U.S. patent application number 13/966150 was filed with the patent office on 2014-06-05 for capsule array devices and methods of use.
This patent application is currently assigned to 10X Technologies, Inc.. The applicant listed for this patent is 10X Technologies, Inc.. Invention is credited to Benjamin Hindson, Serge Saxonov.
Application Number | 20140155295 13/966150 |
Document ID | / |
Family ID | 50101458 |
Filed Date | 2014-06-05 |
United States Patent
Application |
20140155295 |
Kind Code |
A1 |
Hindson; Benjamin ; et
al. |
June 5, 2014 |
CAPSULE ARRAY DEVICES AND METHODS OF USE
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.
Inventors: |
Hindson; Benjamin;
(Pleasanton, CA) ; Saxonov; Serge; (Oakland,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
10X Technologies, Inc. |
Pleasanton |
CA |
US |
|
|
Assignee: |
10X Technologies, Inc.
Pleasanton
CA
|
Family ID: |
50101458 |
Appl. No.: |
13/966150 |
Filed: |
August 13, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61683192 |
Aug 14, 2012 |
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|
61737374 |
Dec 14, 2012 |
|
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61762435 |
Feb 8, 2013 |
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61800223 |
Mar 15, 2013 |
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61840403 |
Jun 27, 2013 |
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61844804 |
Jul 10, 2013 |
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Current U.S.
Class: |
506/16 ;
506/30 |
Current CPC
Class: |
C12N 15/1065 20130101;
B01L 2200/0647 20130101; C12Q 2535/122 20130101; B01L 2400/0677
20130101; B01L 3/508 20130101; B01L 3/502715 20130101; C12Q 1/6806
20130101; B01L 3/523 20130101; C12Q 2563/159 20130101; B01J 19/0046
20130101; C12Q 1/6806 20130101; C12Q 2535/122 20130101; C12Q
2563/159 20130101 |
Class at
Publication: |
506/16 ;
506/30 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1.-77. (canceled)
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.
79. The composition of claim 78, wherein said chemical cross-linker
is a disulfide bond.
80. The composition of claim 78, further comprising a polymer
gel.
81. The composition of claim 80, wherein said polymer gel is a
polyacrylamide gel.
82. The composition of claim 78, wherein said first microcapsule
comprises a bead.
83. The composition of claim 82, wherein said bead is a gel
bead.
84. 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.
85. 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.
86. The composition of claim 85, wherein said reducing agent is
dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).
87. The composition of claim 78, wherein a second microcapsule
comprises said first microcapsule.
88. The composition of claim 87, wherein said second microcapsule
is a droplet.
89. The composition of claim 78, further comprising a nucleic acid
that comprises said oligonucleotide barcode, wherein said nucleic
acid comprises a deoxyuridine triphosphate (dUTP).
90. The composition of claim 78, further comprising a polymerase
unable to accept a deoxyuridine triphosphate (dUTP).
91. The composition of claim 78, further comprising a target
analyte.
92. The composition of claim 91, wherein said target analyte is a
nucleic acid.
93. 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.
94. The composition of claim 93, wherein said nucleic acid is
genomic DNA (gDNA).
95. 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.
96. The composition of claim 78, wherein said oligonucleotide
barcode is coupled to said microcapsule via said chemical
cross-linker.
97. The composition of claim 96, wherein said chemical cross-linker
is a disulfide bond.
98. A composition comprising a degradable gel bead, wherein the
degradable gel bead comprises at least about 1,000,000
oligonucleotide barcodes.
99. The composition of claim 98, wherein the 1,000,000
oligonucleotide barcodes are identical.
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.
101. The method of claim 100, wherein said partition is a well.
102. The method of claim 100, wherein said partition is a
droplet.
103. The method of claim 100, wherein said microcapsule comprises a
polymer gel.
104. The method of claim 103, wherein said polymer gel is a
polyacrylamide.
105. The method of claim 100, wherein said microcapsule comprises a
bead.
106. The method of claim 105, wherein said bead is a gel bead.
107. The method of claim 100, wherein said microcapsule comprises a
chemical cross-linker.
108. The method of claim 107, wherein said chemical cross-linker is
a disulfide bond.
109. 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.
110. 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.
111. The method of claim 110, wherein said reducing agent is
dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).
112. The method of claim 100, wherein a nucleic acid comprises said
oligonucleotide barcode and wherein said nucleic acid comprises a
deoxyuridine triphosphate (dUTP).
113. The method of claim 100, wherein said partition comprises a
polymerase unable to accept a deoxyuridine triphosphate (dUTP).
114. The method of claim 100, further comprising attaching said
oligonucleotide barcode to said target analyte.
115. The method of claim 114, wherein said attaching is completed
with a nucleic acid amplification reaction.
116. The method of claim 100, wherein said target analyte is a
nucleic acid.
117. 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.
118. The method of claim 117, wherein said nucleic acid is genomic
DNA (gDNA).
119. The method of claim 100, wherein said oligonucleotide barcode
is coupled to said microcapsule via a chemical cross-linker.
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.
121. The device of claim 120, wherein said partition is a well.
122. The device of claim 120, wherein said partition is a
droplet.
123. The device of claim 120, wherein said microcapsule comprises a
chemical cross-linker.
124. The device of claim 123, wherein said chemical cross-linker is
a disulfide bond.
125. The device of claim 120, wherein said microcapsule further
comprises a polymer gel.
126. The device of claim 125, wherein said polymer gel is a
polyacrylamide gel.
127. The device of claim 120, wherein said microcapsule comprises a
bead.
128. The device of claim 127, wherein said bead is a gel bead.
129. 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.
130. 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.
131. The device of claim 130, wherein said reducing agent is
dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).
132. The device of claim 120, wherein a nucleic acid comprises said
oligonucleotide barcode and wherein said nucleic acid comprises a
deoxyuridine triphosphate (dUTP).
133. The device of claim 120, wherein said partition comprises a
polymerase unable to accept a deoxyuridine triphosphate (dUTP).
134. The device of claim 120, wherein said partition comprises a
target analyte.
135. The device of claim 134, wherein said target analyte is a
nucleic acid.
136. 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.
137. The device of claim 136, wherein said nucleic acid is genomic
DNA (gDNA).
138. The device of claim 120, wherein said oligonucleotide barcode
is coupled to said microcapsule via a chemical cross-linker.
139. The device of claim 138, wherein said chemical cross-linker is
a disulfide bond.
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.
141. The composition of claim 140, wherein said polymer gel is a
polyacrylamide gel.
142. The composition of claim 140, wherein said first microcapsule
comprises a bead.
143. The composition of claim 142, wherein said bead is a gel
bead.
144. 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.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 61/683,192, filed Aug. 14, 2012; U.S.
Provisional Patent Application No. 61/737,374, filed Dec. 14, 2012;
U.S. Provisional Patent Application No. 61/762,435, filed Feb. 8,
2013; U.S. Provisional Patent Application No. 61/800,223, filed
Mar. 15, 2013; U.S. Provisional Patent Application No. 61/840,403,
filed Jun. 27, 2013; and U.S. Provisional Patent Application No.
61/844,804, filed Jul. 10, 2013, which applications are
incorporated herein by reference in their entireties for all
purposes.
BACKGROUND OF THE INVENTION
[0002] The detection and quantification of analytes is important
for molecular biology and medical applications such as diagnostics.
Genetic testing is particularly useful for a number of diagnostic
methods. For example, disorders that are caused by mutations, such
as cancer, may be detected or more accurately characterized with
DNA sequence information.
[0003] Appropriate sample preparation is often needed prior to
conducting a molecular reaction such as a sequencing reaction. A
starting sample may be a biological sample such as a collection of
cells, tissue, or nucleic acids. When the starting material is
cells or tissue, the sample may need to be lysed or otherwise
manipulated in order to permit the extraction of molecules such as
DNA. Sample preparation may also involve fragmenting molecules,
isolating molecules, and/or attaching unique identifiers to
particular fragments of molecules, among other actions. There is a
need in the art for improved methods and devices for preparing
samples prior to downstream applications.
SUMMARY OF THE INVENTION
[0004] This disclosure provides compositions and methods for a
microcapsule array device.
[0005] An aspect of the disclosure provides a composition
comprising a first microcapsule, wherein: the first microcapsule is
degradable upon the application of a stimulus to the first
microcapsule; and the first microcapsule comprises an
oligonucleotide barcode. In some cases, the first microcapsule may
comprise a chemical cross-linker. The chemical cross-linker, for
example, may be a disulfide bond. In some cases, the composition
may comprise a polymer gel, such as, for example a polyacrylamide
gel. The first microcapsule may comprise a bead. In some cases, the
bead may be a gel bead.
[0006] Moreover, the stimulus may be selected from the group
consisting of a biological, chemical, thermal, electrical,
magnetic, or photo stimulus, and combination thereof. In some
cases, the chemical stimulus may be selected from the group
consisting of a change in pH, a change in ion concentration, and a
reducing agent. The reducing agent may be, for example,
dithiothreitol (DTT) or tris(2-carboxyethyl) phosphine (TCEP).
[0007] A second microcapsule may comprise the first microcapsule.
Moreover, the second microcapsule may be a droplet. In some cases,
the composition may also comprise a nucleic acid that comprises the
oligonucleotide barcode, wherein the nucleic acid comprises a
deoxyuridine triphosphate (dUTP). In some cases, the composition
may comprise a polymerase unable to accept a deoxyuridine
triphosphate (dUTP). Also, the composition may comprise a target
analyte, such as, for example, a nucleic acid. The nucleic acid may
be 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. In
some cases, the nucleic acid may be genomic DNA (gDNA).
[0008] Additionally, the density of the oligonucleotide barcodes
may be at least about 1,000,000 oligonucleotide barcodes per the
first microcapsule. The oligonucleotide barcode may be coupled to
the microcapsule via a chemical cross-linker, such as, for example
a disulfide bond.
[0009] An additional aspect of the disclosure comprises a device
comprising a plurality of partitions, wherein: at least one
partition of the plurality of partitions comprises a microcapsule
comprising an oligonucleotide barcode; and the microcapsule is
degradable upon the application of a stimulus to the microcapsule.
The partition, for example, may be a well or a droplet. In some
cases, the microcapsule comprises a chemical cross-linker such as,
for example, a disulfide bond. Moreover, the microcapsule may
comprise a polymer gel such as, for example, a polyacrylamide gel.
Also, the microcapsule may comprise a bead. In some cases, the bead
may be a gel bead.
[0010] The stimulus may be selected from the group consisting of a
biological, chemical, thermal, electrical, magnetic, or photo
stimulus, and a combination thereof. In some cases, the chemical
stimulus may be selected from the group consisting of a change in
pH, change in ion concentration, and a reducing agent. The reducing
agent, for example, may be dithiothreitol (DTT) or
tris(2-carboxyethyl)phosphine (TCEP).
[0011] Furthermore, a nucleic acid may comprise the oligonucleotide
barcode and the nucleic acid may comprise a deoxyuridine
triphosphate (dUTP). In some cases, the partition may comprise a
polymerase unable to accept a deoxyuridine triphosphate (dUTP).
Additionally, the partition may comprise a target analyte such as,
for example, a nucleic acid. The nucleic acid may be 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. In
some cases, the nucleic acid may be genomic DNA (gDNA). The
oligonucleotide barcode may be coupled to the microcapsule via a
chemical cross-linker. In some cases, the chemical cross-linker may
be a disulfide bond.
[0012] A further aspect of the disclosure provides a method for
sample preparation comprising combining a microcapsule comprising
an oligonucleotide barcode and a target analyte into a partition,
wherein the microcapsule is degradable upon the application of a
stimulus to the microcapsule; and applying the stimulus to the
microcapsule to release the oligonucleotide barcode to the target
analyte. The partition may be, for example, a well or a droplet. In
some cases, the microcapsule may comprise a polymer gel such as,
for example, a polyacrylamide. Moreover, the microcapsule may
comprise a bead. In some cases, the bead may be a gel bead.
Moreover, the microcapsule may comprise a chemical cross-linker
such as, for example, a disulfide bond.
[0013] The stimulus may be selected from the group consisting of a
biological, chemical, thermal, electrical, magnetic, photo
stimulus, and a combination thereof. In some cases, the chemical
stimulus may be selected from the group consisting of a change in
pH, change in ion concentration, and a reducing agent. The reducing
agent may be, for example, dithiothreitol (DTT) or
tris(2-carboxyethyl)phosphine (TCEP).
[0014] Also, a nucleic acid may comprise the oligonucleotide
barcode and the nucleic acid may comprise a deoxyuridine
triphosphate (dUTP). In some cases, the partition may comprise a
polymerase unable to accept a deoxyuridine triphosphate (dUTP).
Moreover, the method may also comprise attaching the
oligonucleotide barcode to the target analyte. The attaching may be
completed, for example, with a nucleic acid amplification reaction.
Moreover, the analyte may be a nucleic acid. In some cases, the
nucleic acid may be 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. In some cases, the nucleic acid may be genomic DNA
(gDNA). Furthermore, the oligonucleotide barcode may be coupled to
the microcapsule via a chemical cross-linker. In some cases, the
chemical cross-linker may be a disulfide bond.
[0015] A further aspect of the disclosure provides a composition
comprising a degradable gel bead, wherein the gel bead comprises at
least about 1,000,000 oligonucleotide barcodes. In some cases, the
1,000,000 oligonucleotide barcodes are identical.
INCORPORATION BY REFERENCE
[0016] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference in their
entireties for all purposes and to the same extent as if each
individual publication, patent, or patent application was
specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The novel features of a device of this disclosure are set
forth with particularity in the appended claims. A better
understanding of the features and advantages of this disclosure
will be obtained by reference to the following detailed description
that sets forth illustrative embodiments, in which the principles
of a device of this disclosure are utilized, and the accompanying
drawings of which:
[0018] FIG. 1A is a schematic representation of a microcapsule or
inner reagent droplet.
