# US 2021/0340596 A1 · Target capture and barcoding in monodisperse droplets

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

- Number: US 2021/0340596 A1
- Document ID: US20210340596A1
- Status: Published application
- Kind: A1
- Application number: 17/281,157
- Filed: 2019-09-27
- Published: 2021-11-04
- Earliest claimed: 2018-09-28
- Assignees: Fluent Biosciences Inc.
- Applicants: Fluent Biosciences Inc.
- Inventors: Robert Meltzer; Kristina Fontanez; Desiree Schenk; Yi Xue
- Web page: https://patentgrep.com/patent/US20210340596A1

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C08L 33/26: Homopolymers or copolymers of acrylamide or methacrylamide
- G01N 33/54313: {the carrier being characterised by its particulate form}
- C12Q 1/6869: Methods for sequencing
- G01N 2458/10: Oligonucleotides as tagging agents for labelling antibodies
- 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
- C08L: COMPOSITIONS OF MACROMOLECULAR COMPOUNDS (compositions based on polymerisable monomers C08F, C08G; artificial filaments or fibres D01F; textile treating compositions D06)
- 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)

## Abstract

The methods and systems described herein provide capture template particles and an improved emulsion droplet-based target capture and barcoding method thereof. The capture template particles and methods disclosed herein allow capturing targets of interest from biological samples, and barcoding of specific nucleic acids contained in the captured targets. The nucleic acids can be contained within living or nonliving structures, including particles, viruses, and cells. The nucleic acids can include, e.g., DNA or RNA.

## Claims

### Claim 1

A capture template particle comprising:
a template particle comprising one or more tethering moieties, wherein each of the one or more tethering moieties is attachable to one or more target-specific elements.

### Claim 2 (depends on 1)

The capture template particle of claim 1, wherein the one or more tethering moieties are selected from a capture moiety, and a capture element genetic identifier moiety.

### Claim 3 (depends on 1)

The capture template particle of claim 1, wherein the target-specific elements are selected from a target-specific capture element, and a target-specific capture element genetic identifier.

### Claim 4 (depends on 1)

The capture template particle of claim 1, wherein the capture moiety comprises an acrylate-terminated hydrocarbon linker with biotin termination, an alkyne-terminated linker with biotin termination, or an alkyne-terminated linker without biotin termination.

### Claim 5 (depends on 4)

The capture template particle of claim 4, wherein the capture moiety is attached to a target- specific capture element.

### Claim 6 (depends on 4)

The capture template particle of claim 4, wherein the target-specific capture element is selected from the group consisting of poly-T polynucleotide sequences, aptamers, and antibodies.

### Claim 7 (depends on 1)

The capture template particle of claim 1, wherein the capture element genetic identifier moiety comprises an acrylate terminated hydrocarbon linker with terminal adapter polynucleotide.

### Claim 8 (depends on 7)

The capture template particle of claim 7, wherein the capture element genetic identifier moiety is attached to a target-specific capture element genetic identifier.

### Claim 9 (depends on 8)

The capture template particle of claim 8, wherein the target-specific capture element genetic identifier an oligonucleotide and comprises elements selected from the group consisting of a universal adaptor sequence, a template-type element, a template-ID element, a target-specific capture element, and combinations thereof.

### Claim 10 (depends on 1)

The capture template particle of claim 1, wherein the template particle further comprises a hydrogel selected from the group consisting of agarose, alginate, polyethylene glycol (PEG), polyacrylamide (PAA), acrylate, an acrylamide/bis-acrylamide copolymer matrix, azide-modified PEG, and combinations thereof.

### Claim 11 (depends on 10)

The capture template particle of claim 10, wherein the template particle is sized to the desired diameter by microfluidic co-flow with immiscible oil.

### Claim 12

A method of target capture and barcoding in monodisperse droplets comprising:
combining a plurality of capture template particles with a first fluid to provide a first mixture, wherein the first fluid comprises a plurality of target particles;
incubating the first mixture to allow association of the plurality of target particles to the plurality of capture template particles, thereby a portion of the plurality of target particles become associated to the capture template particles;
generating a supernatant and pellet from the first mixture, optionally by centrifuging;
removing the supernatant to separate the plurality of capture template particles and associated target particles from the solution of the first mixture;
washing and resuspending the pellet in a reaction buffer to provide a second mixture;
combining the second mixture with a second fluid to provide a third mixture, wherein the second fluid is immiscible with the second mixture; and
shearing the third mixture such that a plurality of the capture template particles are encapsulated in a plurality of monodisperse droplets in the second fluid, thereby providing a plurality of monodisperse droplets comprising reaction buffer, one of the capture template particles, and one of the plurality of target particles associated to said capture template particles.

### Claim 13 (depends on 12)

The method of claim 12, wherein the second fluid comprises an oil.

### Claim 14 (depends on 13)

The method of claim 13, wherein the oil comprises a fluorocarbon oil, a silicone oil, a hydrocarbon oil, or a combination thereof.

### Claim 15 (depends on 14)

The method of claim 14, whereas the first fluid comprises a biological sample.

### Claim 16 (depends on 15)

The method of claim 15, wherein the biological sample is a body fluid.

### Claim 17 (depends on 16)

The method of claim 16, wherein the body fluid comprises target particles selected from the group consisting of circulating cells, cellular components, cell-free nucleic acids, extracellular vesicles, protein antigens, prokaryotic cells, fungi, viruses, and combinations thereof.

### Claim 18 (depends on 17)

The method of claim 17, wherein the body fluid is a human body fluid.

### Claim 19 (depends on 18)

The method of claim 18, wherein the association of the plurality of target particles to the plurality of capture template particles is target-specific.

### Claim 20 (depends on 19)

The method of claim 19, wherein the associated target particles associate with the capture template particles via the target-specific capture element.

### Claim at position 21

21-95. (canceled)