Kits and Flow Cells

Abstract

An example kit includes a flow cell and a cleavage mixture. The exemplary flow cell includes a substrate, a catalytic polymer hydrogel on the substrate, the catalytic polymer hydrogel including a deblocking catalyst, and an amplification primer attached to the catalytic polymer hydrogel. The deblocking catalyst accelerates cleavage of the blocking group of the 3'-OH blocked nucleotide incorporated into the template strand attached to the amplification primer introduced into the flow cell. An example cleavage mixture includes components that initiate cleavage of the blocking group.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 948,605, filed December 16, 2019, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Various procedures in biological or chemical research involve carrying out numerous controlled reactions on a local support surface or inside a given reaction chamber. The designated reactions can then be observed or detected, and subsequent analysis can help to characterize the chemicals involved in the reactions. In some instances, the controlled reactions produce fluorescence, and thus optical systems can be used for detection. In other instances, the controlled reactions change charge, conductivity, or some other electrical property, and thus electronic systems can be used for detection. Introduction

[0003] A first aspect disclosed herein is a kit comprising a flow cell comprising a substrate, a catalytic polymer hydrogel on the substrate, the catalytic polymer hydrogel comprising a deblocking catalyst, and an amplification primer attached to the catalytic polymer hydrogel, the catalyst being introduced into the flow cell and accelerating cleavage of a blocking group of a 3'OH blocked nucleotide incorporated into a template strand attached to the amplification primer, and a cleavage mixture comprising components to initiate cleavage of the blocking group.

[0004] In one example of the first aspect, the deblocking catalyst is selected from the group consisting of an acid catalyst, a base catalyst, an enzyme, a peptide, a DNAzyme, an organic catalyst, and combinations thereof.

[0005] In one example of the first embodiment, the deblocking catalyst is a metal of a metal-ligand complex attached to the polymer hydrogel.

[0006] In one example of the first aspect, the deblocking catalyst is an acid catalyst selected from the group consisting of carboxylic acids, phosphonic acids, sulfonic acids, and combinations thereof.

[0007] In one example of the first aspect, the deblocking catalyst is a base catalyst selected from the group consisting of amines, heterocyclic amines, and combinations thereof.

[0008] In one example of the first embodiment, the unblocking catalyst is an enzyme.

[0009] In one example of the first embodiment, the unblocking catalyst is a peptide.

[0010] In one example of the first embodiment, the unblocking catalyst is a DNAzyme.

[0011] It should be understood that any of the features of the kits disclosed herein may be combined in any desired manner and / or configuration to achieve the advantages described in this disclosure, including, for example, accelerating the deblocking reaction rate.

[0012] In a second aspect disclosed herein, a method includes incorporating individual nucleotides into each nascent strand along the template strand by introducing the incorporation mixture into a flow cell comprising a substrate, a catalytic polymer hydrogel on the substrate, the catalytic polymer hydrogel comprising a deblocking catalyst, and a template strand attached to the catalytic polymer hydrogel, wherein each nucleotide comprises a dye label attached to the base and a 3'OH blocking group attached to the sugar; removing the incorporation mixture; optically imaging the incorporation of each nucleotide; and introducing a cleavage mixture into the flow cell comprising a component that initiates cleavage of the 3'OH blocking group, whereby the deblocking catalyst accelerates cleavage of the 3'OH blocking group.

[0013] In one example of the second embodiment, the catalyst accelerates the removal of the outer protecting group of the 3'OH blocking group, and a reagent in the cleavage mixture removes the inner protecting group of the 3'OH blocking group.

[0014] It should be understood that any of the features of this method may be combined in any desired manner. Further, it should be understood that any combination of the features of this method and / or kit may be used together and / or in combination with any of the examples disclosed herein to achieve the advantages described in this disclosure, including, for example, accelerating the deblocking kinetics.

[0015] A third aspect disclosed herein is a flow cell comprising a substrate, a catalytic polymer hydrogel on the substrate, the catalytic polymer hydrogel comprising a deblocking catalyst selected from the group consisting of: a phosphonic acid; a heterocyclic amine; an enzyme; a peptide; a DNAzyme; a metal of a metal-ligand complex; an organic catalyst selected from the group consisting of urea, thiourea, imidazole, guanidine, 1,8-diazabicyclo(5.4.0)-undec-7-ene, and combinations thereof; and a photoacid generator, and an amplification primer attached to the catalytic polymer hydrogel.

[0016] In one example of the third embodiment, the catalyst is incorporated into a monomer unit of the catalytic polymer hydrogel.

[0017] In one example of the third embodiment, the catalyst is grafted to the initial polymer hydrogel.

[0018] In one example of the third embodiment, the catalytic polymer hydrogel comprises an initial polymer hydrogel, the flow cell further comprises an oligonucleotide attached to the initial polymer hydrogel, and the deblocking catalyst is attached to a complementary oligonucleotide tether hybridized to the oligonucleotide.

[0019] It should be understood that any of the features of the flow cell may be combined in any desired manner. Further, it should be understood that any combination of the features of the flow cell and / or the features of the kits and / or methods may be used together and / or in combination with any of the examples disclosed herein to achieve the advantages described in this disclosure, including, for example, producing a polymer hydrogel that contributes to enhanced deblocking kinetics.

[0020] A fourth aspect disclosed herein is a flow cell comprising a substrate, a polymer hydrogel on the substrate, the polymer hydrogel comprising a first member of a hydrogen bonding pair, a deblocking catalyst attached to the polymer hydrogel via a second member of the hydrogen bonding pair, and an amplification primer attached to the catalytic polymer hydrogel.

[0021] It should be understood that any of the features of this flow cell may be combined in any desired manner. Further, it should be understood that any combination of features of this and / or other flow cells and / or features of kits and / or methods may be used together and / or in combination with any of the examples disclosed herein to achieve the advantages described in this disclosure, including, for example, accelerating the deblocking kinetics.

[0022] A fifth aspect disclosed herein is a method comprising: applying a catalytic polymer hydrogel to a surface of a flow cell substrate, the catalytic polymer hydrogel comprising a deblocking catalyst selected from the group consisting of: a phosphonic acid; a heterocyclic amine; an enzyme; a peptide; a DNAzyme; a metal of a metal-ligand complex; an organic catalyst selected from the group consisting of urea, thiourea, imidazole, guanidine, 1,8-diazabicyclo(5.4.0)-undec-7-ene, and combinations thereof; and a photoacid generator; and attaching an amplification primer to the catalytic polymer hydrogel.

[0023] One example of the fifth embodiment further includes forming a catalytic polymer hydrogel that includes a deblocking catalyst.

[0024] In one example of the fifth embodiment, forming the catalytic polymer hydrogel includes copolymerizing a first monomer that includes a primer-grafted functional group with a second monomer that includes a deblocking catalyst.