[0019] FIG. 1B is a schematic representation of a microcapsule
containing multiple inner reagent droplets.
[0020] FIG. 2A is a schematic illustration of a top down view of an
exemplary microcapsule array.
[0021] FIG. 2B is a schematic illustration of an exemplary side
view of a microcapsule array.
[0022] FIG. 3 is a schematic illustration of a multi-microcapsule
array configuration on a 96-well plate holder.
[0023] FIG. 4A is a schematic flow diagram representative of a
reaction sequence in one microwell of a microwell capsule
array.
[0024] FIG. 4B is similar to 4A, except that it is annotated with
examples of methods that can be performed at each step.
DETAILED DESCRIPTION OF THE INVENTION
[0025] While various embodiments of the invention have been shown
and described herein, it will be obvious to those skilled in the
art that such embodiments are provided by way of example only.
Numerous variations, changes, and substitutions may occur to those
skilled in the art without departing from the invention. It should
be understood that various alternatives to the embodiments of the
invention described herein may be employed.
I. GENERAL OVERVIEW
[0026] The present disclosure provides microwell or other partition
capsule array devices and methods of using such devices. Generally,
the device is an assembly of partitions (e.g., microwells,
droplets) that are loaded with microcapsules, often at a particular
concentration of microcapsules per partition.
[0027] The devices may be particularly useful to perform sample
preparation operations. In some cases, a device subdivides a sample
(e.g., a heterogeneous mixture of nucleic acids, a mixture of
cells, etc.) into multiple partitions such that only a portion of
the sample is present in each partition. For example, a nucleic
acid sample comprising a mixture of nucleic acids may be
partitioned such that no more than one strand of (or molecule of)
nucleic acid is present in each partition. In other examples, a
cell sample may be partitioned such that no more than one cell is
present in each partition.
[0028] Following the partitioning step, any of a number of
different operations may be performed on the subdivided sample
within the device. The partitions may include one or more capsules
that contain one or more reagents (e.g., enzymes, unique
identifiers (e.g., bar codes), antibodies, etc.). In some cases,
the device, a companion device or a user provides a trigger that
causes the microcapsules to release one or more of the reagents
into the respective partition. The release of the reagent may
enable contact of the reagent with the subdivided sample. For
example, if the reagent is a unique identifier such as a barcode,
the sample may be tagged with the unique identifier. The tagged
sample may then be used in a downstream application such as a
sequencing reaction.
[0029] A variety of different reactions and/operations may occur
within a device disclosed herein, including but not limited to:
sample partitioning, sample isolation, binding reactions,
fragmentation (e.g., prior to partitioning or following
partitioning), ligation reactions, and other enzymatic reactions.
The device also may be useful for a variety of different molecular
biology applications including, but not limited to, nucleic acid
sequencing, protein sequencing, nucleic acid quantification,
sequencing optimization, detecting gene expression, quantifying
gene expression, and single-cell analysis of genomic or expressed
markers. Moreover, the device has numerous medical applications.
For example, it may be used for the identification, detection,
diagnosis, treatment, staging of, or risk prediction of various
genetic and non-genetic diseases and disorders including
cancer.
II. MICROCAPSULES
[0030] FIG. 1A is a schematic of an exemplary microcapsule
comprising an internal compartment 120 enveloped by a second layer
130, which is encapsulated by a solid or semi-permeable shell or
membrane 110. In general, the shell separates the internal
compartment(s) from their immediate environment (e.g., interior of
a microwell). The internal compartments, e.g., 120, 130, may
comprise materials such as reagents. As depicted in FIG. 1A, the
reagents 100 may be present in the internal compartment 120.
However, in some cases, the reagents are located in the enveloping
layer 130 or in both compartments. Generally, the microcapsule may
release the inner materials, or a portion thereof, following the
introduction of a particular trigger. The trigger may cause
disruption of the shell layer 110 and/or the internal enveloping
layer 130, thereby permitting contact of the internal compartment
100, 120 with the outside environment, such as the cavity of a
microwell.
[0031] The microcapsule may comprise several fluidic phases and may
comprise an emulsion (e.g. water-in-oil emulsion, oil-in-water
emulsion). A microcapsule may comprise an internal layer 120 that
is immiscible with a second layer 130 enveloping the internal
layer. For example, the internal layer 120 may comprise an aqueous
fluid, while the enveloping layer 130 may be a non-aqueous fluid
such as an oil. Conversely, the internal layer 120 may comprise a
non-aqueous fluid (e.g., oil), and the enveloping layer 130 may
comprise an aqueous fluid. In some cases, the microcapsule does not
comprise an enveloping second layer. Often, the microcapsule is
further encapsulated by a shell layer 110, which may comprise a
polymeric material. In some cases, a microcapsule may comprise a
droplet. In some cases, a microcapsule may be a droplet.
[0032] Droplets and methods for droplet generation, for example,
are described in U.S. Pat. No. RE41,780, which is incorporated
herein by reference in its entirety for all purposes. The device
also may contain a microfluidic element that enables the flow of a
sample and/or microcapsules through the device and distribution of
the sample and/or microcapsules within the partitions.
[0033] The microcapsule can comprise multiple compartments. The
microcapsule may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500,
2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000,
7500, 8000, 8500, 9000, 9500, 10000, or 50000 compartments. In
other cases, the microcapsule comprises less than 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000,
1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500,
7000, 7500, 8000, 8500, 9000, 9500, 10000, or 50000 compartments.
Similarly, each compartment, or a subset thereof, may also be
subdivided into a plurality of additional compartments. In some
cases, each compartment, or subset thereof, is subdivided into at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000,
4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500,
10000, or 50000 compartments. In other cases, each compartment, or
subset thereof, is further subdivided into less than 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 500,
1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000,
6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or 50000
compartments.
[0034] There are several possible distributions of reagent in the
multiple compartments. For example, each compartment (or some
percentage of the total number of compartments) may comprise the
same reagent or the same combination or reagents. In some cases,
each compartment (or some percentage of the total number of
compartments) comprises different reagents or a different
combination of reagents.
[0035] The compartments may be configured in a variety of ways. In
some cases, the microcapsule may comprise multiple concentric
compartments (repeating units of compartments that contain the
preceding compartment), often separated by an immiscible layer. In
such microcapsules, the reagents may be present in alternating
compartments, in every third compartment, or in every fourth
compartment.
[0036] In some cases, most of the compartments with a microcapsule
are not concentric; instead, they exist as separate, self-contained
entities within a microcapsule. FIG. 1B depicts an example of a
microcapsule that contains a plurality of smaller microcapsules
140, each containing a reagent. Like many of the other
microcapsules described herein, the microcapsule may be
encapsulated by an outer shell, often comprising a polymer material
150. The plurality of smaller microcapsules encapsulated within the
larger microcapsule may be physically separated by an immiscible
fluid 160, thereby preventing mixing of reagents before application
of a stimulus and release of reagents into solution. In some cases,
the immiscible fluid is loaded with additional materials or
reagents. In some cases, the plurality of smaller microcapsules are
surrounded by a layer of immiscible fluid (e.g., 170) which is
further surrounded by a fluid 160 that is miscible with the inner
fluid of the microcapsules. For example, the interior microcapsules
180 may comprise an aqueous interior enveloped by an immiscible
(e.g., oil) layer, that is further surrounded by an aqueous layer
160. The miscible compartments (e.g., 160 and 180) may each contain
reagents. They may contain the same reagents (or the same
combination of reagents) or different reagents (or different
combination of reagents). Alternatively, one or some of the
miscible compartments may comprise no reagents.
[0037] The microcapsule may comprise a polymeric shell (see, e.g.,
FIGS. 1 and 2) or multiple polymeric shells. For example, the
microcapsule may comprise multiple polymeric shells layered on top
of each other. In other cases, individual compartments within a
microcapsule comprise a polymeric shell, or a subset of the
compartments may comprise a polymeric shell. For example, all or
some of the smaller compartments 140 in FIG. 1B may comprise a
polymeric shell that separates them from the fluidic interior 160.
The microcapsule may be designed so that a particular reagent is
contained within a compartment that has a polymerized shell, while
a different reagent is within a compartment that is simply
enveloped by an immiscible liquid. For example, a reagent that is
desired to be released upon a heat trigger may be contained within
the compartments that have a heat-sensitive or heat-activatable
polymerized shell, while reagents designed to be released upon a
different trigger may be present in different types of
compartments. In another example, paramagnetic particles may be
incorporated into the capsule shell wall. A magnet or electric
field may then be used to position the capsule to a desired
location. In some cases, a magnetic field (e.g., high frequency
alternating magnetic field) can be applied to such capsules; the
incorporated paramagnetic particles may then transform the energy
of the magnetic field into heat, thereby triggering rupture of the
capsule.
[0038] The microcapsule component of a device of this disclosure
may provide for the controlled and/or timed release of reagents for
sample preparation of an analyte. Microcapsules may be used in
particular for controlled release and transport of varying types of
chemicals, ingredients, pharmaceuticals, fragrances etc. . . . ,
including particularly sensitive reagents such as enzymes and
proteins (see, e.g., D. D. Lewis, "Biodegradable Polymers and Drug
Delivery Systems", M. Chasin and R. Langer, editors (Marcel Decker,
New York, 1990); J. P. McGee et al., J. Control. Release 34 (1995),
77).
[0039] Microcapsules may also provide a means for delivery of
reagents in discrete and definable amounts. Microcapsules may be
used to prevent premature mixing of reagents with the sample, by
segregating the reagents from the sample. Microcapsules also may
ease handling of--and limit contacts with--particularly sensitive
reagents such as enzymes, nucleic acids and other chemicals used in
sample preparation.
[0040] A. Preparation of Microcapsules
[0041] Microcapsules of a device of this disclosure may be prepared
by numerous methods and processes. Preparative techniques may
include pan coating, spray drying, centrifugal extrusion,
emulsion-based methods, and/or microfluidic techniques. Typically,
a method for preparation is chosen based on the desired
characteristics of the microcapsule. For example, shell wall
thickness, permeability, chemical composition of the shell wall,
mechanical integrity of the shell wall and capsule size may be
taken into consideration when choosing a method. Methods of
preparation may also be selected based on the ability to
incorporate specific materials within the capsule such as whether
the core materials (e.g., fluids, reagents, etc.) are aqueous,
organic or inorganic. Additionally, preparation methods can affect
the shape and size of the microcapsule. For example a capsule's
shape, (e.g., spherical, ellipsoidal, etc.), may depend on the
shape of the droplet in the precursor liquid which may be
determined by the viscosity and surface tension of the core liquid,
direction of flow of the emulsion, the choice of surfactants used
in droplet stabilization, as well as physical confinement such as
preparations made in a microchannel or capillary of a particular
size (e.g., a size requiring distortion of the microcapsule in
order for the microcapsule to fit within the microchannel or
capillary.
[0042] Microcapsules may be prepared through emulsification
polymerization, a process in which monomer units at an
aqueous/organic interface in an emulsion polymerize to form a
shell. Reagents are mixed with the aqueous phase of the biphasic
mixture. Vigorous shaking, or sonication of the mixture, creates
droplets containing reagents, which are encased by a polymeric
shell.
[0043] In some cases, microcapsules may be prepared through
layer-by-layer assembly, a process in which negatively and
positively charged polyelectrolytes are deposited onto particles
such as metal oxide cores. Electrostatic interactions between
polyelectrolytes create a polymeric shell around the core. The core
can be subsequently removed via addition of acid, resulting in a
semi-permeable hollow sphere which can be loaded with various
reagents.
[0044] In still further cases, microcapsules may be prepared
through coacervation, a process in which two oppositely charged
polymers in aqueous solution become entangled to form a neutralized
polymer shell wall. One polymer may be contained within an oil
phase, while the other, of opposite charge is contained in an
aqueous phase. This aqueous phase may contain reagents to be
encapsulated. The attraction of one polymer for another can result
in the formation of coascervates. In some embodiments, gelatin and
gum Arabic are components of this preparative method.
[0045] Microcapsules also may be prepared through internal phase
separation, a process in which a polymer is dissolved in a solvent
mixture containing volatile and nonvolatile solvents. Droplets of
the resultant solution are suspended in an aqueous layer, which is
stabilized by continual agitation and the use of surfactants. This
phase may contain reagents to be encapsulated. When the volatile
solvent evaporates, the polymers coalesce to form a shell wall. In
some cases, polymers such as polystyrene, poly(methyl methacrylate)
and poly(tetrahydrofuran) are used to form shell walls.
[0046] Microcapsules also may be prepared through flow focusing
methods, a process in which a microcapillary device is used to
generate double emulsions containing a single internal droplet
encased in a middle fluid which is then dispersed to an outer
fluid. The inner droplet may contain reagents to be encapsulated.
The middle fluid becomes the shell wall, which can be formed via
cross-linking reactions.
[0047] B. Microcapsule Composition
[0048] Microcapsules may comprise a variety of materials with a
wide range of chemical characteristics. Generally, the
microcapsules comprise materials with the ability to form
microcapsules of a desired shape and size and that are compatible
with the reagents to be stored in the microcapsules.
[0049] Microcapsules may comprise a wide range of different
polymers including but not limited to: polymers, heat sensitive
polymers, photosensitive polymers, magnetic polymers, pH sensitive
polymers, salt-sensitive polymers, chemically sensitive polymers,
polyelectrolytes, polysaccharides, peptides, proteins, and/or
plastics. Polymers may include but are not limited to materials
such as poly(N-isopropylacrylamide) (PNIPAAm), poly(styrene
sulfonate) (PSS), poly(allyl amine) (PAAm), poly(acrylic acid)
(PAA), poly(ethylene imine) (PEI), poly(diallyldimethyl-ammonium
chloride) (PDADMAC), poly(pyrolle) (PPy), poly(vinylpyrrolidone)
(PVPON), poly(vinyl pyridine) (PVP), poly(methacrylic acid) (PMAA),
poly(methyl methacrylate) (PMMA), polystyrene (PS),
poly(tetrahydrofuran) (PTHF), poly(phthaladehyde) (PTHF),
poly(hexyl viologen) (PHV), poly(L-lysine) (PLL), poly(L-arginine)
(PARG), poly(lactic-co-glycolic acid) (PLGA).
