WO2021011895A1 - Methods and devices for single cell barcoding - Google Patents

Methods and devices for single cell barcoding Download PDF

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Publication number
WO2021011895A1
WO2021011895A1 PCT/US2020/042604 US2020042604W WO2021011895A1 WO 2021011895 A1 WO2021011895 A1 WO 2021011895A1 US 2020042604 W US2020042604 W US 2020042604W WO 2021011895 A1 WO2021011895 A1 WO 2021011895A1
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Prior art keywords
oligonucleotide
sequence
microfluidic device
hydrodynamic
traps
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PCT/US2020/042604
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French (fr)
Inventor
Benjamin Yellen
Jeffrey BLACKINTON
Bernard Hirschbein
Tyson Clark
Jon ZAWISTOWSKI
Nicolas ROELOFS
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Celldom, Inc.
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Publication of WO2021011895A1 publication Critical patent/WO2021011895A1/en

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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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    • 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
    • B01L3/502707—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 the manufacture of the container or its components
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    • 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
    • B01L3/502761—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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B81—MICROSTRUCTURAL TECHNOLOGY
    • B81C—PROCESSES OR APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OR TREATMENT OF MICROSTRUCTURAL DEVICES OR SYSTEMS
    • B81C1/00—Manufacture or treatment of devices or systems in or on a substrate
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    • C12M23/00—Constructional details, e.g. recesses, hinges
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    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
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    • 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
    • C12Q1/6869—Methods for sequencing
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    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/0054—Means for coding or tagging the apparatus or the reagents
    • B01J2219/00547—Bar codes
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
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    • 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
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0877—Flow chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0883—Serpentine channels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0893—Geometry, shape and general structure having a very large number of wells, microfabricated wells
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • RNA, DNA, protein, antibodies, and other forms of cellular expression in a microfluidic device comprising a plurality of hydrodynamic traps and oligonucleotide arrays, wherein the barcoded analytes (e.g ., the RNA, DNA, protein, antibodies, and other forms of cellular expression released from single cells trapped within the chip) may be analyzed using high throughput DNA sequencers and other analysis instruments. Also disclosed are methods for fabricating the microfluidic device.
  • microfluidic devices comprising: a) a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device; and b) a plurality of oligonucleotide arrays, wherein the interior region of each hydrodynamic trap comprises an oligonucleotide array, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence.
  • microfluidic devices comprising: a) a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device; and b) a plurality of oligonucleotide arrays, wherein the interior region of each hydrodynamic trap comprises an oligonucleotide array, wherein each oligonucleotide array comprises a plurality of features, wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence, and wherein at least a subset of the features comprise a plurality of oligonucleotide molecules comprising a single gene fragment sequence, such that the plurality of gene fragment sequences presented by the subset of features collectively span a full length gene sequence.
  • microfluidic devices comprising: a) a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device; and b) a plurality of oligonucleotide arrays, wherein the interior region of each hydrodynamic trap comprises an oligonucleotide array, wherein each oligonucleotide array comprises a plurality of features, wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence, and wherein at least one feature of the plurality comprises a plurality of oligonucleotide molecules comprising a single full-length gene sequence.
  • each hydrodynamic trap may comprise an entrance region, an interior region, and an exit region that collectively constitute an interior fluid flow path through the hydrodynamic trap that has a fluidic resistance, RT; ii) each hydrodynamic trap in a majority of the hydrodynamic traps may be in fluid communication with one long bypass fluid flow channel having a fluidic resistance, RA, and with one or two short bypass fluid flow channels each having a fluidic resistance that is less than RA, wherein each bypass fluid flow channel connects the exit region of the hydrodynamic trap to the entrance region of another hydrodynamic trap; and iii) fluid flows through an adjacent short bypass channel in a first direction if a hydrodynamic trap is unoccupied, and in a second direction if the hydrodynamic trap is occupied by an object.
  • the ratio RA/RT is at least 1.1. In some embodiments, the ratio RA/RT is at least 1.3. In some embodiments, each hydrodynamic trap comprises at least one constriction that has a spatial dimension that is less than about one half of the smallest dimension of the object. In some embodiments, the ratio RA/RT is at least 1.2 and a capture probability for an individual hydrodynamic trap retaining a suspended object on first contact is at least 0.36. In some embodiments, the ratio RA/RT is at least 1.45 and a capture probability for an individual hydrodynamic trap retaining a suspended object on first contact is at least 0.60.
  • each hydrodynamic trap comprises a frit structure within the exit region, and wherein the frit structure comprises one or more constrictions that have a spatial dimension that is smaller than the smallest dimension of the suspended objects.
  • the plurality of hydrodynamic traps comprises at least 100 traps. In some embodiments, the plurality of hydrodynamic traps comprises at least 1,000 traps. In some embodiments, the plurality of hydrodynamic traps comprises at least 10,000 traps. In some embodiments, an initial trapping efficiency for trapping the suspended objects is at least 80%. In some embodiments, an initial trapping efficiency for trapping the suspended objects is at least 95%.
  • the oligonucleotide molecules in each feature of the oligonucleotide array are covalently attached to a surface or coating layer within the interior region of each hydrodynamic trap. In some embodiments, the oligonucleotide molecules in each feature of the oligonucleotide array are non- covalently tethered to a surface or coating layer within the interior region of each hydrodynamic trap. In some embodiments, the oligonucleotide molecules in each feature of the oligonucleotide array are entrapped within a coating layer in the interior region of each hydrodynamic trap. In some embodiments, the plurality of oligonucleotide arrays is fabricated using a contact printing or stamping technique.
  • the plurality of oligonucleotide arrays is fabricated using an inkjet printing, microarray spotting, or spatially-addressable solid phase synthesis technique.
  • each oligonucleotide array comprises at least 10 features.
  • each oligonucleotide array comprises at least 100 features.
  • each oligonucleotide array comprises at least 1,000 features.
  • the common barcode sequences comprise unique non-overlapping sequences with a Hamming distance that enables demultiplexing with error correction capability.
  • the common barcode sequences comprise a G/C content ranging from 30% to
  • a length of the common barcode sequence ranges from 6 base pairs to 20 bases.
  • the common barcode sequence comprises a unique cell barcode sequence.
  • the unique cell barcode sequence comprises a string of
  • M is
  • the unique cell barcode sequence is the same for each feature within a given oligonucleotide array, and different from that for all of the features in all of the other oligonucleotide arrays. In some embodiments, the unique cell barcode sequence is different for each feature within a given oligonucleotide array, and different from that for all of the features in all of the other oligonucleotide arrays. In some embodiments, the unique cell barcode sequence for each feature of each oligonucleotide array of the plurality of oligonucleotide arrays is known.
  • the oligonucleotides in each feature further comprise a spacer sequence, a cleavage sequence, an adapter sequence, at least one primer sequence, a cell barcode sequence, a molecular index sequence, a molecular recognition sequence, or any combination thereof.
  • a length of the universal primer sequence ranges from 15 bases to 30 bases.
  • a length of the unique molecular index sequence ranges from 5 bases to
  • a length of the molecular recognition sequence ranges from 2 bases to 40 bases. In some embodiments, a length of the spacer sequence ranges from 5 bases to
  • a length of the oligonucleotide molecules ranges from 50 bases to 150 bases.
  • the oligonucleotides in each feature comprise a molecular index sequence that is different for each individual oligonucleotide of the plurality of
  • the oligonucleotides in each feature comprise a molecular recognition sequence that is different for different features of a given oligonucleotide array.
  • the molecular recognition sequence comprises a sequence selected from the group consisting of an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(deoxyguanosine) (oligo-dG) sequence, an oligo(riboguanosine) (oligo-rG) sequence, a complement of an mRNA sequence or fragment thereof, a complement of a tRNA sequence or fragment thereof, a complement of an rRNA sequence or fragment thereof, a complement of a DNA sequence, a complement of a cDNA sequence, a complement of a gene sequence or fragment thereof, and any combination thereof.
  • the plurality of features in each oligonucleotide array of the plurality of oligonucleotide arrays comprise a same set of molecular recognition sequences.
  • a subset of the plurality of oligonucleotide arrays comprises a set of molecular recognition sequences that is different from that in the oligonucleotide arrays of the remainder of the plurality.
  • the average length of a gene fragment oligonucleotide sequence is at least 40 bases.
  • each oligonucleotide array of the plurality comprises a same known set of features and a same known set of gene fragment sequences.
  • each oligonucleotide array of the plurality comprises a known set of features and a known set of gene fragment sequences that are different from those in all other oligonucleotide arrays.
  • a subset of oligonucleotide arrays of the plurality comprises a known set of features and a known set of gene fragment sequences that are different from those in the remainder of the plurality.
  • the average length of the full-length gene sequences presented by the plurality of oligonucleotide arrays is at least 1 kilobase.
  • each oligonucleotide array of the plurality comprises a feature comprising the same known full-length gene sequence.
  • each oligonucleotide array of the plurality comprises a feature comprising a different full-length gene sequence.
  • a subset of oligonucleotide arrays of the plurality comprises a feature comprising a full-length gene sequence that is different from that in the remainder of the plurality.
  • the oligonucleotide arrays in all or a portion of the plurality of oligonucleotide arrays further comprise one or more additional features that comprise a plurality of oligonucleotide molecules comprising one or more known capture probe sequences.
  • the one or more known capture probe sequences are used to capture one or more antigens, antibodies, peptides, proteins, or enzymes that have been labeled with an oligonucleotide sequence that is
  • the hydrodynamic traps are configured to trap single cells.
  • the plurality of oligonucleotides further comprises a cleavage site, a molecular recognition sequence, a random multimer capture sequence, a unique molecular index sequence, a universal primer sequence, an adapter sequence, a spacer sequence, or any combination thereof.
  • the cleavage site comprises a deoxyuridine base.
  • the cleavage site comprises a photocleavable linker.
  • the oligonucleotide molecule is released into solution upon exposure to light or treatment with an enzyme.
  • the oligonucleotide molecule is released into solution upon treatment with combination of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII.
  • UDG Uracil DNA glycosylase
  • the oligonucleotide array in each hydrodynamic trap is unique.
  • the plurality of common barcode sequences for the plurality of hydrodynamic traps comprise unique non- overlapping sequences with a Hamming distance that enables demultiplexing with error correction capability.
  • the common barcode sequences comprise a G/C content ranging from 40% to 60%.
  • a length of the common barcode sequence ranges from 8 base pairs to 20 bases.
  • a length of the universal primer sequence ranges from 15 base pairs to 30 bases.
  • a length of the unique molecular index sequence ranges from 5 bases to 15 bases.
  • a length of the molecular recognition sequence ranges from 2 bases to 40 bases.
  • a length of the spacer sequence ranges from 5 bases to 50 bases. In some embodiments, a length of the oligonucleotide molecules ranges from 50 bases to 150 bases.
  • the plurality of hydrodynamic traps comprises at least 100 traps. In some embodiments, the plurality of hydrodynamic traps comprises at least 1,000 traps. In some embodiments, the plurality of hydrodynamic traps comprises at least 10,000 traps. In some embodiments, the plurality of hydrodynamic traps comprises at least 100,000 traps.
  • the fabricating in step (b) comprises the use of contact printing or stamping to create a replica of the molecular pattern (or its inverse) on the surface. In some embodiments, the fabricating in step (b) comprises the use of an inkjet printing, microarray spotting, or spatially-addressable solid phase synthesis technique. In some embodiments, each
  • each oligonucleotide array comprises at least 1 feature. In some embodiments, each oligonucleotide array comprises at least 10 features. In some embodiments, each oligonucleotide array comprises at least 100 features.
  • the common barcode sequence comprises a unique cell barcode sequence. In some embodiments, the unique cell barcode sequence comprises a string of N“words”, and wherein each“word” comprises a string of M bases. In some embodiments, M is 1 base, 2 bases, 3 bases, or at least 4 bases. In some embodiments, N is at least 10“words”.
  • the unique cell barcode sequence is the same for each feature within a given oligonucleotide array, and different from that for all of the features in all of the other oligonucleotide arrays. In some embodiments, the unique cell barcode sequence is different for each feature within a given oligonucleotide array, and different from that for all of the features in all of the other oligonucleotide arrays. In some embodiments, the unique cell barcode sequence for each feature of each oligonucleotide array of the plurality of
  • the oligonucleotides in each feature comprise a molecular index sequence that is different for each individual oligonucleotide of the plurality of oligonucleotides within a given feature. In some embodiments, the oligonucleotides in each feature comprise a molecular recognition sequence that is different for different features of a given oligonucleotide array.
  • the molecular recognition sequence comprises a sequence selected from the group consisting of an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(deoxyguanosine) (oligo-dG) sequence, an oligo(riboguanosine) (oligo-rG) sequence, a random multimer sequence, a complement of an mRNA sequence or fragment thereof, a complement of a tRNA sequence or fragment thereof, a complement of an rRNA sequence or fragment thereof, a complement of a DNA sequence, a complement of a cDNA sequence, a complement of a gene sequence or fragment thereof, and any combination thereof.
  • hydrodynamic trap of the plurality of traps comprise a same set of molecular recognition sequences.
  • the plurality of molecular recognition sequences in the features of a subset of the plurality of oligonucleotide arrays is different from that in the oligonucleotide arrays of the remainder of the plurality.
  • the substrate is fabricated in silicon
  • the lid is glass
  • the bonding in step (c) comprises coating a thin film of polydimethylsiloxane (PDMS) on the glass lid prior to bonding it to the silicon substrate.
  • PDMS polydimethylsiloxane
  • the substrate is fabricated in silicon
  • the lid comprises a glass substrate with a polydimethylsiloxane (PDMS) layer coated thereon
  • the bonding in step (c) comprises coating and patterning a thin film of hydrogel on the PDMS layer prior to bonding the lid to the silicon substrate.
  • the bonding in step (c) comprises application of a thin coating layer of epoxy to the lid prior to fabricating the plurality of oligonucleotide arrays.
  • the epoxy is a UV-curable epoxy or a heat-curable epoxy.
  • the bonding in step (c) comprises formation of a hydrogel layer between the lid and substrate using UV-induced polymerization of acrylate groups that are attached to both surfaces.
  • the method may further comprise the use of an air plug or oil plug to seal the hydrodynamic traps to reduce transfer of molecular components between hydrodynamic traps following lysis of the single cells in (c).
  • a determination of the number of unique molecular index barcode sequences corresponding to each unique cell barcode sequence is used to quantify how many copies of a given target oligonucleotide were released from a give single cell.
  • the amplifying and sequencing are performed within the microfluidic device.
  • the amplifying and sequencing are performed after eluting the barcoded oligonucleotide molecules from the microfluidic device.
  • the target oligonucleotide molecules comprise mRNA molecules or fragments thereof, tRNA molecules or fragments thereof, rRNA molecules or fragments thereof, RNA molecules or fragments thereof, DNA molecules or fragments thereof, or any combination thereof.
  • the oligonucleotide molecules of the oligonucleotide array disposed in each hydrodynamic trap comprise a cleavage site, and the cleavage site comprises a deoxyuridine base.
  • the oligonucleotide molecules of the oligonucleotide array disposed in each hydrodynamic trap comprise a cleavage site, and the cleavage site comprises a photocleavable linker moiety.
  • the oligonucleotide molecules of the oligonucleotide array disposed in each hydrodynamic trap are released into solution upon exposure to light or treatment with an enzyme.
  • the oligonucleotide molecules are released into solution upon treatment with a combination of Uracil DNA glycosylase (UDG) and the DNA glycosylase- lyase Endonuclease VIII.
  • the primer extension reaction in (e) comprises a reverse transcription reaction or a DNA polymerase reaction.
  • the lysis buffer comprises a chaotropic agent.
  • the chaotropic agent comprises concentrated urea, guanidinium thiocyanate, or any combination thereof.
  • the cells comprise cancer cells.
  • the cells comprise fetal cells.
  • the cells comprise CRISPR-edited cells.
  • the method is used to perform library preparation for a DNA-seq experiment, an RNA-seq experiment, an RNA-seq experiment, an
  • the method is used to perform a cell transfection and gene expression assay.
  • each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device once assembled; b) patterning a plurality of oligonucleotide arrays on an interior surface of the interior regions of the plurality of hydrodynamic traps or on a first surface of a lid, wherein each oligonucleotide array comprises a plurality of features, and wherein all or a portion of the features in an oligonucleotide array comprise a plurality of oligonucleotide molecules comprising a single gene fragment sequence, such that the plurality of gene fragment sequences presented by a plurality of features in the oligonucleotide array collectively span a full-length gene
  • the oligonucleotide molecules in each feature are covalently attached to a coating layer within the interior region of each hydrodynamic trap or on the first surface of the lid. In some embodiments, the
  • each oligonucleotide array comprises at least 10 features. In some embodiments, each oligonucleotide array comprises at least 100 features. In some embodiments, each oligonucleotide array comprises at least 1,000 features. In some embodiments, the average length of a gene fragment oligonucleotide sequence is at least 40 bases.
  • the average length of a gene fragment oligonucleotide sequence is at least 120 bases. In some embodiments, the average length of a gene fragment oligonucleotide sequence is at least 360 bases. In some embodiments, each oligonucleotide array of the plurality comprises a same known set of features and a same known set of gene fragment sequences. In some embodiments, each oligonucleotide array of the plurality comprises a known set of features and a set of known gene fragment sequences that are different from those in all other oligonucleotide arrays.
  • a subset of oligonucleotide arrays of the plurality comprises a known set of features and a set of known gene fragment sequences that are different from those in the remainder of the plurality.
  • the patterning in (b) comprises the use of an inkjet printing, microarray spotting, or spatially-addressable solid phase synthesis technique.
  • the polymerase chain assembly reaction or Gibson assembly reaction is performed without cleaving the gene fragment sequences from the surface or coating layer.
  • the polymerase chain assembly reaction or Gibson assembly reaction further comprises the use of a restriction enzyme to remove partially-assembled gene sequences.
  • the hydrodynamic traps are configured to trap single cells.
  • each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain at least one cell suspended in a fluid passing through the microfluidic device; and ii) a plurality of oligonucleotide arrays, wherein an oligonucleotide array is contained within the interior region of each hydrodynamic trap, wherein each oligonucleotide array comprises a plurality of features, and wherein at least one feature of the plurality comprises a plurality of oligonucleotide molecules each comprising a single full-length gene sequence or fragment thereof; b) flowing a suspension of cells through the microfluidic device, thereby trapping at least one cell in all or a portion of the plurality of hydrodynamic traps; c) cleaving the at least one full length gene sequence or fragment thereof from the oligonucleotide array within the interior region of each hydrodynamic trap; and d) transfecting the at least one cell in each
  • a) providing a microfluidic device comprising: i) a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain at least one cell suspended in a fluid passing through the microfluidic device; and ii) a plurality of oligonucleotide arrays, wherein an oligonucleotide array is contained within the interior region of each hydrodynamic trap, wherein each oligonucleotide array comprises a plurality of features, and wherein at least one feature of the plurality comprises a plurality of oligonucleotide molecules each comprising a single full length gene sequence or fragment thereof, and wherein at least one feature of the plurality comprises multiple copies of an oligonucleotide capture probe sequence; b) flowing a suspension of cells through the microfluidic device
  • oligonucleotide capture probe sequences as a function of a fluid flow rate through the
  • the cleaving in (c) comprises the use of a photo cleavage reaction or restriction enzyme reaction.
  • the transfecting in (d) comprises the use of a chemical transfection agent, an electroporation technique, a lipofection technique, a sonoporation technique, a phototransfection technique, a restriction enzyme, or any combination thereof.
  • the hydrodynamic traps are sealed within the microfluidic device by flowing a hydrogel, oil, or air through the interconnecting fluid channels.
  • the average length of the full-length gene sequences presented by the plurality of oligonucleotide arrays is at least 1 kilobase. In some embodiments, the
  • oligonucleotide arrays in all or a portion of the plurality of oligonucleotide arrays further comprise one or more additional features that comprise a plurality of oligonucleotide molecules comprising one or more known capture probe sequences.
  • the one or more known capture probe sequences are used to capture one or more antigens, antibodies, peptides, proteins, or enzymes that have been labeled with an oligonucleotide sequence that is
  • each hydrodynamic trap comprises an oligonucleotide array disposed on a surface therein, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence; b) introducing cells into the microfluidic device and capturing a single cell in all or a portion of the plurality of hydrodynamic traps; c) introducing a cell lysis buffer comprising reverse transcription reagents into the microfluidic device; d) replacing the cell lysis buffer in the plurality of bypass channels with air or oil to prevent cross contamination between different hydrodynamic traps; e) cleaving the oligonucleotide molecules comprising the common barcode sequence from the
  • each hydrodynamic trap comprises an oligonucleotide array disposed on a surface therein, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence; b) introducing cells into the microfluidic device and capturing a single cell in all or a portion of the plurality of hydrodynamic traps; c) introducing a cell lysis buffer configured to dissociate nuclear membranes and histone complexes, thereby denaturing cellular DNA, into the microfluidic device; d) introducing a DNA polymerization/amplification reaction mixture into the microfluidic device; e) replacing the DNA polymerization/amplification reaction mixture in the plurality
  • RNA-seq and DNA-seq libraries comprising: a) providing a microfluidic device comprising a plurality of
  • each hydrodynamic trap comprises an oligonucleotide array disposed on a surface therein, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence; b) introducing cells into the microfluidic device and capturing a single cell in all or a portion of the plurality of hydrodynamic traps; c) simultaneously or sequentially introducing lysis buffer(s) comprising: (i) reverse transcription reagents, and (ii) DNA polymerization reagents into the microfluidic device; d) replacing the lysis buffer(s) in the plurality of bypass channels with air or oil to prevent cross contamination between different hydrodynamic traps; e) cleaving the oligonucleotide molecules comprising the common barcode sequence from the surface; f) simultaneously or sequentially performing: (i) a reverse transcription reaction on cellular
  • DNA sequences from the microfluidic device for amplification and sequencing are identical to DNA sequences from the microfluidic device for amplification and sequencing.
  • each hydrodynamic trap comprises an oligonucleotide array disposed on a surface therein, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence; b) introducing cells into the microfluidic device and capturing a single cell in all or a portion of the plurality of hydrodynamic traps; c) introducing a non-ionic cell lysis buffer configured to lyse cell membranes to yield pure nuclei into the microfluidic device; d) introducing a mixture comprising a transposase, DNA fragmentation, and DNA polymerization/amplification reagents into the microfluidic chip; e) replacing
  • the common barcode sequence comprises a unique cell barcode sequence.
  • the plurality of oligonucleotide molecules further comprises a spacer sequence, and adapter sequence, a cleavage site, a molecular recognition sequence, a random heptamer capture sequence, a unique molecular index sequence, a universal primer sequence, or any combination thereof.
  • the plurality of oligonucleotide molecules comprises a cleavage site, and wherein the cleavage site comprises a deoxyuridine base.
  • the plurality of oligonucleotide molecules comprises a cleavage site, and wherein the cleavage site comprises a photocleavable linker.
  • the plurality of oligonucleotide molecules within a feature is released into solution upon exposure to light or treatment with an enzyme.
  • the 5’ end of the plurality of oligonucleotide molecules within a feature is released into solution upon treatment with a combination of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase
  • the plurality of oligonucleotide molecules comprises a molecular recognition sequence that is the same for each feature within an oligonucleotide array.
  • the molecular recognition sequence comprises a sequence selected from the group consisting of an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(de
  • oligo(deoxyguanosine) (oligo-dG) sequence
  • oligo(guanosine) (oligo-rG) sequence
  • a random heptamer sequence and any combination thereof.
  • a microfluidic device comprising: i. a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device; ii.
  • each oligonucleotide array of the plurality is disposed on a surface within a hydrodynamic trap of the plurality of hydrodynamic traps, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence and a free 5’ end; and b) an imaging unit configured to acquire high-resolution images of the interior region of each hydrodynamic trap and one or more objects contained therein.
  • the oligonucleotide molecules comprise a cleavage site.
  • the cleavage site comprises a deoxyuridine base. In some embodiments, the cleavage site comprises a photocleavable linker. In some embodiments, the oligonucleotide molecules further comprise a molecular recognition sequence that is the same or different for different features of an oligonucleotide array.
  • the molecular recognition sequence comprises a sequence selected from the group consisting of an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(deoxyguanosine) (oligo-dG) sequence, an oligo(guanosine) (oligo- rG) sequence, a random multimer sequence, a complement of an mRNA sequence or fragment thereof, a complement of a tRNA sequence or fragment thereof, a complement of an rRNA sequence or fragment thereof, a complement of a DNA sequence, a complement of a cDNA sequence, a complement of a gene sequence or fragment thereof, and any combination thereof.
  • the objects comprise cells.
  • the cells comprise cancer cells.
  • the system is configured to perform single cell RNA sequencing library preparation, single cell DNA sequencing library preparation, single cell ATAC sequencing library preparation, protein detection, antibody detection, or any combination thereof.
  • kits comprising: a) a microfluidic device comprising: i. a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device; ii.
  • each oligonucleotide array of the plurality is disposed on a surface within a hydrodynamic trap of the plurality of hydrodynamic traps, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence and a free 5’ end; and b) one or more reagents for performing single cell RNA sequencing library preparation, single cell DNA sequencing library preparation, single cell ATAC sequencing library preparation, protein detection, antibody detection, any combination thereof.
  • FIGS. 1A-B show an example of the hydrodynamic trapping geometry in a ladder configuration and the two flow regimes where the cell capture efficiency is low (FIG. 1A) owing to the fluid flow splitting at the trap entrance or high (FIG. IB) owing to the fluid flow joining at the trap entrance.
  • FIGS. 2A-B show an example of the hydrodynamic trapping geometry in a mesh configuration and the two flow regimes where the cell capture efficiency is low (FIG. 2A) owing to the fluid flow splitting at the trap entrance or high (FIG. 2B) owing to the fluid flow joining at the trap entrance.
  • FIGS. 3A-D show the distribution of trapped cells in the microfluidic device as a function of the flow resistance ratio and trapping efficiency (q).
  • the traps For low efficient traps (FIG. 3A and FIG. 3B), the traps have a greater tendency to miss the cells than when using higher efficiency traps (FIG. 3C and FIG. 3D).
  • FIG. 6 shows a non-limiting example of a cell trapping architecture for a microfluidic device comprising a plurality of oligonucleotides contained inside each trap.
  • FIGS. 7A-B show micrographs of a cell trapping architecture in a microfluidic device both before (FIG. 7A) and after (FIG. 7B) flowing in a fluorescent liquid and then sealing the traps with a plug of air.
  • FIGS. 8A-B show examples of data for the base calls from a sequencing run performed using oligonucleotide barcode primers that are attached to a surface and either contain a cleavable linker (FIG. 8A) or do not contain a cleavable linker (FIG. 8B).
  • FIGS. 8A-B show examples of data for the base calls from a sequencing run performed using oligonucleotide barcode primers that are attached to a surface and either contain a cleavable linker (FIG. 8A) or do not contain a cleavable linker (FIG. 8B).
  • the hallmarks of a successful library preparation reaction were only observed in the base call data of FIG. 8A due to the molecules having been successfully cleaved from the surface, thereby enabling them to participate in the library preparation reactions.
  • FIG. 9 shows non-limiting examples of data for two methods of cleaving biotin- terminated oligonucleotides comprising either a photocleavable linker or a deoxyuridine nucleotide that were spotted on a streptavidin-coated surface.
  • FIG. 10 shows a comparison of surface-tethered oligonucleotides that were exposed to cell culture media for prolonged time intervals as compared to the controls.
  • FIG. 11 shows a non-limiting example of data for an oligonucleotide pattern replication technique.
  • FIGS. 12A-B show another non-limiting example of data for an oligonucleotide pattern replication technique.
  • FIG. 12A Fluorescently labeled, acrylate terminated oligos hybridized to spots of complementary, surface bound oligo on the template surface.
  • FIG. 12B Mirror image observed after oligos transferred to a daughter surface using acrylates and hybridized with a fluorescent, complementary sequence.
  • FIGS. 13A-B show examples of data for an RNA library preparation reaction that was performed in a microfluidic device.
  • the barcode region was constructed by enzymatic ligation of two distinct barcodes (FIG. 13A). Following the sequencing quality control steps, more than 60% of the reads contained the barcode, mapped to the genome, and contained useable biological information (FIG. 13B).
  • FIGS. 14A-B show examples of RNA library preparation and sequencing data.
  • FIG. 15 provides an outline of an RNA-seq workflow.
  • FIG. 16 provides an outline of two different ATAC-seq workflows that can be implemented in the disclosed microfluidic devices.
  • FIG. 17 provides a schematic illustration of a computer system that is programmed or otherwise configured to implement the methods and systems provided herein.
  • microfluidic devices that comprise a plurality of hydrodynamic traps and a plurality of oligonucleotide arrays, where each oligonucleotide array is housed within one of the traps of the plurality of hydrodynamic traps within the device.
  • the hydrodynamic traps of the device may be configured to retain objects, e.g ., cells, beads, or other particles, suspended in a fluid passing through the device.
  • the hydrodynamic traps may comprise all or a portion of the design features disclosed in co-pending PCT International Patent Application
  • the microfluidic devices include a substrate in which all or a portion of the hydrodynamic traps and fluid channels, e.g. , interconnecting fluid channels or bypass fluid channels, are fabricated, and a lid which is bonded to the substrate to seal the hydrodynamic traps and fluid channels of the device.
  • the substrate and/or lid is fabricated from an optically-transparent material such that the microfluidic device is configured for high-resolution imaging of the plurality of hydrodynamic traps for use in monitoring, e.g. , cell growth or phenotypic changes as the cells are exposed to a stimulus such as a therapeutic drug treatment.
  • the oligonucleotide arrays disposed in each hydrodynamic trap comprise barcoded oligonucleotides for performing genotyping or gene expression profiling studies in parallel with phenotypic studies of single cells.
  • each oligonucleotide array comprises a plurality of features, where each feature comprises a plurality of identical oligonucleotide molecules.
  • the oligonucleotide arrays may be fabricated on the underside of a lid, or a coating layer thereon, that is subsequently bonded to a substrate comprising the plurality of hydrodynamic traps and interconnecting fluid flow channels to seal the traps and fluid flow channels.
  • the oligonucleotide arrays may be fabricated on a surface within the hydrodynamic traps, e.g.
  • the oligonucleotide molecules of each feature may comprise oligonucleotide barcode sequences, e.g. , a cell barcode sequence (e.g, a sequence used to barcode molecules derived from a single cell or small clonal population of cells derived from a single cell) and/or a molecular index sequence (e.g, used to count the number of copies of a specific molecule derived from a single cell or clonal population of cells derived from a single cell).
  • a cell barcode sequence e.g, a sequence used to barcode molecules derived from a single cell or small clonal population of cells derived from a single cell
  • a molecular index sequence e.g, used to count the number of copies of a specific molecule derived from a single cell or clonal population of cells derived from a single cell.
  • the oligonucleotide molecules of each feature may further comprise a spacer sequence, an adapter sequence, at least one primer sequence (e.g, a universal primer sequence), a molecular index sequence, a molecular recognition sequence or randomer capture sequence, a cleavage site, or any combination thereof.
  • a primer sequence e.g, a universal primer sequence
  • a molecular index sequence e.g., a molecular index sequence
  • a molecular recognition sequence or randomer capture sequence e.g., a cleavage site, or any combination thereof.
  • the plurality of oligonucleotide arrays may be patterned or fabricated using, e.g, a contact printing or stamping approach.
  • a library of DNA colonies or“features” of the plurality of oligonucleotide arrays
  • a low-cost stamping approach such as that described by R. Mitra and G. Church (1999),“In situ Localized Amplification and Contact
  • the process should allow for the lid to be bonded to the substrate comprising the microfluidic channels and hydrodynamic traps, such that an adequate seal can be formed everywhere without negatively affecting the oligonucleotide array pattern.
  • the plurality of oligonucleotide arrays may be patterned or fabricated using, e.g ., inkjet printing, solid phase synthesis, or other direct oligonucleotide deposition method, and deposited either on the lid (or a coating layer thereof), or directly onto the substrate in the hydrodynamic trap sites or some interior region of the traps (or a coating layer thereon).
  • microfluidic devices comprising barcoded oligonucleotide arrays may be used to perform a variety of single cell genotyping and gene expression profiling studies, e.g. , single cell RNA sequencing (scRNA-seq) library preparation, single cell DNA sequencing (scDNA-seq) library preparation, Assay for
  • Examples of the technical challenges that have required innovative design and development include, but are not limited to, (i) the identification of scalable fabrication techniques for reproducible deposition of oligonucleotide arrays in precise patterns within the plurality of hydrodynamic traps of the disclosed devices, (ii) the identification of bonding processes for attaching a lid to the microfluidic substrate that are compatible with and preserve the integrity of the oligonucleotide arrays, (iii) the identification of attachment chemistries for tethering barcoded oligonucleotide molecules to an interior surface or coating layer of the device that are stable to prolonged exposure of the oligonucleotide arrays to aqueous buffers and cell culture media at elevated temperatures, (iv) the identification of an appropriate combination of oligonucleotide array feature (or spot) size, barcoded oligonucleotide surface density within the array feature(s), and efficient cleavage chemistries that provide for concentrations of barcoded oligonucleotide
  • a number refers to that number plus or minus 10% of that number.
  • the term‘about’ when used in the context of a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value.
  • microfluidic device and“microfluidic chip” (or simply, “device” or“chip”) are used interchangeably.
  • a trap may be used interchangeably and may refer to a feature comprising a constriction in one or two dimensions within a fluid channel that is designed to retain or trap cells or other objects suspended in a fluid.
  • a trap may comprise an entrance region, optionally, an interior region, and an exit region, at least one of which comprises a constriction.
  • an interior region of the trap may be significantly larger in at least one or two dimensions than the entrance region and/or exit region and may be configured to
  • the interior region of a hydrodynamic trap may be used as a cell culture chamber.
  • the term "object” generally refers to a cell or fragment thereof (e.g ., a cellular organelle such as a cell nucleus, mitochondrion, or exosome), an organism (e.g., a bacterium), a bead, a particle, a droplet (e.g, a liquid droplet), or in plural form, may refer to any combination thereof.
  • the term “cell” generally refers to any of a variety of cells known to those of skill in the art. In some instances, the term “cell” may refer to any adherent and non-adherent eukaryotic cell, mammalian cell, a primary or immortalized human cell or cell line, a primary or immortalized rodent cell or cell line, a cancer cell, a normal or diseased human cell derived from any
  • a white blood cell red blood cell, platelet, epithelial cell, endothelial cell, neuron, glial cell, astrocyte, fibroblast, skeletal muscle cell, smooth muscle cell, gamete, or cell from the heart, lungs, brain, liver, kidney, spleen, pancreas, thymus, bladder, stomach, colon, small intestine
  • a distinct cell subset such as an immune cell, a CD8 + T cell, CD4 + T cell, cancer stem cell, Lgr5/6 + stem cell, undifferentiated human stem cell, a human stem cell that has been induced to differentiate, a rare cell (e.g ., a circulating tumor cell (CTC), a circulating epithelial cell, a circulating endothelial cell, a circulating endometrial cell, a bone marrow cell, a progenitor cell, a foam cell, a mesen
  • the term "cell” may refer to an immune cell, e.g, a T cell, a cytotoxic (killer) T cell, a helper T cell, an alpha beta T cell, a gamma delta T cell, a T cell progenitor, a B cell, a B-cell progenitor, a lymphoid stem cell, a myeloid progenitor cell, a lymphocyte, a granulocyte, a Natural Killer cell, a plasma cell, a memory cell, a neutrophil, an eosinophil, a basophil, a mast cell, a monocyte, a dendritic cell, and/or a macrophage, or in plural form, to any combination thereof.
  • an immune cell e.g, a T cell, a cytotoxic (killer) T cell, a helper T cell, an alpha beta T cell, a gamma delta T cell, a T cell progenitor, a B cell, a B
  • the term “bead” generally refers to any type of solid, porous, or hollow spherical, non-spherical, or irregularly-shaped object composed of glass, plastic, ceramic, metal, a polymeric material, or any combination thereof.
  • the term “bead” may refer to a silica bead, a silica gel bead, a controlled pore glass bead, a magnetic bead (e.g, a
  • Sepharose bead, a cellulose bead, a polystyrene bead, etc., or in plural form, may refer to any combination thereof.
  • a bead may comprise tethered or immobilized capture, detection, or barcoding reagents, e.g, antibodies, cytokine-specific antibodies, chemokine- specific antibodies, growth factor-specific antibodies, enzymes, enzyme substrates, avidin or streptavidin, protein A, protein G, other proteins, small molecules, glycoproteins, drug molecules, polysaccharides, fluorophores, oligonucleotides, oligonucleotide aptamers, oligonucleotide barcodes, or any combination thereof.
  • a bead may be a cytokine-sensing bead such as multiplexed Luminex xMAP® immuno-assay beads sold by
  • Thermo Fischer (Waltham, MA), which can be used to detect from 3 to 30 different cytokines and growth factors.
  • the diameter or average diameter of a bead may be at least
  • target molecule may refer to any of a variety of biological molecules that may be tagged and identified using the single cell barcoding methods described herein. Examples include, but are not limited to, genomic or mitochondrial DNA sequences or fragments thereof, gene sequences or fragments thereof, exon sequences or fragments thereof, intron sequences or fragments thereof, RNA sequences or fragments thereof, mRNA sequences or fragments thereof, tRNA sequences or fragments thereof, rRNA sequences or fragments thereof, and microRNA sequences or fragments thereof.
  • target molecule may comprise proteins or other molecules expressed by single cells that may be detected using, e.g, oligonucleotide-labeled antibodies, oligonucleotide-labeled secondary antibodies, oligonucleotide-labeled antigens, and the like.
  • a target recognition sequence may be directed to ( e.g ., complementary to) a specific target molecule, e.g., a specific gene sequence.
  • a target recognition sequence may be directed to a class of target molecules, e.g, mRNA molecules within a single cell.
  • a target recognition sequence may be directed to random oligonucleotide sequences (e.g, where the“target recognition sequence” is itself a set of random sequences, e.g. a set of random heptamer sequences).
  • Methods, devices, and systems for single cell barcoding Methods, devices, and systems are described that may be used for a variety of single cell phenotyping, molecular barcoding, and/or molecular counting studies, including but not limited to DNA sequencing-based studies, RNA sequencing-based studies, gene expression profiling studies, genetically-engineered protein expression studies,“multi-omic” studies, and the like.
  • the devices comprise microfluidic chips that are designed to capture single cells or controlled numbers of cells to form small clusters, and then enable these captured cells to be maintained on chip for prolonged periods of time (e.g, many days) while being exposed to external perturbations, such as drugs, antibodies, or other stimulants.
  • the methods described herein include: a) methods for patterning a plurality of oligonucleotide arrays within the device, e.g, where each hydrodynamic trap contains an oligonucleotide array; b) methods to use these oligonucleotide arrays in the preparation of barcode-tagged sequences that are complementary to the RNA and DNA present in the trapped single cells; c) methods to analyze more than one type of molecular expression pattern, such as a combination of RNA + DNA, RNA + chromatin accessibility, RNA + protein expression; and the like; d) methods that enable these multi-omic measurements to be conducted in either a single pot reaction format or in a format that involves multi-step fluid additions.
  • the systems described herein may comprise: a) a high-content imager to acquire brightfield and/or fluorescent time-lapse images for each cell or cell culture (within the plurality of hydrodynamic traps) over many days, which are used in phenotypic analysis of single cell functional responses to environmental perturbations, and b) a thermal cycler and reagent delivery system that is used to implement the molecular barcoding and/or molecular counting studies, as discussed above, and c) data analysis methods that enable the simultaneous measurement of molecular expression and live-cell images from each cell, as well as methods to graphically present these datasets in a visual, interactive format.
  • Microfluidic device designs for efficient trapping of single cells provide microfluidic devices that enable highly efficient trapping of single cells or other objects by employing designs that exploit a previously unrecognized trait of mesh fluidic networks (see, e.g. , co-pending PCT International Patent Application No. PCT /US20 18/056221 (published as WO 2019/079399 Al)). Tuning the relative fluidic resistances of flow paths in a hydrodynamic fluidic circuit comprising a plurality of trapping features and at least two different types of interconnecting bypass channels ensures that all fluid flow
  • RT hydrodynamic resistance through the trap
  • RB short bypass channel sections
  • RA communal long bypass channel section
  • the traps within the array are populated sequentially in the order that cells are introduced, which in principle allows the disclosed devices to achieve near perfect efficiency in trapping single cells.
  • the disclosed devices are thus ideally suited for handling small cell samples where high trapping efficiencies are critical.
  • FIGS. 1A-B show an example of the hydrodynamic trapping geometry in a ladder configuration and the two flow regimes where the cell capture efficiency is low (FIG. 1 A) owing to the fluid flow splitting at the trap entrance or high (FIG. IB) owing to the fluid flow joining at the trap entrance.
  • FIGS. 2A-B show an example of the hydrodynamic trapping geometry in a mesh configuration and the two flow regimes where the cell capture efficiency is low (FIG. 2A) owing to the fluid flow splitting at the trap entrance or high (FIG. 2B) owing to the fluid flow joining at the trap entrance.
  • the disclosed device designs are based on mesh-like networks of fluid channels.
  • the devices comprise: a) a microfluidic network having at least one inlet and at least one outlet; b) a plurality of microfluidic constrictions (or“traps”), wherein a dimension of the constriction is smaller than a dimension of a suspended object contained within the fluid, and disposed so as to capture suspended objects flowing into the constriction; c) each microfluidic constriction comprising an entrance point or region and an exit point or region, and optionally, an interior region, d) the exit point of said microfluidic constriction is in direct fluidic connection with at least two additional microfluidic constrictions; e) the pressure at the exit point of said microfluidic constriction is higher than the pressure at the entrance point of either downstream microfluidic constriction when said microfluidic constriction has not yet captured a suspended object; and f) the pressure at the exit point of said microfluidic constriction
  • the disclosed microfluidic devices may comprise: a) a plurality of weir-traps disposed between, and in fluid communication with, at least one fluid inlet and at least one fluid outlet, wherein each weir-trap is configured to retain an object suspended in a fluid passing through the microfluidic device, and wherein: i) each weir-trap comprises an entrance region, an optional interior region, and an exit region that collectively constitute an interior fluid flow path through the weir-trap; ii) each weir-trap in a majority of the weir-traps (i.e., all of the weir-traps except for those nearest the at least one fluid inlet or at least one fluid outlet) is in fluid communication with either two or three exterior fluid flow paths (bypass fluid channels) that connect the exit region of a weir-trap to the entrance region of another weir-trap; and iii) a ratio of the fluidic resistance of one exterior fluid flow path (e.g, a longer,
  • the exit region of all or a portion of the weir-traps may comprise a frit to prevent cells or other objects from flowing out of the interior region (or chamber) of the trap.
  • the two or three exterior fluid flow paths may comprise one or two shorter fluid bypass channels comprising a fluidic resistance, R B , which is less than R A . In the case that there are two shorter fluid bypass channels, their fluidic resistance may be the same as each other, or different from each other, but will in either case be less than RA.
  • FIGS. 3A-D show the distribution of trapped cells in the microfluidic device as a function of the flow resistance ratio and trapping efficiency (q).
  • the ratio R A / R T may range from about 0.2 to about 2.0. In some embodiments, the ratio R A / R T may be at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, or at least 2.0. In some embodiments, the ratio R A / R T may be at most 2.0, at most 1.9, at most 1.8, at most 1.7, at most
  • the ratio R A / R T may range from about 0.4 to about
  • ratio R A / R T may have any value within this range, e.g ., about 1.25.
  • the ratio R A /R T is at least 1.2 and a capture probability for an individual hydrodynamic trap retaining a suspended object on first contact is at least 0.36. In some instances, the ratio R A /R T is at least 1.45 and a capture probability for an individual hydrodynamic trap retaining a suspended object on first contact is at least 0.60.
  • the capture probability for an individual weir-trap of the disclosed devices retaining a suspended cell or object on first contact may range from about 0.05 to about 0.99.
  • the capture probability may be at least 0.05, at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 0.95, or at least 0.99.
  • the capture probability may be at most 0.99, at most 0.95, at most 0.9, at most 0.8, at most 0.7, at most 0.6, at most 0.5, at most 0.4, at most 0.3, at most 0.2, at most 0.1, or at most 0.05. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example in some instances the capture probability may range from about 0.2 to about 0.8. Those of skill in the art will recognize that the capture probability may have any value within this range, e.g. , about 0.66.