[0025] In one example of the fifth embodiment, forming the catalytic polymer hydrogel includes synthesizing an initial polymer hydrogel and grafting a catalyst onto the initial polymer hydrogel.

[0026] In one example of the fifth embodiment, forming the catalytic polymer hydrogel includes synthesizing an initial polymer hydrogel, grafting an oligonucleotide to the initial polymer hydrogel, and hybridizing a complementary oligonucleotide tether to the oligonucleotide, wherein the catalyst is attached to the complementary oligonucleotide tether.

[0027] In one example of the fifth embodiment, forming the catalytic polymer hydrogel includes synthesizing an initial polymer hydrogel and attaching a metal-ligand complex to the initial polymer hydrogel, where the metal of the metal-ligand complex is a catalyst.

[0028] It should be understood that any of the features of this method may be combined in any desired manner. Further, it should be understood that any combination of the features of this method and / or other method features and / or kit and / or flow cell features may be used together and / or in combination with any of the examples disclosed herein to achieve the advantages described in this disclosure, including, for example, accelerating the deblocking kinetics. [Brief description of the drawings]

[0029] Features of examples of the present disclosure will become apparent upon reference to the following detailed description and the drawings in which like reference numbers correspond to similar, but perhaps not identical, components. For the sake of brevity, reference numbers or features having previously described functions may or may not be described in conjunction with the other drawings in which they appear.

[0030] [Figure 1] FIG. 1 is a schematic flow diagram showing a multi-step process for deblocking an example of a 3′OH blocked nucleotide.

[0031] [Diagram 2] FIG. 1 is a schematic diagram of an example catalyst attached to an example polymer hydrogel via an example non-covalent bond.

[0032] [Figure 3A] FIG. 2 is a top view of an example of a flow cell.

[0033] [Figure 3B] FIG. 1 is an enlarged partial cross-sectional view of an example flow channel of a flow cell including an example polymer hydrogel disposed within the flow channel.

[0034] [Figure 3C] FIG. 1 is an enlarged partial cross-sectional view of an example flow channel of a flow cell including an example polymer hydrogel disposed within a recess formed in the flow channel. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Some nucleic acid sequencing techniques utilize a 3'OH blocking group linked to the oxygen atom of the sugar molecule in a nucleotide. The 3'OH blocking group can function as a reversible terminator that allows only a single base to be incorporated in each sequencing cycle. More specifically, in some instances, the 3'OH blocking group is removed to initiate the next sequencing cycle. Temporarily terminating nucleotide incorporation allows time to wash away unincorporated nucleotides, excite detectable labels (e.g., fluorescent dyes) attached to incorporated nucleotides, and image the excited detectable labels, to name a few actions.

[0036] Catalytic polymer hydrogels are disclosed herein. The catalyst of the catalytic polymer hydrogel is a reactive molecule or portion thereof that accelerates the cleavage of 3'OH blocking groups from incorporated nucleotides. The catalyst may be selected to be inactive during the incorporation event. The catalyst may be selected for the reaction that occurs during deblocking (after incorporation and imaging), thus accelerating the cleavage of any 3'OH blocking groups. In certain embodiments, the catalyst may be useful for 3'OH blocking groups that tend to be more stable in aqueous solution but exhibit slower deblocking kinetics. In these examples, the catalyst may help reduce each sequencing cycle time, help reduce the total sequencing turnaround time, and help alleviate phasing issues that may arise when deblocking is slow, to name a few advantages.

[0037] During cleavage of the 3'OH blocking group, a series of reactions can occur. Figure 1 shows an example of this series of reactions for the example of a 3'OH blocked nucleotide 10. The 3'OH blocked nucleotide 10 includes a heterocyclic base B, a sugar (shown in Figure 1 as deoxyribose), one or more phosphate groups P, and a 3'OH blocking group 12 attached to the sugar. In this example, the 3'OH blocking group 12 is azidomethyl. In step 1 of Figure 1, a phosphine (where R is H or (CH2) nPR3) where OH (n=1-3) is added to react with the azide. This reaction results in the loss of nitrogen and the formation of an iminophosphorane. In step 2, the iminophosphorane is hydrolyzed to form a hemiaminal ether, and in step 3, the hemiaminal ether is hydrolyzed to expose the 3'OH. ​​For the deblocking shown in FIG. 1, any catalyst that accelerates the hydrolysis of steps 2 and / or 3 of FIG. 1 can be incorporated into or onto the polymer hydrogel, or any catalyst that accelerates the reaction of step 1 of FIG. 1 can be incorporated into or onto the polymer hydrogel. When a catalyst is specifically selected to catalyze step 1 of FIG. 1, the catalyst accelerates the removal of the outer protecting group of the 3'OH blocking group 12 (e.g., the azide group shown in FIG. 1) and a reagent in the cleavage mixture can also be used to remove the inner protecting group of the 3'OH blocking group 12 (e.g., the -CH2- shown in FIG. 1).

[0038] FIG. 1 shows one example of deblocking, however it is understood that the catalyst used may depend on the 3′OH blocking group and the reaction used to cleave the 3′OH blocking group.

[0039] definition

[0040] Terms used herein should be understood to take their ordinary meaning in the relevant art unless otherwise specified. Some terms used herein and their meanings are described below.

[0041] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0042] The terms comprising, including, containing, and the various forms of these terms are synonymous with each other and are intended to be equally broad.

[0043] Terms such as top, bottom, lower, upper, and above are used herein to describe the flow cell and / or various components of the flow cell. It is understood that these directional terms do not imply a particular orientation, but are used to specify the relative orientation between components. The use of directional terms should not be construed as limiting the examples disclosed herein to any particular orientation.

[0044] "Acrylamide monomer" has the structure [ka] or a monomer having the structure containing an acrylamide group. An example of a monomer containing an acrylamide group is azidoacetamidopentylacrylamide: [ka] and N-isopropylacrylamide: [ka] Other acrylamide monomers may also be used, some examples of which are described herein.

[0045] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). The alkyl group can have from 1 to 20 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like. As an example, the designation "C1-C6 alkyl" indicates that there are 1 to 6 carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl.

[0046] As used herein, "alkenyl" refers to a straight or branched hydrocarbon chain containing one or more double bonds. Alkenyl groups can have 2 to 20 carbon atoms. Examples of alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like.

[0047] As used herein, "alkyne" or "alkynyl" refers to a straight or branched hydrocarbon chain containing one or more triple bonds. Alkynyl groups can have from 2 to 20 carbon atoms.

[0048] As used herein, "aralkyl" and "aryl(alkyl)" refer to an aryl group bonded as a substituent through a lower alkylene group. The lower alkylene group and the aryl group of the aralkyl may be substituted or unsubstituted. Examples include, but are not limited to, benzyl, 2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl.

[0049] 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. Examples of aryl groups include phenyl, naphthyl, azulenyl, and anthracenyl.

