Fluidic device including a fluid channel and method for making same

Abstract

In one example, a method of preparing a fluid channel includes covalently bonding a first region of a substrate to a first region of a cover using a first moiety covalently bonded to the first region of the substrate and a second moiety covalently bonded to the first region of the cover, the covalent bond between the first region of the substrate and the first region of the cover suspending the second region of the cover over the second region of the substrate to form the fluid channel.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. patent application Ser. No. 63 / 194,330, filed May 28, 2021, entitled “Fluidic Devices Including Fluidic Channels, and Methods of Making the Same,” the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THEINVENTION The present application relates generally to devices that include fluid channels. [Background technology]

[0003] Fluidic channels are used in many technical applications. For example, certain molecular analyses, such as certain polynucleotide sequencing methods, utilize polynucleotides bound within a fluidic channel (sometimes called a flow cell). For example, oligonucleotide primers (e.g., single-stranded DNA or ssDNA) may be grafted to the fluidic channel and used to amplify target polynucleotides for sequencing. Fluidic channels may be formed by bonding covers, which may be formed from glass or plastic substrates, using adhesives such as pressure-sensitive adhesives or epoxies. However, such adhesives may chemically react with the fluids within the fluidic channel, may fluoresce in a way that prevents detection of the desired fluorescent signal, and / or may be thermally unstable, thus potentially resulting in fluid leakage or other damage. Thus, there is a need for improved methods for making fluidic channels that may, for example, comprise different materials. Summary of the Invention

[0004] The examples provided herein relate to devices that include fluid channels and methods of making the same.

[0005] Some examples herein provide a method for preparing a fluid channel. The method may include covalently bonding a first region of a substrate to a first region of a cover using a first portion covalently bonded to the first region of the substrate and a second portion covalently bonded to the first region of the cover. The covalent bond between the first region of the substrate and the first region of the cover may suspend the second region of the cover above the second region of the substrate to form a fluid channel.

[0006] In some examples, the method further comprises binding the oligonucleotide to the second region of the substrate. In some examples, the oligonucleotide is bound to the second region of the substrate before the first region of the substrate is bound to the first region of the cover. In some examples, the method further comprises protecting the oligonucleotide before the first region of the substrate is covalently bound to the first region of the cover. In some examples, protecting the oligonucleotide comprises depositing a mask on the oligonucleotide. In some examples, the method further comprises removing the mask after the first region of the substrate is covalently bound to the first region of the cover. In some examples, the oligonucleotide is covalently bound to the second region of the substrate using a second moiety covalently bound to the second region of the substrate. In some examples, the oligonucleotide comprises a capture primer. In some examples, the oligonucleotide is bound to the second region of the substrate after the first region of the substrate is bound to the first region of the cover.

[0007] In some examples, covalently bonding the first region of the substrate to the first region of the cover includes selectively applying heat to the first region of the substrate or the first region of the cover. In some examples, the heat is applied using light. In some examples, the light includes infrared or near infrared light.

[0008] In some examples, covalently bonding the first region of the substrate to the first region of the cover includes selectively applying pressure to the first region of the substrate and the first region of the cover.

[0009] In some examples, covalently bonding the first region of the substrate to the first region of the cover includes reacting the first moiety with a second moiety, in some examples, the reaction between the first moiety and the second moiety includes an azide-alkyne [3+2] cycloaddition.

[0010] In some examples, the method further includes disposing an intervening layer between the first region of the substrate and the first region of the cover. Covalently bonding the first region of the substrate to the first region of the cover can include covalently bonding the first region of the substrate to the intervening layer and covalently bonding the first region of the cover to the intervening layer. In some examples, the intervening layer includes a first portion or a second portion. In some examples, covalently bonding the first region of the substrate to the intervening layer including a includes a first azide-alkyne [3+2] cycloaddition. In some examples, covalently bonding the first region of the cover to the intervening layer includes a second azide-alkyne [3+2] cycloaddition.

[0011] In some examples, the first moiety is covalently bonded to a first region of the substrate via a silane, carboxylate, or amidate group, and in some examples, the second moiety is covalently bonded to a second region of the substrate via a silane, carboxylate, or amidate group.

[0012] In some examples, the cover comprises a different material than the substrate. In some examples, the cover comprises at least one material selected from the group consisting of cyclic olefin polymer (COP), cyclic olefin copolymer (COC), glass, silicon, polypropylene (PP), photoresist, polyethylene terephthalate (PET), poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM), and polyethylene (PE). In some examples, the substrate comprises at least one material selected from the group consisting of cyclic olefin polymer (COP), cyclic olefin copolymer (COC), glass, silicon, polypropylene (PP), photoresist, polyethylene terephthalate (PET), poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM), and polyethylene (PE). In some examples, the second region of the cover is separated from the second region of the substrate by about 1 μm to about 1 cm.

[0013] Some examples herein provide a fluidic device including a substrate including a first region and a second region, and a cover including a first region and a second region. The first region of the substrate can be covalently bonded to the first region of the cover using a first portion covalently bonded to the first region of the substrate and a second portion covalently bonded to the first region of the cover. The covalent bond between the first region of the substrate and the first region of the cover suspends the second region of the cover above the second region of the substrate to form a fluidic channel.

[0014] In some examples, the device further comprises an oligonucleotide bound to a second region of the substrate. In some examples, the oligonucleotide is covalently bound to the second region of the substrate using a second moiety covalently bound to the second region of the substrate. In some examples, the oligonucleotide comprises a capture primer.

[0015] In some examples, the first region of the substrate is covalently bonded to the first region of the cover via the product of an azide-alkyne [3+2] cycloaddition reaction between the first moiety and the second moiety.

[0016] In some examples, the method further includes an intervening layer between the first region of the substrate and the first region of the cover, the first region of the substrate being covalently bonded to the intervening layer, and the first region of the cover being covalently bonded to the intervening layer. In some examples, the intervening layer includes a first portion or a second portion. In some examples, the first region of the substrate is covalently bonded to the intervening layer via a product of an azide-alkyne [3+2] cycloaddition reaction. In some examples, the first region of the cover is covalently bonded to the intervening layer via a product of an azide-alkyne [3+2] cycloaddition reaction.

[0017] In some examples, the first moiety is covalently bonded to a first region of the substrate via a silane, carboxylate, or amidate group, and in some examples, the second moiety is covalently bonded to a second region of the cover via a silane, carboxylate, or amidate group.

[0018] In some examples, the cover comprises a different material than the substrate. In some examples, the substrate comprises at least one material selected from the group consisting of cyclic olefin polymer (COP), cyclic olefin copolymer (COC), glass, silicon, polypropylene (PP), photoresist, polyethylene terephthalate (PET), poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM), and polyethylene (PE). In some examples, the cover comprises at least one material selected from the group consisting of cyclic olefin polymer (COP), cyclic olefin copolymer (COC), glass, silicon, polypropylene (PP), photoresist, polyethylene terephthalate (PET), poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM), and polyethylene (PE). In some examples, the second region of the cover is separated from the second region of the substrate by about 1 μm to about 1 cm.

[0019] Some examples herein provide methods of making the above-mentioned device, wherein a first region of the substrate is covalently bonded to a first region of the cover by selectively applying heat to the first region of the substrate or the first region of the cover.

[0020] In some examples, the heat is applied using light, which in some examples may include infrared or near infrared light.

[0021] It should be understood that any respective feature / example of each of the aspects of the present disclosure described herein may be implemented together in any suitable combination, and any feature / example from any one or more of these aspects may be implemented together in any suitable combination with any of the features of the other aspects described herein, in order to achieve the benefits described herein. [Brief description of the drawings]

[0022] [Figure 1A]1A-1C illustrate generally the structures and operations in an exemplary process for creating a fluid channel. [Figure 1B] 1A-1C illustrate generally the structures and operations in an exemplary process for creating a fluid channel. [Figure 1C] 1A-1C illustrate generally the structures and operations in an exemplary process for creating a fluid channel. [Figure 1D] 1A-1C illustrate generally the structures and operations in an exemplary process for creating a fluid channel. [Figure 2A] 1A-1C illustrate generally the structures and operations in an exemplary process for providing oligonucleotides in a fluidic channel. [Figure 2B] 1A-1C illustrate generally the structures and operations in an exemplary process for providing oligonucleotides in a fluidic channel. [Figure 2C] 1A-1C illustrate generally the structures and operations in an exemplary process for providing oligonucleotides in a fluidic channel. [Figure 2D] 1A-1C illustrate generally the structures and operations in an exemplary process for providing oligonucleotides in a fluidic channel. [Figure 2E] 1A-1C illustrate generally the structures and operations in an exemplary process for providing oligonucleotides in a fluidic channel. [Figure 3A] 1A-1C illustrate generally the structures and operations in an exemplary process for providing oligonucleotides in a fluidic channel. [Figure 3B] 1A-1C illustrate generally the structures and operations in an exemplary process for providing oligonucleotides in a fluidic channel. [Figure 3C] 1A-1C illustrate generally the structures and operations in an exemplary process for providing oligonucleotides in a fluidic channel. [Figure 3D] 1A-1C illustrate generally the structures and operations in an exemplary process for providing oligonucleotides in a fluidic channel. [Figure 3E]1A-1C illustrate generally the structures and operations in an exemplary process for providing oligonucleotides in a fluidic channel. [Figure 4A] 1A-1C illustrate generally the structures and operations in an exemplary process for creating a fluid channel. [Figure 4B] 1A-1C illustrate generally the structures and operations in an exemplary process for creating a fluid channel. [Figure 5A] 1A-1D illustrate generally the structures and operations in an exemplary process for bonding a portion to a substrate or cover for use in creating a fluidic channel. [Figure 5B] 1A-1D illustrate generally the structures and operations in an exemplary process for bonding a portion to a substrate or cover for use in creating a fluidic channel. [Figure 6] 13A-13C show schematics of exemplary reactions for bonding moieties to a substrate or cover for use in creating fluidic channels. [Figure 7A] 1A-1C illustrate generally the structures and operations in an exemplary process for creating a fluid channel. [Figure 7B] 1A-1C illustrate generally the structures and operations in an exemplary process for creating a fluid channel. [Figure 7C] 1A-1C illustrate generally the structures and operations in an exemplary process for creating a fluid channel. [Figure 7D] 1A-1C illustrate generally the structures and operations in an exemplary process for creating a fluid channel. [Figure 7E] 1A-1C illustrate generally the structures and operations in an exemplary process for creating a fluid channel. [Figure 7F] 1A-1C illustrate generally the structures and operations in an exemplary process for creating a fluid channel. [Figure 7G] 1A-1C illustrate generally the structures and operations in an exemplary process for creating a fluid channel. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The examples provided herein relate to devices that include fluid channels and methods of making the same.