  • the initial or pre-saturation trapping efficiencies for trapping cells or other objects suspended in a fluid passing through the disclosed weir-trap array devices may range from about 10% to about 100%.
  • the initial or pre-saturation trapping efficiency of the disclosed devices may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%.
  • the initial or pre-saturation trapping efficiency may be at most 99%, at most 98%, at most 95%, at most 90%, at most 80%, at most 70%, at most 60%, at most 50%, at most 40%, at most 30%, at most 20%, or at most 10%. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example the initial or pre- saturation trapping efficiency may range from about 40% to about 99%. Those of skill in the art will recognize that the pre-saturation trapping efficiency may have any value within this range, e.g. , about 97%.
  • the weir-traps of the disclosed microfluidic devices will generally comprise a constriction in at least one dimension, e.g ., an entry point or entrance region comprising a constriction that is smaller than the smallest dimension of the cell or object to be trapped.
  • the constriction in at least one dimension may range in size from about 10% to about 90% of the smallest dimension of the cell or object to be trapped.
  • the constriction may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the smallest dimension of the cell or object to be trapped. In some embodiments, the constriction may be at most 90%, at most 80%, at most 70%, at most 60%, at most 50%, at most 40%, at most 30%, at most 20%, or at most 10% of the smallest dimension of the cell or object to be trapped. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example the constriction may range in size from about 20% to about 70% of the smallest dimension of the cell or object to be trapped. Those of skill in the art will recognize that the constriction may have any value within this range, e.g. , about 33% of the smallest dimension of the cell or object to be trapped.
  • the weir-traps of the disclosed microfluidic devices will generally comprise a
  • constriction in at least one dimension e.g. , an entry point or entrance region comprising a constriction that is smaller than the smallest dimension of the cell or object to be trapped.
  • the constriction in at least one dimension may range in size from about 1 pm to about 100 pm.
  • the constriction in at least one dimension may have a dimension of at least 1 pm, at least 2 pm, at least 3 pm, at least 4 pm, at least 5 pm, at least 6 pm, at least 7 pm, at least 8 pm, at least 9 pm, at least 10 pm, at least 20 pm, at least 30 pm, at least 40 pm, at least 50 pm, at least 60 pm, at least 70 pm, at least 80 pm, at least 90 pm, or at least 100 pm.
  • the constriction in at least one dimension may have a dimension of at most 100 pm, at most 90 pm, at most 80 pm, at most 70 pm, at most 60 pm, at most 50 pm, at most 40 pm, at most 30 pm, at most 20 pm, at most 10 pm, at most 9 pm, at most 8 pm, at most 7 pm, at most 6 pm, at most 5 pm, at most 4 pm, at most 3 pm, at most 2 pm, at most 1 pm.
  • Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example the constriction in at least one dimension may range in size from about 3 pm to about 6 pm. Those of skill in the art will recognize that the constriction may have any dimension within this range, e.g. , about 4.5 pm.
  • the disclosed microfluidic devices may comprise: a) a plurality of weir-traps disposed between, and in fluid communication with, at least one fluid inlet and at least one fluid outlet, wherein each weir-trap is configured to retain an object suspended in a fluid passing through the microfluidic device, and wherein: i) each weir-trap comprises an entrance region, an interior region, and an exit region that collectively constitute an interior fluid flow path through the weir-trap; and ii) the volume of the interior region of the weir trap is greater than the volume of the entrance region or exit region.
  • the weir-trap designs of the disclosed microfluidic devices may comprise an entrance region (or entry point), optionally, an interior region (or chamber), and an exit region (or exit point).
  • the interior region (or chamber), if present, may have any of a variety of cross-sectional shapes within the plane of the microfluidic device.
  • the interior region may have a largely circular shape, elliptical shape, square shape, rectangular shape, triangular shape, hexagonal shape, irregular shape, or any combination thereof.
  • the exit regions of all or a portion of the weir-traps may comprise a frit structure, where the frit structure comprises one or more constrictions that have a spatial dimension that is smaller than the smallest dimension of the suspended objects.
  • the exit region of each weir-trap comprises a frit that forms the boundary of the interior region, and the interior region of the weir- trap is quite large in comparison to the entrance region comprising the constriction used to trap cells or objects suspended in a fluid.
  • the weir-traps in this example again comprise a frit within the exit region of the trap.
  • the disclosed microfluidic devices may comprise: a) a plurality of weir-traps disposed between, and in fluid communication with, at least one fluid inlet and at least one fluid outlet, wherein each weir-trap is configured to retain an object suspended in a fluid passing through the microfluidic device, and wherein: i) each weir-trap comprises a constriction in at least one dimension that is smaller than the smallest dimension of the object; and ii) a ratio of a fluidic resistance of a fluid flow path that bypasses a weir-trap to that for a fluid flow path passing through the weir-trap is at least 0.4.
  • the constriction in at least one dimension may range in size from about 10% to about 90% of the smallest dimension of the cell or object to be trapped.
  • the resistance of the fluid flow path that bypasses the weir-trap to that for the fluid flow path passing through the weir-trap may range from about 0.4 to about 2.0.
  • Non-limiting examples of combinations of constriction dimension (specified in terms of the percentage of the smallest dimension of the cell or object to be trapped) and resistance ratio (R A / R T ) that are included in the present disclosure are (10%, 0.5), (10%, 0.6), (10%, 0.7), (10%, 0.8), (10%, 0.9), (10%, 1.0), (10%, 1.1), (10%, 1.2), (10%, 1.3), (10%, 1.4),
  • the hydrodynamic traps may be configured to trap single cells or sub- cellular organelles, e.g ., nuclei. In some embodiments, the hydrodynamic traps may be configured to trap single cell pairs (i.e., pairs of single cells). In some instances, the
  • hydrodynamic traps may be configured to trap small groups of cells. In some instances, the hydrodynamic traps may be configured to trap groups of cells that may be used to establish organoids. In some instances, the hydrodynamic traps may be configured to function as cell growth chambers for trapping single cells and incubating them under conditions that support cell growth and division, thereby establishing small clonal groups of cells within a hydrodynamic trap.
  • the microfluidic devices disclosed herein may comprise at least two separately fabricated parts (e.g, (i) a substrate that incorporates etched, embossed, or ablated fluid channels, and (ii) a cover or lid) that are subsequently either mechanically clamped together, temporarily adhered together, or
  • the microfluidic devices disclosed herein may comprise three or more separately fabricated parts (e.g, (i) a substrate, (ii) a fluid channel layer, and (iii) a cover or lid) that are subsequently either mechanically clamped together, temporarily adhered together, or permanently bonded together.
  • the microfluidic devices disclosed herein may comprise a removable cover or lid.
  • suitable fabrication techniques include, but are not limited to, conventional machining, CNC machining, injection molding, 3D printing, alignment and lamination of one or more layers of laser- or die-cut polymer film, or any of a number of microfabrication techniques such as photolithography and wet chemical etching, dry etching, deep reactive ion etching (DRIE), or laser micromachining.
  • DRIE deep reactive ion etching
  • all or a portion of the microfluidic devices may be
  • microfluidic devices disclosed herein or the components thereof may be fabricated using any of a variety of materials known to those of skill in the art. In general, the choice of material used will depend on the choice of fabrication technique, and vice versa. Examples of suitable materials include, but are not limited to, silicon, fused-silica, glass, any of a variety of polymers, e.g. polydimethylsiloxane (PDMS; elastomer),
  • PDMS polydimethylsiloxane
  • PMMA polymethylmethacrylate
  • PC polycarbonate
  • PS polystyrene
  • PP polypropylene
  • PE polyethylene
  • HDPE high density polyethylene
  • COP cyclic olefin polymers
  • COC cyclic olefin copolymers
  • PET polyethylene terephthalate
  • PTFE non-stick material
  • any of a variety of photoresists such as SU8 or any other thick film photoresist, or any combination of these materials.
  • all or a portion of the microfluidic device may be fabricated from an optically transparent material to facilitate observation and monitoring (e.g, using high-resolution bright-field and/or fluorescence imaging) of cells or objects entrapped within the device.
  • the different layers in a microfluidic device comprising multiple layers may be fabricated from different materials, e.g, a fluid channel layer may be fabricated from an elastomeric material while the device substrate and a cover plate may be fabricated from glass or another suitable material.
  • the thickness of a fluid channel layer will determine the depth of the fluid channels and microfluidic chambers (e.g,“micro-chambers”, “trapping chambers”, or the interior regions of the traps) within the device and will thus influence the volume of the trapping chambers.
  • the depth of the fluid channels and trapping chambers within the device will determined by the etch depth, embossed depth, or ablation depth, and will thus influence the volume of the trapping chambers.
  • the fluid channels and trapping chambers may have the same depth or different depths.
  • the depth of fluid channels and/or trapping chambers within the disclosed devices may range from about 1 pm and about 1 mm.
  • the depth of the fluid channels and/or trapping chambers may be at least 1 pm, at least 5 pm, at least 10 pm, at least 20 pm, at least 30 pm, at least 40 pm , at least 50 pm, at least 100 pm, at least 200 pm, at least 300 pm, at least 400 pm, at least 500 pm, at least 600 pm, at least 700 pm, at least 800 pm, at least 900 pm, or at least 1 mm.
  • the depth of the fluid channels and/or trapping chambers may be at most 1 mm, at most 900 pm, at most 800 pm, at most 700 pm, at most 600 pm, at most 500 pm, at most 400 pm, at most 300 pm, at most 200 pm, at most 100 pm, at most 50 pm, at most 40 pm, at most 30 pm, at most 20 pm, at most 10 pm, at most 5 pm, or at most 1 pm.
  • the depth of the fluid channels and/or trapping chambers may range from about 50 pm to about 100 pm. Those of skill in the art will recognize that depth of the fluid channels and/or trapping chambers may have any value within this range, for example, about 95 pm.
  • the volumes of the microfluidic chambers may range from about 1,000 pm 3 to about 1 mm 3 .
  • the microfluidic chamber volume may be at least 1,000 pm 3 , at least 10,000 pm 3 , at least 100,000 pm 3 , at least 1,000,000 pm 3 , at least 0.2 mm 3 , at least 0.5 mm 3 , or at least 1 mm 3 .
  • the microfluidic chamber volume is at most 1 mm 3 , at most 0.5 mm 3 , at most 0.2 mm 3 , at most 1,000,000 pm 3 , at most 100,000 pm 3 , at most 10,000 pm 3 , or at most 1,000 pm 3 . Any of the lower and upper values described in this paragraph may be combined to form a range included within the disclosure, for example, the microfluidic chamber volume may range from about 100,000 pm 3 to about 0.2 mm 3 . Those of skill in the art will recognize that the chamber volume may have any value within this range, for example, about 1,250,000 pm 3 .
  • the number of weir-traps and/or microfluidic chambers in the plurality of traps and/or chambers contained within a device of the present disclosure may range from about 1 to about 10 6 , or more. In some embodiments, the number of traps and/or chambers within the device may be at least 1, at least 10, at least 100, at least 1,000, at least 10 4 , at least 10 5 , or at least 10 6 . In some embodiments, the number of traps and/or chambers within the device may be at most 10 6 , at most 10 5 , at most 10 4 , at most 1,000, at most 100, or at most 1.
  • the number of traps and/or chambers within the device may range from about 100 to about 10,000.
  • the number of traps and/or chambers within the device may have any value within this range, for example, about 1,200.
  • the pitch (or spacing) between weir-traps may range from about 100 pm to about 1,000 pm, or more. In some embodiments, the pitch between weir-traps may be at least at least 100 pm, at least 200 pm, at least 300 pm, at least 400 pm, at least 500 pm, at least 600 pm, at least 700 pm, at least 800 pm, at least 900 pm, or at least 1,000 pm. In some embodiments, the pitch between weir-traps may be at most 1,000 pm, at most 900 pm, at most 800 mih, at most 700 mm, at most 600 mih, at most 500 mih, at most 400 mih, at most 300 mih, at most 200 mm, or at most 100 mih.
  • the pitch between weir-traps may range from about 200 pm to about 400 pm.
  • the pitch between weir-traps may have any value within this range, for example, about 220 pm.
  • one or more interior surfaces of the disclosed microfluidic devices may comprise one or more coating layers that, e.g ., facilitate attachment of the oligonucleotide barcode molecules of the oligonucleotide arrays and/or reduce non-specific binding of proteins to the interior surfaces of the device.
  • suitable coating materials may include, but are not limited to, silane coatings, linear or branched polyethylene glycol (PEG) coatings, polydimethylsiloxane (PDMS) coatings, hydrogel coatings, and the like, or any combination of these.
  • the one or more interior surfaces of the disclosed microfluidic devices may comprise one, two, three, four, five, or more than five coating layers.
  • the oligonucleotide barcode molecules of the oligonucleotide arrays may be tethered to (or entrapped within) any one coating layer or tethered to (or entrapped within) any combination of two, three, four, five, or more than five coating layers. In some instances, the oligonucleotide barcode molecules of the oligonucleotide arrays may be embedded within one, two, three, four, five, or more than five coating layers.
  • the bonding of microfluidic device components may comprise the use of a technique that provides a reliable seal while not adversely affecting the integrity of the oligonucleotide arrays disposed within the device.
  • bonding techniques include, but are not limited to, use of adhesive layers or films (e.g, UV-curable adhesives or heat-curable adhesives), thin polydimethylsiloxane (PDMS) layers and coatings thereon, room temperature anodic bonding of glass to silicon, local ultrasonic welding, laser welding, and the like, or any combination thereof.
  • the substrate is fabricated in silicon
  • the lid is glass
  • the bonding step comprises coating a thin film of polydimethylsiloxane (PDMS) on the glass lid prior to bonding it to the silicon substrate.
  • the substrate is fabricated in silicon
  • the lid comprises a glass substrate with a polydimethylsiloxane (PDMS) layer coated thereon
  • the bonding comprises coating and patterning a thin film of hydrogel on the PDMS layer prior to bonding the lid to the silicon substrate.
  • the bonding step comprises application of a thin coating layer of epoxy to the lid prior to fabricating the plurality of oligonucleotide arrays.
  • the epoxy is a UV-curable epoxy or a heat-curable epoxy.
  • the bonding step comprises formation of a hydrogel layer between the lid and substrate using thermal- or UV-induced polymerization of acrylate, methacrylate, or acrylamide groups that are attached to both surfaces.
  • the hydrogel layer between the lid and substrate comprises a cross-linked gel made by Michael addition of terminal thiol groups of a first polymer to acrylic or maleimide groups of a second polymer.
  • one of the polymers is a branched polymer, e.g ., a 4-armed polymer, 8-armed, 12- armed, 16-armed, or 32-armed polymer.
  • the acrylate groups are co-polymers containing both polydimethylsiloxane sequences and acrylate or methacrylate polymer sequences.
  • the substrate and/or the lid further comprise one or more fiducial marks to facilitate alignment of the lid and substrate during assembly.
  • the chips may be further packaged in a substrate carrier that facilitates ease-of-handling and/or facilitates interfacing the microfluidic device with external system components.
  • the substrate carrier (or chip package, housing, etc.) may comprise additional functional components.
  • the substrate carrier may comprise integrated thermal sensors and/or controllers for local temperature control, gas connections that enable interfacing the device with external pressure controllers, and microfluidic interfaces (e.g, fluid connectors) that interface with external fluidics controllers to allow control of fluid flow within the microfluidic devices.
  • the carrier may be comprised of a reusable housing that is machined in metal, such as aluminum or steel, or it may be composed of a single use disposable plastic housing that is fabricated by low-cost manufacturing techniques such as plastic injection molding.
  • the housing may further comprise one or more thermal heating and sensing elements, such as resistive heating elements, thermoelectric modules, or another heat source that can be externally controlled, and may also comprise temperature monitoring capability using, e.g, one or more thermistors, thermocouples, resistive temperature detectors, thermostats, or other temperature sensing devices.
  • the temperature inside the plastic housing may be held at constant temperatures, such as at 4 °C, 21 °C, 37 °C, 42 °C, 50 °C, etc.
  • the temperature may be cycled between different set temperatures (e.g, a set temperature from about 48 °C to about 65 °C for annealing, a set temperature from about 68 °C to about 72 °C for primer extension, and a set temperature from about 94 °C to about 98 °C for denaturation) and cycle times (e.g, cycle times ranging from about 10 seconds per cycle, 20 seconds per cycle, 30 second per cycle, 1 minute per cycle, 2 minutes per cycle, 3 minutes per cycle, 4 minutes per cycle, 5 minutes per cycle, 6 minutes per cycle, 7 minutes per cycle, 8 minutes per cycle, 9 minutes per cycle, 10 minutes per cycle, or more than 10 minutes per cycle (or any cycle time within this range) in applications that require
  • set temperatures e.g, a set temperature from about 48 °C to about 65 °C for annealing, a set temperature from about 68 °C to about 72 °C for primer extension, and a set temperature from about 94 °C to about 98 °C for den
  • the disclosed microfluidic devices may comprise oligonucleotide arrays disposed in all or a portion of the plurality of hydrodynamic traps within the device. In some instances, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% of the hydrodynamic traps within the device may comprise an oligonucleotide array.
  • each oligonucleotide array may comprise from between 1 and 1,000 features or spots. In some instances, each oligonucleotide array may comprise at least 1, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1,000 features or spots.
  • each oligonucleotide array may comprise at most 1,000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 400, at most 300, at most 200, at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, at most 10, or at most 1 feature or spot. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances each oligonucleotide array may comprise from about 10 to about 100 features or spots. Those of skill in the art will recognize that the number of features or spots in the oligonucleotide arrays may have any value within this range, e.g.
  • the number of features or spots in each oligonucleotide array within the device may be the same. In some instances, the number of features or spots in different oligonucleotide arrays within the device may be different.
  • the optimal size of the features or spots in an oligonucleotide array may be determined by the fabrication technique used, the surface density of the resulting tethered oligonucleotide molecules within the feature or spot, the diameter or area of the feature or spot, and/or the volume of the hydrodynamic trap chamber in which the oligonucleotide array is disposed (in order to ensure that an effective concentration of oligonucleotide barcode molecules (or barcoded primers) is compatible with performing molecular biology procedures such as reverse transcription, DNA polymerization, and/or DNA amplification reactions.
  • the average diameter or longest dimension of the oligonucleotide array features or spots may range from about 10 pm to about 1 mm. In some instances, the average diameter or longest dimension of the features or spots is at least 10 pm, at least 15 pm, at least 20 mih, at least 25 mih, at least 30 mih, at least 35 mih, at least 40 mih, at least 45 mih, at least 50 mih, at least 60 mih, at least 70 mih, at least 80 mih, at least 90 mih, at least 100 mih, at least 200 mih, at least 300 mih, at least 400 mih, at least 500 mih, at least 600 mih, at least 700 mih, at least 800 mih, at least 900 mih, or at least 1,000 mih. In some instances, the average diameter or longest dimension of the features or spots is at most 1,000 mih, at most 900 mih, at most 800 mih, at most
  • the average diameter or longest dimension of the features or spots may have any value within this range, e.g ., about 74 pm.
  • the features or spots within an oligonucleotide array may all have the same average diameter or longest dimension. In some instances, different features or spots within an oligonucleotide array may have different average diameters or longest dimensions.
  • the surface density of tethered oligonucleotide barcode molecules or primers within a given feature or spot may range from about 100 molecules per pm 2 to about
  • the surface density of oligonucleotide barcode molecules or primers may be at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000, at least
  • oligonucleotide barcode molecules or primers may be at most
  • the surface density of oligonucleotide barcode molecules or primers may range from about 10,000 molecules per pm 2 to about 90,000 molecules per pm 2 .
  • the surface density of oligonucleotide barcode molecules or primer molecules may have any value within this range, e.g. , about 85,600 molecules per pm 2 .
  • the plurality of oligonucleotide molecules within each feature or spot on the oligonucleotide array may comprise a common barcode sequence, e.g., a cell barcode sequence, that is presented in all of the features of a given oligonucleotide array and provides a unique identifier for determining, e.g, the identity of a single cell from which barcoded DNA molecules, RNA molecules, etc., were derived.
  • the common barcode sequence or unique cell barcode sequence is the same for each feature within a given oligonucleotide array, and different from that for all features in all other oligonucleotide arrays within the device. In some instances, the common barcode sequence or unique cell barcode sequence is different for each feature within a given oligonucleotide array, and different from that for all features in all other oligonucleotide arrays within the device. In some instances, the common barcode sequence or unique cell barcode sequence for each feature of each oligonucleotide array of the plurality of oligonucleotide arrays is known. In some instances, the identity of each oligonucleotide sequence for each feature in the plurality of oligonucleotide arrays may be determined using a sequencing-by-hybridization approach.
  • the unique cell barcode sequence may comprise a string of N“words”, wherein each“word” comprises a string of M bases.
  • M may be 1 base, 2 bases, 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, or at least 10 bases.
  • N is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12, such that the common barcode or unique cell barcode sequence comprises up to at least 12“words”, each“word” comprising a string of up to
  • the common barcode or unique cell barcode may range in length from about 1 base to about 120 bases.
  • the length of the common barcode or unique cell barcode may be at least 1 base, at least 5 bases, at least 10 bases, at least 20 bases, at least 30 bases, at least 40 bases, at least 50 bases, at least 60 bases, at least 70 bases, at least 80 bases, at least 90 bases, at least 100 bases, as least 110 bases, or at least 120 bases.
  • the length of the common barcode or unique cell barcode may be at most 120 bases, at most 110 bases, at most 100 bases, at most 90 bases, at most 80 bases, at most 70 bases, at most 60 bases, at most 50 bases, at most 40 bases, at most 30 bases, at most 20 bases, at most 10 bases, at most 5 bases, or at most 1 base. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the length of the common barcode or unique cell barcode may range from 10 bases to 20 bases. Those of skill in the art will recognize that the length of the common barcode or unique cell barcode may have any value within this range, e.g, 42 bases. [0096] In some instances, the plurality of common barcode sequences or unique cell barcode sequences for the plurality of hydrodynamic traps within a device comprise unique non overlapping sequences with a Hamming distance that enables demultiplexing with error correction capability.
  • the common barcode sequence or unique cell barcode sequence may comprise a G/C content ranging from about 10% to about 90%. In some instances, the common barcode sequence or unique cell barcode sequence may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% G/C content. In some instances, the common barcode sequence or unique cell barcode sequence may comprise at most 90%, at most 80%, at most 70%, at most 60%, at most 50%, at most 40%, at most 30%, at most 20%, or at most 10% G/C content.
  • the common barcode or unique cell barcode may comprise from about 30% to about 70% G/C content.
  • the common barcode or unique cell barcode may comprise any value of G/C content within this range, e.g. , about 54% G/C content.
  • the tethered oligonucleotide barcode molecules or primers of the oligonucleotide arrays may comprise a free 5’ end. In some instances, the tethered
  • oligonucleotide barcode molecules or primers of the oligonucleotide arrays may comprise a free 3’ end.
  • the oligonucleotide barcode molecules within the features of the oligonucleotide arrays may further comprise a spacer sequence, an adapter sequence, at least one primer sequence (e.g, a universal primer sequence), a molecular index sequence, a molecular recognition or target capture sequence, a random heptamer capture sequence, a cleavable moiety or cleavage site, a surface attachment moiety, or any combination thereof.
  • primer sequence e.g, a universal primer sequence
  • oligonucleotide molecules of the array may comprise a molecular recognition or target capture sequence designed to capture specific target molecules, e.g, specific gene sequences or gene fragment sequence, or to capture specific classes of target molecules, e.g, using a poly-T sequences to capture poly-A tagged RNA molecules.
  • the molecular recognition or target capture sequence may comprise a capture sequence designed to capture a broad class of target molecules, e.g, when using a random capture sequence (a“randomer”) to hybridize to genomic DNA.
  • Suitable molecular recognition or target capture sequences include, but are not limited to, an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(deoxyguanosine) (oligo-dG) sequence, an oligo(riboguanosine) (oligo-rG) sequence, a sequence comprising a phosphorothioate oligonucleotide, a sequence comprising a locked nucleic acid, a sequence containing 2-OMe oligonucleotides, a sequence containing modified nucleotides, a complement of an mRNA sequence or fragment thereof, a complement of a tRNA sequence or fragment thereof, a complement of an rRNA sequence or fragment thereof, a complement of a DNA sequence, a complement of a cDNA sequence, a complement of a gene sequence or fragment thereof, or any combination thereof.
  • the molecular recognition or target capture sequence may comprise a random sequence (or“randomer”).
  • the molecular recognition or target capture sequence may comprise, for example, a random tetramer, a random pentamer, a random hexamer, a random heptamer, a random octamer, a random nonamer, a random decamer, a random undecamer, a random dodecamer, or a random multimer, and the like.
  • the molecular recognition or target capture sequence may range in length from about 1 base to about 150 bases.
  • the length of the molecular recognition or target capture sequence may be at least 1 base, at least 2 bases, at least 5 bases, at least 10 bases, at least 20 bases, at least 30 bases, at least 40 bases, at least 50 bases, at least 60 bases, at least 70 bases, at least 80 bases, at least 90 bases, at least 100 bases, as least 110 bases, as least 120 bases, as least 130 bases, as least 140 bases, or at least 150 bases.
  • the length of the molecular recognition or target capture sequence may be at most 150 bases, at most 140 bases, at most 130 bases, at most 120 bases, at most 110 bases, at most 100 bases, at most 90 bases, at most 80 bases, at most 70 bases, at most 60 bases, at most 50 bases, at most 40 bases, at most 30 bases, at most 20 bases, at most 10 bases, at most 5 bases, at most 2 bases, or at most 1 base. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the length of the molecular recognition or target capture sequence may range from 2 bases to 50 bases. Those of skill in the art will recognize that the length of the molecular recognition or target capture sequence may have any value within this range, e.g ., 12 bases.
  • the oligonucleotide molecules in each feature of an oligonucleotide array may comprise a molecular recognition sequence that is the same as that for the other features within the oligonucleotide array. In some instances, the oligonucleotide molecules in each feature of an oligonucleotide array may comprise a molecular recognition sequence that is different from that for the other features within the oligonucleotide array. In some instances, the oligonucleotide molecules in each feature of an oligonucleotide array may comprise a molecular recognition sequence that is the same as that for a subset of other features within the array and different from the remainder of features in the array.
  • the oligonucleotide molecules of the oligonucleotide arrays may comprise a molecular index (or counter) sequence that is unique for each individual oligonucleotide molecule of the plurality of oligonucleotides within a given feature, and where the sequence diversity represented by the plurality of unique molecular index sequences presented within a given feature is large compared to the expected number of target molecules to be derived from a single cell, thereby allowing one to count the number of barcoded target molecules (or complements thereof) derived from a single cell by determining the number of unique molecular index sequences that share the same cell barcode in a sequencing run performed after the barcoded target molecules have been amplified and sequenced.
  • a molecular index (or counter) sequence that is unique for each individual oligonucleotide molecule of the plurality of oligonucleotides within a given feature, and where the sequence diversity represented by the plurality of unique molecular index sequences presented within
  • the molecular index sequence may range in length from about 1 base to about 150 bases.
  • the length of the molecular index sequence may be at least 1 base, at least 2 bases, at least 5 bases, at least 10 bases, at least 20 bases, at least 30 bases, at least 40 bases, at least 50 bases, at least 60 bases, at least 70 bases, at least 80 bases, at least 90 bases, at least 100 bases, as least 110 bases, as least 120 bases, as least 130 bases, as least 140 bases, or at least 150 bases.
  • the length of the molecular index sequence may be at most 150 bases, at most 140 bases, at most 130 bases, at most 120 bases, at most 110 bases, at most 100 bases, at most 90 bases, at most 80 bases, at most 70 bases, at most 60 bases, at most 50 bases, at most 40 bases, at most 30 bases, at most 20 bases, at most 10 bases, at most 5 bases, at most 2 bases, or at most 1 base. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the length of the molecular index sequence may range from 5 bases to 10 bases. Those of skill in the art will recognize that the length of the molecular recognition or target capture sequence may have any value within this range, e.g ., 7 bases.
  • the oligonucleotide barcode molecules of the oligonucleotide arrays disposed within the device may comprise specific functional groups at the 3’ or 5’ end that facilitate attachment of the oligonucleotides to a surface or coating layer.
  • Examples include, but are not limited to, amine-terminated oligonucleotides (for grafting onto surfaces or coating layers functionalized with an epoxide, glicydyl, aldehyde, NHS ester, or other amine-reactive moiety), azide-terminated oligonucleotides (for grafting onto alkyne- functionalized surfaces or coating layers using a type of“click chemistry”), alkyne-terminated oligonucleotides (for grafting onto azide-functionalized surfaces or coating layers), acroxyl- terminated oligonucleotides (for grafting onto acrylamide or methacrylamide functionalized surfaces or coating layers), and biotin-terminated oligonucleotides (for attachment to streptavidin-functionalized surfaces or coating layers), maleimide- or acrylate-terminated oligonucleotides (for grafting onto thiol-functionalized surfaces or coating layers), and thiol-
  • the oligonucleotide barcode molecules of the oligonucleotide arrays disposed within the device may comprise a cleavable moiety or cleavage site that allows the oligonucleotide molecules within one or more features of the oligonucleotide array to be cleaved and released into solution with the hydrodynamic trap, thereby enabling more efficient solution-phase priming of target molecules, reverse transcription, DNA polymerization, DNA amplification, etc.
  • suitable cleavage mechanisms include, but are not limited to, sequences or linkers that are cleaved using light (e.g. , UV light), heat, enzymes (e.g, a USERTM enzyme), reducing or oxidizing agents, a change in pH, or any combination thereof.
  • the cleavage site comprises a deoxyuridine base.
  • USERTM enzyme a mixture of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII
  • UDG Uracil DNA glycosylase
  • apyrimidinic DNA glycosylase-lyase Endonuclease VIII
  • the 5’ end of the oligonucleotide barcode molecules may be released into solution upon treatment with light (e.g, UV light), heat, enzymes (e.g, a USERTM enzyme), reducing or oxidizing agents, a change in pH, or any combination thereof.
  • the 3’ end of the oligonucleotide barcode molecules may be released into solution upon treatment with light (e.g, UV light), heat, enzymes (e.g, a USERTM enzyme), reducing or oxidizing agents, a change in pH, or any combination thereof.
  • the oligonucleotide molecules in every feature of an oligonucleotide array may comprise the same cleavable moiety, e.g, a photocleavable linker, an enzymatically- cleavable linker, a pH-sensitive cleavable linker, a redox-cleavable linker, or a thermally- cleavable linker.
  • a photocleavable linker e.g, an enzymatically- cleavable linker, a pH-sensitive cleavable linker, a redox-cleavable linker, or a thermally- cleavable linker.
  • the oligonucleotide molecules in different features of the oligonucleotide array may comprise different cleavable moieties, e.g, linkers cleaved by orthogonal cleavage mechanisms, so that barcoded oligonucleotide molecules can be selectively released from different features of the array at, for example, different steps in a sequencing library preparation workflow.
  • the oligonucleotide barcode molecules of the oligonucleotide arrays disposed within the device may comprise one or more spacer sequences disposed between the surface or coating layer to which the oligonucleotide molecules are tethered and the remainder of the oligonucleotide sequence or between other functional components of the oligonucleotide sequence (e.g ., between a molecular recognition sequence and a molecular index sequence, etc.).
  • the oligonucleotide barcode molecules of the oligonucleotide arrays disposed within the device may comprise one or more spacer sequences disposed between the surface or coating layer to which the oligonucleotide molecules are tethered and a cleavage site. In some instances, the oligonucleotide barcode molecules of the oligonucleotide arrays disposed within the device may comprise one or more spacer sequences disposed between a cleavage site and the remainder of the remainder of the oligonucleotide sequence.
  • the one or more spacer sequences may range in length from about 1 base to about 50 bases.
  • the length of the spacer sequence may be at least 1 base, at least 2 bases, at least 5 bases, at least 10 bases, at least 15 bases, at least 20 bases, at least 25 bases, at least 30 bases, at least 35 bases, at least 40 bases, at least 45 bases, or at least 50 bases.
  • the length of the spacer sequence may be at at most 50 bases, at most 45 bases, at most 40 bases, at most 35 bases, at most 30 bases, at most 25 bases, at most 20 bases, at most 15 bases, at most 10 bases, at most 5 bases, at most 2 bases, or at most 1 base.
  • the length of the spacer sequence may range from 10 bases to 20 bases.
  • the length of the spacer sequence may have any value within this range, e.g., 14 bases.
  • Adapter sequences In some instances, the oligonucleotide barcode molecules of the oligonucleotide arrays disposed within the device may comprise one or more adapter sequences for facilitating downstream compatibility with Next-Generation sequencing platforms. Examples of suitable adapter sequences include, but are not limited to, the Illumina P5/P7 adapter sequences that bind to the Illumina platform flowcells.
  • the one or more adapter sequences may range in length from about 6 bases to about 30 bases.
  • the length of the adapter sequence may be at least 6 bases, at least 7 bases, at least 8 bases, at least 9 bases, at least 10 bases, at least 11 bases, or at least 12 bases.
  • the length of the adapter sequence may be at most 12 bases, at most 11 bases, at most 10 bases, at most 9 bases, at most 8 bases, at most 7 bases, or at most 6 bases. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the length of the adapter sequence may range from 7 bases to 11 bases.
  • the length of the adapter sequence may have any value within this range, e.g, 10 bases.
  • the oligonucleotide barcode molecules of the oligonucleotide arrays disposed within the device may comprise one or more primer sequences
  • the oligonucleotide barcodes may comprise 1, 2, 3, 4, 5, 6, or more than 6 primer sequences.
  • the one or more primer sequences may range in length from about 10 bases to about 50 bases.
  • the length of the primer sequence may be at least 10 bases, at least 15 bases, at least 20 bases, at least 25 bases, at least 30 bases, at least 35 bases, at least 40 bases, at least 45 bases, or at least 50 bases.
  • the length of the primer sequence may be at most 50 bases, at most 45 bases, at most 40 bases, at most 35 bases, at most 30 bases, at most 25 bases, at most 20 bases, at most 15 bases, or at most 10 bases. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the length of the primer sequence may range from 20 bases to 30 bases. Those of skill in the art will recognize that the length of the primer sequence may have any value within this range, e.g., 24 bases.
  • the oligonucleotide barcode molecules of one or more features within an oligonucleotide array may comprise all or a portion of a gene sequence or fragment thereof.
  • the plurality of gene sequence fragments presented by the one or more features of the oligonucleotide array collectively span a full-length gene sequence.
  • the average length of the gene sequence fragments presented in features of the array may range from about 0.01 kilobase (10 bases) to about 1 kilobase (1,000 bases). In some instances, the average length of the gene sequence fragments may be at least 0.01 kilobase, at least 0.02 kilobase, at least 0.1 kilobase, at least 0.2 kilobase, at least 0.3 kilobase, at least 0.4 kilobase, at least 0.5 kilobase, at least 0.6 kilobase, or at least 0.7 kilobase, at least 0.8 kilobase, at least 0.9 kilobase, or at least 1 kilobase.
  • the average length of the gene sequence fragments may be at most 1 kilobase, at most 0.9 kilobase, at most 0.8 kilobase, at most 0.7 kilobase, at most 0.6 kilobase, at most 0.5 kilobase, at most 0.4 kilobase, at most 0.3 kilobase, at most 0.2 kilobase, or at most 0.1 kilobase. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the average length of gene sequence fragments may range from 0.2 kilobase to 0.9 kilobase.
  • the average length of the gene sequence fragments may have any value within this range, e.g, about 0.56 kilobase.
  • the average length of a full-length gene sequence assembled within the disclosed microfluidic devices using two or more gene sequence fragments presented in the features of an oligonucleotide array disposed within a trap of the device may range from about
  • the average length of the full-length gene sequence may be at least 0.5 kilobase, at least 1 kilobase, at least 1.5 kilobase, at least 2 kilobase, at least 2.5 kilobase, at least 3 kilobase, at least 3.5 kilobase, at least 4 kilobase, at least 4.5 kilobase, at least 5 kilobase, at least 5.5 kilobase, at least 6 kilobase, at least 6.5 kilobase, at least
  • the average length of the full-length gene sequence may be at most 10 kilobase, at most 9.5 kilobase, at most 9 kilobase, at most 8.5 kilobase, at most 8 kilobase, at most 7.5 kilobase, at most 7 kilobase, at most 6.5 kilobase, at most 6 kilobase, at most 5.5 kilobase, at most 5 kilobase, at most 4.5 kilobase, at most 4 kilobase, at most 3.5 kilobase, at most 3 kilobase, at most 2.5 kilobase, at most 2 kilobase, at most 1.5 kilobase, at most 1 kilobase, or at most 0.5 kilobase.
  • the average length of full-length gene sequence may range from 0.2 kilobase to 6 kilobase.
  • the average length of the full-length gene sequence may have any value within this range, e.g ., about 2.8 kilobase.
  • the oligonucleotide molecules of the plurality of arrays within the device may comprise conventional nucleotide building blocks.
  • the oligonucleotide molecules of the plurality of arrays may comprise one or more modified nucleotides as building blocks, e.g. , including but not limited to, locked nucleic acid sequences, ribonucleotides, and phosphorthioated nucleotides.
  • the total length of the oligonucleotide molecules may range from about 20 bases to about 200 bases.
  • the length of the oligonucleotide molecules may be at least 20 bases, at least 30 bases, at least 40 bases, at least 50 bases, at least 60 bases, at least 70 bases, at least 80 bases, at least 90 bases, at least 100 bases, as least 110 bases, at least 120 bases, at least 130 bases, at least 140 bases, at least 150 bases, at least 160 bases, at least 170 bases, at least 180 bases, at least 190 bases, or at least 200 bases.
  • the length of the oligonucleotide molecules may be at most 2000 bases, at most 190 bases, at most 180 bases, at most 170 bases, at most 160 bases, at most 150 bases, at most 140 bases, at most 130 bases, at most 120 bases, at most 110 bases, at most 100 bases, at most 90 bases, at most 80 bases, at most 70 bases, at most 60 bases, at most 50 bases, at most 40 bases, at most 30 bases, or at most 20 bases. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the length of the oligonucleotide molecules may range from 70 bases to 120 bases. Those of skill in the art will recognize that the length of the oligonucleotide molecules may have any value within this range, e.g ., 74 bases.
  • Oligonucleotide barcode array fabrication A variety of techniques may be used to synthesize and/or deposit oligonucleotide arrays within the hydrodynamic traps of the disclosed microfluidic devices (as illustrated in FIG. 6). The plurality of oligonucleotide arrays (or molecular patterns) can be deposited in the plurality of hydrodynamic traps, e.g. , one
  • oligonucleotide array per hydrodynamic trap or in a portion of the interior region of each hydrodynamic trap (FIG. 6), by any of a variety of techniques known to those of skill in the art including, but not limited to, inkjet printing, microarray spotting, solid phase oligonucleotide synthesis (e.g, using a spatially-addressable solid-phase synthesis technique), enzymatic extension, enzymatic ligation of one or more smaller oligonucleotide segments, or a pattern transfer process, such as stamping or contact printing. In some instances, contact printing or stamping may be used to create a replica of the molecular pattern (or its inverse) on a surface.
  • the oligonucleotide barcode sequences may be introduced into the hydrodynamic traps via beads, vesicles, virus particles, or genomic material contained within a cell or virus particle.
  • one or more coating layers are added to an interior surface of the substrate or to a first surface of the lid for the purpose of attaching entrapping the oligonucleotide molecules of the oligonucleotide arrays.
  • the plurality of oligonucleotide arrays may be attached to the underside of the lid used to seal the device and/or deposited directly on the substrate (e.g. within the interior regions of the hydrodynamic traps), and maintained in place through covalent, ionic, and/or affinity interactions with a substrate or lid surface (or a coating layer thereon).
  • the substrate e.g. within the interior regions of the hydrodynamic traps
  • affinity interactions e.g. within the interior regions of the hydrodynamic traps
  • oligonucleotide molecules of the oligonucleotide arrays may be grafted onto the surface by deposition of amine-terminated oligonucleotides onto lids or substrates that are functionalized with an epoxide, glicydyl, aldehyde, NHS ester, or other amine-reactive moiety.
  • the oligonucleotide arrays may be grafted onto the surface by deposition of azide- terminated oligonucleotides onto an alkyne-functionalized substrate or lid, which is a well-known type of“click chemistry”.
  • the oligonucleotide arrays may be grafted onto the surface by deposition of alkyne-terminated oligonucleotides to an azide-functionalized substrate or lid. In some instances, the oligonucleotide arrays may be grafted onto the surface by deposition of an acroxyl -terminated oligonucleotides to an acrylamide or methacrylamide functionalized substrate. In some instances, the oligonucleotide arrays may be grafted onto the surface by deposition of biotin-terminated oligonucleotides on a streptavidin functionalized substrate.
  • the oligonucleotide arrays may be grafted onto the surface by deposition of maleimide- or acrylate-terminated oligonucleotides on a thiol-functionalized substrate or lid. In some instances, the oligonucleotide arrays may be grafted onto the surface by deposition of thiol-terminated oligonucleotides on a maleimide- or acrylate-functionalized substrate or lid.
  • the oligonucleotide arrays may be grafted onto the surface by deposition of unfunctionalized oligonucleotides onto substrates containing grafted cationic surface charges, wherein the oligonucleotides remain fixed in place due to ionic interactions.
  • the oligonucleotides may be generated by solid-phase synthesis on the underside of the lid or directly in the microfluidic substrate.
  • the oligonucleotide arrays may be fabricated using a transfer process comprising: a) generating a master substrate containing a plurality of oligonucleotides that are hybridized to complementary strands that contain an affinity group; and b) then transferring the complementary strands to the underside of a lid or to a surface within the microfluidic substrate by a liquid or solid stamping process, as described in prior references (see, e.g ., A. A. Yu, F. Stellacci, Contact Printing Beyond Surface Roughness: Liquid Supramolecular Nanostamping, Advanced Materials, Volumel9, Issue 24, 2007, pp. 4338-4342
  • the liquid stamping process may involve creating a covalent network of acroxy-terminated DNA molecules inside a matrix of acrylates, methacrylates, polymethacrylates, polymethylmethacrylates, polydimethylsiloxanes, and/or any of their block copolymer combinations in a layer sandwiched between a master and a lid, after which the lid is peeled away from the master, yielding a complementary set of oligonucleotide arrays attached to the lid - the lid subsequently being used to seal the
  • microfluidic device as discussed elsewhere.
  • FIG. 11 shows examples of data for an oligonucleotide pattern replication technique in an acrylamide gel that involves spotting the oligonucleotides onto a template glass chip, and then hybridizing the spotted oligonucleotides to complementary oligonucleotides having an acryoxy- termination.
  • the template chip was then immersed in an acrylamide solution layered between the template chip and a daughter chip, following which the acrylamide is allowed to set and incorporate the acroxy-terminated oligonucleotides into the polymer backbone of the gel.
  • Fluorescence images of the template chip (top and middle rows) and daughter chip (bottom row) surfaces were taken before and after the transfer step.
  • the complementary strands are shown to become incorporated in the acrylamide layer adhering to the daughter chip, demonstrating a DNA pattern transfer mechanism.
  • FIGS. 12A-B shows a process similar to that described in FIG. 11, except that the gel is composed of a block copolymer containing acrylamide and polydimethylsiloxane blocks.
  • FIG. 12A Fluorescently labeled, acrylate terminated oligos hybridized to spots of complementary, surface bound oligo on the template surface.
  • FIG. 12B Mirror image observed after oligos transferred to a daughter surface using acrylates and hybridized with a fluorescent,
  • Methods for performing single cell analysis comprising one or more of: a) providing a microfluidic device according to any of the embodiments described elsewhere herein; b) flowing a suspension of cells through the microfluidic device, thereby trapping single cells in all or a portion of the plurality of hydrodynamic traps; c) flowing a cell lysis and/or molecular reaction buffer (e.g.