[0050] An "amine" or "amino" functional group is -NR a R b R a and R b are each independently hydrogen (e.g., [ka] ), selected from C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclyl, C6-C10 aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

[0051] As used herein, the term "attached" refers to the state in which two things are joined, fixed, adhered, connected, or bound to each other directly or indirectly. For example, a nucleic acid can be attached to a polymer hydrogel by a covalent bond or a non-covalent bond. A covalent bond is characterized by the sharing of electron pairs between atoms. A non-covalent bond is a physical bond that does not involve the sharing of electron pairs, and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, and hydrophobic interactions.

[0052] An "azido" or "azido" functional group refers to -N3.

[0053] The terms "block copolymer" and "blockwise" formed monomer units refer to copolymers formed when two or more monomers cluster together to form a block of repeating units. Each block should have at least one feature not present in an adjacent block and / or at least one block-specific functional group (e.g., azide for attaching a primer, catalyst, etc.).

[0054] As used herein, the term "catalyzed polymer hydrogel" refers to a copolymer having a catalyst incorporated into one of the monomer units, or to an initial polymer hydrogel having a catalyst attached thereto. As used herein, the term "initial polymer hydrogel" refers to a polymerized hydrogel prior to any reaction / interaction to introduce a catalyst.

[0055] As used herein, "carbocyclyl" refers to a non-aromatic ring or ring system containing only carbon atoms in the ring system backbone. When a carbocyclyl is a ring system, two or more rings can be joined together in a fused, bridged, or spiro-linked manner. A carbocyclyl can have any degree of saturation, provided that at least one ring in the ring system is not aromatic. Thus, carbocyclyl includes cycloalkyl, cycloalkenyl, and cycloalkynyl. A carbocyclyl group can have from 3 to 20 carbon atoms. Examples of carbocyclyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicyclo[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl.

[0056] As used herein, the term "carboxylic acid" or "carboxyl" as used herein refers to -COOH.

[0057] As used herein, "cycloalkyl" refers to a monocyclic or polycyclic hydrocarbon ring system that is fully saturated (containing no double or triple bonds). When composed of more than one ring, the rings may be joined together in a fused fashion. Cycloalkyl groups may have 3 to 10 atoms in the ring. In some embodiments, cycloalkyl groups may have 3 to 8 atoms in the ring. Cycloalkyl groups may be unsubstituted or substituted. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0058] As used herein, "cycloalkenyl" or "cycloalkene" means a carbocyclyl ring or ring system having at least one double bond, and none of the rings in the ring system are aromatic. Examples include cyclohexenyl or cyclohexene and norbornenyl or norbornene.

[0059] As used herein, "cycloalkynyl" or "cycloalkyne" refers to a carbocyclyl ring or ring system having at least one triple bond, and wherein none of the rings in the ring system are aromatic. One example is cyclooctyne. Another example is bicyclononyne.

[0060] As used herein, the term "deposition" refers to any suitable application technique, which may be manual or automated, potentially resulting in the modification of surface properties. In general, deposition can be performed using deposition techniques, coating techniques, grafting techniques, and the like. Some specific examples include chemical vapor deposition (CVD), spray coating (e.g., ultrasonic spray coating), spin coating, dunk or dip coating, doctor blade coating, paddle dispensing, flow-through coating, aerosol printing, screen printing, microcontact printing, inkjet printing, and the like.

[0061] As used herein, the term "depression" refers to a discrete, concave feature in a substrate or patterned resin having a surface opening that is at least partially surrounded by an intervening region of the substrate or patterned resin. The depressions can have any of a variety of shapes at their surface openings, including, for example, circular, elliptical, square, polygonal, star-shaped (with any number of vertices), and the like. The cross-section of the depression perpendicular to the surface can be curved, square, polygonal, hyperbolic, conical, angular, and the like. By way of example, the depression can be a hole or two interconnected holes. The depression can also have more complex structures, such as a ridge, a step feature, and the like.

[0062] The term "each," when used in reference to a collection of items, is intended to identify each individual item in the set, but does not necessarily refer to every item in the set. Exceptions may occur where express disclosure or context clearly dictates otherwise.

[0063] As used herein, the term "flow cell" is intended to mean a container having a chamber (e.g., a flow path) in which a reaction can be carried out, an inlet for delivering a reagent to the chamber, and an outlet for removing the reagent from the chamber. In some examples, the chamber allows for detection of a reaction occurring within the chamber. For example, the chamber can include one or more transparent surfaces that allow for optical detection of an array, optically labeled molecules, and the like.

[0064] As used herein, a "flow path" or "channel" may be an area defined between two bonded or otherwise attached components that is selectively capable of receiving a liquid sample. In some examples, a flow path may be defined between a patterned or unpatterned substrate and a lid, and thus in fluid communication with one or more recesses defined in the patterned resin. A flow path may also be defined between two patterned or unpatterned substrate surfaces that are bonded to one another.

[0065] As used herein, "heterocyclic amine" refers to an aromatic ring or ring system (i.e., two or more fused rings which share two adjacent atoms) that contains an amine nitrogen as the one or only heteroatom in the ring backbone.

[0066] As used herein, "heteroaryl" refers to an aromatic ring or ring system (i.e., two or more fused rings that share 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 a 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).

[0067] As used herein, "heterocyclyl" refers to a non-aromatic ring or ring system containing at least one heteroatom in the ring backbone. Heterocyclyls may be joined together in fused, bridged, or spiro-linked fashions. Heterocyclyls may have any degree of saturation, provided that at least one ring in the ring system is not aromatic. In the ring system, the heteroatom may be present in either the non-aromatic ring or the aromatic ring. Heterocyclyl 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). In some examples, the heteroatom is O, N, or S.

[0068] As used herein, the term "hydrazine" or "hydrazinyl" refers to the group -NHNH.

[0069] As used herein, the term "hydrazone" or "hydrazonyl" refers to a compound represented by the formula R a and R b are each independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclyl, C6-C10 aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein. [ka] Refers to the base.

[0070] As used herein, "hydroxy" or "hydroxyl" refers to an --OH group.

[0071] As used herein, the term "intervening region" refers to a region of, for example, a substrate, patterned resin, or other support that separates recesses. For example, an intervening region can separate one recess of an array from another recess of an array. Two recesses that are separated from each other may be discontinuous, i.e., not in physical contact with each other. In many instances, the intervening region is continuous while the recesses are discontinuous, such as when multiple recesses are defined in an otherwise continuous surface. In other instances, the intervening region and the features are discontinuous, such as when multiple grooves are separated by respective intervening regions. The separation provided by the intervening region may be partial separation or complete separation. The intervening region may have a surface material that is different from the surface material of the recesses defined on the surface. For example, the recesses may have a polymer and a primer set present therein, and the intervening region may have neither a polymer nor a primer set present thereon.

