US20140155295A1 - Capsule array devices and methods of use - Google Patents

Capsule array devices and methods of use Download PDF

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Publication number
US20140155295A1
US20140155295A1 US13/966,150 US201313966150A US2014155295A1 US 20140155295 A1 US20140155295 A1 US 20140155295A1 US 201313966150 A US201313966150 A US 201313966150A US 2014155295 A1 US2014155295 A1 US 2014155295A1
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United States
Prior art keywords
microcapsule
dna
composition
nucleic acid
microcapsules
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Abandoned
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US13/966,150
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English (en)
Inventor
Benjamin Hindson
Serge Saxonov
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10X Genomics Inc
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10X Technologies Inc
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Application filed by 10X Technologies Inc filed Critical 10X Technologies Inc
Priority to US13/966,150 priority Critical patent/US20140155295A1/en
Assigned to 10X TECHNOLOGIES, INC. reassignment 10X TECHNOLOGIES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HINDSON, BENJAMIN, SAXONOV, SERGE
Priority to US14/249,673 priority patent/US20140287963A1/en
Publication of US20140155295A1 publication Critical patent/US20140155295A1/en
Priority to KR1020227005065A priority patent/KR102436171B1/ko
Priority to AU2014302277A priority patent/AU2014302277A1/en
Priority to US14/316,383 priority patent/US20140378345A1/en
Priority to CN201910621839.XA priority patent/CN110592182B/zh
Priority to US14/316,463 priority patent/US20140378322A1/en
Priority to EP24158974.6A priority patent/EP4357493A3/en
Priority to US14/316,416 priority patent/US20140378349A1/en
Priority to PCT/US2014/044398 priority patent/WO2014210353A2/en
Priority to MX2015016968A priority patent/MX361481B/es
Priority to JP2016524208A priority patent/JP6563912B2/ja
Priority to EP14817610.0A priority patent/EP3013957B2/en
Priority to US14/316,431 priority patent/US20150005200A1/en
Priority to BR112015032512A priority patent/BR112015032512A8/pt
Priority to EP18189200.1A priority patent/EP3467160A1/en
Priority to CN201480047858.1A priority patent/CN105492607B/zh
Priority to KR1020167002243A priority patent/KR102212234B1/ko
Priority to US14/316,398 priority patent/US20150005199A1/en
Priority to KR1020227028867A priority patent/KR102642680B1/ko
Priority to US14/316,447 priority patent/US10221442B2/en
Priority to CN202311514025.9A priority patent/CN117568449A/zh
Priority to CA2915499A priority patent/CA2915499A1/en
Priority to KR1020217002986A priority patent/KR102366116B1/ko
Assigned to 10X GENOMICS, INC. reassignment 10X GENOMICS, INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: 10X TECHNOLOGIES, INC.
Priority to US14/624,468 priority patent/US9689024B2/en
Priority to US14/624,484 priority patent/US20150224466A1/en
Priority to US14/624,473 priority patent/US9695468B2/en
Assigned to 10X GENOMICS, INC. reassignment 10X GENOMICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SCHNALL-LEVIN, MICHAEL
Priority to US15/598,898 priority patent/US20170321252A1/en
Priority to US15/687,357 priority patent/US20170356027A1/en
Priority to US15/718,764 priority patent/US20180094298A1/en
Priority to US15/718,893 priority patent/US20180051321A1/en
Priority to US15/719,459 priority patent/US10053723B2/en
Priority to US15/847,659 priority patent/US20180112253A1/en
Priority to US15/887,711 priority patent/US20180179580A1/en
Priority to US15/975,468 priority patent/US20180258466A1/en
Priority to US16/052,486 priority patent/US10323279B2/en
Priority to US16/052,431 priority patent/US10273541B2/en
Priority to US16/056,231 priority patent/US11591637B2/en
Priority to US16/212,441 priority patent/US10752949B2/en
Priority to US16/231,142 priority patent/US10584381B2/en
Priority to US16/231,185 priority patent/US10400280B2/en
Priority to US16/242,962 priority patent/US20190203262A1/en
Priority to US16/246,322 priority patent/US10597718B2/en
Priority to US16/294,769 priority patent/US10450607B2/en
Priority to US16/395,090 priority patent/US10669583B2/en
Priority to US16/435,362 priority patent/US10626458B2/en
Priority to US16/435,417 priority patent/US10752950B2/en
Priority to US16/519,863 priority patent/US11078522B2/en
Priority to US16/736,323 priority patent/US12098423B2/en
Priority to US16/844,141 priority patent/US11441179B2/en
Priority to US16/998,425 priority patent/US11035002B2/en
Priority to US16/998,414 priority patent/US11021749B2/en
Priority to AU2020244615A priority patent/AU2020244615B2/en
