CN118804943A

CN118804943A - Droplet forming apparatus and method with fluoropolymer silane coating agent

Info

Publication number: CN118804943A
Application number: CN202380024477.0A
Authority: CN
Inventors: 塔哈加塔·穆克吉; 埃里克·安德森
Original assignee: 10X Genomics Inc
Current assignee: 10X Genomics Inc
Priority date: 2022-03-04
Filing date: 2023-03-03
Publication date: 2024-10-18

Abstract

The present invention provides devices, systems for generating droplets, and methods of use thereof. These devices, systems, and methods have a droplet source region coated with a fluoropolymer silane coating agent that provides a surface coating having a total fluorine weight percent of at least 57%.

Description

Droplet forming apparatus and method with fluoropolymer silane coating agent

Background

Many biomedical applications rely on high throughput assays of samples bound to one or more reagents in droplets or particles. For example, in both research and clinical applications, high throughput gene detection using target-specific reagents can provide information about a sample during drug discovery, biomarker discovery, and clinical diagnostics, among other procedures. Many of these applications rely on the ability to produce a uniform sample. However, a large number of intermolecular interactions may lead to swelling and/or wetting, which may complicate accurate sample preparation. An improved apparatus and method for producing droplets would be beneficial.

Disclosure of Invention

In one aspect, the present invention provides a topcoat compound having the structure of formula I:

Wherein each Y is independently-O (CR ⁹ ₂) p-, wherein p is 1,2, 3, 4, 5, or 6, and each R ⁹ is independently F or C ₁-C₆ perfluoroalkyl, Each n is independently 1, 2, 3, 4,5, 6, OR 7, each Z is independently-OR ¹⁰ -, wherein R ¹⁰ is C ₁-C₆ perfluoroalkylene, m is 1, 2, 3, 4,5, OR 6, each W is independently-C (O) NR _a-、-NR_a C (O) -, -C (O) O-, -OC (O) -, -C (O) S-, -SC (O) -or-O-, R _a is-H, C ₁-C₆ perfluoroalkyl or-F, X ¹ is- [ (CH ₂)_jQ]_s-(CH₂)_j -or optionally substituted C ₁-C₆ alkylene), And X ² is- (CH ₂)_j-[Q(CH₂)_j]_s -or optionally substituted C ₁-C₆ alkylene, wherein Q is-O-, -S-, -NR _b-、-NR_bC(O)-、-C(O)NR_b - -C (O) O-, -C (O) S-or-SC (O) -, R _b is-H or C ₁-C₆ alkyl, each j is independently 1,2, 3, 4, 5 or 6, and S is 1,2, 3, 4, 5 or 6, Each of R ¹、R²、R³、R⁴、R⁵ and R ⁶ is independently optionally substituted C ₁-C₆ alkoxy or C ₁-C₆ alkylene, Wherein at least one of R ¹、R² or R ³, and one of R ⁴、R⁵ or R ⁶ is C ₁-C₆ alkoxy, And each of R ⁷ and R ⁸ is independently F or C ₁-C₆ perfluoroalkyl, and at least one R ⁹ is C ₁-C₆ perfluoroalkyl, or R ¹⁰ is C ₃-C₆ perfluoroalkylene. In certain embodiments, n+n is 4 to 13, e.g., 7 to 13, such as 4 (e.g., 1+3, 2+2, or 3+1), 5 (e.g., 1+4, 2+3, 3+2, or 4+1), 6 (e.g., 1+5, 2+4, 3+3, 4+2, or 5+1), 7 (e.g., 1+6, 2+5, 3+4, 4+3, 5+2, or 6+1), 8 (e.g., 1+7, 2+6, 3+5, 4+4, 5+3, 6+2, or 7+1), 9 (e.g., 2+7, 3+6, 4+5, 5+4, 6+3, or 7+2), 10 (e.g., 4+5, 5+5 ], 6+4 or 7+3), 11 (e.g., 4+7, 5+6, 6+5 or 7+4), 12 (e.g., 5+7, 6+6 or 7+5), or 13 (e.g., 6+7 or 7+6). 5, or 6, or.

In some embodiments, the compound, when bound to a surface, comprises at least 57% total fluorine weight percent. In some embodiments, Y is-OCF ₂-CF(R¹¹) -, wherein R ¹¹ is C ₁-C₆ perfluoroalkyl. In some embodiments, R ¹¹ is CF ₃. In some embodiments, Z is-OCF ₂-CF₂-CF₂-CF₂ -or-OCF ₂-CF₂-CF₂-CF₂-CF_2-. In some embodiments, each of R ¹、R²、R³、R⁴、R⁵ and R ⁶ is methoxy. In some embodiments, the compound has the structure of formula IIa or IIb:

Wherein each n is independently 1, 2, 3, 4, 5, 6, or 7, and m is 1, 2, 3, 4, 5, or 6. In some embodiments, m is 1 to 5, e.g., 3. In some embodiments, n+n is 4 to 13, e.g., 7 to 13, such as 4 (e.g., 1+3, 2+2, or 3+1), 5 (e.g., 1+4, 2+3, 3+2, or 4+1), 6 (e.g., 1+5, 2+4, 3+3, 4+2, or 5+1), 7 (e.g., 1+6, 2+5, 3+4, 4+3, 5+2, or 6+1), 8 (e.g., 1+7, 2+6, 3+5, 4+4, 5+3, 6+2, or 7+1), 9 (e.g., 2+7, 3+6, 4+5, 5+4, 6+3, or 7+2), 10 (e.g., 4+5, 5, 6, or 6+4), 11 (e.g., 4+7, 5, 6+5, or 7+4), 12 (e.g., 5+7, 6+6, or 7+5), or 13 (e.g., 6+7). For example, when the compound has the structure of formula IIIa or IIIb, n+n is 7:

as another example, when the compound has the structure of formula IVa or Ivb, n+n is 10:

as another example, when the compound has the structure of formula Va or Vb, n+n is 10:

The invention also provides a device for generating droplets of a first liquid in a second liquid. The device includes a first channel having a first depth, a first width, a first proximal end, and a first distal end; and a drop source region in fluid communication with the first distal end, the drop source region configured to generate drops of the first liquid in the second liquid. The droplet source region includes a fluorocarbon surface coating having a total fluorine weight percent of at least 57%.

In some embodiments, the fluorocarbon surface coating comprises a surface coating compound having a VI moiety

Wherein each Y is independently-O (CR ⁹ ₂)_p -wherein p is 1,2,3,4, 5, or 6, and each R ⁹ is independently F or C ₁-C₆ perfluoroalkyl, each n is independently 1,2,3,4, 5, 6, or 7, Each Z is independently-OR ¹⁰ -, wherein R ¹⁰ is C ₁-C₆ perfluoroalkylene, m is 1,2, 3, 4,5, OR 6, each of R ⁷ and R ⁸ is independently F OR C ₁-C₆ perfluoroalkyl, And wherein at least one R ⁹ is C ₁-C₆ perfluoroalkyl or R ¹⁰ is C ₃-C₆ perfluoroalkylene. In certain embodiments, n+n is 4 to 13, e.g., 7 to 13, such as 4 (e.g., 1+3, 2+2, or 3+1), 5 (e.g., 1+4, 2+3, 3+2, or 4+1), 6 (e.g., 1+5, 2+4, 3+3, 4+2, or 5+1), 7 (e.g., 1+6, 2+5, 3+4, 4+3, 5+2, or 6+1), 8 (e.g., 1+7, 2+6, 3+5, 4+4, 5+3, 6+2, or 7+1), 9 (e.g., 2+7, 3+6, 4+5, 5+4, 6+3, or 7+2), 10 (e.g., 4+5, 5+5 ], 6+4 or 7+3), 11 (e.g., 4+7, 5+6, 6+5 or 7+4), 12 (e.g., 5+7, 6+6 or 7+5), or 13 (e.g., 6+7 or 7+6). 5, or 6, or.

In some embodiments, the device includes a first reservoir in fluid communication with the first proximal end.

In some embodiments, the device includes a second channel having a second depth, a second width, a second proximal end, and a second distal end, the second channel intersecting the first channel between the first proximal end and the first distal end. In some embodiments, the second channel comprises a fluorocarbon surface coating. In some embodiments, the device includes a second reservoir in fluid communication with the second proximal end.

In some embodiments, the droplet source region includes a shelf region having a second depth and a second width, wherein the second width is greater than the first width, and wherein the first distal end is in fluid communication with the shelf region. In some embodiments, the droplet source region includes a collection reservoir configured to collect droplets and includes at least one wall that forms a stepped region in fluid connection with the shelf region.

In some embodiments, the shelf region includes a fluorocarbon surface coating. In some embodiments, the fluorocarbon surface coating is the reaction product of any of the compounds described and/or disclosed herein (e.g., a compound of formula II) with a hydroxyl group. In some embodiments, the fluorocarbon surface coating comprises VIIa or VIIb moieties:

Wherein each n is independently 1,2, 3, 4, 5, 6, or 7, and m is 1,2, 3, 4, 5, or 6. In some embodiments, m is 1 to 5, e.g., 3. In some embodiments, n+n is 4 to 13, e.g., 7 to 13, such as 4 (e.g., 1+3, 2+2, or 3+1), 5 (e.g., 1+4, 2+3, 3+2, or 4+1), 6 (e.g., 1+5, 2+4, 3+3, 4+2, or 5+1), 7 (e.g., 1+6, 2+5, 3+4, 4+3, 5+2, or 6+1), 8 (e.g., 1+7, 2+6, 3+5, 4+4, 5+3, 6+2, or 7+1), 9 (e.g., 2+7, 3+6, 4+5, 5+4, 6+3, or 7+2), 10 (e.g., 4+5, 5, 6+4, or 7+3), 11 (e.g., 4+7, 5, 6+5, or 7+4), 12 (e.g., 5+7, 6+6, or 7+5), or 13 (e.g., 6+7 or 6). 5, or 6, or. For example, when the fluorocarbon surface coating comprises VIIIa or VIIIb moieties, n+n is 7:

As another example, when the fluorocarbon surface coating comprises IXa or IXb moieties, n+n is 10:

As another example, when the fluorocarbon surface coating comprises Xa or Xb moieties, n+n is 10:

The present invention also provides a method for producing a droplet comprising providing the device described herein, and flowing a first liquid from a first proximal end to a droplet source region to produce a droplet of the first liquid in a second liquid.

In some embodiments, the first liquid is aqueous or miscible with water.

In some embodiments, the device further comprises a second channel having a second depth, a second width, a second proximal end, and a second distal end, the second channel intersecting the first channel between the first proximal end and the first distal end; the second channel includes a third liquid that combines with the first liquid at the intersection such that the droplet includes the first liquid and the third liquid. In some embodiments, the first liquid comprises a support, the third liquid comprises particles, and the liquid droplets comprise the support from the first liquid and the particles from the third liquid.

In some embodiments, the method includes generating droplets at a generation frequency of 130Hz to 150 Hz.

Definition of the definition

In order to facilitate the understanding of the invention, some terms are defined below. The terms defined herein have meanings commonly understood by one of ordinary skill in the art to which the invention pertains. Terms such as "a," "an," and "the" are not intended to refer to only a single entity, but rather include the general class of which a particular instance may be used for illustration. The terminology herein is used to describe specific embodiments of the invention but is not limiting of the invention except as outlined in the claims.

As used herein, the term "about" refers to ±10% of the recited value.

The terms "adapter", "adapter" and "tag" may be used synonymously. The adaptors or tags may be coupled to the polynucleotide sequences to be "tagged" by any method, including ligation, hybridization, or other methods.

As used herein, the term "alkoxy" refers to an-O-alkyl group, wherein the alkoxy group is attached to the remainder of the compound through an oxygen atom.

As used herein, the term "alkyl" refers to a saturated straight or branched chain monovalent hydrocarbon radical containing from 1 to 6 (e.g., from 1 to 3, or from 1 to 5) carbons. In some embodiments, the alkyl group is unbranched (i.e., is a straight chain group); in some embodiments, the alkyl group is branched. The alkyl groups are illustrated by the following examples: methyl, ethyl, n-propyl and isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl, and neopentyl, but are not limited to these examples.

As used herein, the term "alkylene" refers to a saturated straight or branched chain divalent hydrocarbon group containing from 1 to 6 (e.g., from 1 to 3, or from 1 to 5) carbons. In some embodiments, the alkylene group is unbranched (i.e., is a linear group); in some embodiments, the alkylene group is branched. The alkylene groups are illustrated by the following examples: methylene, ethylene, n-and i-propylene, n-butylene, s-butylene, i-butylene and t-butylene, and neopentylene, but are not limited to these examples.

As used herein, the term "barcode" generally refers to a label or identifier that conveys or is capable of conveying information about an analyte. The barcode may be part of the analyte. The barcode may be a tag attached to an analyte (e.g., a nucleic acid molecule) or a combination of the tag plus an inherent property of the analyte (e.g., the size of the analyte or terminal sequence). Bar codes may be unique. Bar codes can take a number of different forms. For example, the bar code may include: a polynucleotide bar code; random nucleic acid and/or amino acid sequences; and synthetic nucleic acid and/or amino acid sequences. The barcode can be attached to the analyte in a reversible or irreversible manner. The barcode may be added to a fragment of, for example, a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before, during, and/or after sequencing of the sample. The bar code may allow individual sequencing reads to be identified and/or quantified in real time.

As used herein, the term "biological particle" generally refers to a discrete biological system derived from a biological sample. The biological particle may be a virus. The biological particles may be cells or derivatives of cells. The biological particles may be organelles from cells. Examples of organelles from cells include, but are not limited to, nuclei, endoplasmic reticulum, ribosomes, golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytosis vesicles, vacuoles, and lysosomes. The biological particles may be rare cells from a population of cells. The biological particles can be any type of cell including, but not limited to, prokaryotic cells, eukaryotic cells, bacteria, fungi, plants, mammalian or other animal cell types, mycoplasma, normal tissue cells, tumor cells, or any other cell type whether derived from a single-cell organism or a multicellular organism. The biological particles may be a component of a cell. The biological particles may be or may include DNA, RNA, organelles, proteins, or any combination thereof. The biological particles may be or include a matrix (e.g., a gel or polymer matrix) comprising cells or one or more components from cells (e.g., cell beads), such as DNA, RNA, organelles, proteins, or any combination thereof from cells. The biological particles may be obtained from a tissue of a subject. The biological particles may be hardened cells. Such sclerosant cells may or may not include cell walls or cell membranes. The biological particles may include one or more components of the cell, but may not include other components of the cell. One example of such a component is the nucleus or another organelle of a cell. The cells may be living cells. Living cells may be capable of culturing, for example, when encapsulated in a gel or polymer matrix, or when comprising a gel or polymer matrix.

As used herein, the term "fluidly connected" refers to a direct connection between at least two device elements (e.g., channels, reservoirs, etc.) that allows fluid to move between such device elements without passing through intermediate elements.

As used herein, the term "fluorocarbon" refers to a hydrocarbon in which at least one hydrogen is replaced with fluorine. In some embodiments, the fluorocarbon comprises-CH ₂F、CHF₂ or CF ₃.

As used herein, the term "genome" generally refers to genomic information from a subject, which may be, for example, at least a portion or all of the genetic information of the subject. The genome may be encoded in DNA or RNA. The genome may comprise coding (protein-encoding) and non-coding regions. The genome may comprise sequences of all chromosomes together in an organism. For example, the human genome has a total of 46 chromosomes. The sequence of all these chromosomes together may constitute the human genome.

As used herein, the term "in fluid communication with … …" refers to a connection between at least two device elements (e.g., channels, reservoirs, etc.) that allows fluid to move between such device elements with or without passing through one or more intermediate device elements.

As used herein, the term "macromolecular composition" generally refers to macromolecules contained within or derived from a biological particle. The macromolecular composition may comprise a nucleic acid. In some cases, the biological particles may be macromolecules. The macromolecular composition may comprise DNA or DNA molecules. The macromolecular composition may comprise RNA or RNA molecules. The RNA may be encoded or non-encoded. The RNA may be, for example, messenger RNA (mRNA), ribosomal RNA (rRNA), or transfer RNA (tRNA). The RNA may be a transcript. The RNA molecules can be (i) Clustered Regularly Interspaced Short Palindromic (CRISPR) RNA molecules (crrnas) or (ii) single guide RNA (sgrnas) molecules. The RNA may be a small RNA less than 200 nucleobases in length, or a large RNA greater than 200 nucleobases in length. The micrornas can include 5.8S ribosomal RNAs (rrnas), 5S rrnas, transfer RNAs (trnas), micrornas (mirnas), small interfering RNAs (sirnas), small nucleolar RNAs (snornas), RNAs that interact with Piwi proteins (pirnas), tRNA-derived micrornas (tsrnas), and small rDNA-derived RNAs (srrnas). The RNA may be double-stranded RNA or single-stranded RNA. The RNA may be circular RNA. The macromolecular composition may comprise a protein. The macromolecular composition may comprise a peptide. The macromolecular component may include a polypeptide or protein. The polypeptide or protein may be extracellular or intracellular. The macromolecular components may also include metabolites. Those skilled in the art will know of these and other suitable macromolecular components (also referred to as analytes) (see U.S. patent nos. 10,011,872 and 10,323,278, and WO/2019/157529, each of which is incorporated herein by reference in its entirety).

As used herein, the term "molecular tag" generally refers to a molecule capable of binding to a macromolecular component. Molecular tags can bind to macromolecular components with high affinity. Molecular tags can bind to macromolecular components with high specificity. The molecular tag may comprise a nucleotide sequence. The molecular tag may comprise an oligonucleotide or polypeptide sequence. The molecular tag may comprise a DNA aptamer. The molecular tag may be or comprise a primer. The molecular tag may be or comprise a protein. The molecular tag may comprise a polypeptide. The molecular tag may be a barcode.

As used herein, the term "oil" generally refers to a liquid that is not miscible with water. The oil may have a density higher or lower than water and/or a viscosity higher or lower than water.

At each position in this specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. In particular, the present disclosure is intended to include each individual sub-combination of the members of such groups and ranges. For example, the term "C ₁-C₆ alkyl" is expressly intended to disclose methyl, ethyl, C ₃ alkyl, C ₄ alkyl, C ₅ alkyl, and C ₆ alkyl individually. Furthermore, when a compound includes substituents in groups or in a plurality of positions disclosed therein, the disclosure is intended to cover individual compounds and groups of compounds (e.g., genus and subgenera) that contain each individual subcombination of members at each position, unless otherwise indicated.

The term "optionally substituted X" (e.g., "optionally substituted alkylene") is intended to be equivalent to "X", wherein X is optionally substituted "(e.g.," alkylene ", wherein the alkyl group is optionally substituted). This does not mean that feature "X" (e.g., alkylene) is itself optional. As described herein, certain compounds of interest may comprise one or more "optionally substituted" moieties. Generally, the term "substituted", whether preceded by the term "optional", means that one or more hydrogens of the designated moiety are replaced with a suitable substituent, e.g., any substituent or group described herein. For example, and without limitation, suitable substituents include halo (e.g., fluoro). Unless otherwise indicated, an "optionally substituted" group may have suitable substituents at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from the specified group, the substituents at each position may be the same or different. For example, in the term "optionally substituted C ₁-C₆ alkyl", any hydrogen bonded to C ₁-C₆ may be replaced with a substituent. Combinations of substituents contemplated by the present disclosure are preferably those that result in the formation of stable or chemically viable compounds. As used herein, the term "stable" refers to such compounds: substantially unchanged when subjected to conditions that allow for its production, detection, and in certain embodiments, its recovery, purification, and use for one or more of the purposes disclosed herein.

The term "particulate component of a cell" refers to a discrete biological system derived from the cell or fragment thereof and having at least one dimension of 0.1 μm (e.g., at least 0.1 μm, at least 1 μm, at least 10 μm, or at least 100 μm). The particulate component of the cell may be, for example, an organelle such as a nucleus, endoplasmic reticulum, ribosome, golgi apparatus, endoplasmic reticulum, chloroplast, endocytic vesicle, exocytosis vesicle, vacuole, lysosome, or mitochondria.