[0072] As used herein, "nitrile oxide" refers to a compound represented by the formula "R a C≡N + O - " group, where R a is as previously defined herein. Examples of preparing nitrile oxides include in situ generation from aldoximes by treatment with chloramide-T, or by the action of base on imidoyl chloride [RC(Cl)=NOH], or from the reaction of hydroxylamine with an aldehyde.

[0073] As used herein, a “nitrone” is a nitrone group having R, R, and R as defined herein. a Groups and R b It can be any of the groups [ka] means a group.

[0074] As used herein, a "nucleotide" comprises a nitrogen-containing heterocyclic base, a sugar, and one or more phosphate groups. A nucleotide is a monomeric unit of a nucleic acid sequence. In RNA, the sugar is ribose, while in DNA, the sugar is deoxyribose, i.e., a sugar lacking the hydroxyl group present at the 2' position of the ribose. The nitrogen-containing heterocyclic base (i.e., nucleobase) can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G), as well as modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), as well as modified derivatives or analogs thereof. The C-1 atom of the deoxyribose is attached to the N-1 of a pyrimidine or the N-9 of a purine. Nucleic acid analogs may have changes in either the phosphate backbone, the sugar, or the nucleobase. Examples of nucleic acid analogs include universal bases or phosphate-sugar backbone analogs, such as, for example, peptide nucleic acids (PNAs).

[0075] "Patterned resin" refers to any polymer that may have recesses defined therein. Specific examples of resins and techniques for patterning resins are further described below.

[0076] As used herein, the term "phosphonic acid" as used herein refers to R-PO3H2.

[0077] As used herein, a "primer" is defined as a single-stranded nucleic acid sequence (e.g., single-stranded DNA or single-stranded RNA). Some primers, referred to herein as amplification primers, serve as starting points for template amplification and cluster generation. Other primers, referred to herein as sequencing primers, serve as starting points for DNA or RNA synthesis. The 5' end of the primer may be modified to allow for coupling reactions with functional groups on a polymer. Primer length may be any number of bases long and may include a variety of non-natural nucleotides. In one example, sequencing primers are short, ranging from 10-60 bases, or 20-40 bases.

[0078] As used herein, a "spacer layer" refers to a material that bonds two components together. In some examples, the spacer layer can be or be in contact with a radiation absorbing material that aids in bonding.

[0079] The term "substrate" refers to a structure onto which various components of the flow cell (e.g., catalytic polymer hydrogel, primer, etc.) can be added. The substrate can be a wafer, a panel, a rectangular sheet, a die, or any other suitable configuration. The substrate is generally rigid and insoluble in aqueous liquids. The substrate can be inert to the chemistry used to modify the recesses or present in the recesses. For example, the substrate can be inert to the chemistry used to form the catalytic polymer hydrogel, to deposit the primer, etc. The substrate can be a single layer structure or a multilayer structure (e.g., including a support and a patterned resin on the support). Examples of suitable substrates are further described below.

[0080] As used herein, the term "sulfonic acid" as used herein refers to -S(=O)2-OH.

[0081] A "thiol" functional group refers to -SH.

[0082] As used herein, the terms "tetrazine" and "tetrazinyl" refer to a six-membered heteroaryl group containing four nitrogen atoms. The tetrazine may be optionally substituted.

[0083] As used herein, "tetrazole" refers to a 5-membered heterocyclic group containing 4 nitrogen atoms. The tetrazole may be optionally substituted.

[0084] Catalytic Polymer Hydrogels

[0085] The polymer backbone in any example of the catalyzed polymer hydrogel can be a semi-rigid polymer material that is permeable to liquids and gases. In some examples, the catalyzed polymer hydrogel comprises an initial polymer hydrogel having a catalyst attached thereto. In these examples, the catalyst is added to the initial polymer hydrogel using a post-polymerization treatment. In other examples, the catalyzed polymer hydrogel can be a copolymer that includes a catalyst in one of its monomeric components.

[0086] Examples of early polymer hydrogels include acrylamide copolymers such as, for example, poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide, PAZAM. PAZAM and several other forms of acrylamide copolymers are represented by the following structure (I): [ka] During the ceremony, R A is selected from the group consisting of azide, optionally substituted amino, optionally substituted alkenyl, optionally substituted alkyne, halogen, optionally substituted hydrazone, optionally substituted hydrazine, carboxyl, hydroxy, optionally substituted tetrazole, optionally substituted tetrazine, nitrile oxide, nitrone, sulfate, and thiol; R B is H or optionally substituted alkyl, R C , R D , and R E are each independently selected from the group consisting of H and optionally substituted alkyl; -(CH2) p each - is optionally substituted; p is an integer ranging from 1 to 50; n is an integer ranging from 1 to 50,000; m is an integer ranging from 1 to 100,000.

[0087] One of ordinary skill in the art will recognize that the arrangement of repeating "n" and "m" features in structure (I) is representative, and that the monomer subunits may be present in any order (e.g., random, block, patterned, or combinations thereof) in the polymer structure.

[0088] The molecular weight of PAZAM and other forms of acrylamide copolymers may range from about 5 kDa to about 1500 kDa, or from about 10 kDa to about 1000 kDa, or in a particular example, may be about 312 kDa.

[0089] In some instances, the PAZAM and other forms of acrylamide copolymers are linear polymers. In some other instances, the PAZAM and other forms of acrylamide copolymers are lightly crosslinked polymers.

[0090] In other examples, the initial polymer hydrogel may be a variation of structure (I). In one example, the acrylamide units are N,N-dimethylacrylamide ( [ka] In this example, the acrylamide unit in structure (I) may be replaced with: [ka] where R D , R E , and R F are each H or C1-C6 alkyl, R G and R H are each C1-C6 alkyl (instead of H as in acrylamide). In this example, q may be an integer ranging from 1 to 100,000. In another example, N,N-dimethylacrylamide may be used in addition to the acrylamide units. In this example, structure (I) contains, in addition to the repeating "n" and "m" features, [ka] wherein R D , R E , and R F are each H or C1-C6 alkyl, R G and R H is each C1-C6 alkyl. In this example, q can be an integer ranging from 1 to 100,000.

[0091] As another example of an initial polymer hydrogel, the repeating "n" feature in structure (I) may be replaced with a heterocyclic azide group-containing monomer having structure (II). [ka] In the formula, R 1 is H or C1-C6 alkyl, R2 is H or C1-C6 alkyl, L is a linker comprising a linear chain having 2-20 atoms selected from the group consisting of carbon, oxygen, and nitrogen, and 10 optional substituents on the carbon atoms and any nitrogen atoms in the linear chain, E is a linear chain comprising 1-4 atoms selected from the group consisting of carbon, oxygen, and nitrogen, and including optional substituents on the carbon atoms and any nitrogen atoms in the linear chain, A is an N-substituted amide having H or C1-C4 alkyl attached to N, and Z is a nitrogen-containing heterocycle. Examples of Z include 5-10 ring members present as single ring structures or fused structures. Some specific examples of Z include pyrrolidinyl, pyridinyl, or pyrimidinyl.