  • a buffer comprising one or more enzymatic components and co-factors, cations, etc ., required for performing reverse-transcription reactions, DNA polymerization (primer extension) reactions, DNA amplification reactions, and the like) through the microfluidic device, thereby releasing target oligonucleotide molecules from the single cell trapped in each trap; d) optionally, flowing in an immiscible fluid such as air or oil that fills bypass channels but does not enter the hydrodynamic traps containing the single cells, and is used to prevent mixing of cellular material from different hydrodynamic traps during molecular barcoding steps; e) cleaving the barcoded oligonucleotide molecules from the substrate to participate in solution phase reactions; f) incubating the microfluidic device under conditions that promote hybridization of one or more target oligonucleotide molecules released by lysis of the single cells to one or more molecular recognition sequences presented in the features of the oligonucleotide array in each
  • hydrodynamic trap g) performing a primer extension reaction within the microfluidic device; h) eluting the barcoded oligonucleotide molecules from each trap from the microfluidic device; i) optionally, eluting the barcoded oligonucleotide molecules from specific traps of the microfluidic device by applying UV light to specific hydrodynamic traps to cleave and subsequently elute only those selected primers from the microfluidic device; and j) amplifying and sequencing the barcoded oligonucleotides to detect the presence of the one or more target oligonucleotide molecules in one or more single cells, wherein the sequence of a unique cell barcode sequence presented in the features of the oligonucleotide array in each hydrodynamic trap is used to identify target oligonucleotide molecules that were released from a given single cell.
  • the hydrodynamic traps of the disclosed microfluidic devices in any of the embodiments described herein (and for any of the methods described herein) may be used for trapping a single cell per trap.
  • the hydrodynamic traps may be used for simultaneously or sequentially trapping a single pair of cells (e.g, two different cells or two cells of the same cell type) or a single cell - bead pair (e.g, wherein the bead comprises tethered oligonucleotide barcode molecules, agonists, antagonists, antigens, etc.) in each trap.
  • the hydrodynamic trap may be used to trap a single cell and may then be incubated under conditions that promote cell growth and division to produce small clonal populations of cells. In some instances, the hydrodynamic traps may be used to trap 1, 2, 3, 4, 5, 6, or more than
  • single cells or small clusters of cells may be trapped and maintained in a viable state for prolonged periods of time while phenotypic traits are monitored by means of high-resolution imaging of the trap array.
  • the cells may be maintained in a viable state within the trap array for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weeks, at least 3 weeks, or at least 4 weeks.
  • phenotypic traits are monitored as the single cells or small clusters of cells in the trap array are subjected to one or more chemical or environmental stimuli, e.g ., exposure to a therapeutic drug or drug candidate, a cytokine, a chemokine, a change in temperature, a change in growth medium, etc.
  • the cells within the trap array are lysed at a pre-determined or random experimental endpoint and barcoding reactions are performed to tag one or more selected target molecule classes.
  • the experimental endpoint may be 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours,
  • the barcoded oligonucleotide molecules are released from the array prior to participating in primer hybridization, reverse-transcription, DNA polymerization, and/or DNA amplification reactions while in other instances, the barcoded oligonucleotide molecules are released after participating in enzymatic reactions.
  • a determination of the number of unique molecular index sequences corresponding to each unique cell barcode sequence is used to quantify how many copies of a given target oligonucleotide were released from a given single cell.
  • the amplifying and sequencing are performed within the microfluidic device. In some instances, the amplifying and sequencing are performed after cleaving and eluting the barcoded oligonucleotide molecules from the microfluidic device.
  • the target oligonucleotide molecules comprise mRNA molecules or fragments thereof, tRNA molecules or fragments thereof, rRNA molecules or fragments thereof, RNA molecules or fragments thereof, DNA molecules or fragments thereof, gene sequences or fragments thereof, or any combination thereof.
  • the target oligonucleotide molecules comprise molecules that can be used in determining the open regions of a chromosome for the purpose of identifying the genes that are being expressed.
  • the target oligonucleotide molecules are those that enable amplification of the entire genome of the trapped cells to determine copy number variation and or single nucleotide variants.
  • the target oligonucleotide molecules are those that capture a subset of RNA transcripts, genes, or gene fragments for targeted molecular
  • the target oligonucleotide molecules are oligonucleotide labels attached to antibodies for protein detection. In some instances, the target oligonucleotide molecules detect DNA or RNA sequences introduced into cells.
  • the disclosed microfluidic devices comprising a substrate, lid, and a plurality of oligonucleotide arrays disposed within the hydrodynamic traps of the device may be used in the preparation of RNA-seq libraries for 3’ or 5’ end counting of the expression profiles of single cells, this approach comprising, a) introducing cells into the microfluidic device and capturing them in single cell per trap format, b) introducing a master mix containing cell lysis and reverse transcription reagents into the microfluidic device, and then rapidly replacing the master mix solution in the bypass channels of the device with an air plug or oil plug to prevent cross contamination between different chambers (as illustrated using a fluorescent dye solution (FIG.
  • the master mix optionally contains USERTM enzymes, which are able to cleave a uridine base within the plurality of barcoded oligonucleotide molecules that are attached to the surface, and has the advantage of enabling these molecules to be released from the surface to participate in faster solution-phase reverse transcription reaction kinetics.
  • the instrument is optionally capable of exposing the plurality of barcoded oligonucleotide arrays to UV or visible wavelengths of light at the dosage required to break photocleavable moieties that are
  • the method further comprises: d) exposing the device substrate to temperatures in the range of 30 to 60 °C to enable full length cDNA to be generated from the primed RNA molecules; and f) unsealing each trap chamber by replacing the air or oil with RT buffer and/or other molecular biology reagents used to further amplify and prepare the molecules for analysis by next generation sequencing.
  • the master mix may comprise i) Triton X-100, ii) Tween-20, iii) molecular crowding agents such as polyethylene glycol, Ficoll, gelatin, iv) RNAse inhibitors, v) dNTPs, vi) RT enzymes, vii) buffer components including Potassium, Cesium, Sodium,
  • oligo-dT terminated primers that may incorporate a cell identification barcode and template switch oligos, or primers that have oligo-rG terminations and may incorporate a cell identification barcode
  • the libraries are prepared by in vitro transcription as described in CEL-seq and CEL-seq2. In other instances, the libraries can be prepared using chemistry similar to Smart-seq, Smart-seq2, and
  • FIGS. 8A-B show examples of data for the base calls in an RNA sequencing run in which the library preparation was performed using biotinylated oligonucleotide barcodes comprising a cleavable linker (FIG. 8A) or no cleavable linker (FIG. 8B) spotted on streptavidin-coated glass slides.
  • a suspension of 5 cells were added to the RT/lysis mix incubated in a flow cell, and then exposed to 365 nm UV light for the first 15 minutes of the RT step.
  • the rGrGrG sequence immediately upstream of the cleaved section was used for template switching and preparing full length transcriptome libraries.
  • Raw fastq files were processed to measure base percentages by read position.
  • FIG. 8A Sequence for the photocleavable oligos contain the universal primer, barcode, and GGG sequence at high percentages, while the sequence data for oligos lacking a photocleavable linker (FIG. 8B) lower efficiency of barcode incorporation and GGG sequence. Sequence reads from the cleaved oligos map to the genome efficiently and predominantly map to the 5’ end of mRNAs.
  • FIGS. 13A-B show examples of data for an RNA library preparation reaction that was performed in a microfluidic device, followed by sequencing of the eluted barcoded
  • the barcode region was constructed by enzymatic ligation of two distinct barcodes (FIG. 13A). Following the sequencing quality control steps, more than 60% of the reads contained the barcode, mapped to the genome, and contained useable biological information (FIG. 13B).
  • FIGS. 14A-B show examples of RNA library preparation and sequencing data.
  • FIG. 14A sequencing quality control metrics.
  • FIG. 14B Plot of the number of genes detected versus the number of reads measured.
  • Single cell barcoding for preparation of DNA-seq libraries In some instances, the disclosed microfluidic devices comprising a substrate, lid, and a plurality of oligonucleotide arrays disposed within the hydrodynamic traps of the device may be used in the preparation of
  • DNA-seq libraries for whole genome amplification, or targeted gene analysis comprising, a) introducing cells into the microfluidic device and capturing them in single cell per trap format, b) introducing a cell lysis buffer into the device which is capable of dissociating the nuclear membrane and histone complexes to fully denature the cellular DNA, c) introducing
  • DNA amplification reaction components into the microfluidic chip to neutralize the cell lysis buffer and replace it with a mixture of enzymes and buffer components that is used in performing polymerase-catalyzed primed template extension reactions needed to incorporate the cell-specific oligonucleotide barcode primers in the DNA library preparation reactions, d) replacing the DNA amplification solution in the bypass channels of the device with an air plug or oil plug to prevent cross contamination between different trap chambers; e) incubating the microfluidic chip at constant temperature for a prolonged time interval (e.g.
  • oligonucleotide molecules from the nuclear compartments of the single cells in each trap; d) sealing the hydrodynamic trap array by flowing an immiscible fluid through the microfluidic device to reduce or eliminate transfer of molecules between the different hydrodynamic traps, e) cleaving the barcode oligonucleotides from the substrate or lid using light, heat, enzymes, reducing or oxidizing agents and/or a change in pH; f) performing primer extension reactions to create barcoded complementary sequences to the cellular genome through the use of either barcode oligonucleotides (primers) comprising a targeted panel of gene capture sequences and/or barcode oligonucleotides comprising unbiased randomer capture sequences, and g) eluting the barcoded complementary sequence molecules from the microfluidic device for amplification, sequencing, identification of gene fragments and/or genomic sequences of interest, and detection of the corresponding cell barcodes for identification of individual cells from which the gene fragments and/or genomic sequences were
  • the cell lysis component is based on the use of harsh detergents, such as sodium dodecyl sulfate (SDS).
  • the cell lysis component is based on the use of chaotropic agents, such as concentrated urea, guanidine hydrochloride, guanidine thiocyanate, or other similar hydrogen bond disrupting material - these chaotropic lysis reagents cause the DNA to condense on glass surfaces, thus ensuring that the unraveled genomic DNA is unable to escape the hydrodynamic trap site during the lysis step.
  • the oligonucleotides in the plurality of oligonucleotide arrays comprise a photocleavable or enzymatically-cleavable site that allows these barcoded primers to be cleaved from the substrate to participate in solution-phase DNA amplification reactions, as shown in FIG. 9.
  • FIG. 9 shows non-limiting examples of data for two methods of cleaving biotin- terminated oligonucleotides comprising either a photocleavable linker or a deoxyuridine nucleotide that were spotted on a streptavidin-coated surface. When the substrate is exposed to UV radiation for 2 minutes, only the photocleavable groups are removed from the surface (top row).
  • the oligonucleotides of plurality of oligonucleotide arrays may comprise a random capture sequence that is used for unbiased whole-genome amplification or may comprise a panel of gene-specific capture sequences that is used for targeted gene analysis.
  • RNA- sequencing and DNA-sequencing library preparation reactions conducted in a single-pot reaction mixture in which the cell lysis components, reverse transcription, and polymerase extension reactions are performed in the same buffers and at the same time.
  • the disclosed methods for performing combined amplification and barcode tagging of DNA and RNA molecules derived from a single cell or small group of trapped cells may comprise one or more of: a) providing a microfluidic device according to any of the embodiments described herein; b) flowing a suspension of cells through the microfluidic device, thereby trapping single cells in all or a portion of the plurality of hydrodynamic traps; c) optionally incubating the microfluidic device under conditions that permit cell growth and division to create a small group or clone of cells in all or a portion of the plurality of hydrodynamic traps; d) flowing a cell lysis and/or molecular reaction buffer through the microfluidic device, thereby releasing target oligonucleotide molecules from the cytoplasmic and nuclear compartments of the single cells or small group of cells contained in each trap; e) sealing the hydrodynamic trap array by flowing in an immiscible fluid through the microfluidic device to reduce or eliminate the transfer of molecules between the different hydro
  • RNA-sequencing library preparation reaction is conducted first by lysing only the cytosolic compartments of the cells, and then the DNA- sequencing library preparation reaction is performed with the addition of subsequent cell lysis reagents and other components required for DNA-amplification.
  • the disclosed methods for performing combined amplification and barcode tagging of DNA and RNA molecules derived from a single cell or small group of trapped cells may comprise one or more of: a) providing a microfluidic device according to any of the embodiments described herein; b) flowing a suspension of cells through the microfluidic device, thereby trapping single cells in all or a portion of the plurality of hydrodynamic traps; c) optionally incubating the microfluidic device under conditions that permit cell growth and division to create a small group or clone of cells in all or a portion of the plurality of hydrodynamic traps; d) flowing a cell lysis buffer and/or molecular reaction buffer through the microfluidic device, thereby releasing target
  • oligonucleotide molecules from the cytoplasmic compartment of the single cells or small group of cells contained in each trap; e) sealing the hydrodynamic trap array by flowing an immiscible fluid through the microfluidic device to reduce or eliminate the transfer of molecules between the different hydrodynamic traps and enable the preparation of complementary barcode-tagged sequences of RNA, f) cleaving the barcode oligonucleotides from the substrate or lid using light, heat, enzymes, reducing or oxidizing agents and/or a change in pH; g) performing primer extension reactions to create barcoded complementary DNA sequences for the cellular RNA using barcode oligonucleotides (primers) comprising oligo-dT, oligo-dG, oligo-rG, or other targeted capture sequences, h) eluting the barcoded complementary sequence molecules from the microfluidic device for amplification, sequencing, identification of the RNA fragments, and detection the cell barcodes for identification of individual cells from which the RNA fragment
  • Disclosed herein are methods for performing combined amplification and barcode tagging of the DNA and RNA from a single cell or small group of trapped cells comprising one or more of: a) providing a microfluidic device according to any of the embodiments described herein; b) flowing a suspension of cells through the microfluidic device, thereby trapping single cells in all or a portion of the plurality of hydrodynamic traps; c) optionally incubating the microfluidic device under conditions that permit cell growth and division to create a small group or clone of cells in all or a portion of the plurality of hydrodynamic traps; d) flowing a cell lysis and/or reaction buffer through the microfluidic devise, thereby releasing target oligonucleotides from the cytoplasmic compartment of the cells contained in each trap; e) sealing the hydrodynamic trap array by flowing an immiscible fluid through the microfluidic device to reduce or eliminate transfer of molecules between the different hydrodynamic traps and enable the preparation of
  • each hydrodynamic trap comprises at least one oligonucleotide array that is used for preparing barcode-tagged RNA molecules for sequencing and at least one oligonucleotide array that is configured to capture antibody-conjugated complementary oligonucleotide molecules in order to establish a spatially-controlled ELISA-like assay directly inside each hydrodynamic trap / cell culture chamber, c) flowing a suspension of cells through the microfluidic device, thereby trapping single cells in all or a portion of the plurality of
  • hydrodynamic traps e) flowing a cell lysis buffer through the microfluidic device, thereby releasing target oligonucleotide molecules from the nuclear compartments of the single cells in each trap, where the cell lysis buffer also comprises the antibody-conjugated complementary oligonucleotide molecules and fluorescently-conjugated secondary antibodies needed for antigen detection; e) sealing the hydrodynamic trap array by flowing an immiscible fluid through the microfluidic device to reduce or eliminate transfer of molecules between the different hydrodynamic traps, f) conducting fluorescent imaging of the cell culture chambers to determine the presence and amount of antibodies in each cell, g) cleaving the barcoded oligonucleotides from the substrate or lid using light, heat, enzymes, reducing or oxidizing agents and/or a change in pH; h) performing primer extension reactions to create barcoded complementary sequences to the cellular RNA that are appended to cell-specific barcodes, and g) eluting the barcoded complementary sequence molecules from the microflu
  • the disclosed microfluidic devices comprising a substrate, lid, and a plurality of oligonucleotide arrays disposed within the hydrodynamic traps of the device may be used in the preparation of ATAC- seq libraries for evaluation of epigenetic information and chromatin accessibility, this approach comprising, a) introducing cells into the microfluidic device containing trap-specific DNA barcodes and capturing them in single cell per trap format, b) introducing a non-ionic cell lysis buffer into the device which is capable of lysing the cell membrane to yield pure nuclei, c) introducing transposase and amplification reaction components into the microfluidic chip to neutralize the cell lysis buffer and replace it with a mixture of enzymes and buffer components that is used in performing tagging and fragmentation of the accessible DNA ( i.e ., tagmentation) and PCR amplification using unique barcoded oligonucleotide primer
  • the tagmentation step is carried out prior to trapping the cellular material and individual or groups of nuclei are captured in the traps, followed by amplification of chromatin-accessible DNA using trap-specific barcoded primers.
  • the transposase molecules are captured in the traps via molecular interactions including, but not limited to, biotin-streptavidin, digoxigenin, antibodies, or similar.
  • the transposase is pre-loaded with barcoded DNA that allows for an additional layer of molecular indexing of tagmented nuclei.
  • the oligonucleotides of the plurality of oligonucleotide arrays comprise a photocleavable site that allows the oligonucleotide barcode primers to be cleaved from the substrate so as to participate in solution-phase DNA amplification reactions.
  • the oligonucleotide molecules of the plurality of oligonucleotide arrays may comprise a random capture sequence that is used for unbiased whole-genome amplification or may comprise of a panel of gene-specific capture sequences that is used for targeted gene analysis.
  • the chromatin-accessible DNA is fragmented via an enzymatic reaction prior to amplification with the trap-specific barcoded primers.
  • the chromatin-accessible DNA is labeled with epigenetic markers such as, but not limited to 5 -methyl cytosine, 6-methyl adenosine, 4-methylcytosine, 5-hydroxymethylcytosine, or 8-oxo-guanine prior to release from the traps.
  • the amplification method is a polymerase chain reaction. In other instances, the amplification method comprises whole genome amplification or isothermal amplification.
  • Non-limiting examples of two different ATAC-seq workflows are outlined in FIG. 16. In the left-hand column of FIG. 16, tagmentation is performed within the microfluidic device.
  • Cells are captured in the hydrodynamic traps of the disclosed microfluidic devices, lysed and contacted with a transposase which has been pre-loaded with sequencing adapters, the regions of open chromatin are tagmented, trap-specific barcoded oligonucleotide primers are released from the oligonucleotide array disposed within the trap, primer extension and amplification reactions are performed on chip, and the barcoded libraries are collected for sequencing.
  • a similar workflow is presented in the right-hand column of FIG.
  • RNA-sequencing library preparation is conducted first, followed by chromatin accessibility measurements conducted using, e.g ., ATAC- seq, by first lysing only the cytosolic compartments of the cells to create barcoded cDNA from the RNA, then lysing nuclei to allow for tagging and fragmentation of chromatin-accessible regions of DNA and subsequent barcoding via amplification using trap-specific barcoded primers.
  • the cDNA is retrieved from the microfluidic device prior to tagmentation. In other instances, the cDNA synthesis and tagmentation and carried out in a single step.
  • RNA-sequencing library preparation and the chromatin accessibility measurements are conducted in a single pot mix by lysing the cells and nucleus simultaneously to create barcoded cDNA from the RNA, and also tagging and fragmentation of chromatin-accessible regions of DNA and subsequent barcoding via amplification using trap- specific barcoded primers.
  • the cDNA is retrieved from the microfluidic devise prior to tagmentation. In other instances, the cDNA synthesis and tagmentation are carried out in a single step.
  • RNA-sequencing library preparation is conducted at the same time as antibody expression analysis by including antibodies bound to oligonucleotides in the RNA- sequencing reaction buffer and fabricating a portion of barcoded oligonucleotides resident in the chamber to generate sequences complementary to the antibody-tethered oligonucleotides.
  • the disclosed methods and devices solve this problem by enabling synthesis of genes in the same locations as where cells are incubated, transfected, and analyzed.
  • the disclosed microfluidic devices which may function as a microfluidic cell culture incubation platform, compartmentalize cells into thousands of discrete trap chambers in a microfluidic chip.
  • Each cell hydrodynamic trap contains many identical copies of a unique synthetic gene that are grafted to a surface of the chamber, and which can be cleaved from the surface at a desired time to facilitate transfection into an adjacent cell.
  • Suitable methods for cleavage include, but are not limited to, photocleavage, enzymatic cleavage ( e.g ., using a restriction enzyme), chemical cleavage, or any other method to cleave the gene sequence at or near the end tethered to the substrate, so that the cells can take up the genes.
  • Suitable methods for transfection include, but are not limited to, electroporation, lipofection, sonoporation, phototransfection, or other means that allow for introduction of the synthetic genes into the cells.
  • the chip may be sandwiched between parallel plate electrodes, which are subjected to >1000 V/cm electric field pulses to porate the cell membranes.
  • a piezoelectric transducer can be coupled to the microfluidic chip in order to achieve sonoporation.
  • light can be delivered to individual chambers with a digital light projector or other photomasking systems in order to porate the cells with high intensity optical pulses. It is also possible to achieve poration by flowing in surfactants or other reagents commonly used in lipotransfection processes. Any of these methods, as well as any combination of these methods, may be used to achieve massively parallel gene transfection inside
  • the disclosed microfluidic devices comprise a plurality of oligonucleotide arrays, where each oligonucleotide array is housed within one of a plurality of hydrodynamic traps within the device that are configured to retain objects, e.g., cells, beads, or other particles, suspended in a fluid passing through the device.
  • the hydrodynamic traps may be configured to trap single cells.
  • each oligonucleotide array comprises a plurality of features, where each feature comprises a plurality of oligonucleotide molecules that comprise a fragment of a gene sequence such that, collectively, the set of gene sequence fragments presented by the features of a given array span an entire gene sequence.
  • the oligonucleotide molecules of each feature may further comprise a spacer sequence, an adapter sequence, at least one primer sequence, or any combination thereof.
  • a polymerase chain assembly reaction or a Gibson assembly reaction may be performed within the plurality of hydrodynamic traps in the assembled device to construct a full-length gene sequence within each hydrodynamic trap.
  • the microfluidic devices comprising a collection of full length gene sequences, e.g ., a different full length gene sequence in each of the traps of the plurality of hydrodynamic traps, may be used to trap cells, transfect cells, express native or genetically engineered proteins corresponding to the collection of full length gene sequences, and/or characterize the functionality of the expressed proteins.
  • the oligonucleotide array disposed within each hydrodynamic trap may be used to construct full length gene sequences in a microfluidic chip.
  • the methods may comprise one or more of: a) providing a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device once assembled; b) patterning a plurality of oligonucleotide arrays on a first surface of a substrate (or a coating layer thereon) or a first surface of a lid (or a coating layer thereon), wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a single gene fragment sequence, such that the plurality of gene fragment sequences presented by the plurality of features
  • oligonucleotide array of the plurality comprises that same known set of features and the same known set of gene fragment sequences.
  • each oligonucleotide array of the plurality comprises a known set of features and gene fragment sequences that are different from those in all other oligonucleotide arrays.
  • a subset of oligonucleotide arrays of the plurality comprise a known set of features and gene fragment sequences that are different from those in the remainder of the plurality.
  • the polymerase chain assembly reaction, enzymatic ligation, or Gibson assembly reaction is performed without cleaving the gene fragment sequences from the array.
  • the polymerase chain assembly reaction, enzymatic ligation, or Gibson assembly reaction further comprises the use of a restriction enzyme to remove partially-assembled gene sequences.
  • each oligonucleotide array of the plurality comprises a feature comprising the same known full-length gene sequence.
  • each oligonucleotide array of the plurality comprises a feature comprising a different full-length gene sequence.
  • a subset of oligonucleotide arrays of the plurality comprises a feature comprising a full-length gene sequence that is different from that in the remainder of the plurality.
  • the oligonucleotide arrays in all or a portion of the plurality of oligonucleotide arrays further comprise one or more additional features that comprise a plurality of oligonucleotide molecules comprising one or more known capture probe sequences.
  • the one or more known capture probe sequences are used to capture one or more antigens, antibodies, peptides, proteins, or enzymes that have been labeled with an oligonucleotide sequence that is complementary to the one or more capture probe sequences.
  • each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain at least one cell suspended in a fluid passing through the microfluidic device; and ii) a plurality of oligonucleotide arrays, wherein an oligonucleotide array is contained within the interior region of each hydrodynamic trap, wherein each oligonucleotide array comprises a plurality of features, and wherein at least one feature of the plurality comprises a plurality of oligonucleotide molecules comprising a single full length gene sequence or fragment thereof; b) flowing a suspension of cells through the microfluidic device, thereby trapping at least one cell in all or a portion of the plurality of hydrodynamic traps; c) cleaving the at least one full length gene sequence or fragment thereof from the oligonucleotide array within the interior region of each hydrodynamic trap; and d) transfecting the at least one cell in each hydrodynamic
  • Also disclosed are methods for performing gene expression assays comprising: a) providing a microfluidic device comprising: i) a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain at least one cell suspended in a fluid passing through the microfluidic device; and ii) a plurality of oligonucleotide arrays, wherein an oligonucleotide array is contained within the interior region of each hydrodynamic trap, wherein each oligonucleotide array comprises a plurality of features, and wherein at least one feature of the plurality comprises a plurality of oligonucleotide molecules comprising a single full length gene sequence or fragment thereof, and wherein at least one feature of the plurality comprises multiple copies of an oligonucleotide capture probe sequence; b) flowing a suspension of cells through the microfluidic device, thereby trap
  • the method may further comprise evaluating the binding affinity of expressed antibodies to antigens captured on the oligonucleotide arrays by monitoring the fluorescence intensity at the locations of the features comprising the oligonucleotide capture probe sequences as a function of a fluid flow rate through the microfluidic device.
  • the cleaving in step may comprise the use of a photo-cleavage reaction or restriction enzyme reaction.
  • the transfecting in step may comprise the use of an electroporation technique, a lipofection technique, a sonoporation technique, a photo-transfection technique, a restriction enzyme, or any combination thereof.
  • the hydrodynamic traps are sealed within the microfluidic device by flowing a hydrogel or air through the interconnecting fluid channels.
  • the oligonucleotide molecules in each feature are covalently attached to the surface or coating layer within the interior region of each hydrodynamic trap.
  • each oligonucleotide molecules in each feature are entrapped within the coating layer in the interior region of each hydrodynamic trap.
  • the oligonucleotides in each feature further comprise a spacer sequence, an adapter sequence, a primer sequence, or any combination thereof.
  • each oligonucleotide array comprises at least 10 features.
  • each oligonucleotide array comprises at least 100 features.
  • each oligonucleotide array comprises at least 1,000 features.
  • each oligonucleotide array of the plurality comprises that same known set of features and the same known set of full-length gene sequences or fragments thereof.
  • each oligonucleotide array of the plurality comprises a known set of features and known set of full- length gene sequences or fragments thereof that are different from those in all other
  • oligonucleotide arrays In some embodiments, a subset of oligonucleotide arrays of the plurality comprises a known set of features and known set of full-length gene sequences or fragments thereof that are different from those in the remainder of the plurality. In some embodiments, the average length of the full gene sequences presented by the plurality of oligonucleotide arrays is at least 1 kilobase pairs. In some embodiments, the average length of the full gene sequences presented by the plurality of oligonucleotide arrays is at least 3 kilobase pairs. In some embodiments, the average length of the full gene sequences presented by the plurality of oligonucleotide arrays is at least 5 kilobase pairs.
  • the oligonucleotide arrays in all or a portion of the plurality of oligonucleotide arrays further comprise one or more additional features that comprise a plurality of oligonucleotide molecules comprising one or more known capture probe sequences.
  • the one or more known capture probe sequences are used to capture one or more antigens, antibodies, peptides, proteins, or enzymes that have been labeled with an oligonucleotide sequence that is complementary to the one or more capture probe sequences.
  • the disclosed methods and devices may be used for high throughput determination of gene sequences that produce therapeutic antibodies with high affinity for desired antigens.
  • a fluorescence binding assay may be conducted in the same trap chambers by using, e.g ., antigen(s) spotted onto a surface of the chambers, introducing fluorescently-labeled secondary antibodies into the chambers, and using fluorescence imaging as a readout.
  • This approach may involve printing antigen spots in each chamber, or hybridizing antigen-functionalized DNA probes to complementary DNA probes included in the oligonucleotide array disposed in each chamber of the device.
  • the disclosed methods and devices may be used to screen at least 1, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1,000 gene sequences per device per experimental run.
  • a method for assembling gene sequences within a microfluidic device, transfecting single cells, and testing for expression of, e.g, an antibody comprises the followings steps:
  • Step 1 Pattern an array of gene fragments in each trap chamber of a microfluidic chip.
  • the gene fragments are designed to enable the construction of a full-length gene sequence within the same chambers by using polymerase chain assembly or the Gibson method. This method will also ensure that many identical copies of the synthesized gene sequence are tethered to the bottom or another surface of the chambers.
  • the construction of the full-length gene from the fragments can be conducted in the open-faced chip, or after the chip is enclosed with a lid.
  • Step 2 Mammalian or bacterial cells are introduced to the device and captured in each of the hydrodynamic trap / cell culture chambers. Thereafter, the gene sequences are cleaved from the substrate with, e.g. , a restriction enzyme or with light, and the co-located cells within the chamber are exposed to electric field pulses or some other transfection agent to cause the cells to uptake the genes.
  • a restriction enzyme or with light e.g. a restriction enzyme or with light
  • Step 3 The cells in each chamber are incubated for sufficient time to allow integration of the gene sequences and expression of the gene product.
  • Step 4 A mixture of DNA-linked antigens and fluorescently labeled secondary antibodies are introduced to the microfluidic chip, after which each chamber is rapidly sealed. The system is incubated to allow DNA-linked antigens to hybridize to predetermined features of the oligonucleotide array in each chamber, and to allow the antibodies produced by the cells in the chambers to bind to the tethered antigens, so that the fluorescently-labeled secondary antibodies condense on the tethered antigen spots in the chambers and produce a visual signal of binding efficiency.
  • the strength of the fluorescence signal m be used to infer the quality of the antibody. It may also be possible to measure the strength of the antibody-antigen binding interaction by shearing off the antibodies at different measured flow rates.
  • Systems and system components are systems configured to perform the described methods using the disclosed microfluidic devices.
  • system components required to perform single cell imaging and correlate image-based phenotypic analysis with single cell molecular data (e.g, sequence data, gene expression data, epigenetic data, and the like) generated using the disclosed devices include, but are not limited to, (i) one or more of the disclosed microfluidic devices, (ii) a high-content imager that is configured to acquire high- resolution images of the array of hydrodynamic traps spanning the entire device in brightfield and/or fluorescence imaging modes within minutes, (iii) a fluidics controller for control of fluid flow and delivery of cells or reagents to the device, (iv) gas and pH controllers for monitoring and control of, e.g, O2 concentration, CO2 concentration, and pH within the device, (v) a temperature controller configured for maintaining a set temperature and/or for thermal cycling,
  • the system may be configured for handling multiple chips in each experiment.
  • Additional system components may also be required to enable temperature control, gas and pH control, and/or liquid handling control of two or more chips in parallel, such that cells can be rapidly loaded into the chips, the arrayed cells can be continuously perturbed with mixtures of cell culture media, drugs or drug candidates, and/or other stimulants, and then cell lysis and molecular biology reagents can be introduced to the chips at the desired endpoint for genomic analysis.
  • external incubators gas and pH controllers, and/or reagent delivery modules
  • reagent delivery modules may also be required to enable temperature control, gas and pH control, and/or liquid handling control of two or more chips in parallel, such that cells can be rapidly loaded into the chips, the arrayed cells can be continuously perturbed with mixtures of cell culture media, drugs or drug candidates, and/or other stimulants, and then cell lysis and molecular biology reagents can be introduced to the chips at the desired endpoint for genomic analysis.
  • High-content imager The high-resolution imaging module (or high-content imager) may be configured to operate in bright-field, dark-field, phase-contrast, and/or fluorescence imaging modes and will comprise: (i) one or more excitation light sources, (ii) sets of excitation and emission filters (or other components for adjusting wavelength settings and bandpass), (iii) one or more detectors, and (iv) other optical components for manipulating the path of light beams as they traverse the optical system.
  • any of a variety of light sources known to those of skill in the art may be used as an excitation light source including, but not limited to, arc lamps, tungsten-halogen lamps, lasers (e.g, argon ion lasers, helium-neon (HeNe) lasers, etc.), diode lasers, light emitting diodes (LEDs), light engines, and the like, or any combination thereof.
  • the imaging module may comprise at least one light source, at least two light sources, at least three light sources, at least four light sources, at least five light sources, or more.
  • Excitation and emission filter sets may comprise any of a variety of optical filters known to those of skill in the art including, but not limited to, optical glass filters (e.g, Schott optical filters), long-pass filters, short-pass filters, interference filters, dichroic reflectors, notch filters, and the like, or any combination thereof.
  • optical glass filters e.g, Schott optical filters
  • long-pass filters e.g., Schott optical filters
  • short-pass filters e.g., interference filters, dichroic reflectors, notch filters, and the like, or any combination thereof.
  • the excitation and/or emission wavelengths (or bandpass) for fluorescence imaging are set and/or adjusted by changing one or more optical filters in the optical path of the system.
  • the excitation and/or emission wavelengths are set and/or adjusted using other components such as diffraction gratings, monochromators, acousto-optic modulators, tunable liquid-crystal filters, and the like.
  • excitation or emission wavelength settings for fluorescence detection and imaging may be independently adjusted and range from about 350 nm to about 900 nm.
  • the excitation or emission wavelength are set at about 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 m, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, 725 nm, 750 nm, 775 nm, 800 nm, 825 nm, 850 nm, 875 nm, or 900 nm.
  • the excitation or emission wavelengths may be set to any value within this range, e.g ., about 620 nm.
  • the bandwidths of the excitation and emission light for fluorescence imaging are independently adjusted and are specified as the specified excitation or emission wavelength ⁇ 2 nm, ⁇ 5 nm, ⁇ 10 nm, ⁇ 20 nm, ⁇ 40 nm, ⁇ 80 nm, or greater.
  • the excitation or emission bandwidths may be set to any value within this range, e.g. , about ⁇ 55 nm.
  • the imaging module may comprise at least one detector, at least two detectors (e.g, for simultaneous capture of fluorescence images at two different emission wavelengths), at least three detectors, at least four detectors, at least five detectors, or more.
  • the imaging system may be configured to capture fluorescence intensity images at two (or more) different emission wavelengths sequentially, e.g, by changing the fluorescence emission filter between image capture steps.
  • the imaging system may be configured to collect fluorescence intensity images at two (or more) different emission wavelengths simultaneously, e.g, by including appropriate dichroic reflectors in the emission light optical path and utilizing a different detector for each emission wavelength.
  • optical components examples include, but are not limited to, lenses or lens systems, prisms, beam-splitters, mirrors, optical fibers, diffractive optical elements for correction of chromatic aberration, etc. These components may be configured, along with light sources, excitation and emission filters (or other components for adjusting wavelength settings and bandpass), and detectors, in any of a variety of optical arrangements known to those of skill in the art.
  • the imaging module may further comprise one or more translation stages or other motion control mechanisms for the purpose of moving the microfluidic device relative to the illumination and/or imaging sub-systems, or vice versa.
  • Fluidics controller may comprise a fluid flow controller or perfusion system that provides programmable control of one or more fluid actuation mechanisms used to drive fluid flow in the microfluidic device.
  • suitable fluid actuation mechanisms for use in the disclosed methods, devices, and systems include application of positive or negative pressure to fluid reservoirs connected to one or more device inlets or outlets, electrokinetic forces, electrowetting forces, passive capillary action, capillary action facilitated through the use of membranes and/or wicking pads, and the like.
  • Control of fluid flow through the disclosed microfluidic devices will often be performed through the use of one or more pumps (or other fluid actuation mechanisms) and one or more valves which, in some embodiments, will be housed externally to the device in a user-controlled instrument module.
  • suitable pumps include, but are not limited to, syringe pumps, programmable syringe pumps, peristaltic pumps, diaphragm pumps, and the like.
  • fluid flow through the system may be controlled by means of applying positive pneumatic pressure at one or more inlets of external reagent and buffer containers connected to the microfluidic device, or at one or more inlets of the microfluidic device itself.
  • fluid flow through the device may be controlled by means of drawing a vacuum at one or more outlets of a waste reservoir connected to the device, or at the one or more outlets of the device.
  • suitable valves include, but are not limited to, check valves,
  • electromechanical two-way or three-way valves pneumatic two-way and three-way valves, and the like.
  • the volumetric flow rate through all or a portion of the microfluidic device may vary from about -10 ml/sec to about +10 ml/sec.
  • the absolute value of the volumetric flow rate may be at least 0.00001 ml/sec, at least 0.0001 ml/sec, at least 0.001 ml/sec, at least 0.01 ml/sec, at least 0.1 ml/sec, at least 1 ml/sec, or at least 10 ml/sec, or more.
  • the absolute value of the volumetric flow rate may be at most 10 ml/sec, at most 1 ml/sec, at most 0.1 ml/sec, at most 0.01 ml/sec, at most 0.001 ml/sec, at most 0.0001 ml/sec, or at most 0.00001 ml/sec.
  • the volumetric flow rate at a given point in time may have any value within this range, e.g. a forward flow rate of 1.2 ml/sec, a reverse flow rate of -0.07 ml/sec, or a value of 0 ml/sec (i.e. stopped flow).
  • gas and pH controllers may comprise gas and pH controllers and related components (e.g. sensors) for maintaining a user- specified percentage of gas, e.g. CO2, or user-specified pH in buffers, growth media, or other fluids being delivered to the microfluidic device.
  • suitable sensors include non- dispersive infrared (NDIR) CO2 sensors (used in conjunction with an attenuated total internal reflection (ATR) optics for dissolved CO2 sensing), metal insulator semiconductor field effect transistor (MOSFET)-type sensors for dissolved CO2 sensing (e.g.
  • CO2 or other gas concentrations, or pH may be monitored directly in the fluid contained within the device. In some embodiments, CO2 or other gas concentrations may be monitored in a gas or atmosphere which is in equilibrium with the fluid within the device.
  • the disclosed cell analysis platforms may further comprise a temperature controller for maintaining a user-specified temperature within the microfluidic device, e.g ., to enable cells to be incubated and maintained for extended periods while under continuous microscopic observation, or for ramping temperature between two or more specified temperatures over two or more specified time intervals.
  • temperature control components that may be incorporated into the microfluidic device or into the instrument system include, but are not limited to, resistive heating elements (e.g. indium tin oxide resistive heating elements), Peltier heating or cooling devices, heat sinks, thermistors, thermocouples, infrared light sources, and the like, which are regulated using electronic feedback loops.
  • the temperature controller may provide for a programmable temperature change at one or more specified, adjustable times prior to performing specific device operational steps. In some instances, the temperature controller may provide for programmable changes in temperature over specified time intervals. In some embodiments, the temperature controller may further provide for cycling of temperatures between two or more set temperatures with specified frequencies and ramp rates so that thermal cycling for amplification reactions may be performed.
  • sequencers may typically comprise a commercially-available sequencer such as those available from Illumina (San Diego, CA), Pacific Biosciences (Menlo park, CA), and Thermo- Fisher Scientific (Waltham, MA).
  • the disclosed systems may comprise one or more processors, computers, or computer systems configured for control the disclosed instrument systems; storage, processing, analysis, and display of the acquired image data; and/or storage, processing, analysis, and display of the generated sequencing data.
  • the one or more processors, computers, and computer systems may be configured for control of other system functions and/or other data acquisition, storage, processing, analysis, or display functions as well.
  • FIG. 17 provides a schematic illustration of a computer system 1701 that is programmed or otherwise configured to implement the methods described elsewhere herein (e.g ., methods for performing single cell barcoding used the disclosed microfluidic devices).
  • the computer system 1701 can regulate various aspects of the disclosed methods and systems, such as, for example, the acquisition and processing of image data and the control of reagent delivery to the device.
  • the computer system 1701 may comprise a local computer system, an electronic device (e.g., a smartphone, laptop, or desktop computer) of a user, or an electronic device of a user that is in communication with a computer system that is remotely located with respect to the electronic device.
  • the computer system 1701 may be a post-classical computer system (e.g, a quantum computing system).
  • the computer system 1701 includes a central processing unit (CPU, also referred to as a “processor” or“computer processor” herein) 1705, which can be a single core or multi core processor, or a plurality of processors for parallel processing.
  • the computer system 1701 also includes memory or a memory location 1710 (e.g, random-access memory, read-only memory, flash memory), electronic storage unit 1715 (e.g, a hard disk), a communication interface 1720 (e.g, a network adapter) for communicating with one or more other systems, and peripheral devices 1725, such as cache, other memory, data storage and/or electronic display adapters.
  • the memory 1710, storage unit 1715, interface 1720 and peripheral devices 1725 are in
  • the storage unit 1715 can be a data storage unit (or data repository) for storing data.
  • the computer system 1701 can be operatively coupled to a computer network (“network”) 1730 with the aid of the communication interface 1720.
  • the network 1730 may be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet.
  • the network 1730 in some cases is a telecommunication and/or data network.
  • the network 1730 can include one or more computer servers, which can enable distributed computing, such as cloud computing.
  • the network 1730, in some cases with the aid of the computer system 1701 can implement a peer-to-peer network, which may enable devices coupled to the computer system 1701 to behave as a client or a server.
  • the CPU 1705 is configured to execute a sequence of machine-readable instructions, which can be embodied in a program or software.
  • the instructions may be stored in a memory location, such as the memory 1710.
  • the instructions can be directed to the CPU 1705, which can subsequently program or otherwise configure the CPU 1705 to implement methods of the present disclosure. Examples of operations performed by the CPU 1705 can include fetch, decode, execute, and writeback.
  • the CPU 1705 may be part of a circuit, such as an integrated circuit.
  • a circuit such as an integrated circuit.
  • One or more other components of the system 1701 may be included in the circuit.
  • the circuit is an application specific integrated circuit (ASIC).
  • ASIC application specific integrated circuit
  • the storage unit 1715 is configured to store files, such as drivers, libraries and saved programs.
  • the storage unit 1715 may store user data, e.g ., user preferences and user programs.
  • the computer system 1701 in some cases can include one or more additional data storage units that are external to the computer system 1701, such as data storage units located on a remote server that is in communication with the computer system 1701 through an intranet or the Internet.
  • the computer system 1701 may communicate with one or more remote computer systems through the network 1730.
  • the computer system 1701 may communicate with a remote computer system of a user (e.g, a cloud server).
  • remote computer systems include personal computers (e.g, a desktop PC), portable personal computers (e.g, a laptop or tablet PC), smart phones (e.g, Apple® iPhone, Android-enabled devices, etc.), or personal digital assistants.
  • the user may access the computer system 1701 via the network 1730.
  • the disclosed systems may further comprise software for: (i) processing and display of image data, (ii) controlling fluid flow, reagent delivery, temperature, gas, and pH for the microfluidic devices, and/or (iii) processing and display of the sequencing data acquired for a plurality of single cell barcoding processes.
  • the methods described herein may be implemented by way of machine (e.g, computer processor) executable code stored on an electronic storage location of a computer system such as that illustrated in FIG. 17 (such as, for example, in memory 1710 or electronic storage unit 1715 of computer system 1701).
  • the machine executable or machine-readable code can be provided in the form of software.
  • the code can be executed by the processor 1705.
  • the code can be retrieved from the storage unit 1715 and stored on the memory 1710 for ready access by the processor 1705.
  • the electronic storage unit 1715 can be precluded, and machine-executable instructions are stored on memory 1710.
  • the code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code. In some instances, the code may be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion. [0191] Aspects of the methods and systems provided herein, such as the computer system 1701, can be embodied in programming. Various aspects of the technology may be thought of as
  • Machine-executable code typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium.
  • Machine-executable code can be stored on an electronic storage unit, such as in memory (e.g ., read-only memory, random-access memory, flash memory) or on a hard disk.
  • Storage type media can include any or all of the tangible memory of the computer system, computer processors, or the like, or associated modules thereof, such as various semiconductor memory devices, tape drives, disk drives, optical drives, and the like, which may provide non- transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks.
  • Such communications may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server.
  • another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links.
  • the physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software.
  • readable medium refer to any medium that participates in providing instructions to a processor for execution.
  • a readable medium such as computer-executable code
  • Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer system or the like that may be used to implement databases.
  • Volatile storage media include dynamic memory, such as main memory of such a computer platform.
  • Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system.
  • Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data
  • Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH- EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.
  • the computer system 1701 may include or be in communication with an electronic display 1135 that comprises a user interface (UI) 1140 for providing, for example, an interface for a user to input instructions, upload data to a computer database, download data from a computer database, etc.
  • UI user interface
  • Examples of UTs include, without limitation, a graphical user interface (GUI) and web-based user interface.