[0050] Often, materials for the microcapsules, particularly the
shells of microcapsules, may enable the microcapsule to be
disrupted with an applied stimulus. For example, a microcapsule may
be prepared from heat sensitive polymers and/or may comprise one or
more shells comprising such heat-sensitive polymers. The
heat-sensitive polymer may be stable under conditions used for
storage or loading. Upon exposure to heat, the heat-sensitive
polymer components may undergo depolymerization, resulting in
disruption to the integrity of the shell and release of the inner
materials of the microcapsule (and/or of the inner microcapsules)
to the outside environment (e.g., the interior of a microwell).
Exemplary heat-sensitive polymers may include, but are not limited
to NIPAAm or PNIPAM hydrogel. The microcapsules may also comprise
one or more types of oil. Exemplary oils include but are not
limited to hydrocarbon oils, fluorinated oils, fluorocarbon oils,
silicone oils, mineral oils, vegetable oils, and any other suitable
oil.
[0051] The microcapsules may also comprise a surfactant, such as an
emulsifying surfactant. Exemplary surfactants include, but are not
limited to, cationic surfactants, non-ionic surfactants, anionic
surfactants, hydrocarbon surfactants or fluorosurfactants. The
surfactant may increase the stability of one or more components of
the microcapsule, such as an inner compartment that comprises an
oil.
[0052] Additionally, the microcapsules may comprise an inner
material that is miscible with materials external to the capsule.
For example, the inner material may be an aqueous fluid and the
sample within the microwell may also be in an aqueous fluid. In
other examples, the microcapsule may comprise powders or
nanoparticles that are miscible with an aqueous fluid. For example,
the microcapsule may comprise such powders or nanoparticles in an
inner compartment. Upon disruption of the microcapsule, such
powders or nanoparticles are released into the external environment
(e.g., interior of microwell) and may mix with an aqueous fluid
(e.g., an aqueous sample fluid).
[0053] Additionally, the microcapsule may comprise a material that
is immiscible with the surrounding environment (e.g., interior of
microwell, sample fluid). In such cases, when the inner emulsion is
released to the surrounding environment, the phase separation
between the inner and outer components may promote mixing, such as
mixing of the inner components with the surrounding fluid. In some
cases, when a microcapsule is triggered to release its contents, a
pressure or force is also released that promotes mixing of internal
and external components.
[0054] The microcapsules may also comprise a polymer within the
interior of the capsule. In some instances this polymer may be a
porous polymer bead that may entrap reagents or combinations of
reagents. In other instances, this polymer may be a bead that has
been previously swollen to create a gel. Examples of polymer based
gels that may be used as inner emulsions of capsules may include,
but are not limited to sodium alginate gel, or poly acrylamide gel
swelled with oligonucleotide bar codes or the like.
[0055] In some cases, a microcapsule may be a gel bead comprising
any of the polymer based gels described herein. Gel bead
microcapsules may be generated, for example, by encapsulating one
or more polymeric precursors into droplets. Upon exposure of the
polymeric precursors to an accelerator (e.g.,
tetramethylethylenediamine (TEMED)), a gel bead may be
generated.
[0056] Analytes and/or reagents, such as oligonucleotide barcodes,
for example, may be coupled/immobilized to the interior surface of
a gel bead (e.g., the interior accessible via diffusion of an
oligonucleotide barcode and/or materials used to generate an
oligonucleotide barcode) and/or the outer surface of a gel bead or
any other microcapsule described herein. Coupling/immobilization
may be via any form of chemical bonding (e.g., covalent bond, ionic
bond) or physical phenomena (e.g., Van der Waals forces,
dipole-dipole interactions, etc.). In some cases,
coupling/immobilization of a reagent to a gel bead or any other
microcapsule described herein may be reversible, such as, for
example, via a labile moiety (e.g., via a chemical cross-linker,
including chemical cross-linkers described herein). Upon
application of a stimulus, the labile moiety may be cleaved and the
immobilized reagent set free. In some cases, the labile moiety is a
disulfide bond. For example, in the case where an oligonucleotide
barcode is immobilized to a gel bead via a disulfide bond, exposure
of the disulfide bond to a reducing agent can cleave the disulfide
bond and free the oligonucleotide barcode from the bead. The labile
moiety may be included as part of a gel bead or microcapsule, as
part of a chemical linker that links a reagent or analyte to a gel
bead or microcapsule, and/or as part of a reagent or analyte.
[0057] A gel bead or any other type of microcapsule described
herein may contain varied numbers of reagents. The density of a
reagent per microcapsule may vary depending on the particular
microcapsule utilized and the particular reagent. For example, a
microcapsule or gel bead may comprise at least about 1; 10; 100;
1,000; 10,000; 100,000; 1,000,000; 5,000,000; 10,000,000,
50,000,000; 100,000,000; 500,000,000; or 1,000,000,000
oligonucleotide barcodes per microcapsule or gel bead. A gel bead
may comprise identical oligonucleotide barcodes or may comprise
differing oligonucleotide barcodes.
[0058] In other example, the microcapsule may comprise one or more
materials that create a net neutral, negative or positive charge on
the outer shell wall of the capsule. In some instances, the charge
of a capsule may aid in preventing or promoting aggregation or
clustering of particles, or adherence or repulsion to parts of the
device.
[0059] In addition, the microcapsule may comprise one or more
materials that cause the outer shell wall of the capsule to be
hydrophilic or hydrophobic. A hydrophilic material that may be used
for capsule shell walls may be poly(N-isopropylacrylamide). A
hydrophobic material that may be used for capsule shell walls may
be polystyrene. In certain instances, a hydrophilic shell wall may
aid in wicking of the capsule into wells comprising aqueous
fluid.
[0060] C. Microcapsule Size and Shape
[0061] A microcapsule may be any of a number of sizes or shapes. In
some cases, the shape of the microcapsule may be spherical,
ellipsoidal, cylindrical, hexagonal or any other symmetrical or
non-symmetrical shape. Any cross-section of the microcapsule may
also be of any appropriate shape, include but not limited to:
circular, oblong, square, rectangular, hexagonal, or other
symmetrical or non-symmetrical shape. In some cases, the
microcapsule may be of a specific shape that complements an opening
(e.g., surface of a microwell) of the device. For example, the
microcapsule may be spherical and the opening of a microwell of the
device may be circular.
[0062] The microcapsules may be of uniform size (e.g., all of the
microcapsules are the same size) or heterogeneous size (e.g., some
of the microcapsules are of different sizes). A dimension (e.g.,
diameter, cross-section, side, etc.) of a microcapsule may be at
least about 0.001 .mu.m, 0.01 .mu.m, 0.1 .mu.m, 0.5 .mu.m, 1 .mu.m,
5 .mu.m, 10 .mu.m, 50 .mu.m, 100 .mu.m, 200 .mu.m, 300 .mu.m, 400
.mu.m, 500 .mu.m, 600 .mu.m, 700 .mu.m, 800 .mu.m, 900 .mu.m or 1
nm. In some cases, the microcapsule comprises a microwell that is
at most about 0.001 .mu.m, 0.01 .mu.m, 0.1 .mu.m, 0.5 .mu.m, 1
.mu.m, 5 .mu.m, 10 .mu.m, 50 .mu.m, 100 .mu.m, 200 .mu.m, 300
.mu.m, 400 .mu.m, 500 .mu.m, 600 .mu.m, 700 .mu.m, 800 .mu.m, 900
.mu.m or 1 nm.
[0063] In some cases, microcapsules are of a size and/or shape so
as to allow a limited number of microcapsules to be deposited in
individual partitions (e.g., microwells, droplets) of the
microcapsule array. Microcapsules may have a specific size and/or
shape such that exactly or no more than 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 capsules fit into an individual microwell; in some cases, on
average 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 capsules fit into an
individual microwell. In still further cases, at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 100, 500, or 1000 capsules fit into an
individual microwell.
[0064] D. Reagents and Reagent Loading
[0065] The devices provided herein may comprise free reagents
and/or reagents encapsulated into microcapsules. The reagents may
be a variety of molecules, chemicals, particles, and elements
suitable for sample preparation reactions of an analyte. For
example, a microcapsule used in a sample preparation reaction for
DNA sequencing of a target may comprise one or more of the
following reagents: enzymes, restriction enzymes (e.g., multiple
cutters), ligase, polymerase (e.g., polymerases that do and do not
recognize dUTPs and/or uracil), fluorophores, oligonucleotide
barcodes, buffers, deoxynucleotide triphosphates (dNTPs) (e.g.
deoxyadenosine triphosphate (dATP), deoxycitidine triphosphate
(dCTP), deoxyguanosine triphosphate (dGTP), deoxythymidine
triphosphate (dTTP), deoxyuridine triphosphate (dUTP)),
deoxynucleotide triphosphates (ddNTPs) and the like. In another
example, a microcapsule used in a sample preparation reaction for
single cell analysis may comprise reagents such as one or more of
the following reagents: lysis buffer, detergent, fluorophores,
oligonucleotide barcodes, ligase, proteases, heat activatable
proteases, protease or nuclease inhibitors, buffer, enzymes,
antibodies, nanoparticles, and the like.
[0066] Exemplary reagents include, but are not limited to: buffers,
acidic solution, basic solution, temperature-sensitive enzymes,
pH-sensitive enzymes, light-sensitive enzymes, metals, metal ions,
magnesium chloride, sodium chloride, manganese, aqueous buffer,
mild buffer, ionic buffer, inhibitor, enzyme, protein, nucleic
acid, antibodies, saccharides, lipid, oil, salt, ion, detergents,
ionic detergents, non-ionic detergents, oligonucleotides,
nucleotides, dNTPs, ddNTPs, deoxyribonucleic acid (DNA),
ribonucleic acid (RNA), peptide nucleic acids, circular DNA (cDNA),
double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), plasmid
DNA, cosmid DNA, chromosomal DNA, genomic DNA (gDNA), viral DNA,
bacterial DNA, mtDNA (mitochondrial DNA), messenger RNA (mRNA),
ribosomal RNA (rRNA), transfer RNA (tRNA), nRNA, short-interfering
RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA
(snoRNA), small Cajul body specific RNA, (scaRNA), microRNA,
double-stranded RNA (dsRNA), ribozyme, riboswitch and viral RNA,
polymerase (e.g., polymerases that do and do not recognize dUTPs
and/or uracil), ligase, restriction enzymes, proteases, nucleases,
protease inhibitors, nuclease inhibitors, chelating agents,
reducing agents (e.g., dithiotheritol (DTT),
2-tris(2-carboxyethyl)phosphine (TCEP)), oxidizing agents,
fluorophores, probes, chromophores, dyes, organics, emulsifiers,
surfactants, stabilizers, polymers, water, small molecules,
pharmaceuticals, radioactive molecules, preservatives, antibiotics,
aptamers, and pharmaceutical drug compounds.
[0067] In some cases, a microcapsule comprises a set of reagents
that have a similar attribute (e.g., a set of enzymes, a set of
minerals, a set of oligonucleotides, a mixture of different
bar-codes, a mixture of identical bar-codes). In other cases, a
microcapsule comprises a heterogeneous mixture of reagents. In some
cases, the heterogeneous mixture of reagents comprises all
components necessary to perform a reaction. In some cases, such
mixture comprises all components necessary to perform a reaction,
except for 1, 2, 3, 4, 5, or more components necessary to perform a
reaction. In some cases, such additional components are contained
within a different microcapsule or within a solution within a
partition (e.g., microwell) of the device.
[0068] Reagents may be pre-loaded into the device (e.g., prior to
introduction of analyte) or post-loaded into the device. They may
be loaded directly into the device; or, in some cases, the reagents
are encapsulated into a microcapsule that is loaded into the
device. In some cases, only microcapsules comprising reagents are
introduced. In other cases, both free reagents and reagents
encapsulated in microcapsules are loaded into the device, either
sequentially or concurrently. In some cases, reagents are
introduced to the device either before or after a particular step.
For example, a lysis buffer reagent may be introduced to the device
following partitioning of a cellular sample into multiple
partitions (e.g., microwells, droplets) within the device. In some
cases, reagents and/or microcapsules comprising reagents are
introduced sequentially such that different reactions or operations
occur at different steps. The reagents (or microcapsules) may be
also be loaded at steps interspersed with a reaction or operation
step. For example, microcapsules comprising reagents for
fragmenting molecules (e.g., nucleic acids) may be loaded into the
device, followed by a fragmentation step, which may be followed by
loading of microcapsules comprising reagents for ligating bar-codes
(or other unique identifiers, e.g., antibodies) and subsequent
ligation of the bar-codes to the fragmented molecules. Additional
methods of loading reagents are described further herein in other
sections.
[0069] E. Molecular `Barcodes`
[0070] It may be desirable to retain the option of identifying and
tracking individual molecules or analytes after or during sample
preparation. In some cases, one or more unique molecular
identifiers, sometimes known in the art as a `molecular barcodes,`
are used as sample preparation reagents. These molecules may
comprise a variety of different forms such as oligonucleotide bar
codes, antibodies or antibody fragments, fluorophores,
nanoparticles, and other elements or combinations thereof.
Depending upon the specific application, molecular barcodes may
reversibly or irreversibly bind to the target analyte and allow for
identification and/or quantification of individual analytes after
recovery from a device after sample preparation.