[0092] As yet another example, the initial polymer hydrogel may comprise repeat units of each of structures (III) and (IV): [ka] In the formula, R 1a , R 2a , R 1b and R 2b are each independently selected from hydrogen, optionally substituted alkyl, or optionally substituted phenyl; R 3a and R 3bare each independently selected from hydrogen, optionally substituted alkyl, optionally substituted phenyl, or optionally substituted C7-C14 aralkyl; L 1 and L 2 are each independently selected from an optionally substituted alkylene linker or an optionally substituted heteroalkylene linker.

[0093] It should be understood that other molecules may be used to form the initial polymer hydrogel as long as they are functionalized to graft oligonucleotide primers. Other examples of suitable polymer layers include colloidal structures such as agarose, or polymer mesh structures such as gelatin, or crosslinked polymer structures such as polyacrylamide polymers and copolymers, silane-free acrylamide (SFA), or azidolyzed versions of SFA. Examples of suitable polyacrylamide polymers can be synthesized from acrylamide and acrylic acid, or acrylic acid containing vinyl groups, or monomers that form a [2+2] photocycloaddition reaction. Still other examples of suitable initial polymer hydrogels include mixed copolymers of acrylamide and acrylate. Examples disclosed herein can utilize a variety of polymer structures containing acrylic monomers (e.g., acrylamide, acrylate, etc.), such as star polymers, star-shaped or star-block polymers, branched polymers including dendrimers, etc. For example, monomers (eg, acrylamide, acrylamide containing a catalyst, etc.) may be randomly or blockwise incorporated into the arms of a star polymer.

[0094] The initial polymer hydrogel may be formed using any suitable copolymerization process. A catalyst may then be attached to the initial polymer hydrogel to form the catalyzed polymer hydrogel. A variety of different post-polymerization techniques can be used to attach the catalyst to the initial polymer hydrogel.

[0095] In one example, a catalyst can be grafted onto the initial polymer hydrogel. In this exemplary method, forming the catalyzed polymer hydrogel includes synthesizing an initial polymer hydrogel (without a catalyst) and grafting a catalyst onto the initial polymer hydrogel. In one example, the grafting includes a click chemistry reaction using, for example, copper-catalyzed click chemistry or strain-promoting catalyst-free click chemistry such as bicyclo[6.1.0]non-4-yne (BCN). In one example, the click chemistry reaction results in the covalent attachment of the catalyst to the initial polymer hydrogel. It should be understood that the reaction that occurs depends on the chemistry of the initial polymer hydrogel and the catalyst.

[0096] Any reactive molecule, functional group, etc. that increases the rate of the chemical reaction that cleaves the 3'OH blocking group from the incorporated nucleotide can be used as a catalyst. It should be understood that any acid catalyst, base catalyst, organic catalyst, enzyme catalyst, peptide catalyst, or DNAzyme catalyst may be grafted to the initial polymer hydrogel. Some of the listed catalysts may be classified into two of the listed catalyst categories. In some specific examples, the deblocking catalyst is selected from the group consisting of phosphonic acids; heterocyclic amines; enzymes; peptides; DNAzymes; metals of metal-ligand complexes; organic catalysts selected from the group consisting of urea, thiourea, imidazole, guanidine, 1,8-diazabicyclo(5.4.0)-undec-7-ene, and combinations thereof; and photoacid generators.

[0097] In some examples, the catalyst is an acid catalyst selected from the group consisting of carboxylic acids, phosphonic acids, sulfonic acids, and combinations thereof. The acid catalyst may catalyze the hydrolysis reaction that occurs during deblocking.

[0098] In another example, the catalyst is a base catalyst selected from the group consisting of amines, heterocyclic amines, and combinations thereof. Lewis bases such as thioethers, triazoles, imidazoles, etc. may be particularly suitable for catalyzing the azidophosphine reaction (step 1 in FIG. 1). The base catalyst may catalyze the hydrolysis reaction that occurs during deblocking.

[0099] An organic catalyst consists of carbon, hydrogen, sulfur and / or other non-metallic elements found in organic compounds and increases the rate of the deprotection chemical reaction. Examples of organic catalysts may be selected from the group consisting of urea, thiourea, imidazole, guanidine, 1,8-diazabicyclo(5.4.0)-undec-7-ene, and combinations thereof.

[0100] The enzyme catalyst may be a hydrolase. Examples of suitable hydrolases include phosphatases, esterases (e.g., acetylcholinesterase, lipase, etc.), sequence-specific proteases (e.g., TEV protease or thrombin), and glycosidases (such as cellulose or amylases that do not degrade ribose). Another suitable hydrolase is carbonic anhydrase. Another enzyme catalyst is a serine protease that can cleave peptide bonds in proteins and catalyze the hydrolysis of amides and esters.

[0101] Other catalysts include DNAzymes, also called deoxyribozymes or DNA enzymes. Examples of DNAzymes include those that target ester group hydrolysis, such as DNA mimics of enzymes such as esterases. In these examples, the deblocking chemical reaction is catalyzed by the DNAzyme.

[0102] Although several examples have been provided, it is believed that any available or engineered enzyme can be used, so long as the enzyme is capable of catalyzing the deprotection of the 3'OH blocking group.

[0103] Examples of peptide catalysts include supramolecular β-barrel esterases with hydrophobic exteriors and histidine-rich pore interiors, self-assembling peptide catalysts, and other catalytic peptide assemblies, in which the deprotection chemical reaction is catalyzed by the peptide.

[0104] As another example, the catalyst may be incorporated via non-covalent attachment to the surface of the initial polymer hydrogel, such as hybridization to initial polymer hydrogel surface grafted oligonucleotides, hydrogen bond arrays, biotin-streptavidin or other similar linkages, metal-ligand complex formation, and the like.

[0105] FIG. 2 shows a schematic of one example of non-covalent attachment of a catalyst. Specifically, FIG. 2 shows an oligonucleotide 14 attached to an initial polymer hydrogel 16, with a catalyst 18 attached to a complementary oligonucleotide tether 20 hybridized to the oligonucleotide 14. The oligonucleotide 14 attached to the initial polymer hydrogel 16 may be grafted (e.g., by click chemistry at a terminal azide) as described herein with reference to FIGS. 3A-3C for an amplification primer. The oligonucleotide 14 may have 5-20 nucleotides (shown as N in FIG. 2) and may be the same or different in sequence from the sequence of the amplification primer (described below). The catalyst 18 is attached to a tether 20 having a sequence complementary to the oligonucleotide 14. The catalyst 18 that may be attached according to the example shown in FIG. 2 may be any of an acid catalyst, a base catalyst, an organic catalyst (e.g., imidazole, guanidine, etc.), a copper catalyst, an enzyme catalyst, a peptide catalyst, or a DNAzyme catalyst.