As used herein, the term "perfluoroalkyl" refers to a monovalent saturated straight or branched monovalent alkyl group containing 1 to 6 (e.g., 1 to 3, or 1 to 5) carbons in which each hydrogen atom has been replaced by fluorine. In some embodiments, the perfluoroalkyl group is unbranched (i.e., is a linear group); in some embodiments, the perfluoroalkyl group is branched.

As used herein, the term "perfluoroalkylene" refers to a divalent saturated straight or branched chain divalent alkylene group containing from 1 to 6 (e.g., from 1 to 3, or from 1 to 5) carbons, and wherein each hydrogen atom has been replaced with fluorine. In some embodiments, the perfluoroalkylene group is unbranched (i.e., is a linear group); in some embodiments, the perfluoroalkylene groups are branched.

As used herein, the term "sample" generally refers to a biological sample of a subject. The biological sample may be a nucleic acid sample or a protein sample. The biological sample may be derived from another sample. The sample may be a tissue sample, such as a biopsy sample, core needle biopsy sample, needle aspirate, or fine needle aspirate. The sample may be a liquid sample, such as a blood sample, a urine sample, or a saliva sample. The sample may be a skin sample. The sample may be a cheek swab. The sample may be a plasma or serum sample. The sample may comprise biological particles, such as cells or viruses, or a population thereof, or the sample may alternatively be free of biological particles. The cell-free sample may comprise a polynucleotide. Polynucleotides may be isolated from a body sample, which may be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal secretions, sputum, feces, and tears.

As used herein, the term "sequencing" generally refers to methods and techniques for determining the sequence of nucleotide bases in one or more polynucleotides. These polynucleotides may be, for example, nucleic acid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (e.g., single stranded DNA). Sequencing may be performed by various systems currently available, such as, but not limited toPacific BiosciencesOxfordOr Life Technologies (ION)) A sequencing system produced. Alternatively or in addition, sequencing may be performed using nucleic acid amplification, polymerase Chain Reaction (PCR) (e.g., digital PCR, quantitative PCR, or real-time PCR), or isothermal amplification. Such systems can provide a plurality of raw genetic data corresponding to genetic information of a subject (e.g., a human) as generated by the systems from a sample provided by the subject. In some examples, such systems provide sequencing reads (also referred to herein as "reads"). Reads may include a sequence of nucleobases corresponding to the sequence of a nucleic acid molecule that has been sequenced. In some cases, the systems and methods provided herein may be used with proteome information.

As used herein, the term "subject" generally refers to an animal such as a mammal (e.g., a human) or an avian (e.g., a bird), or other organism such as a plant. The subject may be a vertebrate, mammal, mouse, primate, ape or human. Animals may include, but are not limited to, farm animals, sports animals, and pets. The subject may be a healthy or asymptomatic individual, an individual having or suspected of having a disease (e.g., cancer) or susceptible to the disease, or an individual in need of treatment or suspected of being in need of treatment. The subject may be a patient.

The term "substantially stationary" as used herein with respect to droplet formation generally refers to a state when the movement of droplets formed in the continuous phase is passive movement (e.g., caused by a density difference between the dispersed phase and the continuous phase).

As used herein, the term "carrier" generally refers to particles that are not biological particles. The particles may be solid or semi-solid particles. The particles may be beads, such as gel beads. The gel beads may include a polymer matrix (e.g., a matrix formed by polymerization or cross-linking). The polymer matrix may include one or more polymers (e.g., polymers having different functional groups or repeating units). The polymers in the polymer matrix may be randomly arranged, for example in a random copolymer, and/or have an ordered structure, for example in a block copolymer. Crosslinking may be achieved via covalent, ionic or induced interactions or physical entanglement. The beads may be macromolecules. Beads may be formed from nucleic acid molecules that are bound together. Beads may be formed via covalent or non-covalent assembly of molecules (e.g., macromolecules) such as monomers or polymers. Such polymers or monomers may be natural or synthetic. Such polymers or monomers may be or include, for example, nucleic acid molecules (e.g., DNA or RNA). The beads may be formed of a polymeric material. The beads may be magnetic or non-magnetic. The beads may be rigid. The beads may be flexible and/or compressible. The beads may be destructible or dissolvable. The beads may be solid particles (e.g., metal-based particles including, but not limited to, iron oxide, gold, or silver) covered with a coating comprising one or more polymers. Such coatings may be destructible or dissolvable.

As used herein, any value provided within a range of values includes both the upper limit and the lower limit, and all possible subranges within such range, as well as specific values falling within such range, whether or not a specific value or a specific subrange is explicitly indicated.

Drawings

Fig. 1A-1B are process flow diagrams showing exemplary embodiments of a synthetic surface coating agent.

Fig. 2 is a graph showing the droplet generation frequency.

Fig. 3 is a schematic diagram showing a droplet generation failure.

Fig. 4 is a photograph of a droplet source region.

Fig. 5A-5B are ¹ HNMR spectra of aminosilanes before and after reaction to form compounds of formula III.

Fig. 6 is a graph showing the average value and standard deviation of water contact angle.

Fig. 7 is a graph showing the average and standard deviation of water contact angles, wherein a commercial coating agent and a fluorocarbon coating agent having a structure of formula III are compared.

Detailed Description

The present invention provides fluorocarbon coating agents, as well as devices having fluoropolymer coatings for forming droplets, and methods of use thereof. The device includes a first channel having a first depth, a first width, a first proximal end, and a first distal end. The device includes a drop source region in fluid communication with the first distal end, the drop source region configured to generate drops of the first liquid in the second liquid. The droplet source region includes a topcoat compound that includes at least 57% fluorine. Exemplary coating agents have the structure of formula I.

The topcoat compounds, devices and methods described herein provide various advantages over other available devices and techniques known in the art. In particular, the topcoat compounds, devices, and methods described herein reduce variability between each droplet formed, produce smaller droplet sizes, and/or generate droplets with a reduced incidence of droplet generation failure. In addition, the topcoat compounds, devices and methods provide for prolonged periods of droplet generation due to the increased robustness of the unique topcoat compounds.

Surface coating agent

The surface coating agent of the present invention may have the structure of formula I:

Wherein each Y is independently-O (CR ⁹ ₂) p-, wherein p is 1,2, 3, 4, 5, or 6, and each R ⁹ is independently F or C ₁-C₆ perfluoroalkyl, Each n is independently 1, 2, 3, 4,5, 6, OR 7, each Z is independently-OR ¹⁰ -, wherein R ¹⁰ is C ₁-C₆ perfluoroalkylene, m is 1, 2, 3, 4,5, OR 6, each W is independently-C (O) NR _a-、-NR_a C (O) -, -C (O) O-, -OC (O) -, -C (O) S-, -SC (O) -or-O-, wherein R _a is-H, C ₁-C₆ perfluoroalkyl or-F, X ¹ is- [ (CH ₂)_jQ]_s-(CH₂)_j -or optionally substituted C ₁-C₆ alkylene), And X ² is- (CH ₂)_j-[Q(CH₂)_j]_s -or optionally substituted C ₁-C₆ alkylene, wherein Q is-O-, -S-, -NR _b-、-NR_bC(O)-、-C(O)NR_b - -C (O) O-, -C (O) S-or-SC (O) -, R _b is-H or C ₁-C₆ alkyl, each j is independently 1,2, 3, 4, 5 or 6, and S is 1,2, 3, 4, 5 or 6, Each of R ¹、R²、R³、R⁴、R⁵ and R ⁶ is independently optionally substituted C ₁-C₆ alkoxy or C ₁-C₆ alkyl, Wherein at least one of R ¹、R² or R ³, and one of R ⁴、R⁵ or R ⁶ is C ₁-C₆ alkoxy, And each of R ⁷ and R ⁸ is independently F or C ₁-C₆ perfluoroalkyl, and wherein at least one R ⁹ is C ₁-C₆ perfluoroalkyl, or R ¹⁰ is C ₃-C₆ perfluoroalkylene. In some embodiments, m is 1 to 5, e.g., 3. In certain embodiments, n+n is 4 to 13, e.g., 7 to 13, such as 4 (e.g., 1+3, 2+2, or 3+1), 5 (e.g., 1+4, 2+3, 3+2, or 4+1), 6 (e.g., 1+5, 2+4, 3+3, 4+2, or 5+1), 7 (e.g., 1+6, 2+5, 3+4, 4+3, 5+2, or 6+1), 8 (e.g., 1+7, 2+6, 3+5, 4+4, 5+3, 6+2, or 7+1), 9 (e.g., 2+7, 3+6, 4+5, 5+4, 6+3, or 7+2), 10 (e.g., 4+5, 5+5 ], 6+4 or 7+3), 11 (e.g., 4+7, 5+6, 6+5 or 7+4), 12 (e.g., 5+7, 6+6 or 7+5), or 13 (e.g., 6+7 or 7+6). 5, or 6, or. In some embodiments, the compound having the structure of formula I may comprise at least 57% total fluorine weight percent when bound to a surface. For example, but not limited to, the compound having the structure of formula I, when bound to a surface, may comprise a total fluorine weight percent of about 57% to 70%. As another non-limiting example, a compound having the structure of formula I, when bound to a surface, may comprise about 63% total fluorine weight percent. It will be appreciated that the alkyl portion of the alkoxy group in R ¹-R⁶ will be cleaved upon binding of the compound to the hydroxylated surface.

In some embodiments, Y is -OCF(CF₃)-CF₂、-OCF(CF₃)-CF₂-CF₂、-OCF(CF₃)-CF₂-CF₂-CF₂、-OCF(CF₃)-CF₂-CF₂-CF₂-CF₂ or-OCF (CF ₃)-CF₂-CF₂-CF₂-CF₂-CF₂, in some embodiments, Y is -OCF₂-CF(CF₃)、-OCF₂-CF₂-CF(CF₃)、-OCF₂-CF₂-CF₂-CF(CF₃)、-OCF₂-CF₂-CF₂-CF₂-CF(CF₃) or-OCF ₂-CF₂-CF₂-CF₂-CF₂-CF(CF₃). In some embodiments, Y is -OCF₂-CF(C₂F₅)、-OCF₂-CF(C₃F₇)、-OCF₂-CF(C₄F₉)、-OCF₂-CF(C₅F₁₁) or-OCF ₂-CF(C₆F₁₃). In some embodiments, Y is -OCF₂-CF₂-CF(C₂F₅)、-OCF₂-CF₂-CF(C₃F₇)、-OCF₂-CF₂-CF(C₄F₉)、-OCF₂-CF₂-CF(C₅F₁₁) or-OCF ₂-CF₂-CF(C₆F₁₃). In some embodiments, Y is -OCF₂-CF₂-CF₂-CF(C₂F₅)、-OCF₂-CF₂-CF₂-CF(C₃F₇)、-OCF₂-CF₂-CF₂-CF(C₄F₉)、-OCF₂-CF₂-CF₂-CF(C₅F₁₁) or-OCF ₂-CF₂-CF₂-CF(C₆F₁₃). In some embodiments, Y is -OCF₂-CF₂-CF₂-CF₂-CF(C₂F₅)、-OCF₂-CF₂-CF₂-CF₂-CF(C₃F₇)、-OCF₂-CF₂-CF₂-CF₂-CF(C₄F₉)、-OCF₂-CF₂-CF₂-CF₂-CF(C₅F₁₁) or-OCF ₂-CF₂-CF₂-CF₂-CF(C₆F₁₃). In some embodiments, Y is -OCF₂-CF₂-CF₂-CF₂-CF₂-CF(C₂F₅)、-OCF₂-CF₂-CF₂-CF₂-CF₂-CF(C₃F₇)、-OCF₂-CF₂-CF₂-CF₂-CF₂-CF(C₄F₉)、-OCF₂-CF₂-CF₂-CF₂-CF₂-CF(C₅F₁₁) or-OCF ₂-CF₂-CF₂-CF₂-CF₂-CF(C₆F₁₃). In some embodiments, Y is -OCF₂-CF(CF₃)-CF₂、-OCF₂-CF(C₂F₅)-CF₂、-OCF₂-CF(C₃F₇)-CF₂、-OCF₂-CF(C₄F₉)-CF₂、-OCF₂-CF(C₅F₁₁)-CF₂ or-OCF ₂-CF(C₆F₁₃)-CF₂.

In some embodiments, Y is -OCF₂-CF(CF₃)-CF₂-CF₂、-OCF₂-CF(C₂F₅)-CF₂-CF₂、-OCF₂-CF(C₃F₇)-CF₂-CF₂、-OCF₂-CF(C₄F₉)-CF₂-CF₂、-OCF₂-CF(C₅F₁₁)-CF₂-CF₂ or-OCF ₂-CF(C₆F₁₃)-CF₂-CF₂. In some embodiments, Y is -OCF₂-CF(CF₃)-CF₂-CF₂-CF₂、-OCF₂-CF(C₂F₅)-CF₂-CF₂-CF₂、-OCF₂-CF(C₃F₇)-CF₂-CF₂-CF₂、-OCF₂-CF(C₄F₉)-CF₂-CF₂-CF₂、-OCF₂-CF(C₅F₁₁)-CF₂-CF₂-CF₂ or-OCF ₂-CF(C₆F₁₃)-CF₂-CF₂-CF₂. In some embodiments, Y is -OCF₂-CF(CF₃)-CF₂-CF₂-CF₂CF₂、-OCF₂-CF(C₂F₅)-CF₂-CF₂-CF₂CF₂、-OCF₂-CF(C₃F₇)-CF₂-CF₂-CF₂CF₂、-OCF₂-CF(C₄F₉)-CF₂-CF₂-CF₂CF₂、-OCF₂-CF(C₅F₁₁)-CF₂-CF₂-CF₂CF₂ or-OCF ₂-CF(C₆F₁₃)-CF₂-CF₂-CF₂CF₂. In some embodiments, Y is -OCF₂-CF₂-CF(CF₃)-CF₂、-OCF₂-CF₂-CF(C₂F₅)-CF₂、-OCF₂-CF₂-CF(C₃F₇)-CF₂、-OCF₂-CF₂-CF(C₄F₉)-CF₂、-OCF₂-CF₂-CF(C₅F₁₁)-CF₂ or-OCF ₂-CF₂-CF(C₆F₁₃)-CF₂. In some embodiments, Y is -OCF₂-CF₂-CF(CF₃)-CF₂-CF₂、-OCF₂-CF₂-CF(C₂F₅)-CF₂-CF₂、-OCF₂-CF₂-CF(C₃F₇)-CF₂-CF₂、-OCF₂-CF₂-CF(C₄F₉)-CF₂-CF₂、-OCF₂-CF₂-CF(C₅F₁₁)-CF₂-CF₂ or-OCF ₂-CF₂-CF(C₆F₁₃)-CF₂-CF₂. In some embodiments, Y is -OCF₂-CF₂-CF(CF₃)-CF₂-CF₂-CF₂、-OCF₂-CF₂-CF(C₂F₅)-CF₂-CF₂-CF₂、-OCF₂-CF₂-CF(C₃F₇)-CF₂-CF₂-CF₂、-OCF₂-CF₂-CF(C₄F₉)-CF₂-CF₂-CF₂、-OCF₂-CF₂-CF(C₅F₁₁)-CF₂-CF₂-CF₂ or-OCF ₂-CF₂-CF(C₆F₁₃)-CF₂-CF₂-CF₂. In some embodiments, Y is -OCF₂-CF₂-CF₂-CF(CF₃)-CF₂、-OCF₂-CF₂-CF₂-CF(C₂F₅)-CF₂、-OCF₂-CF₂-CF₂-CF(C₃F₇)-CF₂、-OCF₂-CF₂-CF₂-CF(C₄F₉)-CF₂、-OCF₂-CF₂-CF₂-CF(C₅F₁₁)-CF₂ or-OCF ₂-CF₂-CF₂-CF(C₆F₁₃)-CF₂. In some embodiments, Y is -OCF₂-CF₂-CF₂-CF(CF₃)-CF₂-CF₂、-OCF₂-CF₂-CF₂-CF(C₂F₅)-CF₂-CF₂、-OCF₂-CF₂-CF₂-CF(C₃F₇)-CF₂-CF₂、-OCF₂-CF₂-CF₂-CF(C₄F₉)-CF₂-CF₂、-OCF₂-CF₂-CF₂-CF(C₅F₁₁)-CF₂-CF₂ or-OCF ₂-CF₂-CF₂-CF(C₆F₁₃)-CF₂-CF₂. In some embodiments, Y is -OCF₂-CF₂-CF₂-CF₂-CF(CF₃)-CF₂、-OCF₂-CF₂-CF₂-CF₂-CF(C₂F₅)-CF₂、-OCF₂-CF₂-CF₂-CF₂-CF(C₃F₇)-CF₂、-OCF₂-CF₂-CF₂-CF₂-CF(C₄F₉)-CF₂、-OCF₂-CF₂-CF₂-CF₂-CF(C₅F₁₁)-CF₂ or-OCF ₂-CF₂-CF₂-CF₂-CF(C₆F₁₃)-CF₂.

In some embodiments, Z is -OCF₂、-OCF₂-CF₂、-OCF₂-CF₂-CF₂、-OCF₂-CF₂-CF₂-CF₂、-OCF₂-CF₂-CF₂-CF₂-CF₂ or-OCF ₂-CF₂-CF₂-CF₂-CF₂-CF₂. In some embodiments of the present invention, in some embodiments, each W is independently-C (O) NR _a-、-NR_a C (O) -, -C (O) O-; -OC (O) -, -C (O) S-, -SC (O) -, or-O-, wherein R _a is-H, C ₁-C₆ perfluoroalkyl or-F, is-C (O) NH-, -C (O) O-, -C (O) -S-and/or-O-. In some embodiments, X ¹ and/or X ² are methylene, ethylene, propylene, butylene, pentylene, and/or hexylene. In some embodiments, X ¹ is -CH₂-O-CH₂-、-CH₂-S-CH₂-、-CH₂-NH-CH₂-、-CH₂-NR_b-CH₂-、-CH₂-NR_b-C(O)-CH₂-、-CH₂-C(O)-NR_b-CH₂-、-CH₂-C(O)-O-CH₂-、-CH₂-C(O)-S-CH₂- or-CH ₂-S-C(O)-CH₂ -, and R _b is-H or C ₁-C₆ alkyl. in some embodiments, X ¹ is -CH₂-CH₂-O-CH₂-、-CH₂-CH₂-S-CH₂-、-CH₂-CH₂-NH-CH₂-、-CH₂-CH₂-NR_b-CH₂-、-CH₂-CH₂-NR_b-C(O)-CH₂-、-CH₂-CH₂-C(O)-NR_b-CH₂-、-CH₂-CH₂-C(O)-O-CH₂-、-CH₂-CH₂-C(O)-S-CH₂- or-CH ₂-CH₂-S-C(O)-CH₂ -, and R _b is-H or C ₁-C₆ alkyl. In some embodiments, X ¹ is -CH₂-CH₂-CH₂-O-CH₂-、-CH₂-CH₂-CH₂-S-CH₂-、-CH₂-CH₂-CH₂-NH-CH₂-、-CH₂-CH₂-CH₂-NR_b-CH₂-、-CH₂-CH₂-CH₂-NR_b-C(O)-CH₂-、-CH₂-CH₂-CH₂-C(O)-NR_b-CH₂-、-CH₂-CH₂-CH₂-C(O)-O-CH₂-、-CH₂-CH₂-CH₂-C(O)-S-CH₂- or-CH ₂-CH₂-CH₂-S-C(O)-CH₂ -, and R _b is-H or C ₁-C₆ alkyl. In some embodiments, X ¹ is -CH₂-CH₂-CH₂-CH₂-O-CH₂-、-CH₂-CH₂-CH₂-CH₂-S-CH₂-、-CH₂-CH₂-CH₂-CH₂-NH-CH₂-、-CH₂-CH₂-CH₂-CH₂-NR_b-CH₂-、-CH₂-CH₂-CH₂-CH₂-NR_b-C(O)-CH₂-、-CH₂-CH₂-CH₂-CH₂-C(O)-NR_b-CH₂-、-CH₂-CH₂-CH₂-CH₂-C(O)-O-CH₂-、-CH₂-CH₂-CH₂-CH₂-C(O)-S-CH₂- or-CH ₂-CH₂-CH₂-CH₂-S-C(O)-CH₂ -, and R _b is-H or C ₁-C₆ alkyl. In some embodiments, X ¹ is -CH₂-CH₂-CH₂-CH₂-CH₂-O-CH₂-、-CH₂-CH₂-CH₂-CH₂-CH₂-S-CH₂-、-CH₂-CH₂-CH₂-CH₂-CH₂-NH-CH₂-、-CH₂-CH₂-CH₂-CH₂-CH₂-NR_b-CH₂-、-CH₂-CH₂-CH₂-CH₂-CH₂-NR_b-C(O)-CH₂-、-CH₂-CH₂-CH₂-CH₂-CH₂-C(O)-NR_b-CH₂-、-CH₂-CH₂-CH₂-CH₂-CH₂-C(O)-O-CH₂-、-CH₂-CH₂-CH₂-CH₂-CH₂-C(O)-S-CH₂- or-CH ₂-CH₂-CH₂-CH₂-CH₂-S-C(O)-CH₂ -, and R _b is-H or C ₁-C₆ alkyl. In some embodiments, X ¹ is -CH₂-CH₂-CH₂-CH₂-CH₂-CH₂O-CH₂-、-CH₂-CH₂-CH₂-CH₂-CH₂-CH₂-S-CH₂-、-CH₂-CH₂-CH₂-CH₂-CH₂-CH₂-NH-CH₂-、-CH₂-CH₂-CH₂-CH₂-CH₂-CH₂-NR_b-CH₂-、-CH₂-CH₂-CH₂-CH₂-CH₂-CH₂-NR_b-C(O)-CH₂-、-CH₂-CH₂-CH₂-CH₂-CH₂-CH₂-C(O)-NR_b-CH₂-、-CH₂-CH₂-CH₂-CH₂-CH₂-CH₂-C(O)-O-CH₂-、-CH₂-CH₂-CH₂-CH₂-CH₂-CH₂-C(O)-S-CH₂- or-CH ₂-CH₂-CH₂-CH₂-CH₂-CH₂-S-C(O)-CH₂ -, and R _b is-H or C ₁-C₆ alkyl.

