# US 2025/0154580 A1 · Enzyme translocators in nanogap with 3' -esters

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

- Number: US 2025/0154580 A1
- Document ID: US20250154580A1
- Status: Published application
- Kind: A1
- Application number: 18/507,587
- Filed: 2023-11-13
- Published: 2025-05-15
- Earliest claimed: 2023-11-13
- Assignees: Robert Bosch GmbH
- Applicants: Robert Bosch GmbH
- Inventors: Nadezda FOMINA; Christopher JOHNSON; Young Shik SHIN; Christoph LANG; Armin DARVISH; Gary YAMA; Abhi BHADRAN; Christian GRUMAZ; Sebastian PILSL; Gabrielle VUKASIN
- Web page: https://patentgrep.com/patent/US20250154580A1

## CPC

- C12Q 1/6869: Methods for sequencing
- C12N 9/1241: {Nucleotidyltransferases (2.7.7)}
- C12Q 1/001: {Enzyme electrodes}
- C12Q 1/6825: Nucleic acid detection involving sensors
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- G01N 27/06: of a liquid (involving electrolysis G01N27/26)
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)

## Abstract

A method for nucleic acid sequencing includes providing at least one device comprising a first electrode and a second electrode separated by a dielectric layer, and a polymerase enzyme attached to the surface of the dielectric layer. The dielectric layer induces an electroactive molecule to interact with the electrodes to complete an electrical circuit. The polymerase enzyme targets a polynucleotide strand to the dielectric layer. A sample including a polynucleotide strand and modified nucleotides having an electroactive label covalently bound to the 3′—OH of a sugar ring of the nucleotide via an ester group is provided to the at least one device. Potentials are applied to each electrode to induce electron flow between the electrodes to produce a measurable electrical signal when an electroactive label is present in the dielectric layer. Electrical signals from the electrodes are detected to determine when a modified nucleotide is present in the dielectric layer.

## Claims

### Claim 1

A method for nucleic acid sequencing, comprising:
providing at least one device comprising:
a first electrode, a second electrode, and a dielectric layer defining a sensing zone between the first electrode and the second electrode; and
a polymerase enzyme attached to the surface of the dielectric layer, wherein the polymerase enzyme targets a polynucleotide strand to the dielectric layer;
providing to the at least one device a sample including a polynucleotide strand and at least one modified nucleotide, the modified nucleotide having an electroactive label covalently bound to 3′-OH of a sugar ring of the nucleotide via an ester group;
applying a first potential to the first electrode and a second potential to the second electrode to induce electron flow between the first and second electrodes to produce a measurable electrical signal when an electroactive label is present in the sensing zone; and
detecting a first signal from the first electrode and a second signal from the second electrode to determine when a modified nucleotide is present in the sensing zone.

### Claim 2 (depends on 1)

The method of claim 1, wherein the polymerase enzyme incorporates a modified nucleotide having an electroactive label covalently bound to 3′-OH of a sugar ring of the nucleotide via an ester group into a polynucleotide strand.

### Claim 3 (depends on 2)

The method of claim 2, wherein the polymerase enzyme cleaves the ester group of the modified nucleotide after the nucleotide is incorporated to remove the electroactive label from the sensing zone.

### Claim 4 (depends on 3)

The method of claim 3, wherein the polymerase enzyme is one of an A family polymerase or a B family polymerase.

### Claim 5 (depends on 1)

The method of claim 1, wherein two or more of dATP, dCTP, dGTP, or dTTP/dUTP is modified with a measurably distinct electroactive label.

### Claim 6 (depends on 1)

The method of claim 1, wherein the modified nucleotide having an electroactive label covalently bound to 3′-OH of the sugar ring of the nucleotide via an ester group has the following formula:
wherein:
X is
is a single bond, a double bond, a triple bond,
Base is Adenine (A), Cytosine (C), Guanine (G), Thymine (T), or Uracil (U),
L (Linker) is absent or is a hydrocarbon chain comprising between 1 and 1000 atoms which may contain heteroatoms such as O, N, and S,
n=1-1000
R1 is H or OH, and
R2 is a redox label.

### Claim 7 (depends on 1)

The method of claim 1, further comprising calculating a differential value between the first signal and the second signal to determine when a modified nucleotide is present in the sensing zone.

