# US 2014/0155295 A1 · Capsule array devices and methods of use

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- Number: US 2014/0155295 A1
- Document ID: US20140155295A1
- Status: Published application
- Kind: A1
- Application number: 13/966,150
- Filed: 2013-08-13
- Published: 2014-06-05
- Earliest claimed: 2012-08-14
- Assignees: 10X Technologies, Inc.
- Applicants: 10X Technologies, Inc.
- Inventors: Benjamin Hindson; Serge Saxonov
- Web page: https://patentgrep.com/patent/US20140155295A1

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- B01J 19/0046: {Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays (synthesis methods per se C40B50/00)}
- B01L 3/502715: {characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces}
- B01L 3/508: {Rigid containers without fluid transport within}
- B01L 3/523: {with means for closing or opening}
- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- B01L 2200/0647: Handling flowable solids, e.g. microscopic beads, cells, particles
- B01L 2400/0677: phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
- C12Q 2535/122: Massive parallel sequencing
- C12Q 2563/159: Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
- 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
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

This disclosure provides microwell capsule array devices. The microwell capsule array devices are generally capable of performing one or more sample preparation operations. Such sample preparation operations may be used as a prelude to one more or more analysis operations. For example, a device of this disclosure can achieve physical partitioning and discrete mixing of samples with unique molecular identifiers within a single unit in preparation for various analysis operations. The device may be useful in a variety of applications and most notably nucleic-acid-based sequencing, detection and quantification of gene expression and single-cell analysis.

## Claims

### Claim at position 1

-77. (canceled)

### Claim 78

A composition comprising a first microcapsule, wherein:
a) said first microcapsule is degradable upon the application of a stimulus to said first microcapsule; and
b) said first microcapsule comprises an oligonucleotide barcode and a chemical cross-linker.

### Claim 79 (depends on 78)

The composition of claim 78, wherein said chemical cross-linker is a disulfide bond.

### Claim 80 (depends on 78)

The composition of claim 78, further comprising a polymer gel.

### Claim 81 (depends on 80)

The composition of claim 80, wherein said polymer gel is a polyacrylamide gel.

### Claim 82 (depends on 78)

The composition of claim 78, wherein said first microcapsule comprises a bead.

### Claim 83 (depends on 82)

The composition of claim 82, wherein said bead is a gel bead.

### Claim 84 (depends on 78)

The composition of claim 78, wherein said stimulus is selected from the group consisting of a biological, chemical, thermal, electrical, magnetic, or photo stimulus, and combination thereof.

### Claim 85 (depends on 78)

The composition of claim 78, wherein said chemical stimulus is selected from the group consisting of a change in pH, a change in ion concentration, and a reducing agent.

### Claim 86 (depends on 85)

The composition of claim 85, wherein said reducing agent is dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).

### Claim 87 (depends on 78)

The composition of claim 78, wherein a second microcapsule comprises said first microcapsule.

### Claim 88 (depends on 87)

The composition of claim 87, wherein said second microcapsule is a droplet.

### Claim 89 (depends on 78)

The composition of claim 78, further comprising a nucleic acid that comprises said oligonucleotide barcode, wherein said nucleic acid comprises a deoxyuridine triphosphate (dUTP).

### Claim 90 (depends on 78)

The composition of claim 78, further comprising a polymerase unable to accept a deoxyuridine triphosphate (dUTP).

### Claim 91 (depends on 78)

The composition of claim 78, further comprising a target analyte.

### Claim 92 (depends on 91)

The composition of claim 91, wherein said target analyte is a nucleic acid.

### Claim 93 (depends on 92)

The composition of claim 92, wherein said nucleic acid is selected from the group consisting of DNA, RNA, dNTPs, ddNTPs, amplicons, synthetic nucleotides, synthetic polynucleotides, polynucleotides, oligonucleotides, peptide nucleic acids, cDNA, dsDNA, ssDNA, plasmid DNA, cosmid DNA, High Molecular Weight (MW) DNA, chromosomal DNA, genomic DNA, viral DNA, bacterial DNA, mtDNA (mitochondrial DNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, ribozyme, riboswitch and viral RNA.

