# US 2021/0239581 A1 · Liquid flourescent dye concentrate for flow cytometry evaluation of virus-size particles and related products and methods

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- Number: US 2021/0239581 A1
- Document ID: US20210239581A1
- Status: Published application
- Kind: A1
- Application number: 16/781,782
- Filed: 2020-02-04
- Published: 2021-08-05
- Earliest claimed: 2020-02-04
- Assignees: Sartorius BioAnalytical Instruments, Inc.
- Applicants: Essen Instruments, Inc. d/b/a Essen BioScience, Inc.
- Inventors: Rebecca K. Montange; Jeffrey W. Steaffens
- Web page: https://patentgrep.com/patent/US20210239581A1

## CPC

- G01N 1/30: Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis}
- G01N 33/50: Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing (measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q)
- C12Q 1/70: involving virus or bacteriophage {(immunoassay for viruses G01N33/56983)}
- G01N 15/1404: Handling flow, e.g. hydrodynamic focusing
- G01N 15/1459: {the analysis being performed on a sample stream}
- G01N 33/56983: {Viruses}
- G01N 33/582: {with fluorescent label}
- G01N 2001/302: {Stain compositions}
- G01N 2015/1438: {Using two lasers in succession}
- 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

A kit and method for flow cytometry include a liquid dye concentrate for fluorescent staining of virus-size particles with a plurality of fluorogenic dyes in a liquid medium. The liquid dye concentrate includes a plurality of fluorogenic dyes and one or both of (i) the liquid medium comprising a liquid mixture including water and liquid phase organic material and (ii) disaccharide dissolved in the liquid medium.

## Claims

### Claim 1

A kit for preparing fluorescently stained fluid samples containing biological material for flow cytometry evaluation for unassociated virus-size particles, the kit comprising:
a liquid dye concentrate in a sealed first container, the liquid dye concentrate comprising a plurality of different fluorogenic dyes in a liquid medium, each different said fluorogenic dye having a different fluorescent emission signature for detection when bound to a said unassociated virus-size particle; and
an aqueous sample dilution liquid for preparing aqueous diluted fluid samples containing biological material for flow cytometry evaluation for unassociated virus-size particles, the aqueous sample dilution liquid being contained in a sealed second container;
and wherein the liquid dye concentrate comprises at least one member selected from the group consisting of:
(i) the liquid medium comprises a liquid mixture including water and liquid phase organic material;
(ii) disaccharide dissolved in the liquid medium; and
(iii) a combination including both (i) and (ii).

### Claim 2 (depends on 1)

The kit of claim 1, wherein the sealed first container and the sealed second container are packaged together within a common packaging enclosure.

### Claim 3 (depends on 1)

The kit of claim 1, wherein the aqueous dilution liquid comprises an aqueous buffer solution.

### Claim 4 (depends on 1)

The kit of claim 1, comprising (i), and wherein the liquid medium is an aqueous liquid medium comprising a major proportion on a molar basis of water and a minor proportion on a molar basis of the liquid phase organic material in the liquid mixture.

### Claim 5 (depends on 4)

The kit of claim 4, wherein the liquid phase organic material comprises dimethyl sulfoxide (DMSO).

### Claim 6 (depends on 5)

The kit of claim 5, wherein the liquid medium comprises the liquid phase organic material in a range of from 10 weight percent to 50 weight percent.

### Claim 7 (depends on 5)

The kit of claim 5, comprising (ii), and wherein the disaccharide comprises one or more than one member selected from the group consisting of trehalose, sucrose, lactose, lactulose, melibiose, melibiulose, cellobiose, nigerose, isomaltose, isomaltulose, maltulose, rutinose, β-laminaribiose, and maltose.

### Claim 8 (depends on 7)

The kit of claim 7, wherein the disaccharide comprises trehalose.

### Claim 9 (depends on 8)

The kit of claim 8, wherein a concentration of the disaccharide in the liquid dye concentrate is in a range of from 1 weight percent to 45 weight percent.

### Claim 10

A fluorescent stain product with multiple fluorogenic dyes for direct fluorescent staining of aqueous fluid samples containing biological materials with multiple fluorogenic dyes for flow cytometry evaluation of stained fluid samples for unassociated virus-size particles, the fluorescent stain product comprising:
a liquid dye concentrate comprising a plurality of different fluorogenic dyes in a liquid medium, each different said fluorogenic dye having a different fluorescent emission signature for detection when bound to a said unassociated virus-size particle; and
a sealed container containing the liquid dye concentrate;
and wherein the liquid dye concentrate comprises at least one member selected from the group consisting of:
(i) the liquid medium comprises a mixture including water and liquid phase organic material;
(ii) disaccharide dissolved in the liquid medium; and
(iii) a combination including both (i) and (ii).