Priority to US17/314,526 priority patent/US11359239B2/en
Priority to US17/353,202 priority patent/US12037634B2/en
Priority to US17/392,610 priority patent/US20220098659A1/en
Priority to US18/186,088 priority patent/US20240002929A1/en
Priority to AU2023203879A priority patent/AU2023203879A1/en
Priority to US18/384,527 priority patent/US20240376519A1/en
Priority to US18/680,949 priority patent/US20250129406A1/en
Priority to US18/760,903 priority patent/US20250027149A1/en
Priority to US18/945,323 priority patent/US20250066851A1/en
Priority to US18/960,947 priority patent/US20250146047A1/en
Abandoned legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/52—Containers specially adapted for storing or dispensing a reagent
    • B01L3/523—Containers specially adapted for storing or dispensing a reagent with means for closing or opening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0677—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2535/00—Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
    • C12Q2535/122—Massive parallel sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/159—Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components

Definitions

  • the stimulus may be selected from the group consisting of a biological, chemical, thermal, electrical, magnetic, or photo stimulus, and a combination thereof.
  • 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).
  • FIG. 1B is a schematic representation of a microcapsule containing multiple inner reagent droplets.
  • FIG. 2A is a schematic illustration of a top down view of an exemplary microcapsule array.
  • FIG. 2B is a schematic illustration of an exemplary side view of a microcapsule array.
  • FIG. 3 is a schematic illustration of a multi-microcapsule array configuration on a 96-well plate holder.
  • FIG. 4B is similar to 4 A, except that it is annotated with examples of methods that can be performed at each step.
  • 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.).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • the reagents 100 may be present in the internal compartment 120 .
  • the reagents are located in the enveloping layer 130 or in both compartments.
  • 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.
  • 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.
  • the internal layer 120 may comprise an aqueous fluid
  • the enveloping layer 130 may be a non-aqueous fluid such as an oil.
  • the internal layer 120 may comprise a non-aqueous fluid (e.g., oil)
  • the enveloping layer 130 may comprise an aqueous fluid.
  • the microcapsule does not comprise an enveloping second layer.
  • the microcapsule is further encapsulated by a shell layer 110 , which may comprise a polymeric material.
  • a microcapsule may comprise a droplet.
  • a microcapsule may be a droplet.
  • 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.
  • 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.
  • 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.
  • each compartment, or a subset thereof may also be subdivided into a plurality of additional compartments.
  • 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.
  • 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.
  • each compartment (or some percentage of the total number of compartments) may comprise the same reagent or the same combination or reagents.
  • each compartment (or some percentage of the total number of compartments) comprises different reagents or a different combination of reagents.
  • the compartments may be configured in a variety of ways.
  • the microcapsule may comprise multiple concentric compartments (repeating units of compartments that contain the preceding compartment), often separated by an immiscible layer.
  • the reagents may be present in alternating compartments, in every third compartment, or in every fourth compartment.
  • FIG. 1B depicts an example of a microcapsule that contains a plurality of smaller microcapsules 140 , each containing a reagent.
  • 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.
  • the immiscible fluid is loaded with additional materials or reagents.
  • 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.
  • 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.
  • the microcapsule may comprise a polymeric shell (see, e.g., FIGS. 1 and 2 ) or multiple polymeric shells.
  • the microcapsule may comprise multiple polymeric shells layered on top of each other.
  • individual compartments within a microcapsule comprise a polymeric shell, or a subset of the compartments may comprise a polymeric shell.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the microcapsules may also comprise a polymer within the interior of the capsule.