[0071] A device of this disclosure may be applicable to nucleic
acid sequencing, protein detection, single molecule analysis and
other methods that require a) precise measurement of the presence
and amount of a specific analyte b) multiplex reactions in which
multiple analytes are pooled for analysis. A device of this
disclosure may utilize the microwells of the microwell array or
other type of partition (e.g., droplets) to physically partition
target analytes. This physical partitioning allows for individual
analytes to acquire one or more molecular barcodes. After sample
preparation, individual analytes may be pooled or combined and
extracted from a device for multiplex analysis. For most
applications, multiplex analysis substantially decreases the cost
of analysis as well as increases through-put of the process, such
as in the case of the nucleic acid sequencing. Molecular barcodes
may allow for the identification and quantification of individual
molecules even after pooling of a plurality of analytes. For
example, with respect to nucleic acid sequencing, molecular
barcodes may permit the sequencing of individual nucleic acids,
even after the pooling of a plurality of different nucleic
acids.
[0072] Oligonucleotide barcodes, in some cases, may be particularly
useful in nucleic acid sequencing. In general, an oligonucleotide
barcode may comprise a unique sequence (e.g., a barcode sequence)
that gives the oligonucleotide barcode its identifying
functionality. The unique sequence may be random or non-random.
Attachment of the barcode sequence to a nucleic acid of interest
may associate the barcode sequence with the nucleic acid of
interest. The barcode may then be used to identify the nucleic acid
of interest during sequencing, even when other nucleic acids of
interest (e.g., comprising different barcodes) are present. In
cases where a nucleic acid of interest is fragmented prior to
sequencing, an attached barcode may be used to identify fragments
as belonging to the nucleic acid of interest during sequencing.
[0073] An oligonucleotide barcode may consist solely of a unique
barcode sequence or may be included as part of an oligonucleotide
of longer sequence length. Such an oligonucleotide may be an
adaptor required for a particular sequencing chemistry and/or
method. For example, such adaptors may include, in addition to an
oligonucleotide barcode, immobilization sequence regions necessary
to immobilize (e.g., via hybridization) the adaptor to a solid
surface (e.g., solid surfaces in a sequencer flow cell channel);
sequence regions required for the binding of sequencing primers;
and/or a random sequence (e.g., a random N-mer) that may be useful,
for example, in random amplification schemes. An adaptor can be
attached to a nucleic acid to be sequenced, for example, by
amplification, ligation, or any other method described herein.
[0074] Moreover, an oligonucleotide barcode, and/or a larger
oligonucleotide comprising an oligonucleotide barcode may comprise
natural nucleic acid bases and/or may comprise non-natural bases.
For example, in cases where an oligonucleotide barcode or a larger
oligonucleotide comprising an oligonucleotide barcode is DNA, the
oligonucleotide may comprise the natural DNA bases adenine,
guanine, cytosine, and thymine and/or may comprise non-natural
bases such as uracil.
[0075] F. Microcapsule-Preparation for Microwell Loading
[0076] Following preparation, reagent loaded microcapsules may be
loaded into a device using a variety of methods. Microcapsules, in
some instances, may be loaded as `dry capsules.` After preparation,
capsules may be separated from a liquid phase using various
techniques, including but not limited to differential
centrifugation, evaporation of the liquid phase, chromatography,
filtration and the like. `Dry capsules` may be collected as a
powder or particulate matter and then deposited into microwells of
the microwell array. Loading `dry capsules` may be a preferred
method in instances in which loading of `wet capsules,` leads to
inefficiencies of loading such as empty wells and poor distribution
of microcapsules across the microwell array.
[0077] Reagent-loaded microcapsules may also be loaded into a
device when the microcapsules are within a liquid phase, and
thereby loaded as `wet capsules.` In some instances, microcapsules
may be suspended in a volatile oil such that the oil can be removed
or evaporated, leaving only the dry capsule in the well. Loading
`wet capsules` may be a preferred method in some instances in which
loading of dry capsules leads to inefficiencies of loading, such as
microcapsule clustering, aggregation and poor distribution of
microcapsules across the microwell array. Additional methods of
loading reagents and microcapsules are described in other sections
of this disclosure.
[0078] The microcapsules also may have a particular density. In
some cases, the microcapsules are less dense than an aqueous fluid
(e.g., water); in some cases, the microcapsules are denser than an
aqueous fluid (e.g., water). In some cases, the microcapsules are
less dense than a non-aqueous fluid (e.g., oil); in some cases, the
microcapsules are denser than a non-aqueous fluid (e.g., oil).
Microcapsules may comprise a density at least about 0.05
g/cm.sup.3, 0.1 cm.sup.3, 0.2 g/cm.sup.3, 0.3 g/cm.sup.3, 0.4
g/cm.sup.3, 0.5 g/cm.sup.3, 0.6 g/cm.sup.3, 0.7 g/cm.sup.3, 0.8
g/cm.sup.3, 0.81 g/cm.sup.3, 0.82 g/cm.sup.3, 0.83 g/cm.sup.3, 0.84
g/cm.sup.3, 0.85 g/cm.sup.3, 0.86 g/cm.sup.3, 0.87 g/cm.sup.3, 0.88
g/cm.sup.3, 0.89 g/cm.sup.3, 0.90 g/cm.sup.3, 0.91 g/cm.sup.3, 0.92
g/cm.sup.3, 0.93 g/cm.sup.3, 0.94 g/cm.sup.3, 0.95 g/cm.sup.3, 0.96
g/cm.sup.3, 0.97 g/cm.sup.3, 0.98 g/cm.sup.3, 0.99 g/cm.sup.3, 1.00
g/cm.sup.3, 1.05 g/cm.sup.3, 1.1 g/cm.sup.3, 1.2 g/cm.sup.3, 1.3
g/cm.sup.3, 1.4 g/cm.sup.3, 1.5 g/cm.sup.3, 1.6 g/cm.sup.3, 1.7
g/cm.sup.3, 1.8 g/cm.sup.3, 1.9 g/cm.sup.3, 2.0 g/cm.sup.3, 2.1
g/cm.sup.3, 2.2 g/cm.sup.3, 2.3 g/cm.sup.3, 2.4 g/cm.sup.3, or 2.5
g/cm.sup.3. In other cases, microcapsule densities may be at most
about 0.7 g/cm.sup.3, 0.8 g/cm.sup.3, 0.81 g/cm.sup.3, 0.82
g/cm.sup.3, 0.83 g/cm.sup.3, 0.84 g/cm.sup.3, 0.85 g/cm.sup.3, 0.86
g/cm.sup.3, 0.87 g/cm.sup.3, 0.88 g/cm.sup.3, 0.89 g/cm.sup.3, 0.90
g/cm.sup.3, 0.91 g/cm.sup.3, 0.92 g/cm.sup.3, 0.93 g/cm.sup.3, 0.94
g/cm.sup.3, 0.95 g/cm.sup.3, 0.96 g/cm.sup.3, 0.97 g/cm.sup.3, 0.98
g/cm.sup.3, 0.99 g/cm.sup.3, 1.00 g/cm.sup.3, 1.05 g/cm.sup.3, 1.1
g/cm.sup.3, 1.2 g/cm.sup.3, 1.3 g/cm.sup.3, 1.4 g/cm.sup.3, 1.5
g/cm.sup.3, 1.6 g/cm.sup.3, 1.7 g/cm.sup.3, 1.8 g/cm.sup.3, 1.9
g/cm.sup.3, 2.0 g/cm.sup.3, 2.1 g/cm.sup.3, 2.2 g/cm.sup.3, 2.3
g/cm.sup.3, 2.4 g/cm.sup.3, or 2.5 g/cm.sup.3. Such densities can
reflect the density of the microcapsule in any particular fluid
(e.g., aqueous, water, oil, etc.)
III. MICROWELL ARRAY
[0079] A. Structure/Features
[0080] A device of this disclosure may be a microwell array
comprising a solid plate containing a plurality of holes, cavities
or microwells in which microcapsules and/or analytes are deposited.
Generally, a fluidic sample (or analyte) is introduced into the
device (e.g., through an inlet) and then travels through a flow
channel which distributes the sample into multiple microwells. In
some cases, additional fluid is introduced into the device as well.
The microwells may comprise microcapsules when the sample is
introduced; or, in some cases, the microcapsules are introduced
into the microwells following introduction of the sample.
[0081] FIG. 2A depicts a prototype microwell array; a sideview is
depicted in FIG. 2B. The microwell array may include a plate 220
that can be made of any suitable material commonly used in a
chemical laboratory, including fused silica, soda lima glass,
borosilicate glass, PMMA, sapphire, silicon, germanium, cyclic
olefin copolymer and cyclic polymer, polyethylenes, polypropylenes,
polyacrylates, polycarbonates, plastics, Topas, and other suitable
substrates known in the art. The plate 220 may initially be a flat
solid plate comprising a regular pattern of microwells 270. The
microwells may be formed by drilling or chemical dissolution or any
other suitable method of machining; however, plates with a desired
hole pattern are preferably molded, e.g. by injection-molding,
embossing, or using a suitable polymer, such as cyclic olefin
copolymer.
[0082] The microwell array may comprise an inlet (200 and 240)
and/or an outlet (210 and 260); in some cases, the microwell array
comprises multiple inlets and/or outlets. A sample (or analyte) or
microcapsules may be introduced to the device via the inlet.
Solutions containing analytes, reagents and/or microcapsules may be
manually applied to the inlet port 200 and 240 (or to a conduit
attached to the inlet port) via a pipette. In some cases, a liquid
handling device is used to introduce analytes, reagents, and/or
microcapsules to the device. Exemplary liquid handling devices may
rely on a pipetting robot, capillary action, or dipping into a
fluid. In some cases, the inlet port is connected to a reservoir
comprising microcapsules or analytes. The inlet port may be
attached to a flow channel 250 that permits distribution of the
analyte, sample, or microcapsules to the microwells in the device.
In some cases, the inlet port may be used to introduce to the
device a fluid (e.g., oil, aqueous) that does not contain
microcapsules or analyte, such as a carrier fluid. The carrier
fluid may be introduced via the inlet port before, during, or
following the introduction of analyte and/or microcapsules. In
cases where the device has multiple inlets, the same sample may be
introduced via the multiple inlets, or each inlet may convey a
different sample. In some cases, one inlet may convey a sample or
analyte to the microwells, while a different inlet conveys free
reagents and/or reagents encapsulated in microcapsules to the
device. The device may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10 inlets and/or outlets.
[0083] In some cases, solutions containing microcapsules and/or
analytes may be pulled through the device via a vacuum manifold
attached to the outlet port 210 and 260. Such manifold may apply a
negative pressure to the device. In other cases, a positive
pressure is used to move sample, analytes, and/or microcapsules
through the device. The area, length, and width of surfaces of 230
according to this disclosure may be varied according to the
requirements of the assay to be performed. Considerations may
include, for example, ease of handling, limitations of the
material(s) of which the surface is formed, requirements of
detection or processing systems, requirements of deposition systems
(e.g. microfluidic systems), and the like. The thickness may
comprise a thickness of at least about 0.001 mm, 0.005 mm, 0.01 mm,
0.05 mm, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm,
0.8 mm, 0.9 mm, 1.0 mm, 2.0 mm, 3.0 mm, 4.0 mm, 5.0 mm, 6.0 mm, 7.0
mm, 8.0 mm, 9.0 mm, 10.0 mm, 11 mm, 12 mm, 13 mm, 14 mm, or 15 mm.
In other cases, microcapsule thickness may be at most 0.001 mm,
0.005 mm, 0.01 mm, 0.05 mm, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm,
0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 2.0 mm, 3.0 mm, 4.0 mm, 5.0
mm, 6.0 mm, 7.0 mm, 8.0 mm, 9.0 mm, 10.0 mm, 11 mm, 12 mm, 13 mm,
14 mm, or 15 mm
[0084] The microwells 270 can be any shape and size suitable for
the assay performed. The cross-section of the microwells may have a
cross-sectional dimension that is circular, rectangular, square,
hexagonal, or other symmetric or non-symmetric shape. In some
cases, the shape of the microwell may be cylindrical, cubic,
conical, frustoconical, hexagonal or other symmetric or
non-symmetric shape. The diameter of the microwells 270 may be
determined by the size of the wells desired and the available
surface area of the plate itself. Exemplary microwells comprise
diameters of at least 0.01 .mu.m, 0.1 .mu.m, 0.2 .mu.m, 0.3 .mu.m,
0.4 .mu.m, 0.5 .mu.m, 1 .mu.m, 10 .mu.m, 25 .mu.m, 50 .mu.m, 75
.mu.m, 100 .mu.m, 200 .mu.m, 300 .mu.m, 400 .mu.m, 500 .mu.m, 600
.mu.m, 700 .mu.m, 800 .mu.m, 900 .mu.m, 1.0 mm. In other cases,
microwell diameters may comprise at most 0.01 .mu.m, 0.1 .mu.m, 0.2
.mu.m, 0.3 .mu.m, 0.4 .mu.m, 0.5 .mu.m, 1 .mu.m, 10 .mu.m, 25
.mu.m, 50 .mu.m, 75 .mu.m, 100 .mu.m, 200 .mu.m, 300 .mu.m, 400
.mu.m, 500 .mu.m, 600 .mu.m, 700 .mu.m, 800 .mu.m, 900 .mu.m or 1.0
mm.
[0085] The capacity (or volume) of each well can be a measure of
the height of the well (the thickness of the plate) and the
effective diameter of each well. The capacity of an individual well
may be selected from a wide range of volumes. In some cases, the
device may comprise a well (or microwell) with a capacity of at
least 0.001 fL, 0.01 fL, 0.1 fL, 0.5 fL, 1 fL, 5 fL, 10 fL, 50 fL,
100 fL, 200 fL, 300 fL, 400 fL, 500 fL, 600 fL, 700 fL, 800 fL, 900
fL, 1 pL, 5 pL, 10 pL, 50 pL, 100 pL, 200 pL, 300 pL, 400 pL, 500
pL, 600 pL, 700 pL, 800 pL, 900 pL, 1 nL, 5 nL, 10 nL, 50 nL, 100
nL, 200 nL, 300 nL, 400 nL, 500 nL, 1 uL, 50 uL, or 100 uL. In
other cases, the microcapsule comprises a microwell that is less
than 0.001 fL, 0.01 fL, 0.1 fL, 0.5 L, 5 fL, 10 fL, 50 fL, 100 fL,
200 fL, 300 fL, 400 fL, 500 fL, 600 fL, 700 fL, 800 fL, 900 fL, 1
pL, 5 pL, 10 pL, 50 pL, 100 pL, 200 pL, 300 pL, 400 pL, 500 pL, 600
pL, 700 pL, 800 pL, 900 pL, 1 nL, 5 nL, 10 nL, 50 nL, 100 nL, 200
nL, 300 nL, 400 nL, 500 nL, 1 uL, 50 uL, or 100 uL.
