a tag-reading period with a tag-reading voltage that tends to capture a tag into a nanopore in the lipid bilayer and an open-channel period with an open-channel voltage that tends to repel the tag. A pre-charging voltage source is connected to an integrating capacitor and a working electrode on a second side of the lipid bilayer during a pre-charging time period, such that the integrating capacitor and the working electrode are charged to a pre-charging voltage. The pre-charging voltage source is disconnected from the integrating capacitor and the working electrode during an integrating time period, such that a voltage of the integrating capacitor and a voltage of the working electrode may vary as a current flows through the nanopore. The pre-charging time period overlaps with a beginning portion of the tag-reading period.

## Claims

### Claim 1

A method of analyzing a molecule in a nanopore, comprising:
applying a liquid voltage to an electrolyte on a first side of a membrane, wherein the liquid voltage comprises a tag-reading period with a tag-reading voltage level that is configured to pull a tag of the molecule or a portion of the molecule into the nanopore in the membrane and an open-channel period with an open-channel voltage level that is configured to repel the tag or the portion of the molecule from the nanopore in the membrane;
connecting a pre-charging voltage source to a capacitor and a working electrode on a second side of the membrane during a pre-charging time period, such that the capacitor and the working electrode are charged to a pre-charging voltage; and
disconnecting the pre-charging voltage source from the capacitor and the working electrode during the tag-reading period, such that a voltage of the capacitor and a voltage of the working electrode may vary as a current flows through the nanopore in the membrane;
wherein the pre-charging time period spans across a transition from the open channel period to the tag-reading period.

### Claim 2 (depends on 1)

The method of claim 1, wherein the pre-charging time period overlaps with an ending portion of the open-channel period and a beginning portion of the tag-reading period.

### Claim 3 (depends on 1)

The method of claim 1, wherein the pre-charging time period overlaps with a beginning portion of the open-channel period.

### Claim 4 (depends on 1)

The method of claim 1, wherein the pre-charging time period overlaps with an ending portion of the tag-reading period.

### Claim 5 (depends on 1)

The method of claim 1, wherein the pre-charging time period spans across a transition from the tag-reading period to the open-channel period.

### Claim 6 (depends on 1)

The method of claim 1, wherein the pre-charging time period has a duration of 100 microseconds to 50 milliseconds.

### Claim 7 (depends on 1)

The method of claim 1, wherein the pre-charging time period has a duration of 50 microseconds to 25 milliseconds.

### Claim 8

A system, instrument or device for analyzing a molecule, comprising:
a voltage source;
a counter electrode coupled to the voltage source;
a pre-charging voltage source;
a capacitor that is configured to connect with the pre-charging voltage source;
a working electrode that is configured configurable to connect with the pre-charging voltage source; and
a controller configured to:
control the voltage source to apply a liquid voltage via the counter electrode to an electrolyte on a first side of a membrane, wherein the liquid voltage comprises a tag-reading period with a tag-reading voltage level that is configured to pull a tag of the molecule or a portion of the molecule into a nanopore in the membrane and an open-channel period with an open-channel voltage level that is configured to repel the tag or the portion of the molecule from the nanopore in the membrane;
connect the pre-charging voltage source to the capacitor and the working electrode on a second side of the membrane during a pre-charging time period, such that the capacitor and the working electrode are charged to a pre-charging voltage;
disconnect the pre-charging voltage source from the capacitor and the working electrode during the tag-reading period, such that a voltage of the capacitor and a voltage of the working electrode may vary as a current flows through the nanopore in the membrane; and
wherein the pre-charging time period spans across a transition from the open channel period to the tag-reading period; and
a memory coupled to the controller and configured to provide the controller with instructions.

### Claim 9 (depends on 8)

The system, instrument or device of claim 8, wherein the pre-charging time period overlaps with an ending portion of the open-channel period and a beginning portion of the tag-reading period.

### Claim 10 (depends on 8)

The system, instrument or device of claim 8, wherein the pre-charging time period overlaps with a beginning portion of the open-channel period.

### Claim 11 (depends on 8)

The system, instrument or device of claim 8, wherein the pre-charging time period overlaps with an ending portion of the tag-reading period.

### Claim 12 (depends on 8)

The system, instrument or device of claim 8, wherein the pre-charging time period spans across a transition from the tag-reading period to the open-channel period.

### Claim 13 (depends on 8)

The system, instrument or device of claim 8, wherein the pre-charging time period has a duration of 100 microseconds to 25 milliseconds.