In some embodiments, X ² is -CH₂-O-CH₂-、-CH₂-S-CH₂-、-CH₂-NH-CH₂-、-CH₂-NR_b-CH₂-、-CH₂-C(O)-NR_b-CH₂-、-CH₂-NR_b-C(O)-CH₂-、-CH₂-C(O)-O-CH₂-、-CH₂-C(O)-S-CH₂- or-CH ₂-S-C(O)-CH₂ -, and R _b is-H or C ₁-C₆ alkyl. In some embodiments, X ² is -CH₂-O-CH₂-CH₂-、-CH₂-S-CH₂-CH₂-、-CH₂-NH-CH₂-CH₂-、-CH₂-NR_b-CH₂-CH₂-、-CH₂-NR_b-C(O)-CH₂-CH₂-、-CH₂-C(O)-NR_b-CH₂-CH₂-、-CH₂-C(O)-O-CH₂-CH₂-、-CH₂-C(O)-S-CH₂-CH₂- or-CH ₂-S-C(O)-CH₂-CH₂ -, and R _b is-H or C ₁-C₆ alkyl. In some embodiments, X ² is -CH₂-O-CH₂-CH₂-CH₂-、-CH₂-S-CH₂-CH₂-CH₂-、-CH₂-NH-CH₂-CH₂-CH₂-、-CH₂-NR_b-CH₂-CH₂-CH₂-、-CH₂-NR_b-C(O)-CH₂CH₂-CH₂-、-CH₂-C(O)-NR_b-CH₂-CH₂-CH₂-、-CH₂-C(O)-O-CH₂-CH₂-CH₂-、-CH₂-C(O)-S-CH₂-CH₂-CH₂- or-CH ₂-S-C(O)-CH₂-CH₂-CH₂ -, and R _b is-H or C ₁-C₆ alkyl. In some embodiments, X ² is -CH₂-O-CH₂-CH₂-CH₂-CH₂-、-CH₂-S-CH₂-CH₂-CH₂-CH₂-、-CH₂-NH-CH₂-CH₂-CH₂-CH₂-、-CH₂-NR_b-CH₂-CH₂-CH₂-CH₂-、-CH₂-NR_b-C(O)-CH₂-CH₂-CH₂-CH₂-、-CH₂-C(O)-NR_b-CH₂-CH₂-CH₂-CH₂-、-CH₂-C(O)-O-CH₂-CH₂-CH₂-CH₂-、CH₂-C(O)-S-CH₂-CH₂-CH₂-CH₂- or-CH ₂-S-C(O)-CH₂-CH₂-CH₂-CH₂ -, and R _b is-H or C ₁-C₆ alkyl. In some embodiments, X ² is -CH₂-O-CH₂-CH₂-CH₂-CH₂-CH₂-、-CH₂-S-CH₂-CH₂-CH₂-CH₂-CH₂-、-CH₂-NH-CH₂-CH₂-CH₂-CH₂-CH₂-、-CH₂-NR_b-CH₂-CH₂-CH₂-CH₂-CH₂-、-CH₂-C(O)-NR_b-CH₂-CH₂-CH₂-CH₂-CH₂-、-CH₂-C(O)-O-CH₂-CH₂-CH₂-CH₂-CH₂-、-CH₂-NR_b-C(O)-CH₂-CH₂-CH₂-CH₂-CH₂-、-CH₂-C(O)-S-CH₂-CH₂-CH₂-CH₂-CH₂- or-CH ₂-S-C(O)-CH₂-CH₂-CH₂-CH₂-CH₂ -, and R _b is-H or C ₁-C₆ alkyl. In some embodiments, X ² is -CH₂-O-CH₂-CH₂-CH₂-CH₂-CH₂-CH₂-、-CH₂-S-CH₂-CH₂-CH₂-CH₂-CH₂-CH₂-、-CH₂-NH-CH₂-CH₂-CH₂-CH₂-CH₂-CH₂-、-CH₂-NR_b-CH₂-CH₂-CH₂-CH₂-CH₂-CH₂-、-CH₂-NR_b-C(O)-CH₂-CH₂-CH₂-CH₂-CH₂-CH₂-、-CH₂-C(O)-NR_b-CH₂-CH₂-CH₂-CH₂-CH₂-CH₂-、-CH₂-C(O)-O-CH₂-CH₂-CH₂-CH₂-CH₂-CH₂-、-CH₂-C(O)-S-CH₂-CH₂-CH₂-CH₂-CH₂-CH₂- or-CH ₂-S-C(O)-CH₂-CH₂-CH₂-CH₂-CH₂-CH₂ -, and R _b is-H or C ₁-C₆ alkyl.

In some embodiments, each of R ¹、R²、R³、R⁴、R⁵ and/or R ⁶ is methoxy, ethoxy 、-OCH₂-CH₂-CH₃、-OCH₂-CH₂-CH₂-CH₃、-OCH₂-CH₂-CH₂-CH₂-CH₃, or-OCH ₂-CH₂-CH₂-CH₂-CH₂-CH₃. In other embodiments, R ¹、R²、R³、R⁴、R⁵ and/or R ⁶ are methyl, ethyl, propyl, butyl, pentyl or hexyl. In some embodiments, each of R ⁷ and/or R ⁸ is F or C ₁-C₆ perfluoroalkyl. For example, but not limited to, R ⁷ and/or R ⁸ are -CF₃、-CF₂-CF₃、-CF₂-CF₂-CF₃、-CF₂-CF₂-CF₂-CF₃、-CF₂-CF₂-CF₂-CF₂-CF₃、-CF₂-CF₂-CF₂-CF₂-CF₂-CF₃.

In some embodiments, the compound has the structure of formula IIa or IIb:

Wherein each n is independently 1,2, 3, 4,5, 6, or 7, and m is 1,2, 3, 4,5, or 6. In some embodiments, m is 1 to 5, e.g., 3. In certain embodiments, n+n is 4 to 13, e.g., 7 to 13, such as 4 (e.g., 1+3, 2+2, or 3+1), 5 (e.g., 1+4, 2+3, 3+2, or 4+1), 6 (e.g., 1+5, 2+4, 3+3, 4+2, or 5+1), 7 (e.g., 1+6, 2+5, 3+4, 4+3, 5+2, or 6+1), 8 (e.g., 1+7, 2+6, 3+5, 4+4, 5+3, 6+2, or 7+1), 9 (e.g., 2+7, 3+6, 4+5, 5+4, 6+3, or 7+2), 10 (e.g., 4+5, 5, 6+4, or 7+3), 11 (e.g., 4+7, 5, 6+5, or 7+4), 12 (e.g., 5+7, 6+6, or 7+5), or 13 (e.g., 6+7 or 6). 5, or 6, or. For example, when the compound has structure IIIa or IIIb, n+n is 7:

Device and method for controlling the same

The device of the invention may comprise the following components: a first channel having a first width, a first height, a proximal end and a distal end, and optionally a drop source region. The drop source region can be in fluid communication with, e.g., fluidly connected to, the distal end. The droplet source region may be used to form droplets comprising cellular or particulate components. The droplet source region can include a fluorocarbon surface coating having a total fluorine weight percent of at least 57%.

Channel

The devices described herein include at least a first channel. The channels may have any suitable geometry. The channels may be open ended (e.g., connected to subsequent channels) or closed. The channel may have any length, width, and height suitable for transporting one or more droplets. For example, the length, width, and height may be at least independently, e.g., 0.1 μm to 10mm (e.g., 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, e.g., 1 to 10 μm, e.g., 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, e.g., 10 to 100 μm, e.g., 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, e.g., 100 μm to 1000 μm, e.g., 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 1000 μm, e.g., 1 μm, 2 μm, 9 μm, 10 μm, e.g., 2mm, 10mm, 7mm, 10mm, 6mm, 10 mm). The volume of the channel may be, for example, 1nL to 10mL (e.g., 1nL, 2nL, 3nL, 4nL, 5nL, 6nL, 7nL, 8nL, 9nL, 10nL, e.g., 10nL to 100nL, e.g., 20nL, 30nL, 40nL, 50nL, 60nL, 70nL, 80nL, 90nL, 100nL, e.g., 100nL to 1 μL, e.g., 200nL, 300nL, 400nL, 500nL, 600nL, 700nL, 800nL, 900nL, 1 μL, e.g., 1 μL to 10 μL, for example 2. Mu.L, 3. Mu.L, 4. Mu.L, 5. Mu.L, 6. Mu.L, 7. Mu.L, 8. Mu.L, 9. Mu.L, 10. Mu.L, for example 10 to 100. Mu.L, for example 20. Mu.L, 30. Mu.L, 40. Mu.L, 50. Mu.L, 60. Mu.L, 70. Mu.L, 80. Mu.L, 90. Mu.L, 100. Mu.L, for example 100. Mu.L to 1mL, for example 200. Mu.L, 300. Mu.L, 400. Mu.L, 500. Mu.L, 600. Mu.L, 700. Mu.L, 800. Mu.L, 900. Mu.L, 1mL, for example 1mL to 10mL, for example 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL, 10 mL). In some embodiments, the cross-sectional dimension of the channel is no greater than 1mm.

Reservoir

The device may comprise a reservoir for storing a liquid reagent. For example, the device may comprise a reservoir for storing liquid flowing in the first channel. As another non-limiting example, the device can include a first reservoir in fluid communication with the first proximal end. A single reservoir may also be connected to multiple channels in the device, for example, when the same liquid is to be introduced at two or more different locations in the device. The reservoir may be of any suitable size, for example, to hold 10 μl to 500mL, for example 10 μl to 300mL, 25 μl to 10mL, 100 μl to 1mL, 40 μl to 300 μl, 1mL to 10mL, or 10mL to 50mL.

Drop source region

The devices described herein may be used to generate droplets. Generally, droplets or particles form in the droplet source region. The droplets or particles may be formed by any suitable method known in the art. Generally, drop formation comprises two liquid phases. The two liquid phases may be, for example, a sample phase and an oil phase. During formation, a plurality of discrete volumes of droplets or particles are formed.

The droplets may be formed by: the liquid is shaken or stirred to form individual droplets, to produce a suspension or emulsion containing individual droplets, or to form droplets by pipetting techniques (e.g., with a needle, etc.). Droplets may be formed using millimeter fluid, micro fluid, or nano fluid droplet generators. Examples of such drop generators include, for example, T-junction drop generators, Y-junction drop generators, in-channel junction drop generators, cross (or "X" -shaped) junction drop generators, flow focused junction drop generators, microcapillary drop generators (e.g., co-current flow or flow focusing), and three-dimensional drop generators. Droplets may be produced using a flow focusing device or using an emulsifying system such as homogenization, membrane emulsification, shear cell emulsification, and fluid emulsification.

The discrete droplets may be encapsulated by a carrier fluid that wets the microchannels. These droplets (sometimes referred to as plugs) form a dispersed phase in which the reaction occurs. Systems using embolization differ from the segmented flow injection analysis in that the reagents in the embolization do not come into contact with the microchannels. In a T-junction, the dispersed phase and the continuous phase are injected from two branches of the "T". Droplets of the dispersed phase are created due to shear and interfacial tension at the fluid-fluid interface. The phase with the lower interfacial tension with the channel walls is the continuous phase. To generate droplets in the flow focusing configuration, the continuous phase is injected through two external channels and the dispersed phase is injected through a central channel into a narrow orifice. Those skilled in the art will recognize other geometric designs for creating droplets. Methods of producing droplets are disclosed in the following documents: song et al, angew.chem.45:7336-7356,2006; mazutis et al, nat. Protoc.8 (5): 870-891, 2013; U.S. patent 9,839,911; U.S. patent publication Nos. 2005/0172476, 2006/0163385 and 2007/0003442; PCT publication nos. WO 2009/005680 and WO 2018/009766. In some embodiments, an electric field or acoustic waves may be used to generate the droplets, for example, as described in PCT publication No. WO 2018/009766.

In one embodiment, the droplet source region includes a shelf region that allows liquid to expand substantially in one dimension (e.g., perpendicular to the flow direction). In some embodiments, the shelf region may include a fluorocarbon surface coating as described below. In some embodiments, the width of the shelf region is greater than the width of the first channel at its distal end. In certain embodiments, the first channel is a channel other than a shelf region, e.g., the shelf region widens as compared to the distal end of the first channel, or widens with a steeper slope or curvature than the distal end of the first channel. In other embodiments, the first channel and shelf region merge into a continuous flow path, e.g., a flow path that widens linearly or non-linearly from its proximal end to its proximal end; in these embodiments, the distal end of the first channel may be considered to be any point along the combined first channel and shelf region. In another embodiment, the drop source region includes a stepped region that provides spatial displacement and allows the liquid to expand in more than one dimension. The spatial displacement may be upward or downward, or both upward and downward, relative to the channel. The selection of the direction may be based on the relative densities of the dispersed phase and the continuous phase, with an upward step when the density of the dispersed phase is less than the continuous phase and a downward step when the density of the dispersed phase is greater than the continuous phase. The drop source region may also include a combination of a shelf region and a step region, for example, the shelf region being disposed between the channel and the step region. An exemplary device of this embodiment is described in WO 2019/040637, the droplet-forming device of which is hereby incorporated by reference.

Without wishing to be bound by theory, in the device of the present invention, droplets of the first liquid may be formed in the second liquid by flowing the first liquid from the distal end into the droplet source region. In embodiments having a shelf region and a stepped region, the first liquid stream expands laterally into a dished shape in the shelf region. As the first liquid stream continues to flow through the shelf region, the stream enters a stepped region where the droplets take a more nearly spherical shape and eventually separate from the liquid stream. As the droplets form, the continuous phase passively flows around the primary droplets, for example into a shelf region, where the continuous phase is reformed as the droplets separate from their liquid stream. Unlike in other systems, droplet formation by this mechanism can occur without externally driving the continuous phase. It will be appreciated that the continuous phase may be driven externally during droplet formation, for example by gentle agitation or vibration, but such movement is not necessary for droplet formation.

In these embodiments, the size of the droplets produced is significantly less sensitive to changes in the liquid properties. For example, the size of the droplets generated is less sensitive to the dispersed phase flow rate. The addition of multiple source regions is also significantly easier from a layout and manufacturing perspective. The addition of additional source regions enables the formation of droplets even in the event that one droplet source region becomes plugged. Drop formation may be controlled by adjusting one or more geometric features of the fluid channel architecture, such as the width, height, and/or spread angle of one or more fluid channels. For example, the droplet size and droplet formation speed may be controlled. In some cases, the number of formation regions at the driving pressure may be increased to increase the throughput of droplet formation.

Passive flow of the continuous phase may occur only around the primary droplets. The droplet or particle source region may also include one or more channels that allow continuous phase flow to a location between the distal end of the first channel and the primary droplet body. These channels allow the continuous phase to flow behind the primary droplets, thereby altering (e.g., increasing or decreasing) the rate of droplet formation. Such channels may be fluidly connected to reservoirs of the droplet or particle source region or to different reservoirs of the continuous phase. Although external driving of the continuous phase is not necessary, external driving may be employed, for example, to pump the continuous phase into the droplet or particle source region via additional channels. Such additional channels may be located on one or both sides of the primary drop, or above or below the plane of the primary drop.

In general, a component of the device (e.g., a channel) may have certain geometric features that at least partially determine the size of a droplet. For example, any of the channels described herein have a depth, a height h ₀, and a width w. The droplet or particle source region may have an expansion angle α. The droplet size may decrease with increasing spread angle. The resulting droplet radius R _d can be predicted from the following relationship of the aforementioned geometric parameters h ₀, w, and α:

As a non-limiting example, for a channel with w=21 μm, h=21 μm, and α=3°, the predicted droplet size is 121 μm. In another example, for a channel with w=25 μm, h=25 μm, and α=5°, the predicted droplet size is 123 μm. In yet another example, for a channel with w=28 μm, h=28 μm, and α=7°, the predicted droplet size is 124 μm. In some cases, the spread angle may be in the range of about 0.5 ° to about 4 °, about 0.1 ° to about 10 °, or about 0 ° to about 90 °. For example, the spread angle may be at least about 0.01°、0.1°、0.2°、0.3°、0.4°、0.5°、0.6°、0.7°、0.8°、0.9°、1°、2°、3°、4°、5°、6°、7°、8°、9°、10°、15°、20°、25°、30°、35°、40°、45°、50°、55°、60°、65°、70°、75°、80°、85° or higher. In some cases, the spread angle may be up to about 89°、88°、87°、86°、85°、84°、83°、82°、81°、80°、75°、70°、65°、60°、55°、50°、45°、40°、35°、30°、25°、20°、15°、10°、9°、8°、7°、6°、5°、4°、3°、2°、1°、0.1°、0.01° or less.

The depth and width of the first channels may be the same, or one may be greater than the other, e.g., the width is greater than the depth, or the first depth is greater than the width. In some embodiments, the depth and/or width is between about 0.1 μm and 1000 μm. In some embodiments, the depth and/or width of the first channel is 1 μm to 750 μm, 1 μm to 500 μm, 1 μm to 250 μm, 1 μm to 100 μm, 1 μm to 50 μm, or 3 μm to 40 μm. In some cases, when the width and length are different, the ratio of width to depth is, for example, 0.1 to 10, e.g., 0.5 to 2 or greater than 3, such as 3 to 10, 3 to 7, or 3 to 5. The width and depth of the first channel may or may not be constant over its length. In particular, the width may increase or decrease near the distal end. Generally, the channels may have any suitable cross-section, such as rectangular, triangular, or circular, or a combination thereof. In particular embodiments, the channel may include a groove along the bottom surface. The width or depth of the channels may also be increased or decreased, for example in discrete portions, to alter the flow rate of the liquid or particles or the arrangement of the particles.

The device of the present invention may further comprise an additional channel intersecting the first channel between the proximal and distal ends thereof, e.g., one or more second channels having a second depth, a second width, a second proximal end and a second distal end, the second channels intersecting the first channel between the first proximal and first distal ends. In some embodiments, the second channel may include a fluorocarbon surface coating as described below.