  • An algorithm can be implemented through the use of one or more algorithms, e.g ., an algorithm comprising instructions for acquiring and/or processing image.
  • An algorithm can be implemented by way of software upon execution by the central processing unit 1705.
  • the systems may use machine learning and/or computer vision to drive the high-content imager and/or to analyze the resulting datasets.
  • the system may also comprise user-friendly interfaces that enable exploration of the combination datasets comprising live cell imaging data and data obtained from barcoded RNA- and DNA-sequencing reactions.
  • GPU computing may comprise the use of GPU computing, /. e. , the use of a GPU (graphics processing unit) as a co-processor to accelerate the CPU performance for, e.g. , image segmentation to identify cells, beads, fiducial marks, trap address numbers, and other features or objects in images of the chip, and/or for machine learning-based algorithms for data processing and analytics.
  • GPU processing may be used to drive both hardware function and for analysis of data.
  • the GPU accelerates applications running on the CPU by off-loading some of the compute-intensive and time- consuming portions of the code while the rest of the application still runs on the CPU.
  • the image segmentation and data analytics algorithms run faster by harnessing the massively parallel processing power of the GPU.
  • a typical CPU may comprise, e.g. , four to eight CPU cores, while the GPU may consist of hundreds of smaller cores.
  • This massively parallel architecture is what provides the GPU with its high computational performance capabilities.
  • the software running on the disclosed systems may harness the performance of the parallel GPU architecture using, e.g. , CUDA - a parallel programming model invented by NVIDIA (Santa Clara, CA).
  • suitable GPUs include, but are not limited to, the NVIDIA GeForce®, NVIDIA Quadro®, and NVIDIA Tesla®.
  • kits comprising one or more of the microfluidic devices disclosed herein and/or the assay components (e.g, enzymes, nucleotides, primers, buffers, oligonucleotide barcoded antibodies, surface coating solutions, or other reagents) required to perform any of the methods disclosed herein.
  • assay components e.g, enzymes, nucleotides, primers, buffers, oligonucleotide barcoded antibodies, surface coating solutions, or other reagents
  • kits may comprise an RNA-seq kit for the consumer that includes the reagents required to: 1) lyse the cells, and 2) perform reverse transcription in the chips.
  • kit components may include, but are not limited to, a) lysis detergents, including but not limited to 0.01-1% Tween-20, 0.01-1% Triton X-100, b) 5 mM - 500 mM Tris-HCl, c)
  • 7.5 - 750 mM chloride salt including but not limited to NaCl, KC1, CsCl, d) 0.33 - 33 mM magnesium chloride (MgCb), e) 1-100 mM dithiothreitol (DTT), f) 0.05 - 5 mM guanosine triphosphate (GTP), g) 0.1 - 10 mM dNTPs, h) 4 - 400 U reverse transcriptase, i) crowding agents and additives, including but not limited to, 0.4 - 40% Ficoll PM-400, 0.005 - 0.5% gelatin, j) 1-100 U RNAase inhibitor, k) 0.05 - 5U USER Enzyme, and/or 1) 0.1 - 10 mM of either oligo- dT, oligo-dG, or oligo-rG.
  • MgCb magnesium chloride
  • DTT dithiothreitol
  • GTP 0.05
  • kits may comprise a DNA-seq kit for the consumer that includes the reagents required to: 1) lyse the cells, and 2) perform primer extension reactions in the chips.
  • kit components may include, but are not limited to, a) lysis detergents, including but not limited to 0.01-1% Tween-20, 0.01-1% Triton X-100, b) 5 mM - 500 mM Tris-HCl, c) 7.5 - 750 mM chloride salt, including but not limited to NaCl, KC1, CsCl, d) 0.33 - 33 mM magnesium chloride (MgCb), e) 1-100 mM dithiothreitol (DTT), f) 0.05 - 5 mM guanosine triphosphate (GTP), g) 0.1 - 10 mM dNTPs, h) crowding agents and additives, including but not limited to,
  • kits may comprise a pre-amplification kit for the consumer that includes the reagents required to: 1) remove single stranded primers, and 2) further amplify the barcoded cDNA molecules.
  • Suitable components may include, but are not limited to, a) 2 - 200 U Exonuclease I, b) 0.5 - 5X Kapa High Fidelity Buffer, c) 0.04 - 4 U Kapa High Fidelity DNA polymerase, d) 0.03 - 3mM dNTPs, e) 0.33 - 33 mM magnesium chloride(MgCh), and/or 1) 0.1 - 10 pM pre-amplification oligo.
  • a suspension of cancer cells derived from a cell line or a patient biopsy sample is introduced to the microfluidic device, and single cells are captured in the hydrodynamic trap array followed by multi-day exposure to a drug therapy while imaging the cells multiple times per day.
  • the RNA-seq workflow is applied to prepare transcriptome analysis of the surviving clones in order to understand the gene expression pathways that are up-regulated or down-regulated in the drug resistance clones.
  • the barcodes of the oligonucleotide array are stable for multiple days in the presence of cell culture media, as illustrated in FIG. 10, which shows a comparison of surface- tethered oligonucleotides that were exposed to cell culture media for 3 days (top row), 8 days
  • the barcoded cDNA molecules derived from the surviving clones are exported off the chip, further amplified, and then analyzed by sequencing at a depth of 10,000 to 100,000 reads per cell in a 3’ or 5’ transcript end counting application as commonly done in RNA-seq workflows.
  • the data sets generated by these experiments will reveal the relative composition of the protein encoding genes that make up the single cell transcriptome, and thereby enable the identification of highly variable genes that could be employed as biomarkers for targeting the drug resistant survival pathways of cancer cells.
  • cancer cells from a patient biopsy sample or cell line are introduced to the microfluidic device, captured in single cell per hydrodynamic trap format, and grown for several days while under continuous exposure to a drug therapy.
  • whole genome analysis is conducted on the surviving clones.
  • the barcoded DNA fragments of the surviving clones are retrieved from the chip, further amplified, and prepared for high resolution sequencing.
  • just the protein encoding genes can be selectively amplified in a pool for whole exome sequencing.
  • a typical sequencing run would target 1 million to 100 million reads from each surviving clone, which is needed for high resolution coverage of the genome to determine the presence of single nucleotide variants or copy number variants that are present in the surviving drug-resistant clones.
  • Cancer cells from a patient biopsy sample or cell line are introduced to the microfluidic device, captured in single cell per hydrodynamic trap format, and grown for several days while under continuous exposure to a drug therapy.
  • the chip Upon reaching the experimental endpoint, the chip is subjected both to high resolution whole transcriptome amplification and also high-resolution whole genome analysis on the surviving clones.
  • the genomic analysis would allow for identification of not only the genomic variants involved in the drug resistant fraction, but also how these variants regulate the gene expression profile of the surviving clones, thus enabling the identification of driver mutations and potential drug targets.
  • Certain types of neurological diseases such as Alzheimer’s and Amyotrophic Lateral Sclerosis (ALS) have pathologies that are associated with protein misfolding in cells, as well as the formation of plaques that can be directly observed with fluorescent immunostaining.
  • the combination of high throughput image-based phenotyping and massively parallel single cell RNA expression analysis can be used to determine the relationship between the cells containing these protein malformations and their gene expression.
  • this assay may also benefit from the use of CRISPR and other gene editing tools for use in gain- or loss-of-function inserts that can be directly measured along with the imaging-based phenotyping and whole
  • a pooled CRISPR screen may be used along with high resolution RNA expression analysis and multi-day time lapse imaging to determine the gain- or loss-of-function inserts that affect cell growth, drug survival, invasiveness, and other phenotypes relevant to oncology drug development.
  • Cancer cells may be subjected to a massively parallel pooled CRISPR screen to perform single gene knockouts or single gene inserts across the entire genome, then these cells are arrayed in the microfluidic chips in single cell per hydrodynamic trap format, exposed to a drug while monitoring the cellular responses from image-based phenotypes, including, for example, their growth rates, mobility, and/or polarity of focal adhesions.
  • single cell gene expression libraries may be prepared that can not only be used to determine which CRISPR modification(s) were made on that cell, but also the relative distribution of protein encoding genes that the cell is currently transcribing in response to that particular set of CRISPR modifications.
  • Single cell pairs consisting of a single cancer cell paired with a T-cell are formed on the chip by hydrodynamic trapping, then exposed to a therapeutic drug, and subjected to massively parallel whole transcriptome analysis for the purpose of identifying the gene expression patterns underling the cell pairs that have high vs. low cytolytic activity.
  • a suspension of cancer cells is first introduced into the device and captured in single cancer cell per hydrodynamic trap format.
  • a cell biopsy sample is dissociated and then introduced into the device such that there are multiple cells captured in each hydrodynamic trap in a manner that mimics the diversity of the original tumor biopsy. These cells are allowed to grow into organoids and are then exposed to a drug therapy for multiple days while monitoring the organoid development. At the experimental endpoint, high resolution RNA- and DNA-sequencing analysis is conducted to determine the upregulated genes and mutational landscape of the surviving organoids.
  • the virus In various types of viral infections, such as Herpes and HIV, the virus is known to evade the immune system by hiding in a dormant state inside of infected cells. These latently infected cells periodically become activated and are the source of chronic infections in patients.
  • the disclosed single cell analysis platform can be used to organize latently infected immune cells in a hydrodynamic trap array, and then study the viral latency activation processes using fluorescent imaging.
  • high resolution whole transcriptome analysis can be used to compare the gene expression patterns in various phenotypic fractions, including cells that remain in a dormant latent state, as well as the cells that become activated when exposed to the stimulant.
  • the master mix also contains the necessary reagents for detection of one or more proteins using the oligonucleotide array present inside each chamber to capture primary antibodies conjugated to DNA fragments that are complementary to some of the features in the oligonucleotide array and a secondary antibody that is conjugated to fluorescent moieties, which are used to provide a fluorescent signature indicating the presence and amount of antibodies produced by that cell.
  • a secondary antibody that is conjugated to fluorescent moieties, which are used to provide a fluorescent signature indicating the presence and amount of antibodies produced by that cell.
  • the deposition process used to fabricate oligonucleotide arrays in the disclosed devices may involve depositing between 0.1 and 10 femtomole of oligonucleotides per spot, which can be dissolved in various carrier liquids at volumes ranging from 10 - 1000 picoliters.
  • r and 0 are the radius of the spherical cap and the contact angle, assumed to be in the range of 30° - 60°, which is a typical value for a hydrophilic surface. Based on these assumptions and the print volume of 100 pL drops, the spot diameter is calculated to be in the range of 110 - 130 pm.
  • the plurality of oligonucleotides disposed within the array in each hydrodynamic trap of the device may comprise a cleavable moiety that is immediately upstream of a triple-rG section, which is designed to participate in the template switching processes that are commonly employed in reverse transcription molecular biology workflows.
  • the plurality of oligonucleotides may also comprise one or more unique molecular identifiers, cellular barcodes, or other identifiers that can indicate the spatial location of the spot within the oligonucleotide array and/or provide a dictionary for other molecular features contained therein.
  • the plurality of oligonucleotides may also contain a common sequence that is used for primer hybridization and polymerase amplification reactions that may take place inside the microfluidic device or externally after the molecules are retrieved from the chip.
  • This proposed 5’ end counting workflow will involve introducing a mixture of RT enzymes and buffer components, primers that have a 3’ dT termination as well as common sequences for amplification, and other enzymes or components needed to initiate the molecular biology reactions (e.g, USERTM enzymes, lights, DTT).
  • the barcoded mRNA from the cells contained in the device will be retrieved, further amplified and purified, and sequenced to determine the barcode, transcript, and genome mapping
  • FIG. 15 comprises the steps of (1) depositing barcodes into the microfluidic device traps (or chambers), (2) capturing single cells in the chambers, (3) cleaving barcode oligos from a chamber surface, and flowing in lysis and reverse transcription master mix to initiate the RNA-seq library preparation reaction, (4) eluting the barcoded molecules from the chip and amplifying the cDNA, and (5) preparing the cDNA for analysis on the sequencer.
  • the plurality of oligonucleotides contained with the oligonucleotide arrays of the disclosed devices may be constructed by ligating together two different barcode basis sets in every combination. This reduces the cost for purchasing the pre-constructed oligo set used for inkjet printing of the arrays.
  • the master mix used in the disclosed methods may comprise lysis reagents that can lyse both the cytoplasm and nucleus, reverse transcription enzymes, buffers, and components required to cleave the oligonucleotide barcoded primers off the surface to initiate reverse transcription, transposases, DNA polymerases, and buffer components, and other additives required to initiate barcode-tagging of the accessible chromatin sites for ATAC-seq, and may be compatible with a temperature profile that is suitable for performing RNA-Seq and ATAC-Seq simultaneously at one fixed temperature, or sequentially by changing the temperature for different stages of the library preparation reaction.

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Abstract

Microfluidic devices are described that comprise a plurality of hydrodynamic traps configured to trap cells or other particles, where all or a portion of the plurality of hydrodynamic traps incorporate an array of oligonucleotide barcode sequences for barcoding molecules derived from single cells or clonally-amplified populations of single cells. Also described are methods for fabricating and using the disclosed microfluidic devices.

Description

PCT PATENT APPLICATION
METHODS AND DEVICES FOR SINGLE CELL BARCODING
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Application No. 62/875,648, filed on July 18, 2019, of U.S. Provisional Application No. 62/875,692, filed on July 18, 2019, of U.S. Provisional Application No. 63/017,178, filed on April 29, 2020, and of U.S. Provisional Application No. 63/017,187, filed on April 29, 2020, each of which applications are incorporated herein by reference.
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with support from the United States Government under Grant Numbers R43GM128472, 1R43GM128472-01A1, R44GM122142, 2R44GM 122149-02, and R21GM131279 from the National Institutes of Health. The government has certain rights in the invention.
BACKGROUND OF THE INVENTION
[0003] Methods, devices, and systems for performing single-cell analysis have become increasingly important in genomics, transcriptomics, proteomics, and metabolomics studies due to the heterogeneity often seen in both eukaryotic and prokaryotic cell populations. The ability to perform genomic, transcriptomic, proteomic, and metabolomic analyses on single cells would enable the discovery of important cellular processes that may be masked when studying bulk populations of cells.
SUMMARY OF THE INVENTION
[0004] Disclosed herein are methods for barcoding and analyzing RNA, DNA, protein, antibodies, and other forms of cellular expression in a microfluidic device comprising a plurality of hydrodynamic traps and oligonucleotide arrays, wherein the barcoded analytes ( e.g ., the RNA, DNA, protein, antibodies, and other forms of cellular expression released from single cells trapped within the chip) may be analyzed using high throughput DNA sequencers and other analysis instruments. Also disclosed are methods for fabricating the microfluidic device.
[0005] Disclosed herein are microfluidic devices comprising: a) a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device; and b) a plurality of oligonucleotide arrays, wherein the interior region of each hydrodynamic trap comprises an oligonucleotide array, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence.
[0006] Also disclosed are microfluidic devices comprising: a) a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device; and b) a plurality of oligonucleotide arrays, wherein the interior region of each hydrodynamic trap comprises an oligonucleotide array, wherein each oligonucleotide array comprises a plurality of features, wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence, and wherein at least a subset of the features comprise a plurality of oligonucleotide molecules comprising a single gene fragment sequence, such that the plurality of gene fragment sequences presented by the subset of features collectively span a full length gene sequence.
[0007] Further disclosed are microfluidic devices comprising: a) a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device; and b) a plurality of oligonucleotide arrays, wherein the interior region of each hydrodynamic trap comprises an oligonucleotide array, wherein each oligonucleotide array comprises a plurality of features, wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence, and wherein at least one feature of the plurality comprises a plurality of oligonucleotide molecules comprising a single full-length gene sequence.
[0008] In any of the microfluidic device embodiments disclosed herein, i) each hydrodynamic trap may comprise an entrance region, an interior region, and an exit region that collectively constitute an interior fluid flow path through the hydrodynamic trap that has a fluidic resistance, RT; ii) each hydrodynamic trap in a majority of the hydrodynamic traps may be in fluid communication with one long bypass fluid flow channel having a fluidic resistance, RA, and with one or two short bypass fluid flow channels each having a fluidic resistance that is less than RA, wherein each bypass fluid flow channel connects the exit region of the hydrodynamic trap to the entrance region of another hydrodynamic trap; and iii) fluid flows through an adjacent short bypass channel in a first direction if a hydrodynamic trap is unoccupied, and in a second direction if the hydrodynamic trap is occupied by an object. In some embodiments, the ratio RA/RT is at least 1.1. In some embodiments, the ratio RA/RT is at least 1.3. In some embodiments, each hydrodynamic trap comprises at least one constriction that has a spatial dimension that is less than about one half of the smallest dimension of the object. In some embodiments, the ratio RA/RT is at least 1.2 and a capture probability for an individual hydrodynamic trap retaining a suspended object on first contact is at least 0.36. In some embodiments, the ratio RA/RT is at least 1.45 and a capture probability for an individual hydrodynamic trap retaining a suspended object on first contact is at least 0.60. In some embodiments, each hydrodynamic trap comprises a frit structure within the exit region, and wherein the frit structure comprises one or more constrictions that have a spatial dimension that is smaller than the smallest dimension of the suspended objects. In some embodiments, the plurality of hydrodynamic traps comprises at least 100 traps. In some embodiments, the plurality of hydrodynamic traps comprises at least 1,000 traps. In some embodiments, the plurality of hydrodynamic traps comprises at least 10,000 traps. In some embodiments, an initial trapping efficiency for trapping the suspended objects is at least 80%. In some embodiments, an initial trapping efficiency for trapping the suspended objects is at least 95%. In some embodiments, the oligonucleotide molecules in each feature of the oligonucleotide array are covalently attached to a surface or coating layer within the interior region of each hydrodynamic trap. In some embodiments, the oligonucleotide molecules in each feature of the oligonucleotide array are non- covalently tethered to a surface or coating layer within the interior region of each hydrodynamic trap. In some embodiments, the oligonucleotide molecules in each feature of the oligonucleotide array are entrapped within a coating layer in the interior region of each hydrodynamic trap. In some embodiments, the plurality of oligonucleotide arrays is fabricated using a contact printing or stamping technique. In some embodiments, the plurality of oligonucleotide arrays is fabricated using an inkjet printing, microarray spotting, or spatially-addressable solid phase synthesis technique. In some embodiments, each oligonucleotide array comprises at least 10 features. In some embodiments, each oligonucleotide array comprises at least 100 features. In some embodiments, each oligonucleotide array comprises at least 1,000 features. In some embodiments, the common barcode sequences comprise unique non-overlapping sequences with a Hamming distance that enables demultiplexing with error correction capability. In some embodiments, the common barcode sequences comprise a G/C content ranging from 30% to
70%. In some embodiments, a length of the common barcode sequence ranges from 6 base pairs to 20 bases. In some embodiments, the common barcode sequence comprises a unique cell barcode sequence. In some embodiments, the unique cell barcode sequence comprises a string of
N“words”, and wherein each“word” comprises a string of M bases. In some embodiments, M is
1 base, 2 bases, 3 bases, or at least 4 bases. In some embodiments, N is at least 5“words”. In some embodiments, the unique cell barcode sequence is the same for each feature within a given oligonucleotide array, and different from that for all of the features in all of the other oligonucleotide arrays. In some embodiments, the unique cell barcode sequence is different for each feature within a given oligonucleotide array, and different from that for all of the features in all of the other oligonucleotide arrays. In some embodiments, the unique cell barcode sequence for each feature of each oligonucleotide array of the plurality of oligonucleotide arrays is known.
In some embodiments, the oligonucleotides in each feature further comprise a spacer sequence, a cleavage sequence, an adapter sequence, at least one primer sequence, a cell barcode sequence, a molecular index sequence, a molecular recognition sequence, or any combination thereof. In some embodiments, a length of the universal primer sequence ranges from 15 bases to 30 bases.
In some embodiments, a length of the unique molecular index sequence ranges from 5 bases to
15 bases. In some embodiments, a length of the molecular recognition sequence ranges from 2 bases to 40 bases. In some embodiments, a length of the spacer sequence ranges from 5 bases to
50 bases. In some embodiments, a length of the oligonucleotide molecules ranges from 50 bases to 150 bases. In some embodiments, the oligonucleotides in each feature comprise a molecular index sequence that is different for each individual oligonucleotide of the plurality of
oligonucleotides within a given feature. In some embodiments, the oligonucleotides in each feature comprise a molecular recognition sequence that is different for different features of a given oligonucleotide array. In some embodiments, the molecular recognition sequence comprises a sequence selected from the group consisting of an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(deoxyguanosine) (oligo-dG) sequence, an oligo(riboguanosine) (oligo-rG) sequence, a complement of an mRNA sequence or fragment thereof, a complement of a tRNA sequence or fragment thereof, a complement of an rRNA sequence or fragment thereof, a complement of a DNA sequence, a complement of a cDNA sequence, a complement of a gene sequence or fragment thereof, and any combination thereof. In some embodiments, the plurality of features in each oligonucleotide array of the plurality of oligonucleotide arrays comprise a same set of molecular recognition sequences. In some embodiments, a subset of the plurality of oligonucleotide arrays comprises a set of molecular recognition sequences that is different from that in the oligonucleotide arrays of the remainder of the plurality. In some embodiments, the average length of a gene fragment oligonucleotide sequence is at least 40 bases. In some embodiments, each oligonucleotide array of the plurality comprises a same known set of features and a same known set of gene fragment sequences. In some embodiments, each oligonucleotide array of the plurality comprises a known set of features and a known set of gene fragment sequences that are different from those in all other oligonucleotide arrays. In some embodiments, a subset of oligonucleotide arrays of the plurality comprises a known set of features and a known set of gene fragment sequences that are different from those in the remainder of the plurality. In some embodiments, the average length of the full-length gene sequences presented by the plurality of oligonucleotide arrays is at least 1 kilobase. In some embodiments, each oligonucleotide array of the plurality comprises a feature comprising the same known full-length gene sequence. In some embodiments, each oligonucleotide array of the plurality comprises a feature comprising a different full-length gene sequence. In some embodiments, a subset of oligonucleotide arrays of the plurality comprises a feature comprising a full-length gene sequence that is different from that in the remainder of the plurality. In some embodiments, the oligonucleotide arrays in all or a portion of the plurality of oligonucleotide arrays further comprise one or more additional features that comprise a plurality of oligonucleotide molecules comprising one or more known capture probe sequences. In some embodiments, the one or more known capture probe sequences are used to capture one or more antigens, antibodies, peptides, proteins, or enzymes that have been labeled with an oligonucleotide sequence that is
complementary to the one or more capture probe sequences. In some embodiments, the hydrodynamic traps are configured to trap single cells.
[0009] Disclosed herein are methods for fabricating a microfluidic device comprising: a) providing a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device once assembled; b) fabricating a plurality of oligonucleotide arrays on interior surfaces of the interior regions of the plurality of hydrodynamic traps or on a first surface of a lid, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence and a free 5’ end or 3’ end; and c) bonding the first surface of the lid to the substrate to seal the plurality of hydrodynamic traps and interconnecting fluid flow channels in the assembled device such that the interior region of each hydrodynamic trap comprises an oligonucleotide array. In some embodiments, the plurality of oligonucleotides further comprises a cleavage site, a molecular recognition sequence, a random multimer capture sequence, a unique molecular index sequence, a universal primer sequence, an adapter sequence, a spacer sequence, or any combination thereof. In some embodiments, the cleavage site comprises a deoxyuridine base. In some embodiments, the cleavage site comprises a photocleavable linker. In some embodiments, the oligonucleotide molecule is released into solution upon exposure to light or treatment with an enzyme. In some embodiments, the oligonucleotide molecule is released into solution upon treatment with combination of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII. In some embodiments, the common barcode sequence for the
oligonucleotide array in each hydrodynamic trap is unique. In some embodiments, the plurality of common barcode sequences for the plurality of hydrodynamic traps comprise unique non- overlapping sequences with a Hamming distance that enables demultiplexing with error correction capability. In some embodiments, the common barcode sequences comprise a G/C content ranging from 40% to 60%. In some embodiments, a length of the common barcode sequence ranges from 8 base pairs to 20 bases. In some embodiments, a length of the universal primer sequence ranges from 15 base pairs to 30 bases. In some embodiments, a length of the unique molecular index sequence ranges from 5 bases to 15 bases. In some embodiments, a length of the molecular recognition sequence ranges from 2 bases to 40 bases. In some embodiments, a length of the spacer sequence ranges from 5 bases to 50 bases. In some embodiments, a length of the oligonucleotide molecules ranges from 50 bases to 150 bases. In some embodiments, the plurality of hydrodynamic traps comprises at least 100 traps. In some embodiments, the plurality of hydrodynamic traps comprises at least 1,000 traps. In some embodiments, the plurality of hydrodynamic traps comprises at least 10,000 traps. In some embodiments, the plurality of hydrodynamic traps comprises at least 100,000 traps. In some embodiments, the fabricating in step (b) comprises the use of contact printing or stamping to create a replica of the molecular pattern (or its inverse) on the surface. In some embodiments, the fabricating in step (b) comprises the use of an inkjet printing, microarray spotting, or spatially-addressable solid phase synthesis technique. In some embodiments, each
oligonucleotide array comprises at least 1 feature. In some embodiments, each oligonucleotide array comprises at least 10 features. In some embodiments, each oligonucleotide array comprises at least 100 features. In some embodiments, the common barcode sequence comprises a unique cell barcode sequence. In some embodiments, the unique cell barcode sequence comprises a string of N“words”, and wherein each“word” comprises a string of M bases. In some embodiments, M is 1 base, 2 bases, 3 bases, or at least 4 bases. In some embodiments, N is at least 10“words”. In some embodiments, the unique cell barcode sequence is the same for each feature within a given oligonucleotide array, and different from that for all of the features in all of the other oligonucleotide arrays. In some embodiments, the unique cell barcode sequence is different for each feature within a given oligonucleotide array, and different from that for all of the features in all of the other oligonucleotide arrays. In some embodiments, the unique cell barcode sequence for each feature of each oligonucleotide array of the plurality of
oligonucleotide arrays is known. In some embodiments, the oligonucleotides in each feature comprise a molecular index sequence that is different for each individual oligonucleotide of the plurality of oligonucleotides within a given feature. In some embodiments, the oligonucleotides in each feature comprise a molecular recognition sequence that is different for different features of a given oligonucleotide array. In some embodiments, the molecular recognition sequence comprises a sequence selected from the group consisting of an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(deoxyguanosine) (oligo-dG) sequence, an oligo(riboguanosine) (oligo-rG) sequence, a random multimer sequence, a complement of an mRNA sequence or fragment thereof, a complement of a tRNA sequence or fragment thereof, a complement of an rRNA sequence or fragment thereof, a complement of a DNA sequence, a complement of a cDNA sequence, a complement of a gene sequence or fragment thereof, and any combination thereof.
In some embodiments, the plurality of features in the oligonucleotide arrays in each
hydrodynamic trap of the plurality of traps comprise a same set of molecular recognition sequences. In some embodiments, the plurality of molecular recognition sequences in the features of a subset of the plurality of oligonucleotide arrays is different from that in the oligonucleotide arrays of the remainder of the plurality. In some embodiments, the substrate is fabricated in silicon, the lid is glass, and the bonding in step (c) comprises coating a thin film of polydimethylsiloxane (PDMS) on the glass lid prior to bonding it to the silicon substrate. In some embodiments, the substrate is fabricated in silicon, the lid comprises a glass substrate with a polydimethylsiloxane (PDMS) layer coated thereon, and the bonding in step (c) comprises coating and patterning a thin film of hydrogel on the PDMS layer prior to bonding the lid to the silicon substrate. In some embodiments, the bonding in step (c) comprises application of a thin coating layer of epoxy to the lid prior to fabricating the plurality of oligonucleotide arrays. In some embodiments, the epoxy is a UV-curable epoxy or a heat-curable epoxy. In some embodiments, the bonding in step (c) comprises formation of a hydrogel layer between the lid and substrate using UV-induced polymerization of acrylate groups that are attached to both surfaces.
[0010] Disclosed herein are methods for performing single cell analysis, the methods
comprising: a) providing a microfluidic device according to any of the embodiments described herein; b) flowing a suspension of cells through the microfluidic device, thereby trapping single cells in all or a portion of the plurality of hydrodynamic traps; c) flowing a lysis buffer through the microfluidic device, thereby releasing target oligonucleotide molecules from the single cell trapped in each trap; d) incubating the microfluidic device under conditions that promote hybridization of one or more target oligonucleotide molecules released by the single cells to one or more molecular recognition sequences presented in the features of the oligonucleotide array in each trap; e) performing a primer extension reaction within the microfluidic device; f) optionally, eluting barcoded oligonucleotide molecules corresponding to complementary copies of the one or more target oligonucleotide molecules released by the single cell in each trap from the microfluidic device; and g) amplifying and sequencing the barcoded oligonucleotides to detect the presence of the one or more target oligonucleotide molecules in one or more single cells, wherein the sequence of a unique cell barcode sequence presented in the features of the oligonucleotide array in each hydrodynamic trap is used to identify target oligonucleotide molecules that were released from a given single cell. In some embodiments, method may further comprise a cleavage step to release barcoded oligonucleotide primers from the
oligonucleotide arrays disposed within each hydrodynamic trap in the plurality of hydrodynamic traps within the microfluidic device. In some embodiments, the method may further comprise the use of an air plug or oil plug to seal the hydrodynamic traps to reduce transfer of molecular components between hydrodynamic traps following lysis of the single cells in (c). In some embodiments, a determination of the number of unique molecular index barcode sequences corresponding to each unique cell barcode sequence is used to quantify how many copies of a given target oligonucleotide were released from a give single cell. In some embodiments, the amplifying and sequencing are performed within the microfluidic device. In some embodiments, the amplifying and sequencing are performed after eluting the barcoded oligonucleotide molecules from the microfluidic device. In some embodiments, the target oligonucleotide molecules comprise mRNA molecules or fragments thereof, tRNA molecules or fragments thereof, rRNA molecules or fragments thereof, RNA molecules or fragments thereof, DNA molecules or fragments thereof, or any combination thereof. In some embodiments, the oligonucleotide molecules of the oligonucleotide array disposed in each hydrodynamic trap comprise a cleavage site, and the cleavage site comprises a deoxyuridine base. In some embodiments, the oligonucleotide molecules of the oligonucleotide array disposed in each hydrodynamic trap comprise a cleavage site, and the cleavage site comprises a photocleavable linker moiety. In some embodiments, the oligonucleotide molecules of the oligonucleotide array disposed in each hydrodynamic trap are released into solution upon exposure to light or treatment with an enzyme. In some embodiments, the oligonucleotide molecules are released into solution upon treatment with a combination of Uracil DNA glycosylase (UDG) and the DNA glycosylase- lyase Endonuclease VIII. In some embodiments, the primer extension reaction in (e) comprises a reverse transcription reaction or a DNA polymerase reaction. In some embodiments, the lysis buffer comprises a chaotropic agent. In some embodiments, the chaotropic agent comprises concentrated urea, guanidinium thiocyanate, or any combination thereof. In some embodiments, the cells comprise cancer cells. In some embodiments, the cells comprise fetal cells. In some embodiments, the cells comprise CRISPR-edited cells. In some embodiments, the method is used to perform library preparation for a DNA-seq experiment, an RNA-seq experiment, an
ATAC-seq experiment, protein detection, antibody detection or any combination thereof. In some embodiments, the method is used to perform a cell transfection and gene expression assay.
[0011] Disclosed herein are methods for construction of full-length gene sequences in a microfluidic chip, the methods comprising: a) providing a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device once assembled; b) patterning a plurality of oligonucleotide arrays on an interior surface of the interior regions of the plurality of hydrodynamic traps or on a first surface of a lid, wherein each oligonucleotide array comprises a plurality of features, and wherein all or a portion of the features in an oligonucleotide array comprise a plurality of oligonucleotide molecules comprising a single gene fragment sequence, such that the plurality of gene fragment sequences presented by a plurality of features in the oligonucleotide array collectively span a full-length gene sequence; c) bonding the first surface of the lid to the substrate to seal the plurality of hydrodynamic traps and interconnecting fluid flow channels in the assembled device such that the interior region of each hydrodynamic trap comprises an oligonucleotide array; and d) performing a polymerase chain assembly reaction or a
Gibson assembly reaction within the plurality of hydrodynamic traps to construct a full-length gene sequence within each hydrodynamic trap. In some embodiments, the oligonucleotide molecules in each feature are covalently attached to a coating layer within the interior region of each hydrodynamic trap or on the first surface of the lid. In some embodiments, the
oligonucleotide molecules in each feature are entrapped within the coating layer. In some embodiments, the oligonucleotides in each feature further comprise a spacer sequence, a cleavage site, a molecular recognition sequence, a random heptamer sequence, a unique molecular index sequence, an adapter sequence, a primer sequence, or any combination thereof. In some embodiments, each oligonucleotide array comprises at least 10 features. In some embodiments, each oligonucleotide array comprises at least 100 features. In some embodiments, each oligonucleotide array comprises at least 1,000 features. In some embodiments, the average length of a gene fragment oligonucleotide sequence is at least 40 bases. In some embodiments, the average length of a gene fragment oligonucleotide sequence is at least 120 bases. In some embodiments, the average length of a gene fragment oligonucleotide sequence is at least 360 bases. In some embodiments, each oligonucleotide array of the plurality comprises a same known set of features and a same known set of gene fragment sequences. In some embodiments, each oligonucleotide array of the plurality comprises a known set of features and a set of known gene fragment sequences that are different from those in all other oligonucleotide arrays. In some embodiments, a subset of oligonucleotide arrays of the plurality comprises a known set of features and a set of known gene fragment sequences that are different from those in the remainder of the plurality. In some embodiments, the patterning in (b) comprises the use of an inkjet printing, microarray spotting, or spatially-addressable solid phase synthesis technique. In some embodiments, the polymerase chain assembly reaction or Gibson assembly reaction is performed without cleaving the gene fragment sequences from the surface or coating layer. In some embodiments, the polymerase chain assembly reaction or Gibson assembly reaction further comprises the use of a restriction enzyme to remove partially-assembled gene sequences. In some embodiments, the hydrodynamic traps are configured to trap single cells.
[0012] Disclosed herein are methods for performing cell transfection, the methods comprising: a) providing a microfluidic device comprising: i) a substrate comprising a plurality of
hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain at least one cell suspended in a fluid passing through the microfluidic device; and ii) a plurality of oligonucleotide arrays, wherein an oligonucleotide array is contained within the interior region of each hydrodynamic trap, wherein each oligonucleotide array comprises a plurality of features, and wherein at least one feature of the plurality comprises a plurality of oligonucleotide molecules each comprising a single full-length gene sequence or fragment thereof; b) flowing a suspension of cells through the microfluidic device, thereby trapping at least one cell in all or a portion of the plurality of hydrodynamic traps; c) cleaving the at least one full length gene sequence or fragment thereof from the oligonucleotide array within the interior region of each hydrodynamic trap; and d) transfecting the at least one cell in each hydrodynamic trap with the at least one full length gene sequences or fragment thereof.
[0013] Disclosed herein are methods for performing gene expression assays, the methods comprising: a) providing a microfluidic device comprising: i) a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain at least one cell suspended in a fluid passing through the microfluidic device; and ii) a plurality of oligonucleotide arrays, wherein an oligonucleotide array is contained within the interior region of each hydrodynamic trap, wherein each oligonucleotide array comprises a plurality of features, and wherein at least one feature of the plurality comprises a plurality of oligonucleotide molecules each comprising a single full length gene sequence or fragment thereof, and wherein at least one feature of the plurality comprises multiple copies of an oligonucleotide capture probe sequence; b) flowing a suspension of cells through the microfluidic device, thereby trapping at least one cell in all or a portion of the plurality of hydrodynamic traps; c) cleaving the at least one full length gene sequence or fragment thereof from the oligonucleotide array within the interior region of each hydrodynamic trap; d) transfecting the at least one cell in each hydrodynamic trap with the at least one full length gene sequence or fragment thereof; e) incubating the at least one cell in the plurality of hydrodynamic traps under conditions that promote cell division to create a clonal population of cells in each hydrodynamic trap that express a gene product for the at least one full length gene sequence or fragment thereof; f) flowing a mixture comprising a cell lysis buffer, at least one oligonucleotide-labeled antigen, at least one fluorescently-labeled antibody, at least one fluorescently-labeled secondary antibody, or any combination thereof, through the microfluidic device, wherein the oligonucleotide label of the at least one oligonucleotide-labeled antigen is complementary to the at least one capture probe feature on the oligonucleotide array in each hydrodynamic trap; g) sealing the hydrodynamic traps within the microfluidic device; and h) imaging the hydrodynamic traps in the microfluidic device to detect the presence of the gene product for the at least one full length gene sequence or fragment thereof by monitoring fluorescence intensity at the location of the at least one feature comprising the oligonucleotide capture probe sequence. In some embodiments, the method further comprises evaluating the binding affinity of expressed antibodies to antigens captured on the oligonucleotide arrays by monitoring the fluorescence intensity at the locations of the features comprising the
oligonucleotide capture probe sequences as a function of a fluid flow rate through the
microfluidic device. In some embodiments, the cleaving in (c) comprises the use of a photo cleavage reaction or restriction enzyme reaction. In some embodiments, the transfecting in (d) comprises the use of a chemical transfection agent, an electroporation technique, a lipofection technique, a sonoporation technique, a phototransfection technique, a restriction enzyme, or any combination thereof. In some embodiments, the hydrodynamic traps are sealed within the microfluidic device by flowing a hydrogel, oil, or air through the interconnecting fluid channels. In some embodiments, the average length of the full-length gene sequences presented by the plurality of oligonucleotide arrays is at least 1 kilobase. In some embodiments, the
oligonucleotide arrays in all or a portion of the plurality of oligonucleotide arrays further comprise one or more additional features that comprise a plurality of oligonucleotide molecules comprising one or more known capture probe sequences. In some embodiments, the one or more known capture probe sequences are used to capture one or more antigens, antibodies, peptides, proteins, or enzymes that have been labeled with an oligonucleotide sequence that is
complementary to the one or more capture probe sequences.
[0014] Disclosed herein are methods of preparing single cell RNA-seq libraries, the methods comprising: a) providing a microfluidic device comprising a plurality of hydrodynamic traps and a plurality of bypass channels, wherein each hydrodynamic trap comprises an oligonucleotide array disposed on a surface therein, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence; b) introducing cells into the microfluidic device and capturing a single cell in all or a portion of the plurality of hydrodynamic traps; c) introducing a cell lysis buffer comprising reverse transcription reagents into the microfluidic device; d) replacing the cell lysis buffer in the plurality of bypass channels with air or oil to prevent cross contamination between different hydrodynamic traps; e) cleaving the oligonucleotide molecules comprising the common barcode sequence from the surface; f) performing a reverse transcription reaction on cellular RNA molecules using the oligonucleotide molecules comprising the common barcode sequences as primers to create complementary cDNA copies; and g) eluting the barcoded cDNA copies from the microfluidic device for amplification and sequencing.
[0015] Disclosed herein are methods for preparing single cell DNA-seq libraries, the methods comprising: a) providing a microfluidic device comprising a plurality of hydrodynamic traps and a plurality of bypass channels, wherein each hydrodynamic trap comprises an oligonucleotide array disposed on a surface therein, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence; b) introducing cells into the microfluidic device and capturing a single cell in all or a portion of the plurality of hydrodynamic traps; c) introducing a cell lysis buffer configured to dissociate nuclear membranes and histone complexes, thereby denaturing cellular DNA, into the microfluidic device; d) introducing a DNA polymerization/amplification reaction mixture into the microfluidic device; e) replacing the DNA polymerization/amplification reaction mixture in the plurality of bypass channels with air or oil to prevent cross contamination between different hydrodynamic traps; f) cleaving the oligonucleotide molecules comprising the common barcode sequence from the surface; g) performing primer extension reactions using the oligonucleotide molecules comprising the common barcode sequences as primers to create complementary copies of cellular DNA sequences; and h) eluting the barcoded complementary copies of cellular DNA sequences from the microfluidic device for amplification and sequencing.
[0016] Disclosed herein are methods for preparing single cell RNA-seq and DNA-seq libraries, the methods comprising: a) providing a microfluidic device comprising a plurality of
hydrodynamic traps and a plurality of bypass channels, wherein each hydrodynamic trap comprises an oligonucleotide array disposed on a surface therein, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence; b) introducing cells into the microfluidic device and capturing a single cell in all or a portion of the plurality of hydrodynamic traps; c) simultaneously or sequentially introducing lysis buffer(s) comprising: (i) reverse transcription reagents, and (ii) DNA polymerization reagents into the microfluidic device; d) replacing the lysis buffer(s) in the plurality of bypass channels with air or oil to prevent cross contamination between different hydrodynamic traps; e) cleaving the oligonucleotide molecules comprising the common barcode sequence from the surface; f) simultaneously or sequentially performing: (i) a reverse transcription reaction on cellular RNA molecules using the oligonucleotide molecules comprising the common barcode sequences as primers to create complementary cDNA copies, and (ii) a DNA polymerization reaction using the oligonucleotide molecules comprising the common barcode as primers to create complementary copies of cellular
DNA sequences; and g) eluting the barcoded cDNA copies and complementary copies of cellular
DNA sequences from the microfluidic device for amplification and sequencing.
[0017] Disclosed herein are methods for preparing single cell ATAC-seq libraries, the methods comprising: a) providing a microfluidic device comprising a plurality of hydrodynamic traps and a plurality of bypass channels, wherein each hydrodynamic trap comprises an oligonucleotide array disposed on a surface therein, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence; b) introducing cells into the microfluidic device and capturing a single cell in all or a portion of the plurality of hydrodynamic traps; c) introducing a non-ionic cell lysis buffer configured to lyse cell membranes to yield pure nuclei into the microfluidic device; d) introducing a mixture comprising a transposase, DNA fragmentation, and DNA polymerization/amplification reagents into the microfluidic chip; e) replacing the transposase,
DNA fragmentation, and DNA polymerization/amplification reagents in the plurality of bypass channels with air or oil to prevent cross contamination between different hydrodynamic traps; f) cleaving the oligonucleotide molecules comprising the common barcode sequence from the surface; g) performing DNA polymerization/amplification to amplify fragmented DNA using the oligonucleotide molecules comprising the common barcode as primers, and h) eluting the barcoded complementary copies of the fragmented DNA from the microfluidic device for amplification and sequencing. In some embodiments, the common barcode sequence comprises a unique cell barcode sequence. In some embodiments, the plurality of oligonucleotide molecules further comprises a spacer sequence, and adapter sequence, a cleavage site, a molecular recognition sequence, a random heptamer capture sequence, a unique molecular index sequence, a universal primer sequence, or any combination thereof. In some embodiments, the plurality of oligonucleotide molecules comprises a cleavage site, and wherein the cleavage site comprises a deoxyuridine base. In some embodiments, the plurality of oligonucleotide molecules comprises a cleavage site, and wherein the cleavage site comprises a photocleavable linker. In some embodiments, the plurality of oligonucleotide molecules within a feature is released into solution upon exposure to light or treatment with an enzyme. In some embodiments, the 5’ end of the plurality of oligonucleotide molecules within a feature is released into solution upon treatment with a combination of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase
Endonuclease VIII. In some embodiments, the plurality of oligonucleotide molecules comprises a molecular recognition sequence that is the same for each feature within an oligonucleotide array. In some embodiments, the molecular recognition sequence comprises a sequence selected from the group consisting of an oligo(deoxythymidine) (oligo-dT) sequence, an
oligo(deoxyguanosine) (oligo-dG) sequence, an oligo(guanosine) (oligo-rG) sequence, a random heptamer sequence, and any combination thereof.
[0018] Disclosed herein are systems comprising: a) a microfluidic device comprising: i. a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device; ii. a plurality of oligonucleotide arrays, wherein each oligonucleotide array of the plurality is disposed on a surface within a hydrodynamic trap of the plurality of hydrodynamic traps, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence and a free 5’ end; and b) an imaging unit configured to acquire high-resolution images of the interior region of each hydrodynamic trap and one or more objects contained therein. In some embodiments, the oligonucleotide molecules comprise a cleavage site. In some
embodiments, the cleavage site comprises a deoxyuridine base. In some embodiments, the cleavage site comprises a photocleavable linker. In some embodiments, the oligonucleotide molecules further comprise a molecular recognition sequence that is the same or different for different features of an oligonucleotide array. In some embodiments, the molecular recognition sequence comprises a sequence selected from the group consisting of an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(deoxyguanosine) (oligo-dG) sequence, an oligo(guanosine) (oligo- rG) sequence, a random multimer sequence, a complement of an mRNA sequence or fragment thereof, a complement of a tRNA sequence or fragment thereof, a complement of an rRNA sequence or fragment thereof, a complement of a DNA sequence, a complement of a cDNA sequence, a complement of a gene sequence or fragment thereof, and any combination thereof.