[0106] Suitable copper catalysts may include copper(I) catalysts such as copper(I) acetate, copper(I) bromide, copper(I) chloride, copper(I) iodide, bis(1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene)copper(I) tetrafluoroborate, [bis[(tetrabutylammonium iodide)copper(I) iodide], [bis(trimethylsilyl)acetylene](hexafluoroacetylacetonato)copper(I), bromotris(triphenylphosphine)copper(I), chloro(1,5-cyclooctadiene)copper(I) dimer, copper(I) 3-methylsalicylate, and the like.

[0107] One example of a method for forming the catalytic polymer hydrogel 16' shown in Figure 2 includes synthesizing an initial polymer hydrogel 16 (not including a catalyst 18), grafting an oligonucleotide 14 onto the initial polymer hydrogel 16, and hybridizing a complementary oligonucleotide tether 20 to the oligonucleotide 14, where the catalyst 18 is attached to the complementary oligonucleotide tether 20.

[0108] Another example of non-covalent attachment of catalyst 18 to the initial polymer hydrogel 16 includes a hydrogen bond array. In this example, catalyst 18 is hydrogen bonded to polymer hydrogel 16. Any hydrogen bond pair can be used where one member of the hydrogen bond pair is covalently attached to the initial polymer hydrogel and the other member of the hydrogen bond pair is attached to catalyst 18. Examples of hydrogen bond pairs include O...H, NH...N, and CH...N, to name a few.

[0109] Yet another example of non-covalent attachment of catalyst 18 to the initial polymer hydrogel 16 includes metal-ligand complex formation. In one example, catalyst 18 is the metal of a metal-ligand complex attached to the initial polymer hydrogel 16. Catalyst 18 may be any suitable catalytic metal, such as, for example, copper, palladium, ruthenium, etc. The catalytic metal forms a complex with a ligand attached to the initial polymer hydrogel 16. An example of a metal-ligand complex includes copper(II) complexes with ligands such as bis(2-pyridylmethyl)-amine or pyridine-functionalized cyclodextrin.

[0110] One example of a method for forming this example of a catalytic polymer hydrogel 16' includes synthesizing an initial polymer hydrogel 16 and attaching a metal-ligand complex to the initial polymer hydrogel 16, where the metal of the metal-ligand complex is a catalyst 18.

[0111] Still other catalysts 18 that can be attached to the initial polymer hydrogel 16 include photoacid generators. When a photoacid generator is used as the catalyst 18, catalytic activity can be initiated or enhanced upon exposure to an external stimulus, such as photons of a wavelength consistent with the particular photoacid generator.

[0112] The photoacid generator catalyst may be attached to the polymer hydrogel 16' by guest-host chemistry, where the catalyst 18 is the guest and the host is capable of i) attaching to the polymer hydrogel 16' and ii) retaining the guest until stimulated by an external stimulus. Examples of host compounds include cucurbituril and cyclodextrin. An example of a guest type of photoacid generator catalyst is a blue / visible light based photoacid generator. Photoinitiated catalysts (e.g., photoacid generators) can also be used as guests in guest-host examples.

[0113] The guest-host molecules may be attached to the nascent polymer hydrogel 16 using any of the post-polymerization techniques disclosed herein (eg, grafting, non-covalent attachment).

[0114] Although several examples have been provided, it should be understood that the catalyst 18 attached to the initial polymer hydrogel 16 may depend in part on the chemical nature of the initial polymer hydrogel 16 and the chemical nature of the blocking group 12 included in the nucleotide 10 that is introduced into the catalytic polymer hydrogel 16'.

[0115] In other examples disclosed herein, no initial polymer hydrogel 16 is utilized. Rather, the product of the copolymerization is a catalyzed polymer hydrogel 16'. In these examples, the catalyst 18 is incorporated into the monomer units used during copolymerization.

[0116] In these examples, catalyst 18 may be part of any of the acrylamide monomer units described herein for initial polymer hydrogel 16. In one example, R A can be a carboxyl group, which is an example of an acid catalyst. In another example, R2 in structure (II) can be a C1-C6 alkyl, and catalyst 18 can be incorporated as an end group of the C1-C6 alkyl chain. The catalyst containing monomer units may be copolymerized (randomly or blockwise) with any of the acrylamide monomer units described herein, so long as one of the units contains a primer-grafted functionality. An example of a catalyst containing acrylamide monomer units that may be used is shown in structure (V): [ka] In the formula, R 4 is H or CH3, and X is a functional group that functions as a catalyst. In some examples, the catalyst (X) can be any example of an acid catalyst, base catalyst, or organic catalyst disclosed herein. In some examples, a linking group (such as an alkyl group, a short poly(ethylene glycol) chain, etc.) can be placed between NH and the catalyst (X).

[0117] Other examples of catalyst containing monomer units that can be used include (meth)acrylic monomers (eg, acrylic acid, methacrylic acid) having catalyst (X) attached thereto.

[0118] In one exemplary method, forming the catalytic polymer hydrogel 16' includes copolymerizing a first monomer that includes a primer-grafted functional group (e.g., azide) with a second monomer that includes a catalyst 18. In some examples, a third acrylamide monomer unit may also be used in the copolymerization process. An example of a copolymer formed by this method is shown in Structure (VI): [ka] In the formula, R A, R B , R C , R D , and R E and p is as described for Structure (I), X is as described for Structure (V), n and r are independently in the range of 5 mol % to 30 mol %, and m is the remaining mol %.

[0119] In one example, the surface concentration of catalyst 18 can range from about 0.01% to about 50% of the repeat units of polymer hydrogel 16'. These concentrations can exceed practical catalyst solution concentrations, which contributes to the high efficiency of the examples disclosed herein.

[0120] In yet other examples, the nucleophile-reactive functional group can be used as an anchor for catalyst 18 or to generate the catalyst in situ. In some examples, the nucleophile-reactive functional group is a cyclic sulfonate ester (such as a sultone ring) that can undergo a ring-opening reaction upon exposure to a nucleophile, optionally under basic (high pH) conditions, such as pH 9 or higher. An example of a ring-opening reaction of a sultone is as follows: [ka]

[0121] In one example, this ring-opening reaction may occur during deblocking such that a catalyst 18, such as an acid or base, is generated in situ. The nucleophile reactive functional groups can be considered catalyst precursors attached to the catalytic polymer hydrogel 16'.

[0122] In another example, "Nu" is catalyst 18, and the ring-opening reaction forms a covalent bond with what was previously a sultone. This reaction may be formed prior to sequencing, such that catalyst 18 is present when deblocking is performed.

[0123] Other examples of nucleophile-reactive functional groups have the structures: [ka] wherein (a) Y is SO and Y' is CH; or (b) Y and Y' are both C(O). In other embodiments, the nucleophile-reactive functional group is [ka] Suitable nucleophiles include primary alkyl amines and alkyl alcohols.

[0124] In some embodiments, the monomer containing a nucleophile reactive functional group is as follows: [ka] In a particular example, the monomer is: [ka] It is.