Each of the first proximal end and the second proximal end is in fluid communication with or configured to be in fluid communication with a liquid source, e.g., fluidly connected to a liquid source, e.g., a reservoir integral with or coupled to the device (e.g., via a conduit). For example, the second channel may include a second reservoir in fluid communication with the second proximal end. The inclusion of one or more channel intersections allows for separation of liquid from or introduction of liquid into the first channel, e.g., liquid that combines with or does not combine with liquid in the first channel, e.g., to form a sheath flow. The channel can intersect the first channel at any suitable angle, for example between 5 ° and 135 °, such as between 75 ° and 115 °, or between 85 ° and 95 °, relative to the centerline of the first channel. Additional channels may similarly be present to allow for the introduction of additional liquids or additional flows of the same liquid. The plurality of channels may intersect the first channel on the same side or on different sides of the first channel. When multiple channels intersect on different sides, the channels may intersect along the length of the first channel to allow liquid to be introduced at the same point. Alternatively, the channels may intersect at different points along the length of the first channel. In some cases, a channel configured to direct a liquid containing a plurality of particles may contain one or more grooves in one or more surfaces of the channel for directing the plurality of particles toward a drop forming fluid connection. For example, such channeling may increase the individual occupancy of the generated droplets or particles. These additional channels may have any of the structural features discussed above for the first channel.

The device may comprise a plurality of first channels, for example to increase the rate of droplet or particle formation. Generally, by increasing the number of droplet or particle source regions in the device, the flux can be significantly increased. For example, assuming that the liquid flow rates are substantially the same, a device having five droplet or particle source regions may produce five times as many droplets or particles as a device having only one droplet or particle source region. The device may have as many drop or particle source regions as the size of the liquid source (e.g., reservoir) actually allows. For example, the device may have at least about 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, or more droplet or particle source regions. The inclusion of multiple drop or particle source regions may be desirable to include channels that intersect but do not intersect, e.g., the flow paths are in different planes. The plurality of first channels may be in fluid communication with, e.g. fluidly connected to, the individual source reservoirs and/or the individual droplet or particle source regions. In other embodiments, two or more first channels are in fluid communication with the same fluid source, e.g., are fluidly connected to the same fluid source, e.g., wherein a plurality of first channels branch from a single upstream channel. The droplet or particle source region can include a plurality of inlets in fluid communication with the first proximal end, and a plurality of outlets (e.g., a plurality of outlets in fluid communication with the collection region) (e.g., fluidly connected to the first proximal end and in fluid communication with the plurality of outlets). The number of inlets and the number of outlets in the droplet or particle source region may be the same (e.g., there may be 3 to 10 inlets and/or 3 to 10 outlets). Alternatively or in addition, the flux of droplet or particle formation may be increased by increasing the flow rate of the first liquid. In some cases, the throughput of droplet or particle formation may be increased by providing a plurality of single droplet or particle forming devices, such as devices having a first channel and a droplet or particle source region, such as parallel droplet or particle forming devices, in a single device.

The width of the shelf region may be 0.1 μm to 1000 μm. In specific embodiments, the width of the shelf is from 1 μm to 750 μm, from 10 μm to 500 μm, from 10 μm to 250 μm, or from 10 μm to 150 μm. The width of the shelf region may be constant along its length, for example forming a rectangular shape. Alternatively, the width of the shelf region may increase along its length away from the distal end of the first channel. Such an increase may be linear, non-linear, or a combination thereof. In certain embodiments, the shelf is widened relative to the width of the distal end of the first channel by 5% to 10,000%, for example at least 300% (e.g., 10% to 500%, 100% to 750%, 300% to 1000%, or 500% to 1000%). The depth of the shelf may be the same as or different from the first channel. For example, the bottom of the first channel at its distal end and the bottom of the shelf region may be coplanar. Alternatively, there may be a step or ramp where the distal end meets the shelf region. The distal end may also have a depth greater than the shelf region such that the first channel forms a recess in the shelf region. The depth of the shelf may be 0.1 μm to 1000 μm, for example 1 μm to 750 μm, 1 μm to 500 μm, 1 μm to 250 μm, 1 μm to 100 μm, 1 μm to 50 μm, or 3 μm to 40 μm. In some embodiments, the depth is substantially constant along the length of the shelf. Alternatively, the depth of the shelf is inclined, e.g. downwardly or upwardly, from the distal end of the liquid channel to the stepped region. For example, the final depth of the inclined shelf may be 5% to 1000%, such as 10% to 750%, 10% to 500%, 50% to 500%, 60% to 250%, 70% to 200%, or 100% to 150% greater than the shortest depth. The total length of the shelf region may be at least about 0.1 μm to about 1000 μm, such as 0.1 μm to 750 μm, 0.1 μm to 500 μm, 0.1 μm to 250 μm, 0.1 μm to 150 μm, 1 μm to 150 μm, 10 μm to 150 μm, 50 μm to 150 μm, 100 μm to 150 μm, 10 μm to 80 μm, or 10 μm to 50 μm. In certain embodiments, the side walls of the shelf region (i.e., those defining the width) may not be parallel to one another. In other embodiments, the wall of the shelf region may narrow from the distal end of the first channel toward the stepped region. For example, the width of the shelf region near the distal end of the first channel may be large enough to support droplet formation. In other embodiments, the shelf region is not substantially rectangular, e.g., is not rectangular or is not rectangular with rounded corners or chamfers.

The stepped region includes a spatial displacement (e.g., depth). Typically, the displacement occurs at an angle of about 90 ° (e.g., between 85 ° and 95 °). Other angles are also possible, such as 10 ° to 90 °, such as 20 ° to 90 °, 45 ° to 90 °, or 70 ° to 90 °. The spatial displacement of the stepped region may be of any suitable size to be accommodated on the device, as the final extent of displacement does not affect the performance of the device. Preferably, the displacement is several times the diameter of the droplet being formed. In certain embodiments, the displacement is from about 1 μm to about 10cm, such as at least 10 μm, at least 40 μm, at least 100 μm, or at least 500 μm, such as 40 μm to 600 μm. In some example embodiments, the displacement is at least 40 μm, at least 45 μm, at least 50 μm, at least 55 μm, at least 60 μm, at least 65 μm, at least 70 μm, at least 75 μm, at least 80 μm, at least 85 μm, at least 90 μm, at least 95 μm, at least 100 μm, at least 110 μm, at least 120 μm, at least 130 μm, at least 140 μm, at least 150 μm, at least 160 μm, at least 170 μm, at least 180 μm, at least 190 μm, at least 200 μm, at least 220 μm, at least 240 μm, at least 260 μm, at least 280 μm, at least 300 μm, at least 320 μm, at least 340 μm, at least 360 μm, at least 380 μm, at least 400 μm, at least 420 μm, at least 440 μm, at least 460 μm, at least 480 μm, at least 500 μm, at least 540 μm, at least 560 μm, at least 580 μm, or at least 600 μm. In some cases, the depth of the stepped region is substantially constant. Alternatively, the depth of the stepped region may increase away from the shelf region, for example, to allow the sinking or floating droplets to roll off of the spatial displacement as they form. The stepped region may also increase in depth in two dimensions relative to the shelf region, e.g., both above and below the plane of the shelf region. The stepped region may have an inlet and/or an outlet for adding a continuous phase, flowing a continuous phase, or removing a continuous phase and/or droplets.

Although the dimensions of the device may be described as width or depth, the channels, shelf regions, and step regions may be disposed in any plane. For example, the width of the shelf may be in the x-y plane, the x-z plane, the y-z plane, or any plane therebetween. Further, the drop source region (e.g., including the shelf region) may be laterally spaced in the x-y plane relative to the first channel, or located above or below the first channel. Similarly, the droplet source region (e.g., including the step region) may be laterally spaced in the x-y plane, e.g., relative to the shelf region, or above or below the shelf region. The spatial displacement in the step region may be oriented in any plane suitable to allow the nascent droplet to form a spherical shape. The fluidic components may also be in different planes as long as connectivity and other dimensional requirements are met.

In some cases, the device of the invention comprises a collection area, for example a volume for collecting the formed droplets or particles. The collection area may be a reservoir containing the continuous phase or may be any other suitable structure on or in the device, such as a channel, shelf or cavity. For reservoirs or other elements used in collection, the walls may be smooth and not include orthogonal elements that would impede droplet or particle movement. For example, the walls may not include any features that at least partially protrude or recess from the surface. However, it should be understood that such elements may have an upper or lower limit. The formed droplets or particles may move out of the path of the next droplet or particle being formed under the force of gravity (up or down depending on the relative densities of the droplet or particle and the continuous phase). Alternatively or in addition, the formed droplets or particles may be moved out of the way of the next droplet or particle being formed by an external force (e.g., gentle agitation, flowing continuous phase, or vibration) applied to the liquid in the collection region. Similarly, there may be a reservoir for liquid flowing in an additional channel, such as a channel intersecting the first channel. A single reservoir may also be connected to multiple channels in the device, for example, when the same liquid is to be introduced at two or more different locations in the device. A waste reservoir or overflow reservoir may also be included to collect waste or overflow as droplets or particles are formed. Alternatively, the device may be configured to cooperate with a liquid source, which may be an external reservoir, such as a vial, tube or bag. Similarly, the device may be configured to mate with a separate component that houses the reservoir. The reservoir may be of any suitable size, for example, to hold 10 μl to 500mL, for example 10 μl to 300mL, 25 μl to 10mL, 100 μl to 1mL, 40 μl to 300 μl, 1mL to 10mL, or 10mL to 50mL. When there are multiple reservoirs, each reservoir may be the same or different sizes.

In addition to the components discussed above, the apparatus of the present invention may include additional components. In some cases, the microfluidic systems described herein may include one or more liquid flow cells to direct the flow of one or more liquids (such as an aqueous liquid and/or a second liquid that is immiscible with the aqueous liquid). In some cases, the liquid flow unit may include a compressor to provide positive pressure at an upstream location to direct liquid flow from the upstream location to a downstream location. In some cases, the liquid flow unit may include a pump to provide a negative pressure at the downstream location to direct the flow of liquid from the upstream location to the downstream location. In some cases, the liquid flow unit may include both a compressor and a pump, each in a different location. In some cases, the liquid flow unit may comprise different devices located at different positions. The liquid flow unit may comprise an actuator. In some cases, where the second liquid is substantially stationary, the reservoir may maintain a constant pressure field at or near each droplet or particle source region. The device may also include various valves to control the flow of liquid along the channel, or to allow liquid or droplets or particles to be introduced or removed from the device. Suitable valves are known in the art. Valves that may be used in the devices of the present invention include diaphragm valves, solenoid valves, pinch valves, or combinations thereof. The valve can be controlled manually, electrically, magnetically, hydraulically, pneumatically, or by a combination of these. The device may also include an integral liquid pump or may be connectable to a pump to allow pumping into the first channel and any other channels requiring flow. Examples of pressure pumps include syringes, peristaltic pumps, diaphragm pumps, and vacuum sources. Other pumps may employ centrifugal or electrodynamic forces. Alternatively, liquid movement may be controlled by gravity, capillary action, or surface treatment. Multiple pumps and mechanisms for forcing the movement of the liquid may be employed in a single device. The device may also include one or more vents to allow pressure equalization, and one or more filters to remove particulates or other unwanted components from the liquid. The device may also include one or more inlets and/or outlets, for example, for introducing liquid and/or removing droplets or particles. Such additional components may be initiated or monitored by one or more controllers or computers operatively coupled to the apparatus (e.g., by being integrated with the apparatus, physically connected (mechanically or electrically), or by being wired or wirelessly connected).

Alternatively or in addition to controlling droplet or particle formation via microfluidic channel geometry, one or more piezoelectric elements may be used to control droplet or particle formation. The piezoelectric element may be positioned inside the channel (i.e., in contact with the fluid in the channel), outside the channel (i.e., isolated from the fluid), or a combination of both. In some cases, the piezoelectric element may be located at the outlet of the channel, e.g., where the channel connects to a reservoir or other channel to serve as a droplet or particle generation point. For example, the piezoelectric element may be integrated with the channel or coupled or otherwise secured to the channel. Examples of fasteners include, but are not limited to, complementary threads, form-fit paired fasteners, hooks and loops, latches, threads, screws, staples, clips, clamps, tines, rings, brads, rubber bands, rivets, grommets, pins, ties, snaps, adhesives (e.g., glue), tape, vacuum, seals, magnets, welding, or combinations thereof. In some cases, piezoelectric material may be deposited on the chip. In some cases, the piezoelectric element may be built into the channel. Alternatively or in addition, the piezoelectric element may be connected to the reservoir or channel, or may be a component of the reservoir or channel, such as a wall. In some cases, the piezoelectric element may further include an aperture therethrough such that liquid may pass through upon actuation of the piezoelectric element, or the device may include an aperture operatively coupled to the piezoelectric element.

The piezoelectric element may have various shapes and sizes. The piezoelectric element may have a shape or cross-section that is circular, triangular, square, rectangular, or partial, or a combination of these shapes. The piezoelectric element may have a thickness of from about 100 micrometers (fm) to about 100 millimeters (mm). The piezoelectric element may have a dimension (e.g., cross-section) of at least about 1 mm. For example, the piezoelectric element may be formed of lead zirconate titanate, zinc oxide, barium titanate, potassium niobate, sodium tungstate, ba2NaNb5O5, and Pb2KNb5O 15. For example, the piezoelectric element may be a piezoelectric crystal. The piezoelectric element may contract when a voltage is applied and return to its original state when no voltage is applied. Alternatively, the piezoelectric element may expand when a voltage is applied, and return to its original state when no voltage is applied. Alternatively or in addition, applying a voltage to the piezoelectric element may cause mechanical stress, vibration, bending, deformation, compression, decompression, expansion, and/or a combination of these movements in its structure, and vice versa (e.g., applying some form of mechanical stress or pressure on the piezoelectric element may produce a voltage). In some cases, the piezoelectric element may include a composite of both piezoelectric material and non-piezoelectric material. An interdigital transducer (IDT) can also be patterned on top of a piezoelectric element to generate an acoustic wave of a particular frequency, depending on the size and distance of the finger on the IDT.

In some cases, the piezoelectric element may be in a first state, such as an equilibrium state, when no charge is applied. When an electrical charge is applied to the piezoelectric element, the piezoelectric element may bend back, pulling a portion of the first channel outward and drawing more of the first fluid (such as from a reservoir of the first fluid) into the first channel via the negative pressure. When the charge changes, the piezoelectric element may bend in the other direction (e.g., inwardly toward the channel contents), pushing a portion of the first channel inwardly, and pushing (e.g., at least partially via displacement) a volume of the first fluid, thereby generating droplets of the first fluid in the second fluid. After propelling the droplet, the piezoelectric element may return to the first state. The cycle may be repeated to generate more droplets or particles. In some cases, each cycle may generate a plurality of droplets or particles (e.g., a volume of the first fluid that is propelled breaks as it enters the second fluid to form a plurality of discrete droplets). A plurality of droplets or particles may be collected in the second channel for continued transport to a different location (e.g., reservoir), direct harvesting, and/or storage.

While the above non-limiting examples describe the piezoelectric element bending in response to the application of an electrical charge, upon application of an electrical charge the piezoelectric element may also undergo or undergo vibration, bending, deformation, compression, decompression, expansion, other mechanical stresses, and/or combinations of these movements, which movements may be transferred to the first channel.

In some cases, the channel may include multiple piezoelectric elements that work independently or cooperatively to achieve a desired formation (e.g., advancement) of a droplet or particle. For example, the first channel of the device may be coupled to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 piezoelectric elements. In one example, a separate piezoelectric element may be operatively coupled to (or be part of) each side wall of the channel. In another example, the plurality of piezoelectric elements may be positioned adjacent to one another along an axis parallel to the direction of flow in the first channel. Alternatively or in addition, a plurality of piezoelectric elements may surround the first channel. For example, a plurality of piezoelectric elements may each be in electrical communication with the same controller or one or more different controllers. The generation flux of droplets or particles may be increased by increasing the point of generation of droplets or particles, such as increasing the number of connections between the first fluid channel and the second fluid channel. For example, each first fluid channel may comprise a piezoelectric element for controlling the generation of droplets or particles at each generation point. The piezoelectric element may be actuated to promote the formation of droplets or particles and/or the flow of droplets or particles.

The frequency of the application of charge to the piezoelectric element can be adjusted to control the rate of droplet or particle generation. For example, the frequency of droplet or particle generation may increase as the frequency of alternating charge increases. In addition, the material of the piezoelectric element, the number of piezoelectric elements in the channel, the location of the piezoelectric element, the strength of the applied charge, the hydrodynamic forces of the respective fluids, and other factors may be adjusted to control the generation of droplets or particles and/or the size of the generated droplets or particles. For example, without wishing to be bound by a particular theory, if the applied charge strength increases, the mechanical stress experienced by the piezoelectric element may increase, which may increase the effect on the deformation of the first channel structure, thereby increasing the volume of the first fluid being propelled, causing an increase in droplet or particle size.

In one non-limiting example, a first channel may carry a first fluid (e.g., aqueous) and a second channel may carry a second liquid (e.g., oil) that is immiscible with the first fluid. The two fluids may communicate at a junction. In some cases, the first fluid in the first channel may include suspended particles. These particles may be a support, biological particle, cell bead, or any combination thereof (e.g., a combination of a support and a cell, or a combination of a support and a cell bead, etc.). The discrete droplets generated may contain particles, such as when one or more particles are suspended in a volume of a first fluid that is advanced into a second fluid. Alternatively, the discrete droplets generated may comprise more than one particle. Alternatively, the discrete droplets generated may not contain any particles. For example, in some cases, the discrete droplets generated may comprise one or more biological particles, wherein the first fluid in the first channel comprises a plurality of biological particles.

Alternatively or in addition, one or more piezoelectric elements can be used to acoustically control drop formation.

The piezoelectric element may be operably coupled to a first end of a buffer substrate (e.g., glass). The second end of the buffer substrate, opposite the first end, may include an acoustic lens. In some cases, the acoustic lens may have a spherical (e.g., hemispherical) cavity. In other cases, the acoustic lens may be a different shape and/or include one or more other objects for focusing the acoustic waves. The second end of the buffer substrate and/or the acoustic lens may be in contact with the first fluid in the first channel. Alternatively, the piezoelectric element may be operatively coupled to a portion (e.g., a wall) of the first channel without an intermediate substrate. The piezoelectric element may be in electrical communication with the controller. The piezoelectric element may be responsive to (e.g., excited by) a voltage driven at an RF frequency. In some embodiments, the piezoelectric element may be made of zinc oxide (ZnO).

The frequency of the voltage applied to the piezoelectric element may be driven from about 5 megahertz (MHz) to about 300MHz, for example, about 5MHz, about 6MHz, about 7MHz, about 9MHz, about 10MHz, about 20MHz, about 30MHz, about 40MHz, about 50MHz, about 60MHz, about 70MHz, about 80MHz, about 90MHz, about 100MHz, about 110MHz, about 120MHz, about 130MHz, about 140MHz, about 150MHz, about 160MHz, about 170MHz, about 180MHz, about 190MHz, about 200MHz, about 210MHz, about 220MHz, about 230MHz, about 240MHz, about 250MHz, about 260MHz, about 270MHz, about 280MHz, about 290MHz, or about 300MHz. Alternatively, the RF energy may have a frequency range of less than about 5MHz or greater than about 300MHz. It will be appreciated that the necessary voltage and/or RF frequency at which the voltage is driven may vary with the characteristics (e.g., efficiency) of the piezoelectric element.

