# US 11,906,509 B2 · Nanopore-matched protein shuttle for molecular characterization and methodology for data analysis thereof

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

- Number: US 11,906,509 B2
- Document ID: US11906509B2
- Status: Granted
- Kind: B2
- Application number: 18/122,887
- Filed: 2023-03-17
- Published: 2023-11-02
- Granted: 2024-02-20
- Earliest claimed: 2017-07-17
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard College
- Inventors: Lene V. Hau; Jene A. Golovchenko
- Web page: https://patentgrep.com/patent/US11906509B2

## CPC

- B82Y 15/00: Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
- 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)}
- G01N 33/6872: {Intracellular protein regulatory factors and their receptors, e.g. including ion channels}
- C12Q 1/6869: Methods for sequencing
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- 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

Systems and methods are provided for characterizing shuttle capture events in a nanopore sensor. The method first collects time-dependent current blockage signatures for at least one bias voltage. The method then identifies each signature as corresponding to a permanent or transient event. The method then generates a protein dynamics landscape (PDL) for the transient event signatures. The PDL comprises a set of histograms of nanopore current data and characterizes current through the nanopore during shuttle capture events. The method can then comprise identifying an entrance level blockage value based on the permanent event signatures. Permanent event captures can be determined by time duration which is larger than a certain threshold time value. Applying a voltage between the fluidic chambers above a threshold voltage level can be used to control that the vast majority of events are permanent.

## Claims

### Claim 1

A method for characterizing shuttle capture in a nanopore sensor, comprising:
collecting a plurality of time-dependent current blockage signatures for a selected bias voltage;
calculating, based on the plurality of time-dependent current blockage signatures, a duration of a shuttle capture event and an average blockage percentage for the shuttle capture event;
determining a blockage level based on a distribution characteristic of the plurality of time-dependent current blockage signatures.

### Claim 2 (depends on 1)

The method of claim 1, wherein the determining further comprises whether the duration of the shuttle capture event falls in a range between a pre-defined minimum duration and a pre-defined maximum duration.

### Claim 3 (depends on 1)

The method of claim 1, wherein the determining further comprises identifying a particular blockage event as permanent if the duration of the particular blockage event is larger than a threshold time.

### Claim 4 (depends on 3)

The method of claim 3, wherein the threshold time is any period of time larger than 500 milliseconds.

### Claim 5 (depends on 2)

The method of claim 2, wherein the pre-defined minimum duration is 0 milliseconds and the pre-defined maximum duration is 2 milliseconds.

### Claim 6 (depends on 5)

The method of claim 5, wherein the plurality of time-dependent current blockage signatures provides a blockage peak at 40% current blockage.

### Claim 7 (depends on 2)

The method of claim 2, wherein the pre-defined minimum duration is 0 milliseconds and the pre-defined maximum duration is 5 milliseconds.

### Claim 8 (depends on 7)

The method of claim 7, wherein the plurality of time-dependent current blockage signatures provides blockage peaks at 45% and 52% current blockage.

### Claim 9 (depends on 2)

The method of claim 2, wherein the pre-defined minimum duration is 0 milliseconds and the pre-defined maximum duration is 10 milliseconds.

### Claim 10 (depends on 9)

The method of claim 9, wherein the plurality of time-dependent current blockage signatures provides blockage peaks at 40%, 45%, and 52% current blockage.

### Claim 11 (depends on 2)

The method of claim 2, wherein the pre-defined minimum duration is 0 milliseconds and the pre-defined maximum duration is 30 milliseconds.

### Claim 12 (depends on 11)

The method of claim 11, wherein the plurality of time-dependent current blockage signatures provides a blockage peak at 57% current blockage.

### Claim 13 (depends on 2)

The method of claim 2, wherein the pre-defined minimum duration is 0 milliseconds and the pre-defined maximum duration is 100 milliseconds.

### Claim 14 (depends on 13)

The method of claim 13, wherein the plurality of time-dependent current blockage signatures provides blockage peaks at 40%, 45%, 52%, and 57% current blockage.

### Claim 15 (depends on 2)

The method of claim 2, wherein the pre-defined minimum duration is 0 milliseconds and the pre-defined maximum duration is 500 milliseconds.

### Claim 16 (depends on 15)

The method of claim 15, wherein the plurality of time-dependent current blockage signatures provides blockage peaks at 40%, 45%, 52%, and 57% current blockage.

### Claim 17 (depends on 2)

The method of claim 2, wherein the pre-defined minimum duration is 0 milliseconds and the pre-defined maximum duration is 1000 milliseconds.

### Claim 18 (depends on 17)

The method of claim 17, wherein the plurality of time-dependent current blockage signatures provides a blockage peak at 80% current blockage.

### Claim 19 (depends on 2)

The method of claim 2, further comprising identifying an expected protein molecule orientation based on at least one of a comparison of protein dynamics landscapes (PDLs) for transient and permanent events and a presence or absence of blockage peaks for individual spectra with the PDLs for transient and permanent events, wherein each PDL comprises a collection of blockage spectra, wherein each of the collection of blockage spectra comprises a histogram of blockage values for events of a selected maximum duration.