# US 11,079,355 B2 · Ultrasound-assisted electrochemical distinction of normal and cancerous cells

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- Number: US 11,079,355 B2
- Document ID: US11079355B2
- Status: Granted
- Kind: B2
- Application number: 15/960,542
- Filed: 2018-04-23
- Published: 2018-09-06
- Granted: 2021-08-03
- Earliest claimed: 2017-04-24
- Assignees: Nano Hesgarsazan Salamat Arya
- Applicants: Mohammad Abdolahad; Mohammad Ali Khayamian; Saeid Ansaryan; Majid Baniassadi
- Inventors: Mohammad Abdolahad; Mohammad Ali Khayamian; Saeid Ansaryan; Majid Baniassadi
- Web page: https://patentgrep.com/patent/US11079355B2

## CPC

- G01N 27/48: using polarography, i.e. measuring changes in current under a slowly-varying voltage
- A61B 5/1473: invasive, e.g. introduced into the body by a catheter
- C23C 14/0005: {Separation of the coating from the substrate}
- C23C 14/022: {by means of bombardment with energetic particles or radiation}
- C23C 14/205: {by cathodic sputtering}
- C23C 14/3464: {using more than one target (C23C14/56 takes precedence)}
- C23C 14/5806: {Thermal treatment}
- C23C 14/5846: {Reactive treatment}
- C25B 1/02: Hydrogen or oxygen
- C25B 9/17: Cells comprising dimensionally-stable non-movable electrodes; Assemblies of constructional parts thereof
- C25B 11/051: Electrodes formed of electrocatalysts on a substrate or carrier
- C25B 11/052: Electrodes comprising one or more electrocatalytic coatings on a substrate
- C25B 11/055: characterised by the substrate or carrier material
- C25B 11/057: consisting of a single element or compound
- C25B 11/063: Valve metal, e.g. titanium
- C25B 11/091: consisting of at least one catalytic element and at least one catalytic compound; consisting of two or more catalytic elements or catalytic compounds
- G01N 27/327: Biochemical electrodes {, e.g. electrical or mechanical details for in vitro measurements}
- G01N 33/4833: {of solid biological material, e.g. tissue samples, cell cultures (tissue in vivo A61B5/00; cell suspensions G01N33/48735)}
- G01N 33/49: Blood {(chemical methods for determining blood cell populations G01N33/5094; chemical analysis of blood groups or blood types G01N33/80)}
- G03F 7/70: {Microphotolithographic exposure; Apparatus therefor (photo-masks G03F1/00)}
- 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)
- A61B: DIAGNOSIS; SURGERY; IDENTIFICATION (usefulness limited to only animals A61D)
- C23C: COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL (making metal-coated products by extrusion B21C23/22; covering with metal by connecting pre-existing layers to articles, see the relevant places, e.g. B21D39/00, B23K; metallising of glass C03C; metallising mortars, concrete, artificial stone, ceramics or natural stone C04B41/00; enamelling of, or applying a vitreous layer to, metals C23D; treating metal surfaces or coating of metals by electrolysis or electrophoresis C25D; single-crystal film growth C30B; by metallising textiles D06M11/83; decorating textiles by locally metallising D06Q1/04)
- C25B: ELECTROLYTIC OR ELECTROPHORETIC PROCESSES FOR THE PRODUCTION OF COMPOUNDS OR NON-METALS; APPARATUS THEREFOR (anodic or cathodic protection C23F13/00; single-crystal growth C30B)
- G03F: PHOTOMECHANICAL PRODUCTION OF TEXTURED OR PATTERNED SURFACES, e.g. FOR PRINTING, FOR PROCESSING OF SEMICONDUCTOR DEVICES; MATERIALS THEREFOR; ORIGINALS THEREFOR; APPARATUS SPECIALLY ADAPTED THEREFOR; (phototypographic composing devices B41B; photosensitive materials or processes for photographic purposes G03C; electrophotography, sensitive layers or processes therefor G03G)