The first fluid and the second fluid may remain separated at or near the connection via an immiscible barrier prior to application of the voltage to the piezoelectric element. The voltage, when applied to the piezoelectric element, can generate an acoustic wave (e.g., a sound wave) propagating in the buffer substrate. The buffer substrate (such as glass) may be any material capable of transmitting sound waves. The acoustic lens of the buffer substrate may focus the acoustic wave at an immiscible interface between the two immiscible fluids. The acoustic lens may be positioned such that the interface is located at the focal plane of the acoustic wave convergence. Upon impact of the acoustic pulse on the barrier, the pressure of the acoustic wave may cause a volume of the first fluid to be propelled into the second fluid, thereby generating a volume of droplets or particles of the first fluid in the second fluid. In some cases, multiple droplets or particles may be generated per actuation (e.g., a volume of a first fluid being actuated breaks as it enters a second fluid to form multiple discrete droplets or particles). After ejection of the droplet or particle, the immiscible interface may return to its original state. The application of voltage to the piezoelectric element may then be repeated to then generate more droplets or particles. A plurality of droplets or particles may be collected in the second channel for continued transport to a different location (e.g., reservoir), direct harvesting, and/or storage. Advantageously, the droplets or particles produced may have a substantially uniform size, velocity (upon ejection) and/or directionality.

In some cases, the device may include a plurality of piezoelectric elements that work independently or cooperatively to achieve a desired formation (e.g., advancement) of a droplet or particle. For example, the first channel may be coupled to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 piezoelectric elements. In one example, the plurality of piezoelectric elements may be positioned adjacent to one another along an axis parallel to the first channel. Alternatively or in addition, a plurality of piezoelectric elements may surround the first channel. In some cases, the plurality of piezoelectric elements may each be in electrical communication with the same controller or one or more different controllers. The plurality of piezoelectric elements may each emit sound waves from the same buffer substrate or one or more different buffer substrates. In some cases, a single buffer substrate may include multiple acoustic lenses located at different locations.

In some cases, the first channel may be in communication with the third channel. The third channel may carry the first fluid (such as from a reservoir of the first fluid) to the first channel. The third channel may comprise one or more piezoelectric elements, for example, as described herein in the device. As described elsewhere herein, the third channel may carry a first fluid having one or more particles (e.g., a support, biological particles, etc.) and/or one or more reagents suspended in the fluid. Alternatively or in addition, the device may comprise one or more other channels in communication with the first channel and/or the second channel.

The number and duration of the voltage pulses applied to the piezoelectric element can be adjusted to control the rate of droplet or particle generation. For example, the frequency of droplet or particle generation may increase as the number of voltage pulses increases. In addition, the material and size of the piezoelectric elements, the material and size of the buffer substrate, the material, size, and shape of the acoustic lenses, the number of piezoelectric elements, the number of buffer substrates, the number of acoustic lenses, the respective positions of the one or more piezoelectric elements, the respective positions of the one or more buffer substrates, the respective positions of the one or more acoustic lenses, the dimensions (e.g., length, width, height, deployment angle) of the respective channels, the voltage levels applied to the piezoelectric elements, the hydrodynamic forces of the respective fluids, and other factors may be adjusted to control the rate of droplet or particle generation and/or the size of the droplets or particles generated.

The discrete droplets generated may contain particles, such as when one or more supports are suspended in a volume of a first fluid that is advanced into a second fluid. Alternatively, the discrete droplets generated may comprise more than one particle. Alternatively, the discrete droplets generated may not contain any particles. For example, in some cases, the discrete droplets generated may comprise one or more biological particles, wherein the first fluid in the first channel further comprises a suspension of a plurality of biological particles.

In some cases, droplets or particles formed using piezoelectric elements may be collected in a collection region disposed below the droplet or particle generation point. The collection region may be configured to house a fluid source to keep the formed droplets or particles isolated from each other. The collection area used after the formation of the droplets or particles assisted by the piezoelectric or acoustic elements may contain a continuous circulation of oil, for example, using a paddle mixer, a conveyor system or a magnetic stirring rod. Alternatively, the collection region may contain one or more chemically reactive agents which may provide a coating on the droplets or particles to ensure isolation, for example polymerisation reactions such as thermally or photo-initiated polymerisation reactions.

In addition to the components discussed above, the apparatus of the present invention may include additional components. For example, the channel may include a filter to prevent debris from entering the device. In some cases, the microfluidic systems described herein may include one or more liquid flow cells to direct the flow of one or more liquids (such as an aqueous liquid and/or a second liquid that is immiscible with the aqueous liquid). In some cases, the liquid flow unit may include a compressor to provide positive pressure at an upstream location to direct liquid flow from the upstream location to a downstream location. In some cases, the liquid flow unit may include a pump to provide a negative pressure at the downstream location to direct the flow of liquid from the upstream location to the downstream location. In some cases, the liquid flow unit may include both a compressor and a pump, each in a different location. In some cases, the liquid flow unit may comprise different devices located at different positions. The liquid flow cell 27 may comprise an actuator. In some cases, where the second liquid is substantially stationary, the reservoir may maintain a constant pressure field at or near each drop formation region. The device may also include various valves to control the flow of liquid along the channel, or to allow liquid or droplets to be introduced or removed from the device. Suitable valves are known in the art. Valves that may be used in the devices of the present invention include diaphragm valves, solenoid valves, pinch valves, or combinations thereof. The valve can be controlled manually, electrically, magnetically, hydraulically, pneumatically, or by a combination of these. The device may also include an integral liquid pump or may be connectable to a pump to allow pumping into the first channel and any other channels requiring flow. Examples of pressure pumps include syringes, peristaltic pumps, diaphragm pumps, and vacuum sources. Other pumps may employ centrifugal or electrodynamic forces. Alternatively, liquid movement may be controlled by gravity, capillary action, or surface treatment. Multiple pumps and mechanisms for forcing the movement of the liquid may be employed in a single device. The device may also include one or more vents to allow pressure equalization, and one or more filters to remove particulates or other unwanted components from the liquid. The device may also include one or more inlets and/or outlets, for example, for introducing liquid and/or removing liquid droplets. Such additional components may be initiated or monitored by one or more controllers or computers operatively coupled to the apparatus (e.g., by being integrated with the apparatus, physically connected (mechanically or electrically), or by being wired or wirelessly connected).

Surface particles

The surface of the device may include a material, coating, or surface texture that determines the physical characteristics of the device. In particular, the flow of liquid through the device of the present invention may be controlled by the surface characteristics of the device (e.g., wettability of the liquid contacting surface). In some cases, a portion of the device (e.g., a channel or filter) may have a surface that is wetted to facilitate liquid flow (e.g., in the channel) or assist the first liquid in forming droplets (e.g., if formed) in the second liquid (e.g., in the channel).

Wettability is the ability of a liquid to remain in contact with a solid surface, which can be measured as a function of water contact angle. The water contact angle of a material may be measured by any suitable method known in the art, such as static hydrostatic, pendant drop, dynamic hydrostatic, dynamic Wilhelmy, filament meniscus and Washburn equation capillary rise. The wettability of each surface may be adapted to produce droplets of the first liquid in the second liquid.

For example, the portion of the device carrying the aqueous phase (e.g., the channel) may have a surface material or coating that is hydrophilic or more hydrophilic than another portion of the device, e.g., comprising a material or coating having a water contact angle less than or equal to about 90 °, and/or the exterior of the device surrounding the channel may have a surface material or coating that is hydrophobic or more hydrophobic than the channel, e.g., comprising a material or coating having a water contact angle greater than 70 ° (e.g., greater than 90 °, greater than 95 °, greater than 100 ° (e.g., 95 ° to 120 ° or 100 ° to 10 °). In certain embodiments, portions of the device may include a material or surface coating that reduces or prevents wetting by the aqueous phase. The device may be designed with a single type of material or coating over the entire device. For example, the device may be designed with a single type of material or coating having the structure of formula I as described above. Alternatively, the device may have separate regions of different materials or coatings. Surface texture may also be used to control fluid flow.

The device surface characteristics may be characteristics of a natural surface (i.e., surface characteristics of a base material used to make the device) or characteristics of a surface treatment. Non-limiting examples of surface treatments include, for example, surface coatings and surface textures. In some embodiments, the surface coating may have a total fluorine weight percent of about 57% to 70%. As a non-limiting example, the surface may include a fluorocarbon surface coating having VI moieties

Wherein each Y is independently-O (CR ⁹ ₂)_p -and p is 1,2, 3, 4, 5, or 6, and each R ⁹ is independently F or C ₁-C₆ perfluoroalkyl, each n is independently 1,2, 3, 4, 5, 6, or 7, Each Z is independently-OR ¹⁰ -, wherein R ¹⁰ is C ₁-C₆ perfluoroalkylene, m is 1,2, 3, 4,5, OR 6, each of R ⁷ and R ⁸ is independently F OR C ₁-C₆ perfluoroalkyl, wherein at least one R ⁹ is C ₁-C₆ perfluoroalkyl, or R ¹⁰ is C ₃-C₆ perfluoroalkylene. In some embodiments, m is 1 to 5, e.g., 3. In certain embodiments, n+n is 4 to 13, e.g., 7 to 13, such as 4 (e.g., 1+3, 2+2, or 3+1), 5 (e.g., 1+4, 2+3, 3+2, or 4+1), 6 (e.g., 1+5, 2+4, 3+3, 4+2, or 5+1), 7 (e.g., 1+6, 2+5, 3+4, 4+3, 5+2, or 6+1), 8 (e.g., 1+7, 2+6, 3+5, 4+4, 5+3, 6+2, or 7+1), 9 (e.g., 2+7, 3+6, 4+5, 5+4, 6+3, or 7+2), 10 (e.g., 4+5, 5+5 ], 6+4 or 7+3), 11 (e.g., 4+7, 5+6, 6+5 or 7+4), 12 (e.g., 5+7, 6+6 or 7+5), or 13 (e.g., 6+7 or 7+6). 5, or 6, or. In some embodiments, the fluorocarbon surface coating has VIIa or VIIb moieties:

Wherein each n is independently 1,2,3,4, 5,6, or 7, and m is 1,2,3,4, 5, or 6. In some embodiments, m is 1 to 5, e.g., 3. In certain embodiments, n+n is 4 to 13, for example 7, such as 4 (e.g., 1+3, 2+2, or 3+1), 5 (e.g., 1+4, 2+3, 3+2, or 4+1), 6 (e.g., 1+5, 2+4, 3+3, 4+2, or 5+1), 7 (e.g., 1+6, 2+5, 3+4, 4+3, 5+2, or 6+1), 8 (e.g., 1+7, 2+6, 3+5, 4+4, 5+3, 6+2, or 7+1), 9 (e.g., 2+7, 3+6, 4+5, 5+4, or 7+2), 10 (e.g., 4+5, 5, 6+4, or 7+3), 11 (e.g., 4+7, 5+6, 6+5, or 7+4), 12 (e.g., 5+7, 6+ 7+5), or 13 (e.g., 6+7, or 7). 5, or 6, or, for example, when the fluorocarbon surface coating has VIIIa or VIIIb moieties, n+n is 7:

in one approach, the device surface characteristics may be attributed to one or more surface coatings present in the device portion. In some embodiments, other portions of the device include a hydrophobic coating that includes a fluoropolymer (e.g., Glass treatments), silanes, siloxanes, silicones, or other coatings known in the art. Other coatings include those deposited from a precursor vapor phase, such as: diundecyl-1, 2-tetrahydrododecyl dimethyl tris (dimethylaminosilane), eicosyl-1, 2-tetrahydrododecyl trichlorosilane (C12) heptadecafluoro-1, 2-tetrahydrodecyl trichlorosilane (C10), nonafluoro-1, 2-tetrahydrohexyl tris (dimethylamino) silane, 3,3,3,4,4,5,5,6,6-nonafluorohexyl trichlorosilane tridecafluoro-1, 2-tetrahydrooctyl trichlorosilane (C8), bis (tridecafluoro-1, 2-tetrahydrooctyl) dimethylsilyloxymethyl chlorosilane, nonafluorohexyltriethoxysilane (C6), dodecyltrichlorosilane (DTS), dimethyldichlorosilane (DDMS) or 10-undecenyltrichlorosilane (V11), pentafluorophenylpropyl trichlorosilane (C5). In some embodiments, the hydrophilic coating comprises polymers such as polysaccharides, polyethylene glycols, polyamines, and polycarboxylic acids. Hydrophilic surfaces can also be created by oxygen plasma treatment of certain materials.

The coated surface may be formed by depositing a metal oxide onto the surface of the device. Exemplary metal oxides that may be used to coat the surface include, but are not limited to, al ₂O₃、TiO₂、SiO₂, or combinations thereof. Other metal oxides that can be used for surface modification are known in the art. The metal oxide may be deposited onto the surface by standard deposition techniques including, but not limited to, atomic Layer Deposition (ALD), physical Vapor Deposition (PVD) (e.g., sputtering), chemical Vapor Deposition (CVD), or laser deposition. Other deposition techniques for coating a surface (e.g., liquid-based deposition) are known in the art. For example, an atomic layer of Al ₂O₃ may be deposited on a surface by contacting it with Trimethylaluminum (TMA) and water. In some embodiments, the fluorocarbon surface coating can be the reaction product of any of the compounds described herein with hydroxyl groups. For example, and without limitation, one or more of the compounds described herein may interact with hydroxyl groups on the surface of the device and/or a portion of the device, wherein plasma activation and/or plasma functionalization techniques may be performed to form a fluorocarbon surface coating.

In another approach, the device surface characteristics may be attributable to surface texture. For example, the surface may have a nanotexture, e.g., the surface has nanosurface features such as pyramids or pillars that alter the wettability of the surface. The nanotextured surface may be hydrophilic, hydrophobic, or superhydrophobic, e.g., having a water contact angle greater than 150 °. Exemplary superhydrophobic materials include manganese oxide polystyrene (MnO ₂/PS) nanocomposites, zinc oxide polystyrene (ZnO/PS) nanocomposites, precipitated calcium carbonate, carbon nanotube structures, and silica nanocomposites. The superhydrophobic coating can also include a low surface energy material (e.g., an inherently hydrophobic material) and a surface roughness (e.g., a photolithographic technique that etches the material by patterning openings in a mask using laser ablation techniques, plasma etching techniques). Examples of low surface energy materials include fluorocarbon materials such as Polytetrafluoroethylene (PTFE), fluorinated Ethylene Propylene (FEP), ethylene Tetrafluoroethylene (ETFE), ethylene Chlorotrifluoroethylene (ECTFE), perfluoroalkoxyalkane (PFA), poly (chlorotrifluoroethylene) (CTFE), perfluoroalkoxyalkane (PFA), and poly (vinylidene fluoride) (PVDF). Other superhydrophobic surfaces are known in the art.

In some cases, the water contact angle of the hydrophilic or more hydrophilic material or coating is less than or equal to about 90 °, e.g., less than 80 °, 70 °, 60 °, 50 °, 40 °, 30 °, 20 °, or 10 °, e.g., 90 °, 85 °, 80 °, 75 °, 70 °, 65 °, 60 °, 55 °, 50 °, 45 °, 40 °, 35 °, 30 °,25 °, 20 °,15 °,10 °,9 °,8 °,7 °,6 °,5 °,4 °,3 °,2 °,1 °, or 0 °. In some cases, the water contact angle of the hydrophobic or more hydrophobic material or coating is at least 70 °, e.g., at least 80 °, at least 85 °, at least 90 °, at least 95 °, or at least 100 ° (e.g., about 100 °, 101 °, 102 °, 103 °, 104 °, 105 °, 106 °, 107 °, 108 °, 109 °, 110 °, 115 °, 120 °, 125 °, 130 °, 135 °, 140 °, 145 °, or about 150 °).

The difference in water contact angle between the hydrophilic or more hydrophilic material or coating and the hydrophobic or more hydrophobic material or coating may be 5 ° to 100 °, for example 5 ° to 80 °,5 ° to 60 °,5 ° to 50 °,5 ° to 40 °,5 ° to 30 °,5 ° to 20 °,10 ° to 75 °, 15 ° to 70 °,20 ° to 65 °, 25 ° to 60 °,30 ° to 50 °,35 ° to 45 °, for example 5 °, 6 °, 7 °, 8 °,9 °,10 °, 15 °,20 °, 25 °,30 °,35 °,40 °, 45 °, 50 °, 55 °, 60 °, 65 °, 70 °, 75 °, 80 °, 85 °, 90 °, 95 °, or 100 °.

In some embodiments, the fluorocarbon surface coating produces a coated surface that is sufficiently hydrophobic to produce a water contact angle of at least 105 ° during droplet generation, e.g., to reduce asymmetric droplet generation. For example, a fluorocarbon surface coating may produce the following hydrophobic coatings: the hydrophobic coating produces a water contact angle of about 105 ° to about 150 ° (e.g., 105 ° to 120 °, 120 ° to 130 °, 130 ° to 140 °, or 140 ° to 150 °). The water contact angle can vary based on the concentration of the fluorocarbon surface coating applied to the device or a portion of the device (e.g., surface, channel, reservoir, etc.). For example, when the concentration of the fluorocarbon coating agent is about 0.2 wt%, the water contact angle may be about 105 ° to about 113 °. As another example, when the concentration of the fluorocarbon surface coating agent is about 0.5 wt%, the water contact angle may be about 109 ° to about 114 °.

The discussion above centers on water contact angle. It should be understood that the liquid employed in the apparatus and method of the present invention may not be water or even aqueous. Thus, the actual contact angle of the liquid on the device surface may be different from the water contact angle. In addition, when a material or coating is not incorporated into the device of the present invention, the determination of the water contact angle of the material or coating may be performed on the material or coating.

Particles

The invention includes devices that employ particles (e.g., particles used in assays). For example, particles configured to have an analyte moiety (e.g., a barcode, a nucleic acid, a binding molecule (e.g., a protein, peptide, aptamer, antibody or antibody fragment), an enzyme, a substrate, a droplet, etc.) may be included in a droplet containing an analyte to modify the analyte and/or detect the presence or concentration of the analyte. In some embodiments, the particles are synthetic particles (e.g., supports, such as gel beads).

For example, the droplet may contain one or more analyte portions, e.g., a unique identifier, such as a bar code. The analyte moiety (e.g., a bar code) may be introduced into the droplet prior to, after, or concurrent with the formation of the droplet. Delivering analyte moieties (e.g., barcodes) to a particular droplet allows for the subsequent characterization of individual samples (e.g., biological particles) to the particular droplet. The analyte moiety (e.g., a barcode) may be delivered to the droplet, e.g., on a nucleic acid (e.g., an oligonucleotide) via any suitable mechanism. Analyte moieties (e.g., barcoded nucleic acids (e.g., oligonucleotides)) may be introduced into the droplet via particles (such as microcapsules). In some cases, an analyte moiety (e.g., a barcoded nucleic acid (e.g., an oligonucleotide)) may be initially associated with a particle (e.g., a microcapsule) and then released upon application of a stimulus that allows the analyte moiety (e.g., a nucleic acid (e.g., an oligonucleotide)) to dissociate or release from the particle.

The particles (e.g., support) can be porous, non-porous, hollow (e.g., microcapsules), solid, semi-fluid, and/or combinations of the foregoing properties. In some cases, the particles (e.g., support) may be dissolvable, rupturable, and/or degradable. In some cases, the particles (e.g., support) may be non-degradable. In some cases, the particles (e.g., support) may be a gel support. The gel beads may be hydrogel beads. Gel beads may be formed from molecular precursors (such as polymers or monomeric species). The semi-solid particles (e.g., support) may be a liposome support. The solid particles (e.g., support) may comprise a metal, wherein the metal includes iron oxide, gold, and silver. In some cases, the particles (e.g., support) may be silica beads. In some cases, the particles (e.g., support) may be rigid. In other cases, the particles (e.g., support) may be flexible and/or compressible.