[0086] There may be variability in the volume of fluid in different
microwells in the array. More specifically, the volume of different
microwells may vary by at least (or at most) plus or minus 1%, 2%,
3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,
200%, 300%, 400%, 500%, or 1000% across a set of microwells. For
example, a microwell may comprise a volume of fluid that is at most
80% of the fluid volume within a second microwell.
[0087] Based on the dimension of individual microwells and the size
of the plate, the microwell array may comprise a range of well
densities. In some examples, a plurality of microwells may have a
density of at least about 2,500 wells/cm.sup.2, at least about
1,000 wells/cm.sup.2. In some cases, the plurality of wells may
have a density of at least 10 wells/cm.sup.2. In other cases, the
well density may comprise at least 10 wells/cm.sup.2, 50
wells/cm.sup.2, 100 wells/cm.sup.2, 500 wells/cm.sup.2, 1000
wells/cm.sup.2, 5000 wells/cm.sup.2, 10000 wells/cm.sup.2, 50000
wells/cm.sup.2, or 100000 wells/cm.sup.2. In other cases, the well
density may be less than 100000 wells/cm.sup.2, 10000
wells/cm.sup.2, 5000 wells/cm.sup.2, 1000 wells/cm.sup.2, 500
wells/cm.sup.2, or 100 wells/cm.sup.2.
[0088] In some cases, the interior surface of the microwells
comprises a hydrophilic material that preferably accommodates an
aqueous sample; in some cases, the region between the microwells is
composed of a hydrophobic material that may preferentially attract
a hydrophobic sealing fluid described herein.
[0089] Multiple microwell arrays, e.g., FIG. 2B may be arranged
within a single device. FIG. 3, 300. For example, discrete
microwell array slides may be arrayed in parallel on a plate
holder. In some cases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25,
50 or 100 microwell arrays are arrayed in parallel. In other cases,
at most 100, 50, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 devices are
arrayed in parallel. The microwell arrays within a common device
may be manipulated simultaneously or sequentially. For example,
arrayed devices may be loaded with samples or capsules
simultaneously or sequentially.
[0090] B. Microwell Array Fluids
[0091] The microwell array may comprise any of a number of
different fluids including aqueous, non-aqueous, oils, and organic
solvents, such as alcohols. In some cases, the fluid is used to
carry a component, e.g., reagent, microcapsule, or analyte, to a
target location such as microwells, output port, etc. In other
cases, the fluid is used to flush the system. In still other cases,
the fluid may be used to seal the microwells.
[0092] Any fluid or buffer that is physiologically compatible with
the analytes (e.g., cells, molecules) or reagents used in the
device may be used. In some cases, the fluid is aqueous (buffered
or not buffered). For example, a sample comprising a population of
cells suspended in a buffered aqueous solution may be introduced
into the microwell array, allowed to flow through the device, and
distributed to the microwells. In other cases, the fluid passing
through the device is nonaqueous (e.g., oil). Exemplary non-aqueous
fluids include but are not limited to: oils, non-polar solvent,
hydrocarbon oil, decane (e.g., tetradecane or hexadecane),
fluorocarbon oil, fluorinated oil, silicone oil, mineral oil, or
other oil.
[0093] Often, the microcapsules are suspended in a fluid that is
compatible with the components of the shell of the microcapsule.
Fluids including but not limited to water, alcohols, hydrocarbon
oils or fluorocarbon oils are particularly useful fluids for
suspending and flowing microcapsules through the microarray
device.
[0094] C. Further Partitioning and Sealing
[0095] After the analyte, free reagents, and/or microcapsules are
loaded into the device and distributed to the microwells, a sealing
fluid may be used to further partition or isolate them within the
microwells. The sealing fluid may also be used to seal the
individual wells. The sealing fluid may be introduced through the
same inlet port that was used to introduce the analyte, reagents
and/or microcapsules. But in some cases, the sealing fluid is
introduced to the device by a separate inlet port, or through
multiple separate inlet ports.
[0096] Often, the sealing fluid is a non-aqueous fluid (e.g., oil).
When the sealing fluid flows through the microwell array device, it
may displace excess aqueous solution (e.g., solution comprising
analytes, free reagents and/or microcapsules) from individual
microwells, thereby potentially removing aqueous bridges between
adjacent microwells. The wells themselves, as described herein, may
comprise a hydrophilic material that enables wicking of the aqueous
fluids (e.g., sample fluid, microcapsule fluid) into individual
wells. In some cases, regions external to the wells comprise
hydrophobic material, again to encourage the positioning of the
aqueous fluid into the interior of the microwells.
[0097] The sealing fluid may either remain in the device or be
removed. The sealing fluid may be removed, e.g., by flowing through
the outlet port. In other cases, the sealing oil may comprise a
volatile oil that can be removed by the application of heat. Once
the sealing fluid is removed, analytes, free reagents and/or
microcapsules may be physically partitioned from one another in the
microwells.
[0098] A fluid may be selected such that its density is equal to,
greater than or less than the density of the microcapsules. For
example, the microcapsules may be denser than the sealing oil
and/or aqueous fluid of the sample and reagents, thereby enabling
the microcapsules to remain in the microwells as the sealing oil
flows through the device. In another example, the capsules may be
less dense than the aqueous fluid of the sample or the fluid that
the microcapsules are suspended in, as described herein, thereby
facilitating movement and distribution of the capsules across the
plurality of microwells in a device.
[0099] In the case of microcapsules comprising paramagnetic
material, a magnetic field may be used to load or direct the
capsules into the microwells. A magnetic field may also be used to
retain such microcapsules within the wells while the wells are
being filled with sample, reagent, and/or sealing fluids. The
magnetic field may also be used to remove capsule shells from the
wells, particularly following rupture of the capsules.
[0100] In some cases, the sealing fluid may remain in the
microwells when operations or reactions are conducted therein. The
presence of the sealing fluid may act to further partition,
isolate, or seal the individual microwells. In other cases, the
sealing fluid may act as a carrier for the microcapsules. For
example, sealing fluid comprising microcapsules may be introduced
to the device to facilitate distribution of the microcapsules to
the individual microwells. For such applications, the sealing fluid
may be denser than the microcapsules in order to encourage more
even distribution of the microcapsules to the microwells. Upon
application of a stimulus, the microcapsules within the sealing
fluid may release reagents to the microwell. In some cases, the
sealing fluid may comprise a chemical or other agent capable of
traveling from the sealing fluid to a well (e.g., by leaching or
other mechanism) and triggering capsule rupture, where the capsule
is present within the microwell or within the sealing fluid.
[0101] Methods other than those involving sealing fluids may also
be used to seal the microwells following the loading of the
analyte, free reagents, and/or microcapsules. For example, the
microwells may be sealed with a laminate, tape, plastic cover,
oils, waxes, or other suitable material to create an enclosed
reaction chamber. The sealants described herein may protect the
contents of the microwells from evaporation or other unintended
consequences of the reactions or operations. Prevention of
evaporation may be particularly necessary when heat is applied to
the device, e.g., when heat is applied to stimulate microcapsule
release.
[0102] In some cases, the laminate seal may also allow recovery of
contents from individual wells. In this case, a single well of
interest may be unsealed (e.g., by removal of the laminate seal) at
a given time in order to enable further analysis of an analyte such
as by MALDI mass spectrometry. Such applications may be useful in a
number of settings, including high-throughput drug screening.
III. LOADING STEP(S)
[0103] As described herein, analytes, free reagents, and/or
microcapsules may be loaded into the present device in any
appropriate manner or order. The loading may be random or
non-random. In some cases, a precise number of analytes and/or
microcapsules are loaded into each individual microwell. In some
cases, a precise number of analytes and/or microcapsules are loaded
into a particular subset of microwells in the plate. In still other
cases, an average number of analytes and/or micrcocapsules are
loaded into each individual microwell. Furthermore, as described
herein, in some cases, "dry" microcapsules are loaded into the
device, while in other cases "wet" microcapsules are loaded into
the device. In some cases, a combination of "dry" and "wet"
microcapsules and/or reagents are loaded into the device, either
simultaneously or sequentially.
[0104] As mentioned herein, the loading of the device may occur in
any order and may occur in multiple stages. In some cases, the
microcapsules are pre-loaded into the device, prior to the loading
of the analyte. In other cases, the microcapsules and analyte are
loaded concurrently. In still other cases, the analytes are loaded
before the microcapsules are loaded.
[0105] The microcapsules and/or analytes may be loaded in multiple
stages or multiple times. For example, microcapsules may be loaded
into the device both prior to and after analytes are loaded into
the device. The microcapsules that are pre-loaded (e.g., loaded
prior to the analyte introduction) may comprise the same reagents
as the microcapsules loaded after the analyte introduction. In
other cases, the pre-loaded microcapsules contain reagents that are
different from the reagents within the microcapsules loaded after
analyte introduction. In some cases, at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 15, or 20 different sets of microcapsules are loaded onto
the device. In some cases, the different sets of microcapsules are
loaded sequentially; or, different sets of microcapsules may also
be loaded simultaneously. Similarly, multiple sets of analytes can
be loaded into the device. In some cases, at least 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 15, or 20 different sets of analytes are loaded
onto the device. In some cases, the different sets of analytes are
loaded sequentially; or, different sets of analytes may also be
loaded simultaneously.
[0106] This disclosure provides devices comprising certain numbers
of microcapsules and/or analytes loaded per well. In some cases, at
most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, or 100
microcapsules and/or analytes are loaded into each individual
microwell. In some cases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 30, 40, 50, 75, or 100 microcapsules and/or analytes are
loaded into each individual microwell. In some cases, on average,
at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, or
100 microcapsules and/or analytes are loaded into each individual
microwell. In other cases, on average, at least 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 15, 20, 30, 40, 50, 75, or 100 microcapsules and/or
analytes are loaded into each individual microwell. In some cases,
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, or 100
microcapsules and/or analytes are loaded into each individual
microwell.
[0107] Analytes and/or microcapsules may be applied in a quantity
that allows a desired number of analytes to be deposited into an
individual microwell. For example, terminal dilution of analytes,
such as cells, may achieve the loading of one cell per one
microwell or any desired number of analytes per microwell. In some
cases, a Poisson distribution is used to direct or predict the
final concentration of analytes or microcapsules per well.
[0108] The microcapsules may be loaded into the microarray device
in a particular pattern. For example, certain sections of the
device may comprise microcapsules containing a particular reagent
(e.g., unique bar-code, enzyme, antibody, antibody subclass, etc.),
while other sections of the device may comprise microcapsules
containing a different reagent (e.g., a different bar-code,
different enzyme, different antibody different antibody subclass,
etc.). In some cases, the microcapsules in one section of the array
may contain control reagents. For example, they may contain
positive controls that include a control analyte and necessary
materials for a reaction. Or, in some cases, the microcapsules
contain negative control reagents such as deactivated enzyme, or a
synthetic oligonucleotide sequence that is resistant to
fragmentation. In some cases, negative control reagents may control
for the specificity of the sample preparation reaction etc. In
other cases, the negative control microcapsules may comprise the
same reagents present in other microcapsules except that the
negative control microcapsule may lack a certain reagent (e.g.,
lysis buffer, polymerase, etc.).
[0109] The analytes/sample also may be loaded into the microarray
device in a particular pattern. For example, certain sections of
the device may comprise particular analytes, such as control
analytes or analytes deriving from a particular source. This may be
used in combination with specific loading of bar codes into known
well locations. This feature may allow mapping of specific
locations on the array to sequence data, thereby reducing the
number of bar codes to be used for labeling reactions.
[0110] In cases where a partition is a droplet, an analyte and
reagents may be combined within the droplet with the aid of a
microfluidic device. For example, a droplet may be generated that
comprises a gel bead (e.g., comprising an oligonucleotide barcode)
a nucleic acid analyte, and any other desired reagents. The gel
bead, nucleic acid analyte, and reagents in an aqueous phase may be
combined at a junction of two or more channels of a microfluidic
device. At a second junction of two or more channels of the
microfluidic device, a droplet comprising the resulting mixture may
be generated by contacting the aqueous mixture of reagents, gel
bead, and nucleic acid analyte with an oil continuous phase.
IV. MICROCAPSULE STIMULI
[0111] Various different stimuli may be used to trigger release of
reagents from the microcapsules, or from internal compartments
therein. In some cases, a microcapsule is degradable. Generally,
the trigger may cause disruption or degradation of the shell or
membrane enveloping the microcapsule, disruption or degradation of
the interior of a microcapsule, and/or disruption or degradation of
any chemical bonds that immobilize a reagent to the microcapsule.
Exemplary triggers include but are not limited to: chemical
triggers, bulk changes, biological triggers, light triggers,
thermal triggers, magnetic triggers, and any combination thereof.
See, e.g., Esser-Kahn et al., (2011) Macromolecules 44: 5539-5553;
Wang et al., (2009) ChemPhysChem 10:2405-2409;
[0112] A. Chemical Stimuli and Bulk Changes
[0113] Numerous chemical triggers may be used to trigger the
disruption or degradation of the microcapsules. Examples of these
chemical changes may include, but are not limited to pH-mediated
changes to the shell wall, disintegration of the shell wall via
chemical cleavage of crosslink bonds, triggered depolymerization of
the shell wall, and shell wall switching reactions. Bulk changes
may also be used to trigger disruption of the microcapsules.