### Claim 11

A method for flow cytometry evaluation of biological material for unassociated virus-size particles, the method comprising:
providing an aqueous preliminary fluid sample with biological material in an aqueous liquid for flow cytometry evaluation for unassociated virus-size particles;
providing a liquid dye concentrate comprising a plurality of different fluorogenic dyes in a liquid medium, each different said fluorogenic dye having a different fluorescent emission signature for detection when bound to a said unassociated virus-size particle;
preparing an aqueous stained fluid sample, comprising staining the preliminary fluid sample with liquid dye concentrate;
subjecting the stained fluid sample to flow cytometry in a flow cytometer to detect and count occurrences of the unassociated virus-size particles stained with at least one of the fluorogenic dyes;
and wherein the method comprises at least one member selected from the group consisting of:
(i) the liquid medium comprising liquid phase organic material;
(ii) the liquid dye concentrate comprising disaccharide dissolved in the liquid medium;
(iii) the providing the liquid dye concentrate being in the absence of reconstituting the fluorogenic dyes from a dry form into the liquid medium; and
(iv) combinations including two or more of any of (i)-(iii).

### Claim 12 (depends on 11)

The method of claim 11, wherein the staining the preliminary fluid sample comprises combining the preliminary fluid sample and the liquid dye concentrate at a volume ratio of volume of the liquid dye concentrate to volume of the preliminary fluid sample in a range of from 1:125 to 1:24.

### Claim 13 (depends on 11)

The method of claim 11, comprising the providing the liquid dye concentrate being in the absence of reconstituting the fluorogenic dyes from a dry form into the liquid medium.

### Claim 14 (depends on 11)

The method of claim 11, wherein the providing the liquid dye concentrate comprises unsealing a first sealed container containing the liquid dye concentrate and removing from the unsealed container a quantity of the liquid dye concentrate for combining with the preliminary fluid sample during the staining, and wherein the first sealed container is in a kit, the kit comprising:
the liquid dye concentrate in a sealed first container, the liquid dye concentrate comprising a plurality of different fluorogenic dyes in a liquid medium, each different said fluorogenic dye having a different fluorescent emission signature for detection when bound to a said unassociated virus-size particle; and
an aqueous sample dilution liquid for preparing aqueous diluted fluid samples containing biological material for flow cytometry evaluation for unassociated virus-size particles, the aqueous sample dilution liquid being contained in a sealed second container.

### Claim 15 (depends on 14)

The method of claim 14, comprising:
providing a plurality of said preliminary fluid samples each with a portion of a biological material from a stock source and each diluted to different dilution ratios with the aqueous sample dilution liquid;
and separately subjecting each said stained fluid sample to the flow cytometry.

### Claim 16 (depends on 11)

The method of claim 11, wherein the stained fluid sample comprises a concentration of the liquid phase organic material in a range of from 0.25 weight percent to 2 weight percent.

### Claim 17 (depends on 16)

The method of claim 16, wherein the stained fluid sample comprises a concentration of the disaccharide in a range of from 0.1 weight percent to 1 weight percent.

### Claim 18 (depends on 17)

The method of claim 17, wherein the flow cytometry comprises:
hydrodynamically focusing a flow of the stained fluid sample with a sheath fluid; and
flowing the hydrodynamically focused stained fluid sample through a flow cell in which the flowing hydrodynamically focused stained fluid sample is subjected to excitation radiation to stimulate a fluorescent emission response from each of a first said fluorogenic dye and a second said fluorogenic dye on the unassociated virus-size particles stained with both the first fluorogenic dye and the second fluorogenic dye; and
detecting for both a first fluorescent emission signature of the first said fluorogenic stain staining a said unassociated virus-size particle and a second fluorescent emission signature of the second said fluorogenic stain staining a said unassociated virus-size particle;
wherein, the flowing comprises maintaining a flow rate of the hydrodynamically focused stained fluid sample through the flow cell in a range of from 300 nanoliters per minute to 6000 nanoliters per minute; and
separately detecting for each of the first fluorescent emission signature and the second fluorescent emission signature and time correlating detection of the first fluorescent emission signature and detection of the second fluorescent emission signature to determine a detection event indicative of an occurrence of a said unassociated virus-size particle stained with both the first fluorogenic dye and the second fluorogenic dye.

### Claim 19 (depends on 18)

The method of claim 18, wherein the unassociated virus-size particles have a particle size in a range of from 10 nanometers to 1 micron.

### Claim 20

A stained fluid sample for flow cytometry evaluation for unassociated virus-size particles, the stained fluid sample comprising:
an aqueous liquid medium;
a biological material of interest for flow cytometry evaluation for the unassociated virus-size particles dispersed in the aqueous liquid medium; and
a plurality of different fluorogenic dyes dispersed in the aqueous liquid medium, each said fluorogenic dye having a different fluorescent emission signature and each containing at least one aromatic group susceptible to pi stacking, the plurality of different fluorogenic dyes including at least a first fluorogenic dye with a first fluorescent emission signature and a second fluorogenic dye with a second fluorescent emission signature that is different than the first fluorescent emission signature;
and wherein:
the aqueous liquid medium comprises a liquid organic material at a concentration in a range of from 0.25 weight percent to 2 weight percent; and
the stained fluid sample comprises a disaccharide dissolved in the aqueous liquid medium and the stained fluid sample comprises the disaccharide at a concentration in a range of from 0.1 weight percent to 3 weight percent.