The particles (e.g., support) may comprise natural materials and/or synthetic materials. For example, the particles (e.g., support) may comprise natural polymers, synthetic polymers, or both natural and synthetic polymers. Examples of natural polymers include proteins and sugars such as deoxyribonucleic acid, rubber, cellulose, starch (e.g., amylose, amylopectin), proteins, enzymes, polysaccharides, silk, polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan, psyllium, gum arabic, agar, gelatin, shellac, karaya, xanthan, corn gum, guar gum, karaya, agarose, alginic acid, alginate, or natural polymers thereof. Examples of synthetic polymers include acrylic, nylon, silicone, spandex (spandex), viscose rayon, polycarboxylic acid, polyvinyl acetate, polyacrylamide, polyacrylate, polyethylene glycol, polyurethane, polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene, polycarbonate, polyethylene terephthalate, poly (chlorotrifluoroethylene), poly (ethylene oxide), poly (ethylene terephthalate), polyethylene, polyisobutylene, poly (methyl methacrylate), poly (formaldehyde), polyoxymethylene, polypropylene, polystyrene, poly (tetrafluoroethylene), poly (vinyl acetate), poly (vinyl alcohol), poly (vinyl chloride), poly (vinylidene fluoride), poly (vinyl fluoride), and/or combinations (e.g., copolymers) thereof. The support may also be formed of materials other than polymers including lipids, micelles, ceramics, glass-ceramics, material composites, metals, other inorganic materials, and the like.

In some cases, the particles (e.g., support) may comprise molecular precursors (e.g., monomers or polymers) that may form a polymer network via polymerization of the molecular precursors. In some cases, the precursor may be an already polymerized species capable of undergoing further polymerization (e.g., via chemical crosslinks). In some cases, the precursor may include one or more of an acrylamide or methacrylamide monomer, oligomer, or polymer. In some cases, the particles (e.g., support) may comprise prepolymers, which are oligomers capable of further polymerization. For example, polyurethane beads can be prepared using a prepolymer. In some cases, the particles (e.g., support) may comprise separate polymers that may be further polymerized together. In some cases, particles (e.g., supports) may be generated via polymerization of different precursors such that they comprise mixed polymers, copolymers, and/or block copolymers. In some cases, the particles (e.g., support) may comprise covalent or ionic bonds between polymer precursors (e.g., monomers, oligomers, linear polymers), oligonucleotides, primers, and other entities. In some cases, the covalent bond may be a carbon-carbon bond or a thioether bond.

Crosslinking may be permanent or reversible, depending on the particular crosslinking agent used. Reversible crosslinking may allow linearization or dissociation of the polymer under appropriate conditions. In some cases, reversible crosslinking may also allow for reversible attachment of materials bound to the support surface. In some cases, the crosslinker may form disulfide bonds. In some cases, the disulfide-forming chemical cross-linking agent may be cystamine or a modified cystamine.

The particles (e.g., support) may be of uniform size or non-uniform size. In some cases, the particles (e.g., support) may have a diameter of at least about 1 micrometer (μm), 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1mm, or more. In some cases, the diameter of the particles (e.g., support) may be less than about 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1mm, or less. In some cases, the diameter of the particles (e.g., support) may be in the range of about 40 μm to 75 μm, 30 μm to 75 μm, 20 μm to 75 μm, 40 μm to 85 μm, 40 μm to 95 μm, 20 μm to 100 μm, 10 μm to 100 μm, 1 μm to 100 μm, 20 μm to 250 μm, or 20 μm to 500 μm. The size of the particles (e.g. support, e.g. gel beads) used to generate the droplets is typically similar to the cross section (width or depth) of the first channel. In some cases, the gel beads are greater than the width and/or depth of the first channel and/or shelf, e.g., at least 1.5 times, 2 times, 3 times, or 4 times the width and/or depth of the first channel and/or shelf.

In certain embodiments, the particles (e.g., support) may be provided as a population or plurality of particles (e.g., support) having a relatively monodisperse size distribution. Where it may be desirable to provide a relatively consistent amount of reagent within a droplet, maintaining a relatively consistent particle (e.g., support) characteristic (such as size) may contribute to overall consistency. In particular, the particles (e.g., supports) described herein can have a size distribution with a coefficient of variation of their cross-sectional dimensions of less than 50%, less than 40%, less than 30%, less than 20%, and in some cases less than 15%, less than 10%, less than 5%, or less.

The particles may have any suitable shape. Examples of particle (e.g., support) shapes include, but are not limited to, spherical, non-spherical, elliptical, oblong, amorphous, circular, cylindrical, and variations thereof.

Particles (e.g., supports) injected or otherwise introduced into the droplets may contain releasably, cleavable, or reversibly attachable analyte moieties (e.g., barcodes). Particles (e.g., supports) injected or otherwise introduced into the droplets may contain activatable analyte moieties (e.g., barcodes). The particles (e.g., support) injected or otherwise introduced into the droplets may be degradable, rupturable, or dissolvable particles, such as dissolvable beads.

Particles (e.g., a support) within the channel can flow in a substantially regular flow profile (e.g., at a regular flow rate). Such regular flow profiles may allow droplets to include individual particles (e.g., a support) and individual cells or other biological particles when formed. Such regular flow profiles may allow droplets to have a dual occupancy of greater than 5％、10％、20％、30％、40％、50％、60％、70％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％ or 99% of the population (e.g., droplets having at least one support and at least one cell or other biological particle). In some embodiments, the droplet has a 1:1 double occupancy of greater than 5％、10％、20％、30％、40％、50％、60％、70％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％ or 99% of the population (i.e., the droplet has exactly one particle (e.g., support) and exactly one cell or other biological particle). Such regular flow patterns and devices that can be used to provide such regular flow patterns are provided, for example, in U.S. patent publication No. 2015/0292988, which is incorporated herein by reference in its entirety.

As discussed above, the analyte moiety (e.g., a barcode) may be releasably, cleavable, or reversibly attached to the particle (e.g., a support) such that the analyte moiety (e.g., a barcode) may be released or releasable by cleavage of the linkage between the barcode molecule and the particle (e.g., a support), or may be released by degradation of the particle (e.g., a support) itself, thereby allowing the barcode to be accessed by other reagents or by other reagents, or both. Releasable analyte moieties (e.g., barcodes) may sometimes be referred to as activatable analyte moieties (e.g., activatable barcodes) because they are available for reaction once released. Thus, for example, an activatable analyte moiety (e.g., an activatable barcode) may be activated by releasing the analyte moiety (e.g., a barcode) from a particle (e.g., a support (or other suitable type of droplet as described herein)). Other activatable configurations are also contemplated in the context of the described methods and systems.

In addition to or instead of a cleavable linkage between a particle (e.g., support) and an associated moiety, such as a barcode-containing nucleic acid (e.g., oligonucleotide), the particle (e.g., support) may be degradable, cleavable, or dissolvable upon exposure to one or more stimuli (e.g., temperature change, pH change, exposure to a particular chemical species or chemical phase, exposure to light, reducing agent, etc.). In some cases, the particles (e.g., support) may be dissolvable such that the material component of the particles (e.g., support) degrades or dissolves when exposed to a particular chemical species or environmental change (such as a temperature change or pH change). In some cases, the gel beads may degrade or dissolve under elevated temperature and/or alkaline conditions. In some cases, the particles (e.g., support) may be thermally degradable such that the particles (e.g., support) degrade when the particles (e.g., support) are exposed to an appropriate temperature change (e.g., heat). Degradation or dissolution of a particle (e.g., a support) associated with a species (e.g., a nucleic acid, e.g., an oligonucleotide, e.g., a barcoded oligonucleotide) can result in release of the species from the particle (e.g., support). It will be appreciated from the above disclosure that particle (e.g., support) degradation may refer to dissociation of bound or entrained species from the particle (e.g., support), with and without concomitant structural degradation of the physical particle (e.g., support) itself. For example, entrained species may be released from the particles (e.g., support) by, for example, osmotic pressure differences due to chemical environmental changes. For example, particle (e.g., support) pore size changes due to osmotic pressure differences can generally occur without structural degradation of the particles (e.g., support) themselves. In some cases, an increase in pore size due to osmotic swelling of the particles (e.g., support or microcapsules (liposomes)) may allow release of the species entrained within the particles. In other cases, osmotic shrinkage of the particles may result in better retention of entrained species by the particles (e.g., support) due to the reduced pore size.

Degradable particles (e.g., supports) can be introduced into the droplets such that upon application of an appropriate stimulus, the particles (e.g., supports) degrade within the droplets and any associated species (e.g., nucleic acids, oligonucleotides, or fragments thereof) are released within the droplets. The free species (e.g., nucleic acids, oligonucleotides, or fragments thereof) may interact with other reagents contained in the droplets. For example, polyacrylamide beads containing cystamine and linked to a barcode sequence via disulfide bonds can be combined with a reducing agent within droplets of a water-in-oil emulsion. Within the droplet, the reducing agent can break down various disulfide bonds, causing the particles (e.g., support) to degrade and the barcode sequence to be released into the aqueous internal environment of the droplet. In another example, heating a droplet containing a particle (e.g., support) bound analyte moiety (e.g., barcode) in an alkaline solution can also cause the particle (e.g., support) to degrade, and the attached barcode sequence to be released into the aqueous internal environment of the droplet.

Any suitable number of analyte moieties (e.g., molecular tag molecules (e.g., primers, barcoded oligonucleotides, etc.) may be associated with the particles (e.g., support) such that the analyte moieties (e.g., molecular tag molecules (e.g., primers, e.g., barcoded oligonucleotides, etc.) are present in the droplet at a predefined concentration after release from the particles. Such predefined concentrations may be selected to facilitate certain reactions, such as amplification, for generating a sequencing library within a droplet. In some cases, the predefined concentration of the primer may be limited by the method of generating the oligonucleotide-bearing particle (e.g., support).

Additional reagents may be included as part of the particles (e.g., analyte moieties), and/or may be included in solution or dispersed in the droplets, e.g., to activate, mediate, or otherwise participate in a reaction (e.g., a reaction between the analyte and the analyte moieties).

Biological sample

The droplets of the present disclosure may comprise biological particles (e.g., cells or particulate components thereof, e.g., organelles such as nuclei or mitochondria) and/or macromolecular components thereof (e.g., components of cells (e.g., intracellular or extracellular proteins, nucleic acids, glycans, or lipids) or cellular products (e.g., secreted products)). Analytes (e.g., components or products thereof) from biological particles may be considered biological analytes. In some embodiments, biological particles (e.g., cells or products thereof) are contained in a droplet, e.g., with one or more particles (e.g., a support) having an analyte moiety. In some embodiments, the biological particles (e.g., cells and/or components or products thereof) may be encapsulated within a gel, such as via polymerization of droplets comprising the biological particles and a precursor capable of polymerizing or gelling.

In the case of encapsulated biological particles (e.g., cells or particulate components thereof), the biological particles may be contained in a droplet containing a lysing agent to release the contents of the biological particles (e.g., the contents containing one or more analytes (e.g., biological analytes)) within the droplet. In such cases, the lysing agent may be contacted with the biological particle suspension at the same time as or immediately prior to introducing the biological particles into the droplet or particle source region, e.g., through one or more additional channels upstream or proximal to the second channel, or a third channel upstream or proximal to the second droplet or particle source region. Examples of lysing agents include bioactive agents, such as, for example, lysing enzymes for lysing different cell types (e.g., gram positive or negative bacteria, plants, yeast, mammals, etc.), such as lysozyme, leucopeptidase, lysostaphin, labiase, rhizoctonia solani lyase (kitalase), lywallase, and a variety of other lysing enzymes available from, for example, sigma-Aldrich, inc. (St Louis, MO), as well as other commercially available lysing enzymes. Additionally or alternatively, other lysing agents may be included in droplets having biological particles (e.g., cells or particulate components thereof) to cause the contents of the biological particles to be released into the droplets. For example, in some cases, a surfactant-based lysis solution may be used to lyse cells, but these may be less desirable for emulsion-based systems where surfactants may interfere with stable emulsions. In some cases, the lysis solution may contain nonionic surfactants, such as TRITON X-100 and TWEEN 20. In some cases, the lysis solution may contain ionic surfactants such as sodium dodecyl sarcosinate and Sodium Dodecyl Sulfate (SDS). In some embodiments, the lysis solution is hypotonic, thereby lysing the cells by osmotic shock. Electroporation, thermal, acoustic or mechanical cell disruption may also be used in certain situations, for example, to form non-emulsion based droplets, such as encapsulated biological particles, which may be in addition to or instead of droplet formation, wherein any pore size of the encapsulate is sufficiently small to retain a nucleic acid fragment of a desired size after cell disruption.

In addition to lysing agents, other agents may also be included in the droplets with the biological particles, including, for example, dnase and rnase inactivating agents or inhibitors, such as proteinase K, chelating agents (such as EDTA), and other agents for removing or otherwise reducing the negative activity or impact of different cell lysate components on subsequent nucleic acid processing. In addition, in the case of encapsulated biological particles (e.g., cells or particulate components thereof), the biological particles can be exposed to an appropriate stimulus to release the biological particles or their contents from the microcapsules within the droplets. For example, in some cases, chemical stimuli may be contained in the droplets along with the encapsulated biological particles to allow for degradation of the encapsulation matrix and release of the cells or their contents into the larger droplets. In some cases, the stimulus may be the same as the stimulus described elsewhere herein for releasing an analyte moiety (e.g., an oligonucleotide) from its corresponding particle (e.g., a support). In alternative aspects, this may be a different and non-overlapping stimulus so as to allow the encapsulated biological particles to be released into the droplet at a different time than the analyte moiety (e.g., oligonucleotide) is released into the same droplet.

Additional reagents (such as endonucleases) may also be included in the droplets with the biological particles to fragment the DNA of the biological particles, DNA polymerase and dntps used to amplify the nucleic acid fragments of the biological particles, and attach barcode molecular tags to the amplified fragments. Other reagents may also include reverse transcriptases (including enzymes having terminal transferase activity), primers and oligonucleotides, and switch oligonucleotides (also referred to herein as "switch oligonucleotides" or "template switch oligonucleotides") that may be used for template switching. In some cases, template switching may be used to increase the length of the cDNA. In some cases, template switching may be used to supplement a predefined nucleic acid sequence to the cDNA. In the example of template switching, the cDNA may be produced by reverse transcription of a template (e.g., cellular mRNA), where a reverse transcriptase having terminal transferase activity may add additional nucleotides, such as poly-C, to the cDNA in a template-independent manner. The transition oligonucleotide may comprise a sequence complementary to an additional nucleotide, such as poly-G. An additional nucleotide on the cDNA (e.g., polyC) may hybridize to an additional nucleotide on the switch oligonucleotide (e.g., polyG), whereby the reverse transcriptase may use the switch oligonucleotide as a template to further extend the cDNA. The template switching oligonucleotide may comprise a hybridization region and a template region. The hybridization region may comprise any sequence capable of hybridizing to a target. In some cases, as previously described, the hybridization region comprises a series of G bases to complement the overhanging C base at the 3' end of the cDNA molecule. The series of G bases can include 1G base, 2G bases, 3G bases, 4G bases, 5G bases, or more than 5G bases. The template sequence may comprise any sequence to be incorporated into the cDNA. In some cases, the template region comprises at least 1 (e.g., at least 2, 3, 4, 5, or more) tag sequences and/or functional sequences. The transition oligonucleotide may comprise deoxyribonucleic acid; ribonucleic acid; modified nucleic acids, including 2-aminopurine, 2, 6-diaminopurine (2-amino-dA), inverted dT, 5-methyl dC, 2' -deoxyinosine, super T (5-hydroxybutyrine-2 ' -deoxyuridine), super G (8-aza-7-deazaguanosine), locked Nucleic Acids (LNA), unlocked nucleic acids (UNA, e.g., UNA-A, UNA-U, UNA-C, UNA-G), iso-dG, iso-dC, 2' fluoro bases (e.g., fluoro C, fluoro U, fluoro A, and fluoro G), or any combination.

In some cases, the transition oligonucleotide may be 2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250 nucleotides or more in length.

In some cases, the transition oligonucleotide may be at least about 2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249 or 250 nucleotides in length or more.

In some cases, the transition oligonucleotide may be up to 2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249 or 250 nucleotides in length.

Once the contents of the cells are released into their respective droplets, the macromolecular components contained therein (e.g., macromolecular components of biological particles such as RNA, DNA, or proteins) may be further processed within these droplets.

As described above, the macromolecular components (e.g., bioanalyte) of each biological particle (e.g., cell or microparticle component thereof) may have unique identifiers (e.g., barcodes) such that, when characterizing those macromolecular components, components from a heterogeneous population of cells may have been mixed and dispersed or dissolved in a common liquid at that time, any given component (e.g., bioanalyte) may trace back to the biological particle (e.g., cell) from which the component was obtained. The ability to attribute features to individual biological particles or groups of biological particles is provided by assigning unique identifiers Fu Te to individual biological particles or groups of biological particles. Unique identifiers, for example in the form of nucleic acid barcodes, may be assigned or associated with individual biological particles (e.g., cells or particulate components thereof) or populations of biological particles (e.g., cells or particulate components thereof) to tag or label macromolecular components (and thus features) of biological particles with these unique identifiers. These unique identifiers can then be used to attribute the composition and characteristics of the biological particles to individual biological particles or groups of biological particles. As described in the systems and methods herein, this can be achieved by forming droplets (via particles, e.g., a support) that include individual biological particles or groups of biological particles having unique identifiers.

In some aspects, the unique identifier is provided in the form of an oligonucleotide, and the nucleic acid molecule comprises a nucleic acid barcode sequence that may be linked or otherwise associated with the nucleic acid content of the individual biological particle, or with other components of the biological particle, particularly with fragments of such nucleic acids. The oligonucleotides are spaced apart such that the nucleic acid barcode sequences contained therein are identical between the oligonucleotides in a given droplet, but the oligonucleotides may and do have different barcode sequences between different droplets, or at least represent a large number of different barcode sequences on all droplets in a given assay. In some aspects, only one nucleic acid barcode sequence may be associated with a given droplet, but in some cases, there may be two or more different barcode sequences.

The nucleic acid barcode sequence may comprise from 6 to about 20 or more nucleotides within the oligonucleotide sequence. In some cases, the barcode sequence may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or more in length. In some cases, the barcode sequence may be at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or more in length. In some cases, the barcode sequence may be up to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or less in length. These nucleotides may be completely contiguous, i.e. in a single stretch of adjacent nucleotides, or they may be divided into two or more separate subsequences separated by 1 or more nucleotides. In some cases, the separate barcode sequences may be about 4 to about 16 nucleotides in length. In some cases, the barcode sequence may be 4,5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode sequence may be at least 4,5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode sequence may be up to 4,5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or less.

The analyte moiety (e.g., oligonucleotide) in the droplet may also include other functional sequences useful in processing nucleic acid from the biological particles contained in the droplet. These sequences include, for example, targeting or random/universal amplification primer sequences for amplifying genomic DNA from individual biological particles within a droplet, while attaching an associated barcode sequence, sequencing primer or primer recognition site, hybridization or detection sequences, for example, for identifying the presence of these sequences or for pulling down any of the nucleic acids of a barcode or many other potential functional sequences.

Other mechanisms of forming droplets containing oligonucleotides may also be employed, including, for example, coalescing two or more droplets (one of which contains an oligonucleotide), or microdispersing an oligonucleotide into a droplet (e.g., a droplet within a microfluidic device).

In one example, particles (e.g., supports) are provided that each include a plurality of the above-described barcoded oligonucleotides releasably attached to the support, wherein all oligonucleotides attached to a particular support will include the same nucleic acid barcode sequence, but represent a plurality of different barcode sequences in the population of supports used. In some embodiments, hydrogel beads (e.g., supports with a polyacrylamide polymer matrix) are used as solid carriers and delivery vehicles for oligonucleotides into droplets, as they are capable of carrying a large number of oligonucleotide molecules, and can be configured to release those oligonucleotides upon exposure to a specific stimulus, as described elsewhere herein. In some cases, the population of supports provides a diverse barcode sequence library comprising at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 different barcode sequences or more. In addition, each support may have a large number of attached oligonucleotide molecules. In particular, the number of oligonucleotide molecules comprising a barcode sequence on each support can be at least about 1,000 oligonucleotide molecules, at least about 5,000 oligonucleotide molecules, at least about 10,000 oligonucleotide molecules, at least about 50,000 oligonucleotide molecules, at least about 100,000 oligonucleotide molecules, at least about 500,000 oligonucleotide molecules, at least about 1,000,000 oligonucleotide molecules, at least about 5,000,000 oligonucleotide molecules, at least about 10,000,000 oligonucleotide molecules, at least about 50,000,000 oligonucleotide molecules, at least about 100,000,000 oligonucleotide molecules, and in some cases at least about billions of oligonucleotide molecules, or more.