[0125] Flow Cell

[0126] The polymer hydrogel 16 and incorporated catalyst 18 can be used in a flow cell 22, an example of which is shown in Figure 3 A. The flow cell 22 includes a substrate 24 and a catalytic polymer hydrogel 16' on the substrate 24.

[0127] The substrate 24 may be a single layer / material. Examples of suitable single layer substrates include epoxy siloxanes, glass, modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene with other materials, polypropylene, polyethylene, polybutylene, polyurethane, polytetrafluoroethylene (such as TEFLON® from Chemours), cyclic olefin / cycloolefin polymers (COP) (such as ZEONOR® from Zeon), polyimides, etc.), nylon (polyamide), ceramic / ceramic oxides, silica, fused silica, or silica-based materials, aluminum silicate, silicon and modified silicon (e.g., boron doped p+ silicon), silicon nitride (Si3N4), silicon oxide (SiO2), tantalum pentoxide (Ta2O5), or other tantalum oxides (TaO x), hafnium oxide (HfO2), carbon, metal, inorganic glass, and the like. The substrate 24 may also be a multi-layer structure. Some examples of multi-layer structures include glass or silicon having a coating layer of tantalum oxide or other ceramic oxide on the surface. Other examples of multi-layer structures include a support (e.g., glass or silicon) having a patterned resin thereon. Yet other examples of multi-layer substrates may include silicon-on-insulator (SOI) substrates.

[0128] In one example, the substrate 24 may have a rectangular sheet or panel having a diameter ranging from about 2 mm to about 300 mm, or a maximum dimension of about 10 feet or less (~3 meters). In one example, the substrate 24 is a wafer having a diameter ranging from about 200 mm to about 300 mm. In another example, the substrate 24 is a die having a width ranging from about 0.1 mm to about 10 mm. Although exemplary dimensions are shown, it should be understood that a substrate 24 having any suitable dimensions may be used. In another example, a panel may be used that is a rectangular support having a surface area larger than a 300 mm circular wafer.

[0129] In the example shown in FIG. 3A, the flow cell 22 includes channels 26. Although several channels 26 are shown, it should be understood that any number of channels 26 (e.g., a single channel 26, four channels 26, etc.) may be included in the flow cell 22. Each channel 26 is an area defined between two attached components (e.g., a substrate 24 and a lid or two substrates 24) into which a fluid (e.g., as described herein) may be introduced and removed therefrom. Each channel 26 may be separated from each other channel 26 such that a fluid introduced into any particular channel 26 does not flow into any adjacent channel 26. Some examples of fluids introduced into the channels 26 may introduce reaction components (e.g., polymerase, sequencing primers, nucleotides, etc.), washes, deblocking agents, etc.

[0130] The channels 26 may be defined in the substrate 24 using any suitable technique, depending in part on the material of the substrate 24. In one example, the channels 26 are etched into the glass substrate 24. In another example, the channels 26 may be patterned into the resin of the multi-layer substrate 24 using photolithography, nanoimprint lithography, or the like. In yet another example, a separation material (not shown) may be applied to the substrate 24 such that the separation material defines the walls of the channels 26 and the substrate 24 defines the bottom of the channels 26.

[0131] In one example, the channels 26 have a rectangular configuration. The length and width of the channels 26 may be less than the length and width, respectively, of the substrate 24, such that a portion of the substrate surface surrounding the channels 26 may be available for attachment to a lid (not shown) or another substrate 24. In some cases, the width of each channel 26 may be at least about 1 mm, at least about 2.5 mm, at least about 5 mm, at least about 7 mm, at least about 10 mm, or more. In some cases, the length of each lane 20 may be at least about 10 mm, at least about 25 mm, at least about 50 mm, at least about 100 mm, or more. The width and / or length of each channel 26 may be greater than, less than, or between the values ​​specified above. In another example, the channels 26 are square (e.g., 10 mm x 10 mm).

[0132] The depth of each channel 26 may be a monolayer thick if microcontact printing, aerosol printing, or inkjet printing is used to deposit the separation material that defines the channel walls. In other examples, the depth of each channel 26 can be about 1 μm, about 10 μm, about 50 μm, about 100 μm, or more. In one example, the depth may range from about 10 μm to about 100 μm. In another example, the depth is about 5 μm or less. It should be understood that the depth of each channel 26 may be greater than, less than, or between the values ​​specified above.

[0133] Various examples of structures within the flow channel 26 of the flow cell 22 are shown in Figures 3B and 3C.

[0134] 3B, the flow cell 22 includes a single layer substrate 24A and a flow channel 26 defined within the single layer substrate 24A. In this example, a catalytic polymer hydrogel 16' is disposed within the flow channel 26.

[0135] To introduce the catalytic polymer hydrogel 16' into the flow channels 26, a mixture of the catalytic polymer hydrogel 16' may be created and then applied to the substrate 24 (having the flow channels 26 defined therein). In one example, the catalytic polymer hydrogel 16' may be present in a mixture (e.g., with water or ethanol and water). The mixture may then be applied to the substrate surface (including within the flow channels 26) using spin coating, or immersion or dip coating, or flow of material under positive or negative pressure, or another suitable technique. These types of techniques deposit the catalytic polymer hydrogel 16' uniformly on the substrate 24 (e.g., within the flow channels 26 and on the interstitial regions 28). Other selective deposition techniques (including, e.g., masks, controlled printing techniques, etc.) may be used to specifically deposit the catalytic polymer hydrogel 16' within the flow channels 26 but not onto the interstitial regions 28.

[0136] In some examples, the substrate surface (including the portion exposed to the flow channel 26) may be activated and then the mixture (including the catalytic polymer hydrogel 16') may be applied thereto. In one example, a silane or silane derivative (e.g., norbornene silane) may be deposited on the substrate surface using vapor deposition, spin coating, or other deposition methods. In another example, the substrate surface may be exposed to plasma ashing to generate surface-activating agents (e.g., -OH groups) that can adhere to the catalytic polymer hydrogel 16'.

[0137] Depending on the chemical nature of the catalytic polymer hydrogel 16', the applied mixture may be subjected to a curing process. In one example, curing may occur at temperatures ranging from room temperature (e.g., about 25° C.) to about 95° C. for times ranging from about 1 millisecond to about several days.

[0138] Polishing may then be performed to remove the catalyzed polymer hydrogel 16' from the interstitial regions 28 around the channels 26, while leaving the catalyzed polymer hydrogel 16' at least substantially intact on the surfaces of the channels 26.

[0139] In some instances, the as-deposited catalyzed polymer hydrogel 16' already has the catalyst 18 attached thereto, and therefore no additional processing is performed to introduce the catalyst 18 into the flow channels 26. One example of this is when the catalyst 18 is part of a monomer unit of the polymer hydrogel backbone. Another example of this is when the catalyst 18 is attached after polymerization. Thus, one exemplary method disclosed herein includes forming a catalyzed polymer hydrogel 16' that includes the catalyst 18 and applying the catalyzed polymer hydrogel 16' to a surface of a substrate 24.