[0024] For example, provided herein is a method of forming a fluidic channel that includes covalently bonding a cover to a substrate, either directly or through an intervening layer to which both the cover and substrate are covalently bonded. The cover, substrate, and any intervening layer may include respective moieties that can react with each other without the need for a solvent or catalyst. For example, the moieties may include click chemistry moieties that perform an azide-alkyne [3+2] cycloaddition reaction with each other. However, it will be understood that any suitable chemistry that forms a covalent bond may be used, such as thermally driven non-click reactions (e.g., epoxy-amine reactions). In some examples, another portion of the substrate may be attached to an oligonucleotide, e.g., a capture primer, such as may be used to amplify a target polynucleotide. For example, the cover may be covalently bonded to the substrate such that a portion of the cover is suspended above a portion of the substrate to which the oligonucleotide is attached. The fluidic channel provided by the covalent bond between the cover and substrate may be used to flow a fluid, e.g., a fluid containing a target polynucleotide, a polymerase, a nucleotide, a reagent, etc., over the oligonucleotide. The covalent bond between the cover and substrate may be expected to be substantially unreactive with the fluid in the fluidic channel. Additionally or alternatively, such covalent bonds between the cover and the substrate may be expected to not fluoresce in a manner that would prevent detection of the desired fluorescent signal. Additionally or alternatively, such covalent bonds between the cover and the substrate may be expected to be more thermally stable than conventional adhesives, and thus prevent leakage or other damage. Thus, fluidic devices including the fluidic channels of the present invention may be expected to be particularly robust and useful for handling many different types of fluids.

[0025] We first provide a brief explanation of some of the terms used herein, then we describe some example devices that contain fluidic channels and methods of making the same.

[0026] term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The use of the term "including" and other forms such as "include", "includes" and "included" is not limiting. The use of the term "having" and other forms such as "have", "has" and "had" is not limiting. As used herein, whether in a transitional phrase or in the body of a claim, the terms "comprise" and "comprising" should be interpreted as having an open-ended meaning. That is, the above terms should be interpreted as synonymous with the phrase "having at least" or "comprising at least". For example, when used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the recited features or components, but may include additional features or components.

[0027] As used throughout this specification, the terms "substantially," "approximately," and "about" are used to describe and take into account small variations due to processing variations, etc. For example, small variations can refer to ±5% or less, such as ±2% or less, such as ±1% or less, such as ±0.5% or less, such as ±0.2% or less, such as ±0.1% or less, such as ±0.05% or less.

[0028] As used herein, the terms "covalently coupled" or "covalently bonded" refer to the formation of a chemical bond characterized by the sharing of electron pairs between atoms. For example, a covalently bonded molecule refers to a molecule that forms a chemical bond with a substrate as compared to attachment to the surface by other means, e.g., non-covalent bonds such as electrostatic interactions.

[0029] As used herein, "C a ~C b " or "C a-b " refers to the number of carbon atoms in a particular group. That is, a group can contain from "a" to "b" (inclusive) carbon atoms. Thus, for example, "C1-C4 alkyl" or "C 1~4 Alkyl" or "C 1~4 An "alkyl" group refers to all alkyl groups having 1 to 4 carbons, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)-, and (CH3)3C-.

[0030] As used herein, the term "halogen" or "halo" means fluorine, chlorine, bromine, or iodine, with fluorine and chlorine being examples.

[0031] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). An alkyl group may have 1 to 20 carbon atoms (wherever indicated herein, a numerical range such as "1 to 20" refers to each integer within the given range. For example, "1 to 20 carbon atoms" means that an alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 20 carbon atoms, but this definition also covers occurrences of the term "alkyl" (where no numerical range is specified). An alkyl group may also be a medium sized alkyl having 1 to 9 carbon atoms. An alkyl group may also be a lower alkyl having 1 to 4 carbon atoms. An alkyl group may also be a "C 1~4 By way of example only, "C 1~4 Alkyl" or "C 1~4 "Alkyl" indicates that there are 1 to 4 carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like.

[0032] As used herein, "alkenyl" refers to a straight or branched hydrocarbon chain containing one or more double bonds. Alkenyl groups can have from 2 to 20 carbon atoms, although this definition also covers occurrences of the term "alkenyl" where no numerical range is specified. Alkenyl groups can also be medium-sized alkenyls having from 2 to 9 carbon atoms. Alkenyl groups can also be lower alkenyls having from 2 to 4 carbon atoms. An alkenyl group is defined as a "C 2~4 Alkenyl" or similar designations. 2~4"Alkenyl" indicates that there are 2 to 4 carbon atoms in the alkenyl chain, i.e., the alkenyl chain is selected from the group consisting of ethenyl, propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl, buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl, 1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl, buta-1,2-dienyl, and buta-1,2-dien-4-yl. Exemplary alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, and hexenyl.

[0033] Groups containing alkenyl groups include optionally substituted alkenyl, cycloalkenyl, and heterocycloalkenyl groups.

[0034] As used herein, "alkynyl" refers to a straight or branched hydrocarbon chain containing one or more triple bonds. An alkynyl group can have from 2 to 20 carbon atoms, although this definition also covers occurrences of the term "alkynyl" where no numerical range is specified. An alkynyl group can also be a medium sized alkynyl having from 2 to 9 carbon atoms. An alkynyl group can also be a lower alkynyl having from 2 to 4 carbon atoms. An alkynyl group can also be a "C 2~4 By way of example only, "C 2~4 alkynyl" or "C 2~4 "Alkynyl" indicates that there are 2 to 4 carbon atoms in the alkynyl chain, i.e., the alkynyl chain is selected from the group consisting of ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl, and 2-butynyl. Typical alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl.

[0035] Groups containing alkynyl groups include optionally substituted alkynyl, cycloalkynyl, and heterocycloalkynyl groups.

[0036] As used herein, "aryl" refers to an aromatic ring or ring system (i.e., two or more fused rings sharing two adjacent carbon atoms) that contains only carbon in the ring backbone. When aryl is a ring system, all rings in the system are aromatic rings. Aryl groups can have from 6 to 18 carbon atoms, although this definition also covers occurrences of the term "aryl" where no numerical range is specified. In some instances, aryl groups have from 6 to 10 carbon atoms. An aryl group is defined as "C 6~10 Aryl, C6 or C 10 Examples of aryl groups include, but are not limited to, phenyl, naphthyl, azulenyl, and anthracenyl.

[0037] As used herein, "heterocycle" refers to a cyclic compound that contains carbon atoms along with another atom (heteroatom), such as nitrogen, oxygen, or sulfur. The heterocycle can be aromatic (heteroaryl) or aliphatic. An aliphatic heterocycle can be fully saturated or can contain one or more double bonds, e.g., the heterocycle can be a heterocycloalkyl. The heterocycle can contain a single heterocycle or multiple fused heterocycles.

[0038] As used herein, "heteroaryl" refers to an aromatic ring or ring system (i.e., two or more fused rings sharing two adjacent atoms) containing one or more heteroatoms, i.e., elements other than carbon, including but not limited to nitrogen, oxygen, and sulfur, in the ring backbone. When heteroaryl is a ring system, all rings in the system are aromatic rings. Heteroaryl groups can have 5 to 18 ring members (i.e., the number of atoms that make up the ring backbone, including carbon atoms and heteroatoms), although this definition also covers occurrences of the term "heteroaryl" where no numerical range is specified. In some examples, heteroaryl groups have 5 to 10 ring members or 5 to 7 ring members. Heteroaryl groups can be designated as "5-7 membered heteroaryl," "5-10 membered heteroaryl," or similar designations. Examples of heteroaryl rings include, but are not limited to, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothienyl.

