# US 10,481,144 B2 · Nanopore sequencing using n-mers

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- Number: US 10,481,144 B2
- Document ID: US10481144B2
- Status: Granted
- Kind: B2
- Application number: 15/654,395
- Filed: 2017-07-19
- Published: 2018-03-15
- Granted: 2019-11-19
- Earliest claimed: 2009-04-10
- Assignees: Pacific Biosciences of California, Inc.
- Applicants: Pacific Biosciences of California, Inc.
- Inventors: Stephen Turner; Benjamin Flusberg
- Web page: https://patentgrep.com/patent/US10481144B2

## 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)}
- B82Y 5/00: Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
- B82Y 15/00: Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- G01N 27/447: using electrophoresis
- G01N 27/44791: {Microapparatus (sample containers with integrated microfluidic structures B01L3/5027)}
- C12Q 2565/631: being a biochannel or pore
- 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)
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- 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

The invention relates to devices and methods for nanopore sequencing. The invention provides devices having an array of nanopores. The devices comprise an upper fluidic region in fluidic contact with the nanopores. The upper fluidic region comprises and upper electrode. Below each nanopore, and in fluidic contact with each nanopore is a discrete fluidic region. There is no direct fluidic contact between these discrete fluidic regions. The devices have an advantage of lower levels of electrical cross-talk between nanopores in the array for improved nucleic acid sequencing.

## Claims

### Claim 1

A device for nucleic acid sequencing comprising:
an array of at least 100 nanopores,
above the array of nanopores, in fluidic contact with the array of nanopores, an upper fluidic region comprising an upper electrode, the upper fluidic region comprising nucleic acid molecules,
below the array of nanopores, an array of discrete fluidic regions, each discrete fluidic region in fluidic contact with a nanopore, and each discrete fluidic region comprising a lower electrode, wherein there is no direct fluidic contact between the discrete fluidic regions, and
below the array of discrete fluidic regions, an array of electronic circuits, each electronic circuit comprising an amplifier in electrical contact with the fluidic region above it,
wherein voltage is applied between the upper electrode and the lower electrodes, resulting in translocation of a nucleic acid molecule through a plurality of the nanopores, and whereby a measured electrical signal is used to determine sequences of a plurality of nucleic acid molecules.

### Claim 2 (depends on 1)

The device of claim 1 wherein the device comprises a semiconductor substrate in which the electronic circuits are formed.

### Claim 3 (depends on 2)

The device of claim 2 wherein the electronic circuits comprise CMOS circuits.

### Claim 4 (depends on 1)

The device of claim 1 wherein the electronic circuits further comprise analog to digital converters, memory, or clock circuits.

### Claim 5 (depends on 1)

The device of claim 1 wherein each discrete reservoir has two electrodes, one acting as a drive electrode, and one acting as a measurement electrode.

### Claim 6 (depends on 1)

The device of claim 1 wherein the nanopores comprise protein nanopores.

### Claim 7 (depends on 6)

The device of claim 6 wherein the protein nanopores comprise alpha-hemolysin proteins.

### Claim 8 (depends on 6)

The device of claim 6 wherein the protein nanopores comprise MspA proteins.

### Claim 9 (depends on 1)

The device of claim 1 wherein the protein nanopores are within biological membranes.

### Claim 10 (depends on 1)

The device of claim 1 wherein the nanopores comprise solid state nanopores.

### Claim 11 (depends on 1)

The device of claim 1 wherein the nanopores comprise hybrid solid state-protein nanopores.

### Claim 12 (depends on 1)

The device of claim 1 wherein each discrete fluidic region has a cross sectional dimension from 1 micron to 10 microns.

### Claim 13 (depends on 1)

The device of claim 1 wherein the upper fluidic region further comprises enzymes for control of translocation rate.

### Claim 14 (depends on 13)

The device of claim 13 wherein the enzymes for control of translocation rate comprise polymerases.

### Claim 15 (depends on 13)

The device of claim 13 wherein the enzymes for control of translocation rate comprise exonucleases.

### Claim 16 (depends on 13)

The device of claim 13 wherein the enzymes for control of translocation rate comprise helicases.

### Claim 17 (depends on 1)

The device of claim 1 wherein the nucleic acid comprises DNA.

### Claim 18 (depends on 1)

The device of claim 1 wherein the device comprises a substrate comprising a semiconductor component bound to an insulator component.

### Claim 19 (depends on 1)

The device of claim 1 comprising from 100 to 10,000 nanopores.