  • this polymer may be a porous polymer bead that may entrap reagents or combinations of reagents.
  • 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.
  • the microcapsule comprises a microwell that is at most about 0.001 ⁇ m, 0.01 ⁇ m, 0.1 ⁇ m, 0.5 ⁇ m, 1 ⁇ m, 5 ⁇ m, 10 ⁇ m, 50 ⁇ m, 100 ⁇ m, 200 ⁇ m, 300 ⁇ m, 400 ⁇ m, 500 ⁇ m, 600 ⁇ m, 700 ⁇ m, 800 ⁇ m, 900 ⁇ m or 1 nm.
  • 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).
  • an aqueous fluid e.g., water
  • a non-aqueous fluid e.g., oil
  • the microcapsules are denser than a non-aqueous fluid (e.g., oil).
  • 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.
  • 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.
  • a precise number of analytes and/or microcapsules are loaded into each individual microwell.
  • a precise number of analytes and/or microcapsules are loaded into a particular subset of microwells in the plate.
  • an average number of analytes and/or micrcocapsules are loaded into each individual microwell.
  • “dry” microcapsules are loaded into the device, while in other cases “wet” microcapsules are loaded into the device.
  • a combination of “dry” and “wet” microcapsules and/or reagents are loaded into the device, either simultaneously or sequentially.
  • the loading of the device may occur in any order and may occur in multiple stages.
  • the microcapsules are pre-loaded into the device, prior to the loading of the analyte.
  • the microcapsules and analyte are loaded concurrently.
  • the analytes are loaded before the microcapsules are loaded.
  • microcapsules and/or analytes may be loaded in multiple stages or multiple times.
  • 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
  • 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.
  • the different sets of microcapsules are loaded sequentially; or, different sets of microcapsules may also be loaded simultaneously.
  • 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.
  • 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.
  • 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.
  • the microcapsules may be loaded into the microarray device in a particular pattern.
  • 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.).
  • 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.
  • the microcapsules contain negative control reagents such as deactivated enzyme, or a synthetic oligonucleotide sequence that is resistant to fragmentation.
  • negative control reagents may control for the specificity of the sample preparation reaction etc.
  • 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.).
  • the analytes/sample also may be loaded into the microarray device in a particular pattern.
  • 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.
  • 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.
  • a microcapsule is degradable.
  • 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;
  • a change in pH of the solution 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.
  • 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.
  • cross-linkers e.g., disulfide bonds
  • 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.
  • a reducing agent such as beta-mercaptoethanol, dithiotheritol (DTT), or 2-tris(2-carboxyethyl)phosphine (TCEP)
  • DTT dithiotheritol
  • TCEP 2-tris(2-carboxyethyl)phosphine
  • enzymes may be added to cleave peptide bonds within the microcapsules, thereby resulting in cleavage of shell wall cross linkers.
  • 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.
  • Biological stimuli may also be used to trigger disruption or degradation of microcapsules.
  • 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.
  • 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.
  • 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.
  • the proteases may be heat-activated.
  • 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.
  • 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.
  • the heat may increase the internal pressure of the inner components of the capsule such that the capsule ruptures or explodes.
  • 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.
  • a device of this disclosure may comprise magnetic particles for either purpose.
  • incorporation of Fe3O4 nanoparticles into polyelectrolyte containing capsules triggers rupture in the presence of an oscillating magnetic field stimulus.
  • 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.
  • a device of this disclosure may be used in combination with any apparatus or device that provides such trigger or stimulus.
  • 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.
  • the stimulus is a biological enzyme
  • the enzyme may be injected into a device such that it is deposited into each microwell.
  • a device may be used in combination with a magnetic or electric plate.
  • 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.
  • a droplet may comprise a degradable gel bead (e.g., a gel bead comprising chemical cross-linkers, such as, for example, disulfide bonds).
  • a chemical stimulus e.g., a reducing agent
  • a chemical stimulus e.g., a reducing agent
  • 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.
  • 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.
  • 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.
  • a device may be used in combination with a PCR thermocycler.
  • 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.