[0039] As used herein, "cycloalkyl" means a fully saturated carbocyclyl ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0040] As used herein, "cycloalkenyl" or "cycloalkene" refers to a carbocyclyl ring or ring system having at least one double bond, and none of the rings in the ring system are aromatic. An example is cyclohexenyl or cyclohexene. Another example is norbornene or norbornenyl.

[0041] As used herein, "heterocycloalkenyl" or "heterocycloalkene" refers to a carbocyclyl ring or ring system having at least one heteroatom in the ring backbone with at least one double bond, and none of the rings in the ring system are aromatic. In some examples, the heterocycloalkenyl or heterocycloalkene ring or ring system is 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered.

[0042] As used herein, "cycloalkynyl" or "cycloalkyne" means a carbocyclyl ring or ring system having at least one triple bond, wherein none of the rings in the ring system are aromatic. An example is cyclooctyne. Another example is biclononyne. Another example is dibenzocyclooctyne (DBCO).

[0043] As used herein, "heterocycloalkynyl" or "heterocycloalkyne" refers to a carbocyclyl ring or ring system having at least one heteroatom in the ring backbone, with at least one triple bond, and none of the rings in the ring system are aromatic. In some examples, the heterocycloalkynyl or heterocycloalkyne ring or ring system is 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered.

[0044] As used herein, "heterocycloalkyl" refers to a non-aromatic ring or ring system containing at least one heteroatom in the ring backbone. Heterocycloalkyls may be joined together in a fused, bridged, or spiro-linked fashion. Heterocycloalkyls may have any degree of saturation, provided that at least one heterocycloalkyl ring in the ring system is not aromatic. Heterocycloalkyl groups may have 3 to 20 ring members (i.e., the number of atoms that make up the ring backbone, including carbon atoms and heteroatoms), although this definition also covers occurrences of the term "heterocycloalkyl" where no numerical range is specified. Heterocycloalkyl groups may also be medium-sized heterocycloalkyls having 3 to 10 ring members. Heterocycloalkyl groups may also be heterocycloalkyls having 3 to 6 ring members. Heterocycloalkyl groups may be designated as "3 to 6 membered heterocycloalkyls" or similar designations. In some 6-membered monocyclic heterocycloalkyls, the heteroatoms are selected from one to three of O, N, or S. In some 5-membered monocyclic heterocycloalkyls, the heteroatoms are selected from 1 or 2 heteroatoms selected from O, N, or S.Examples of heterocycloalkyl rings include azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiapanyl, piperidinyl, piperazinyl, dioxapiperazinyl, pyrrolidinyl, pyrrolidionyl, pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxathinyl, 1,4-oxathinyl, 1,4-oxathiyl, 2H-1,2-oxazinyl, trioxanyl, hexahydro-1, Examples include, but are not limited to, 3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithiolyl, 1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolidinonyl, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl, thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, and tetrahydroquinoline.

[0045] As used herein, a substituent is derived from an unsubstituted parent group in which one or more hydrogen atoms have been replaced by another atom or group. Unless otherwise indicated, when a group is deemed to be "substituted", it means that the group is substituted with one or more substituents independently selected from the following: C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 heteroalkyl, C3-C7 carbocyclyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), C3-C7-carbocyclyl-C1-C6-alkyl ...). 5-10 membered heterocyclyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 5-10 membered heterocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), aryl (halo, C1 aryl(C1-C6)alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy); aryl(C1-C6)alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy); 5-10 membered heteroaryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy); 5-10 membered heteroaryl halo(C1-C6)alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), halo, cyano, hydroxy, C1-C6 alkoxy, C1-C6 alkoxy(C1-C6)alkyl (i.e., ether), aryloxy, sulfhydryl (mercapto), halo(C1-C6)alkyl (e.g., -CF3), halo(C1-C6)alkoxy (e.g., -OCF3), C1-C6 alkylthio, arylthio, amino,Amino (C1-C6) alkyl, nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, acyl, cyanato, isocyanato, thiocyanato, isothiocyanato, sulfinyl, sulfonyl, and oxo (=O). When a group is described as being "optionally substituted," if the group is substituted, it can be substituted with the above-listed substituents.

[0046] When the compounds disclosed herein have at least one stereocenter, they may exist as individual enantiomers or diastereomers, or as mixtures of such isomers, including racemates. Separation of individual isomers or selective synthesis of individual isomers can be achieved by application of various methods well known to those skilled in the art. When it is understood that the compounds disclosed herein exist in tautomeric forms, all tautomeric forms are included within the scope of the structures shown. Unless otherwise indicated, all such isomers and mixtures thereof are included within the scope of the compounds disclosed herein. Furthermore, the compounds disclosed herein may exist in one or more crystalline or amorphous forms. Unless otherwise indicated, all such forms are included within the scope of the compounds disclosed herein, including any polymorphic forms. In addition, some of the compounds disclosed herein may form solvates with water (i.e., hydrates) or common organic solvents. Unless otherwise indicated, such solvates are included within the scope of the compounds disclosed herein.

[0047] As used herein, the term "nucleotide" is intended to mean a molecule that includes a sugar and at least one phosphate group, and in some instances also includes a nucleobase. A nucleotide that lacks a nucleobase may be referred to as "abasic." Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (GT ... deoxyadenosine monophosphate (UTP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), and deoxythymidine monophosphate (DTMP).These include deoxythymidine diphosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), and deoxyuridine triphosphate (dUTP).

[0048] As used herein, the term "nucleotide" is also intended to encompass any nucleotide analog, which is a type of nucleotide that contains a modified nucleobase, sugar and / or phosphate moiety as compared to naturally occurring nucleotides. Exemplary modified nucleobases include inosine, xanthate, hypoxanthate, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil ... These include cytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyl adenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, and the like. As is known in the art, certain nucleotide analogs cannot become incorporated into polynucleotides, such as nucleotide analogs such as adenosine 5'-phosphosulfate. A nucleotide can include any suitable number of phosphates, such as 3, 4, 5, 6, or more than 6 phosphates.

[0049] As used herein, the term "polynucleotide" refers to a molecule that comprises a sequence of nucleotides that are linked together. A polynucleotide is a non-limiting example of a polymer. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), and analogs thereof. A polynucleotide may be a single-stranded sequence of nucleotides, such as RNA or single-stranded DNA, a double-stranded sequence of nucleotides, such as double-stranded DNA, DNA that folds to form a hairpin that is partially single-stranded and partially double-stranded, a double-stranded fusion in which there are molecules that are non-covalently linked to each other (e.g., via reversible hydrogen bonds), and / or may comprise a mixture of single-stranded and double-stranded sequences of nucleotides. Double stranded DNA (dsDNA) includes genomic DNA, and PCR and amplification products. Single stranded DNA (ssDNA) can be converted to dsDNA and vice versa. Polynucleotides can include non-naturally occurring DNA such as enantiomeric DNA. The exact sequence of nucleotides in a polynucleotide can be known or unknown. The following are examples of polynucleotides: genes or gene fragments (e.g., probes, primers, expressed sequence tags (ESTs), or serial analysis of gene expression (SAGE) tags), genomic DNA, genomic DNA fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, synthetic polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers, or amplified copies of any of the foregoing.

[0050] As used herein, the term "target polynucleotide" is intended to mean a polynucleotide that is the subject of an analysis or action. The analysis or action includes subjecting the polynucleotide to amplification, sequencing, and / or other procedures. The target polynucleotide may include additional nucleotide sequences to the target sequence being analyzed. For example, the target polynucleotide may include one or more adapters, including adapters that function as primer binding sites, that flank the target polynucleotide sequence being analyzed. The target polynucleotide hybridized to the capture primer may include nucleotides that extend beyond the 5' or 3' end of the capture oligonucleotide, such that not all of the target polynucleotide is suitable for extension. In certain examples, multiple target polynucleotides may have first and second adapters that are the same as each other, although they may have different sequences from each other. Two adapters that may flank a particular target polynucleotide sequence may have the same sequence as each other, or complementary sequences to each other, or the two adapters may have different sequences. Thus, a species in a plurality of target polynucleotides may include, for example, a region of known sequence flanked by regions of unknown sequence that are evaluated by sequencing (e.g., SBS). In some examples, the target polynucleotide carries an adapter at a single end, and such an adapter may be located at either the 3' or 5' end of the target polynucleotide. The target polynucleotide may be used without an adapter, in which case the primer binding sequence may directly use a sequence present in the target polynucleotide.

[0051] The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein. The difference in the terminology is not intended to indicate any particular difference in size, sequence, or other properties, unless otherwise specified. For clarity of explanation, when describing a particular method or composition that includes several polynucleotide species, different terms may be used to distinguish one species of polynucleotide from another species.