[0114] A change in pH of the solution, particularly a decrease in
pH, may trigger disruption via a number of different mechanisms.
The addition of acid may cause degradation or disassembly of the
shell wall through a variety of mechanisms. Addition of protons may
disassemble cross-linking of polymers in the shell wall, disrupt
ionic or hydrogen bonds in the shell wall, or create nanopores in
the shell wall to allow the inner contents to leak through to the
exterior. In some examples, the microcapsule comprises
acid-degradable chemical cross-linkers such a ketals. A decrease in
pH, particular to a pH lower than 5, may induce the ketal to
convert to a ketone and two alcohols and facilitate disruption of
the microcapsule. In other examples, the microcapsules may comprise
one or more polyelectrolytes (e.g., PAA, PAAm, PSS, etc.) that are
pH sensitive. A decrease in pH may disrupt the ionic- or
hydrogen-bonding interactions of such microcapsules, or create
nanopores therein. In some cases, microcapsules comprising
polyelectrolytes comprise a charged, gel-based core that expands
and contracts upon a change of pH.
[0115] Removal of cross-linkers (e.g., disulfide bonds) within the
microcapsules can also be accomplished through a number of
mechanisms. In some examples, various chemicals can be added to a
solution of microcapsules that induce either oxidation, reduction
or other chemical changes to polymer components of the shell wall.
In some cases, a reducing agent, such as beta-mercaptoethanol,
dithiotheritol (DTT), or 2-tris(2-carboxyethyl)phosphine (TCEP), is
added such that disulfide bonds in a microcapsule shell wall are
disrupted. In addition, enzymes may be added to cleave peptide
bonds within the microcapsules, thereby resulting in cleavage of
shell wall cross linkers.
[0116] Depolymerization can also be used to disrupt the
microcapsules. A chemical trigger may be added to facilitate the
removal of a protecting head group. For example, the trigger may
cause removal of a head group of a carbonate ester or carbamate
within a polymer, which in turn causes depolymerization and release
of reagents from the inside of the capsule.
[0117] Shell wall switching reactions may be due to any structural
change to the porosity of the shell wall. The porosity of a shell
wall may be modified, for example, by the addition of azo dyes or
viologen derivatives. Addition of energy (e.g., electricity, light)
may also be used to stimulate a change in porosity.
[0118] In yet another example, a chemical trigger may comprise an
osmotic trigger, whereby a change in ion or solute concentration of
microcapsule solution induces swelling of the capsule. Swelling may
cause a buildup of internal pressure such that the capsule ruptures
to release its contents.
[0119] It is also known in the art that bulk or physical changes to
the microcapsule through various stimuli also offer many advantages
in designing capsules to release reagents. Bulk or physical changes
occur on a macroscopic scale, in which capsule rupture is the
result of mechano-physical forces induced by a stimulus. These
processes may include, but are not limited to pressure induced
rupture, shell wall melting, or changes in the porosity of the
shell wall.
[0120] B. Biological Stimuli
[0121] Biological stimuli may also be used to trigger disruption or
degradation of microcapsules. Generally, biological triggers
resemble chemical triggers, but many examples use biomolecules, or
molecules commonly found in living systems such as enzymes,
peptides, saccharides, fatty acids, nucleic acids and the like. For
example, microcapsules may comprise polymers with peptide
cross-links that are sensitive to cleavage by specific proteases.
More specifically, one example may comprise a microcapsule
comprising GFLGK peptide cross links. Upon addition of a biological
trigger such as the protease Cathepsin B, the peptide cross links
of the shell well are cleaved and the contents of the capsule are
released. In other cases, the proteases may be heat-activated. In
another example, microcapsules comprise a shell wall comprising
cellulose. Addition of the hydrolytic enzyme chitosan serves as
biologic trigger for cleavage of cellulosic bonds, depolymerization
of the shell wall, and release of its inner contents.
[0122] C. Thermal Stimuli
[0123] The microcapsules may also be induced to release their
contents upon the application of a thermal stimulus. A change in
temperature can cause a variety changes to the microcapsule. A
change in heat may cause melting of a microcapsule such that the
shell wall disintegrates. In other cases, the heat may increase the
internal pressure of the inner components of the capsule such that
the capsule ruptures or explodes. In still other cases, the heat
may transform the capsule into a shrunken dehydrated state. The
heat may also act upon heat-sensitive polymers within the shell of
a microcapsule to cause disruption of the microcapsule.
[0124] In one example, a microcapsule comprises a thermo-sensitive
hydrogel shell encapsulating one or more emulsified reagent
particles. Upon the application of heat, such as above 35 C, the
hydrogel material of the outer shell wall shrinks. The sudden
shrinkage of the shell ruptures the capsule and allows the reagents
of the inside of the capsule to squirt out in the sample
preparation solution in the microwell.
[0125] In some cases, the shell wall may comprise a diblock
polymer, or a mixture of two polymers, with different heat
sensitivities. One polymer may be particularly likely to shrink
after the application of heat, while the other is more heat-stable.
When heat is applied to such shell wall, the heat-sensitive polymer
may shrink, while the other remains intact, causing a pore to form.
In still other cases, a shell wall may comprise magnetic
nanoparticles. Exposure to a magnetic field may cause the
generation of heat, leading to rupture of the microcapsule.
[0126] D. Magnetic Stimuli
[0127] Inclusion of magnetic nanoparticles to the shell wall of
microcapsules may allow triggered rupture of the capsules as well
as guide the particles in an array. A device of this disclosure may
comprise magnetic particles for either purpose. In one example,
incorporation of Fe3O4 nanoparticles into polyelectrolyte
containing capsules triggers rupture in the presence of an
oscillating magnetic field stimulus.
[0128] E. Electrical and Light Stimuli
[0129] A microcapsule may also be disrupted or degraded as the
result of electrical stimulation. Similar to magnetic particles
described in the previous section, electrically sensitive particles
can allow for both triggered rupture of the capsules as well as
other functions such as alignment in an electric field, electrical
conductivity or redox reactions. In one example, microcapsules
containing electrically sensitive material are aligned in an
electric field such that release of inner reagents can be
controlled. In other examples, electrical fields may induce redox
reactions within the shell wall itself that may increase
porosity.
[0130] A light stimulus may also be used to disrupt the
microcapsules. Numerous light triggers are possible and may include
systems that use various molecules such as nanoparticles and
chromophores capable of absorbing photons of specific ranges of
wavelengths. For example, metal oxide coatings can be used as
capsule triggers. UV irradiation of polyelectrolyte capsules coated
with SiO2/TiO2 may result in disintegration of the capsule wall. In
yet another example, photo switchable materials such as azobenzene
groups may be incorporated in the shell wall. Upon the application
of UV or visible light, chemicals such as these undergo a
reversible cis-to-trans isomerization upon absorption of photons.
In this aspect, incorporation of photo switches result in a shell
wall that may disintegrate or become more porous upon the
application of a light trigger.
[0131] F. Application of Stimuli
[0132] A device of this disclosure may be used in combination with
any apparatus or device that provides such trigger or stimulus. For
example, if the stimulus is thermal, a device may be used in
combination with a heated or thermally controlled plate, which
allows heating of the microwells and may induce the rupture of
capsules. Any of a number of heat transfers may be used for thermal
stimuli, including but not limited to applying heat by radiative
heat transfer, convective heat transfer, or conductive heat
transfer. In other cases, if the stimulus is a biological enzyme,
the enzyme may be injected into a device such that it is deposited
into each microwell. In another aspect, if the stimulus is a
magnetic or electric field, a device may be used in combination
with a magnetic or electric plate.
[0133] A chemical stimulus may be added to a partition and may
exert its function at various times after contacting a chemical
stimulus with a microcapsule. The speed at which a chemical
stimulus exerts its effect may vary depending on, for example, the
amount/concentration of a chemical stimulus contacted with a
microcapsule and/or the particular chemical stimulus used. For
example, a droplet may comprise a degradable gel bead (e.g., a gel
bead comprising chemical cross-linkers, such as, for example,
disulfide bonds). Upon droplet formation, a chemical stimulus
(e.g., a reducing agent) may be included in the droplet with the
gel bead. The chemical stimulus may degrade the gel bead
immediately on contact with the gel bead, soon after (e.g., about
0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 min) contact with the gel
bead, or at a later time. In some cases, degradation of the gel
bead may occur before, during, or after a further processing step,
such as, for example, a thermal cycling step as described
herein.
V. SAMPLE PREPARATION, REACTION AND RECOVERY
[0134] After application of the stimulus, rupturing of capsules and
release of the reagents, the sample preparation reaction may
proceed in a device. Reactions within a device may be incubated for
various periods of times depending on the reagents used in the
sample reactions. A device may also be used in combination with
other devices that aid in the sample preparation reaction. For
example, if PCR amplification is desired, a device may be used in
combination with a PCR thermocycler. In some cases, a thermocycler
may comprise a plurality of wells. In cases where partitions are
droplets, the droplets may be entered into the wells of the
thermocycler. In some cases, each well may comprise multiple
droplets, such that when thermal cycling is initiated, multiple
droplets are thermal cycled in each well. In another example, if
the reaction requires agitation, a device may be used in
combination with a shaking apparatus.
[0135] Following the completion of the sample preparation reaction,
the analytes and products of the sample reactions may be recovered.
In some cases, a device may utilize a method comprising the
application of liquid or gas to flush out the contents of the
individual microwells. In one example, the liquid comprises an
immiscible carrier fluid that preferentially wets the microwell
array material. It may also be immiscible with water so as to flush
the reaction products out of the well. In another example, the
liquid may be an aqueous fluid that can be used to flush out the
samples out of the wells. After flushing of the contents of the
microwells, the contents of the microwells are pooled for a variety
of downstream analyses and applications.
VI. APPLICATIONS
[0136] FIG. 4A provides a general flow of many of the methods of
the present disclosure; and FIG. 4B provides a generally annotated
version of 4A. One or more microcapsule(s) that contain reagents
410 may be pre-loaded into microwells, followed by addition of an
analyte, which, in this particular Figure, is a nucleic acid
analyte 420. The microwells may then be sealed 430 by any method,
such as by application of a sealing fluid. The inlet and outlet
ports may also be sealed, for example to prevent evaporation.
Following these steps, a stimulus (e.g., heat, chemical,
biological, etc.) may be applied to the microwells in order to
disrupt the microcapsules 460 and trigger release of the reagents
450 to the interior of the microwell. Subsequently, an incubation
step 440 may occur in order to enable the reagents perform a
particular function such as lysis of cells, digestion of protein,
fragmentation of high molecular weight nucleic acids, or ligation
of oligonucleotide bar codes. Following the incubation step (which
is optional), the contents of the microwells may be recovered
either singly or in bulk.
[0137] A. Analytes
[0138] A device of this disclosure may have a wide variety of uses
in the manipulation, preparation, identification and/or
quantification of analytes. In some cases, the analyte is a cell or
population of cells. The population of cells may be homogeneous
(e.g., from a cell line, of the same cell type, from the same type
of tissue, from the same organ, etc.) or heterogenous (mixture of
different types of cells). The cells may be primary cells, cell
lines, recombinant cells, primary cells, encapsulated cells, free
cells, etc.
[0139] The analytes may also be molecules, including but not
limited to: polypeptides, proteins, antibodies, enzymes, nucleic
acids, saccharides, small molecules, drugs, and the like. Examples
of nucleic acids include but are not limited to: 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 (e.g., retroviral RNA).
[0140] In some cases, the analytes are pre-mixed with one or more
additional materials, such as one or more reagents (e.g., ligase,
protease, polymerase) prior to being loaded into the device. In
some cases, the analytes are pre-mixed with microcapsules
comprising one or more reagents prior to being loaded onto the
device.
[0141] The samples may be derived from a variety of sources
including human, mammal, non-human mammal, ape, monkey, chimpanzee,
plant, reptilian, amphibian, avian, fungal, viral or bacterial
sources. Samples such as cells, nucleic acids and proteins may also
be obtained from a variety of clinical sources such as biopsies,
aspirates, blood draws, urine samples, formalin fixed embedded
tissues and the like.
[0142] A device of this disclosure may also enable the analytes to
be tagged or tracked in order to permit subsequent identification
of an origin of the analytes. This feature is in contrast with
other methods that use pooled or multiplex reactions and that only
provide measurements or analyses as an average of multiple samples.
Here, the physical partitioning and assignment of a unique
identifier to individual analytes allows acquisition of data from
individual samples and is not limited to averages of samples.
[0143] In some examples, nucleic acids or other molecules derived
from a single cell may share a common tag or identifier and
therefore may be later identified as being derived from that cell.
Similarly, all of the fragments from a single strand of nucleic
acid may be tagged with the same identifier or tag, thereby
permitting subsequent identification of fragments with similar
phasing or linkage on the same strand. In other cases, gene
expression products (e.g., mRNA, protein) from an individual cell
may be tagged in order to quantify expression. In still other
cases, the device can be used as a PCR amplification control. In
such cases, multiple amplification products from a PCR reaction can
be tagged with the same tag or identifier. If the products are
later sequenced and demonstrate sequence differences, differences
among products with the same identifier can then be attributed to
PCR error.
[0144] The analytes may be loaded onto the device before, after, or
during loading of the microcapsules and/or free reagents. In some
cases, the analytes are encapsulated into microcapsules before
loading into the microcapsule array. For example, nucleic acid
analytes may be encapsulated into a microcapsule, which is then
loaded onto the device and later triggered to release the analytes
into an appropriate microwell.