### Claim 94 (depends on 93)

The composition of claim 93, wherein said nucleic acid is genomic DNA (gDNA).

### Claim 95 (depends on 78)

The composition of claim 78, wherein the density of said oligonucleotide barcodes is at least about 1,000,000 oligonucleotide barcodes per said first microcapsule.

### Claim 96 (depends on 78)

The composition of claim 78, wherein said oligonucleotide barcode is coupled to said microcapsule via said chemical cross-linker.

### Claim 97 (depends on 96)

The composition of claim 96, wherein said chemical cross-linker is a disulfide bond.

### Claim 98

A composition comprising a degradable gel bead, wherein the degradable gel bead comprises at least about 1,000,000 oligonucleotide barcodes.

### Claim 99 (depends on 98)

The composition of claim 98, wherein the 1,000,000 oligonucleotide barcodes are identical.

### Claim 100

A method for sample preparation, comprising:
a) combining a microcapsule comprising an oligonucleotide barcode and a target analyte into a partition, wherein said microcapsule is degradable upon the application of a stimulus to said microcapsule; and
b) applying said stimulus to said microcapsule to release said oligonucleotide barcode to said target analyte.

### Claim 101 (depends on 100)

The method of claim 100, wherein said partition is a well.

### Claim 102 (depends on 100)

The method of claim 100, wherein said partition is a droplet.

### Claim 103 (depends on 100)

The method of claim 100, wherein said microcapsule comprises a polymer gel.

### Claim 104 (depends on 103)

The method of claim 103, wherein said polymer gel is a polyacrylamide.

### Claim 105 (depends on 100)

The method of claim 100, wherein said microcapsule comprises a bead.

### Claim 106 (depends on 105)

The method of claim 105, wherein said bead is a gel bead.

### Claim 107 (depends on 100)

The method of claim 100, wherein said microcapsule comprises a chemical cross-linker.

### Claim 108 (depends on 107)

The method of claim 107, wherein said chemical cross-linker is a disulfide bond.

### Claim 109 (depends on 100)

The method of claim 100, wherein said stimulus is selected from the group consisting of a biological, chemical, thermal, electrical, magnetic, photo stimulus, and a combination thereof.

### Claim 110 (depends on 109)

The method of claim 109, wherein said chemical stimulus is selected from the group consisting of a change in pH, change in ion concentration, and a reducing agent.

### Claim 111 (depends on 110)

The method of claim 110, wherein said reducing agent is dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).

### Claim 112 (depends on 100)

The method of claim 100, wherein a nucleic acid comprises said oligonucleotide barcode and wherein said nucleic acid comprises a deoxyuridine triphosphate (dUTP).

### Claim 113 (depends on 100)

The method of claim 100, wherein said partition comprises a polymerase unable to accept a deoxyuridine triphosphate (dUTP).

### Claim 114 (depends on 100)

The method of claim 100, further comprising attaching said oligonucleotide barcode to said target analyte.

### Claim 115 (depends on 114)

The method of claim 114, wherein said attaching is completed with a nucleic acid amplification reaction.

### Claim 116 (depends on 100)

The method of claim 100, wherein said target analyte is a nucleic acid.

### Claim 117 (depends on 116)

The method of claim 116, wherein said nucleic acid is selected from the group consisting of DNA, RNA, dNTPs, ddNTPs, amplicons, synthetic nucleotides, synthetic polynucleotides, polynucleotides, oligonucleotides, peptide nucleic acids, cDNA, dsDNA, ssDNA, plasmid DNA, cosmid DNA, High Molecular Weight (MW) DNA, chromosomal DNA, genomic DNA, viral DNA, bacterial DNA, mtDNA (mitochondrial DNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, ribozyme, riboswitch and viral RNA.

### Claim 118 (depends on 117)

The method of claim 117, wherein said nucleic acid is genomic DNA (gDNA).

