# US 11,299,727 B2 · System for analyzing single cell

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- Number: US 11,299,727 B2
- Document ID: US11299727B2
- Status: Granted
- Kind: B2
- Application number: 16/329,682
- Filed: 2017-03-06
- Published: 2019-06-27
- Granted: 2022-04-12
- Earliest claimed: 2016-09-05
- Assignees: Hitachi, Ltd.
- Applicants: Hitachi, Ltd.
- Inventors: Tomoyuki Sakai; Masataka Shirai
- Web page: https://patentgrep.com/patent/US11299727B2

## CPC

- C12N 15/1013: {by using magnetic beads}
- C12M 47/06: {Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material (lysis of microorganisms C12N1/06; extracting or separating nucleic acids from biological samples C12N15/1003)}
- C12N 15/09: Recombinant DNA-technology
- C12N 15/1006: {by means of a solid support carrier, e.g. particles, polymers}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}

## Abstract

A device is provided for capturing a nucleic acid in a single cell, having: a two-dimensional array comprising a substrate, a plurality of single cell capturing holes provided on one surface of the substrate, and a nucleic acid capturing region comprising, on the inside of the substrate, a nucleic acid capturing body, which is configured to capture the nucleic acids extracted from the individual cells respectively captured by the single cell capturing hole; a flow channel which is provided adjacent to the nucleic acid capturing region of the substrate, and is configured to discharge a solution in the nucleic acid capturing region; and a cylindrical structure body which is arranged on the substrate at the time of introducing a cell suspension and encloses a plurality of the single cell capturing holes, wherein the cylindrical structure body is removed after the cells are captured by the single cell capturing holes.

## Claims

### Claim 1

A method for capturing a nucleic acid in each of a plurality of single cells from a cell suspension comprising a plurality of cells, comprising:
(i) preparing a system for analyzing a nucleic acid in a single cell, the system comprising: a two-dimensional array comprising a substrate, a plurality of single cell capturing holes provided on an upper surface of the substrate, and a nucleic acid capturing region comprising, on an inside of the substrate, a nucleic acid capturing body, which is configured to capture the nucleic acids extracted from individual cells respectively captured by the single cell capturing hole; a means for introducing a cell suspension to the upper surface of the substrate, on which the single cell capturing holes are provided; a flow channel which is provided adjacent to the nucleic acid capturing region of the substrate, and is configured to discharge a solution in the nucleic acid capturing region; a means for applying a negative pressure from the surface opposite the upper surface of the substrate on which the single cell capturing holes are provided; a cylindrical structure body enclosing the plurality of single cell capturing holes; and a mechanism which is configured to arrange the cylindrical structure body on the upper surface of the substrate when introducing the cell suspension by bringing the cylindrical structure body into contact with the upper surface of the substrate and to remove the cylindrical structure body after capturing the cells by the single cell capturing holes;
(ii) introducing the cell suspension to the cylindrical structure body in a state in which the cylindrical structure body is arranged on the upper surface of the substrate;
(iii) applying a negative pressure from the surface opposite the upper surface of the substrate on which the single cell capturing holes are provided to capture the cells by the single cell capturing holes;
(iv) removing the cylindrical structure body; and
(v) lysing the captured cell, and capturing the nucleic acid by the nucleic acid capturing body.

### Claim 2 (depends on 1)

The method of claim 1, wherein when the cylindrical structure body is arranged on the upper surface of the substrate, the system further comprises a means for controlling a distance between the upper surface on which the single cell capturing holes of the substrate are provided and an end of the cylindrical structure body.

### Claim 3 (depends on 2)

The method of claim 2, wherein the means for controlling the distance is a fixing tool provided in the cylindrical structure body.

### Claim 4 (depends on 2)

The method of claim 2, wherein the means for controlling the distance is a mechanism which is configured to control movement of the cylindrical structure body.

### Claim 5 (depends on 1)

The method of claim 1, wherein the nucleic acid is mRNA.

### Claim 6 (depends on 1)

The method of claim 1, wherein the nucleic acid capturing body is magnetic beads.

### Claim 7 (depends on 1)

The method of claim 1, wherein when the cylindrical structure body is arranged on the upper surface of the substrate, the mechanism is configured to control a distance between the upper surface of the substrate, on which the single cell capturing holes are provided, and an end of the cylindrical structure body, or
when the cylindrical structure body is arranged on the upper surface of the substrate, the cylindrical structure body has a fixing tool for controlling the distance between the upper surface on which the single cell capturing holes of the substrate are provided and the end of the cylindrical structure body.

### Claim 8 (depends on 1)

The method of claim 1, wherein the means for introducing the cell suspension is provided with a means for controlling an amount of the cell suspension to be dispensed.