# US 12,181,460 B2 · Semiconductor nanosensing device with multilayer graphene sheet for sequencing or sensing nucleic acids

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 12,181,460 B2
- Document ID: US12181460B2
- Status: Granted
- Kind: B2
- Application number: 18/039,209
- Filed: 2021-11-29
- Published: 2024-01-04
- Granted: 2024-12-31
- Earliest claimed: 2020-11-30
- Assignees: Gentroma, Inc.
- Applicants: Gentroma Inc.
- Inventors: Krista Fretes
- Web page: https://patentgrep.com/patent/US12181460B2

## 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 30/00: Nanotechnology for materials or surface science, e.g. nanocomposites
- G01N 27/3278: {involving nanosized elements, e.g. nanogaps or nanoparticles (nanopores G01N33/48721; magnetic beads G01N27/745)}
- G01N 27/44791: {Microapparatus (sample containers with integrated microfluidic structures B01L3/5027)}
- 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

## Abstract

A nanosensing device is disclosed for high throughput nucleic acid sequencing or sensing. The device is a silicon chip, having a silicon nitride top layer, an opening through the chip, a hole in the top layer, and a graphene sheet over the top layer and hole. A graphene layer with a p-type crinkle ruga is placed over the hole, and one of more nanopores in the graphene layer may be provided over the hole. A nucleic acid strand transported to the chip may be translocated through a nanopore and the hole by microfluidic forces. An electrical potential across the nanopores can be measured with a patch clamp amplifier to make nucleobase assignments and sequence the DNA. Alternatively, there are no nanopores and the interaction with the crinkle ruga is used to sense the presence of a nucleic acid polymer.

## Claims

### Claim 1

A sequencing chip for sequencing a strand of nucleic acids comprising
a. a chip formed from a substrate fabricated from silicon about 1.0 mm to about 10 mm wide, about 1.0 mm to about 10 mm long, with a thickness of about 50 μm to 500 μm;
b. wherein the substrate has a top layer of silicon nitride (Si3N4) with a thickness of 20 nm to 500 nm;
c. wherein the substrate optionally has a bottom layer of silicon nitride with a thickness of 20 nm to 500 nm;
d. wherein the formed chip has at least one hole drilled by a microscopic method in the top layer of silicon nitride and that penetrates the entire thickness of the chip, and wherein the hole is 50 nm to 1000 nm wide and 50 nm to 1000 nm long, wherein the hole is in the shape of a circle or simple polygon;
e. wherein a multilayer graphene sheet about 1-60 nm thick is affixed to the Si3N4 layer on the top substrate in a latitudinal orientation, and wherein the graphene sheet is subjected to lateral compression to cause a p-type crinkle ruga having a crease to form thereon over the at least one hole; and
f. wherein a pair of electrodes is provided on opposite ends of each hole on the graphene sheet, where the electrodes are connected to electrical detection equipment capable of measuring an ionic current across the graphene sheet and wherein fluctuations in the ionic current of a nucleic acid strand as it interacts with the graphene sheet are measured by the electrical detection equipment and converted into nucleobase assignments.

### Claim 2 (depends on 1)

The sequencing chip of claim 1, wherein the graphene sheet comprises one or more nanopores about 0.3 to about 10 nm in diameter centered over the hole, such that a nucleic acid polymer strand can translocate through at least one nanopore and wherein fluctuations in the ionic current of a nucleic acid strand translocating through at least one nanopore are measured by the electrical detection equipment and converted into nucleobase assignments.

### Claim 3 (depends on 2)

A method of detecting nucleobases in a strand of nucleic acids, comprising a sequencing chip with a graphene sheet containing at least one or more nanopores according to claim 2 and directing a saline solution of the nucleic acid comprising a strand of nucleobases toward a nanopore in the graphene sheet, wherein the nucleobase strand translocates through the pore and interacts with the layers of the multilayer graphene sheet, and wherein a patch clamp amplifier connected to the electrodes at each hole measures changes in the ionic current and detects each nucleobase in the nucleic acid strand translocating through the nanopore and the nucleic acid sequence is assigned.

### Claim 4 (depends on 1)

The sequencing chip of claim 1, wherein the graphene sheet has no nanopores and wherein the fluctuations in the ionic current across the at least one hole caused by a nucleic acid polymer interacting with the crinkle ruga on the graphene sheet are measured by the electrical detection equipment and converted into nucleobase assignments.

### Claim 5 (depends on 4)

A method of sensing nucleobases in a strand of nucleic acids, comprising a sequencing chip with a graphene sheet according to claim 4 and directing a saline solution of the nucleic acid comprising a strand of nucleobases toward the crinkle ruga on the graphene sheet, wherein the nucleobase strand interacts with the multilayer graphene sheet, and wherein a patch clamp amplifier connected to the electrodes at each hole measures changes in the ionic current and electrical characteristics of the nucleic acid strand to detect each nucleobase in the nucleic acid strand and the nucleic acid sequence is assigned.

### Claim 6 (depends on 1)

The sequencing chip of claim 1, wherein the substrate is about 1.5 mm to about 3.0 mm wide and about 1.5 mm to about 3.0 mm long.

### Claim 7 (depends on 1)

The sequencing chip of claim 1, wherein the substrate has a top layer of silicon nitride (Si3N4) with a thickness of about 200 nm.

### Claim 8 (depends on 1)

The sequencing chip of claim 1 wherein the substrate has a bottom layer of silicon nitride with a thickness of 100 nm to 300 nm or the substrate has a bottom layer of silicon nitride with a thickness of about 200 nm.

### Claim 9 (depends on 1)

The sequencing chip of claim 1 wherein the substrate has a bottom layer of silicon nitride with a thickness of about 200 nm.

### Claim 10 (depends on 1)

The sequencing chip of claim 1, wherein the at least one hole is selected from at least one of the group consisting of a circle, a triangle, a square, a rectangle, a hexagon and an hourglass shape.

### Claim 11 (depends on 1)

The sequencing chip of claim 1, further comprising a grid of holes in the chip, wherein the grid is up to a up to a 100×100 grid, or up to a 20×20 grid, or up to a 10×10 grid.

### Claim 12 (depends on 11)

The sequencing chip of claim 11, further comprising an array of electrodes.

### Claim 13 (depends on 1)

The sequencing chip of claim 1, further comprising one or more nucleic acid probes bound to the multilayer graphene sheet.

### Claim 14 (depends on 1)

The sequencing chip of claim 1 further comprising at least one opening defining the base of a square frustopyramid penetrating from a bottom surface of the substrate into a top surface of the substrate of the chip, wherein the opening does not penetrate the top layer of silicon nitride, and wherein the top of the square frustopyramid is a square window about 5 μm to about 100 μm on each side.

### Claim 15 (depends on 1)

The sequencing chip of claim 1 wherein the at least one hole in the top layer of silicon nitride centered over the window is about 350 nm to about 500 nm wide and about 350 nm to about 1000 nm long.