In some embodiments, the objects comprise cells. In some embodiments, the cells comprise cancer cells. In some embodiments, the system is configured to perform single cell RNA sequencing library preparation, single cell DNA sequencing library preparation, single cell ATAC sequencing library preparation, protein detection, antibody detection, or any combination thereof.
[0019] Disclosed herein are kits comprising: a) a microfluidic device comprising: i. a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device; ii. a plurality of oligonucleotide arrays, wherein each oligonucleotide array of the plurality is disposed on a surface within a hydrodynamic trap of the plurality of hydrodynamic traps, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence and a free 5’ end; and b) one or more reagents for performing single cell RNA sequencing library preparation, single cell DNA sequencing library preparation, single cell ATAC sequencing library preparation, protein detection, antibody detection, any combination thereof.
INCORPORATION BY REFERENCE
[0020] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference 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
[0021] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
[0022] FIGS. 1A-B show an example of the hydrodynamic trapping geometry in a ladder configuration and the two flow regimes where the cell capture efficiency is low (FIG. 1A) owing to the fluid flow splitting at the trap entrance or high (FIG. IB) owing to the fluid flow joining at the trap entrance.
[0023] FIGS. 2A-B show an example of the hydrodynamic trapping geometry in a mesh configuration and the two flow regimes where the cell capture efficiency is low (FIG. 2A) owing to the fluid flow splitting at the trap entrance or high (FIG. 2B) owing to the fluid flow joining at the trap entrance.
[0024] FIGS. 3A-D show the distribution of trapped cells in the microfluidic device as a function of the flow resistance ratio and trapping efficiency (q). For low efficient traps (FIG. 3A and FIG. 3B), the traps have a greater tendency to miss the cells than when using higher efficiency traps (FIG. 3C and FIG. 3D).
[0025] FIG. 4 shows a non-limiting example of a cell trapping architecture for a microfluidic device that has a trapping ratio that is approximately calculated as: RA/RT = 0.42.
[0026] FIG. 5 shows a non-limiting example of a cell trapping architecture for a microfluidic device that has a trapping ratio that is approximately calculated as: RA/RT = 1.2.
[0027] FIG. 6 shows a non-limiting example of a cell trapping architecture for a microfluidic device comprising a plurality of oligonucleotides contained inside each trap. [0028] FIGS. 7A-B show micrographs of a cell trapping architecture in a microfluidic device both before (FIG. 7A) and after (FIG. 7B) flowing in a fluorescent liquid and then sealing the traps with a plug of air.
[0029] FIGS. 8A-B show examples of data for the base calls from a sequencing run performed using oligonucleotide barcode primers that are attached to a surface and either contain a cleavable linker (FIG. 8A) or do not contain a cleavable linker (FIG. 8B). The hallmarks of a successful library preparation reaction were only observed in the base call data of FIG. 8A due to the molecules having been successfully cleaved from the surface, thereby enabling them to participate in the library preparation reactions.
[0030] FIG. 9 shows non-limiting examples of data for two methods of cleaving biotin- terminated oligonucleotides comprising either a photocleavable linker or a deoxyuridine nucleotide that were spotted on a streptavidin-coated surface.
[0031] FIG. 10 shows a comparison of surface-tethered oligonucleotides that were exposed to cell culture media for prolonged time intervals as compared to the controls.
[0032] FIG. 11 shows a non-limiting example of data for an oligonucleotide pattern replication technique.
[0033] FIGS. 12A-B show another non-limiting example of data for an oligonucleotide pattern replication technique. FIG. 12A: Fluorescently labeled, acrylate terminated oligos hybridized to spots of complementary, surface bound oligo on the template surface. FIG. 12B: Mirror image observed after oligos transferred to a daughter surface using acrylates and hybridized with a fluorescent, complementary sequence.
[0034] FIGS. 13A-B show examples of data for an RNA library preparation reaction that was performed in a microfluidic device. The barcode region was constructed by enzymatic ligation of two distinct barcodes (FIG. 13A). Following the sequencing quality control steps, more than 60% of the reads contained the barcode, mapped to the genome, and contained useable biological information (FIG. 13B).
[0035] FIGS. 14A-B show examples of RNA library preparation and sequencing data.
[0036] FIG. 15 provides an outline of an RNA-seq workflow.
[0037] FIG. 16 provides an outline of two different ATAC-seq workflows that can be implemented in the disclosed microfluidic devices.
[0038] FIG. 17 provides a schematic illustration of a computer system that is programmed or otherwise configured to implement the methods and systems provided herein.
DETAILED DESCRIPTION OF THE INVENTION
[0039] Disclosed herein are microfluidic devices that comprise a plurality of hydrodynamic traps and a plurality of oligonucleotide arrays, where each oligonucleotide array is housed within one of the traps of the plurality of hydrodynamic traps within the device. The hydrodynamic traps of the device may be configured to retain objects, e.g ., cells, beads, or other particles, suspended in a fluid passing through the device. In some instances, the hydrodynamic traps may comprise all or a portion of the design features disclosed in co-pending PCT International Patent Application
Publication No. WO 2019/079399 Al. The microfluidic devices include a substrate in which all or a portion of the hydrodynamic traps and fluid channels, e.g. , interconnecting fluid channels or bypass fluid channels, are fabricated, and a lid which is bonded to the substrate to seal the hydrodynamic traps and fluid channels of the device. In some instances, the substrate and/or lid is fabricated from an optically-transparent material such that the microfluidic device is configured for high-resolution imaging of the plurality of hydrodynamic traps for use in monitoring, e.g. , cell growth or phenotypic changes as the cells are exposed to a stimulus such as a therapeutic drug treatment. In some instances, the oligonucleotide arrays disposed in each hydrodynamic trap comprise barcoded oligonucleotides for performing genotyping or gene expression profiling studies in parallel with phenotypic studies of single cells.
[0040] In some instances, each oligonucleotide array comprises a plurality of features, where each feature comprises a plurality of identical oligonucleotide molecules. In some instances, the oligonucleotide arrays may be fabricated on the underside of a lid, or a coating layer thereon, that is subsequently bonded to a substrate comprising the plurality of hydrodynamic traps and interconnecting fluid flow channels to seal the traps and fluid flow channels. In some instances, the oligonucleotide arrays may be fabricated on a surface within the hydrodynamic traps, e.g. , a surface within an interior region of the hydrodynamic traps, or a coating layer thereon, prior to bonding of a lid to seal the traps and fluid flow channels. In some instances, the oligonucleotide molecules of each feature may comprise oligonucleotide barcode sequences, e.g. , a cell barcode sequence (e.g, a sequence used to barcode molecules derived from a single cell or small clonal population of cells derived from a single cell) and/or a molecular index sequence (e.g, used to count the number of copies of a specific molecule derived from a single cell or clonal population of cells derived from a single cell). In some instances, the oligonucleotide molecules of each feature may further comprise a spacer sequence, an adapter sequence, at least one primer sequence (e.g, a universal primer sequence), a molecular index sequence, a molecular recognition sequence or randomer capture sequence, a cleavage site, or any combination thereof.
[0041] Also disclosed herein are methods for fabricating and using said microfluidic devices. In some instances, the plurality of oligonucleotide arrays may be patterned or fabricated using, e.g, a contact printing or stamping approach. For example, in some instances a library of DNA colonies (or“features” of the plurality of oligonucleotide arrays) may be created on a substrate, and identical copies transferred to a microfluidic lid using a low-cost stamping approach such as that described by R. Mitra and G. Church (1999),“In situ Localized Amplification and Contact
Replication of Many Individual DNA Molecules”, Nucleic Acid Research 27(24):e34. The process should allow for the lid to be bonded to the substrate comprising the microfluidic channels and hydrodynamic traps, such that an adequate seal can be formed everywhere without negatively affecting the oligonucleotide array pattern. In some instances, the plurality of oligonucleotide arrays may be patterned or fabricated using, e.g ., inkjet printing, solid phase synthesis, or other direct oligonucleotide deposition method, and deposited either on the lid (or a coating layer thereof), or directly onto the substrate in the hydrodynamic trap sites or some interior region of the traps (or a coating layer thereon). This process should also allow a lid to be bonded to the substrate, such that an adequate seal can be formed everywhere without negatively affecting the oligonucleotide array pattern. The disclosed microfluidic devices comprising barcoded oligonucleotide arrays may be used to perform a variety of single cell genotyping and gene expression profiling studies, e.g. , single cell RNA sequencing (scRNA-seq) library preparation, single cell DNA sequencing (scDNA-seq) library preparation, Assay for
Transposase-Accessible Chromatin using Sequencing (ATAC-seq), protein detection, antibody detection, etc., or any combination thereof, as will be discussed in more detail below.
[0042] The development of the disclosed microfluidic devices and methods for their use have required the Applicant to overcome a variety of technical hurdles and unpredictable outcomes.
Examples of the technical challenges that have required innovative design and development include, but are not limited to, (i) the identification of scalable fabrication techniques for reproducible deposition of oligonucleotide arrays in precise patterns within the plurality of hydrodynamic traps of the disclosed devices, (ii) the identification of bonding processes for attaching a lid to the microfluidic substrate that are compatible with and preserve the integrity of the oligonucleotide arrays, (iii) the identification of attachment chemistries for tethering barcoded oligonucleotide molecules to an interior surface or coating layer of the device that are stable to prolonged exposure of the oligonucleotide arrays to aqueous buffers and cell culture media at elevated temperatures, (iv) the identification of an appropriate combination of oligonucleotide array feature (or spot) size, barcoded oligonucleotide surface density within the array feature(s), and efficient cleavage chemistries that provide for concentrations of barcoded oligonucleotide primers upon release with the trap chambers that are compatible with reverse transcription and
DNA polymerization / amplification procedures, and (v) methods for sealing and un-sealing the trap chambers within the device to prevent cross-contamination between different trap chambers during cell lysis steps, reverse transcription steps, DNA polymerization / amplification steps, etc.. [0043] Definitions: Unless otherwise defined, all of the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field to which this disclosure belongs.
[0044] As used in this specification and the appended claims, the singular forms“a”,“an”, and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass“and/or” unless otherwise stated.
[0045] As used herein, the term‘about’ a number refers to that number plus or minus 10% of that number. The term‘about’ when used in the context of a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value.
[0046] As used herein, the phrases“microfluidic device” and“microfluidic chip” (or simply, “device” or“chip”) are used interchangeably.
[0047] As used herein, the terms "trap", "trapping feature",“trapping chamber”, "cell trap", and "weir-trap" may be used interchangeably and may refer to a feature comprising a constriction in one or two dimensions within a fluid channel that is designed to retain or trap cells or other objects suspended in a fluid. In some instances, a trap may comprise an entrance region, optionally, an interior region, and an exit region, at least one of which comprises a constriction.
In some instances, an interior region of the trap may be significantly larger in at least one or two dimensions than the entrance region and/or exit region and may be configured to
compartmentalize individual cells that have been trapped. In some instances, the interior region of a hydrodynamic trap may be used as a cell culture chamber.
[0048] As used herein, the term "object" generally refers to a cell or fragment thereof ( e.g ., a cellular organelle such as a cell nucleus, mitochondrion, or exosome), an organism (e.g., a bacterium), a bead, a particle, a droplet (e.g, a liquid droplet), or in plural form, may refer to any combination thereof.
[0049] As used herein, the term "cell" generally refers to any of a variety of cells known to those of skill in the art. In some instances, the term "cell" may refer to any adherent and non-adherent eukaryotic cell, mammalian cell, a primary or immortalized human cell or cell line, a primary or immortalized rodent cell or cell line, a cancer cell, a normal or diseased human cell derived from any
[0050] of a variety of different organs or tissue types (e.g, a white blood cell, red blood cell, platelet, epithelial cell, endothelial cell, neuron, glial cell, astrocyte, fibroblast, skeletal muscle cell, smooth muscle cell, gamete, or cell from the heart, lungs, brain, liver, kidney, spleen, pancreas, thymus, bladder, stomach, colon, small intestine), a distinct cell subset such as an immune cell, a CD8+ T cell, CD4+ T cell,
Figure imgf000021_0001
cancer stem cell, Lgr5/6+ stem cell, undifferentiated human stem cell, a human stem cell that has been induced to differentiate, a rare cell ( e.g ., a circulating tumor cell (CTC), a circulating epithelial cell, a circulating endothelial cell, a circulating endometrial cell, a bone marrow cell, a progenitor cell, a foam cell, a mesenchymal cell, or a trophoblast), an animal cell (e.g., mouse, rat, pig, dog, cow, or horse), a plant cell, a yeast cell, a fungal cell, a bacterial cell, an algae cell, an adherent or non-adherent prokaryotic cell, or in plural form, any combination thereof. In some instances, the term "cell" may refer to an immune cell, e.g, a T cell, a cytotoxic (killer) T cell, a helper T cell, an alpha beta T cell, a gamma delta T cell, a T cell progenitor, a B cell, a B-cell progenitor, a lymphoid stem cell, a myeloid progenitor cell, a lymphocyte, a granulocyte, a Natural Killer cell, a plasma cell, a memory cell, a neutrophil, an eosinophil, a basophil, a mast cell, a monocyte, a dendritic cell, and/or a macrophage, or in plural form, to any combination thereof.
[0051] As used herein, the term "bead" generally refers to any type of solid, porous, or hollow spherical, non-spherical, or irregularly-shaped object composed of glass, plastic, ceramic, metal, a polymeric material, or any combination thereof. In some instances, the term "bead" may refer to a silica bead, a silica gel bead, a controlled pore glass bead, a magnetic bead (e.g, a
Dynabead), a Wang resin bead, a Merrifield resin bead, an agarose bead, a Sephadex bead, a
Sepharose bead, a cellulose bead, a polystyrene bead, etc., or in plural form, may refer to any combination thereof. In some instances, a bead may comprise tethered or immobilized capture, detection, or barcoding reagents, e.g, antibodies, cytokine-specific antibodies, chemokine- specific antibodies, growth factor-specific antibodies, enzymes, enzyme substrates, avidin or streptavidin, protein A, protein G, other proteins, small molecules, glycoproteins, drug molecules, polysaccharides, fluorophores, oligonucleotides, oligonucleotide aptamers, oligonucleotide barcodes, or any combination thereof. In some instances, a bead may be a cytokine-sensing bead such as multiplexed Luminex xMAP® immuno-assay beads sold by
Thermo Fischer (Waltham, MA), which can be used to detect from 3 to 30 different cytokines and growth factors. In some instances, the diameter or average diameter of a bead may be at least
0.5 pm, at least 1 pm, at least 5 pm, at least 10 pm, at least 15 pm, at least 20 pm, at least 25 pm, at least 30 pm, at least 35 pm, at least 40 pm, at least 45 pm, or at least 50 pm.
[0052] As used herein, the phrase“target molecule” may refer to any of a variety of biological molecules that may be tagged and identified using the single cell barcoding methods described herein. Examples include, but are not limited to, genomic or mitochondrial DNA sequences or fragments thereof, gene sequences or fragments thereof, exon sequences or fragments thereof, intron sequences or fragments thereof, RNA sequences or fragments thereof, mRNA sequences or fragments thereof, tRNA sequences or fragments thereof, rRNA sequences or fragments thereof, and microRNA sequences or fragments thereof. In some instances, target molecule may comprise proteins or other molecules expressed by single cells that may be detected using, e.g, oligonucleotide-labeled antibodies, oligonucleotide-labeled secondary antibodies, oligonucleotide-labeled antigens, and the like.
[0053] As used herein, the phrases“target recognition sequence” and“capture sequence” are used interchangeably. In some instances, a target recognition sequence may be directed to ( e.g ., complementary to) a specific target molecule, e.g., a specific gene sequence. In some instances, a target recognition sequence may be directed to a class of target molecules, e.g, mRNA molecules within a single cell. In some instances, a target recognition sequence may be directed to random oligonucleotide sequences (e.g, where the“target recognition sequence” is itself a set of random sequences, e.g. a set of random heptamer sequences).
[0054] Methods, devices, and systems for single cell barcoding: Methods, devices, and systems are described that may be used for a variety of single cell phenotyping, molecular barcoding, and/or molecular counting studies, including but not limited to DNA sequencing-based studies, RNA sequencing-based studies, gene expression profiling studies, genetically-engineered protein expression studies,“multi-omic” studies, and the like. The devices comprise microfluidic chips that are designed to capture single cells or controlled numbers of cells to form small clusters, and then enable these captured cells to be maintained on chip for prolonged periods of time (e.g, many days) while being exposed to external perturbations, such as drugs, antibodies, or other stimulants.
[0055] The methods described herein include: a) methods for patterning a plurality of oligonucleotide arrays within the device, e.g, where each hydrodynamic trap contains an oligonucleotide array; b) methods to use these oligonucleotide arrays in the preparation of barcode-tagged sequences that are complementary to the RNA and DNA present in the trapped single cells; c) methods to analyze more than one type of molecular expression pattern, such as a combination of RNA + DNA, RNA + chromatin accessibility, RNA + protein expression; and the like; d) methods that enable these multi-omic measurements to be conducted in either a single pot reaction format or in a format that involves multi-step fluid additions.
[0056] The systems described herein may comprise: a) a high-content imager to acquire brightfield and/or fluorescent time-lapse images for each cell or cell culture (within the plurality of hydrodynamic traps) over many days, which are used in phenotypic analysis of single cell functional responses to environmental perturbations, and b) a thermal cycler and reagent delivery system that is used to implement the molecular barcoding and/or molecular counting studies, as discussed above, and c) data analysis methods that enable the simultaneous measurement of molecular expression and live-cell images from each cell, as well as methods to graphically present these datasets in a visual, interactive format. [0057] Microfluidic device designs for efficient trapping of single cells: As noted above, in one aspect the present disclosure provides microfluidic devices that enable highly efficient trapping of single cells or other objects by employing designs that exploit a previously unrecognized trait of mesh fluidic networks (see, e.g. , co-pending PCT International Patent Application No. PCT /US20 18/056221 (published as WO 2019/079399 Al)). Tuning the relative fluidic resistances of flow paths in a hydrodynamic fluidic circuit comprising a plurality of trapping features and at least two different types of interconnecting bypass channels ensures that all fluid flow
streamlines go through the trap, thereby ensuring that every cell is forced into the first trap that it encounters. This phenomenon is achieved by adjusting the hydrodynamic resistance through the trap, RT (i.e., the fluidic resistance of the entire trap geometry spanning the distance from the entry point to the exit point of a single trap) relative to the fluidic resistance through one or more short bypass channel sections, RB, and a communal long bypass channel section, RA, with the requirement that RT < RA. After a cell has been trapped, the local ratio of fluidic resistances changes in a manner such that the direction of fluid flow in the adjoining bypass channels reverses and flows away from the cell trap, thereby causing the next approaching cell to move towards the next available trap. In this manner, the traps within the array are populated sequentially in the order that cells are introduced, which in principle allows the disclosed devices to achieve near perfect efficiency in trapping single cells. The disclosed devices (non-limiting examples are shown in FIGS. 1A-B and FIGS. 2A-B) are thus ideally suited for handling small cell samples where high trapping efficiencies are critical.
[0058] FIGS. 1A-B show an example of the hydrodynamic trapping geometry in a ladder configuration and the two flow regimes where the cell capture efficiency is low (FIG. 1 A) owing to the fluid flow splitting at the trap entrance or high (FIG. IB) owing to the fluid flow joining at the trap entrance.
[0059] FIGS. 2A-B show an example of the hydrodynamic trapping geometry in a mesh configuration and the two flow regimes where the cell capture efficiency is low (FIG. 2A) owing to the fluid flow splitting at the trap entrance or high (FIG. 2B) owing to the fluid flow joining at the trap entrance.
[0060] The disclosed device designs are based on mesh-like networks of fluid channels. In some aspects, the devices comprise: a) a microfluidic network having at least one inlet and at least one outlet; b) a plurality of microfluidic constrictions (or“traps”), wherein a dimension of the constriction is smaller than a dimension of a suspended object contained within the fluid, and disposed so as to capture suspended objects flowing into the constriction; c) each microfluidic constriction comprising an entrance point or region and an exit point or region, and optionally, an interior region, d) the exit point of said microfluidic constriction is in direct fluidic connection with at least two additional microfluidic constrictions; e) the pressure at the exit point of said microfluidic constriction is higher than the pressure at the entrance point of either downstream microfluidic constriction when said microfluidic constriction has not yet captured a suspended object; and f) the pressure at the exit point of said microfluidic constriction is lower than the pressure at the entrance point of at least one of the downstream microfluidic constriction when said microfluidic constriction has captured a suspended object. In some instances, the exit region of the constrictions or traps may comprise a frit, e.g ., a series of columnar features having a spacing that is sufficiently small to prevent cells or other objects from leaving an interior region of the constriction or trap.
[0061] In some instances, the disclosed microfluidic devices may comprise: a) a plurality of weir-traps disposed between, and in fluid communication with, at least one fluid inlet and at least one fluid outlet, wherein each weir-trap is configured to retain an object suspended in a fluid passing through the microfluidic device, and wherein: i) each weir-trap comprises an entrance region, an optional interior region, and an exit region that collectively constitute an interior fluid flow path through the weir-trap; ii) each weir-trap in a majority of the weir-traps (i.e., all of the weir-traps except for those nearest the at least one fluid inlet or at least one fluid outlet) is in fluid communication with either two or three exterior fluid flow paths (bypass fluid channels) that connect the exit region of a weir-trap to the entrance region of another weir-trap; and iii) a ratio of the fluidic resistance of one exterior fluid flow path (e.g, a longer, communal fluid bypass channel) to that of the interior fluid flow path through the trap (i.e., RA / RT) is at least 0.4. In some embodiments, the exit region of all or a portion of the weir-traps may comprise a frit to prevent cells or other objects from flowing out of the interior region (or chamber) of the trap. In some embodiments, the two or three exterior fluid flow paths (bypass fluid channels) may comprise one or two shorter fluid bypass channels comprising a fluidic resistance, RB, which is less than RA. In the case that there are two shorter fluid bypass channels, their fluidic resistance may be the same as each other, or different from each other, but will in either case be less than RA.
[0062] FIGS. 3A-D show the distribution of trapped cells in the microfluidic device as a function of the flow resistance ratio and trapping efficiency (q). For low efficient traps (FIG. 3A ((RA/RT) = 0.25) and FIG. 3B (RA/RT = 0.42)), the traps have a greater tendency to miss the cells than when using higher efficiency traps (FIG. 3C (RA/RT = 1.20) and FIG. 3D (RA/RT = 1.45)).
[0063] In some embodiments, the ratio RA / RT may range from about 0.2 to about 2.0. In some embodiments, the ratio RA / RT may be at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, or at least 2.0. In some embodiments, the ratio RA / RT may be at most 2.0, at most 1.9, at most 1.8, at most 1.7, at most
1.6, at most 1.5, at most 1.4, at most 1.3, at most 1.2, at most 1.1, at most 1.0, at most 0.9, at most 0.8, at most 0.7, at most 0.6, at most 0.5, at most 0.4, at most 0.3, or at most 0.2. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, the ratio RA / RT may range from about 0.4 to about
1.6. Those of skill in the art will recognize that the ratio RA / RT may have any value within this range, e.g ., about 1.25.
[0064] In some instances, the ratio RA/RT is at least 1.2 and a capture probability for an individual hydrodynamic trap retaining a suspended object on first contact is at least 0.36. In some instances, the ratio RA/RT is at least 1.45 and a capture probability for an individual hydrodynamic trap retaining a suspended object on first contact is at least 0.60.
[0065] The capture probability for an individual weir-trap of the disclosed devices retaining a suspended cell or object on first contact (i.e., the first time that a cell or object encounters a weir- trap within the device) may range from about 0.05 to about 0.99. For example, in some instances, the capture probability may be at least 0.05, at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 0.95, or at least 0.99. In some instances, the capture probability may be at most 0.99, at most 0.95, at most 0.9, at most 0.8, at most 0.7, at most 0.6, at most 0.5, at most 0.4, at most 0.3, at most 0.2, at most 0.1, or at most 0.05. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example in some instances the capture probability may range from about 0.2 to about 0.8. Those of skill in the art will recognize that the capture probability may have any value within this range, e.g. , about 0.66.
[0066] The initial or pre-saturation trapping efficiencies for trapping cells or other objects suspended in a fluid passing through the disclosed weir-trap array devices may range from about 10% to about 100%. For example, in some instances, the initial or pre-saturation trapping efficiency of the disclosed devices may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%. In some instances, the initial or pre-saturation trapping efficiency may be at most 99%, at most 98%, at most 95%, at most 90%, at most 80%, at most 70%, at most 60%, at most 50%, at most 40%, at most 30%, at most 20%, or at most 10%. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example the initial or pre- saturation trapping efficiency may range from about 40% to about 99%. Those of skill in the art will recognize that the pre-saturation trapping efficiency may have any value within this range, e.g. , about 97%. [0067] The weir-traps of the disclosed microfluidic devices will generally comprise a constriction in at least one dimension, e.g ., an entry point or entrance region comprising a constriction that is smaller than the smallest dimension of the cell or object to be trapped. In some embodiments, the constriction in at least one dimension may range in size from about 10% to about 90% of the smallest dimension of the cell or object to be trapped. In some
embodiments, the constriction may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the smallest dimension of the cell or object to be trapped. In some embodiments, the constriction may be at most 90%, at most 80%, at most 70%, at most 60%, at most 50%, at most 40%, at most 30%, at most 20%, or at most 10% of the smallest dimension of the cell or object to be trapped. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example the constriction may range in size from about 20% to about 70% of the smallest dimension of the cell or object to be trapped. Those of skill in the art will recognize that the constriction may have any value within this range, e.g. , about 33% of the smallest dimension of the cell or object to be trapped.
[0068] The weir-traps of the disclosed microfluidic devices will generally comprise a
constriction in at least one dimension, e.g. , an entry point or entrance region comprising a constriction that is smaller than the smallest dimension of the cell or object to be trapped. In some embodiments, the constriction in at least one dimension may range in size from about 1 pm to about 100 pm. For example, in some embodiments, the constriction in at least one dimension may have a dimension of at least 1 pm, at least 2 pm, at least 3 pm, at least 4 pm, at least 5 pm, at least 6 pm, at least 7 pm, at least 8 pm, at least 9 pm, at least 10 pm, at least 20 pm, at least 30 pm, at least 40 pm, at least 50 pm, at least 60 pm, at least 70 pm, at least 80 pm, at least 90 pm, or at least 100 pm. In some embodiments, the constriction in at least one dimension may have a dimension of at most 100 pm, at most 90 pm, at most 80 pm, at most 70 pm, at most 60 pm, at most 50 pm, at most 40 pm, at most 30 pm, at most 20 pm, at most 10 pm, at most 9 pm, at most 8 pm, at most 7 pm, at most 6 pm, at most 5 pm, at most 4 pm, at most 3 pm, at most 2 pm, at most 1 pm. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example the constriction in at least one dimension may range in size from about 3 pm to about 6 pm. Those of skill in the art will recognize that the constriction may have any dimension within this range, e.g. , about 4.5 pm.
[0069] In some instances, the disclosed microfluidic devices may comprise: a) a plurality of weir-traps disposed between, and in fluid communication with, at least one fluid inlet and at least one fluid outlet, wherein each weir-trap is configured to retain an object suspended in a fluid passing through the microfluidic device, and wherein: i) each weir-trap comprises an entrance region, an interior region, and an exit region that collectively constitute an interior fluid flow path through the weir-trap; and ii) the volume of the interior region of the weir trap is greater than the volume of the entrance region or exit region.
[0070] The weir-trap designs of the disclosed microfluidic devices may comprise an entrance region (or entry point), optionally, an interior region (or chamber), and an exit region (or exit point). The interior region (or chamber), if present, may have any of a variety of cross-sectional shapes within the plane of the microfluidic device. For example, the interior region may have a largely circular shape, elliptical shape, square shape, rectangular shape, triangular shape, hexagonal shape, irregular shape, or any combination thereof. In some instances, the exit regions of all or a portion of the weir-traps may comprise a frit structure, where the frit structure comprises one or more constrictions that have a spatial dimension that is smaller than the smallest dimension of the suspended objects.
[0071] FIG. 4 illustrates a mesh network trapping geometry that has a trapping ratio that is approximately calculated as: RA/RT = 0.42. In this example, the exit region of each weir-trap comprises a frit that forms the boundary of the interior region, and the interior region of the weir- trap is quite large in comparison to the entrance region comprising the constriction used to trap cells or objects suspended in a fluid.
[0072] FIG. 5 illustrates a mesh network trapping geometry that has a trapping ratio that is approximately calculated as: RA/RT = 1.2. The weir-traps in this example again comprise a frit within the exit region of the trap.
[0073] In some instances, the disclosed microfluidic devices may comprise: a) a plurality of weir-traps disposed between, and in fluid communication with, at least one fluid inlet and at least one fluid outlet, wherein each weir-trap is configured to retain an object suspended in a fluid passing through the microfluidic device, and wherein: i) each weir-trap comprises a constriction in at least one dimension that is smaller than the smallest dimension of the object; and ii) a ratio of a fluidic resistance of a fluid flow path that bypasses a weir-trap to that for a fluid flow path passing through the weir-trap is at least 0.4. In some instances, as noted above, the constriction in at least one dimension may range in size from about 10% to about 90% of the smallest dimension of the cell or object to be trapped. For any of these instances in which the constriction in at least one dimension ranges in size from about 10% to about 90% of the smallest dimension of the cell or object to be trapped, the resistance of the fluid flow path that bypasses the weir-trap to that for the fluid flow path passing through the weir-trap (RA / RT) may range from about 0.4 to about 2.0. Non-limiting examples of combinations of constriction dimension (specified in terms of the percentage of the smallest dimension of the cell or object to be trapped) and resistance ratio (RA / RT) that are included in the present disclosure are (10%, 0.5), (10%, 0.6), (10%, 0.7), (10%, 0.8), (10%, 0.9), (10%, 1.0), (10%, 1.1), (10%, 1.2), (10%, 1.3), (10%, 1.4),
(10%, 1.5), (10%, 1.6), (10%, 1.7), (10%, 1.8), (10%, 1.9), (10%, 2.0), (20%, 0.5), (20%, 0.6),
(20%, 0.7), (20%, 0.8), (20%, 0.9), (20%, 1.0), (20%, 1.1), (20%, 1.2), (20%, 1.3), (20%, 1.4),
(20%, 1.5), (20%, 1.6), (20%, 1.7), (20%, 1.8), (20%, 1.9), (20%, 2.0), (30%, 0.5), (30%, 0.6),
(30%, 0.7), (30%, 0.8), (30%, 0.9), (30%, 1.0), (30%, 1.1), (30%, 1.2), (30%, 1.3), (30%, 1.4),
(30%, 1.5), (30%, 1.6), (30%, 1.7), (30%, 1.8), (30%, 1.9), (30%, 2.0), (40%, 0.5), (40%, 0.6),
(40%, 0.7), (40%, 0.8), (40%, 0.9), (40%, 1.0), (40%, 1.1), (40%, 1.2), (40%, 1.3), (40%, 1.4),
(40%, 1.5), (40%, 1.6), (40%, 1.7), (40%, 1.8), (40%, 1.9), (40%, 2.0), (50%, 0.5), (50%, 0.6),
(50%, 0.7), (50%, 0.8), (50%, 0.9), (50%, 1.0), (50%, 1.1), (50%, 1.2), (50%, 1.3), (50%, 1.4),
(50%, 1.5), (50%, 1.6), (50%, 1.7), (50%, 1.8), (50%, 1.9), (50%, 2.0), (60%, 0.5), (60%, 0.6),
(60%, 0.7), (60%, 0.8), (60%, 0.9), (60%, 1.0), (60%, 1.1), (60%, 1.2), (60%, 1.3), (60%, 1.4),
(60%, 1.5), (60%, 1.6), (60%, 1.7), (60%, 1.8), (60%, 1.9), and (60%, 2.0).
[0074] In some instances, the hydrodynamic traps may be configured to trap single cells or sub- cellular organelles, e.g ., nuclei. In some embodiments, the hydrodynamic traps may be configured to trap single cell pairs (i.e., pairs of single cells). In some instances, the
hydrodynamic traps may be configured to trap small groups of cells. In some instances, the hydrodynamic traps may be configured to trap groups of cells that may be used to establish organoids. In some instances, the hydrodynamic traps may be configured to function as cell growth chambers for trapping single cells and incubating them under conditions that support cell growth and division, thereby establishing small clonal groups of cells within a hydrodynamic trap.
[0075] Microfluidic device fabrication: In some embodiments, the microfluidic devices disclosed herein may comprise at least two separately fabricated parts (e.g, (i) a substrate that incorporates etched, embossed, or ablated fluid channels, and (ii) a cover or lid) that are subsequently either mechanically clamped together, temporarily adhered together, or
permanently bonded together. In some embodiments, the microfluidic devices disclosed herein may comprise three or more separately fabricated parts (e.g, (i) a substrate, (ii) a fluid channel layer, and (iii) a cover or lid) that are subsequently either mechanically clamped together, temporarily adhered together, or permanently bonded together. In some embodiments, the microfluidic devices disclosed herein may comprise a removable cover or lid. Examples of suitable fabrication techniques include, but are not limited to, conventional machining, CNC machining, injection molding, 3D printing, alignment and lamination of one or more layers of laser- or die-cut polymer film, or any of a number of microfabrication techniques such as photolithography and wet chemical etching, dry etching, deep reactive ion etching (DRIE), or laser micromachining. In some embodiments, all or a portion of the microfluidic devices may be
3D printed or casted from an elastomeric material.
[0076] The microfluidic devices disclosed herein or the components thereof ( e.g ., the substrate and/or lid) may be fabricated using any of a variety of materials known to those of skill in the art. In general, the choice of material used will depend on the choice of fabrication technique, and vice versa. Examples of suitable materials include, but are not limited to, silicon, fused-silica, glass, any of a variety of polymers, e.g. polydimethylsiloxane (PDMS; elastomer),
polymethylmethacrylate (PMMA), polycarbonate (PC), polystyrene (PS), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), polyimide, cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET), epoxy resins, a non-stick material such as teflon (PTFE), any of a variety of photoresists such as SU8 or any other thick film photoresist, or any combination of these materials.
[0077] In some embodiments, all or a portion of the microfluidic device (e.g, the cover or lid) may be fabricated from an optically transparent material to facilitate observation and monitoring (e.g, using high-resolution bright-field and/or fluorescence imaging) of cells or objects entrapped within the device. In some embodiments, the different layers in a microfluidic device comprising multiple layers may be fabricated from different materials, e.g, a fluid channel layer may be fabricated from an elastomeric material while the device substrate and a cover plate may be fabricated from glass or another suitable material.
[0078] As indicated above, in some embodiments the thickness of a fluid channel layer will determine the depth of the fluid channels and microfluidic chambers (e.g,“micro-chambers”, “trapping chambers”, or the interior regions of the traps) within the device and will thus influence the volume of the trapping chambers. In some embodiments, e.g, where fluid channels and trapping features are etched, embossed, or ablated into a substrate, the depth of the fluid channels and trapping chambers within the device will determined by the etch depth, embossed depth, or ablation depth, and will thus influence the volume of the trapping chambers. In some
embodiments, e.g, where fluid channels and trapping features are etched, embossed, or ablated into a substrate, the fluid channels and trapping chambers may have the same depth or different depths.
[0079] In general, the depth of fluid channels and/or trapping chambers within the disclosed devices may range from about 1 pm and about 1 mm. In some embodiments, the depth of the fluid channels and/or trapping chambers may be at least 1 pm, at least 5 pm, at least 10 pm, at least 20 pm, at least 30 pm, at least 40 pm , at least 50 pm, at least 100 pm, at least 200 pm, at least 300 pm, at least 400 pm, at least 500 pm, at least 600 pm, at least 700 pm, at least 800 pm, at least 900 pm, or at least 1 mm. In some embodiments, the depth of the fluid channels and/or trapping chambers may be at most 1 mm, at most 900 pm, at most 800 pm, at most 700 pm, at most 600 pm, at most 500 pm, at most 400 pm, at most 300 pm, at most 200 pm, at most 100 pm, at most 50 pm, at most 40 pm, at most 30 pm, at most 20 pm, at most 10 pm, at most 5 pm, or at most 1 pm. Any of the lower and upper values described in this paragraph may be combined to form a range included within the disclosure, for example, the depth of the fluid channels and/or trapping chambers may range from about 50 pm to about 100 pm. Those of skill in the art will recognize that depth of the fluid channels and/or trapping chambers may have any value within this range, for example, about 95 pm.
[0080] In general, the volumes of the microfluidic chambers ( e.g ., the hydrodynamic traps or trapping chambers) used in the disclosed devices may range from about 1,000 pm3 to about 1 mm3. In some embodiments, the microfluidic chamber volume may be at least 1,000 pm3, at least 10,000 pm3, at least 100,000 pm3, at least 1,000,000 pm3, at least 0.2 mm3, at least 0.5 mm3, or at least 1 mm3. In some embodiments, the microfluidic chamber volume is at most 1 mm3, at most 0.5 mm3, at most 0.2 mm3, at most 1,000,000 pm3, at most 100,000 pm3, at most 10,000 pm3, or at most 1,000 pm3. Any of the lower and upper values described in this paragraph may be combined to form a range included within the disclosure, for example, the microfluidic chamber volume may range from about 100,000 pm3 to about 0.2 mm3. Those of skill in the art will recognize that the chamber volume may have any value within this range, for example, about 1,250,000 pm3.
[0081] In some embodiments, the number of weir-traps and/or microfluidic chambers in the plurality of traps and/or chambers contained within a device of the present disclosure may range from about 1 to about 106, or more. In some embodiments, the number of traps and/or chambers within the device may be at least 1, at least 10, at least 100, at least 1,000, at least 104, at least 105, or at least 106. In some embodiments, the number of traps and/or chambers within the device may be at most 106, at most 105, at most 104, at most 1,000, at most 100, or at most 1.
Any of the lower and upper values described in this paragraph may be combined to form a range included within the disclosure, for example, the number of traps and/or chambers within the device may range from about 100 to about 10,000. Those of skill in the art will recognize that the number of traps and/or chambers within the device may have any value within this range, for example, about 1,200.
[0082] In some embodiments, the pitch (or spacing) between weir-traps may range from about 100 pm to about 1,000 pm, or more. In some embodiments, the pitch between weir-traps may be at least at least 100 pm, at least 200 pm, at least 300 pm, at least 400 pm, at least 500 pm, at least 600 pm, at least 700 pm, at least 800 pm, at least 900 pm, or at least 1,000 pm. In some embodiments, the pitch between weir-traps may be at most 1,000 pm, at most 900 pm, at most 800 mih, at most 700 mm, at most 600 mih, at most 500 mih, at most 400 mih, at most 300 mih, at most 200 mm, or at most 100 mih. Any of the lower and upper values described in this paragraph may be combined to form a range included within the disclosure, for example, the pitch between weir-traps may range from about 200 pm to about 400 pm. Those of skill in the art will recognize that the pitch between weir-traps may have any value within this range, for example, about 220 pm.
[0083] In some instances, one or more interior surfaces of the disclosed microfluidic devices may comprise one or more coating layers that, e.g ., facilitate attachment of the oligonucleotide barcode molecules of the oligonucleotide arrays and/or reduce non-specific binding of proteins to the interior surfaces of the device. Examples of suitable coating materials may include, but are not limited to, silane coatings, linear or branched polyethylene glycol (PEG) coatings, polydimethylsiloxane (PDMS) coatings, hydrogel coatings, and the like, or any combination of these. In some instances, the one or more interior surfaces of the disclosed microfluidic devices may comprise one, two, three, four, five, or more than five coating layers. In some instances, the oligonucleotide barcode molecules of the oligonucleotide arrays may be tethered to (or entrapped within) any one coating layer or tethered to (or entrapped within) any combination of two, three, four, five, or more than five coating layers. In some instances, the oligonucleotide barcode molecules of the oligonucleotide arrays may be embedded within one, two, three, four, five, or more than five coating layers.
[0084] In general, the bonding of microfluidic device components (e.g, a substrate and a lid that seals the hydrodynamic traps and interconnecting fluid channels) to create the assembled devices may comprise the use of a technique that provides a reliable seal while not adversely affecting the integrity of the oligonucleotide arrays disposed within the device. Examples of bonding techniques that may be used include, but are not limited to, use of adhesive layers or films (e.g, UV-curable adhesives or heat-curable adhesives), thin polydimethylsiloxane (PDMS) layers and coatings thereon, room temperature anodic bonding of glass to silicon, local ultrasonic welding, laser welding, and the like, or any combination thereof.
[0085] In some instances, the substrate is fabricated in silicon, the lid is glass, and the bonding step comprises coating a thin film of polydimethylsiloxane (PDMS) on the glass lid prior to bonding it to the silicon substrate. In some instances, the substrate is fabricated in silicon, the lid comprises a glass substrate with a polydimethylsiloxane (PDMS) layer coated thereon, and the bonding comprises coating and patterning a thin film of hydrogel on the PDMS layer prior to bonding the lid to the silicon substrate. In some instances, the bonding step comprises application of a thin coating layer of epoxy to the lid prior to fabricating the plurality of oligonucleotide arrays. In some instances, the epoxy is a UV-curable epoxy or a heat-curable epoxy. In some instances, the bonding step comprises formation of a hydrogel layer between the lid and substrate using thermal- or UV-induced polymerization of acrylate, methacrylate, or acrylamide groups that are attached to both surfaces. In some instances, the hydrogel layer between the lid and substrate comprises a cross-linked gel made by Michael addition of terminal thiol groups of a first polymer to acrylic or maleimide groups of a second polymer. In some instances, one of the polymers is a branched polymer, e.g ., a 4-armed polymer, 8-armed, 12- armed, 16-armed, or 32-armed polymer. In some instances, the acrylate groups are co-polymers containing both polydimethylsiloxane sequences and acrylate or methacrylate polymer sequences.
[0086] In some embodiments, the substrate and/or the lid further comprise one or more fiducial marks to facilitate alignment of the lid and substrate during assembly.
[0087] In some cases, the chips may be further packaged in a substrate carrier that facilitates ease-of-handling and/or facilitates interfacing the microfluidic device with external system components. In some instances, the substrate carrier (or chip package, housing, etc.) may comprise additional functional components. For example, in some instances, the substrate carrier may comprise integrated thermal sensors and/or controllers for local temperature control, gas connections that enable interfacing the device with external pressure controllers, and microfluidic interfaces (e.g, fluid connectors) that interface with external fluidics controllers to allow control of fluid flow within the microfluidic devices. The carrier may be comprised of a reusable housing that is machined in metal, such as aluminum or steel, or it may be composed of a single use disposable plastic housing that is fabricated by low-cost manufacturing techniques such as plastic injection molding. As noted, the housing may further comprise one or more thermal heating and sensing elements, such as resistive heating elements, thermoelectric modules, or another heat source that can be externally controlled, and may also comprise temperature monitoring capability using, e.g, one or more thermistors, thermocouples, resistive temperature detectors, thermostats, or other temperature sensing devices. In some instances, the temperature inside the plastic housing may be held at constant temperatures, such as at 4 °C, 21 °C, 37 °C, 42 °C, 50 °C, etc. (or any temperature within this range), for long time intervals, e.g, about 10 minutes, 30 minutes, 1 hour, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more than 4 weeks (or any time interval within this range). In some instances, the temperature may be cycled between different set temperatures (e.g, a set temperature from about 48 °C to about 65 °C for annealing, a set temperature from about 68 °C to about 72 °C for primer extension, and a set temperature from about 94 °C to about 98 °C for denaturation) and cycle times (e.g, cycle times ranging from about 10 seconds per cycle, 20 seconds per cycle, 30 second per cycle, 1 minute per cycle, 2 minutes per cycle, 3 minutes per cycle, 4 minutes per cycle, 5 minutes per cycle, 6 minutes per cycle, 7 minutes per cycle, 8 minutes per cycle, 9 minutes per cycle, 10 minutes per cycle, or more than 10 minutes per cycle (or any cycle time within this range) in applications that require
PCR amplification.