### Claim 8

A system for nucleic acid sequencing comprising:
at least one device including:
at least one electrochemical sensor comprising a first electrode, a second electrode, and a dielectric layer defining a sensing zone between the first electrode and the second electrode; and a polymerase enzyme attached to the surface of the dielectric layer, wherein the polymerase enzyme targets a polynucleotide strand to the sensing zone; and
a controller configured to:
direct a first current through the first electrode and a second current through the second electrode to induce electron flow between the first and second electrodes to produce a measurable electrical signal when an electroactive label is present in the sensing zone;
provide to the at least one device a sample including a polynucleotide strand and at least one modified nucleotide, the modified nucleotide having an electroactive label covalently bound to 3′-OH of a sugar ring of the nucleotide via an ester group; and
detect the first current flowing from the first electrode and the second current flowing from the second electrode.

### Claim 9 (depends on 8)

The system of claim 8, wherein the polymerase enzyme incorporates a modified nucleotide having an electroactive label covalently bound to 3′-OH of a sugar ring of the nucleotide via an ester group into a polynucleotide strand, and wherein the polymerase enzyme cleaves the ester group of the modified nucleotide after the nucleotide is incorporated to remove the electroactive label from the sensing zone.

### Claim 10 (depends on 9)

The system of claim 9, wherein the polymerase enzyme is one of an A family polymerase or a B family polymerase.

### Claim 11 (depends on 8)

The system of claim 8, wherein the measurable electrical signal is produced while the electroactive label is still incorporated into the enzyme-template-dNTP complex and while the electroactive label is within the sensing zone.

### Claim 12 (depends on 8)

The system of claim 8, wherein the controller is further configured to calculate a differential value between the first current and the second current to determine when a modified nucleotide is present in the sensing zone.

### Claim 13 (depends on 8)

The system of claim 8, wherein the modified nucleotide having an electroactive label covalently bound to 3′-OH of the sugar ring of the nucleotide via an ester group has the following formula:
wherein:
X is
is a single bond, a double bond, a triple bond,
Base is Adenine (A), Cytosine (C), Guanine (G), Thymine (T), or Uracil (U),
L (Linker) is absent or a hydrocarbon chain comprising between 1 and 1000 atoms which may contain heteroatoms such as O, N, and S,
n=1-1000
R1 is H or OH, and
R2 is a redox label.

### Claim 14 (depends on 8)

The system of claim 8, wherein the at least one device is a plurality of devices.

### Claim 15

A method for forming a system for nucleic acid sequencing, the method comprising:
providing a first electrode and a second electrode;
positioning a dielectric layer between the first and second electrodes to define a sensing zone;
attaching a polymerase enzyme to the surface of the dielectric layer, wherein the polymerase enzyme incorporates a modified nucleotide having an electroactive label covalently bound to 3′-OH of a sugar ring of the nucleotide via an ester group into a polynucleotide strand; and
configuring the electrodes to detect a change in current when an electroactive label is present within the sensing zone.

### Claim 16 (depends on 15)

The method of claim 15, further comprising generating two of dATP, dCTP, dGTP, and dTTP/dUTP each of the two modified with a measurably distinct electroactive label covalently bound to 3′-OH of a sugar ring of the dATP, dCTP, dGTP, and dTTP/dUTP via an ester group.

### Claim 17 (depends on 15)

The method of claim 15, further comprising generating dATP, dCTP, dGTP, and dTTP/dUTP each modified with a measurably distinct electroactive label covalently bound to the 3′-OH of a sugar ring of the dATP, dCTP, dGTP, and dTTP/dUTP via an ester group.

### Claim 18 (depends on 15)

The method of claim 15, wherein the polymerase enzyme cleaves the ester group of the modified nucleotide after the nucleotide is incorporated to remove the electroactive label from the sensing zone.

### Claim 19 (depends on 15)

The method of claim 15, further comprising configuring a controller to:
direct a first current through the first electrode and a second current through the second electrode to induce electron flow between the first and second electrodes;
direct exposure of the polymerase enzyme to a sample including the polynucleotide strand and dNTPs modified with an electroactive label covalently bound to 3′-OH group of the sugar ring of the nucleotide via an ester group; and
detect the first current flowing from the first electrode and the second current flowing from the second electrode.

### Claim 20 (depends on 19)

The method of claim 19, wherein the controller is further configured to calculate a differential value between the first current and the second current to determine when a modified nucleotide is present in the sensing zone.