In addition, when a population of supports is included in a droplet, the resulting population of droplets can also include a diverse barcode library including at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 different barcode sequences. Further, each droplet in the population can comprise at least about 1,000 oligonucleotide molecules, at least about 5,000 oligonucleotide molecules, at least about 10,000 oligonucleotide molecules, at least about 50,000 oligonucleotide molecules, at least about 100,000 oligonucleotide molecules, at least about 500,000 oligonucleotide molecules, at least about 1,000,000 oligonucleotide molecules, at least about 5,000,000 oligonucleotide molecules, at least about 10,000,000 oligonucleotide molecules, at least about 50,000,000 oligonucleotide molecules, at least about 100,000,000 oligonucleotide molecules, and in some cases at least about billions of oligonucleotide molecules.

In some cases, it may be desirable to incorporate multiple different barcodes within a given droplet that are attached to a single or multiple particles (e.g., a support) within the droplet. For example, in some cases, mixed but known sets of barcode sequences may provide greater assurance of authentication in subsequent processing, e.g., by providing a stronger barcode address or attribution to a given droplet as a duplicate acknowledgement or independent acknowledgement of output from the given droplet.

The oligonucleotide may be capable of being released from the particle (e.g., the support) upon application of a particular stimulus. In some cases, the stimulus may be a light stimulus, such as by cleavage of a photolabile bond, thereby releasing the oligonucleotide. In other cases, thermal stimulation may be used, wherein an increase in the temperature of the particle (e.g., support) environment will result in bond cleavage, or other release of the oligonucleotide from the particle (e.g., support). In still other cases, chemical stimuli are used to cleave the binding of the oligonucleotide to the support or otherwise cause release of the oligonucleotide from the particle (e.g., support). In one instance, such compositions include the polyacrylamide matrices described above for encapsulating biological particles, and can be degraded by exposure to a reducing agent, such as Dithiothreitol (DTT), to release the attached oligonucleotides.

The droplets described herein may comprise one or more biological particles (e.g., cells or particulate components thereof), one or more particles carrying a barcode (e.g., a support), or at least both one biological particle and one particle carrying a barcode (e.g., a support). In some cases, the droplets may be unoccupied, containing neither biological particles nor particles carrying a barcode (e.g., a support). As previously described, by controlling the flow characteristics of each liquid combined at the drop source region, and controlling the geometry of the drop source region, drop formation can be optimized to achieve a desired level of particle (e.g., support, biological particle, or both) occupancy within the generated drop.

Method for producing droplets

The methods described herein may include generating droplets. The methods disclosed herein can generally produce emulsions, i.e., droplets of a dispersed phase in a continuous phase. For example, the liquid droplet may comprise a first liquid and the other liquid may be a second liquid. The first liquid may be substantially immiscible with the second liquid. In some cases, the first liquid may be an aqueous liquid or may be substantially miscible with water. Droplets produced according to the methods disclosed herein can combine a variety of liquids. For example, the liquid droplets may combine the first liquid and the third liquid. The first liquid may be substantially miscible with the third liquid. The second liquid may be an oil, as described herein.

A variety of applications require assessment of the presence and quantification of different biological particles or organism types within a population of biological particles, including, for example, microbiome analysis and characterization, environmental testing, food safety testing, epidemiological analysis, for example, in contaminant traceability, and the like.

The methods described herein may allow for the production of one or more droplets comprising a single particle (e.g., a support) and/or a single biological particle (e.g., a cell or particulate component thereof) and having a uniform and predictable droplet size. The method also allows sorting and/or generating one or more droplets comprising a single biological particle (e.g., a cell or a particulate component thereof) and more than one particle (e.g., a support), one or more droplets comprising more than one biological particle (e.g., a cell or a particulate component thereof) and a single particle (e.g., a support), and/or one or more droplets comprising more than one biological particle (e.g., a cell) and more than one particle (e.g., a support). The method may also allow for increased throughput of droplet sorting and/or formation.

Generally, the droplets are formed by allowing a first liquid to flow into a second liquid in the droplet source region, for example, in the case of spontaneous formation of droplets as described herein. These droplets may include an aqueous liquid dispersed phase within a non-aqueous continuous phase, such as an oil phase. In some cases, droplet formation may occur without externally driven movement of the continuous phase (e.g., the second liquid, such as oil). As discussed above, although the continuous phase is not necessary for droplet formation, it may still be externally driven. Emulsion systems for producing stable droplets in a non-aqueous (e.g., oil) continuous phase are described in detail in, for example, U.S. patent No. 9,012,390, which is incorporated herein by reference in its entirety for all purposes. Alternatively or in addition, the droplet may comprise a microvesicle, for example, having an internal liquid center or core and an external barrier surrounding it. In some cases, the droplets may include a porous matrix capable of entraining and/or retaining material within its matrix. The droplets may be collected in a substantially stationary liquid volume, for example, by using the buoyancy of the formed droplets to move them out of the path of the primary droplets (up or down, depending on the relative densities of the droplets and the continuous phase). Alternatively or in addition, the formed droplets may actively move out of the path of the primary droplets, for example using a gentle flow of the continuous phase (e.g., a liquid stream or a mildly agitated liquid).

Dispensing a carrier, such as a particle (e.g., a support carrying a barcoded oligonucleotide) or a biological particle (e.g., a cell or particulate component thereof), into discrete droplets can generally be accomplished by introducing a flowing stream of particles (e.g., a support) in an aqueous liquid into a flowing stream or non-flowing reservoir of a non-aqueous liquid such that droplets are generated. In some cases, the occupancy rate of the resulting droplets (e.g., the number of particles (e.g., supports) in each droplet) may be controlled by providing an aqueous stream of particles (e.g., supports) at a particular concentration or frequency and sorting the droplets in a suitable manner. In some cases, the occupancy rate of the resulting droplets may also be controlled by adjusting one or more geometric features at the droplet formation point (such as the width of the fluid channel carrying the particles (e.g., support)) relative to the diameter of the given particles (e.g., support), followed by sorting the droplets to provide a uniform population within the separate channels of the collection region.

Where droplets containing individual particles (e.g., a support) are desired, the relative flow rates of the liquids may be selected such that, on average, each droplet contains less than one particle (e.g., a support) to ensure that those already occupied droplets are occupied primarily individually. In some embodiments, the relative flow rates of the liquids may be selected such that a majority of the droplets are occupied, e.g., only a small percentage of the droplets are allowed to be unoccupied. The flow and channel architecture may be controlled to ensure that the individually occupied droplets have a desired number, unoccupied droplets are less than a certain level, and/or the multiple occupied droplets are less than a certain level.

When the methods described herein further include generating droplets, the devices described herein can be operated such that a majority of the occupied droplets include no more than one biological particle (e.g., a cell or particulate component thereof) in each occupied droplet. In some cases, the droplet sorting and/or forming process is performed such that less than 25% of the occupied droplets contain more than one biological particle (e.g., multiple occupied droplets), and in many cases, less than 20% of the occupied droplets have more than one biological particle. In some cases, less than 10% or even less than 5% of the occupied droplets include more than one biological particle in each droplet.

For example, from a cost and/or efficiency standpoint, it may be desirable to avoid creating an excessive number of empty droplets. However, while this may be achieved by providing a sufficient number of particles (e.g., a support) into the droplet or particle source region, poisson distribution may, among other things, increase the number of droplets that may include multiple biological particles. Thus, up to about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less of the generated droplets may be unoccupied. In some cases, the flow of one or more particles or liquids into the droplet or particle source region may be directed such that, in many cases, no more than about 50% of the generated droplets, no more than about 25% of the generated droplets, or no more than about 10% of the generated droplets are unoccupied. In addition, in the case of forming too many empty droplets, it is desirable to sort out droplets that are not empty for subsequent use. In addition, in the case of forming too many droplets that do not contain the desired material but are not empty, it is desirable to sort out the droplets that do not contain the desired material for subsequent use. The flow may be controlled so as to present a non-poisson distribution of individually occupied droplets while providing a lower level of unoccupied droplets. The above ranges of unoccupied droplets can be achieved while still providing any of the individual occupancy rates described above. For example, in many cases, droplets resulting using the devices and methods described herein have multiple occupancy rates of less than about 25%, less than about 20%, less than about 15%, less than about 10%, and in many cases less than about 5%, while unoccupied droplets are less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than a percentage.

The flow of the first fluid may be such that the droplet comprises a single particle (e.g., a support). In certain embodiments, the yield of droplets comprising individual particles is at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

It should be understood that the occupancy rates described above also apply to droplets comprising both biological particles (e.g., cells or particulate components thereof) and a support. Occupied droplets (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of occupied droplets) can include both support and biological particles. Particles (e.g., supports) within a channel (e.g., a particle channel) can flow in a substantially regular flow profile (e.g., at a regular flow rate) to provide droplets having individual particles (e.g., supports) and individual cells or other biological particles when formed and/or sorted. Such regular flow profiles may allow droplets to have a dual occupancy of greater than 5％、10％、20％、30％、40％、50％、60％、70％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％ or 99% (e.g., droplets having at least one support and at least one cell or biological particle). Such regular flow profiles may allow droplets to have a 1:1 double occupancy of greater than 5％、10％、20％、30％、40％、50％、60％、70％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％ or 99% (e.g., droplets having at least one support and at least one cell or biological particle). Such regular flow patterns and devices that can be used to provide such regular flow patterns are provided, for example, in U.S. patent publication No. 2015/0292988, which is incorporated herein by reference in its entirety.

In some cases, additional particles may be used to deliver additional reagents to the droplets. In such cases, it may be advantageous to introduce different particles (e.g., supports) from different support sources (e.g., containing different related reagents) through different channel inlets into a common channel or droplet or particle source region (e.g., proximal to or upstream of the droplet or particle source region) or droplet formation intersection. In such cases, the flow rate and/or frequency of each of the different particle (e.g., support) sources into the channel or fluidic connection may be controlled to provide a desired ratio of particles (e.g., support) from each source, while optionally ensuring that a desired pairing or combination of such particles (e.g., support) is formed into droplets having a desired number of biological particles.

The droplets or particles described herein can have a small volume, for example, a value of less than about 10 microliters (μl), 5 μl,1 μl, 900 picoliters (pL), 800pL, 700pL, 600pL, 500pL, 400pL, 300pL, 200pL, 100pL, 50pL, 20pL, 10pL, 1pL, 500 nanoliters (nL), 100nL, 50nL, or less. For example, the total volume of the droplets or particles can be less than about 1000pL, 900pL, 800pL, 700pL, 600pL, 500pL, 400pL, 300pL, 200pL, 100pL, 50pL, 20pL, 10pL, 1pL, or less. Where the droplet further comprises particles (e.g., a support or microcapsule), it is to be understood that the volume of sample liquid within the droplet can be less than about 90% of the above-described volume, less than about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% of the above-described volume (e.g., the above-described volume of the dispensed liquid), such as 1% to 99%, 5% to 95%, 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60%, such as 1% to 5%, 5% to 10%, 10% to 15%, 15% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85% to 90%, 90% to 95%, or 95% to 100% of the above-described volume.

Any suitable number of droplets or particles may be generated. For example, in the methods described herein, a plurality of droplets or particles may be generated, including at least about 1,000 droplets or particles, at least about 5,000 droplets or particles, at least about 10,000 droplets or particles, at least about 50,000 droplets or particles, at least about 100,000 droplets or particles, at least about 500,000 droplets or particles, at least about 1,000,000 droplets or particles, at least about 5,000,000 droplets or particles, at least about 10,000,000 droplets or particles, at least about 50,000,000 droplets or particles, at least about 100,000,000 droplets or particles, at least about 500,000,000 droplets or particles, at least about 1,000,000,000 droplets or particles, or more. Further, the plurality of droplets may include both unoccupied droplets (e.g., empty droplets) and occupied droplets.

The droplets or particles may be polydisperse, monodisperse, or substantially monodisperse (e.g., have a uniform diameter distribution). A plurality of droplets or particles is substantially monodisperse where the diameter distribution of the droplets or particles is such that no more than about 10%, about 5%, about 4%, about 3%, about 2%, about 1% or less of the droplets or particles have a diameter greater than or less than about 20%, about 30%, about 50%, about 75%, about 80%, about 90%, about 95%, about 99% or more of the average diameter of all droplets or particles.

Fluid to be dispersed into droplets may be delivered from a reservoir to a droplet source region. Alternatively, the fluid to be dispersed into droplets is formed in situ by combining two or more fluids in the device. For example, the fluid to be dispersed may be formed by combining one fluid comprising one or more reagents with one or more other fluids comprising one or more reagents. In these embodiments, mixing the fluid streams may cause a chemical reaction. For example, when particles are employed, a fluid having a reagent that breaks the particles may be associated with the particles, e.g., immediately upstream of the droplet generation region. In these embodiments, the particles may be cells, which may be combined with a lysing agent (such as a surfactant). When particles (e.g., supports) are employed, the particles (e.g., supports) may dissolve or chemically degrade, such as by changing the pH (acid or base), redox potential (e.g., adding an oxidizing or reducing agent), enzymatic activity, salt or ion concentration, or other mechanism.

The first fluid is conveyed through the first channel at a flow rate sufficient to generate droplets in the droplet source region. The faster flow rate of the first fluid generally increases the rate of droplet generation; at a sufficiently high rate, however, the first fluid will form a jet that may not break up into droplets. Typically, the flow rate of the first fluid through the first channel may be between about 0.01 μL/min to about 100 μL/min, such as between 0.1 μL/min to 50 μL/min, between 0.1 μL/min to 10 μL/min, or between 1 μL/min to 5 μL/min. In some cases, the flow rate of the first liquid may be between about 0.04 μL/min and about 40 μL/min. In some cases, the flow rate of the first liquid may be between about 0.01 μL/min and about 100 μL/min. Alternatively, the flow rate of the first liquid may be less than about 0.01 μl/min. Alternatively, the flow rate of the first liquid may be greater than about 40 μl/min, for example 45μL/min、50μL/min、55μL/min、60μL/min、65μL/min、70μL/min、75μL/min、80μL/min、85μL/min、90μL/min、95μL/min、100μL/min、110μL/min、120μL/min、130μL/min、140μL/min、150μL/min, or greater. At lower flow rates (such as flow rates less than or equal to about 10 μl/min), the droplet radius may not depend on the flow rate of the first liquid. Alternatively or in addition, the droplet radius may be independent of the flow rate of the first liquid for any of the aforementioned flow rates.

Typical droplet or particle formation rates for individual channels in the device of the invention are between 0.1Hz and 10,000Hz, for example between 1Hz and 1000Hz, or between 1Hz and 500 Hz. The use of a plurality of first channels may increase the rate of droplet or particle formation by increasing the number of formation sites. For example, but not limited to, the droplet or particle formation rate of a single channel may be 130Hz to 150Hz.

As discussed above, droplet or particle formation may occur without externally driven continuous phase motion. In such embodiments, the continuous phase flows in response to displacement or other forces of the pre-feed stream of the first fluid. Channels may be present in the droplet or particle source region (e.g., including the shelf region) to allow the continuous phase to be transported more rapidly around the first fluid. Such increased transport of the continuous phase may increase the rate of droplet or particle formation. Alternatively, the continuous phase may be actively transported. For example, the continuous phase may be actively transported into a droplet or particle source region (e.g., including a shelf region) to increase the rate of droplet or particle formation; the continuous phase may be actively conveyed to form a sheath flow around the first fluid as it exits the distal end; or the continuous phase may be actively transported to remove droplets or particles from the formation point.

Additional factors that affect the rate of droplet or particle formation include the viscosity of the first fluid and the continuous phase, wherein increasing the viscosity of either fluid decreases the rate of droplet or particle formation. In certain embodiments, the viscosity of the first fluid and/or the continuous phase is between 0.5cP and 10 cP. In addition, lower interfacial tension results in slower droplet or particle formation. In certain embodiments, the interfacial tension is between 0.1mN/m and 100mN/m, e.g., 1mN/m to 100mN/m, or 2mN/m to 60mN/m. The depth of the shelf region may also be used to control the rate of droplet or particle formation, with shallower depths resulting in faster rates of formation.

These methods can be used to produce droplets or particles having diameters in the range of 1 μm to 500 μm (e.g., 1 μm to 250 μm, 5 μm to 200 μm, 5 μm to 150 μm, or 12 μm to 125 μm). Factors affecting droplet or particle size include formation rate, cross-sectional dimensions of the distal end of the first channel, depth of shelf, and fluid properties and dynamic effects such as interfacial tension, viscosity, and flow rate.

The first liquid may be aqueous and the second liquid may be oil (or vice versa). Examples of oils include perfluorinated oils, mineral oils, and silicone oils. For example, the fluorinated oil may include a fluorosurfactant for stabilizing the resulting droplets (e.g., inhibiting subsequent coalescence of the resulting droplets). Examples of particularly useful liquids and fluorosurfactants are described, for example, in U.S. patent No. 9,012,390, which is incorporated herein by reference in its entirety for all purposes. Specific examples include hydrofluoroethers such as HFE 7500, 7300, 7200 or 7100. Suitable liquids are those described in US2015/0224466 and US 62/522,292, the liquids of these patents being hereby incorporated by reference. In some cases, the liquid includes additional components, such as particles, e.g., cells or gel beads. As discussed above, the first fluid or continuous phase may include reagents for performing various reactions, such as nucleic acid amplification, cleavage, or bead lysis. The first liquid or continuous phase may include additional components that stabilize or otherwise affect the droplets or particles or components within the droplets. Such additional components include surfactants, antioxidants, preservatives, buffers, antibiotics, salts, dispersants, enzymes, nanoparticles, and sugars.

The devices and methods of the present disclosure may be used in a variety of applications, such as processing a single analyte (e.g., a biological analyte, e.g., RNA, DNA, or protein) or multiple analytes (e.g., a biological analyte, e.g., DNA and RNA, DNA and protein, RNA and protein, or RNA, DNA and protein) from a single cell. For example, a biological particle (e.g., a cell or virus) may be formed in a droplet, and one or more analytes (e.g., biological analytes) from the biological particle (e.g., a cell) may be modified or detected (e.g., analyte moieties bound, labeled, or otherwise modified) for subsequent processing. The plurality of analytes may be from a single cell. The process may enable, for example, proteomic, transcriptomic, and/or genomic analysis of the cell or population thereof (e.g., simultaneous proteomic, transcriptomic, and/or genomic analysis of the cell or population thereof).

Methods of modifying an analyte include providing a plurality of particles (e.g., a support) in a liquid carrier (e.g., an aqueous carrier); providing a sample containing an analyte (e.g., as part of a cell or component or product thereof) in a sample liquid; and using the device to bind the liquids and form analyte droplets containing one or more particles and one or more analytes (e.g., as part of one or more cells or components or products thereof). This isolation of one or more particles from the analyte (e.g., a biological analyte associated with a cell) in the droplet enables labeling of discrete portions of a large heterologous sample (e.g., a single cell within a heterologous population). Once labeled or otherwise modified, the droplets or particles may then be sorted or combined (e.g., by breaking the emulsion), and the resulting liquid may be analyzed to determine a variety of characteristics associated with each of the plurality of single cells.

In a particular embodiment, the invention features a method of producing a droplet of an analyte using a device having a particle channel and a sample channel that intersect proximal to the droplet or particle source region. Particles having an analyte portion in a liquid carrier flow through the particle channel from proximal to distal (e.g., toward the droplet or particle source region), and a sample liquid containing the analyte flows through the sample channel from proximal to distal (e.g., toward the droplet or particle source region) until the two liquids meet and combine at the intersection of the sample channel and the particle channel upstream (and/or proximal) of the droplet or particle source region. The combination of the liquid carrier and the sample liquid produces an analyte liquid. For example, but not limited to, a first liquid from a first channel comprising a support may interact with a third liquid from a second channel having biological particles such that the liquid droplets comprise the first liquid and the third liquid to produce an analyte liquid having both the support and the biological particles. In some embodiments, the two liquids are miscible (e.g., they both contain solutes dissolved in water or an aqueous buffer). The combining of the two liquids can occur at a controlled relative rate such that the analyte liquid has a desired volume ratio of particle liquid to sample liquid, a desired numerical ratio of particles to cells, or a combination thereof (e.g., one particle per 50pL per cell). Analyte droplets are formed when analyte liquid flows through the droplet or particle source region into a spacer liquid (e.g., a liquid that is not miscible with the analyte liquid, such as an oil). Alternatively or in addition, the analyte droplets may accumulate in the collection region (e.g., as a substantially stationary population). In some cases, accumulation of the population of droplets may occur by a gentle flow of fluid within the collection region, e.g., to move the formed droplets out of the path of the primary droplets.