[0088] Oligonucleotide array design: The disclosed microfluidic devices may comprise oligonucleotide arrays disposed in all or a portion of the plurality of hydrodynamic traps within the device. In some instances, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% of the hydrodynamic traps within the device may comprise an oligonucleotide array.
[0089] In some instances, each oligonucleotide array may comprise from between 1 and 1,000 features or spots. In some instances, each oligonucleotide array may comprise at least 1, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1,000 features or spots. In some instances, each oligonucleotide array may comprise at most 1,000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 400, at most 300, at most 200, at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, at most 10, or at most 1 feature or spot. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances each oligonucleotide array may comprise from about 10 to about 100 features or spots. Those of skill in the art will recognize that the number of features or spots in the oligonucleotide arrays may have any value within this range, e.g. , about 18 features or spots. In some instances, the number of features or spots in each oligonucleotide array within the device may be the same. In some instances, the number of features or spots in different oligonucleotide arrays within the device may be different.
[0090] In some instances, the optimal size of the features or spots in an oligonucleotide array may be determined by the fabrication technique used, the surface density of the resulting tethered oligonucleotide molecules within the feature or spot, the diameter or area of the feature or spot, and/or the volume of the hydrodynamic trap chamber in which the oligonucleotide array is disposed (in order to ensure that an effective concentration of oligonucleotide barcode molecules (or barcoded primers) is compatible with performing molecular biology procedures such as reverse transcription, DNA polymerization, and/or DNA amplification reactions.
[0091] In some instances, the average diameter or longest dimension of the oligonucleotide array features or spots may range from about 10 pm to about 1 mm. In some instances, the average diameter or longest dimension of the features or spots is at least 10 pm, at least 15 pm, at least 20 mih, at least 25 mih, at least 30 mih, at least 35 mih, at least 40 mih, at least 45 mih, at least 50 mih, at least 60 mih, at least 70 mih, at least 80 mih, at least 90 mih, at least 100 mih, at least 200 mih, at least 300 mih, at least 400 mih, at least 500 mih, at least 600 mih, at least 700 mih, at least 800 mih, at least 900 mih, or at least 1,000 mih. In some instances, the average diameter or longest dimension of the features or spots is at most 1,000 mih, at most 900 mih, at most 800 mih, at most
700 mih, at most 600 mih, at most 500 mih, at most 400 mih, at most 300 mih, at most 200 mih, at most 100 mih, at most 90 mih, at most 80 mih, at most 70 mih, at most 60 mih, at most 50 mih, at most 45 pm, at most 40 mih, at most 35 mih, at most 30 mih, at most 25 mih, at most 20 pm, at most 15 mih, or at most 10 pm Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the average diameter or longest dimension of the features or spots may range from about 50 pm to about 100 pm. Those of skill in the art will recognize that the average diameter or longest dimension of the features or spots may have any value within this range, e.g ., about 74 pm. In some instances, the features or spots within an oligonucleotide array may all have the same average diameter or longest dimension. In some instances, different features or spots within an oligonucleotide array may have different average diameters or longest dimensions.
[0092] In some instances, the surface density of tethered oligonucleotide barcode molecules or primers within a given feature or spot may range from about 100 molecules per pm2 to about
100,000 molecules per pm2. In some instances, the surface density of oligonucleotide barcode molecules or primers may be at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000, at least
10,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 60,000, at least
70,000, at least 80,000, at least 90,000, or at least 100,000 molecules per pm2. In some instances, the surface density of oligonucleotide barcode molecules or primers may be at most
100,000, at most 90,000, at most 80,000, at most 70,000, at most 60,000, at most 50,000, at most
40,000, at most 30,000, at most 20,000, at most 10,000, at most 9,000, at most 8,000, at most
7,000, at most 6,000, at most 5,000, at most 4,000, at most 3,000, at most 2,000, at most 1,000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 400, at most 300, at most
200, or at most 100 molecules per pm2. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the surface density of oligonucleotide barcode molecules or primers may range from about 10,000 molecules per pm2 to about 90,000 molecules per pm2. Those of skill in the art will recognize that the surface density of oligonucleotide barcode molecules or primer molecules may have any value within this range, e.g. , about 85,600 molecules per pm2. [0093] Common barcode or unique cell barcode sequences: In some instances, the plurality of oligonucleotide molecules ( e.g ., molecules comprising identical copies of a given oligonucleotide sequence) within each feature or spot on the oligonucleotide array may comprise a common barcode sequence, e.g., a cell barcode sequence, that is presented in all of the features of a given oligonucleotide array and provides a unique identifier for determining, e.g, the identity of a single cell from which barcoded DNA molecules, RNA molecules, etc., were derived. In some instances, the common barcode sequence or unique cell barcode sequence is the same for each feature within a given oligonucleotide array, and different from that for all features in all other oligonucleotide arrays within the device. In some instances, the common barcode sequence or unique cell barcode sequence is different for each feature within a given oligonucleotide array, and different from that for all features in all other oligonucleotide arrays within the device. In some instances, the common barcode sequence or unique cell barcode sequence for each feature of each oligonucleotide array of the plurality of oligonucleotide arrays is known. In some instances, the identity of each oligonucleotide sequence for each feature in the plurality of oligonucleotide arrays may be determined using a sequencing-by-hybridization approach.
[0094] In some instances, the unique cell barcode sequence may comprise a string of N“words”, wherein each“word” comprises a string of M bases. In some instances, M may be 1 base, 2 bases, 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, or at least 10 bases. In some instances, N is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12, such that the common barcode or unique cell barcode sequence comprises up to at least 12“words”, each“word” comprising a string of up to
10 bases.
[0095] In some instances, the common barcode or unique cell barcode may range in length from about 1 base to about 120 bases. In some instances, the length of the common barcode or unique cell barcode may be at least 1 base, at least 5 bases, at least 10 bases, at least 20 bases, at least 30 bases, at least 40 bases, at least 50 bases, at least 60 bases, at least 70 bases, at least 80 bases, at least 90 bases, at least 100 bases, as least 110 bases, or at least 120 bases. In some instances, the length of the common barcode or unique cell barcode may be at most 120 bases, at most 110 bases, at most 100 bases, at most 90 bases, at most 80 bases, at most 70 bases, at most 60 bases, at most 50 bases, at most 40 bases, at most 30 bases, at most 20 bases, at most 10 bases, at most 5 bases, or at most 1 base. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the length of the common barcode or unique cell barcode may range from 10 bases to 20 bases. Those of skill in the art will recognize that the length of the common barcode or unique cell barcode may have any value within this range, e.g, 42 bases. [0096] In some instances, the plurality of common barcode sequences or unique cell barcode sequences for the plurality of hydrodynamic traps within a device comprise unique non overlapping sequences with a Hamming distance that enables demultiplexing with error correction capability.
[0097] In some instances, the common barcode sequence or unique cell barcode sequence may comprise a G/C content ranging from about 10% to about 90%. In some instances, the common barcode sequence or unique cell barcode sequence may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% G/C content. In some instances, the common barcode sequence or unique cell barcode sequence may comprise at most 90%, at most 80%, at most 70%, at most 60%, at most 50%, at most 40%, at most 30%, at most 20%, or at most 10% G/C content. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the common barcode or unique cell barcode may comprise from about 30% to about 70% G/C content. Those of skill in the art will recognize that the common barcode or unique cell barcode may comprise any value of G/C content within this range, e.g. , about 54% G/C content.
[0098] In some instances, the tethered oligonucleotide barcode molecules or primers of the oligonucleotide arrays may comprise a free 5’ end. In some instances, the tethered
oligonucleotide barcode molecules or primers of the oligonucleotide arrays may comprise a free 3’ end.
[0099] Other oligonucleotide sequence components: In some instances, the oligonucleotide barcode molecules within the features of the oligonucleotide arrays may further comprise a spacer sequence, an adapter sequence, at least one primer sequence (e.g, a universal primer sequence), a molecular index sequence, a molecular recognition or target capture sequence, a random heptamer capture sequence, a cleavable moiety or cleavage site, a surface attachment moiety, or any combination thereof.
[0100] Molecular recognition or target capture sequences: In many instances, the
oligonucleotide molecules of the array may comprise a molecular recognition or target capture sequence designed to capture specific target molecules, e.g, specific gene sequences or gene fragment sequence, or to capture specific classes of target molecules, e.g, using a poly-T sequences to capture poly-A tagged RNA molecules. In some instances, the molecular recognition or target capture sequence may comprise a capture sequence designed to capture a broad class of target molecules, e.g, when using a random capture sequence (a“randomer”) to hybridize to genomic DNA. Examples of suitable molecular recognition or target capture sequences include, but are not limited to, an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(deoxyguanosine) (oligo-dG) sequence, an oligo(riboguanosine) (oligo-rG) sequence, a sequence comprising a phosphorothioate oligonucleotide, a sequence comprising a locked nucleic acid, a sequence containing 2-OMe oligonucleotides, a sequence containing modified nucleotides, a complement of an mRNA sequence or fragment thereof, a complement of a tRNA sequence or fragment thereof, a complement of an rRNA sequence or fragment thereof, a complement of a DNA sequence, a complement of a cDNA sequence, a complement of a gene sequence or fragment thereof, or any combination thereof.
[0101] In some instances, the molecular recognition or target capture sequence may comprise a random sequence (or“randomer”). In some instances, the molecular recognition or target capture sequence may comprise, for example, a random tetramer, a random pentamer, a random hexamer, a random heptamer, a random octamer, a random nonamer, a random decamer, a random undecamer, a random dodecamer, or a random multimer, and the like.
[0102] In some instances, the molecular recognition or target capture sequence may range in length from about 1 base to about 150 bases. In some instances, the length of the molecular recognition or target capture sequence may be at least 1 base, at least 2 bases, at least 5 bases, at least 10 bases, at least 20 bases, at least 30 bases, at least 40 bases, at least 50 bases, at least 60 bases, at least 70 bases, at least 80 bases, at least 90 bases, at least 100 bases, as least 110 bases, as least 120 bases, as least 130 bases, as least 140 bases, or at least 150 bases. In some instances, the length of the molecular recognition or target capture sequence may be at most 150 bases, at most 140 bases, at most 130 bases, at most 120 bases, at most 110 bases, at most 100 bases, at most 90 bases, at most 80 bases, at most 70 bases, at most 60 bases, at most 50 bases, at most 40 bases, at most 30 bases, at most 20 bases, at most 10 bases, at most 5 bases, at most 2 bases, or at most 1 base. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the length of the molecular recognition or target capture sequence may range from 2 bases to 50 bases. Those of skill in the art will recognize that the length of the molecular recognition or target capture sequence may have any value within this range, e.g ., 12 bases.
[0103] In some instances, the oligonucleotide molecules in each feature of an oligonucleotide array may comprise a molecular recognition sequence that is the same as that for the other features within the oligonucleotide array. In some instances, the oligonucleotide molecules in each feature of an oligonucleotide array may comprise a molecular recognition sequence that is different from that for the other features within the oligonucleotide array. In some instances, the oligonucleotide molecules in each feature of an oligonucleotide array may comprise a molecular recognition sequence that is the same as that for a subset of other features within the array and different from the remainder of features in the array. [0104] Molecular index sequences: In some instances, the oligonucleotide molecules of the oligonucleotide arrays may comprise a molecular index (or counter) sequence that is unique for each individual oligonucleotide molecule of the plurality of oligonucleotides within a given feature, and where the sequence diversity represented by the plurality of unique molecular index sequences presented within a given feature is large compared to the expected number of target molecules to be derived from a single cell, thereby allowing one to count the number of barcoded target molecules (or complements thereof) derived from a single cell by determining the number of unique molecular index sequences that share the same cell barcode in a sequencing run performed after the barcoded target molecules have been amplified and sequenced. One non limiting example of an approach to stochastic barcoding of molecules derived from cells has been described by Fan, et al. (2015), "Combinatorial labeling of single cells for gene expression cytometry", Science 347(6222): 1258367.
[0105] In some instances, the molecular index sequence may range in length from about 1 base to about 150 bases. In some instances, the length of the molecular index sequence may be at least 1 base, at least 2 bases, at least 5 bases, at least 10 bases, at least 20 bases, at least 30 bases, at least 40 bases, at least 50 bases, at least 60 bases, at least 70 bases, at least 80 bases, at least 90 bases, at least 100 bases, as least 110 bases, as least 120 bases, as least 130 bases, as least 140 bases, or at least 150 bases. In some instances, the length of the molecular index sequence may be at most 150 bases, at most 140 bases, at most 130 bases, at most 120 bases, at most 110 bases, at most 100 bases, at most 90 bases, at most 80 bases, at most 70 bases, at most 60 bases, at most 50 bases, at most 40 bases, at most 30 bases, at most 20 bases, at most 10 bases, at most 5 bases, at most 2 bases, or at most 1 base. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the length of the molecular index sequence may range from 5 bases to 10 bases. Those of skill in the art will recognize that the length of the molecular recognition or target capture sequence may have any value within this range, e.g ., 7 bases.
[0106] Surface attachment moieties: In some instances, the oligonucleotide barcode molecules of the oligonucleotide arrays disposed within the device may comprise specific functional groups at the 3’ or 5’ end that facilitate attachment of the oligonucleotides to a surface or coating layer. Examples include, but are not limited to, amine-terminated oligonucleotides (for grafting onto surfaces or coating layers functionalized with an epoxide, glicydyl, aldehyde, NHS ester, or other amine-reactive moiety), azide-terminated oligonucleotides (for grafting onto alkyne- functionalized surfaces or coating layers using a type of“click chemistry”), alkyne-terminated oligonucleotides (for grafting onto azide-functionalized surfaces or coating layers), acroxyl- terminated oligonucleotides (for grafting onto acrylamide or methacrylamide functionalized surfaces or coating layers), and biotin-terminated oligonucleotides (for attachment to streptavidin-functionalized surfaces or coating layers), maleimide- or acrylate-terminated oligonucleotides (for grafting onto thiol-functionalized surfaces or coating layers), and thiol- terminated oligonucleotides (for grafting onto maleimide- or acrylate-functionalized surfaces of coating layers).
[0107] Cleavable moieties or cleavage sites: In some instances, the oligonucleotide barcode molecules of the oligonucleotide arrays disposed within the device may comprise a cleavable moiety or cleavage site that allows the oligonucleotide molecules within one or more features of the oligonucleotide array to be cleaved and released into solution with the hydrodynamic trap, thereby enabling more efficient solution-phase priming of target molecules, reverse transcription, DNA polymerization, DNA amplification, etc. Examples of suitable cleavage mechanisms include, but are not limited to, sequences or linkers that are cleaved using light ( e.g. , UV light), heat, enzymes (e.g, a USER™ enzyme), reducing or oxidizing agents, a change in pH, or any combination thereof.
[0108] In some instances, the cleavage site comprises a deoxyuridine base. Treatment with USER™ enzyme (a mixture of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII) catalyzes the excision of the uracil base, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact. The lyase activity of Endonuclease VIII then breaks the phosphodiester backbone at the 3 ' and 5 ' sides of the abasic site.
[0109] In some instances, the 5’ end of the oligonucleotide barcode molecules may be released into solution upon treatment with light (e.g, UV light), heat, enzymes (e.g, a USER™ enzyme), reducing or oxidizing agents, a change in pH, or any combination thereof. In some instances, the 3’ end of the oligonucleotide barcode molecules may be released into solution upon treatment with light (e.g, UV light), heat, enzymes (e.g, a USER™ enzyme), reducing or oxidizing agents, a change in pH, or any combination thereof.
[0110] In some instances, the oligonucleotide molecules in every feature of an oligonucleotide array may comprise the same cleavable moiety, e.g, a photocleavable linker, an enzymatically- cleavable linker, a pH-sensitive cleavable linker, a redox-cleavable linker, or a thermally- cleavable linker. In some instances, the oligonucleotide molecules in different features of the oligonucleotide array may comprise different cleavable moieties, e.g, linkers cleaved by orthogonal cleavage mechanisms, so that barcoded oligonucleotide molecules can be selectively released from different features of the array at, for example, different steps in a sequencing library preparation workflow.
[0111] Spacer sequences: In some instances, the oligonucleotide barcode molecules of the oligonucleotide arrays disposed within the device may comprise one or more spacer sequences disposed between the surface or coating layer to which the oligonucleotide molecules are tethered and the remainder of the oligonucleotide sequence or between other functional components of the oligonucleotide sequence ( e.g ., between a molecular recognition sequence and a molecular index sequence, etc.). In some instances, the oligonucleotide barcode molecules of the oligonucleotide arrays disposed within the device may comprise one or more spacer sequences disposed between the surface or coating layer to which the oligonucleotide molecules are tethered and a cleavage site. In some instances, the oligonucleotide barcode molecules of the oligonucleotide arrays disposed within the device may comprise one or more spacer sequences disposed between a cleavage site and the remainder of the remainder of the oligonucleotide sequence.
[0112] In some instances, the one or more spacer sequences may range in length from about 1 base to about 50 bases. In some instances, the length of the spacer sequence may be at least 1 base, at least 2 bases, at least 5 bases, at least 10 bases, at least 15 bases, at least 20 bases, at least 25 bases, at least 30 bases, at least 35 bases, at least 40 bases, at least 45 bases, or at least 50 bases. In some instances, the length of the spacer sequence may be at at most 50 bases, at most 45 bases, at most 40 bases, at most 35 bases, at most 30 bases, at most 25 bases, at most 20 bases, at most 15 bases, at most 10 bases, at most 5 bases, at most 2 bases, or at most 1 base. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the length of the spacer sequence may range from 10 bases to 20 bases. Those of skill in the art will recognize that the length of the spacer sequence may have any value within this range, e.g., 14 bases.
[0113] Adapter sequences: In some instances, the oligonucleotide barcode molecules of the oligonucleotide arrays disposed within the device may comprise one or more adapter sequences for facilitating downstream compatibility with Next-Generation sequencing platforms. Examples of suitable adapter sequences include, but are not limited to, the Illumina P5/P7 adapter sequences that bind to the Illumina platform flowcells.
[0114] In some instances, the one or more adapter sequences may range in length from about 6 bases to about 30 bases. In some instances, the length of the adapter sequence may be at least 6 bases, at least 7 bases, at least 8 bases, at least 9 bases, at least 10 bases, at least 11 bases, or at least 12 bases. In some instances, the length of the adapter sequence may be at most 12 bases, at most 11 bases, at most 10 bases, at most 9 bases, at most 8 bases, at most 7 bases, or at most 6 bases. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the length of the adapter sequence may range from 7 bases to 11 bases. Those of skill in the art will recognize that the length of the adapter sequence may have any value within this range, e.g, 10 bases. [0115] Primer sequences: In some instances, the oligonucleotide barcode molecules of the oligonucleotide arrays disposed within the device may comprise one or more primer sequences
( e.g ., universal primer sequences) for facilitating amplification and sequencing reactions. In some instances, the oligonucleotide barcodes may comprise 1, 2, 3, 4, 5, 6, or more than 6 primer sequences.
[0116] In some instances, the one or more primer sequences may range in length from about 10 bases to about 50 bases. In some instances, the length of the primer sequence may be at least 10 bases, at least 15 bases, at least 20 bases, at least 25 bases, at least 30 bases, at least 35 bases, at least 40 bases, at least 45 bases, or at least 50 bases. In some instances, the length of the primer sequence may be at most 50 bases, at most 45 bases, at most 40 bases, at most 35 bases, at most 30 bases, at most 25 bases, at most 20 bases, at most 15 bases, or at most 10 bases. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the length of the primer sequence may range from 20 bases to 30 bases. Those of skill in the art will recognize that the length of the primer sequence may have any value within this range, e.g., 24 bases.
[0117] Gene sequence fragments: In some instances, the oligonucleotide barcode molecules of one or more features within an oligonucleotide array may comprise all or a portion of a gene sequence or fragment thereof. In some instances, the plurality of gene sequence fragments presented by the one or more features of the oligonucleotide array collectively span a full-length gene sequence.
[0118] In some instances, the average length of the gene sequence fragments presented in features of the array may range from about 0.01 kilobase (10 bases) to about 1 kilobase (1,000 bases). In some instances, the average length of the gene sequence fragments may be at least 0.01 kilobase, at least 0.02 kilobase, at least 0.1 kilobase, at least 0.2 kilobase, at least 0.3 kilobase, at least 0.4 kilobase, at least 0.5 kilobase, at least 0.6 kilobase, or at least 0.7 kilobase, at least 0.8 kilobase, at least 0.9 kilobase, or at least 1 kilobase. In some instances, the average length of the gene sequence fragments may be at most 1 kilobase, at most 0.9 kilobase, at most 0.8 kilobase, at most 0.7 kilobase, at most 0.6 kilobase, at most 0.5 kilobase, at most 0.4 kilobase, at most 0.3 kilobase, at most 0.2 kilobase, or at most 0.1 kilobase. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the average length of gene sequence fragments may range from 0.2 kilobase to 0.9 kilobase. Those of skill in the art will recognize that the average length of the gene sequence fragments may have any value within this range, e.g, about 0.56 kilobase. [0119] In some instances, the average length of a full-length gene sequence assembled within the disclosed microfluidic devices using two or more gene sequence fragments presented in the features of an oligonucleotide array disposed within a trap of the device may range from about
0.5 kilobase to about 10 kilobase. In some instances, the average length of the full-length gene sequence may be at least 0.5 kilobase, at least 1 kilobase, at least 1.5 kilobase, at least 2 kilobase, at least 2.5 kilobase, at least 3 kilobase, at least 3.5 kilobase, at least 4 kilobase, at least 4.5 kilobase, at least 5 kilobase, at least 5.5 kilobase, at least 6 kilobase, at least 6.5 kilobase, at least
7 kilobase, at least 7.5 kilobase, at least 8 kilobase, at least 8.5 kilobase, at least 9 kilobase, at least 9.5 kilobase, or at least 10 kilobase. In some instances, the average length of the full-length gene sequence may be at most 10 kilobase, at most 9.5 kilobase, at most 9 kilobase, at most 8.5 kilobase, at most 8 kilobase, at most 7.5 kilobase, at most 7 kilobase, at most 6.5 kilobase, at most 6 kilobase, at most 5.5 kilobase, at most 5 kilobase, at most 4.5 kilobase, at most 4 kilobase, at most 3.5 kilobase, at most 3 kilobase, at most 2.5 kilobase, at most 2 kilobase, at most 1.5 kilobase, at most 1 kilobase, or at most 0.5 kilobase. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the average length of full-length gene sequence may range from 0.2 kilobase to 6 kilobase. Those of skill in the art will recognize that the average length of the full-length gene sequence may have any value within this range, e.g ., about 2.8 kilobase.
[0120] Oligonucleotide composition & total length: In some instances, the oligonucleotide molecules of the plurality of arrays within the device may comprise conventional nucleotide building blocks. In some instances, the oligonucleotide molecules of the plurality of arrays may comprise one or more modified nucleotides as building blocks, e.g. , including but not limited to, locked nucleic acid sequences, ribonucleotides, and phosphorthioated nucleotides.
[0121] In some instances, the total length of the oligonucleotide molecules may range from about 20 bases to about 200 bases. In some instances, the length of the oligonucleotide molecules may be at least 20 bases, at least 30 bases, at least 40 bases, at least 50 bases, at least 60 bases, at least 70 bases, at least 80 bases, at least 90 bases, at least 100 bases, as least 110 bases, at least 120 bases, at least 130 bases, at least 140 bases, at least 150 bases, at least 160 bases, at least 170 bases, at least 180 bases, at least 190 bases, or at least 200 bases. In some instances, the length of the oligonucleotide molecules may be at most 2000 bases, at most 190 bases, at most 180 bases, at most 170 bases, at most 160 bases, at most 150 bases, at most 140 bases, at most 130 bases, at most 120 bases, at most 110 bases, at most 100 bases, at most 90 bases, at most 80 bases, at most 70 bases, at most 60 bases, at most 50 bases, at most 40 bases, at most 30 bases, or at most 20 bases. Any of the lower and upper values described in this paragraph may be combined to form a range included within the present disclosure, for example, in some instances the length of the oligonucleotide molecules may range from 70 bases to 120 bases. Those of skill in the art will recognize that the length of the oligonucleotide molecules may have any value within this range, e.g ., 74 bases.
[0122] Oligonucleotide barcode array fabrication: A variety of techniques may be used to synthesize and/or deposit oligonucleotide arrays within the hydrodynamic traps of the disclosed microfluidic devices (as illustrated in FIG. 6). The plurality of oligonucleotide arrays (or molecular patterns) can be deposited in the plurality of hydrodynamic traps, e.g. , one
oligonucleotide array per hydrodynamic trap, or in a portion of the interior region of each hydrodynamic trap (FIG. 6), by any of a variety of techniques known to those of skill in the art including, but not limited to, inkjet printing, microarray spotting, solid phase oligonucleotide synthesis (e.g, using a spatially-addressable solid-phase synthesis technique), enzymatic extension, enzymatic ligation of one or more smaller oligonucleotide segments, or a pattern transfer process, such as stamping or contact printing. In some instances, contact printing or stamping may be used to create a replica of the molecular pattern (or its inverse) on a surface. In some instances, the oligonucleotide barcode sequences may be introduced into the hydrodynamic traps via beads, vesicles, virus particles, or genomic material contained within a cell or virus particle. In some instances, one or more coating layers are added to an interior surface of the substrate or to a first surface of the lid for the purpose of attaching entrapping the oligonucleotide molecules of the oligonucleotide arrays.
[0123] The plurality of oligonucleotide arrays may be attached to the underside of the lid used to seal the device and/or deposited directly on the substrate (e.g. within the interior regions of the hydrodynamic traps), and maintained in place through covalent, ionic, and/or affinity interactions with a substrate or lid surface (or a coating layer thereon). In some instances, the
oligonucleotide molecules of the oligonucleotide arrays may be grafted onto the surface by deposition of amine-terminated oligonucleotides onto lids or substrates that are functionalized with an epoxide, glicydyl, aldehyde, NHS ester, or other amine-reactive moiety. In some instances, the oligonucleotide arrays may be grafted onto the surface by deposition of azide- terminated oligonucleotides onto an alkyne-functionalized substrate or lid, which is a well-known type of“click chemistry”. In some instances, the oligonucleotide arrays may be grafted onto the surface by deposition of alkyne-terminated oligonucleotides to an azide-functionalized substrate or lid. In some instances, the oligonucleotide arrays may be grafted onto the surface by deposition of an acroxyl -terminated oligonucleotides to an acrylamide or methacrylamide functionalized substrate. In some instances, the oligonucleotide arrays may be grafted onto the surface by deposition of biotin-terminated oligonucleotides on a streptavidin functionalized substrate. In some instances, the oligonucleotide arrays may be grafted onto the surface by deposition of maleimide- or acrylate-terminated oligonucleotides on a thiol-functionalized substrate or lid. In some instances, the oligonucleotide arrays may be grafted onto the surface by deposition of thiol-terminated oligonucleotides on a maleimide- or acrylate-functionalized substrate or lid. In some instances, the oligonucleotide arrays may be grafted onto the surface by deposition of unfunctionalized oligonucleotides onto substrates containing grafted cationic surface charges, wherein the oligonucleotides remain fixed in place due to ionic interactions. In some instances, the oligonucleotides may be generated by solid-phase synthesis on the underside of the lid or directly in the microfluidic substrate.
[0124] In some instances, the oligonucleotide arrays may be fabricated using a transfer process comprising: a) generating a master substrate containing a plurality of oligonucleotides that are hybridized to complementary strands that contain an affinity group; and b) then transferring the complementary strands to the underside of a lid or to a surface within the microfluidic substrate by a liquid or solid stamping process, as described in prior references (see, e.g ., A. A. Yu, F. Stellacci, Contact Printing Beyond Surface Roughness: Liquid Supramolecular Nanostamping, Advanced Materials, Volumel9, Issue 24, 2007, pp. 4338-4342
https://doi.org/10.1002/adma.200701068). In some instances, the liquid stamping process may involve creating a covalent network of acroxy-terminated DNA molecules inside a matrix of acrylates, methacrylates, polymethacrylates, polymethylmethacrylates, polydimethylsiloxanes, and/or any of their block copolymer combinations in a layer sandwiched between a master and a lid, after which the lid is peeled away from the master, yielding a complementary set of oligonucleotide arrays attached to the lid - the lid subsequently being used to seal the
microfluidic device as discussed elsewhere.
[0125] FIG. 11 shows examples of data for an oligonucleotide pattern replication technique in an acrylamide gel that involves spotting the oligonucleotides onto a template glass chip, and then hybridizing the spotted oligonucleotides to complementary oligonucleotides having an acryoxy- termination. The template chip was then immersed in an acrylamide solution layered between the template chip and a daughter chip, following which the acrylamide is allowed to set and incorporate the acroxy-terminated oligonucleotides into the polymer backbone of the gel.
Fluorescence images of the template chip (top and middle rows) and daughter chip (bottom row) surfaces were taken before and after the transfer step. The complementary strands are shown to become incorporated in the acrylamide layer adhering to the daughter chip, demonstrating a DNA pattern transfer mechanism.
[0126] FIGS. 12A-B shows a process similar to that described in FIG. 11, except that the gel is composed of a block copolymer containing acrylamide and polydimethylsiloxane blocks. FIG. 12A: Fluorescently labeled, acrylate terminated oligos hybridized to spots of complementary, surface bound oligo on the template surface. FIG. 12B: Mirror image observed after oligos transferred to a daughter surface using acrylates and hybridized with a fluorescent,
complementary sequence.
[0127] Methods for performing single cell analysis: Disclosed herein are methods for performing single cell analysis, the methods comprising one or more of: a) providing a microfluidic device according to any of the embodiments described elsewhere herein; b) flowing a suspension of cells through the microfluidic device, thereby trapping single cells in all or a portion of the plurality of hydrodynamic traps; c) flowing a cell lysis and/or molecular reaction buffer ( e.g. , a buffer comprising one or more enzymatic components and co-factors, cations, etc ., required for performing reverse-transcription reactions, DNA polymerization (primer extension) reactions, DNA amplification reactions, and the like) through the microfluidic device, thereby releasing target oligonucleotide molecules from the single cell trapped in each trap; d) optionally, flowing in an immiscible fluid such as air or oil that fills bypass channels but does not enter the hydrodynamic traps containing the single cells, and is used to prevent mixing of cellular material from different hydrodynamic traps during molecular barcoding steps; e) cleaving the barcoded oligonucleotide molecules from the substrate to participate in solution phase reactions; f) incubating the microfluidic device under conditions that promote hybridization of one or more target oligonucleotide molecules released by lysis of the single cells to one or more molecular recognition sequences presented in the features of the oligonucleotide array in each
hydrodynamic trap; g) performing a primer extension reaction within the microfluidic device; h) eluting the barcoded oligonucleotide molecules from each trap from the microfluidic device; i) optionally, eluting the barcoded oligonucleotide molecules from specific traps of the microfluidic device by applying UV light to specific hydrodynamic traps to cleave and subsequently elute only those selected primers from the microfluidic device; and j) amplifying and sequencing the barcoded oligonucleotides to detect the presence of the one or more target oligonucleotide molecules in one or more single cells, wherein the sequence of a unique cell barcode sequence presented in the features of the oligonucleotide array in each hydrodynamic trap is used to identify target oligonucleotide molecules that were released from a given single cell.
[0128] In some instances, the hydrodynamic traps of the disclosed microfluidic devices in any of the embodiments described herein (and for any of the methods described herein) may be used for trapping a single cell per trap. In some instances, the hydrodynamic traps may be used for simultaneously or sequentially trapping a single pair of cells (e.g, two different cells or two cells of the same cell type) or a single cell - bead pair (e.g, wherein the bead comprises tethered oligonucleotide barcode molecules, agonists, antagonists, antigens, etc.) in each trap. In some instances, the hydrodynamic trap may be used to trap a single cell and may then be incubated under conditions that promote cell growth and division to produce small clonal populations of cells. In some instances, the hydrodynamic traps may be used to trap 1, 2, 3, 4, 5, 6, or more than
6 single cells and/or beads in total per trap.
[0129] In some instances, single cells or small clusters of cells may be trapped and maintained in a viable state for prolonged periods of time while phenotypic traits are monitored by means of high-resolution imaging of the trap array. In some instances, the cells may be maintained in a viable state within the trap array for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weeks, at least 3 weeks, or at least 4 weeks. In some instances, phenotypic traits are monitored as the single cells or small clusters of cells in the trap array are subjected to one or more chemical or environmental stimuli, e.g ., exposure to a therapeutic drug or drug candidate, a cytokine, a chemokine, a change in temperature, a change in growth medium, etc. In some instances, the cells within the trap array are lysed at a pre-determined or random experimental endpoint and barcoding reactions are performed to tag one or more selected target molecule classes. In some instances, the experimental endpoint may be 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours,
1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, or any time point within this range.
[0130] In some instances, the barcoded oligonucleotide molecules are released from the array prior to participating in primer hybridization, reverse-transcription, DNA polymerization, and/or DNA amplification reactions while in other instances, the barcoded oligonucleotide molecules are released after participating in enzymatic reactions.
[0131] In some instances, a determination of the number of unique molecular index sequences corresponding to each unique cell barcode sequence is used to quantify how many copies of a given target oligonucleotide were released from a given single cell. In some instances, the amplifying and sequencing are performed within the microfluidic device. In some instances, the amplifying and sequencing are performed after cleaving and eluting the barcoded oligonucleotide molecules from the microfluidic device. In some instances, the target oligonucleotide molecules comprise mRNA molecules or fragments thereof, tRNA molecules or fragments thereof, rRNA molecules or fragments thereof, RNA molecules or fragments thereof, DNA molecules or fragments thereof, gene sequences or fragments thereof, or any combination thereof. In some instances, the target oligonucleotide molecules comprise molecules that can be used in determining the open regions of a chromosome for the purpose of identifying the genes that are being expressed. In some instances, the target oligonucleotide molecules are those that enable amplification of the entire genome of the trapped cells to determine copy number variation and or single nucleotide variants. In some instances, the target oligonucleotide molecules are those that capture a subset of RNA transcripts, genes, or gene fragments for targeted molecular
identification, including whole exome analysis. In some instances, the target oligonucleotide molecules are oligonucleotide labels attached to antibodies for protein detection. In some instances, the target oligonucleotide molecules detect DNA or RNA sequences introduced into cells.
[0132] Single cell barcoding for preparation ofRNA-seq libraries: In some instances, the disclosed microfluidic devices comprising a substrate, lid, and a plurality of oligonucleotide arrays disposed within the hydrodynamic traps of the device may be used in the preparation of RNA-seq libraries for 3’ or 5’ end counting of the expression profiles of single cells, this approach comprising, a) introducing cells into the microfluidic device and capturing them in single cell per trap format, b) introducing a master mix containing cell lysis and reverse transcription reagents into the microfluidic device, and then rapidly replacing the master mix solution in the bypass channels of the device with an air plug or oil plug to prevent cross contamination between different chambers (as illustrated using a fluorescent dye solution (FIG. 7B) instead of master mix to fill the trapping chambers of the device shown in FIG. 7A), and c) cleaving the barcode oligonucleotides from the interior surfaces of the traps by means of chemical cleavage, enzymatic cleavage, and/or photocleavage techniques. In some instances, the master mix optionally contains USER™ enzymes, which are able to cleave a uridine base within the plurality of barcoded oligonucleotide molecules that are attached to the surface, and has the advantage of enabling these molecules to be released from the surface to participate in faster solution-phase reverse transcription reaction kinetics. In some instances, the instrument is optionally capable of exposing the plurality of barcoded oligonucleotide arrays to UV or visible wavelengths of light at the dosage required to break photocleavable moieties that are
incorporated into the backbone of the oligonucleotide sequences, which is an alternative approach to release the barcode oligo primers from the surface and enable solution-phase reverse transcription reactions. The method further comprises: d) exposing the device substrate to temperatures in the range of 30 to 60 °C to enable full length cDNA to be generated from the primed RNA molecules; and f) unsealing each trap chamber by replacing the air or oil with RT buffer and/or other molecular biology reagents used to further amplify and prepare the molecules for analysis by next generation sequencing. [0133] In some instances, the master mix may comprise i) Triton X-100, ii) Tween-20, iii) molecular crowding agents such as polyethylene glycol, Ficoll, gelatin, iv) RNAse inhibitors, v) dNTPs, vi) RT enzymes, vii) buffer components including Potassium, Cesium, Sodium,
Chlorine, Magnesium, and other common salts, viii) optionally, USER™ enzymes for enzymatic cleaving of oligonucleotide arrays that contain at least one uridine base, and ix) oligo-dT terminated primers that may incorporate a cell identification barcode and template switch oligos, or primers that have oligo-rG terminations and may incorporate a cell identification barcode
(these dT and rG primers also containing the same or different amplification sites used for subsequent library preparation steps), or any combination thereof. In some instances, the libraries are prepared by in vitro transcription as described in CEL-seq and CEL-seq2. In other instances, the libraries can be prepared using chemistry similar to Smart-seq, Smart-seq2, and
Smart-seq3, Quartz-seq, as well as other methods of RNA library preparation known to those skilled in the art.
[0134] FIGS. 8A-B show examples of data for the base calls in an RNA sequencing run in which the library preparation was performed using biotinylated oligonucleotide barcodes comprising a cleavable linker (FIG. 8A) or no cleavable linker (FIG. 8B) spotted on streptavidin-coated glass slides. A suspension of 5 cells were added to the RT/lysis mix incubated in a flow cell, and then exposed to 365 nm UV light for the first 15 minutes of the RT step. The rGrGrG sequence immediately upstream of the cleaved section was used for template switching and preparing full length transcriptome libraries. Raw fastq files were processed to measure base percentages by read position. FIG. 8A: Sequence for the photocleavable oligos contain the universal primer, barcode, and GGG sequence at high percentages, while the sequence data for oligos lacking a photocleavable linker (FIG. 8B) lower efficiency of barcode incorporation and GGG sequence. Sequence reads from the cleaved oligos map to the genome efficiently and predominantly map to the 5’ end of mRNAs.
[0135] FIGS. 13A-B show examples of data for an RNA library preparation reaction that was performed in a microfluidic device, followed by sequencing of the eluted barcoded
complementary sequences on a sequencer. The barcode region was constructed by enzymatic ligation of two distinct barcodes (FIG. 13A). Following the sequencing quality control steps, more than 60% of the reads contained the barcode, mapped to the genome, and contained useable biological information (FIG. 13B).
[0136] FIGS. 14A-B show examples of RNA library preparation and sequencing data. FIG.
14A: sequencing quality control metrics. FIG. 14B: Plot of the number of genes detected versus the number of reads measured. [0137] Single cell barcoding for preparation of DNA-seq libraries: In some instances, the disclosed microfluidic devices comprising a substrate, lid, and a plurality of oligonucleotide arrays disposed within the hydrodynamic traps of the device may be used in the preparation of
DNA-seq libraries for whole genome amplification, or targeted gene analysis, this approach comprising, a) introducing cells into the microfluidic device and capturing them in single cell per trap format, b) introducing a cell lysis buffer into the device which is capable of dissociating the nuclear membrane and histone complexes to fully denature the cellular DNA, c) introducing
DNA amplification reaction components into the microfluidic chip to neutralize the cell lysis buffer and replace it with a mixture of enzymes and buffer components that is used in performing polymerase-catalyzed primed template extension reactions needed to incorporate the cell-specific oligonucleotide barcode primers in the DNA library preparation reactions, d) replacing the DNA amplification solution in the bypass channels of the device with an air plug or oil plug to prevent cross contamination between different trap chambers; e) incubating the microfluidic chip at constant temperature for a prolonged time interval ( e.g. , one hour to many hours), or thermal cycling the microfluidic chip for at least 5 cycles to amplify the DNA, and f) unsealing each trap chamber by replacing the air or oil with buffer and/or other molecular biology reagents used to further amplify and prepare the molecules for analysis by next generation sequencing.
[0138] Disclosed herein are methods for performing highly parallel or massively parallel targeted or whole genome amplification of the genomic DNA of single cells, the methods comprising: a) providing a microfluidic device according to any of the embodiments described herein; b) flowing a suspension of cells through the microfluidic device, thereby trapping single cells in all or a portion of the plurality of hydrodynamic traps; c) flowing a cell lysis and/or molecular reaction buffer through the microfluidic device, thereby releasing target
oligonucleotide molecules from the nuclear compartments of the single cells in each trap; d) sealing the hydrodynamic trap array by flowing an immiscible fluid through the microfluidic device to reduce or eliminate transfer of molecules between the different hydrodynamic traps, e) cleaving the barcode oligonucleotides from the substrate or lid using light, heat, enzymes, reducing or oxidizing agents and/or a change in pH; f) performing primer extension reactions to create barcoded complementary sequences to the cellular genome through the use of either barcode oligonucleotides (primers) comprising a targeted panel of gene capture sequences and/or barcode oligonucleotides comprising unbiased randomer capture sequences, and g) eluting the barcoded complementary sequence molecules from the microfluidic device for amplification, sequencing, identification of gene fragments and/or genomic sequences of interest, and detection of the corresponding cell barcodes for identification of individual cells from which the gene fragments and/or genomic sequences were derived. [0139] In some instances, the cell lysis component is based on alkaline lysis mechanisms (pH
>12). In some instances, the cell lysis component is based on the use of harsh detergents, such as sodium dodecyl sulfate (SDS). In some instances, the cell lysis component is based on the use of chaotropic agents, such as concentrated urea, guanidine hydrochloride, guanidine thiocyanate, or other similar hydrogen bond disrupting material - these chaotropic lysis reagents cause the DNA to condense on glass surfaces, thus ensuring that the unraveled genomic DNA is unable to escape the hydrodynamic trap site during the lysis step.
[0140] In some instances, the oligonucleotides in the plurality of oligonucleotide arrays comprise a photocleavable or enzymatically-cleavable site that allows these barcoded primers to be cleaved from the substrate to participate in solution-phase DNA amplification reactions, as shown in FIG. 9. FIG. 9 shows non-limiting examples of data for two methods of cleaving biotin- terminated oligonucleotides comprising either a photocleavable linker or a deoxyuridine nucleotide that were spotted on a streptavidin-coated surface. When the substrate is exposed to UV radiation for 2 minutes, only the photocleavable groups are removed from the surface (top row). When the substrate is exposed to USER™ enzyme for 45 minutes, only the uridine containing oligonucleotides are removed from the surface (bottom row). In some instances, the oligonucleotides of plurality of oligonucleotide arrays may comprise a random capture sequence that is used for unbiased whole-genome amplification or may comprise a panel of gene-specific capture sequences that is used for targeted gene analysis.
[0141] Single cell barcoding for combined RNA-seq and DNA-seq library preparation in a one- pot reaction: In some instances, the disclosed microfluidic devices may be used for RNA- sequencing and DNA-sequencing library preparation reactions conducted in a single-pot reaction mixture in which the cell lysis components, reverse transcription, and polymerase extension reactions are performed in the same buffers and at the same time. The disclosed methods for performing combined amplification and barcode tagging of DNA and RNA molecules derived from a single cell or small group of trapped cells may comprise one or more of: a) providing a microfluidic device according to any of the embodiments described herein; b) flowing a suspension of cells through the microfluidic device, thereby trapping single cells in all or a portion of the plurality of hydrodynamic traps; c) optionally incubating the microfluidic device under conditions that permit cell growth and division to create a small group or clone of cells in all or a portion of the plurality of hydrodynamic traps; d) flowing a cell lysis and/or molecular reaction buffer through the microfluidic device, thereby releasing target oligonucleotide molecules from the cytoplasmic and nuclear compartments of the single cells or small group of cells contained in each trap; e) sealing the hydrodynamic trap array by flowing in an immiscible fluid through the microfluidic device to reduce or eliminate the transfer of molecules between the different hydrodynamic traps and enable the preparation of complementary barcode-tagged sequences of RNA and DNA in a single pot reaction, f) cleaving the barcode oligonucleotides from the substrate or lid using light, heat, enzymes, reducing or oxidizing agents and/or a change in pH; g) performing primer extension reactions to create barcoded complementary DNA sequences for the cellular genomic DNA, and barcoded complementary DNA sequences for the cellular RNA, using either barcode oligonucleotides (primers) comprising a targeted panel of gene capture sequences, barcode oligonucleotides (primers) comprising unbiased randomer capture sequences, and/or barcode oligonucleotides (primers) comprising oligo-dT, oligo-dG, oligo-rG, or other targeted capture sequences, and h) eluting the barcoded complementary sequence molecules from the microfluidic device for amplification, sequencing, identification of the RNA molecules, gene fragments and/or genomic sequences of interest, and detection of the corresponding cell barcodes for identification of individual cells from which the RNA molecules, gene fragments and/or genomic sequences were derived.