## Abstract

A method for cancer diagnosis is disclosed. The method includes forming a plurality of cultured cells on an electrochemical biosensor placing the electrochemical biosensor in a medium solution comprising a cell culture solution of a plurality of biological cells, measuring a first electrochemical response from the electrochemical biosensor with the plurality of cultured cells, forming a plurality of stimulated cells on the electrochemical biosensor by ultrasonically stimulating of the plurality of cultured cells, measuring a second electrochemical response from the electrochemical biosensor with the plurality of stimulated cells, and detecting presence of cancer cells responsive to a difference between the first electrochemical response and the second electrochemical response being less than a threshold. Where, the first electrochemical response includes an electrochemical response of the plurality of cultured cells and the second electrochemical response includes an electrochemical response of the plurality of stimulated cells.

## Claims

### Claim 1

A method for detecting cancer cells, comprising:
preparing an electrochemical-ultrasonic system, comprising:
fabricating an electrochemical biosensor, the electrochemical biosensor comprising an integrated three-electrodes array patterned on a nanoroughened surface of a substrate, the integrated three-electrodes array comprising a working electrode, a counter electrode, and a reference electrode;
culturing a plurality of biological cells on the electrochemical biosensor comprising attaching the plurality of biological cells onto the working electrode by placing the electrochemical biosensor in a medium solution, the medium solution comprising a cell culture solution of the plurality of biological cells;
connecting the electrochemical biosensor with the plurality of cultured cells to an electrochemical stimulator-analyzer system, the electrochemical stimulator-analyzer system configured to measure electrochemical responses; and
exposing the electrochemical biosensor with the plurality of cultured cells to an ultrasonic system;
generating a plurality of microbubbles on the electrochemical biosensor with the plurality of cultured cells comprising electrolysis of the medium solution by applying an instantaneous electrical potential in the medium solution utilizing a cyclic voltammetry technique using the electrochemical stimulator-analyzer system, applying the instantaneous electrical potential in the medium solution comprising applying a DC signal with a voltage between −2 V and −0.5 V for a time duration less than 1 seconds on the biosensor with the plurality of cultured cells;
measuring a first electrochemical response from the electrochemical biosensor with the plurality of cultured cells using the electrochemical stimulator-analyzer system, the first electrochemical response comprising an electrochemical response of the plurality of cultured cells;
forming a plurality of stimulated cells on the electrochemical biosensor by applying ultrasonic waves to the plurality of cultured cells and the plurality of microbubbles using the ultrasonic system;
measuring a second electrochemical response from the electrochemical biosensor with the plurality of stimulated cells using the electrochemical stimulator-analyzer system, the second electrochemical response comprising an electrochemical response of the plurality of stimulated cells; and
detecting presence of cancer cells responsive to a difference between the first electrochemical response and the second electrochemical response being less than a threshold.

### Claim 2

A method for detecting cancer cells, comprising:
forming a plurality of cultured cells on an electrochemical biosensor by placing the electrochemical biosensor in a medium solution comprising a cell culture solution of a plurality of biological cells;
measuring a first electrochemical response from the electrochemical biosensor with the plurality of cultured cells, the first electrochemical response comprising a first cyclic voltammetry (CV) pattern comprising a first set of measured electrical currents versus a range of applied electrical potentials comprising a respective first current peak;
generating a plurality of microbubbles on the electrochemical biosensor with the plurality of cultured cells comprising electrolysis of the medium solution by applying an instantaneous electrical potential to the electrochemical biosensor with the plurality of cultured cells via a cyclic voltammetry technique using an electrochemical stimulator-analyzer system;
forming a plurality of stimulated cells on the electrochemical biosensor by applying ultrasonic waves to the plurality of cultured cells and the plurality of microbubbles using an ultrasonic system;
measuring a second electrochemical response from the electrochemical biosensor with the plurality of stimulated cells, the second electrochemical response comprising a second CV pattern comprising a second set of measured electrical currents versus the range of applied electrical potentials comprising a respective second current peak; and
detecting presence of cancer cells responsive to a difference between the first electrochemical response and the second electrochemical response being less than a threshold, the difference between the first electrochemical response and the second electrochemical response comprising a peak to peak
Δ
⁢
⁢
I
I
,
the peak to peak
Δ
⁢
⁢
I
I
being defined by:
peak to peak
Δ
⁢
⁢
I
I
=
I
US
⁢
⁢
off
-
I
US
⁢
⁢
on
I
US
⁢
⁢
off
where IUSoff comprises the first current peak and IUSon comprises the second current peak.