[0052] As used herein, "polymerase" is intended to mean an enzyme having an active site that assembles polynucleotides by polymerizing nucleotides into polynucleotides. A polymerase can bind to a primed single-stranded target polynucleotide and grow it by successively adding nucleotides to the primer to form a "complementary copy" polynucleotide with a sequence complementary to that of the target polynucleotide. Another polymerase, or the same polymerase, can then form a copy of the target nucleotide by forming a complementary copy of the complementary copy polynucleotide. A DNA polymerase can bind to a target polynucleotide and then move downstream of the target polynucleotide while it grows it by successively adding nucleotides to the free hydroxyl group at the 3' end of the polynucleotide chain (amplicon growth). A DNA polymerase can synthesize a complementary DNA molecule from a DNA template, and an RNA polymerase can synthesize an RNA molecule from a DNA template (transcription). A polymerase can use a short RNA or DNA strand (primer) to initiate strand growth. Some polymerases can displace the strand upstream of the site where they add a base to the strand. Such polymerases may also be said to be strand displacing, which means they have the activity of removing the complementary strand from the template strand that is read by the polymerase. Exemplary polymerases with strand displacement activity include, but are not limited to, Bst (Bacillus stearothermophilus) polymerase, exo-Klenow polymerase, or a large fragment of sequencing grade T7 exo-polymerase. Some polymerases cleave the strand in front of them and effectively replace it with the growing strand (5' exonuclease activity). Some polymerases have the activity of degrading the strand behind them (3' exonuclease activity). Some useful polymerases have been mutated or otherwise modified to reduce or eliminate 3' and / or 5' exonuclease activity.

[0053] As used herein, the term "primer" is defined as a polynucleotide to which nucleotides can be added via a free 3'OH group. The length of the primer can be any suitable number of bases long and can include a suitable combination of natural and / or non-natural nucleotides. The target polynucleotide can include an "adapter" that hybridizes to the primer (having a sequence complementary to the primer) and can be amplified to generate a complementary copy polynucleotide by adding a nucleotide to the free 3'OH group of the primer. "Capture primer" refers to a primer that is bound to a substrate. In some examples, the capture primer is a P5 and P7 primer commercially available from Illumina, Inc. (San Diego, CA). In some examples, the primer (such as the primer, or the P5 or P7 primer) includes a linker or spacer at the 5' end. Such a linker or spacer can be included to allow chemical or enzymatic cleavage or to impart some other desired property, for example, to allow covalent attachment to the substrate or to act as a spacer to position the cleavage site at an optimal distance from the solid support. In certain cases, 10 spacer nucleotides may be positioned between the attachment points of the P5 or P7 primer to the polymer or solid support. In some examples, a poly-T spacer is used, although other nucleotides and combinations thereof may also be used. In one example, the spacer is a 6T-10T spacer. In some examples, the linker includes a cleavable nucleotide that includes a chemically cleavable functional group, such as a vicinal diol or an allyl T.

[0054] As used herein, the term "amplicon" when used in reference to a nucleic acid refers to a product of the replication of a nucleic acid, which product has a nucleotide sequence that is substantially the same as or complementary to at least a portion of the nucleotide sequence of the nucleic acid. "Amplification" and "amplifying" refer to the process of creating an amplicon of a polynucleotide. A first amplicon of a target polynucleotide can typically be a complementary copy. Additional amplicons are copies made from the target polynucleotide or the first amplicon after the generation of the first amplicon. Subsequent amplicons can have a sequence that is substantially complementary to or substantially identical to the target polynucleotide. It will be understood that a small number of mutations of a polynucleotide (e.g., due to amplification artifacts) may occur when generating an amplicon of that polynucleotide.

[0055] As used herein, the term "silane" refers to an organic or inorganic compound containing one or more silicon atoms. A non-limiting example of an inorganic silane compound is SiH4 or a halogenated SiH4 in which hydrogen is replaced by one or more halogen atoms. A non-limiting example of an organosilane compound is XR C -Si(OR D )3, where X is a non-hydrolyzable organic group such as amino, vinyl, epoxy, methacrylate, sulfur, alkyl, alkenyl, or alkynyl. R C is a spacer, e.g., -(CH2) n wherein n is 0 to 1000, and each R D are independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted 5-10 membered heteroaryl, and optionally substituted 5-10 membered heterocyclyl, as defined herein. In some examples, the silane is selected from XR C -Si(OR D )3-OR DThe oxygen atom of the group is adjacent to the organosilane compound XR C -Si(OR D )3. Additionally, the silane compound can be crosslinked to attach to the silicon atom of the surface. C -Si(OR D ) three moieties may be covalently attached to the substrate surface. Thus, in some examples, the described silanes may have the following structure:

[0056] [ka]

[0057] As used herein, the term "silane" can include a mixture of different silane compounds. In some examples, X is a norbornenyl group. In some examples, X is a bicyclononyl group. In some examples, X is an alkene or alkyne-containing group. In some examples, X is an alkene or alkyne. In some examples, R C The linker is C 2~6 It is an alkylene group.

[0058] As used herein, the term "substrate" refers to a material that includes a solid support. The substrate may include a polymer that defines a solid support or is disposed on a solid support. Exemplary substrate materials may include glass, silica, plastic, quartz, metal, metal oxide, organo-silicates (e.g., polyhedral organic silsesquioxanes (POSS)), polyacrylates, tantalum oxide, complementary metal oxide semiconductor (CMOS), or combinations thereof. An example of a POSS may be that described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776-778, which is incorporated herein by reference in its entirety. Illustratively, POSS-containing monomers can be polymerized to rapidly reach a gel point to provide a POSS resin (a polymer functionalized to include POSS), on which functionalization of soft materials can be performed. In some examples, the substrate used in this application includes a silica-based substrate, such as glass, fused silica, or other silica-containing materials. In some examples, the substrate may include silicon, silicon nitride, or hydrogenated silicone. In some examples, the substrate used in this application includes plastic materials or components such as polyethylene, polystyrene, poly(vinyl chloride), polypropylene, nylon, polyester, polycarbonate, and poly(methyl methacrylate). Examples of plastic materials include poly(methyl methacrylate), polystyrene, and cyclic olefin polymer substrates. In some examples, the substrate is or includes a silica-based material or a plastic material, or a combination thereof. In certain examples, the substrate has at least one surface that includes glass or a silicon-based polymer. In some examples, the substrate may include a metal. In some such examples, the metal is gold. In some examples, the substrate has at least one surface that includes a metal oxide. In one example, the surface includes tantalum oxide or tin oxide.Acrylamides, enones, or acrylates may also be utilized as substrate materials or components. Other substrate materials may include, but are not limited to, gallium arsenide, indium phosphide, aluminum, ceramic, polyimide, quartz, resins, polymers, quartz, resins, polymers, and copolymers. In some examples, the substrate and / or substrate surface may be or include quartz. In some other examples, the substrate and / or substrate surface may be or include a semiconductor, such as GaAs or ITO. The above list is intended to illustrate, but not limit, the present application. The substrate may include a single material or multiple different materials. The substrate may be a composite or laminate. In some examples, the substrate includes an organosilicate material. The substrate may be flat, round, spherical, rod-shaped, or any other suitable shape. The substrate may be rigid or flexible. In some examples, the substrate is a bead or a flow cell.

[0059] In some examples, the surface is a patterned surface. A "patterned surface" refers to an arrangement of different regions in or on an exposed layer of a substrate. For example, one or more of the regions can be features in which one or more capture primers are present. The features can be separated by gap regions in which no capture primers are present. In some examples, the pattern can be an xy format of features in rows and columns. In some examples, the pattern can be a repeating arrangement of features and / or gap regions. In some examples, the pattern can be a random arrangement of features and / or gap regions. In some examples, the substrate comprises an array of wells (recesses) on the surface. The wells can be provided with substantially vertical sidewalls. In some examples, the substrate comprises an array of posts (protrusions) on the surface. The wells and posts can be fabricated as commonly known in the art using a variety of techniques, including but not limited to photolithography, stamping techniques, molding techniques, nanoimprint lithography, and microetching techniques. As will be appreciated in the art, the techniques used will depend on the composition and shape of the array substrate. Illustratively, posts having a diameter of about 50 nm to about 500 nm may be referred to as nanoposts and may have a height of similar dimensions to the diameter.

[0060] The features within the patterned surface of the substrate may include wells (e.g., wells such as microwells or nanowells, or posts such as nanoposts) patterned with covalently attached gels such as poly(N-(5-azidoacetamylpentyl)acrylamide-co-acrylamide) (PAZAM) on glass, silicon, plastic, or other suitable materials in an array. This process creates a gel pad used for sequencing, which may be stable over many cycles of sequencing operations. Covalently attaching the polymer to the wells may be useful to maintain the gel on the structured features throughout the life of the structured substrate during various applications. However, in many instances, the gel does not need to be covalently attached to the wells. For example, in some conditions, silane free acrylamide (SFA), which is not primarily covalently attached to any portion of the structured substrate, may be used as the gel material.

[0061] In a particular example, a structured substrate can be created by patterning a suitable material with wells (e.g., microwells or nanowells), coating the patterned material with a gel material (e.g., PAZAM, SFA, or chemically modified variants thereof, such as the azido form of SFA (azido-SFA)), and polishing the gel-coated material, for example, by chemical or mechanical polishing, thereby retaining the gel in the wells but removing or inactivating substantially all of the gel from the interstitial regions of the surface of the structured substrate between the wells. Primers can then be bound to the gel material. A solution containing a plurality of target polynucleotides (e.g., a fragmented human genome or a portion thereof) can then be contacted with the polished substrate such that individual target polynucleotides are seeded into individual wells through interaction with primers attached to the gel material, but the target polynucleotides do not occupy the interstitial regions due to the absence or inactivity of the gel material. Amplification of the target polynucleotides can be confined to the wells due to the absence or inactivity of the gel in the interstitial regions, preventing outward migration of clusters. The process is easily manufacturable, scalable, and utilizes conventional micro- or nano-fabrication methods.

