# US 10,871,482 B2 · Target detection with nanopore and a polymer scaffold complex

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

- Number: US 10,871,482 B2
- Document ID: US10871482B2
- Status: Granted
- Kind: B2
- Application number: 15/160,647
- Filed: 2016-05-20
- Published: 2016-09-15
- Granted: 2020-12-22
- Earliest claimed: 2013-05-06
- Assignees: Ontera Inc.
- Applicants: Ontera Inc.
- Inventors: Trevor J. Morin; Daniel Alexander Heller; William B. Dunbar
- Web page: https://patentgrep.com/patent/US10871482B2

## 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 27/4473: {by electric means}
- G01N 27/44791: {Microapparatus (sample containers with integrated microfluidic structures B01L3/5027)}
- G01N 33/5302: {Apparatus specially adapted for immunological test procedures}
- G01N 33/536: with immune complex formed in liquid phase
- G01N 33/54306: {Solid-phase reaction mechanisms}
- G01N 33/54366: {Apparatus specially adapted for solid-phase testing}
- G01N 33/56911: {Bacteria}
- G01N 33/56983: {Viruses}
- G01N 33/575: for cancer
- 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

Provided are methods for detecting a target molecule or particle suspected to be present in a sample, comprising (a) contacting the sample with (i) a fusion molecule comprising a ligand capable of binding to the target molecule or particle and a binding domain, and (ii) a polymer scaffold comprising at least one binding motif to which the binding domain is capable of binding, under conditions that allow the target molecule or particle to bind to the ligand and the binding domain to bind to the binding motif; (b) loading the polymer into a device comprising a pore that separates an interior space of the device into two volumes, and configuring the device to pass the polymer through the pore from one volume to the other volume, wherein the device further comprises a sensor adjacent to the pore configured to identify objects passing through the pore; and (c) determining, with the sensor, whether the fusion molecule or particle bound to the binding motif is bound to the target molecule or particle, thereby detecting the presence of the target molecule or particle in the sample.

## Claims

### Claim 1

A method for detecting a target molecule or particle suspected to be present in a sample, comprising:
(a) contacting the sample with (i) a fusion molecule comprising a ligand capable of binding to the target molecule or particle and a binding domain, and (ii) a polymer scaffold comprising a polymer and at least one binding motif to which the binding domain is capable of binding, under conditions that allow the target molecule or particle to bind to the ligand and the binding domain to bind to the binding motif;
(b) loading the polymer scaffold into a device comprising a pore that separates an interior space of the device into two volumes, and
(c) passing the polymer scaffold bound to a fusion molecule through the pore from one volume to the other volume, wherein the device further comprises a sensor adjacent to the pore capable of detecting objects passing through the pore; and
(d) determining, with the sensor, whether the polymer scaffold bound to a fusion molecule is bound to the target molecule or particle, thereby detecting the presence of the target molecule or particle in the sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein the target molecule is selected from the group consisting of a protein, a peptide, a nucleic acid, a chemical compound, an ion, and an element.

### Claim 3 (depends on 1)

The method of claim 1, wherein the target particle is selected from the group consisting of a protein complex or aggregate, a protein/nucleic acid complex, a fragmented or fully assembled virus, a bacterium, a cell, and a cellular aggregate.

### Claim 4 (depends on 1)

The method of claim 1, wherein step (a) is performed prior to step (b).

### Claim 5 (depends on 1)

The method of claim 1, wherein step (b) is performed prior to step (a).

### Claim 6 (depends on 1)

The method of claim 1, further comprising applying a condition suspected to alter the binding between the target molecule or particle and the ligand, and carrying out the determination again.

### Claim 7 (depends on 6)

The method of claim 6, wherein the condition is selected from the group consisting of removing the target molecule or particle from the sample, adding an agent that competes with the target molecule or particle, or the ligand for binding, and changing the pH, salt, or temperature.

### Claim 8 (depends on 1)

The method of claim 1, wherein the binding motif comprises a chemical modification for binding to the binding domain.

### Claim 9 (depends on 8)

The method of claim 8, wherein the chemical modification is selected from the group consisting of acetylation, methylation, SUMOylation, glycosylation, phosphorylation and oxidation.

### Claim 10 (depends on 1)

The method of claim 1, wherein the polymer is at least one of a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a peptide nucleic acid (PNA), a DNA/RNA hybrid, and a polypeptide.

### Claim 11 (depends on 1)

The method of claim 1, wherein the binding domain is selected from the group consisting of a helix-turn-helix, a zinc finger, a leucine zipper, a winged helix, a winged helix turn helix, a helix-loop-helix, and a high mobility group box (HMG-box).

### Claim 12 (depends on 1)

The method of claim 1, wherein the binding domain is selected from the group consisting of locked nucleic acids (LNAs), peptide nucleic acids (PNAs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPRs), and aptamers.

### Claim 13 (depends on 1)

The method of claim 1, wherein the ligand is selected from the group consisting of an antibody, an epitope, a hormone, a neurotransmitter, a cytokine, a growth factor, a cell recognition molecule, and a receptor.

### Claim 14 (depends on 1)

The method of claim 1, wherein the binding domain and the ligand are linked, via a covalent bond, a hydrogen bond, an ionic bond, a metallic bond, a van der Waals force, a hydrophobic interaction, or a planar stacking interaction, or are translated as a continuous polypeptide, to form the fusion molecule.

### Claim 15 (depends on 1)

The method of claim 1, further comprising contacting the sample with a detectable label capable of binding to the target molecule or particle, or the target molecule or particle/ligand complex.

### Claim 16 (depends on 1)

The method of claim 1, wherein the polymer comprises at least two units of the binding motif.

### Claim 17 (depends on 1)

The method of claim 1, wherein the polymer comprises at least two different binding motifs; the sample is in contact with at least two fusion molecules each comprising a different binding domain capable of binding to each binding motif and a different ligand capable of binding to a different target molecule or particle; and the sensor is capable of detecting whether the fusion molecule bound to each binding motif is bound to a target molecule or particle.

### Claim 18 (depends on 1)

The method of claim 1, wherein the sensor comprises electrodes to apply a voltage differential between the two volumes.

### Claim 19 (depends on 1)

The method of claim 1, wherein the device comprises an upper chamber, a middle chamber and a lower chamber,
wherein the upper chamber is in communication with the middle chamber through a first pore, and the middle chamber is in communication with the lower chamber through a second pore;
wherein the first pore and second pore are about 1 nm to about 100 nm in diameter, and are about 10 nm to about 1000 nm apart from each other; and
wherein each of the chambers comprises an electrode connected to a power supply.

### Claim 20 (depends on 1)

The method of claim 1, wherein the device comprises an upper chamber, a middle chamber and a lower chamber,
wherein the upper chamber is in communication with the middle chamber through a first pore, and the middle chamber is in communication with the lower chamber through a second pore;
wherein the first pore and second pore are about 100 nm to about 10000 nm in diameter, and are about 10 nm to about 1000 nm apart from each other; and
wherein each of the chambers comprises an electrode for connecting to a power supply.

### Claim 21 (depends on 1)

The method of claim 1, further comprising moving the polymer in a reversed direction after the binding motif passes through the pore to detect, again, whether the fusion molecule bound to each binding motif is bound to a target molecule or particle.