[0142] Single cell barcoding for RNA-seq and DNA-seq library preparation using a multi addition workflow: In some embodiments, the RNA-sequencing library preparation reaction is conducted first by lysing only the cytosolic compartments of the cells, and then the DNA- sequencing library preparation reaction is performed with the addition of subsequent cell lysis reagents and other components required for DNA-amplification. The disclosed methods for performing combined amplification and barcode tagging of DNA and RNA molecules derived from a single cell or small group of trapped cells may comprise one or more of: a) providing a microfluidic device according to any of the embodiments described herein; b) flowing a suspension of cells through the microfluidic device, thereby trapping single cells in all or a portion of the plurality of hydrodynamic traps; c) optionally incubating the microfluidic device under conditions that permit cell growth and division to create a small group or clone of cells in all or a portion of the plurality of hydrodynamic traps; d) flowing a cell lysis buffer and/or molecular reaction buffer through the microfluidic device, thereby releasing target
oligonucleotide molecules from the cytoplasmic compartment of the single cells or small group of cells contained in each trap; e) sealing the hydrodynamic trap array by flowing an immiscible fluid through the microfluidic device to reduce or eliminate the transfer of molecules between the different hydrodynamic traps and enable the preparation of complementary barcode-tagged sequences of RNA, f) cleaving the barcode oligonucleotides from the substrate or lid using light, heat, enzymes, reducing or oxidizing agents and/or a change in pH; g) performing primer extension reactions to create barcoded complementary DNA sequences for the cellular RNA using barcode oligonucleotides (primers) comprising oligo-dT, oligo-dG, oligo-rG, or other targeted capture sequences, h) eluting the barcoded complementary sequence molecules from the microfluidic device for amplification, sequencing, identification of the RNA fragments, and detection the cell barcodes for identification of individual cells from which the RNA fragments were derived, i) flowing a nuclear lysis buffer through the microfluidic device that contains chaotropic agents, such as guanidine hydrochloride, guanidine thiocyanate, urea, or some other hydrogen-bond breaking solvent; j) flowing enzymes and other components required for DNA amplification through the microfluidic device; k) sealing the hydrodynamic trap array by flowing an immiscible fluid through the microfluidic device to reduce or eliminate transfer of molecules between the different hydrodynamic traps and enable the preparation of complementary barcode- tagged sequences of DNA; 1) performing primer extension reactions to create barcoded complementary DNA sequences of gene fragments or genomic DNA sequences using barcode oligonucleotides (primers) comprising a targeted panel of gene capture sequences and/or unbiased randomer capture sequences; and m) eluting the barcode-tagged complementary DNA sequence molecules from the microfluidic device for amplification, sequencing, identification of gene fragments or genomic DNA sequences, and detection of the corresponding cell barcodes for identification of individual cells from which the gene fragments or genomic DNA sequences were derived.
[0143] Disclosed herein are methods for performing combined amplification and barcode tagging of the DNA and RNA from a single cell or small group of trapped cells, comprising one or more of: a) providing a microfluidic device according to any of the embodiments described herein; b) flowing a suspension of cells through the microfluidic device, thereby trapping single cells in all or a portion of the plurality of hydrodynamic traps; c) optionally incubating the microfluidic device under conditions that permit cell growth and division to create a small group or clone of cells in all or a portion of the plurality of hydrodynamic traps; d) flowing a cell lysis and/or reaction buffer through the microfluidic devise, thereby releasing target oligonucleotides from the cytoplasmic compartment of the cells contained in each trap; e) sealing the hydrodynamic trap array by flowing an immiscible fluid through the microfluidic device to reduce or eliminate transfer of molecules between the different hydrodynamic traps and enable the preparation of complementary barcode-tagged sequences of RNA, f) cleaving the barcoded oligonucleotides from the substrate of lid using light, heat, enzymes, reducing or oxidizing agents, and/or a change in pH; g) performing primer extension reactions to create barcoded complementary DNA sequences of the cellular RNA using barcode oligonucleotides (primers) comprising oligo-dT, oligo-dG, oligo-rG, or other targeted capture sequences, h) eluting the molecules from the microfluidic device for amplification, sequencing, identification of the RNA fragments, and detection of the corresponding cell barcodes for identification of individual cells from which the RNA fragments were derived, i) flowing in a nuclear lysis and reaction buffer containing barcode-loaded transposases to insert barcodes and amplification primer sequences at random into genomic DNA, j) amplifying genomic DNA using the amplification primer sequences, and k) eluting the amplified, barcoded DNA molecules for sequencing and identification of gene fragments and barcodes.
[0144] Single cell barcodingfor analysis of gene expression and antibody production: Disclosed herein are methods for performing combined amplification and barcode tagging of the RNA and fluorescence-based antibody expression analysis in single cells, the methods comprising one or more of: a) providing a microfluidic device according to any of the embodiments described herein, wherein each hydrodynamic trap comprises at least one oligonucleotide array that is used for preparing barcode-tagged RNA molecules for sequencing and at least one oligonucleotide array that is configured to capture antibody-conjugated complementary oligonucleotide molecules in order to establish a spatially-controlled ELISA-like assay directly inside each hydrodynamic trap / cell culture chamber, c) flowing a suspension of cells through the microfluidic device, thereby trapping single cells in all or a portion of the plurality of
hydrodynamic traps; e) flowing a cell lysis buffer through the microfluidic device, thereby releasing target oligonucleotide molecules from the nuclear compartments of the single cells in each trap, where the cell lysis buffer also comprises the antibody-conjugated complementary oligonucleotide molecules and fluorescently-conjugated secondary antibodies needed for antigen detection; e) sealing the hydrodynamic trap array by flowing an immiscible fluid through the microfluidic device to reduce or eliminate transfer of molecules between the different hydrodynamic traps, f) conducting fluorescent imaging of the cell culture chambers to determine the presence and amount of antibodies in each cell, g) cleaving the barcoded oligonucleotides from the substrate or lid using light, heat, enzymes, reducing or oxidizing agents and/or a change in pH; h) performing primer extension reactions to create barcoded complementary sequences to the cellular RNA that are appended to cell-specific barcodes, and g) eluting the barcoded complementary sequence molecules from the microfluidic device for amplification and sequencing.
[0145] Single cell barcoding for ATAC-seq library preparation: In some instances, the disclosed microfluidic devices comprising a substrate, lid, and a plurality of oligonucleotide arrays disposed within the hydrodynamic traps of the device may be used in the preparation of ATAC- seq libraries for evaluation of epigenetic information and chromatin accessibility, this approach comprising, a) introducing cells into the microfluidic device containing trap-specific DNA barcodes and capturing them in single cell per trap format, b) introducing a non-ionic cell lysis buffer into the device which is capable of lysing the cell membrane to yield pure nuclei, c) introducing transposase and amplification reaction components into the microfluidic chip to neutralize the cell lysis buffer and replace it with a mixture of enzymes and buffer components that is used in performing tagging and fragmentation of the accessible DNA ( i.e ., tagmentation) and PCR amplification using unique barcoded oligonucleotide primers present in each trap, d) replacing the DNA amplification solution in the bypass channels of the device with an air plug or oil plug to prevent cross contamination between different trap chambers e) incubating the microfluidic chip at a constant temperature for a prolonged time interval ( e.g ., one hour to many hours), or thermal cycling the microfluidic chip for at least 5 cycles to amplify the fragmented
DNA, and f) unsealing each trap chamber by replacing the air or oil with buffer and/or other molecular biology reagents used to further amplify and prepare the molecules for analysis by next generation sequencing.
[0146] In some instances, the tagmentation step is carried out prior to trapping the cellular material and individual or groups of nuclei are captured in the traps, followed by amplification of chromatin-accessible DNA using trap-specific barcoded primers. In some instances, the transposase molecules are captured in the traps via molecular interactions including, but not limited to, biotin-streptavidin, digoxigenin, antibodies, or similar. In some instances, the transposase is pre-loaded with barcoded DNA that allows for an additional layer of molecular indexing of tagmented nuclei. In some instances, the oligonucleotides of the plurality of oligonucleotide arrays comprise a photocleavable site that allows the oligonucleotide barcode primers to be cleaved from the substrate so as to participate in solution-phase DNA amplification reactions. In some instances, the oligonucleotide molecules of the plurality of oligonucleotide arrays may comprise a random capture sequence that is used for unbiased whole-genome amplification or may comprise of a panel of gene-specific capture sequences that is used for targeted gene analysis. In some instances, the chromatin-accessible DNA is fragmented via an enzymatic reaction prior to amplification with the trap-specific barcoded primers. In some instances, the chromatin-accessible DNA is labeled with epigenetic markers such as, but not limited to 5 -methyl cytosine, 6-methyl adenosine, 4-methylcytosine, 5-hydroxymethylcytosine, or 8-oxo-guanine prior to release from the traps. In some instances, the amplification method is a polymerase chain reaction. In other instances, the amplification method comprises whole genome amplification or isothermal amplification. Non-limiting examples of two different ATAC-seq workflows are outlined in FIG. 16. In the left-hand column of FIG. 16, tagmentation is performed within the microfluidic device. Cells are captured in the hydrodynamic traps of the disclosed microfluidic devices, lysed and contacted with a transposase which has been pre-loaded with sequencing adapters, the regions of open chromatin are tagmented, trap-specific barcoded oligonucleotide primers are released from the oligonucleotide array disposed within the trap, primer extension and amplification reactions are performed on chip, and the barcoded libraries are collected for sequencing. A similar workflow is presented in the right-hand column of FIG.
16, in which the tagmentation step is performed in bulk solution, and individual cell nuclei (or groups of cell nuclei) are trapped within the microfluidic device and subjected to trap-specific barcoding reactions.
[0147] Additional methods of use: In some instances, RNA-sequencing library preparation is conducted first, followed by chromatin accessibility measurements conducted using, e.g ., ATAC- seq, by first lysing only the cytosolic compartments of the cells to create barcoded cDNA from the RNA, then lysing nuclei to allow for tagging and fragmentation of chromatin-accessible regions of DNA and subsequent barcoding via amplification using trap-specific barcoded primers. In some instances, the cDNA is retrieved from the microfluidic device prior to tagmentation. In other instances, the cDNA synthesis and tagmentation and carried out in a single step.
[0148] In some instances, RNA-sequencing library preparation and the chromatin accessibility measurements, e.g. , ATAC-seq, are conducted in a single pot mix by lysing the cells and nucleus simultaneously to create barcoded cDNA from the RNA, and also tagging and fragmentation of chromatin-accessible regions of DNA and subsequent barcoding via amplification using trap- specific barcoded primers. In some instances, the cDNA is retrieved from the microfluidic devise prior to tagmentation. In other instances, the cDNA synthesis and tagmentation are carried out in a single step.
[0149] In some instances, RNA-sequencing library preparation is conducted at the same time as antibody expression analysis by including antibodies bound to oligonucleotides in the RNA- sequencing reaction buffer and fabricating a portion of barcoded oligonucleotides resident in the chamber to generate sequences complementary to the antibody-tethered oligonucleotides.
[0150] Cell transfection and gene expression assays: Also disclosed herein are methods and microfluidic devices for performing cell transfection and gene expression assays. Today, commercial entities are able to produce tens of thousands of synthetic genes in a massively parallel format, however at present there are no massively parallel screening tools that can determine which genes produce the best antibodies. The state of the art in the field of“targeted antibody optimization” (TAO) is to synthesize thousands of targeted variants - these
modifications are designed to subtly change the binding epitope of the antibody in a manner that allows for more efficient sampling of parameter space. The TAO approach is preferable to stochastic directed evolution processes, which often produce nonsense variants and require larger pools and phage displays in the antibody screening process. However, current methods are unable to efficiently test the activity of the synthesized genes in a highly parallelized,
miniaturized format. Current approaches typically deliver one synthesized gene to each well of a 96-well plate, after which the desired antibody is expressed in bacterial or mammalian culture systems, and finally the activity of the antibody is analyzed in fluorescence binding assay. This approach requires a significant quantity of synthesized genes for each well, and a multi-step process to transfect the genes into the cells and then evaluate the secreted product.
[0151] The disclosed methods and devices solve this problem by enabling synthesis of genes in the same locations as where cells are incubated, transfected, and analyzed. Specifically, the disclosed microfluidic devices which may function as a microfluidic cell culture incubation platform, compartmentalize cells into thousands of discrete trap chambers in a microfluidic chip.
Each cell hydrodynamic trap (culture chamber) contains many identical copies of a unique synthetic gene that are grafted to a surface of the chamber, and which can be cleaved from the surface at a desired time to facilitate transfection into an adjacent cell. Suitable methods for cleavage include, but are not limited to, photocleavage, enzymatic cleavage ( e.g ., using a restriction enzyme), chemical cleavage, or any other method to cleave the gene sequence at or near the end tethered to the substrate, so that the cells can take up the genes. Suitable methods for transfection include, but are not limited to, electroporation, lipofection, sonoporation, phototransfection, or other means that allow for introduction of the synthetic genes into the cells.
In some instances, for example, the chip may be sandwiched between parallel plate electrodes, which are subjected to >1000 V/cm electric field pulses to porate the cell membranes.
Alternatively, a piezoelectric transducer can be coupled to the microfluidic chip in order to achieve sonoporation. Alternatively, light can be delivered to individual chambers with a digital light projector or other photomasking systems in order to porate the cells with high intensity optical pulses. It is also possible to achieve poration by flowing in surfactants or other reagents commonly used in lipotransfection processes. Any of these methods, as well as any combination of these methods, may be used to achieve massively parallel gene transfection inside
miniaturized cell culture chambers.
[0152] As discussed elsewhere herein, the disclosed microfluidic devices comprise a plurality of oligonucleotide arrays, where each oligonucleotide array is housed within one of a plurality of hydrodynamic traps within the device that are configured to retain objects, e.g., cells, beads, or other particles, suspended in a fluid passing through the device. In some instances, the hydrodynamic traps may be configured to trap single cells.
[0153] In some instances, each oligonucleotide array comprises a plurality of features, where each feature comprises a plurality of oligonucleotide molecules that comprise a fragment of a gene sequence such that, collectively, the set of gene sequence fragments presented by the features of a given array span an entire gene sequence. In some instances, the oligonucleotide molecules of each feature may further comprise a spacer sequence, an adapter sequence, at least one primer sequence, or any combination thereof. In some instances, a polymerase chain assembly reaction or a Gibson assembly reaction may be performed within the plurality of hydrodynamic traps in the assembled device to construct a full-length gene sequence within each hydrodynamic trap. In some instances, the microfluidic devices comprising a collection of full length gene sequences, e.g ., a different full length gene sequence in each of the traps of the plurality of hydrodynamic traps, may be used to trap cells, transfect cells, express native or genetically engineered proteins corresponding to the collection of full length gene sequences, and/or characterize the functionality of the expressed proteins.
[0154] In some instances of the disclosed methods and devices, the oligonucleotide array disposed within each hydrodynamic trap may be used to construct full length gene sequences in a microfluidic chip. In some embodiments, the methods may comprise one or more of: a) providing a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device once assembled; b) patterning a plurality of oligonucleotide arrays on a first surface of a substrate (or a coating layer thereon) or a first surface of a lid (or a coating layer thereon), wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a single gene fragment sequence, such that the plurality of gene fragment sequences presented by the plurality of features collectively span a full length gene sequence; c) bonding the first surface of the lid to the substrate to seal the plurality of hydrodynamic traps and interconnecting fluid flow channels in the assembled device such that the interior region of each hydrodynamic trap comprises an oligonucleotide array; and d) performing a polymerase chain assembly reaction, enzymatic ligation, or a Gibson assembly reaction within the plurality of hydrodynamic traps to construct a full length gene sequence within each hydrodynamic trap. In some instances, each
oligonucleotide array of the plurality comprises that same known set of features and the same known set of gene fragment sequences. In some instances, each oligonucleotide array of the plurality comprises a known set of features and gene fragment sequences that are different from those in all other oligonucleotide arrays. In some instances, a subset of oligonucleotide arrays of the plurality comprise a known set of features and gene fragment sequences that are different from those in the remainder of the plurality. In some instances, the polymerase chain assembly reaction, enzymatic ligation, or Gibson assembly reaction is performed without cleaving the gene fragment sequences from the array. In some instances, the polymerase chain assembly reaction, enzymatic ligation, or Gibson assembly reaction further comprises the use of a restriction enzyme to remove partially-assembled gene sequences. In some instances, each oligonucleotide array of the plurality comprises a feature comprising the same known full-length gene sequence. In some instances, each oligonucleotide array of the plurality comprises a feature comprising a different full-length gene sequence. In some instances, a subset of oligonucleotide arrays of the plurality comprises a feature comprising a full-length gene sequence that is different from that in the remainder of the plurality. In some instances, the oligonucleotide arrays in all or a portion of the plurality of oligonucleotide arrays further comprise one or more additional features that comprise a plurality of oligonucleotide molecules comprising one or more known capture probe sequences.
In some instances, the one or more known capture probe sequences are used to capture one or more antigens, antibodies, peptides, proteins, or enzymes that have been labeled with an oligonucleotide sequence that is complementary to the one or more capture probe sequences.
[0155] Disclosed herein are methods for performing cell transfection, the methods comprising: a) providing a microfluidic device comprising: i) a substrate comprising a plurality of
hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain at least one cell suspended in a fluid passing through the microfluidic device; and ii) a plurality of oligonucleotide arrays, wherein an oligonucleotide array is contained within the interior region of each hydrodynamic trap, wherein each oligonucleotide array comprises a plurality of features, and wherein at least one feature of the plurality comprises a plurality of oligonucleotide molecules comprising a single full length gene sequence or fragment thereof; b) flowing a suspension of cells through the microfluidic device, thereby trapping at least one cell in all or a portion of the plurality of hydrodynamic traps; c) cleaving the at least one full length gene sequence or fragment thereof from the oligonucleotide array within the interior region of each hydrodynamic trap; and d) transfecting the at least one cell in each hydrodynamic trap with the at least one full length gene sequences or fragment thereof.
[0156] Also disclosed are methods for performing gene expression assays, the methods comprising: a) providing a microfluidic device comprising: i) a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain at least one cell suspended in a fluid passing through the microfluidic device; and ii) a plurality of oligonucleotide arrays, wherein an oligonucleotide array is contained within the interior region of each hydrodynamic trap, wherein each oligonucleotide array comprises a plurality of features, and wherein at least one feature of the plurality comprises a plurality of oligonucleotide molecules comprising a single full length gene sequence or fragment thereof, and wherein at least one feature of the plurality comprises multiple copies of an oligonucleotide capture probe sequence; b) flowing a suspension of cells through the microfluidic device, thereby trapping at least one cell in all or a portion of the plurality of hydrodynamic traps; c) cleaving the at least one full length gene sequence or fragment thereof from the oligonucleotide array within the interior region of each hydrodynamic trap; d) transfecting the at least one cell in each hydrodynamic trap with the at least one full length gene sequence or fragment thereof; e) incubating the at least one cell in the plurality of hydrodynamic traps under conditions that promote cell division to create a clonal population of cells in each hydrodynamic trap that express a gene product for the at least one full length gene sequence or fragment thereof; f) flowing a mixture comprising a cell lysis buffer, at least one oligonucleotide-labeled antigen, at least one fluorescently-labeled antibody, at least one fluorescently-labeled secondary antibody, or any combination thereof, through the microfluidic device, wherein the at least oligonucleotide label is complementary to the at least one capture probe feature on the oligonucleotide array in each hydrodynamic trap; g) sealing the hydrodynamic traps within the microfluidic device; and h) imaging the hydrodynamic traps in the microfluidic device to detect the presence of the gene product for the at least one full length gene sequence or fragment thereof by monitoring fluorescence intensity at the location of the at least one feature comprising the oligonucleotide capture probe sequence. In some embodiments, the method may further comprise evaluating the binding affinity of expressed antibodies to antigens captured on the oligonucleotide arrays by monitoring the fluorescence intensity at the locations of the features comprising the oligonucleotide capture probe sequences as a function of a fluid flow rate through the microfluidic device.
[0157] In some embodiments, the cleaving in step may comprise the use of a photo-cleavage reaction or restriction enzyme reaction. In some embodiments, the transfecting in step may comprise the use of an electroporation technique, a lipofection technique, a sonoporation technique, a photo-transfection technique, a restriction enzyme, or any combination thereof. In some embodiments, the hydrodynamic traps are sealed within the microfluidic device by flowing a hydrogel or air through the interconnecting fluid channels. In some embodiments, the oligonucleotide molecules in each feature are covalently attached to the surface or coating layer within the interior region of each hydrodynamic trap. In some embodiments, the oligonucleotide molecules in each feature are entrapped within the coating layer in the interior region of each hydrodynamic trap. In some embodiments, the oligonucleotides in each feature further comprise a spacer sequence, an adapter sequence, a primer sequence, or any combination thereof. In some embodiments, each oligonucleotide array comprises at least 10 features. In some embodiments, each oligonucleotide array comprises at least 100 features. In some embodiments, each oligonucleotide array comprises at least 1,000 features. In some embodiments, each
oligonucleotide array of the plurality comprises that same known set of features and the same known set of full-length gene sequences or fragments thereof. In some embodiments, each oligonucleotide array of the plurality comprises a known set of features and known set of full- length gene sequences or fragments thereof that are different from those in all other
oligonucleotide arrays. In some embodiments, a subset of oligonucleotide arrays of the plurality comprises a known set of features and known set of full-length gene sequences or fragments thereof that are different from those in the remainder of the plurality. In some embodiments, the average length of the full gene sequences presented by the plurality of oligonucleotide arrays is at least 1 kilobase pairs. In some embodiments, the average length of the full gene sequences presented by the plurality of oligonucleotide arrays is at least 3 kilobase pairs. In some embodiments, the average length of the full gene sequences presented by the plurality of oligonucleotide arrays is at least 5 kilobase pairs. In some embodiments, the oligonucleotide arrays in all or a portion of the plurality of oligonucleotide arrays further comprise one or more additional features that comprise a plurality of oligonucleotide molecules comprising one or more known capture probe sequences. In some embodiments, the one or more known capture probe sequences are used to capture one or more antigens, antibodies, peptides, proteins, or enzymes that have been labeled with an oligonucleotide sequence that is complementary to the one or more capture probe sequences.
[0158] In a specific application, for example, the disclosed methods and devices may be used for high throughput determination of gene sequences that produce therapeutic antibodies with high affinity for desired antigens. After the cells are transfected and begin producing antibodies, a fluorescence binding assay may be conducted in the same trap chambers by using, e.g ., antigen(s) spotted onto a surface of the chambers, introducing fluorescently-labeled secondary antibodies into the chambers, and using fluorescence imaging as a readout. This approach may involve printing antigen spots in each chamber, or hybridizing antigen-functionalized DNA probes to complementary DNA probes included in the oligonucleotide array disposed in each chamber of the device. Following introduction of the fluorescent probes (e.g, fluorescently-labeled secondary antibodies) and incubation of the device for a period of time, it is possible to qualitatively and/or quantitatively measure the binding activity of a specific antibody by analyzing the fluorescent signal of the antigen spots in those chambers. By determining which chambers produce the brightest spots, it will be possible to infer which synthetic genes that produced the best antibodies. In some instances, the disclosed methods and devices may be used to screen at least 1, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1,000 gene sequences per device per experimental run. [0159] One non-limiting example of a method for assembling gene sequences within a microfluidic device, transfecting single cells, and testing for expression of, e.g, an antibody, comprises the followings steps:
[0160] Step 1. Pattern an array of gene fragments in each trap chamber of a microfluidic chip. The gene fragments are designed to enable the construction of a full-length gene sequence within the same chambers by using polymerase chain assembly or the Gibson method. This method will also ensure that many identical copies of the synthesized gene sequence are tethered to the bottom or another surface of the chambers. The construction of the full-length gene from the fragments can be conducted in the open-faced chip, or after the chip is enclosed with a lid.
[0161] Step 2. Mammalian or bacterial cells are introduced to the device and captured in each of the hydrodynamic trap / cell culture chambers. Thereafter, the gene sequences are cleaved from the substrate with, e.g. , a restriction enzyme or with light, and the co-located cells within the chamber are exposed to electric field pulses or some other transfection agent to cause the cells to uptake the genes.
[0162] Step 3. The cells in each chamber are incubated for sufficient time to allow integration of the gene sequences and expression of the gene product.
[0163] Step 4. A mixture of DNA-linked antigens and fluorescently labeled secondary antibodies are introduced to the microfluidic chip, after which each chamber is rapidly sealed. The system is incubated to allow DNA-linked antigens to hybridize to predetermined features of the oligonucleotide array in each chamber, and to allow the antibodies produced by the cells in the chambers to bind to the tethered antigens, so that the fluorescently-labeled secondary antibodies condense on the tethered antigen spots in the chambers and produce a visual signal of binding efficiency. The strength of the fluorescence signal m be used to infer the quality of the antibody. It may also be possible to measure the strength of the antibody-antigen binding interaction by shearing off the antibodies at different measured flow rates.
[0164] Systems and system components: Also disclosed are systems configured to perform the described methods using the disclosed microfluidic devices. Examples of system components required to perform single cell imaging and correlate image-based phenotypic analysis with single cell molecular data (e.g, sequence data, gene expression data, epigenetic data, and the like) generated using the disclosed devices include, but are not limited to, (i) one or more of the disclosed microfluidic devices, (ii) a high-content imager that is configured to acquire high- resolution images of the array of hydrodynamic traps spanning the entire device in brightfield and/or fluorescence imaging modes within minutes, (iii) a fluidics controller for control of fluid flow and delivery of cells or reagents to the device, (iv) gas and pH controllers for monitoring and control of, e.g, O2 concentration, CO2 concentration, and pH within the device, (v) a temperature controller configured for maintaining a set temperature and/or for thermal cycling,
(vi) a sequencer, and (vii) one or more processors or computers, or any combination thereof. In some instances, the system may be configured for handling multiple chips in each experiment.
[0165] Additional system components ( e.g ., external incubators, gas and pH controllers, and/or reagent delivery modules) may also be required to enable temperature control, gas and pH control, and/or liquid handling control of two or more chips in parallel, such that cells can be rapidly loaded into the chips, the arrayed cells can be continuously perturbed with mixtures of cell culture media, drugs or drug candidates, and/or other stimulants, and then cell lysis and molecular biology reagents can be introduced to the chips at the desired endpoint for genomic analysis.
[0166] High-content imager: The high-resolution imaging module (or high-content imager) may be configured to operate in bright-field, dark-field, phase-contrast, and/or fluorescence imaging modes and will comprise: (i) one or more excitation light sources, (ii) sets of excitation and emission filters (or other components for adjusting wavelength settings and bandpass), (iii) one or more detectors, and (iv) other optical components for manipulating the path of light beams as they traverse the optical system.
[0167] Any of a variety of light sources known to those of skill in the art may be used as an excitation light source including, but not limited to, arc lamps, tungsten-halogen lamps, lasers (e.g, argon ion lasers, helium-neon (HeNe) lasers, etc.), diode lasers, light emitting diodes (LEDs), light engines, and the like, or any combination thereof. In some instances, the imaging module may comprise at least one light source, at least two light sources, at least three light sources, at least four light sources, at least five light sources, or more.
[0168] Excitation and emission filter sets may comprise any of a variety of optical filters known to those of skill in the art including, but not limited to, optical glass filters (e.g, Schott optical filters), long-pass filters, short-pass filters, interference filters, dichroic reflectors, notch filters, and the like, or any combination thereof. In some instances, the excitation and/or emission wavelengths (or bandpass) for fluorescence imaging are set and/or adjusted by changing one or more optical filters in the optical path of the system. In some instances, the excitation and/or emission wavelengths (and bandpass) are set and/or adjusted using other components such as diffraction gratings, monochromators, acousto-optic modulators, tunable liquid-crystal filters, and the like.
[0169] In some instances, excitation or emission wavelength settings for fluorescence detection and imaging may be independently adjusted and range from about 350 nm to about 900 nm. In some instances, the excitation or emission wavelength are set at about 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 m, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, 725 nm, 750 nm, 775 nm, 800 nm, 825 nm, 850 nm, 875 nm, or 900 nm. Those of skill in the art will recognize that the excitation or emission wavelengths may be set to any value within this range, e.g ., about 620 nm.
[0170] In some instances, the bandwidths of the excitation and emission light for fluorescence imaging are independently adjusted and are specified as the specified excitation or emission wavelength ± 2 nm, ± 5 nm, ± 10 nm, ± 20 nm, ± 40 nm, ± 80 nm, or greater. Those of skill in the art will recognize that the excitation or emission bandwidths may be set to any value within this range, e.g. , about ± 55 nm.
[0171] Any of a variety of detectors and image sensors known to those of skill in the art may be used including, but not limited to, photodiodes, avalanche photodiodes, photodiode arrays, photomultipliers, CCD or CMOS image sensors and cameras, and the like, or any combination thereof. In some instances, the imaging module may comprise at least one detector, at least two detectors (e.g, for simultaneous capture of fluorescence images at two different emission wavelengths), at least three detectors, at least four detectors, at least five detectors, or more. In some optical designs, the imaging system may be configured to capture fluorescence intensity images at two (or more) different emission wavelengths sequentially, e.g, by changing the fluorescence emission filter between image capture steps. In some optical designs, the imaging system may be configured to collect fluorescence intensity images at two (or more) different emission wavelengths simultaneously, e.g, by including appropriate dichroic reflectors in the emission light optical path and utilizing a different detector for each emission wavelength.
[0172] Examples of other optical components that may be utilized in the imaging module include, but are not limited to, lenses or lens systems, prisms, beam-splitters, mirrors, optical fibers, diffractive optical elements for correction of chromatic aberration, etc. These components may be configured, along with light sources, excitation and emission filters (or other components for adjusting wavelength settings and bandpass), and detectors, in any of a variety of optical arrangements known to those of skill in the art.
[0173] In some instances, the imaging module may further comprise one or more translation stages or other motion control mechanisms for the purpose of moving the microfluidic device relative to the illumination and/or imaging sub-systems, or vice versa.
[0174] Fluidics controller: In some instances, the disclosed instrument systems (or cell analysis platforms) may comprise a fluid flow controller or perfusion system that provides programmable control of one or more fluid actuation mechanisms used to drive fluid flow in the microfluidic device. Examples of suitable fluid actuation mechanisms for use in the disclosed methods, devices, and systems include application of positive or negative pressure to fluid reservoirs connected to one or more device inlets or outlets, electrokinetic forces, electrowetting forces, passive capillary action, capillary action facilitated through the use of membranes and/or wicking pads, and the like.
[0175] Control of fluid flow through the disclosed microfluidic devices will often be performed through the use of one or more pumps (or other fluid actuation mechanisms) and one or more valves which, in some embodiments, will be housed externally to the device in a user-controlled instrument module. Examples of suitable pumps include, but are not limited to, syringe pumps, programmable syringe pumps, peristaltic pumps, diaphragm pumps, and the like. In some instances, fluid flow through the system may be controlled by means of applying positive pneumatic pressure at one or more inlets of external reagent and buffer containers connected to the microfluidic device, or at one or more inlets of the microfluidic device itself. In some instances, fluid flow through the device may be controlled by means of drawing a vacuum at one or more outlets of a waste reservoir connected to the device, or at the one or more outlets of the device. Examples of suitable valves include, but are not limited to, check valves,
electromechanical two-way or three-way valves, pneumatic two-way and three-way valves, and the like.
[0176] Different fluid flow rates may be utilized at different points in the microfluidic device operating sequence. For example, in some instances of the disclosed methods, devices, and systems, the volumetric flow rate through all or a portion of the microfluidic device may vary from about -10 ml/sec to about +10 ml/sec. In some embodiments, the absolute value of the volumetric flow rate may be at least 0.00001 ml/sec, at least 0.0001 ml/sec, at least 0.001 ml/sec, at least 0.01 ml/sec, at least 0.1 ml/sec, at least 1 ml/sec, or at least 10 ml/sec, or more. In some embodiments, the absolute value of the volumetric flow rate may be at most 10 ml/sec, at most 1 ml/sec, at most 0.1 ml/sec, at most 0.01 ml/sec, at most 0.001 ml/sec, at most 0.0001 ml/sec, or at most 0.00001 ml/sec. The volumetric flow rate at a given point in time may have any value within this range, e.g. a forward flow rate of 1.2 ml/sec, a reverse flow rate of -0.07 ml/sec, or a value of 0 ml/sec (i.e. stopped flow).
[0177] Gas and pH controllers: In some embodiments, the disclosed cell analysis platforms may comprise gas and pH controllers and related components (e.g. sensors) for maintaining a user- specified percentage of gas, e.g. CO2, or user-specified pH in buffers, growth media, or other fluids being delivered to the microfluidic device. Examples of suitable sensors include non- dispersive infrared (NDIR) CO2 sensors (used in conjunction with an attenuated total internal reflection (ATR) optics for dissolved CO2 sensing), metal insulator semiconductor field effect transistor (MOSFET)-type sensors for dissolved CO2 sensing (e.g. , having Pt-NiO thin films as the active CO2 sensing material deposited on the gate electrode), CCh-sensitive electrodes (e.g, Mettler Toledo’s InPro 5000i dissolved CO2 sensor series), pH-sensitive electrodes, pads immersed in the fluid, which produce a color change corresponding to the amount of dissolved
CO2 or the pH in the fluid such as those sold under the tradename Presens® sensor spots
[PreSens Precision Sensing, GmbH, Regensburg, Germany], and the like. For control of CO2 and pH, suitable sensors are used in a feedback loop to control aci d/base titrations and CO2 injection.
In some embodiments, CO2 or other gas concentrations, or pH, may be monitored directly in the fluid contained within the device. In some embodiments, CO2 or other gas concentrations may be monitored in a gas or atmosphere which is in equilibrium with the fluid within the device.
[0178] Temperature controllers: In some embodiments, the disclosed cell analysis platforms may further comprise a temperature controller for maintaining a user-specified temperature within the microfluidic device, e.g ., to enable cells to be incubated and maintained for extended periods while under continuous microscopic observation, or for ramping temperature between two or more specified temperatures over two or more specified time intervals. Examples of temperature control components that may be incorporated into the microfluidic device or into the instrument system include, but are not limited to, resistive heating elements (e.g. indium tin oxide resistive heating elements), Peltier heating or cooling devices, heat sinks, thermistors, thermocouples, infrared light sources, and the like, which are regulated using electronic feedback loops.
[0179] In some instances, the temperature controller may provide for a programmable temperature change at one or more specified, adjustable times prior to performing specific device operational steps. In some instances, the temperature controller may provide for programmable changes in temperature over specified time intervals. In some embodiments, the temperature controller may further provide for cycling of temperatures between two or more set temperatures with specified frequencies and ramp rates so that thermal cycling for amplification reactions may be performed.
[0180] Sequencers: In some instances, the disclosed systems may further comprise one or more sequencers, which may typically comprise a commercially-available sequencer such as those available from Illumina (San Diego, CA), Pacific Biosciences (Menlo park, CA), and Thermo- Fisher Scientific (Waltham, MA).
[0181] Processors and computers: In some instances, the disclosed systems may comprise one or more processors, computers, or computer systems configured for control the disclosed instrument systems; storage, processing, analysis, and display of the acquired image data; and/or storage, processing, analysis, and display of the generated sequencing data. In some instances, the one or more processors, computers, and computer systems may be configured for control of other system functions and/or other data acquisition, storage, processing, analysis, or display functions as well.
[0182] FIG. 17 provides a schematic illustration of a computer system 1701 that is programmed or otherwise configured to implement the methods described elsewhere herein ( e.g ., methods for performing single cell barcoding used the disclosed microfluidic devices). The computer system 1701 can regulate various aspects of the disclosed methods and systems, such as, for example, the acquisition and processing of image data and the control of reagent delivery to the device.
The computer system 1701 may comprise a local computer system, an electronic device (e.g., a smartphone, laptop, or desktop computer) of a user, or an electronic device of a user that is in communication with a computer system that is remotely located with respect to the electronic device. The computer system 1701 may be a post-classical computer system (e.g, a quantum computing system).
[0183] The computer system 1701 includes a central processing unit (CPU, also referred to as a “processor” or“computer processor” herein) 1705, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1701 also includes memory or a memory location 1710 (e.g, random-access memory, read-only memory, flash memory), electronic storage unit 1715 (e.g, a hard disk), a communication interface 1720 (e.g, a network adapter) for communicating with one or more other systems, and peripheral devices 1725, such as cache, other memory, data storage and/or electronic display adapters. The memory 1710, storage unit 1715, interface 1720 and peripheral devices 1725 are in
communication with the CPU 1705 through a communication bus (solid lines), such as a motherboard. The storage unit 1715 can be a data storage unit (or data repository) for storing data. The computer system 1701 can be operatively coupled to a computer network (“network”) 1730 with the aid of the communication interface 1720. The network 1730 may be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 1730 in some cases is a telecommunication and/or data network. The network 1730 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1730, in some cases with the aid of the computer system 1701, can implement a peer-to-peer network, which may enable devices coupled to the computer system 1701 to behave as a client or a server.
[0184] The CPU 1705 is configured to execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1710. The instructions can be directed to the CPU 1705, which can subsequently program or otherwise configure the CPU 1705 to implement methods of the present disclosure. Examples of operations performed by the CPU 1705 can include fetch, decode, execute, and writeback.
[0185] The CPU 1705 may be part of a circuit, such as an integrated circuit. One or more other components of the system 1701 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).
[0186] The storage unit 1715 is configured to store files, such as drivers, libraries and saved programs. The storage unit 1715 may store user data, e.g ., user preferences and user programs. The computer system 1701 in some cases can include one or more additional data storage units that are external to the computer system 1701, such as data storage units located on a remote server that is in communication with the computer system 1701 through an intranet or the Internet.
[0187] The computer system 1701 may communicate with one or more remote computer systems through the network 1730. For instance, the computer system 1701 may communicate with a remote computer system of a user (e.g, a cloud server). Examples of remote computer systems include personal computers (e.g, a desktop PC), portable personal computers (e.g, a laptop or tablet PC), smart phones (e.g, Apple® iPhone, Android-enabled devices, etc.), or personal digital assistants. The user may access the computer system 1701 via the network 1730.
[0188] Software & algorithms: As discussed above, in some instances, the disclosed systems may further comprise software for: (i) processing and display of image data, (ii) controlling fluid flow, reagent delivery, temperature, gas, and pH for the microfluidic devices, and/or (iii) processing and display of the sequencing data acquired for a plurality of single cell barcoding processes.
[0189] In some instances, the methods described herein may be implemented by way of machine (e.g, computer processor) executable code stored on an electronic storage location of a computer system such as that illustrated in FIG. 17 (such as, for example, in memory 1710 or electronic storage unit 1715 of computer system 1701). The machine executable or machine-readable code can be provided in the form of software. During use, the code can be executed by the processor 1705. In some cases, the code can be retrieved from the storage unit 1715 and stored on the memory 1710 for ready access by the processor 1705. In some situations, the electronic storage unit 1715 can be precluded, and machine-executable instructions are stored on memory 1710.
[0190] In some instances, the code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code. In some instances, the code may be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion. [0191] Aspects of the methods and systems provided herein, such as the computer system 1701, can be embodied in programming. Various aspects of the technology may be thought of as
“products” or“articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as in memory ( e.g ., read-only memory, random-access memory, flash memory) or on a hard disk.
“Storage” type media can include any or all of the tangible memory of the computer system, computer processors, or the like, or associated modules thereof, such as various semiconductor memory devices, tape drives, disk drives, optical drives, and the like, which may provide non- transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks.
Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible“storage” media, terms such as computer or machine
“readable medium” refer to any medium that participates in providing instructions to a processor for execution.
[0192] Hence, a readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer system or the like that may be used to implement databases. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data
communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH- EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.
[0193] The computer system 1701 may include or be in communication with an electronic display 1135 that comprises a user interface (UI) 1140 for providing, for example, an interface for a user to input instructions, upload data to a computer database, download data from a computer database, etc. Examples of UTs include, without limitation, a graphical user interface (GUI) and web-based user interface.
[0194] In some instances, the methods and systems of the present disclosure may be
implemented through the use of one or more algorithms, e.g ., an algorithm comprising instructions for acquiring and/or processing image. An algorithm can be implemented by way of software upon execution by the central processing unit 1705.
[0195] In some instances, the systems may use machine learning and/or computer vision to drive the high-content imager and/or to analyze the resulting datasets. The system may also comprise user-friendly interfaces that enable exploration of the combination datasets comprising live cell imaging data and data obtained from barcoded RNA- and DNA-sequencing reactions.
[0196] GPU computing: In some instances, the disclosed systems may comprise the use of GPU computing, /. e. , the use of a GPU (graphics processing unit) as a co-processor to accelerate the CPU performance for, e.g. , image segmentation to identify cells, beads, fiducial marks, trap address numbers, and other features or objects in images of the chip, and/or for machine learning-based algorithms for data processing and analytics. In some instances, GPU processing may be used to drive both hardware function and for analysis of data. The GPU accelerates applications running on the CPU by off-loading some of the compute-intensive and time- consuming portions of the code while the rest of the application still runs on the CPU. The image segmentation and data analytics algorithms run faster by harnessing the massively parallel processing power of the GPU. A typical CPU may comprise, e.g. , four to eight CPU cores, while the GPU may consist of hundreds of smaller cores. This massively parallel architecture is what provides the GPU with its high computational performance capabilities. In some instances, the software running on the disclosed systems may harness the performance of the parallel GPU architecture using, e.g. , CUDA - a parallel programming model invented by NVIDIA (Santa Clara, CA). Examples of suitable GPUs include, but are not limited to, the NVIDIA GeForce®, NVIDIA Quadro®, and NVIDIA Tesla®.
[0197] Kits: Disclosed herein are kits comprising one or more of the microfluidic devices disclosed herein and/or the assay components (e.g, enzymes, nucleotides, primers, buffers, oligonucleotide barcoded antibodies, surface coating solutions, or other reagents) required to perform any of the methods disclosed herein.
[0198] In some instances, for example, the kits may comprise an RNA-seq kit for the consumer that includes the reagents required to: 1) lyse the cells, and 2) perform reverse transcription in the chips. Suitable kit components may include, but are not limited to, a) lysis detergents, including but not limited to 0.01-1% Tween-20, 0.01-1% Triton X-100, b) 5 mM - 500 mM Tris-HCl, c)
7.5 - 750 mM chloride salt, including but not limited to NaCl, KC1, CsCl, d) 0.33 - 33 mM magnesium chloride (MgCb), e) 1-100 mM dithiothreitol (DTT), f) 0.05 - 5 mM guanosine triphosphate (GTP), g) 0.1 - 10 mM dNTPs, h) 4 - 400 U reverse transcriptase, i) crowding agents and additives, including but not limited to, 0.4 - 40% Ficoll PM-400, 0.005 - 0.5% gelatin, j) 1-100 U RNAase inhibitor, k) 0.05 - 5U USER Enzyme, and/or 1) 0.1 - 10 mM of either oligo- dT, oligo-dG, or oligo-rG.
[0199] In some instances, the kits may comprise a DNA-seq kit for the consumer that includes the reagents required to: 1) lyse the cells, and 2) perform primer extension reactions in the chips. Suitable kit components may include, but are not limited to, a) lysis detergents, including but not limited to 0.01-1% Tween-20, 0.01-1% Triton X-100, b) 5 mM - 500 mM Tris-HCl, c) 7.5 - 750 mM chloride salt, including but not limited to NaCl, KC1, CsCl, d) 0.33 - 33 mM magnesium chloride (MgCb), e) 1-100 mM dithiothreitol (DTT), f) 0.05 - 5 mM guanosine triphosphate (GTP), g) 0.1 - 10 mM dNTPs, h) crowding agents and additives, including but not limited to,
0.4 - 40% Ficoll PM-400, 0.005 - 0.5% gelatin, i) 1-100 U RNAase inhibitor, j) 0.05 - 5U USER Enzyme, and/or k) 0.1 - 10 pM of either oligo-dT, oligo-dG, or oligo-rG.