[0062] Patterned substrates may include, for example, etched wells in a slide or chip. The pattern of well etching and geometry may be of a variety of different shapes and sizes, and such features may be physically or functionally separable from one another. Particularly useful substrates with such structural features include patterned substrates that allow selection of solid particle sizes, such as microspheres. An example of a patterned substrate with these properties is the etched substrate used in conjunction with BEAD ARRAY technology (Illumina, Inc., San Diego, Calif.). Nano-imprint lithography (NIL) may be used to provide the wells.

[0063] In some examples, the substrates described herein form at least a part of a flow cell, are located within a flow cell, or are coupled to a flow cell. A flow cell may include a flow chamber that is divided into multiple lanes or multiple sectors. Examples of flow cells and substrates for the manufacture of flow cells that can be used in the methods and compositions described herein include, but are not limited to, those commercially available from Illumina, Inc. (San Diego, CA).

[0064] As used herein, the term "fluidic channel" refers to an elongated, at least partially enclosed structure through which a fluid may flow, e.g., through which a fluid may be directed. A fluidic channel may have a length, a width, and a height. The width and height together may define a cross-sectional area of ​​the fluidic channel. The cross-section of the fluidic channel may have any suitable shape, e.g., fully curved, partially curved, fully polygonal, or partially polygonal. Illustratively, the cross-section of the fluidic channel may be circular, elliptical, square, rectangular, etc. The fluid may substantially fill the cross-sectional area of ​​the fluidic channel. The fluid may flow along the length of the fluidic channel. The fluidic channel may be formed by a cover bonded to a substrate. A flow cell is a non-limiting example of a fluidic channel.

[0065] As used herein, the term "fluidic device" refers to a device that includes at least one fluid channel, and can optionally include multiple fluid channels.

[0066] As used herein, the term "cover" refers to a substrate that can be bonded to another substrate to form a fluid channel. Thus, the cover can include any of the materials described elsewhere herein that can be included in the substrate. The cover can include the same material as the substrate to which it is bonded, or can include one or more materials that are different from the substrate to which it is bonded. The cover can be bonded directly to the substrate or to an intervening layer that is bonded to the substrate. Although regions of the cover may be described and illustrated as being "over" the substrate, this is intended only to mean that the cover and substrate are spaced apart from one another, and does not imply a particular spatial orientation of the cover relative to the substrate. The cover can include a recess configured such that when the cover is bonded to the substrate, the recess is spaced apart from the substrate to provide a fluid channel. Conversely, the substrate can include a recess configured such that when the cover is bonded to the substrate, the recess is spaced apart from the substrate to provide a fluid channel.

[0067] As used herein, the term "intervening layer" refers to an element that can be bonded to the substrate and the cover to bond the substrate to the cover. The intervening layer can include or consist essentially of a polymer.

[0068] As used herein, the term "polymer" refers to a molecule that includes many repeating subunits or units. Non-limiting examples of polymeric structures include linear, branched, or hyperbranched polymers. Non-limiting examples of linear polymers include block copolymers or random / statistical copolymers. Non-limiting examples of branched polymers include star polymers, star or star block polymers that contain both hydrophobic and hydrophilic segments, H-shaped polymers that contain both hydrophobic and hydrophilic segments, dumbbell polymers, comb polymers, brush polymers, dendronized polymers, ladders, and dendrimers. The polymers may be crosslinked or lightly crosslinked. The polymers described herein may be linear, branched, hyperbranched, or dendritic. The polymers described herein may be in the form of polymer nanoparticles. Other examples of polymer architectures include, but are not limited to, ring block polymers and coil-ring-coil polymers. Polymers with two or more types of repeating units may be arranged as block copolymers, random copolymers, or alternating copolymers, or mixtures thereof. The final copolymer structure may be of different architectures, including, for example, random copolymers, block copolymers, comb polymers, or star polymer architectures. Different classes of polymer backbones include, but are not limited to, polyacrylamides, polyacrylates, polyurethanes, polysiloxanes, silicones, polyacroleins, polyphosphazenes, polyisocyanates, polyols, polysaccharides, polypeptides, and combinations thereof. In some examples, the polymer comprises a polyacrylamide backbone. In some other examples, the polymer comprises a polyacrylate backbone. In yet some other examples, the polymer comprises a polyurethane backbone. In yet some other examples, the polymer comprises a polyphosphazene backbone. In yet some other examples, the polymer comprises a dendrimer backbone. The polymer may comprise one or more moieties that can react with one or more other moieties to form covalent bonds.

[0069] As used herein, the term "adduct" is intended to mean the product of a chemical reaction between two or more molecules, where the product contains all of the atoms of the reacted molecules.

[0070] As used herein, the term "linker" is intended to mean a molecule or molecules that attach one element to another. For example, a linker may attach a molecule to a substrate. A linker may be covalent or non-covalent. Non-limiting examples of covalent linkers include alkyl chains, polyethers, amides, esters, aryl groups, polyaryls, and the like. Non-limiting examples of non-covalent linkers include host-guest complexation, cyclodextrin / norbornene, adamantane ring with β-CD, complexation, DNA hybridization interactions, streptavidin / biotin, and the like.

[0071] As used herein, the term "mask" is intended to mean a first element that prevents a second element from chemically reacting.

[0072] As used herein, the term "selectively" is intended to mean substantially affecting only the object of an action that is "selectively" performed on the object.

[0073] Fluidic device including a fluid channel and method for making same As noted above and described in more detail below, the fluidic device can include fluidic channels formed using covalent bonds between moieties covalently bonded to the substrate and moieties covalently bonded to the cover. In some examples, the moieties undergo an azide-alkyne [3+2] cycloaddition reaction, which can be carried out without the use of catalysts or solvents, although it will be understood that catalysts and / or solvents, and / or other reactions between the moieties can be used. For example, a thermally driven non-click reaction (e.g., an epoxyamine reaction) can be used to covalently bond the substrate to the cover directly or through an intervening layer.

[0074] 1A-1D are schematic illustrations of structures and operations in an exemplary process for fabricating a fluid channel. Referring now to FIG. 1A, the substrate 110 may include a first region 111 and a second region 112. The cover 120 may include a first region 121 and a second region 122. The first region 111 of the substrate 110 may be bonded to a first portion 113. The first region 121 of the cover 120 may be bonded to a second portion 123. The first region 121 of the cover 120 may be covalently bonded to the first region 111 of the substrate 110 using the first portion 113 and the second portion 123. The covalent bond between the first region 111 of the substrate 110 and the first region 121 of the cover 120 may suspend the second region 122 of the cover 120 above the second region 112 of the substrate 110 to form a fluid channel.

[0075] The first portion 113 and the second portion 123 may be used to directly bond the first region 111 of the substrate 110 to the first region 121 of the cover 120, for example in a manner illustrated in FIG. 1B, or alternatively may be used to indirectly bond the first region 111 of the substrate 110 to the first region 121 of the cover 120, for example via an intervening layer, in a manner further described below with reference to FIGS. 4A-4B. Illustratively, in the non-limiting example shown in FIG. 1B, the first portion 113 may react directly with the second portion 123 to covalently bond the first region 111 of the substrate to the second region 121 of the cover 120. It will be understood that any suitable first portion 113 may react with any suitable second portion 123. FIG. 1C illustrates a fluidic device 100 including a fluidic channel 101 resulting from a reaction between the first portion 113 and the second portion 123. Non-limiting examples of moieties 113, 123 are provided below. Exemplary methods and chemical groups that can be used to attach the first moiety 113 to the substrate 110 and the second moiety 123 to the cover 120 are described with reference to Figures 5A-5B.

[0076] As intended to be suggested by the downward arrow in FIG. 1B, covalently bonding the first region 111 of the substrate 110 to the first region 121 of the cover 120 may include (i) selectively applying heat to the first region of the substrate or the first region of the cover, (ii) applying pressure to the first region of the substrate and the first region of the cover, or (iii) applying both heat and pressure. Heat, for example, may increase the rate at which the first region 111 of the substrate 110 becomes covalently bonded to the first region 121 of the cover 120. Heat may be applied using light, or any other heat source. Illustratively, the light may include or consist essentially of one or more infrared or near-infrared wavelengths. For example, a collimated light source (such as a laser or light emitting diode) may be robotically and / or optically controlled to illuminate substantially only the first region 111 and / or the first region 121. The pressure can be applied, for example, by holding the substrate 110 in place and pressing the cover 120 against the substrate 110, either by robotically or manually, or by holding the cover 120 in place and pressing the substrate 110 against the cover 120, either by robotically or manually.