The apparatus useful for analysis may be characterized by any combination of the elements described herein. For example, various droplet or particle source regions may be employed in the design of the device for analysis. In some embodiments, the analyte droplets are formed at a droplet or particle source region having a shelf region where the analyte liquid expands in at least one dimension as it passes through the droplet or particle source region. Any of the shelf regions described herein may be used in the analyte droplet sorting and/or forming methods provided herein. Additionally or alternatively, the droplet or particle source region may have a stepped region at or distal to (e.g., within or distal to) the droplet or particle source region. In some embodiments, the analyte droplets are formed without externally driven continuous phase flow (e.g., by cross flow of one or more liquids at the droplet or particle source region). Alternatively, the analyte droplets are formed in the presence of an externally driven continuous phase flow.

Devices that may be used to form droplets may be characterized by multiple droplet formation, sorting, and/or collection areas (e.g., as separate parallel circuits) in or out of fluid communication with each other. For example, such devices may have 2 to 100, 3 to 50, 4 to 40, 5 to 30, 6 to 24, 8 to 18, or 9 to 12, e.g., 2 to 6, 6 to 12, 12 to 18, 18 to 24, 24 to 36, 36 to 48, or 48 to 96, e.g., 2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48 or more drop formation, sorting, and/or collection regions configured to produce analyte drops.

The source reservoir may store liquid prior to and during droplet or particle formation. In some embodiments, devices useful in forming analyte droplets include one or more particle reservoirs proximally connected to one or more particle channels. The particle suspension may be stored in a particle reservoir prior to formation of the analyte droplets. The particle reservoir may be configured to store particles containing the analyte moiety. For example, the particle reservoir may include a coating that prevents adsorption or binding (e.g., specific or non-specific binding) of the particles or analyte portions, for example. Additionally or alternatively, the particle reservoir may be configured to minimize degradation of the analyte moiety (e.g., by including a nuclease, e.g., a dnase or rnase) or the particle matrix itself, respectively.

Additionally or alternatively, the device includes one or more sample reservoirs proximally connected to the one or more sample channels. Prior to analyte droplet sorting and/or formation, a sample comprising cells and/or other reagents useful for analysis and/or droplet sorting and/or formation may be stored in a sample reservoir. The sample reservoir may be configured to reduce degradation of the sample components, for example, by including a nuclease (e.g., dnase or rnase).

The methods of the invention comprise applying the sample and/or particles to the device, e.g., (a) by pipetting the sample liquid or component or concentrate thereof into a sample reservoir, and/or (b) by pipetting a liquid carrier (e.g., an aqueous carrier) and/or particles into a particle reservoir. In some embodiments, the method involves first pipetting a liquid carrier (e.g., an aqueous carrier) and/or particles into a particle reservoir prior to pipetting the sample liquid or a component or concentrate thereof into the sample reservoir.

The sample reservoir and/or particle reservoir may be incubated under conditions suitable to maintain or promote the activity of its contents until droplet or particle formation and sorting is initiated or started.

Sorting and/or formation of droplets or particles of a biological analyte as provided herein may be used in a variety of applications. In particular, by using the methods and devices described to sort and/or form droplets of biological analyte, a user can perform standard downstream processing methods to barcode a heterogeneous population of cells, or perform single cell nucleic acid sequencing.

In a method of barcoding a population of cells, an aqueous sample having a population of cells is combined with a biological analyte particle having a nucleic acid primer sequence and a barcode in an aqueous carrier at the intersection of a sample channel and a particle channel to form a reaction liquid. The reaction liquid, as it passes through the droplet or particle source region, encounters a spacer liquid (e.g., spacer oil) under droplet formation conditions to form a plurality of reaction droplets in the reaction liquid, each reaction droplet having one or more particles and one or more cells. The reaction droplets are incubated under conditions sufficient to allow for the addition of a barcode to the nucleic acid of the cells in the reaction droplets or particles. In some embodiments, conditions sufficient for barcoding are thermally optimized for nucleic acid replication, transcription, and/or amplification. For example, the reaction droplets may be incubated at a temperature configured to enable reverse transcription of RNA produced by cells in the droplets into DNA with reverse transcriptase. Additionally or alternatively, the reaction droplets may be cycled through a range of temperatures to facilitate amplification, for example, as in Polymerase Chain Reaction (PCR). Thus, in some embodiments, one or more nucleotide amplification reagents (e.g., PCR reagents) (e.g., primers, nucleotides, and/or polymerase) are included in the reaction droplets. Any one or more reagents for nucleic acid replication, transcription and/or amplification may be provided to the reaction droplets by the aqueous sample, the liquid carrier, or both. In some embodiments, one or more reagents for nucleic acid replication, transcription and/or amplification are in an aqueous sample.

The present invention provides methods of single cell nucleic acid sequencing, wherein a heterogeneous population of cells can be characterized by their respective gene expression, e.g., relative to other cells of the population. Methods discussed herein and known in the art as cell-barcode addition may be part of the single cell nucleic acid sequencing methods provided herein. After barcoding, the nucleic acid transcripts that have been barcoded are sequenced and the sequences can be processed, analyzed and stored according to known methods. In some embodiments, the methods are capable of generating a genomic library comprising gene expression data for any individual cell within a heterologous population.

Alternatively, the ability of the methods described herein to sequester single cells or particulate components thereof in a reaction droplet enables applications beyond the scope of genomic characterization. For example, a reaction droplet comprising a single cell or a particulate component thereof and various analyte moieties capable of binding to different proteins may allow the single cell or particulate component thereof to be detectably labeled to provide relative protein expression data. In some embodiments, the analyte moiety is an antigen binding molecule (e.g., an antibody or fragment thereof), wherein each antibody clone is detectably labeled (e.g., with a fluorescent label having a different emission wavelength). Binding of the antibody to the protein may occur within the reaction droplet, and the cells may then be analyzed for bound antibody according to known methods to generate a protein expression library. After detection of the analyte using the methods provided herein, other methods known in the art may be employed to characterize cells within the heterogeneous population. In one example, subsequent operations that can be performed after sorting and/or droplet formation can include formation of amplified products, purification (e.g., via Solid Phase Reversible Immobilization (SPRI)), further processing (e.g., shearing, ligating functional sequences, and subsequent amplification (e.g., via PCR)). These operations may be performed in batches (e.g., outside of the droplet). An exemplary use of droplets formed and/or sorted using the methods of the present invention is to perform nucleic acid amplification, such as Polymerase Chain Reaction (PCR), wherein the reagents necessary to perform the amplification are contained within a first fluid. Where the droplets are droplets in an emulsion, the emulsion may be broken and the contents of the droplets then combined for use in additional operations. Additional reagents that may be included in the droplet along with the support carrying the barcode may include oligonucleotides for blocking ribosomal RNA (rRNA) and nucleases for digesting genomic DNA from cells. Alternatively, rRNA removers may be applied during additional processing operations. The configuration of the constructs generated by this method can help minimize (or avoid) sequencing of the poly-T sequence and/or sequence the 5' end of the polynucleotide sequence during sequencing. The amplification products (e.g., the first amplification product and/or the second amplification product) can be sequenced for sequence analysis. In some cases, amplification may be performed using a partial hairpin sequencing amplification (PHASE) method.

Device manufacturing method

The microfluidic devices of the present disclosure may be fabricated in any of a variety of conventional ways. The devices may be made in whole or in part of a polymeric material such as polyethylene or polyethylene derivatives, such as Cyclic Olefin Copolymer (COC), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate, polystyrene, polypropylene, polyvinyl chloride, polytetrafluoroethylene, polyoxymethylene, polyetheretherketone, polycarbonate, polystyrene, and the like, or they may be made in whole or in part of an inorganic material such as silicon or other silica-based materials, for example glass, quartz, fused silica, borosilicate glass, metal, ceramic, and combinations thereof. The polymer device component may be manufactured using any of a variety of processes including soft lithography, embossing techniques, micromachining, such as laser machining, or in some aspects injection molding of layer components including defined channels and other structures, such as reservoirs, integrated features, and the like. In some aspects, the structure including the reservoirs and channels can be manufactured using, for example, injection molding techniques to create a polymeric structure. In such cases, the laminate layer may be adhered to the molded structured part by off-the-shelf methods including thermal lamination, solvent-based lamination, sonic welding, and the like.

It should be appreciated that structures composed of inorganic materials may also be fabricated using known techniques. For example, channels and other structures may be micro-machined into the surface or etched into the surface using standard photolithographic techniques. In some aspects, microfluidic devices or components thereof may be fabricated using three-dimensional printing techniques to fabricate channels or other structures of the devices and/or discrete components thereof.

Other embodiments

While the application has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains and as may be applied to the essential features hereinbefore set forth. Other embodiments are within the claims.

Examples

Example 1

Referring now to fig. 1A-1B, one exemplary embodiment of a synthetic surface coating agent is illustrated. In one embodiment, the one-step amide formation may be performed by reacting the perfluorodiester with (MeO) ₃Si-CH₂CH₂NH₂ for 4 hours at room temperature. In some embodiments, the reaction includes one or more simple solvent extraction steps, and the product is obtained in greater than 95% purity and greater than 80% yield.

In some embodiments, the product having structure IIIa or IIIb:

The value of n+n is 7. As another example, the value of n+n is equal to 4 to 13 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13). Fig. 5A to 5B show NMR data of aminosilanes before and after reaction with diester IIIb.

Example 2

Referring now to fig. 2, an exemplary embodiment of droplet generation frequency is illustrated. Fluorocarbon surface coating agents are applied to the droplet source region of the device in the form of a liquid solution via a coating mechanism such as dip coating, spray coating, or flow through deposition using pressure flow or capillary flow, and after washing with a suitable solvent, cured at elevated temperature and humidity. In one embodiment, the surface coating agent having the structure of formula IIIb is applied to the source region of the device droplets as shown in fig. 4 at a range of concentrations of 0.05 weight (wt)%, 0.1 wt%, 0.2 wt%, 0.5 wt%, 0.75 wt%, etc. For each of a range of concentrations including 0.05 wt%, 0.1 wt%, 0.2 wt%, 0.5 wt% and 0.75 wt%, a range of 130Hz to 150Hz is achieved. For 0.05 wt.%, the droplet generation frequency was 138.605+/-4.57207Hz. For 0.1 wt.%, the droplet generation frequency was 136.337+/-7.27377Hz. For 0.2 wt.%, the droplet generation frequency was 140.683+/-2.69305Hz. For 0.5 wt.%, the droplet generation frequency was 140.468+/-1.35875Hz. For 0.75 wt.%, the droplet generation frequency was 139.648+/-2.0347Hz. The surface coating agent having the structure of formula I has a reduced standard deviation when compared to the commercially available fluorocarbon surface coating agent designated compound A, has an average value of 135.69+/-4.65357Hz, and exhibits data points below 130 Hz.

Example 3

Referring now to fig. 3, one exemplary embodiment of a droplet generation failure is illustrated. The drop generation failure may include one or more wetting failures and/or one or more asymmetric generation. The surface coating agent having the structure of formula I is applied to the device and/or a portion of the device at a range of concentrations including 0.05 wt%, 0.1 wt%, 0.2 wt%, 0.5 wt% and 0.75 wt%. In one embodiment, a range of concentrations including 0.05 wt%, 0.1 wt%, 0.2 wt%, 0.5 wt% and 0.75 wt% each have reduced wetting failure as compared to commercially available fluorocarbon surface coating agents (noted as compound a). In one embodiment, a range of concentrations including 0.05 wt%, 0.1 wt%, 0.2 wt%, 0.5 wt% and 0.75 wt% each have reduced asymmetric generation compared to compound a. Both wetting failure and asymmetric generation are reduced due to the creation of a coating that causes the water contact angle to become greater.

Example 4

Referring now to fig. 4, one exemplary embodiment of a drop source region is illustrated. The device is included with a first channel 401 and a second channel 402. The drop source region includes a shelf region and a step region. The second channel, shelf region and step region are coated with a compound of formula I. The droplets produced comprise the support from the first channel 403.

Example 5

Fig. 6 is a graph showing the average value and standard deviation of water contact angle. As a control, a commercially available fluorocarbon surface coating agent (compound a) was used. The surface coating agent having the structure of formula I is applied to the surface at a concentration of 0.2% and 0.5%. The water contact angle was measured before washing the surface, and after washing the surface and storing at 4 ℃, 20 ℃, 80 ℃ or room temperature. It was found that for all concentrations of the surface coating agent having the structure of formula I and all storage temperatures, the water contact angle increases under both pre-wash and post-wash conditions when compared to compound a.

Example 6

Fig. 7 is a graph showing the average and standard deviation of water contact angles for commercial coating agents and compounds having the structure of formula IIIb. Each coating was prepared by immersing the plasma cleaned slide in a 0.5 wt% HFE-7100 solution of the coating agent for 2 minutes. The slide was allowed to dry and then soaked in HFE-7100 for 1 minute to wash the membrane. The slides were cured in an environmental chamber at 60℃and 60% relative humidity for 30 minutes. The contact angle was measured on the cured film (not washed) and then measured again after washing the film in HFE-7100 for 10 minutes to remove any uncured coating agent.

Other embodiments are also within the scope of the claims.

Claims

1. A surface coating compound having the structure of formula I:

wherein:

Each Y is independently-O (CR ⁹ ₂)_p -, where p is 1, 2, 3, 4, 5, or 6, and each R ⁹ is independently F or C ₁-C₆ perfluoroalkyl;

Each n is independently 1,2,3,4, 5, 6, or 7;

Each Z is independently-OR ¹⁰ -, wherein R ¹⁰ is C ₁-C₆ perfluoroalkylene;

m is 1,2, 3, 4, 5 or 6;

Each W is independently-C (O) NR _a-、-NR_a C (O) -, -C (O) O-; -OC (O) -, -C (O) S-, -SC (O) -, or-O-, wherein R _a is-H, C ₁-C₆ perfluoroalkyl or-F;

X ¹ is- [ (CH ₂)_jQ]_s-(CH₂)_j -or optionally substituted C ₁-C₆ -alkylene) and X ² is- (CH ₂)_j-[Q(CH₂)_j]_s -or optionally substituted C ₁-C₆ -alkylene), wherein Q is-O-, -S-, -NR _b-、-NR_bC(O)-、-C(O)NR_b -, -C (O) O-, -C (O) S-or-SC (O) -, R _b is-H or C ₁-C₆ alkyl, each j is independently 1,2, 3,4, 5, or 6, and S is 1,2, 3,4, 5, or 6;

Each of R ¹、R²、R³、R⁴、R⁵ and R ⁶ is independently optionally substituted C ₁-C₆ alkoxy or C ₁-C₆ alkyl, wherein at least one of R ¹、R² or R ³ and one of R ⁴、R⁵ or R ⁶ are C ₁-C₆ alkoxy; and

Each of R ⁷ and R ⁸ is independently F or C ₁-C₆ perfluoroalkyl, and

Wherein at least one R ⁹ is C ₁-C₆ perfluoroalkyl, or R ¹⁰ is C ₃-C₆ perfluoroalkylene.

2. The compound of claim 1, wherein the compound provides a total fluorine weight percent of at least 57% when bound to a surface.

3. The compound of any one of the preceding claims, wherein Y is-OCF ₂-CF(R¹¹) -, wherein R ¹¹ is C ₁-C₆ perfluoroalkyl.

4. A compound according to claim 3 wherein R ¹¹ is CF ₃.

5. The compound of any one of the preceding claims, wherein Z is-OCF ₂-CF₂-CF₂-CF₂ -or-OCF ₂-CF₂-CF₂-CF₂-CF₂ -.

6. The compound of any one of the preceding claims, wherein each of R ¹、R²、R³、R⁴、R⁵ and R ⁶ is methoxy.

7. The compound of claim 1, having the structure:

wherein each n is independently 1,2, 3, 4, 5, 6, or 7.

8. The compound of claim 1, having the structure:

Wherein each n is independently 1,2,3, 4, 5, 6, or 7; and

M is 1,2, 3, 4, 5 or 6.

9. The compound of any one of claims 1 to 8, wherein the sum of n and n is 4 to 13.

10. The compound of claim 9, wherein the sum of n and n is 7.

11. The compound of claim 9, wherein the sum of n and n is 10.

12. The compound of claim 9, wherein the sum of n and n is 13.

13. The compound of any one of claims 1 to 12, wherein m is 3.

14. An apparatus for producing droplets of a first liquid in a second liquid, the apparatus comprising:

a) A first channel having a first depth, a first width, a first proximal end, and a first distal end; and

B) A droplet source region in fluid communication with the first distal end, the droplet source region configured to generate droplets of the first liquid in the second liquid, wherein the droplet source region comprises a fluorocarbon surface coating having a total fluorine weight percentage of at least 57%.

15. The apparatus of claim 14, wherein the fluorocarbon surface coating comprises a surface coating compound having a moiety of formula VI:

wherein:

Each n is independently 1,2,3,4, 5, 6, or 7;

m is 1,2, 3, 4, 5 or 6;

Each of R ⁷ and R ⁸ is independently F or C ₁-C₆ perfluoroalkyl,

16. The device of claim 14, further comprising a first reservoir in fluid communication with the first proximal end.

17. The device of claim 14, further comprising a second channel having a second depth, a second width, a second proximal end, and a second distal end, the second channel intersecting the first channel between the first proximal end and the first distal end.

18. The device of claim 17, wherein the second channel comprises the fluorocarbon surface coating.

19. The device of claim 17, further comprising a second reservoir in fluid communication with the second proximal end.

20. The apparatus of claim 14, wherein the droplet source region comprises:

i) A shelf region having a second depth and a second width, wherein the second width is greater than the first width, and wherein the first distal end is in fluid communication with the shelf region; and

Ii) a collection area configured for collecting the droplets and comprising at least one wall forming a stepped area in fluid connection with the shelf area.

21. The apparatus of claim 20, wherein the shelf region comprises the fluorocarbon surface coating.

22. The apparatus of claim 15, wherein the fluorocarbon surface coating comprises the following: wherein each n is independently 1,2, 3, 4, 5, 6, or 7.

23. The apparatus of claim 15, wherein the fluorocarbon surface coating comprises the following:

Wherein each n is independently 1,2,3, 4, 5, 6, or 7; and

M is 1,2, 3, 4, 5 or 6.

24. The device of any one of claims 14 to 23, wherein the sum of n and n is 4 to 13.

25. The compound of claim 24, wherein the sum of n and n is 7.

26. The compound of claim 24, wherein the sum of n and n is 10.

27. The compound of claim 24, wherein the sum of n and n is 13.

28. The device of any one of claims 14 to 27, wherein m is 3.

29. The device of claim 14, wherein the fluorocarbon surface coating is the reaction product of a compound of any one of claims 1 to 13 with a hydroxyl group.

30. A method of producing droplets, comprising:

a) Providing the apparatus of any one of claims 14 to 29; and

B) A first liquid is flowed from the first proximal end toward the drop source region to create drops of the first liquid in a second liquid.

31. The method of claim 30, wherein the first liquid is aqueous or miscible with water.

32. The method of claim 30, wherein the device is the device of claim 14; the second channel includes a third liquid that combines with the first liquid at an intersection; and wherein the liquid droplets comprise the first liquid and the third liquid.

33. The method of claim 30, wherein the first liquid comprises a support, the third liquid comprises particles, and the droplets comprise the support from the first liquid and the particles from the third liquid.

34. The method of claim 30, wherein the droplets are produced at a production frequency of 130Hz to 150 Hz.