### Claim 3 (depends on 2)

The method of claim 2, wherein detecting the presence of cancer cells comprises detecting presence of breast cancer cells responsive to the peak to peak
Δ
⁢
I
I
being less than the threshold comprising a value of 0.15.

### Claim 4 (depends on 3)

The method of claim 3, wherein:
the threshold comprises a value of 0.05 for the ultrasonically stimulating of the plurality of cultured cells with an intensity of less than 1 W/cm2 and a duration of less than 5 seconds, and
the threshold comprises a value of 0.15 for the ultrasonically stimulating of the plurality of cultured cells with an intensity of more than 1 W/cm2 and a duration of less than 5 seconds.

### Claim 5 (depends on 2)

The method of claim 2, wherein each of the measuring the first electrochemical response from the electrochemical biosensor with the plurality of cultured cells and measuring the second electrochemical response from the electrochemical biosensor with the plurality of stimulated cells comprises measuring an electrochemical response via the cyclic voltammetry technique using the electrochemical stimulator-analyzer system.

### Claim 6 (depends on 5)

The method of claim 5, wherein the electrochemical stimulator-analyzer system comprises a potentiostat.

### Claim 7 (depends on 2)

The method of claim 2, wherein forming the plurality of cultured cells on the electrochemical biosensor comprises:
placing the electrochemical biosensor at the bottom of a chamber; and
filling the chamber with the medium solution.

### Claim 8 (depends on 2)

The method of claim 2, wherein generating the plurality of microbubbles on the electrochemical biosensor with the plurality of cultured cells comprises applying a DC signal with a voltage between −2 V and −0.5 V for a time duration less than 1 seconds on the biosensor with the plurality of cultured cells using a potentiostat device.

### Claim 9 (depends on 2)

The method of claim 2, wherein forming the plurality of stimulated cells on the electrochemical biosensor comprises:
ultrasonically stimulating the plurality of cultured cells responsive to applying ultrasonic waves to the plurality of cultured cells on the electrochemical biosensor in the medium solution; and
inducing an acoustic cavitation in the plurality of cultured cells by the plurality of microbubbles responsive to applying ultrasonic waves to the plurality of microbubbles on the electrochemical biosensor in the medium solution.

### Claim 10 (depends on 2)

The method of claim 2, wherein forming the plurality of stimulated cells on the electrochemical biosensor comprises:
exposing the medium solution containing the electrochemical biosensor with the plurality of cultured cells to the ultrasonic system; and
applying ultrasonic waves to the medium solution with a frequency range between 10 KHz and 100 KHz for a time duration between 2s and 10 s to the plurality of cultured cells using the ultrasonic system.

### Claim 11 (depends on 10)

The method of claim 10, wherein exposing the medium solution containing the electrochemical biosensor with the plurality of cultured cells to the ultrasonic system comprises placing an ultrasonic horn above the electrochemical biosensor with the plurality of cultured cells, the ultrasonic horn being connected to an ultrasonic generator.

### Claim 12 (depends on 10)

The method of claim 10, wherein ultrasonic ultrasonically stimulating the plurality of cultured cells comprises applying ultrasonic waves using the ultrasonic system with an intensity between 0.5 w/cm2 and 5 w/cm2.