[0077] As illustrated in Figures 1A-1C, the second region 112 of the substrate 110 may optionally include oligonucleotides 114, 115, such as capture primers having orthogonal sequences. Any such oligonucleotides 114, 115 may be optionally protected using a mask 132 during the reaction between the portions 113, 123. The mask 132 may be removed to complete the preparation of the fluidic device 100 illustrated in Figure 1C. The fluidic channels provided by the second regions 112, 122 may be used to carry fluids across any oligonucleotides 114, 115 that may be bound to the second region of the substrate. Such fluids may include, for example, target polynucleotides to be amplified using the oligonucleotides 114, 115, as well as polymerases and nucleotides used during such amplification. Examples of methods for attaching oligonucleotides 114, 115 to second region 112 of substrate 110 in a manner compatible with attaching substrate 110 to cover 120 are described with reference to Figures 2A-2E, 3A-3E, and 7A-7E. However, it will be understood that any other elements may be attached to second region 112 of substrate 110 and / or any other surface within device 100, such as second region 122 of cover 120 and / or sidewalls 123, 124 that attach first region 121 of cover 120 to second region 122 of cover.

[0078] In the non-limiting example illustrated in FIG. 1C, the second region 122 of the cover 120 may be recessed relative to the first region 121 and coupled to the first region via sidewalls 123, 124, while the second region 112 of the substrate 110 may be substantially planar with the first region 111 (FIG. 1A). However, it will be understood that in other examples (not specifically illustrated), the second region 112 of the substrate 110 may instead be recessed relative to the first region 111 via similar sidewalls, while the second region 122 of the cover 120 may be substantially planar with the first region 121. In yet other examples (not specifically illustrated), the second region 112 of the substrate 110 may be recessed relative to the first region 111 via similar sidewalls, and the second region 122 of the cover 120 may be recessed relative to the first region 121 via similar sidewalls. In any such configuration, when the first regions 111, 121 are directly or indirectly coupled to one another in the manner as provided herein, the recess or recess and sidewall together may provide a space at least partially surrounding the second regions 112, 122. Such a space may provide a fluid channel 101 that may be used to convey fluid across the second region 112 of the substrate 110. The second region 122 of the cover 120 may be separated from the second region 112 of the substrate 110 by any suitable distance, for example, about 1 m to about 1 cm, for example, about 1 μm to about 500 μm, or about 10 μm to about 100 μm. The sidewalls 123, 124 may be separated from one another by any suitable distance, for example, about 1 μm to about 1 cm, for example, about 1 μm to about 500 μm, or about 10 μm to about 100 μm.

[0079] In some examples, a plurality of such recesses and sidewalls are provided in the substrate 110 and / or the cover 120 to form a plurality of fluid channels 101, such as a plurality of flow cells. For example, FIG. 1D illustrates a plan view of an assembly between the substrate 110 and the cover 120 that includes a plurality of such fluid channels 101 in which fluids can flow independently in a manner intended as suggested by the upward arrows. The fluid device 100 illustrated in FIG. 1C can illustrate a cross-section designated as "FIG. 1C" in FIG. 1D. However, it will be understood that the fluid device 100 illustrated in FIG. 1C need not necessarily be part of an assembly as illustrated in FIG. 1C.

[0080] In some examples, the reaction between the first portion 113 and the second portion 123 as illustrated in FIG. 1B includes an azide-alkyne [3+2] cycloaddition, which is also referred to as a Huisgen cycloaddition. Thus, the first region 111 can covalently bond to the first region 121 via the product of such an azide-alkyne [3+2] cycloaddition reaction. In some examples, one of the first portion 113 and the second portion 123 may include an azide (N3), and the other of the first portion 113 and the second portion 123 may include a dibenzocyclooctyne (DBCO) having the following structure:

[0081] [Chemical formula] wherein one of R1 and R2 is H and the other is a bond to the substrate 110 (when the first portion 113 includes DBCO) or the cover 120 (when the second portion 123 includes DBCO), and wherein when R2 is not directly bonded to X, X is CH2, O, S, or NH, and when R2 is directly bonded to X, X is CH or N. The azide can be bonded to the substrate 110 (when the first portion 113 includes the azide) or the cover 120 (when the second portion 123 includes the azide) via a suitable bond such as an alkyl. The azide can react with the dibenzocyclooctyne to form a cyclic adduct having the following structure:

[0082] [ka] where R3 is a bond to the substrate 110 (if the second portion 123 comprises DBCO) or the cover 120 (if the first portion 113 comprises DBCO). Such reactions, or other azide-alkyne [3+2] cycloaddition reactions, can optionally be carried out without the use of solvents and / or catalysts. Additionally or alternatively, the azide-alkyne [3+2] cycloaddition reactions can optionally be facilitated using heat (e.g., from light) and / or pressure in a manner such as that described with reference to FIG. 1B.

[0083] It will be understood that DBCO represents a non-limiting example of an alkyne that may be used in the azide-alkyne [3+2] cycloaddition reaction between the first moiety 113 and the second moiety 123. It will also be understood that the azide-alkyne [3+2] cycloaddition reaction represents a non-limiting example of a suitable reaction between the first moiety 113 and the second moiety 123 for covalently bonding the first region 111 of the substrate 110 to the first region 121 of the cover 120. Other examples of alkynes that may be used in the azide-alkyne [3+2] cycloaddition reaction between the first moiety 113 and the second moiety 123 include bicyclononyne (BCN) or a derivative thereof, difluorocyclooctyne (DIFO) or a derivative thereof, dibenzocyclooctyne (DIBO) or a derivative thereof, and the like. Some non-limiting examples of strained cyclooctynes ​​that can be used in the azide-alkyne [3+2] cycloaddition reaction between the first moiety 113 and the second moiety 123 include the following, where R represents the connection to the substrate or cover:

[0084] [ka]

[0085] For further details regarding exemplary reactions between cycloalkynes and azides that may be adapted for use in the present devices and methods, see Dommerholt et al., "Strain-promoted 1,3-dipolar cycloaddition of cycloalkines and organic azides," Top. Curr. Chem. (Z) 374:16, 20 pages (2016), the entire contents of which are incorporated herein by reference. However, any surface-bound reactant that forms a bond via a cycloaddition reaction can be used, such as aryl azides and pentafluoroalkynes. Surface-bound reactants that form bonds via other types of addition reactions, such as the thermally driven reaction of primary amines with epoxy groups, are also understood.

[0086] As described above with reference to Figures 1A-1C, the oligonucleotides 114, 115 can be bound to the second region 112 of the substrate 110 in such a manner that they contact the fluid flowing through the fluidic channel 101. Such oligonucleotides can be bound to the second region 112 in any suitable manner and at any suitable time with respect to binding to the cover 120 of the substrate 110. Figures 2A-2E illustrate the structures and operations in an exemplary process for providing oligonucleotides in a fluidic channel. Referring now to Figure 2A, the first region 111 and the second region 112 of the substrate 110 can be bound to the first portion 113. To inhibit premature reaction of the first portion 113 in the first region 111 of the substrate 110, the first region 111 can be protected using a mask 211, such as a partially opaque material patterned in any suitable manner, for example using photolithography, spray coating with a stencil, inkjet printing, aerosol printing, etc. In one non-limiting example, the mask 211 comprises a photoresist patterned by photolithography. As illustrated in FIG. 2B, the substrate 110 can then be contacted with a fluid 220 comprising oligonucleotides 114, 115 each bound to a second portion 123. Optionally, the oligonucleotides 115 comprise respective cleavage portions 116, such as 8-oxo-G, which can be cleaved under suitable conditions, such as UV light, chemistry, enzymes, etc. As illustrated in FIG. 2C, the second portions 123 bound to the respective oligonucleotides 114, 115 can react with portions 113 in the second region 112, thus covalently binding the oligonucleotides to the second region 112. The mask 211 can then be suitably removed in a manner as illustrated in FIG. 2D. A mask 132, such as a photolithographically patterned coating, can then be applied over the oligonucleotides 114, 115 in the second region 112 in a manner illustrated in FIG. 2E to protect the oligonucleotides before the first region 111 of the substrate 110 is covalently bonded to the first region 121 of the cover 120.After the first regions 111 of the substrate 110 are covalently bonded to the first regions 121 of the cover 120 in the manner described with reference to FIG. 1C, the mask 132 may be removed.

[0087] It will be understood that the second region 112 of the substrate 110 may be bonded to a portion other than the first portion 113. For example, FIGS. 3A-3E illustrate the structure and operation of another exemplary process for providing oligonucleotides in a fluidic channel. In an example such as that described with reference to FIGS. 3A-3E, the second region 112 of the substrate 110 may instead be bonded to a second portion 123 that is used to bond the oligonucleotides to the second region of the substrate. Referring now to FIG. 3A, the first region 111 and the second region 112 of the substrate 110 may be bonded to the first portion 113. To prevent premature reaction of the first portion 113 in the first region 111 of the substrate 110, the first region 111 may be protected with a mask 311, such as a photolithographically patterned photoresist or other patterned material as described with reference to the mask 211 illustrated in FIGS. 2A-2C. As illustrated in FIG. 3B, the polymer 312 may be disposed over and in contact with the mask 311 in the first region 111 and the moieties 113 in the second region 112. The polymer 312 may include a second moiety 123. Although not specifically illustrated in FIG. 3B, it will be understood that the second moiety 123 of the polymer 312 may react with the first moieties 113 in the region 112, thus covalently bonding the polymer to the region 112 of the substrate 110. Such reaction may be performed at any suitable time, for example, when or shortly after the polymer 312 is disposed over the moieties 113, or after the substrate 110 is bonded to the cover 120. In one non-limiting example, the polymer 312 may include a PAZAM that includes an azide moiety 123, and the moieties 113 bonded to the substrate 110 may include a DBCO moiety that reacts with the azide moiety to form a covalent bond. In another non-limiting example, substrate 110 may include a PAZAM that is deposited on a solid support and includes an azide moiety 113, and polymer 312 may include a DBCO moiety 123 that reacts with the azide moiety to form a covalent bond.