### Claim 119 (depends on 100)

The method of claim 100, wherein said oligonucleotide barcode is coupled to said microcapsule via a chemical cross-linker.

### Claim 120

A device comprising a plurality of partitions, wherein:
a) at least one partition of the plurality of partitions comprises a microcapsule comprising an oligonucleotide barcode; and
b) said microcapsule is degradable upon the application of a stimulus to said microcapsule.

### Claim 121 (depends on 120)

The device of claim 120, wherein said partition is a well.

### Claim 122 (depends on 120)

The device of claim 120, wherein said partition is a droplet.

### Claim 123 (depends on 120)

The device of claim 120, wherein said microcapsule comprises a chemical cross-linker.

### Claim 124 (depends on 123)

The device of claim 123, wherein said chemical cross-linker is a disulfide bond.

### Claim 125 (depends on 120)

The device of claim 120, wherein said microcapsule further comprises a polymer gel.

### Claim 126 (depends on 125)

The device of claim 125, wherein said polymer gel is a polyacrylamide gel.

### Claim 127 (depends on 120)

The device of claim 120, wherein said microcapsule comprises a bead.

### Claim 128 (depends on 127)

The device of claim 127, wherein said bead is a gel bead.

### Claim 129 (depends on 120)

The device of claim 120, wherein said stimulus is selected from the group consisting of a biological, chemical, thermal, electrical, magnetic, or photo stimulus, and a combination thereof.

### Claim 130 (depends on 129)

The device of claim 129, wherein said chemical stimulus is selected from the group consisting of a change in pH, change in ion concentration, and a reducing agent.

### Claim 131 (depends on 130)

The device of claim 130, wherein said reducing agent is dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).

### Claim 132 (depends on 120)

The device of claim 120, wherein a nucleic acid comprises said oligonucleotide barcode and wherein said nucleic acid comprises a deoxyuridine triphosphate (dUTP).

### Claim 133 (depends on 120)

The device of claim 120, wherein said partition comprises a polymerase unable to accept a deoxyuridine triphosphate (dUTP).

### Claim 134 (depends on 120)

The device of claim 120, wherein said partition comprises a target analyte.

### Claim 135 (depends on 134)

The device of claim 134, wherein said target analyte is a nucleic acid.

### Claim 136 (depends on 135)

The device of claim 135, wherein said nucleic acid is selected from the group consisting of DNA, RNA, dNTPs, ddNTPs, amplicons, synthetic nucleotides, synthetic polynucleotides, polynucleotides, oligonucleotides, peptide nucleic acids, cDNA, dsDNA, ssDNA, plasmid DNA, cosmid DNA, High Molecular Weight (MW) DNA, chromosomal DNA, genomic DNA, viral DNA, bacterial DNA, mtDNA (mitochondrial DNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, ribozyme, riboswitch and viral RNA.

### Claim 137 (depends on 136)

The device of claim 136, wherein said nucleic acid is genomic DNA (gDNA).

### Claim 138 (depends on 120)

The device of claim 120, wherein said oligonucleotide barcode is coupled to said microcapsule via a chemical cross-linker.

### Claim 139 (depends on 138)

The device of claim 138, wherein said chemical cross-linker is a disulfide bond.

### Claim 140

A composition comprising a first microcapsule, wherein:
a) said first microcapsule is degradable upon the application of a stimulus to said first microcapsule; and
b) said first microcapsule comprises an oligonucleotide barcode and a polymer gel.

### Claim 141 (depends on 140)

The composition of claim 140, wherein said polymer gel is a polyacrylamide gel.

### Claim 142 (depends on 140)

The composition of claim 140, wherein said first microcapsule comprises a bead.

### Claim 143 (depends on 142)

The composition of claim 142, wherein said bead is a gel bead.

### Claim 144 (depends on 140)

The composition of claim 140, wherein said stimulus is selected from the group consisting of a biological, chemical, thermal, electrical, magnetic, or photo stimulus, and combination thereof.