[0145] Any analytes, such as DNA or cells, may be loaded in
solution or as analytes encapsulated in a capsule. In some cases,
homogeneous or heterogeneous populations of molecules (e.g.,
nucleic acids, proteins, etc.) are encapsulated into microcapsules
and loaded onto the device. In some cases, homogeneous or
heterogeneous populations of cells are encapsulated into
microcapsules and loaded onto the device. The microcapsules may
comprise a random or specified number of cells and/or molecules.
For example, the microcapsules may comprise no more than 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500,
1000, 5000, or 10000 cells and/or molecules per microcapsule. In
other examples, the microcapsules comprise at least 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000,
5000, or 10000 cells and/or molecules per microcapsule. Fluidic
techniques and any other techniques may be used to encapsulate the
cells and/or molecules into the microcapsules.
[0146] Generally, the methods and compositions provided herein are
useful for preparation of an analyte prior to a down-stream
application such as a sequencing reaction. Often, a sequencing
method is classic Sanger sequencing. Sequencing methods may
include, but are not limited to: high-throughput sequencing,
pyrosequencing, sequencing-by-synthesis, single-molecule
sequencing, nanopore sequencing, sequencing-by-ligation,
sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene
Expression (Helicos), Next generation sequencing, Single Molecule
Sequencing by Synthesis (SMSS)(Helicos), massively-parallel
sequencing, Clonal Single Molecule Array (Solexa), shotgun
sequencing, Maxim-Gilbert sequencing, primer walking, and any other
sequencing methods known in the art.
[0147] There are numerous examples of applications that may be
conducted instead of, or in conjunction with, a sequencing
reaction, including but not limited to: biochemical analyses,
proteomics, immunoassays, profiling/fingerprinting of specific cell
types, pharmaceutical screening, bait-capture experiments,
protein-protein interaction screens and the like.
[0148] B. Assignment of Unique Identifiers to Analytes
[0149] The devices disclosed herein may be used in applications
that involve the assignment of unique identifiers, or molecular bar
codes, to analytes. Often, the unique identifier is a bar-code
oligonucleotide that is used to tag the analytes; but, in some
cases, different unique identifiers are used. For example, in some
cases, the unique identifier is an antibody, in which case the
attachment may comprise a binding reaction between the antibody and
the analyte (e.g., antibody and cell, antibody and protein,
antibody and nucleic acid). In other cases, the unique identifier
is a dye, in which case the attachment may comprise intercalation
of the dye into the analyte molecule (such as intercalation into
DNA or RNA) or binding to a probe labeled with the dye. In still
other cases, the unique identifier may be a nucleic acid probe, in
which case the attachment to the analyte may comprise a
hybridization reaction between the nucleic acid and the analyte. In
some cases, the reaction may comprise a chemical linkage between
the identifier and the analyte. In other cases, the reaction may
comprise addition of a metal isotope, either directly to the
analyte or by a probe labeled with the isotope.
[0150] Often, the method comprises attaching oligonucleotide bar
codes to nucleic acid analytes through an enzymatic reaction such
as a ligation reaction. For example, the ligase enzyme may
covalently attach a DNA bar code to fragmented DNA (e.g., high
molecular-weight DNA). Following the attachment of the bar-codes,
the molecules may be subjected to a sequencing reaction. However,
other reactions may be used as well. For example, oligonucleotide
primers containing bar code sequences may be used in amplification
reactions (e.g., PCR, qPCR, reverse-transcriptase PCR, digital PCR,
etc.) of the DNA template analytes, thereby producing tagged
analytes. After assignment of bar codes to individual analytes, the
contents of individual microwells may be recovered via the outlet
port in the device for further analyses.
[0151] The unique identifiers (e.g., oligonucleotide bar-codes,
antibodies, probes, etc.) may be introduced to the device randomly
or nonrandomly. In some cases, they are introduced at an expected
ratio of unique identifiers to microwells. For example, the unique
identifiers may be loaded so that more than about 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, or 200000 unique
identifiers are loaded per microwell. In some cases, the unique
identifiers may be loaded so that less than about 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, or 200000 unique
identifiers are loaded per microwell. In some cases, the average
number of unique identifiers loaded per microwell is less than, or
greater than, about 0.0001, 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, or 200000 unique
identifiers per microwell.
[0152] The unique identifiers also may be loaded so that a set of
one or more identical identifiers are introduced to a particular
well. Such sets may also be loaded so that each microwell contains
a different set of identifiers. For example, a population of
microcapsules may be prepared such that a first microcapsule in the
population comprises multiple copies of identical unique
identifiers (e.g., nucleic acid bar codes, etc.) and a second
microcapsule in the population comprises multiple copies of a
unique identifier that differs from within the first microcapsule.
In some cases, the population of microcapsules may comprise
multiple microcapsules (e.g., greater than 2, 3, 4, 5, 6, 7, 8, 9,
10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 500, 1000, 5000, 10000,
100000, 1000000, 10000000, 100000000, or 1000000000 microcapsules),
each containing multiple copies of a unique identifier that differs
from that contained in the other microcapsules. In some cases, the
population may comprise greater than 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25, 30, 35, 40, 45, 50, 100, 500, 1000, 5000, 10000,
100000, 1000000, 10000000, 100000000, or 1000000000 microcapsules
with identical sets of unique identifiers. In some cases, the
population may comprise greater than 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25, 30, 35, 40, 45, 50, 100, 500, 1000, 5000, 10000,
100000, 1000000, 10000000, 100000000, or 1000000000 microcapsules,
wherein the microcapsules each comprise a different combination of
unique identifiers. For example, in some cases the different
combinations overlap, such that a first microcapsule may comprise,
e.g., unique identifiers A, B, and C, while a second microcapsule
may comprise unique identifiers A, B, and D. In another example,
the different combinations do not overlap, such that a first
microcapsule may comprise, e.g., unique identifiers A, B, and C,
while a second microcapsule may comprise unique identifiers D, E,
and F.
[0153] The unique identifiers may be loaded into the device at an
expected or predicted ratio of unique identifiers per analyte
(e.g., strand of nucleic acid, fragment of nucleic acid, protein,
cell, etc.) In some cases, the unique identifiers are loaded in the
microwells so that more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 50, 100, 500, 1000, 5000, 10000, or 200000 unique identifiers
are loaded per individual analyte in the microwell. In some cases,
the unique identifiers are loaded in the microwells so that less
than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000,
5000, 10000, or 200000 unique identifiers are loaded per individual
analyte in the microwell. In some cases, the average number of
unique identifiers loaded per analyte is less than, or greater
than, about 0.0001, 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 20, 50, 100, 500, 1000, 5000, 10000, or 200000 unique
identifiers per analyte. When more than one identifier is present
per analyte, such identifiers may be copies of the same identifier,
or multiple different identifiers. For example, the attachment
process may be designed to attach multiple identical identifiers to
a single analyte, or multiple different identifiers to the
analyte.
[0154] The unique identifiers may be used to tag a wide range of
analytes, including cells or molecules. For example, unique
identifiers (e.g., bar code oligonucleotides) may be attached to
whole strands of nucleic acids or to fragments of nucleic acids
(e.g., fragmented genomic DNA, fragmented RNA). The unique
identifiers (e.g., antibodies, oligonucleotides) may also bind to
cells, include the external surface of a cell, a marker expressed
on the cell or components within the cell such as organelles, gene
expression products, genomic DNA, mitochondrial DNA, RNA, mRNA, or
proteins. The unique identifiers also may be designed to bind or
hybridize nucleic acids (e.g., DNA, RNA) present in permeabilized
cells, which may or may not be otherwise intact.
[0155] The unique identifiers may be loaded onto the device either
singly or in combination with other elements (e.g., reagents,
analytes). In some cases, free unique identifiers are pooled with
the analytes and the mixture is loaded into the device. In some
cases, unique identifiers encapsulated in microcapsules are pooled
with the analytes, prior to loading of the mixture onto the device.
In still other cases, free unique identifiers are loaded into the
microwells prior to, during (e.g., by separate inlet port), or
following the loading of the analytes. In still other cases, unique
identifiers encapsulated in microcapsules are loaded into the
microwells prior to, concurrently with (e.g., by separate inlet
port), or after loading of the analytes.
[0156] In many applications, it may be important to determine
whether individual analytes each receive a different unique
identifier (e.g., oligonucleotide bar code). If the population of
unique identifiers introduced into the device is not significantly
diverse, different analytes may possibly be tagged with identical
identifiers. The devices disclosed herein may enable detection of
analytes tagged with the same identifier. In some cases, a
reference analyte may be included with the population of analytes
introduced into the device. The reference analyte may be, for
example, a nucleic acid with a known sequence and a known quantity.
After the population of analytes is loaded and partitioned in the
device, unique identifiers may be attached to the analytes, as
described herein. If the unique identifiers are oligonucleotide bar
codes and the analytes are nucleic acids, the tagged analytes may
subsequently be sequenced and quantified. These methods may
indicate if one or more fragments and/or analytes may have been
assigned an identical bar code.
[0157] A method disclosed herein may comprise loading the device
with the reagents necessary for the assignment of bar codes to the
analytes. In the case of ligation reactions, reagents including,
but not limited to, ligase enzyme, buffer, adapter
oligonucleotides, a plurality of unique identifier DNA bar codes
and the like may be loaded into the device. In the case of
enrichment, reagents including but not limited to a plurality of
PCR primers, oligonucleotides containing unique identifying
sequence, or bar code sequence, DNA polymerase, DNTPs, and buffer
and the like may be loaded into the device. The reagents may be
loaded as free reagents or as reagents encapsulated in
microcapsules.
[0158] C. Nucleic Acid Sequencing
[0159] Nucleic acid sequencing may begin with the physical
partitioning of sample analytes into microwells at a particular
density (e.g., about 1 analyte per microwell or other density
described herein). When nucleic acid bar codes are assigned to
individual analytes, it may then be possible to track individual
molecules during subsequent steps such as subsequent amplification
and/or sequencing steps, even if the analytes are later pooled
together and treated en masse.
[0160] a. Nucleic Acid Phasing
[0161] The devices provided herein may be used to prepare analytes
(e.g., nucleic acid analytes) in such a manner that enables phasing
or linkage information to be subsequently obtained. Such
information may allow for the detection of linked genetic
variations in sequences, including genetic variations (e.g., SNPs,
mutations, indels, copy number variations, transversions,
translocations, inversions, etc.) that are separated by long
stretches of nucleic acids. These variations may exist in either a
cis or trans relationship. In cis relationships, two or more
genetic variations may exist in the same polynucleic acid molecule
or strand. In trans relationships, two or more genetic variations
may exist on multiple nucleic acid molecules or strands.
[0162] A method of determining nucleic acid phasing may comprise
loading a nucleic acid sample (e.g., a nucleic acid sample that
spans a given locus or loci) into a device disclosed herein,
distributing the sample such that at most one molecule of nucleic
acid is present per microwell, and fragmenting the sample within
the microwells. The method may further comprise attaching unique
identifiers (e.g., bar codes) to the fragmented nucleic acids as
described herein, recovering the nucleic acids in bulk, and
performing a subsequent sequencing reaction on the samples in order
to detect genetic variations, such as two different genetic
variations. The detection of genetic variations tagged with two
different bar codes may indicate that the two genetic variations
are derived from two separate strands of DNA, reflecting a trans
relationship. Conversely, the detection of two different genetic
variations tagged with the same bar codes may indicate that the two
genetic variations are from the same strand of DNA, reflecting a
cis relationship.
[0163] Phase information may be important for the characterization
of the analyte, particularly if the analyte derives from a subject
at risk of, having, or suspected of a having a particular disease
or disorder (e.g., hereditary recessive disease such as Cystic
Fibrosis, cancer, etc.). The information may be able to distinguish
between the following possibilities: (1) two genetic variations
within the same gene on the same strand of DNA and (2) two genetic
variations within the same gene but located on separate strands of
DNA. Possibility (1) may indicate that one copy of the gene is
normal and the individual is free of the disease, while possibility
(2) may indicate that the individual has or will develop the
disease, particularly if the two genetic variations are damaging to
the function of the gene when present within the same gene copy.
Similarly, the phasing information may also be able to distinguish
between the following possibilities: (1) two genetic variations,
each within a different gene on the same strand of DNA and (2) two
genetic variations, each within a different gene but located on
separate strands of DNA.
[0164] b. Cell-Specific Information
[0165] The devices provided herein may be used to prepare cellular
analytes in such a manner that enables cell-specific information to
be subsequently obtained. Such information may enable detection of
genetic variations (e.g., SNPs, mutations, indels, copy number
variations, transversions, translocations, inversions, etc.) on a
cell-by-cell basis, thereby enabling a determination of whether the
genetic variation(s) are present in the same cell or two different
cells.
[0166] A method of determining nucleic acid cell-specific
information may comprise loading a cellular sample (e.g., a
cellular sample from a subject) into a device disclosed herein,
distributing the sample such that at most one cell is present per
microwell, lysing the cells, and then tagging the nucleic acids
within the cells with unique identifiers using a method described
herein. In some cases, microcapsules comprising unique identifiers
are loaded in the microwell array device (either before, during, or
after the loading of the cellular analytes) in such a manner that
each cell is contacted with a different microcapsule. The resulting
tagged nucleic acids can then be pooled, sequenced, and used to
trace the origin of the nucleic acids. Nucleic acids with identical
unique identifiers may be determined to originate from the same
cell, while nucleic acids with different unique identifiers may be
determined to originate from different cells.
[0167] In a more specific example, the methods herein may be used
to detect the distribution of oncogenic mutations across a
population of cancer tumor cells. In this example, some of the
cells may have a mutation, or amplification, of an oncogene (e.g.,
HER2, BRAF, EGFR, KRAS) on two strands of DNA (homozygous), while
others may be heterozygous for the mutation, while still other
cells may be wild-type and comprise no mutations or other variation
in the oncogene. The methods described herein may be able to detect
these differences, and also may enable quantification of the
relative numbers of homozygous, heterozygous, and wild-type cells.
Such information may be used to stage a particular cancer or to
monitor the progression of the cancer over time.