[0088] The substrate 110 may then be contacted with a fluid 320 including oligonucleotides 114, 115 each bound to a first portion 113 in a manner as illustrated in FIG. 3C. Optionally, the oligonucleotides 115 include respective cleavage portions 116, such as 8-oxo-G, that may be cleaved under suitable conditions, such as UV light, chemistry, enzymes, etc. As illustrated in FIG. 3D, the first portions 113 bound to the respective oligonucleotides 114, 115 may react with the second portions 123 of the polymer 312, thus binding the oligonucleotides to the second regions 112. The mask 311 may then be suitably removed in a manner as illustrated in FIG. 3E, thereby removing any portion of the polymer 312 disposed on such mask. A mask 132 may then be applied over the oligonucleotides 114, 115 in the second regions 112 in a manner as described with reference to FIG. 2E, to protect the oligonucleotides before the first region 111 of the substrate 110 is covalently bound to the first region 121 of the cover 120. After the first regions 111 of the substrate 110 are covalently bonded to the first regions 121 of the cover 120 in the manner described with reference to FIG. 1C, the mask 132 may be removed.

[0089] Although the examples as described with reference to Figures 2A-2E and 3A-3E include binding the oligonucleotides 114, 115 to the second region 112 of the substrate 110 before the first region 111 of the substrate is bound to the first region 121 of the cover, it will be understood that the oligonucleotides may alternatively be bound to the second region of the substrate after the first region of the substrate is bound to the first region of the cover. For example, the second region 112 of the substrate 110 may be bound to a portion that may be contacted with a fluid, including oligonucleotides bound to a portion that may react with a substructure in the second region 112, after the first regions 111, 121 are bound to each other. Illustratively, Figures 7A-7G generally illustrate the structure and operation in another exemplary process for creating a fluid channel. Referring now to Figure 7A, the first region 111 and the second region 112 of the substrate 110 may be bound to a first portion 113. To prevent premature reaction of the first portions 113 in the second regions 112 of the substrate 110, the second regions 112 may be protected with a mask 711, such as a photolithographically patterned photoresist or other patterned material as described with reference to the mask 211 illustrated in Figures 2A-2C.

[0090] As illustrated in FIG. 7B, the unprotected first portions 113 in the first region 111 can be removed, while the first portions 113 in the region 112 remain protected by the mask 711. As illustrated in FIG. 7C, another set of first portions 713 can be bonded to the first region 111 from which the first portions 113 were removed. As illustrated in FIG. 7D and FIG. 7E, the second portions 713 bonded to the cover 120 can be reacted with the first portions 123 to covalently bond the cover to the substrate 110. As illustrated in FIG. 7F, the mask 711 can be removed to expose the first portions 113 in the region 112. The substrate 110 can then be contacted with a fluid containing oligonucleotides 114, 115 each bonded to a moiety (such as the portion 713) that reacts with a moiety that bonds the oligonucleotide to the second region 112, in a manner similar to that described with reference to FIG. 3C. In one non-limiting example, the substrate 110 can include a layer of PAZAM, the first portion 113 can include an azide of PAZAM, the first portion 713 structure can include a strained cycloalkyne bonded to the substrate 110 using silanization, and the second portion 123 can include the azide.

[0091] It will also be understood that Figures 1A-1D illustrate only one example of how the first region 121 of the cover 120 can be covalently bonded to the first region 111 of the substrate 110 using the first portion 113 and the second portion 123. Other examples illustratively can include disposing an intervening layer between the first region of the substrate and the first region of the cover. Covalently bonding the first region of the substrate to the first region of the cover can include covalently bonding the first region of the substrate to the intervening layer and covalently bonding the first region of the cover to the intervening layer. For example, Figures 4A-4B generally illustrate the structure and operation in another exemplary process for creating a fluid channel. Now referring to Figure 4A, the substrate 110 can include a first region 111 and a second region 112, and the cover 120 can include a first region 121 and a second region 122. The first region 111 of the substrate 110 may be bonded to the first portion 113, and the first region 121 of the cover 120 may also be bonded to the first portion 113. As illustrated in FIG. 4A , an intervening layer 430 (e.g., a polymer) may be disposed between the first region 111 of the substrate 110 and the first region of the cover 120. The intervening layer 430 may include the second portion 123. Alternatively, the first region 111 of the substrate 110 may be bonded to the second portion 123, and the first region 121 of the cover 120 may also be bonded to the second portion 123, and the intervening layer 430 may include the first portion 113. In any such example, the first region 121 of the cover 120 may be covalently bonded to the first region 111 of the substrate 110 using the first portion 113 and the second portion 123, and the covalent bond between the first region 111 of the substrate 110 and the first region 121 of the cover 120 may suspend the second region 122 of the cover 120 above the second region 112 of the substrate 110 to form a fluidic channel. For example, in a manner as illustrated in FIG. 4B, the substrate 110 and the first portion 113 of the cover 120 may react with the second portion 123 of the intervening layer 430 to provide a device 100 including a fluidic channel 101′ similar to that described with reference to FIG. 1C. It will be understood that any suitable number of such fluidic channels 101′ may be formed to provide an assembly as described with reference to FIG. 1D.

[0092] Moieties 113 and 123 may be bonded to the substrate, cover, and / or polymer (such as the intervening layer) in any suitable manner. Illustratively, first moiety 113 or second moiety 123 may be covalently bonded to substrate 110, cover 120, intervening layer 430, and / or polymer 312 via silane, carboxylate, or amidate groups, respectively. For example, FIGS. 5A-5B generally illustrate structures and operations in an illustrative process for bonding moieties to a substrate or cover for use in creating a fluidic channel. Referring now to FIG. 5A, in some examples, the surface of substrate 110 may include -H. The -H may be converted to hydroxyl (-OH), as illustrated in FIG. 5A, using any suitable process. It will be understood that some substrates may not necessarily include -H and may be suitably treated to obtain hydroxyl groups on their surfaces. The hydroxyl group may then be bonded to a silane group (silanized), carboxylate group, or amidate group to which the first portion 113 or the second portion 123 is bonded. In a non-limiting example illustrated in FIG. 5A, the hydroxyl group bonded to the substrate 110 is bonded to a silane group that includes an alkyl (R) group and a linker (L) to the first portion 113. Now referring to FIG. 5B, in some examples, the surface of the cover 120 may include -H. The -H may be converted to hydroxyl (-OH) using any suitable process, as illustrated in FIG. 5B. It will be understood that some covers may not necessarily include -H and may be suitably treated to obtain hydroxyl groups on their surface. The hydroxyl group may then be bonded to a silane group (silanized), carboxylate group, or amidate group to which the first portion 113 or the second portion 123 is bonded, for example, in a manner as illustrated in FIG. 6. In the non-limiting example illustrated in FIG. 5B, a hydroxyl group bonded to the cover 120 is bonded to a silane group that includes an alkyl (R) group and a linker (L) to the second portion 123 .

[0093] In one non-limiting example, any alkyl C-H bonds on the surface of substrate 110 or cover 120 can be converted to C-OH bonds using confined photocatalytic oxidation (CPO) and then silanized in a manner similar to that described in Gan et al., "Photoactivation of alkyl C-H and silanization: A simple and general route for preparing high-density primary amines for inert polymer surfaces for protein immobilization," Biomacromolecules 10(5):1238-1243 (2009), the entire contents of which are incorporated herein by reference. In examples in which moiety 113 or 123 is attached to a polymer, such as polymer 312 or intervening layer 430, such a polymer can be formed using a monomer that includes moiety 113 or moiety 123, or a precursor of such moiety, which precursor is then converted to the moiety.

[0094] It will be appreciated that the cover 120 can comprise a different material than the substrate 110 and yet be robust and securely bonded to the substrate in a manner that can reduce or avoid drawbacks associated with conventional adhesives, such as chemical reactions with fluids in the fluidic channels, fluorescence that can interfere with detection of the desired fluorescent signal, and / or thermal instability. In some examples, the substrate 110 can comprise at least one material selected from the group consisting of cyclic olefin polymer (COP), cyclic olefin copolymer (COC), glass, silicon, polypropylene (PP), photoresist, polyethylene terephthalate (PET), poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM), and polyethylene (PE). Regardless of the material used for the substrate 110, the cover 120 may comprise at least one material selected from the group consisting of cyclic olefin polymer (COP), cyclic olefin copolymer (COC), glass, silicon, polypropylene (PP), photoresist, polyethylene terephthalate (PET), poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM), and polyethylene (PE). Such materials of the substrate and cover may be suitably bonded to first or second moieties 113, 123, which may be used to covalently bond the substrate to the cover in a manner as provided herein.

