# US 2025/0146973 A1 · Label-free methods of sensing

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- Number: US 2025/0146973 A1
- Document ID: US20250146973A1
- Status: Published application
- Kind: A1
- Application number: 18/730,176
- Filed: 2023-01-20
- Published: 2025-05-08
- Earliest claimed: 2022-01-21
- Assignees: Board of Regents, The University of Texas System
- Applicants: Board of Regents, The University of Texas System
- Inventors: Georgios ALEXANDRAKIS; Scott RENKES
- Web page: https://patentgrep.com/patent/US20250146973A1

## CPC

- G01N 27/44791: {Microapparatus (sample containers with integrated microfluidic structures B01L3/5027)}
- G01N 33/6872: {Intracellular protein regulatory factors and their receptors, e.g. including ion channels}
- G01N 21/554: {detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance}
- G01N 27/4473: {by electric means}
- 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 33/54373: {involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings}
- G01N 33/5438: {Electrodes}
- G01N 33/56977: {HLA or MHC typing}
- 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
- G01N 2333/7051: T-cell receptor (TcR)-CD3 complex
- G01N 2333/70539: MHC-molecules, e.g. HLA-molecules
- 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

Methods of sensing are disclosed. In some embodiments, a method comprises providing a sensor. The sensor comprises a first layer having a nanohole structure, and a second layer having a nanopore. The nanohole structure is aligned with the nanopore in a translocation direction. The method further comprises contacting a test sample comprising an analyte with the first layer of the sensor. The nanohole structure is irradiated with light, and the analyte is optically trapped in the nanohole structure. The method further comprises applying a first electric field (DC) across the nanopore to draw analyte into the nanopore, and then applying a second electric field (pulsed, modulated or AC) across the nanopore. The method further comprises measuring a change in current and/or phase across the nanopore during application of the second electric field or measuring at least one kinetic parameter of the analyte within the nanopore after removing the second field.

## Claims

### Claim 1

A method of sensing comprising:
providing a sensor comprising:
a first layer having at least one single nanohole structure or at least one dual nanohole structure, and
a second layer having at least one nanopore,
wherein the single nanohole structure comprises only one nanohole,
wherein the dual nanohole structure comprises a first nanohole and a second nanohole connected by a gap, and
wherein the one nanohole or the gap of the first layer is aligned with the nanopore of the second layer in a direction corresponding to a translocation direction across the first and second layers;
providing a test sample comprising an analyte;
contacting the test sample with the first layer of the sensor;
irradiating the single nanohole structure or the dual nanohole structure of the first layer of the sensor with a beam of electromagnetic radiation;
optically trapping the analyte in the single nanohole structure or in the dual nanohole structure and/or in the gap of the first layer of the sensor;
applying a first electric field across the nanopore to draw one or more of the analytes into the nanopore, wherein the first electric field comprises a direct current (DC) electric field;
applying a second electric field across the nanopore after applying the first electric field, wherein the second electric field comprises a pulsed, modulated, or alternating current (AC) electric field; and
measuring one or more of:
change in current and/or phase across the nanopore during application of the second electric field while the analyte is optically trapped and/or during one or more translocation events of the analyte through the nanopore; or
at least one kinetic parameter of the analyte within the nanopore after removing or turning off the second electric field.

### Claim 2 (depends on 1)

The method of claim 1, wherein the at least one kinetic parameter is measured while the analyte decelerates or comes to a stop while optically trapped.

### Claim 3 (depends on 1)

The method of claim 1, wherein the at least one kinetic parameter comprises one or more of the following: equilibrium dissociation constant (Kd), binding on-rate (kon), binding off-rate (koff), and bound fraction.

### Claim 4 (depends on 1)

The method of claim 1, wherein measuring change in current and/or phase further comprises determining a charge of a translocating analyte.

### Claim 5 (depends on 1)

The method of claim 1, wherein measuring change in current and/or phase further comprises determining a dielectric constant of a translocating analyte.

### Claim 6 (depends on 1)

The method of claim 1 further comprising:
measuring a surface plasmon resonance of the single nanohole structure or the dual nanohole structure after optically trapping the analyte in the single nanohole structure or in the dual nanohole structure and/or in the gap of the first layer of the sensor.

### Claim 7 (depends on 6)

The method of claim 6, wherein measuring the surface plasmon resonance further comprises determining the mass of an optically trapped analyte.

### Claim 8 (depends on 1)

The method of claim 1, wherein the analyte comprises complexed and/or non-complexed biomolecules.

### Claim 9 (depends on 1)

The method of claim 1, wherein the test sample is a biological sample obtained from an animal or human subject.

### Claim 10 (depends on 9)

The method of claim 9, wherein the analyte comprises a pMHC or pMHC component.

### Claim 11 (depends on 9)

The method of claim 9, wherein the analyte comprises a HLA-A2 pHMC or HLA-A2 pMHC component.

### Claim 12 (depends on 9)

The method of claim 9, wherein the analyte comprises a TCRm antibody.

### Claim 13 (depends on 9)

The method of claim 9, where in the analyte comprises a TCRm antibody against a HLA-A2 pHMC or against a HLA-A2 pHMC component.

### Claim 14 (depends on 1)

The method of claim 1, wherein the analyte comprises an inorganic nanoparticle.

### Claim 15 (depends on 1)

The method of claim 1, wherein the test sample is concentrated prior to contacting the test sample with the first layer of the sensor.

### Claim 16 (depends on 15)

The method of claim 15, wherein the test sample is concentrated using isotachophoresis (ITP).

### Claim 17 (depends on 15)

The method of claim 15, wherein the test sample is concentrated using an ITP microchannel structure.

### Claim 18 (depends on 17)

The method of claim 17, wherein the ITP microchannel structure is disposed over the first layer of the sensor.

### Claim 19 (depends on 18)

The method of claim 18, wherein the ITP microchannel structure forms a unitary chip with the first layer and the second layer of the sensor.