[0168] In some examples, this disclosure provides methods of
identifying mutations in two different oncogenes (e.g., KRAS and
EGFR). If the same cell comprises genes with both mutations, this
may indicate a more aggressive form of cancer. In contrast, if the
mutations are located in two different cells, this may indicate
that the cancer is more benign, or less advanced.
[0169] The following is another specific example of cell-specific
sequence determination. In this example, a plurality of cells, such
as from a tumor biopsy, is loaded into a device. Single cells from
the sample are deposited into individual wells and labeled with a
DNA bar code.
[0170] Loading of cells into a device may be achieved through
non-random loading. Parameters for non-random loading of analytes,
such as cells, may be understood using an interference function
such that:
" fraction multi - occupancy " = 1 - [ ( 1 - 1 N ) + p N ] C
##EQU00001##
where
[0171] P=probability that a particular cell will attempt but not
fit in the well (measure of interference)
[0172] N=number of wells
[0173] L=number of labels=barcodes
[0174] C=number of cells
[0175] As part of sample preparation reactions, cells may be lysed
and many subsequent reactions are possible, including RNA
amplification, DNA amplification or antibody screening for
different target proteins and genes in individual cells. After the
reaction, the contents of the cells may be pooled together and
could be further analyzed, such as by DNA sequencing. With each
cell assigned a unique barcode, further analyses may be possible
including but not limited to quantification of different gene
levels or nucleic acid sequencing of individual cells. In this
example, it may be determined whether the tumor comprises cells
with different genetic backgrounds (e.g., cancer clones and
subclones). The relative number of each type of cell may also be
calculated.
[0176] c. Amplification Control
[0177] As disclosed herein, the device can be used for purposes of
controlling for amplification errors, such as PCR errors. For
example, a nucleic acid sample may be partitioned into the
microwells of the device. Following partitioning, the sample may be
subjected to a PCR amplification reaction within the microwells.
The PCR products within a microwell may be tagged with the same
unique identifier, using a method described herein. If the products
are later sequenced and demonstrate sequence differences,
differences among products with the same identifier can then be
attributed to PCR error.
[0178] d. Gene-Expression Products Analysis
[0179] In other applications, a device may be used to detect gene
product (e.g., protein, mRNA) expression levels in a sample, often
on a cell-by-cell basis. A sample may comprise individual cells, a
pool of mRNA extract from cells, or other collection of gene
products. In some instances, single cells may be loaded into
microwells. In other instances, a pool of mRNA or other gene
product may be loaded such that a desired quantity of mRNA
molecules is loaded into individual microwells.
[0180] The methods provided herein may be particularly useful for
RNA analysis. For example, using the methods provided herein,
unique identifiers may be assigned to mRNA analytes either directly
or to cDNA products of a reverse transcription reaction performed
on the mRNA analytes. The reverse transcription reaction may be
conducted within the microwells of the device following loading of
the analytes. Reagents for the reaction may include but are not
limited to reverse transcriptase, DNA polymerase enzyme, buffer,
dNTPs, oligonucleotide primers, oligonucleotide primers containing
bar code sequences and the like. One or more reagents may be loaded
into microcapsules or loaded freely in solution into the device or
a combination thereof. Sample preparation may then be conducted,
such as by fragmenting the cDNA and attaching unique identifiers to
the fragments. After sample preparation and recovery, the nucleic
acid products of the reaction may be further analyzed, such as by
sequencing.
[0181] Additionally, a device may be used to characterize multiple
cell markers, similar to a flow cytometer. Any cell marker may be
characterized, including cell-surface markers (e.g., extracellular
proteins, transmembrane markers) and markers located within the
internal portion of a cell (e.g., RNA, mRNA, microRNA, multiple
copies of genes, proteins, alternative splicing products, etc.).
For example, cells may be partitioned within the device, as
described herein, so that at most one cell is present within a
microwell. Cell markers such as nucleic acids (e.g., RNA) may be
extracted and/or fragmented prior to being labeled with a unique
identifier (e.g., molecular bar code). Or, alternatively, the
nucleic acids may be labeled with a unique identifier without being
extracted and/or fragmented. The nucleic acids may then be
subjected to further analysis such as sequencing reactions designed
to detect multiple gene expression products. Such analysis may be
useful in a number of fields. For example, if the starting cells
are immune cells (e.g., T cells, B cells, macrophages, etc.), the
analysis may provide information regarding multiple expressed
markers and enable immunophenotyping of the cells, for example by
identifying different CD markers of the cells (e.g., CD3, CD4, CD8,
CD19, CD20, CD56, etc.). Such markers can provide insights into the
function, character, class, or relative maturity of the cell. Such
markers can also be used in conjunction with markers that are not
necessarily immunophenotyping markers, such as markers of
pathogenic infection (e.g., viral or bacterial protein, DNA, or
RNA). In some cases, the device may be used to identify at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95, 100, 200, 500, 700, 1000, 5000, 10000,
50000, or 100000 different gene expression products or other form
of cellular markers on a single-cell basis. Often, such methods do
not comprise use of dyes or probes (e.g., fluorescent probes or
dyes).
[0182] Gene expression product analysis may be useful in numerous
fields including immunology, cancer biology (e.g., to characterize
the existence, type, stage, aggressiveness, or other characteristic
of cancerous tissue), stem cell biology (e.g., in order to
characterize the differentiation state of a stem cell, potency of a
stem cell, cellular type of a stem cell, or other features of a
stem cell), microbiology, and others. The gene expression analysis
may also be used in drug screening applications, for example to
evaluate the effect of a particular drug or agent on the gene
expression profile of particular cells.
VII. TERMINOLOGY
[0183] The terminology used therein is for the purpose of
describing particular embodiments only and is not intended to be
limiting of a device of this disclosure. As used herein, the
singular forms "a", "an" and "the" are intended to include the
plural forms as well, unless the context clearly indicates
otherwise. Furthermore, to the extent that the terms "including",
"includes", "having", "has", "with", or variants thereof are used
in either the detailed description and/or the claims, such terms
are intended to be inclusive in a manner similar to the term
"comprising".
[0184] Several aspects of a device of this disclosure are described
above with reference to example applications for illustration. It
should be understood that numerous specific details, relationships,
and methods are set forth to provide a full understanding of a
device. One having ordinary skill in the relevant art, however,
will readily recognize that a device can be practiced without one
or more of the specific details or with other methods. This
disclosure is not limited by the illustrated ordering of acts or
events, as some acts may occur in different orders and/or
concurrently with other acts or events. Furthermore, not all
illustrated acts or events are required to implement a methodology
in accordance with this disclosure.
[0185] Ranges can be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint. The
term "about" as used herein refers to a range that is 15% plus or
minus from a stated numerical value within the context of the
particular usage. For example, about 10 would include a range from
8.5 to 11.5.
[0186] The term microwell array, as used herein, generally refers
to a predetermined spatial arrangement of microwells. Microwell
array devices that comprise a microcapsule may also be referred to
as "microwell capsule arrays." Further, the term "array" may be
used herein to refer to multiple arrays arranged on a surface, such
as would be the case where a surface has multiple copies of an
array. Such surfaces bearing multiple arrays may also be referred
to as "multiple arrays" or "repeating arrays."
Example 1
Single Cell DNA Sequencing
[0187] A microwell capsule array is prepared to perform nucleic
acid sequencing on individual human B-cells taken from a blood
sample. Approximately 15,000 cells are harvested and used for
loading into the device. A device of this disclosure and containing
150,000 microwells is used. Each well is cylindrical in shape
having a diameter of 125 um and a height of 125 um, allowing at
most 1 capsule to be loaded per well. Microcapsules made through
emulsion polymerization with a PNIPAM hydrogel shell wall are
created such that the microcapsules have a diameter of 100 um for
loading in the device. The microcapsules are created such that the
PNIPAM shell contains magnetic iron particles. The outer surface of
the shell is then chemically coupled to a antibody specific to a
transmembrane B cell receptor on the outside of a B cell.
[0188] During the preparation process of capsules, reagents are
simultaneously loaded into the capsules. Reagents necessary for
cell lysis and labeling individual DNA strands of the cells with
DNA barcodes are loaded into capsules. Reagents for cell lysis
include a mild non-ionic detergent, buffer and salt. Reagents for
the addition of DNA bar codes to genomic DNA included restriction
enzymes, ligase, and >10,000,000 unique DNA oligonucleotides are
loaded into capsules. Capsules are designed to be sensitive to
rupture at greater than 65 C.
[0189] Capsules are prepared to be applied to the microcapsule
array. The array is placed on a magnetic temperature controlled hot
plate. Microcapsules are added to a sample of B cells such that one
B cell is able to bind to one capsule. Capsule-cell conjugates are
applied in aqueous carrier solution in a quantity in excess to the
relative number of wells. Gentle pipetting of capsules-cells into
the inlet port followed by application of a vacuum manifold to the
outlet port distributes the capsules throughout the device. A
magnetic field is applied through the plate. Excess capsule-cell
solution is removed via pipetting through the outlet port. Each
capsule-cell conjugate is trapped and positioned in individual
wells via the magnetic field.
[0190] After the cells and capsules are loaded in the device, a
carrier oil (or sealing fluid) is applied to the device to remove
any excess aqueous solution bridging adjacent microwells. The
carrier oil applied to the inlet and excess oil is recovered at the
outlet with a vacuum manifold. After the carrier oil is applied,
the inlet and outlet ports are sealed with tape.
[0191] The device is then heated, via the magnetic temperature
controlled hot plate, to a temperature of 70 C for 10 min to allow
for capsule rupture and cell lysis. The hot plate is then switched
to 37 C, for restriction and ligation, for up to 1 hour.
[0192] After the sample preparation reaction is completed, the
contents of the wells are recovered. The inlet and outlet ports of
the device are unsealed and nitrogen gas is applied to the device
to flush out the individual components of the microwells. The
sample is collected in bulk via a pipette at the outlet port, while
the magnetic field retains ruptured capsule shells in individual
microwells.
[0193] The sample is then sequenced using a multiplex sequencing
strategy known in the art. Bar coding of individual cells allows
for sequencing information to be gained for individual cells rather
than as an average of multiple cells. Based upon the number of
cells sequenced and bar codes assigned, SNP cell-specific
information is gained. Moreover, the number of reads for individual
bar codes can be counted to provide insight into the distribution
of different types of cells with varying genetic backgrounds,
within the original population of B cells.
Example 2
DNA Single Strand Sequencing
[0194] A microwell capsule array is prepared to perform nucleic
acid sequencing on individual strands of DNA isolated from a
population of human skin cells. Cells are lysed using detergent and
heat and approximately 15,000 copies of diploid DNA are
precipitated via chloroform/ethanol extraction. A resuspension of
DNA is loaded into the device with approximately 10,000 copies of
haploid DNA. A device of this disclosure, with 300,000 microwells
is used. Each well is cylindrical in shape having a diameter of 125
um and a height of 125 um, allowing at most 1 capsule to be loaded
per well. Microcapsules made through emulsion polymerization with a
PNIPAM hydrogel shell wall are created to a specification of a
sphere with a diameter of 100 um for loading into the device.
[0195] During the preparation of the microcapsules, reagents are
simultaneously loaded into the capsules. The reagents include
reagents necessary for labeling individual DNA strands with DNA
barcodes, including restriction enzymes, ligase, and >10,000,000
unique DNA oligonucleotides. Capsules designed to be sensitive to
rupture at greater than 65 C are used for the encapsulation.
[0196] Capsules are applied aqueous carrier solution in an excess
to the relative number of wells. Gentle pipetting of capsules into
the inlet followed by application of a vacuum manifold to the
outlet distributed the capsules throughout the device. After excess
capsule solution is removed, a suspension of DNA in buffer is
applied to the device in a similar fashion as the capsules.
[0197] After the DNA strands and capsules are loaded in the device,
a carrier oil is applied to the device to remove any excess aqueous
solution bridging adjacent microwells. The carrier oil is applied
to the inlet port and excess oil is recovered at the outlet port
with a vacuum manifold. After the carrier oil is applied, the inlet
and outlet ports are sealed with tape.
[0198] The device is then placed on a temperature controlled hot
plate and heated to temperature of 70 C for 10 min to allow for
capsule rupture. Reagents are released into the sample preparation
reaction. The hot plate is then switched to 37 C, for restriction
and ligation, for up to 1 hour.
[0199] After the sample preparation reaction is completed, the
inlet and outlet ports of the device are unsealed and nitrogen gas
is applied to the device to flush out the individual components of
the microwells. The sample products, en bulk, are collected via
pipette at the outlet port.
[0200] The sample is then sequenced to sufficient coverage (e.g.,
500) using a multiplex sequencing strategy known in the art. Bar
coding of individual DNA strands allows for sequencing information
to be gained from individual strands rather than as an average of
entire sample of DNA. Based upon the number of DNA strands
sequenced and bar codes assigned, SNP phasing/haplotyping
information is gained and many repetitive regions of DNA can be
resolved. In addition, a substantial boost in accuracy can be
gained by discarding mutations that appear randomly with respect to
haplotypes, as those are likely to be sequencing errors.
[0201] It should be understood from the foregoing that, while
particular implementations have been illustrated and described,
various modifications may be made thereto and are contemplated
herein. It is also not intended that the invention be limited by
the specific examples provided within the specification. While the
invention has been described with reference to the aforementioned
specification, the descriptions and illustrations of the preferable
embodiments herein are not meant to be construed in a limiting
sense. Furthermore, it shall be understood that all aspects of the
invention are not limited to the specific depictions,
configurations or relative proportions set forth herein which
depend upon a variety of conditions and variables. Various
modifications in form and detail of the embodiments of the
invention will be apparent to a person skilled in the art. It is
therefore contemplated that the invention shall also cover any such
modifications, variations and equivalents. It is intended that the
following claims define the scope of the invention and that methods
and structures within the scope of these claims and their
equivalents be covered thereby.
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