[0095] In one specific, non-limiting example, the substrate 110 may be silicon on tantalum oxide (Si / TaO x), and the cover 120 can include COC or COP. The substrate 110 can be bonded to azide using a process including, for example, hydroxylating the Si surface using a heated solution containing NH4OH and hydrogen peroxide, followed by silanizing the hydroxylated Si surface with azidopropyltrimethoxysilane using spin coating or a silane oven. The cover 120 can be bonded to DBCO using a process including, for example, mild peroxidation, such as sonication in a solution containing Cu(OAc)2 and hydrogen peroxide, followed by mild reduction cleaning, such as sonication in a solution containing NaBH4 and methanol, followed by silanizing the hydroxylated COC or COP surface with DBCOPEG silane using spin coating or a silane oven.

[0096] Methods of using a fluidic device including a fluidic channel - Patents.com As described elsewhere herein, oligonucleotides can be bound to regions of a substrate within a flow channel in a manner as described with reference to, for example, Figures 1A-1D, 2A-2E, 3A-3E, 4A-4B, or 7A-7G. Oligonucleotides bound to a substrate in a manner as described herein can be used in a variety of amplification techniques. Exemplary techniques that can be used include, but are not limited to, polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), or random prime amplification (RPA), or combinations thereof. In some examples, one or more primers used for amplification can be bound to the substrate. Formats utilizing two or more attached primers allow for bridge amplification (BridgeAmp) or kinetic exclusion amplification (ExAmp), where an amplicon can form a bridge-like structure between two attached primers that flank the copied template sequence. Amplification can also be performed with one amplification primer attached to a substrate and a second primer in solution (eg, emulsion PCR).

[0097] Additionally or alternatively, the oligonucleotides bound to the substrate in the manner described herein can be used to determine the sequence of the target polynucleotide. For example, the target polynucleotide can be bound (e.g., hybridized) to one of the multiple primers covalently bound to the substrate in the manner described herein. The target polynucleotide can be amplified using the multiple primers to form a cluster of substrate-bound amplicons. The cluster of substrate-bound amplicons is contacted with a labeled nucleotide (e.g., a fluorescently labeled nucleotide) and a polymerase, which generates a detectable signal (e.g., fluorescence) while the nucleotide is incorporated by the polymerase, and such a signal can be used to identify the nucleotide, thereby determining the nucleotide sequence of the target polynucleotide.

[0098] Further comments It should be understood that any respective feature / example of each of the aspects of the present disclosure described herein may be implemented together in any suitable combination, and any feature / example from any one or more of these aspects may be implemented together in any suitable combination with any of the features of the other aspects described herein, in order to achieve the benefits described herein.

[0099] While various illustrative examples have been described above, it will be apparent to one skilled in the art that various changes and modifications can be made herein without departing from the present invention. It is intended that the appended claims cover all such changes and modifications that fall within the true spirit and scope of the present invention.

Claims

1. 1. A method of preparing a fluidic channel, comprising: covalently bonding a first region of a substrate to a first region of a cover with a first moiety covalently bonded to the first region of the substrate and a second moiety covalently bonded to the first region of the cover; A covalent bond between the first region of the substrate and the first region of the cover suspends the second region of the cover over the second region of the substrate to form a fluid channel.

2. The method of claim 1 , further comprising binding oligonucleotides to the second region of the substrate.

3. The method of claim 2 , wherein the oligonucleotides are attached to the second region of the substrate before the first region of the substrate is attached to the first region of the cover.

4. The method of claim 2 , further comprising protecting the oligonucleotides before the first region of the substrate is covalently bonded to the first region of the cover.

5. The method of claim 4 , wherein protecting the oligonucleotide comprises depositing a mask onto the oligonucleotide.

6. The method of claim 5 , further comprising removing the mask after the first region of the substrate is covalently bonded to the first region of the cover.

7. The method of claim 2 , wherein the oligonucleotide is covalently attached to the second region of the substrate using a second moiety that is covalently attached to the second region of the substrate.

8. The method of claim 2 , wherein the oligonucleotide comprises a capture primer.

9. The method of claim 2 , wherein the oligonucleotides are attached to the second region of the substrate after the first region of the substrate is attached to the first region of the cover.

10. 2. The method of claim 1, wherein covalently bonding the first region of the substrate to the first region of the cover comprises selectively applying heat to the first region of the substrate or the first region of the cover.

11. The method of claim 10 , wherein the heat is applied using light.

12. The method of claim 11 , wherein the light comprises infrared or near infrared wavelengths.

13. The method of claim 1 , wherein covalently bonding the first region of the substrate to the first region of the cover comprises applying pressure to the first region of the substrate and the first region of the cover.

14. The method of claim 1 , wherein covalently bonding the first region of the substrate to the first region of the cover comprises reacting the first moiety with the second moiety.

15. 15. The method of claim 14, wherein the reaction between the first moiety and the second moiety comprises an azide-alkyne [3+2] cycloaddition.

16. and disposing an intervening layer between the first region of the substrate and the first region of the cover, wherein covalently bonding the first region of the substrate to the first region of the cover comprises: covalently bonding the first region of the substrate to the intermediate layer; covalently bonding the first region of the cover to the intermediate layer.

17. The method of claim 16 , wherein the intermediate layer comprises the first portion or the second portion.

18. 17. The method of claim 16, wherein covalently bonding the first region of the substrate to the intervening layer comprising a comprises a first azide-alkyne [3+2] cycloaddition.

19. The method of claim 16 , wherein covalently bonding the first region of the covering to the intervening layer comprising a comprises a second azide-alkyne [3+2] cycloaddition.

20. The method of claim 1 , wherein the first moiety is covalently attached to the first region of the substrate via a silane group, a carboxylate group, or an amidate group.

21. The method of claim 1 , wherein the second moiety is covalently attached to the second region of the substrate via a silane group, a carboxylate group, or an amidate group.

22. The method of claim 1 , wherein the cover comprises a different material than the substrate.

23. 2. The method of claim 1, wherein the cover comprises at least one material selected from the group consisting of cyclic olefin polymer (COP), cyclic olefin copolymer (COC), glass, silicon, polypropylene (PP), photoresist, polyethylene terephthalate (PET), poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM), and polyethylene (PE).

24. 2. The method of claim 1, wherein the substrate comprises at least one material selected from the group consisting of cyclic olefin polymer (COP), cyclic olefin copolymer (COC), glass, silicon, polypropylene (PP), photoresist, polyethylene terephthalate (PET), poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM), and polyethylene (PE).

25. The method of claim 1 , wherein the second region of the cover is separated from the second region of the substrate by about 1 μm to about 1 cm.

26. A fluidic device, comprising: a substrate including a first region and a second region; a cover including a first region and a second region; the first region of the substrate is covalently bonded to the first region of the cover using a first moiety that is covalently bonded to the first region of the substrate and a second moiety that is covalently bonded to the first region of the cover; A fluidic device, wherein the covalent bond between the first region of the substrate and the first region of the cover suspends the second region of the cover above the second region of the substrate to form a fluidic channel.

27. 27. The device of claim 26, further comprising an oligonucleotide bound to the second region of the substrate.

28. 28. The device of claim 27, wherein the oligonucleotides are covalently attached to the second region of the substrate using a second moiety that is covalently attached to the second region of the substrate.

29. The device of claim 27 , wherein the oligonucleotide comprises a capture primer.

30. 27. The device of claim 26, wherein the first region of the substrate is covalently bonded to the first region of the cover via a product of the azide-alkyne [3+2] cycloaddition reaction between the first moiety and the second moiety.

31. 30. The device of any one of claims 26 to 29, further comprising an intervening layer between the first region of the substrate and the first region of the cover, the first region of the substrate being covalently bonded to the intervening layer, and the first region of the cover being covalently bonded to the intervening layer.

32. 32. The device of claim 31 , wherein the intervening layer comprises the first portion or the second portion.

33. 32. The device of claim 31, wherein the first region of the substrate is covalently bonded to the intervening layer via a product of the azide-alkyne [3+2] cycloaddition reaction.

34. 32. The device of claim 31, wherein the first region of the cover is covalently bonded to the intervening layer via the product of the azide-alkyne [3+2] cycloaddition reaction.

35. 27. The device of claim 26, wherein the first moiety is covalently bonded to the first region of the substrate via a silane group, a carboxylate group, or an amidate group.

36. 27. The device of claim 26, wherein the second moiety is covalently bonded to the second region of the cover via a silane group, a carboxylate group, or an amidate group.

37. 27. The device of claim 26, wherein the cover comprises a different material than the substrate.

38. 27. The device of claim 26, wherein the substrate comprises at least one material selected from the group consisting of cyclic olefin polymer (COP), cyclic olefin copolymer (COC), glass, silicon, polypropylene (PP), photoresist, polyethylene terephthalate (PET), poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM), and polyethylene (PE).

39. 27. The device of claim 26, wherein the cover comprises at least one material selected from the group consisting of cyclic olefin polymer (COP), cyclic olefin copolymer (COC), glass, silicon, polypropylene (PP), photoresist, polyethylene terephthalate (PET), poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM), and polyethylene (PE).

40. 27. The device of claim 26, wherein the second region of the cover is separated from the second region of the substrate by about 1 μm to about 1 cm.

41. 27. The method of making the device of claim 26, wherein the first region of the substrate is covalently bonded to the first region of the cover using selective application of heat to the first region of the substrate or the first region of the cover.

42. 42. The method of claim 41 , wherein the heat is applied using light.

43. 43. The method of claim 42, wherein the light comprises infrared or near infrared wavelengths.

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