[0200] In some instances, the kits may comprise a pre-amplification kit for the consumer that includes the reagents required to: 1) remove single stranded primers, and 2) further amplify the barcoded cDNA molecules. Suitable components may include, but are not limited to, a) 2 - 200 U Exonuclease I, b) 0.5 - 5X Kapa High Fidelity Buffer, c) 0.04 - 4 U Kapa High Fidelity DNA polymerase, d) 0.03 - 3mM dNTPs, e) 0.33 - 33 mM magnesium chloride(MgCh), and/or 1) 0.1 - 10 pM pre-amplification oligo.
EXAMPLES
[0201] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.
Prophetic Example 1 Identification of drug resistant cells from image-based phenotyping coupled to gene expression analysis of the drug-resistant phenotypes
[0202] A suspension of cancer cells derived from a cell line or a patient biopsy sample is introduced to the microfluidic device, and single cells are captured in the hydrodynamic trap array followed by multi-day exposure to a drug therapy while imaging the cells multiple times per day. At the experimental endpoint, which may be up to 1 week or longer, the RNA-seq workflow is applied to prepare transcriptome analysis of the surviving clones in order to understand the gene expression pathways that are up-regulated or down-regulated in the drug resistance clones. The barcodes of the oligonucleotide array are stable for multiple days in the presence of cell culture media, as illustrated in FIG. 10, which shows a comparison of surface- tethered oligonucleotides that were exposed to cell culture media for 3 days (top row), 8 days
(middle row), and 9 days (bottom row), compared to the controls (left column) that were not exposed to cell culture media. The DNA is visualized by hybridizing a fluorescent complement.
The barcoded cDNA molecules derived from the surviving clones are exported off the chip, further amplified, and then analyzed by sequencing at a depth of 10,000 to 100,000 reads per cell in a 3’ or 5’ transcript end counting application as commonly done in RNA-seq workflows. The data sets generated by these experiments will reveal the relative composition of the protein encoding genes that make up the single cell transcriptome, and thereby enable the identification of highly variable genes that could be employed as biomarkers for targeting the drug resistant survival pathways of cancer cells.
Prophetic Example 2 Identification of drug resistant cells from image-based phenotyping coupled to whole genome or whole exome analysis of the drug-resistant phenotypes
[0203] As described in example 1, cancer cells from a patient biopsy sample or cell line are introduced to the microfluidic device, captured in single cell per hydrodynamic trap format, and grown for several days while under continuous exposure to a drug therapy. Upon reaching the experimental endpoint, whole genome analysis is conducted on the surviving clones. The barcoded DNA fragments of the surviving clones are retrieved from the chip, further amplified, and prepared for high resolution sequencing. Optionally, just the protein encoding genes can be selectively amplified in a pool for whole exome sequencing. A typical sequencing run would target 1 million to 100 million reads from each surviving clone, which is needed for high resolution coverage of the genome to determine the presence of single nucleotide variants or copy number variants that are present in the surviving drug-resistant clones.
Prophetic Example 3 Identification of drug resistant cells from image-based phenotyping coupled to combined DNA + RNA analysis of the drug-resistant phenotypes
[0204] Cancer cells from a patient biopsy sample or cell line are introduced to the microfluidic device, captured in single cell per hydrodynamic trap format, and grown for several days while under continuous exposure to a drug therapy. Upon reaching the experimental endpoint, the chip is subjected both to high resolution whole transcriptome amplification and also high-resolution whole genome analysis on the surviving clones. The genomic analysis would allow for identification of not only the genomic variants involved in the drug resistant fraction, but also how these variants regulate the gene expression profile of the surviving clones, thus enabling the identification of driver mutations and potential drug targets.
Prophetic Example 4 Identification of neurological disease states in cells from image-based phenotyping coupled to RNA expression analysis
[0205] Certain types of neurological diseases, such as Alzheimer’s and Amyotrophic Lateral Sclerosis (ALS), have pathologies that are associated with protein misfolding in cells, as well as the formation of plaques that can be directly observed with fluorescent immunostaining. The combination of high throughput image-based phenotyping and massively parallel single cell RNA expression analysis can be used to determine the relationship between the cells containing these protein malformations and their gene expression. Optionally, this assay may also benefit from the use of CRISPR and other gene editing tools for use in gain- or loss-of-function inserts that can be directly measured along with the imaging-based phenotyping and whole
transcriptome measurements.
Prophetic Example 5 Pooled CRISPR screen with high resolution RNA expression analysis.
[0206] A pooled CRISPR screen may be used along with high resolution RNA expression analysis and multi-day time lapse imaging to determine the gain- or loss-of-function inserts that affect cell growth, drug survival, invasiveness, and other phenotypes relevant to oncology drug development. Cancer cells may be subjected to a massively parallel pooled CRISPR screen to perform single gene knockouts or single gene inserts across the entire genome, then these cells are arrayed in the microfluidic chips in single cell per hydrodynamic trap format, exposed to a drug while monitoring the cellular responses from image-based phenotypes, including, for example, their growth rates, mobility, and/or polarity of focal adhesions. At the experimental endpoint, single cell gene expression libraries may be prepared that can not only be used to determine which CRISPR modification(s) were made on that cell, but also the relative distribution of protein encoding genes that the cell is currently transcribing in response to that particular set of CRISPR modifications.
Prophetic Example 6 Trapping of cancer cell + T-cell pairs, followed by massively parallel whole transcriptome analysis.
[0207] Single cell pairs consisting of a single cancer cell paired with a T-cell are formed on the chip by hydrodynamic trapping, then exposed to a therapeutic drug, and subjected to massively parallel whole transcriptome analysis for the purpose of identifying the gene expression patterns underling the cell pairs that have high vs. low cytolytic activity. A suspension of cancer cells is first introduced into the device and captured in single cancer cell per hydrodynamic trap format.
Next, a suspension of T-cells is introduced into the device in order to organize single cell pairs.
These cell pairs are exposed to a drug therapy or antibody cocktail, and then monitored by time- lapse imaging. At the experimental endpoint, the whole trap array is subjected to high resolution whole transcriptome analysis, which is used to identify the genetic markers that are upregulated in the cell pairs that have high vs. low cytolytic activity.
Prophetic Example 7 High throughput organization of patient derived tumor organoids for use in drug screening and biomarker analysis.
[0208] A cell biopsy sample is dissociated and then introduced into the device such that there are multiple cells captured in each hydrodynamic trap in a manner that mimics the diversity of the original tumor biopsy. These cells are allowed to grow into organoids and are then exposed to a drug therapy for multiple days while monitoring the organoid development. At the experimental endpoint, high resolution RNA- and DNA-sequencing analysis is conducted to determine the upregulated genes and mutational landscape of the surviving organoids.
Prophetic Example 8 High throughput immune screening of viral latency and the gene expression circuits involved in latency activation.
[0209] In various types of viral infections, such as Herpes and HIV, the virus is known to evade the immune system by hiding in a dormant state inside of infected cells. These latently infected cells periodically become activated and are the source of chronic infections in patients. The disclosed single cell analysis platform can be used to organize latently infected immune cells in a hydrodynamic trap array, and then study the viral latency activation processes using fluorescent imaging. At the experimental endpoint, high resolution whole transcriptome analysis can be used to compare the gene expression patterns in various phenotypic fractions, including cells that remain in a dormant latent state, as well as the cells that become activated when exposed to the stimulant.
Prophetic Example 9 Combined RNA-seq and secretome analysis of single cells using a protein array assembled in each hydrodynamic trap.
[0210] In synthetic biology applications, it is necessary to determine which genes are involved in creating cell lines that become prolific producers of biologies. In this example, a pool of CRISPR edits can be conducted on a cell line, after which the cells are introduced to the microfluidic device and captured in single cell per hydrodynamic trap format. At the desired experimental endpoint, the master mix is introduced into the device, and then followed by an air plug to seal off each chamber. The master mix also contains the necessary reagents for detection of one or more proteins using the oligonucleotide array present inside each chamber to capture primary antibodies conjugated to DNA fragments that are complementary to some of the features in the oligonucleotide array and a secondary antibody that is conjugated to fluorescent moieties, which are used to provide a fluorescent signature indicating the presence and amount of antibodies produced by that cell. When combining these proteomic, fluorescence-based measurements with the gene expression analysis of the same cells, as well as detection of the
CRISPR barcodes, it becomes possible to determine the efficacy of specific CRISPR edits in relation to the production of specific biologic molecules.
Prophetic Example 10 Targeted optimization of antibodies using massively parallel gene transfection arrays and in situ secretome analysis.
[0211] In some biological applications, it is necessary to transfect single cells with functional genes, and then determine the phenotypic response of these cells. One non-limiting example of such an application is the determination of the rate of antibody production and affinity of the expressed antibodies against a desired antigen target for single transfected cells. Towards this application, it is possible to use Gibson assembly to form full-length gene sequences inside each hydrodynamic trap of the disclosed microfluidic devices, flow single cells into the hydrodynamic trap array, cleave the full-length gene sequences from an interior surface of the hydrodynamic trap and use gene transfection methods, such as chemical perturbation or electroporation methods, to transfect the cells with the gene sequences present in the same traps, and finally measure the antibodies that the transfected cells produce in an ELISA-like assay as discussed in Example 9. The trap chambers associated with a strong fluorescent signal for antibody detection will be used to enable a more targeted method for identifying the gene sequences that yield more prolific, higher affinity producers of biologies.
Prophetic Example 11 Optimizing the spotting conditions to deposit the correct number of oligonucleotides for performing standard molecular biology reactions.
[0212] One of the goals in device fabrication is to achieve a primer concentration inside the sealed hydrodynamic traps at a range that is consistent with performing standard molecular biology reactions, e.g. , the target primer concentrations are the range of 0.10 - 10.0 uM. In some instances, the deposition process used to fabricate oligonucleotide arrays in the disclosed devices may involve depositing between 0.1 and 10 femtomole of oligonucleotides per spot, which can be dissolved in various carrier liquids at volumes ranging from 10 - 1000 picoliters. As a guide for depositing the correct amount of DNA and carrier fluid, the size of the printed spot can be estimated as a spherical cap that has a volume and surface area of:
Figure imgf000076_0001
A cap = 27GG2 (1 - COS(0))
Here, r and 0 are the radius of the spherical cap and the contact angle, assumed to be in the range of 30° - 60°, which is a typical value for a hydrophilic surface. Based on these assumptions and the print volume of 100 pL drops, the spot diameter is calculated to be in the range of 110 - 130 pm.
Prophetic Example 12 5’ end counting RNA-seq application.
In one non-limiting example of the utility of the disclosed microfluidic devices, the plurality of oligonucleotides disposed within the array in each hydrodynamic trap of the device may comprise a cleavable moiety that is immediately upstream of a triple-rG section, which is designed to participate in the template switching processes that are commonly employed in reverse transcription molecular biology workflows. The plurality of oligonucleotides may also comprise one or more unique molecular identifiers, cellular barcodes, or other identifiers that can indicate the spatial location of the spot within the oligonucleotide array and/or provide a dictionary for other molecular features contained therein. The plurality of oligonucleotides may also contain a common sequence that is used for primer hybridization and polymerase amplification reactions that may take place inside the microfluidic device or externally after the molecules are retrieved from the chip. This proposed 5’ end counting workflow will involve introducing a mixture of RT enzymes and buffer components, primers that have a 3’ dT termination as well as common sequences for amplification, and other enzymes or components needed to initiate the molecular biology reactions (e.g, USER™ enzymes, lights, DTT). The barcoded mRNA from the cells contained in the device will be retrieved, further amplified and purified, and sequenced to determine the barcode, transcript, and genome mapping
characteristics. One example of the proposed workflow is outlined in FIG. 15, which comprises the steps of (1) depositing barcodes into the microfluidic device traps (or chambers), (2) capturing single cells in the chambers, (3) cleaving barcode oligos from a chamber surface, and flowing in lysis and reverse transcription master mix to initiate the RNA-seq library preparation reaction, (4) eluting the barcoded molecules from the chip and amplifying the cDNA, and (5) preparing the cDNA for analysis on the sequencer.
Prophetic Example 13 Low cost method for constructing primer sets.
[0213] In some instances, the plurality of oligonucleotides contained with the oligonucleotide arrays of the disclosed devices may be constructed by ligating together two different barcode basis sets in every combination. This reduces the cost for purchasing the pre-constructed oligo set used for inkjet printing of the arrays.
Prophetic Example 14 RNA-seq + ATAC-seq in a single pot mixture.
[0214] In some instances, the master mix used in the disclosed methods may comprise lysis reagents that can lyse both the cytoplasm and nucleus, reverse transcription enzymes, buffers, and components required to cleave the oligonucleotide barcoded primers off the surface to initiate reverse transcription, transposases, DNA polymerases, and buffer components, and other additives required to initiate barcode-tagging of the accessible chromatin sites for ATAC-seq, and may be compatible with a temperature profile that is suitable for performing RNA-Seq and ATAC-Seq simultaneously at one fixed temperature, or sequentially by changing the temperature for different stages of the library preparation reaction.
[0215] While preferred embodiments of the present 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 will now 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 in any combination in practicing the invention. 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.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A microfluidic device comprising:
a) a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device; and
b) a plurality of oligonucleotide arrays, wherein the interior region of each hydrodynamic trap comprises an oligonucleotide array, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence.
2. A microfluidic device comprising:
a) a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device; and
b) a plurality of oligonucleotide arrays, wherein the interior region of each hydrodynamic trap comprises an oligonucleotide array, wherein each oligonucleotide array comprises a plurality of features, wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence, and wherein at least a subset of the features comprise a plurality of oligonucleotide molecules comprising a single gene fragment sequence, such that the plurality of gene fragment sequences presented by the subset of features collectively span a full length gene sequence.
3. A microfluidic device comprising:
a) a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device; and
b) a plurality of oligonucleotide arrays, wherein the interior region of each hydrodynamic trap comprises an oligonucleotide array, wherein each oligonucleotide array comprises a plurality of features, wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence, and wherein at least one feature of the plurality comprises a plurality of oligonucleotide molecules comprising a single full- length gene sequence.
4. The microfluidic device of any one of claims 1 to 3, wherein:
i) each hydrodynamic trap comprises an entrance region, an interior region, and an exit region that collectively constitute an interior fluid flow path through the hydrodynamic trap that has a fluidic resistance, RT;
ii) each hydrodynamic trap in a majority of the hydrodynamic traps is in fluid
communication with one long bypass fluid flow channel having a fluidic resistance, RA, and with one or two short bypass fluid flow channels each having a fluidic resistance that is less than RA, wherein each bypass fluid flow channel connects the exit region of the hydrodynamic trap to the entrance region of another hydrodynamic trap; and
iii) fluid flows through an adjacent short bypass channel in a first direction if a hydrodynamic trap is unoccupied, and in a second direction if the hydrodynamic trap is occupied by an object.
5. The microfluidic device of any one of claims 1 to 4, wherein the ratio RA/RT is at least 1.1.
6. The microfluidic device of any one of claims 1 to 5, wherein the ratio RA/RT is at least 1.3.
7. The microfluidic device of any one of claims 1 to 6, wherein each hydrodynamic trap comprises at least one constriction that has a spatial dimension that is less than about one half of the smallest dimension of the object.
8. The microfluidic device of any one of claims 1 to 7, wherein the ratio RA/RT is at least 1.2 and a capture probability for an individual hydrodynamic trap retaining a suspended object on first contact is at least 0.36.
9. The microfluidic device of any one of claims 1 to 8, wherein the ratio RA/RT is at least 1.45 and a capture probability for an individual hydrodynamic trap retaining a suspended object on first contact is at least 0.60.
10. The microfluidic device of any one of claims 1 to 9, wherein each hydrodynamic trap comprises a frit structure within the exit region, and wherein the frit structure comprises one or more constrictions that have a spatial dimension that is smaller than the smallest dimension of the suspended objects.
11. The microfluidic device of any one of claims 1 to 10, wherein the plurality of hydrodynamic traps comprises at least 100 traps.
12. The microfluidic device of any one of claims 1 to 11, wherein the plurality of hydrodynamic traps comprises at least 1,000 traps.
13. The microfluidic device of any one of claims 1 to 12, wherein the plurality of hydrodynamic traps comprises at least 10,000 traps.
14. The microfluidic device of any one of claims 1 to 13, wherein an initial trapping efficiency for trapping the suspended objects is at least 80%.
15. The microfluidic device of any one of claims 1 to 14, wherein the oligonucleotide molecules in each feature of the oligonucleotide array are covalently attached to a surface or coating layer within the interior region of each hydrodynamic trap.
16. The microfluidic device of any one of claims 1 to 15, wherein the oligonucleotide molecules in each feature of the oligonucleotide array are non-covalently tethered to a surface or coating layer within the interior region of each hydrodynamic trap.
17. The microfluidic device of any one of claims 1 to 15, wherein the oligonucleotide molecules in each feature of the oligonucleotide array are entrapped within a coating layer in the interior region of each hydrodynamic trap.
18. The microfluidic device of any one of claims 1 to 17, wherein the plurality of oligonucleotide arrays is fabricated using a contact printing or stamping technique.
19. The microfluidic device of any one of claims 1 to 17, wherein the plurality of oligonucleotide arrays is fabricated using an inkjet printing, microarray spotting, or spatially-addressable solid phase synthesis technique.
20. The microfluidic device of any one of claims 1 to 19, wherein each oligonucleotide array comprises at least 10 features.
21. The microfluidic device of any one of claims 1 to 20, wherein each oligonucleotide array comprises at least 100 features.
22. The microfluidic device of any one of claims 1 to 21, wherein each oligonucleotide array comprises at least 1,000 features.
23. The microfluidic device of any one of claims 1 to 22, wherein the common barcode sequences comprise unique non-overlapping sequences with a Hamming distance that enables demultiplexing with error correction capability.
24. The microfluidic device of any one of claims 1 to 23, wherein the common barcode sequences comprise a G/C content ranging from 30% to 70%.
25. The microfluidic device of any one of claims 1 to 24, wherein a length of the common barcode sequence ranges from 6 base pairs to 20 bases.
26. The microfluidic device of any one of claims 1 to 25, wherein the common barcode sequence comprises a unique cell barcode sequence.
27. The microfluidic device of claim 26, wherein the unique cell barcode sequence comprises a string of N“words”, and wherein each“word” comprises a string of M bases.
28. The microfluidic device of claim 27, wherein M is 1 base, 2 bases, 3 bases, or at least 4 bases.
29. The microfluidic device of claim 27 or claim 28, wherein N is at least 5“words”.
30. The microfluidic device of claim 27, wherein the unique cell barcode sequence is the same for each feature within a given oligonucleotide array, and different from that for all of the features in all of the other oligonucleotide arrays.
31. The microfluidic device of claim 27, wherein the unique cell barcode sequence is different for each feature within a given oligonucleotide array, and different from that for all of the features in all of the other oligonucleotide arrays.
32. The microfluidic device of any one of claims 26 to 31, wherein the unique cell barcode sequence for each feature of each oligonucleotide array of the plurality of oligonucleotide arrays is known.
33. The microfluidic device of any one of claims 1 to 32, wherein the oligonucleotides in each feature further comprise a spacer sequence, a cleavage sequence, an adapter sequence, at least one primer sequence, a cell barcode sequence, a molecular index sequence, a molecular recognition sequence, or any combination thereof.
34. The microfluidic device of claim 33, wherein a length of the universal primer sequence ranges from 15 bases to 30 bases.
35. The microfluidic device of claim 33 or claim 34, wherein a length of the unique molecular index sequence ranges from 5 bases to 15 bases.
36. The microfluidic device of any one of claims 33 to 35, wherein a length of the molecular recognition sequence ranges from 2 bases to 40 bases.
37. The microfluidic device of any one of claims 33 to 36, wherein a length of the spacer sequence ranges from 5 bases to 50 bases.
38. The microfluidic device of any one of claims 1 to 37, wherein a length of the oligonucleotide molecules ranges from 50 bases to 150 bases.
39. The microfluidic device of any one of claims 33 to 38, wherein the oligonucleotides in each feature comprise a molecular index sequence that is different for each individual oligonucleotide of the plurality of oligonucleotides within a given feature.
40. The microfluidic device of any one of claims 33 to 39, wherein the oligonucleotides in each feature comprise a molecular recognition sequence that is different for different features of a given oligonucleotide array.
41. The microfluidic device of any one of claims 33 to 40, wherein the molecular recognition sequence comprises a sequence selected from the group consisting of an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(deoxyguanosine) (oligo-dG) sequence, an oligo(riboguanosine) (oligo-rG) sequence, a complement of an mRNA sequence or fragment thereof, a complement of a tRNA sequence or fragment thereof, a complement of an rRNA sequence or fragment thereof, a complement of a DNA sequence, a complement of a cDNA sequence, a complement of a gene sequence or fragment thereof, and any combination thereof.
42. The microfluidic device of any one of claims 1 to 41, wherein the plurality of features in each oligonucleotide array of the plurality of oligonucleotide arrays comprise a same set of molecular recognition sequences.
43. The microfluidic device of any one of claims 1 to 41, wherein a subset of the plurality of oligonucleotide arrays comprises a set of molecular recognition sequences that is different from that in the oligonucleotide arrays of the remainder of the plurality.
44. The microfluidic device of any one of claims 2 or 4 to 43, wherein the average length of a gene fragment oligonucleotide sequence is at least 40 bases.
45. The microfluidic device of any one of claims 2 or 4 to 44, wherein each oligonucleotide array of the plurality comprises a known set of features and a known set of gene fragment sequences that are different from those in all other oligonucleotide arrays.
46. The microfluidic device of any one of 3 to 45, wherein the average length of the full-length gene sequences presented by the plurality of oligonucleotide arrays is at least 1 kilobase.
47. The microfluidic device of any one of claims 3 to 46, wherein each oligonucleotide array of the plurality comprises a feature comprising the same known full-length gene sequence.
48. The microfluidic device of any one of claims 3 to 46, wherein each oligonucleotide array of the plurality comprises a feature comprising a different full-length gene sequence.
49. The microfluidic device of any one of claims 3 to 46, wherein a subset of oligonucleotide arrays of the plurality comprises a feature comprising a full-length gene sequence that is different from that in the remainder of the plurality.
50. The microfluidic device of any one of claims 1 to 49, wherein the oligonucleotide arrays in all or a portion of the plurality of oligonucleotide arrays further comprise one or more additional features that comprise a plurality of oligonucleotide molecules comprising one or more known capture probe sequences.
51. The microfluidic device of claim 50, wherein the one or more known capture probe sequences are used to capture one or more antigens, antibodies, peptides, proteins, or enzymes that have been labeled with an oligonucleotide sequence that is complementary to the one or more capture probe sequences.
52. The microfluidic device of any one of claims 1 to 51, wherein the hydrodynamic traps are configured to trap single cells.
53. A method for fabricating a microfluidic device comprising:
a) providing a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device once assembled;
b) fabricating a plurality of oligonucleotide arrays on interior surfaces of the interior regions of the plurality of hydrodynamic traps or on a first surface of a lid, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence and a free 5’ end or 3’ end; and
c) bonding the first surface of the lid to the substrate to seal the plurality of hydrodynamic traps and interconnecting fluid flow channels in the assembled device such that the interior region of each hydrodynamic trap comprises an oligonucleotide array.
54. The method of claim 53, wherein the plurality of oligonucleotides further comprises a cleavage site, a molecular recognition sequence, a random multimer capture sequence, a unique molecular index sequence, a universal primer sequence, an adapter sequence, a spacer sequence, or any combination thereof.
55. The method of claim 54, wherein the cleavage site comprises a deoxyuridine base.
56. The method of claim 54, wherein the cleavage site comprises a photocleavable linker.
57. The method of any one of claims 53 to 56, wherein the oligonucleotide molecule is released into solution upon exposure to light or treatment with an enzyme.
58. The method of claim 57, wherein the oligonucleotide molecule is released into solution upon treatment with combination of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII.
59. The method of any one of claims 53 to 58, wherein the common barcode sequence for the oligonucleotide array in each hydrodynamic trap is unique.
60. The method of any one of claims 53 to 59, wherein the plurality of common barcode sequences for the plurality of hydrodynamic traps comprise unique non-overlapping sequences with a Hamming distance that enables demultiplexing with error correction capability.
61. The method of any one of claims 53 to 60, wherein the common barcode sequences comprise a G/C content ranging from 30% to 70%.
62. The method of any one of claims 53 to 61, wherein a length of the common barcode sequence ranges from 8 base pairs to 20 bases.
63. The method of any one of claims 54 to 62, wherein a length of the universal primer sequence ranges from 15 base pairs to 30 bases.
64. The method of any one of claims 54 to 63, wherein a length of the unique molecular index sequence ranges from 5 bases to 15 bases.
65. The method of any one of claims 54 to 64, wherein a length of the molecular recognition sequence ranges from 2 bases to 40 bases.
66. The method of any one of claims 54 to 65, wherein a length of the spacer sequence ranges from 5 bases to 50 bases.
67. The method of any one of claims 53 to 66, wherein a length of the oligonucleotide molecules ranges from 50 bases to 150 bases.
68. The method of any one of claims 53 to 67, wherein the plurality of hydrodynamic traps comprises at least 100 traps.
69. The method of any one of claims 53 to 68, wherein the plurality of hydrodynamic traps comprises at least 1,000 traps.
70. The method of any one of claims 53 to 69, wherein the plurality of hydrodynamic traps comprises at least 10,000 traps.
71. The method of any one of claims 53 to 70, wherein the fabricating in step (b) comprises the use of contact printing or stamping to create a replica of the molecular pattern (or its inverse) on the surface.
72. The method of any one of claims 53 to 71, wherein the fabricating in step (b) comprises the use of an inkjet printing, microarray spotting, or spatially-addressable solid phase synthesis technique.
73. The method of any one of claims 53 to 72, wherein each oligonucleotide array comprises at least 1 feature.
74. The method of any one of claims 53 to 73, wherein each oligonucleotide array comprises at least 10 features.
75. The method of any one of claims 53 to 74, wherein each oligonucleotide array comprises at least 100 features.
76. The method of any one of claims 53 to 75, wherein the common barcode sequence comprises a unique cell barcode sequence.
77. The method of claim 76, wherein the unique cell barcode sequence is the same for each feature within a given oligonucleotide array, and different from that for all of the features in all of the other oligonucleotide arrays.
78. The method of claim 76, wherein the unique cell barcode sequence is different for each feature within a given oligonucleotide array, and different from that for all of the features in all of the other oligonucleotide arrays.
79. The method of any one of claims 76 to 78, wherein the unique cell barcode sequence for each feature of each oligonucleotide array of the plurality of oligonucleotide arrays is known.
80. The method of any one of claims 53 to 79, wherein the oligonucleotides in each feature comprise a molecular index sequence that is different for each individual oligonucleotide of the plurality of oligonucleotides within a given feature.
81. The method of claim 80, wherein the oligonucleotides in each feature comprise a molecular recognition sequence that is different for different features of a given oligonucleotide array.
82. The method of any one of claims 53 to 81, wherein the molecular recognition sequence comprises a sequence selected from the group consisting of an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(deoxyguanosine) (oligo-dG) sequence, an oligo(riboguanosine) (oligo-rG) sequence, a random multimer sequence, a complement of an mRNA sequence or fragment thereof, a complement of a tRNA sequence or fragment thereof, a complement of an rRNA sequence or fragment thereof, a complement of a DNA sequence, a complement of a cDNA sequence, a complement of a gene sequence or fragment thereof, and any combination thereof.
83. The method of any one of claims 53 to 82, wherein the plurality of features in the oligonucleotide arrays in each hydrodynamic trap of the plurality of traps comprise a same set of molecular recognition sequences.
84. The method of any one of claims 53 to 83, wherein the substrate is fabricated in silicon, the lid is glass, and the bonding in step (c) comprises coating a thin film of polydimethylsiloxane (PDMS) on the glass lid prior to bonding it to the silicon substrate.
85. The method of any one of claims 53 to 84, wherein the substrate is fabricated in silicon, the lid comprises a glass substrate with a polydimethylsiloxane (PDMS) layer coated thereon, and the bonding in step (c) comprises coating and patterning a thin film of hydrogel on the PDMS layer prior to bonding the lid to the silicon substrate.
86. The method of any one of claims 53 to 85, wherein the bonding in step (c) comprises formation of a hydrogel layer between the lid and substrate using UV-induced polymerization of acrylate groups that are attached to both surfaces.
87. A method for performing single cell analysis, the method comprising:
a) providing a microfluidic device according to any one of claims 1 to 55;
b) flowing a suspension of cells through the microfluidic device, thereby trapping single cells in all or a portion of the plurality of hydrodynamic traps;
c) flowing a lysis buffer through the microfluidic device, thereby releasing target oligonucleotide molecules from the single cell trapped in each trap;
d) incubating the microfluidic device under conditions that promote hybridization of one or more target oligonucleotide molecules released by the single cells to one or more molecular recognition sequences presented in the features of the oligonucleotide array in each trap;
e) performing a primer extension reaction within the microfluidic device; f) optionally, eluting barcoded oligonucleotide molecules corresponding to
complementary copies of the one or more target oligonucleotide molecules released by the single cell in each trap from the microfluidic device; and
g) amplifying and sequencing the barcoded oligonucleotides to detect the presence of the one or more target oligonucleotide molecules in one or more single cells, wherein the sequence of a unique cell barcode sequence presented in the features of the oligonucleotide array in each hydrodynamic trap is used to identify target oligonucleotide molecules that were released from a given single cell.
88. The method of claim 87, further comprising a cleavage step to release barcoded
oligonucleotide primers from the oligonucleotide arrays disposed within each hydrodynamic trap in the plurality of hydrodynamic traps within the microfluidic device.
89. The method of claim 87 or claim 88, further comprising the use of an air plug or oil plug to seal the hydrodynamic traps to reduce transfer of molecular components between hydrodynamic traps following lysis of the single cells in (c).
90. The method of any one of claims 87 to 89, wherein a determination of the number of unique molecular index barcode sequences corresponding to each unique cell barcode sequence is used to quantify how many copies of a given target oligonucleotide were released from a give single cell.
91. The method of any one of claims 87 to 90, wherein the amplifying and sequencing are performed within the microfluidic device.
92. The method of any one of claims 87 to 91, wherein the amplifying and sequencing are performed after eluting the barcoded oligonucleotide molecules from the microfluidic device.
93. The method of any one of claims 87 to 92, wherein the target oligonucleotide molecules comprise mRNA molecules or fragments thereof, tRNA molecules or fragments thereof, rRNA molecules or fragments thereof, RNA molecules or fragments thereof, DNA molecules or fragments thereof, or any combination thereof.
94. The method of any one of claims 87 to 93, wherein the oligonucleotide molecules of the oligonucleotide array disposed in each hydrodynamic trap comprise a cleavage site, and the cleavage site comprises a deoxyuridine base.
95. The method of any one of claims 87 to 93, wherein the oligonucleotide molecules of the oligonucleotide array disposed in each hydrodynamic trap comprise a cleavage site, and the cleavage site comprises a photocleavable linker moiety.
96. The method of any one of claims 87 to 95, wherein the oligonucleotide molecules of the oligonucleotide array disposed in each hydrodynamic trap are released into solution upon exposure to light or treatment with an enzyme.
97. The method of claim 96, wherein the oligonucleotide molecules are released into solution upon treatment with a combination of Uracil DNA glycosylase (UDG) and the DNA glycosylase- lyase Endonuclease VIII.
98. The method of any one of claims 87 to 97, wherein the primer extension reaction in (e) comprises a reverse transcription reaction or a DNA polymerase reaction.
99. The method of any one of claims 87 to 98, wherein the lysis buffer comprises a chaotropic agent.
100. The method of claim 99, wherein the chaotropic agent comprises concentrated urea, guanidinium thiocyanate, or any combination thereof.
101. The method of any one of claims 87 to 100, wherein the cells comprise cancer cells.
102. The method of any one of claims 87 to 100, wherein the cells comprise fetal cells.
103. The method of any one of claims 87 to 100, wherein the cells comprise CRISPR-edited cells.
104. The method of any one of claims 87 to 103, wherein the method is used to perform library preparation for a DNA-seq experiment, an RNA-seq experiment, an ATAC-seq experiment, protein detection, antibody detection or any combination thereof.
105. The method of any one of claims 87 to 103, wherein the method is used to perform a cell transfection and gene expression assay.
106. A method for construction of full-length gene sequences in a microfluidic chip, the method comprising:
a) providing a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device once assembled;
b) patterning a plurality of oligonucleotide arrays on an interior surface of the interior regions of the plurality of hydrodynamic traps or on a first surface of a lid, wherein each oligonucleotide array comprises a plurality of features, and wherein all or a portion of the features in an oligonucleotide array comprise a plurality of oligonucleotide molecules comprising a single gene fragment sequence, such that the plurality of gene fragment sequences presented by a plurality of features in the oligonucleotide array collectively span a full-length gene sequence;
c) bonding the first surface of the lid to the substrate to seal the plurality of hydrodynamic traps and interconnecting fluid flow channels in the assembled device such that the interior region of each hydrodynamic trap comprises an oligonucleotide array; and d) performing a polymerase chain assembly reaction or a Gibson assembly reaction within the plurality of hydrodynamic traps to construct a full-length gene sequence within each hydrodynamic trap.
107. A method for performing cell transfection, the method comprising:
a) providing a microfluidic device comprising:
i) a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain at least one cell suspended in a fluid passing through the microfluidic device; and ii) a plurality of oligonucleotide arrays, wherein an oligonucleotide array is contained within the interior region of each hydrodynamic trap, wherein each oligonucleotide array comprises a plurality of features, and wherein at least one feature of the plurality comprises a plurality of oligonucleotide molecules each comprising a single full-length gene sequence or fragment thereof; b) flowing a suspension of cells through the microfluidic device, thereby trapping at least one cell in all or a portion of the plurality of hydrodynamic traps;
c) cleaving the at least one full length gene sequence or fragment thereof from the oligonucleotide array within the interior region of each hydrodynamic trap; and d) transfecting the at least one cell in each hydrodynamic trap with the at least one full length gene sequences or fragment thereof.
108. A method for performing gene expression assays, the method comprising:
a) providing a microfluidic device comprising:
i) a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain at least one cell suspended in a fluid passing through the microfluidic device; and ii) a plurality of oligonucleotide arrays, wherein an oligonucleotide array is contained within the interior region of each hydrodynamic trap, wherein each oligonucleotide array comprises a plurality of features, and wherein at least one feature of the plurality comprises a plurality of oligonucleotide molecules each comprising a single full length gene sequence or fragment thereof, and wherein at least one feature of the plurality comprises multiple copies of an oligonucleotide capture probe sequence;
b) flowing a suspension of cells through the microfluidic device, thereby trapping at least one cell in all or a portion of the plurality of hydrodynamic traps;
c) cleaving the at least one full length gene sequence or fragment thereof from the oligonucleotide array within the interior region of each hydrodynamic trap;
d) transfecting the at least one cell in each hydrodynamic trap with the at least one full length gene sequence or fragment thereof;
e) incubating the at least one cell in the plurality of hydrodynamic traps under conditions that promote cell division to create a clonal population of cells in each hydrodynamic trap that express a gene product for the at least one full length gene sequence or fragment thereof;
f) flowing a mixture comprising a cell lysis buffer, at least one oligonucleotide-labeled antigen, at least one fluorescently-labeled antibody, at least one fluorescently-labeled secondary antibody, or any combination thereof, through the microfluidic device, wherein the oligonucleotide label of the at least one oligonucleotide-labeled antigen is
complementary to the at least one capture probe feature on the oligonucleotide array in each hydrodynamic trap;
g) sealing the hydrodynamic traps within the microfluidic device; and
h) imaging the hydrodynamic traps in the microfluidic device to detect the presence of the gene product for the at least one full length gene sequence or fragment thereof by monitoring fluorescence intensity at the location of the at least one feature comprising the oligonucleotide capture probe sequence.
109. A method of preparing single cell RNA-seq libraries, the method comprising:
a) providing a microfluidic device comprising a plurality of hydrodynamic traps and a plurality of bypass channels, wherein each hydrodynamic trap comprises an
oligonucleotide array disposed on a surface therein, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence;
b) introducing cells into the microfluidic device and capturing a single cell in all or a portion of the plurality of hydrodynamic traps; c) introducing a cell lysis buffer comprising reverse transcription reagents into the microfluidic device;
d) replacing the cell lysis buffer in the plurality of bypass channels with air or oil to prevent cross contamination between different hydrodynamic traps;
e) cleaving the oligonucleotide molecules comprising the common barcode sequence from the surface;
f) performing a reverse transcription reaction on cellular RNA molecules using the oligonucleotide molecules comprising the common barcode sequences as primers to create complementary cDNA copies; and
g) eluting the barcoded cDNA copies from the microfluidic device for amplification and sequencing.
110. A method for preparing single cell DNA-seq libraries, the method comprising:
a) providing a microfluidic device comprising a plurality of hydrodynamic traps and a plurality of bypass channels, wherein each hydrodynamic trap comprises an
oligonucleotide array disposed on a surface therein, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence;
b) introducing cells into the microfluidic device and capturing a single cell in all or a portion of the plurality of hydrodynamic traps;
c) introducing a cell lysis buffer configured to dissociate nuclear membranes and histone complexes, thereby denaturing cellular DNA, into the microfluidic device;
d) introducing a DNA polymerization/amplification reaction mixture into the microfluidic device;
e) replacing the DNA polymerization/amplification reaction mixture in the plurality of bypass channels with air or oil to prevent cross contamination between different hydrodynamic traps;
f) cleaving the oligonucleotide molecules comprising the common barcode sequence from the surface;
g) performing primer extension reactions using the oligonucleotide molecules comprising the common barcode sequences as primers to create complementary copies of cellular DNA sequences; and h) eluting the barcoded complementary copies of cellular DNA sequences from the microfluidic device for amplification and sequencing.
111. A method for preparing single cell RNA-seq and DNA-seq libraries, the method
comprising:
a) providing a microfluidic device comprising a plurality of hydrodynamic traps and a plurality of bypass channels, wherein each hydrodynamic trap comprises an
oligonucleotide array disposed on a surface therein, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence;
b) introducing cells into the microfluidic device and capturing a single cell in all or a portion of the plurality of hydrodynamic traps;
c) simultaneously or sequentially introducing lysis buffer(s) comprising: (i) reverse transcription reagents, and (ii) DNA polymerization reagents into the microfluidic device; d) replacing the lysis buffer(s) in the plurality of bypass channels with air or oil to prevent cross contamination between different hydrodynamic traps;
e) cleaving the oligonucleotide molecules comprising the common barcode sequence from the surface;
f) simultaneously or sequentially performing: (i) a reverse transcription reaction on cellular RNA molecules using the oligonucleotide molecules comprising the common barcode sequences as primers to create complementary cDNA copies, and (ii) a DNA polymerization reaction using the oligonucleotide molecules comprising the common barcode as primers to create complementary copies of cellular DNA sequences; and g) eluting the barcoded cDNA copies and complementary copies of cellular DNA sequences from the microfluidic device for amplification and sequencing.
112. A method for preparing single cell ATAC-seq libraries, the method comprising:
a) providing a microfluidic device comprising a plurality of hydrodynamic traps and a plurality of bypass channels, wherein each hydrodynamic trap comprises an
oligonucleotide array disposed on a surface therein, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence;
b) introducing cells into the microfluidic device and capturing a single cell in all or a portion of the plurality of hydrodynamic traps; c) introducing a non-ionic cell lysis buffer configured to lyse cell membranes to yield pure nuclei into the microfluidic device;
d) introducing a mixture comprising a transposase, DNA fragmentation, and DNA polymerization/amplification reagents into the microfluidic chip;
e) replacing the transposase, DNA fragmentation, and DNA polymerization/amplification reagents in the plurality of bypass channels with air or oil to prevent cross contamination between different hydrodynamic traps;
f) cleaving the oligonucleotide molecules comprising the common barcode sequence from the surface;
g) performing DNA polymerization/amplification to amplify fragmented DNA using the oligonucleotide molecules comprising the common barcode as primers, and
h) eluting the barcoded complementary copies of the fragmented DNA from the microfluidic device for amplification and sequencing.
113. The method of any one of claims 109 to 112, wherein the common barcode sequence comprises a unique cell barcode sequence.
114. The method of any one of claims 109 to 113, wherein the plurality of oligonucleotide molecules further comprises a spacer sequence, and adapter sequence, a cleavage site, a molecular recognition sequence, a random heptamer capture sequence, a unique molecular index sequence, a universal primer sequence, or any combination thereof
115. The method of any one of claims 109 to 114, wherein the plurality of oligonucleotide molecules comprises a cleavage site, and wherein the cleavage site comprises a deoxyuridine base.
116. The method of any one of claims 109 to 114, wherein the plurality of oligonucleotide molecules comprises a cleavage site, and wherein the cleavage site comprises a photocleavable linker.
117. The method of any one of claims 109 to 116, wherein the plurality of oligonucleotide molecules within a feature is released into solution upon exposure to light or treatment with an enzyme.
118. The method of claim 117, wherein the plurality of oligonucleotide molecules within a feature is released into solution upon treatment with a combination of Uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endonuclease VIII.
119. The method of any one of claims 109 to 118, wherein the plurality of oligonucleotide molecules comprises a molecular recognition sequence that is the same for each feature within an oligonucleotide array.
120. The method of claim 119, wherein the molecular recognition sequence comprises a sequence selected from the group consisting of an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(deoxyguanosine) (oligo-dG) sequence, an oligo(guanosine) (oligo-rG) sequence, a random multimer sequence, and any combination thereof.
121. A system comprising:
a) a microfluidic device comprising:
i. a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device;
ii. a plurality of oligonucleotide arrays, wherein each oligonucleotide array of the plurality is disposed on a surface within a hydrodynamic trap of the plurality of hydrodynamic traps, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence and a free 5’ end; and b) an imaging unit configured to acquire high-resolution images of the interior region of each hydrodynamic trap and one or more objects contained therein.
122. The system of claim 121, wherein the oligonucleotide molecules comprise a cleavage site.
123. The system of claim 122, wherein the cleavage site comprises a deoxyuridine base.
124. The system of claim 122, wherein the cleavage site comprises a photocleavable linker.
125. The system of any one of claims 121 to 124, wherein the oligonucleotide molecules further comprise a molecular recognition sequence that is the same or different for different features of an oligonucleotide array.
126. The system of claim 125, wherein the molecular recognition sequence comprises a sequence selected from the group consisting of an oligo(deoxythymidine) (oligo-dT) sequence, an oligo(deoxyguanosine) (oligo-dG) sequence, an oligo(guanosine) (oligo-rG) sequence, a random heptamer sequence, a complement of an mRNA sequence or fragment thereof, a complement of a tRNA sequence or fragment thereof, a complement of an rRNA sequence or fragment thereof, a complement of a DNA sequence, a complement of a cDNA sequence, a complement of a gene sequence or fragment thereof, and any combination thereof.
127. The system of any one of claims 121 to 126, wherein the objects comprise cells.
128. The system of claim 127, wherein the cells comprise cancer cells.
129. The system of any one of claims 121 to 128, wherein the system is configured to perform single cell RNA sequencing library preparation, single cell DNA sequencing library preparation, single cell ATAC sequencing library preparation, protein detection, antibody detection, or any combination thereof.
130. A kit comprising:
a) a microfluidic device comprising:
i. a substrate comprising a plurality of hydrodynamic traps and interconnecting fluid flow channels, wherein each hydrodynamic trap comprises an entrance region, an interior region, and an exit region, and is configured to retain an object suspended in a fluid passing through the microfluidic device;
ii. a plurality of oligonucleotide arrays, wherein each oligonucleotide array of the plurality is disposed on a surface within a hydrodynamic trap of the plurality of hydrodynamic traps, wherein each oligonucleotide array comprises a plurality of features, and wherein each feature comprises a plurality of oligonucleotide molecules comprising a common barcode sequence; and
b) one or more reagents for performing single cell RNA sequencing library preparation, single cell DNA sequencing library preparation, single cell ATAC sequencing library preparation, or any combination thereof.
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