  • a device may be used in combination with a shaking apparatus.
  • 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.
  • 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.
  • 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.
  • 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.
  • gene expression products e.g., mRNA, protein
  • 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.
  • any analytes such as DNA or cells, may be loaded in solution or as analytes encapsulated in a capsule.
  • homogeneous or heterogeneous populations of molecules e.g., nucleic acids, proteins, etc.
  • the microcapsules may comprise a random or specified number of cells and/or molecules.
  • 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.
  • 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.
  • 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.
  • the devices disclosed herein may be used in applications that involve the assignment of unique identifiers, or molecular bar codes, to analytes.
  • the unique identifier is a bar-code oligonucleotide that is used to tag the analytes; but, in some cases, different unique identifiers are used.
  • 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).
  • 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.
  • 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.
  • the reaction may comprise a chemical linkage between the identifier and the analyte.
  • the reaction may comprise addition of a metal isotope, either directly to the analyte or by a probe labeled with the isotope.
  • the method comprises attaching oligonucleotide bar codes to nucleic acid analytes through an enzymatic reaction such as a ligation reaction.
  • the ligase enzyme may covalently attach a DNA bar code to fragmented DNA (e.g., high molecular-weight DNA).
  • the molecules may be subjected to a sequencing reaction.
  • other reactions may be used as well.
  • 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.
  • the contents of individual microwells may be recovered via the outlet port in the device for further analyses.
  • the unique identifiers 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • analyte e.g., strand of nucleic acid, fragment of nucleic acid, protein, cell, etc.
  • 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.
  • 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.
  • such identifiers may be copies of the same identifier, or multiple different identifiers.
  • the attachment process may be designed to attach multiple identical identifiers to a single analyte, or multiple different identifiers to the analyte.
  • the unique identifiers may be used to tag a wide range of analytes, including cells or molecules.
  • unique identifiers e.g., bar code oligonucleotides
  • the unique identifiers 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.
  • 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.
  • elements e.g., reagents, analytes.
  • analytes 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.
  • 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.
  • 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.
  • a method disclosed herein may comprise loading the device with the reagents necessary for the assignment of bar codes to the analytes.
  • 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.
  • 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.
  • 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).
  • a density e.g., about 1 analyte per microwell or other density described herein.
  • 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.
  • analytes e.g., nucleic acid analytes
  • 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, in
  • 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.
  • 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.
  • 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.
  • 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.
  • genetic variations e.g., SNPs, mutations, indels, copy number variations, transversions, translocations, inversions, etc.
  • 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.
  • 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.
  • the methods herein may be used to detect the distribution of oncogenic mutations across a population of cancer tumor cells.
  • 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.
  • 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.
  • oncogenes e.g., KRAS and EGFR
  • 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.
  • 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:
  • 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.
  • the contents of the cells may be pooled together and could be further analyzed, such as by DNA sequencing.
  • further analyses may be possible including but not limited to quantification of different gene levels or nucleic acid sequencing of individual cells.
  • 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.
  • 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.
  • single cells may be loaded into microwells.
  • a pool of mRNA or other gene product may be loaded such that a desired quantity of mRNA molecules is loaded into individual microwells.
  • RNA analysis may be particularly useful for RNA analysis.
  • 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.
  • the nucleic acid products of the reaction may be further analyzed, such as by sequencing.
  • 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.).
  • cell-surface markers e.g., extracellular proteins, transmembrane markers
  • markers located within the internal portion of a cell e.g., RNA, mRNA, microRNA, multiple copies of genes, proteins, alternative splicing products, etc.
  • 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).
  • 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).
  • 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.
  • such methods do not comprise use of dyes or probes (e.g., fluorescent probes or dyes).
  • 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.
  • microwell array 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.”
  • 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.”
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

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US13/966,150 US20140155295A1 (en) 2012-08-14 2013-08-13 Capsule array devices and methods of use
US14/249,673 US20140287963A1 (en) 2012-08-14 2014-04-10 Capsule Array Devices and Methods of Use
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US14/624,468 Division US9689024B2 (en) 2012-08-14 2015-02-17 Methods for droplet-based sample preparation
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