[0140] In other examples, the as-deposited polymer hydrogel is an initial polymer hydrogel 16 without catalyst 18 attached thereto. Because the initial polymer hydrogel 16 does not include catalyst 18, additional processing is performed to introduce catalyst 18 into the initial polymer hydrogel 16 and form a catalyzed polymer hydrogel 16' within the channels 26. In these examples, the initial polymer hydrogel 16 can be deposited within the channels 26, polished, and then the catalyst 18 can be introduced into the initial polymer hydrogel 16 using any of the post-polymerization attachment techniques described herein. Because the initial polymer hydrogel 16 is present within the channels 26 and not on the interstitial regions 28, the catalyst 18 preferentially attaches to the initial polymer hydrogel 16 within the channels 26.

[0141] The flow cell 22 further includes an amplification primer 30 attached to the catalytic polymer hydrogel 16'. The following discussion regarding primer attachment includes the catalytic polymer hydrogel 16'. It should be understood that when the initial polymer hydrogel 16 is introduced into the flow path 26, the catalyst 18 may be introduced before or after the amplification primer 30. Thus, the discussion regarding primer attachment is also applicable to the initial polymer hydrogel 16.

[0142] A grafting process can be performed to graft the amplification primer 30 to the catalytic polymer hydrogel 16' in the flow channel 26. In one example, the amplification primer 30 can be immobilized to the catalytic polymer hydrogel 16' by a single-point covalent attachment at or near the 5' end of the primer 30. This attachment i) frees the adapter-specific portion of the primer 30 to anneal to its cognate sequenceable nucleic acid fragment and ii) frees the 3' hydroxyl group for primer extension. Any suitable covalent attachment can be used for this purpose. Examples of terminal primers that can be used include alkyne-terminated primers (e.g., which can be attached to an azide surface moiety of the catalytic polymer hydrogel 16'), phospho-thioate-terminated primers (e.g., which can be attached to a bromine surface moiety of the catalytic polymer hydrogel 16'), or azide-terminated primers (e.g., which can be attached to an alkyne surface moiety of the catalytic polymer hydrogel 16').

[0143] Specific examples of suitable primers 30 include the P5 and P7 primers used on the surface of commercially available flow cells sold by Illumina Inc. for sequencing on HISEQ™, HISEQX™, MISEQ™, MISEQDX™, MINISEQ™, NEXTSEQ™, NEXTSEQDX™, NOVASEQ™, GENOME ANALYZER™, ISEQ™, and other instrument platforms.

[0144] In one example, grafting may include flow-through deposition (e.g., using a temporarily or permanently attached lid), dunk coating, spray coating, paddle dispensing, or another suitable method of attaching the primer 30 to the catalytic polymer hydrogel 16' in the flow channel 26. Each of these exemplary techniques may utilize a primer solution or mixture, which may include the primer 30, water, a buffer, and a catalyst. The primer 30 reacts with the reactive groups of the catalytic polymer hydrogel 16' in the flow channel 26 by any of the grafting methods and has no affinity for the surrounding substrate 24. Thus, the primer 30 selectively grafts to the catalytic polymer hydrogel 16' in the flow channel 26.

[0145] 3C, flow cell 22 includes a multi-layer substrate 24B including a support 32 and a patterned material 34 disposed on support 32. Patterned material 34 defines recesses 36 separated by intervening regions 28. Recesses 36 are disposed within each of flow channels 26.

[0146] 3C, patterned material 34 is disposed on support 32. It should be understood that any material that can be selectively deposited, or deposited and patterned to form recesses 36 and interstitial regions 28, may be used for patterned material 34.

[0147] As an example of the patterning material 34, an inorganic oxide may be selectively applied to the support 32 by vapor deposition, aerosol printing, or inkjet printing. Examples of suitable inorganic oxides include tantalum oxide (e.g., Ta2O5), aluminum oxide (e.g., Al2O3), silicon oxide (e.g., SiO2), hafnium oxide (e.g., HfO2), and the like.

[0148] As another example of the patterning material 34, a resin may be applied to the support 32 and then patterned. Suitable deposition techniques include chemical vapor deposition, dip coating, dunk coating, spin coating, spray coating, paddle dispensing, ultrasonic spray coating, doctor blade coating, aerosol printing, screen printing, microcontact printing, and the like. Suitable patterning techniques include photolithography, nanoimprint lithography (NIL), stamping techniques, embossing techniques, molding techniques, microetching techniques, printing techniques, and the like. Some examples of suitable resins include polyhedral oligomeric silsesquioxane resin (POSS)-based resins, non-POSS epoxy resins, poly(ethylene glycol) resins, polyether resins (e.g., ring-opened epoxies), acrylic resins, acrylate resins, methacrylate resins, amorphous fluoropolymer resins (e.g., CYTOP® from Bellex), and combinations thereof.

[0149] As used herein, the term "polyhedral oligomeric silsesquioxane" (POSS) refers to a hybrid intermediate between silica (SiO2) and silicone (R2SiO) (e.g., RSiO 1.5 ) refers to a chemical composition having the formula [RSiO 3 / 2 ] n where the R groups can be the same or different. Examples of R groups in POSS include epoxy, azide / azide, thiol, poly(ethylene glycol), norbornene, tetrazine, acrylate, and / or methacrylate, or further, for example, alkyl, aryl, alkoxy, and / or haloalkyl groups. The resin composition disclosed herein may include one or more different cage or core structures as monomer units. The polyhedral structure may be: [ka] It may be a T8 structure such as [ka] This monomer unit typically has eight arms of functional groups R1 to R8.

[0150] The monomer unit may be, for example, [ka] T having 10 silicon atoms and 10 R groups, such as 10 or may have a cage structure, e.g. [ka] T having 12 silicon atoms and 12 R groups, such as 12 Alternatively, the POSS-based material may have a cage structure called T6, T 14 , or T 16 In the examples disclosed herein, the average cage content can be adjusted during synthesis and / or controlled by purification methods, and a distribution of cage sizes of the monomer units can be used.

[0151] In some examples of POSS disclosed herein, R1-R8 or R 10 or R 12 At least one of R1 to R8 or R 10 or R 12 may or may not be the same, and in some examples, R1 to R8 or R 10 or R 12 At least one of R1 to R8 or R 10 or R 12At least one other of the functional groups is a non-epoxy functional group. The non-epoxy functional group may be (a) a reactive group that is orthogonally reactive to the epoxy group (i.e., reacts under different conditions than the epoxy group) that serves as a handle for attaching the resin to an amplification primer, polymer, or polymerization agent; or (b) a group that modulates the mechanical or functional properties of the resin, such as, for example, surface energy tuning. In some examples, the non-epoxy functional group is selected from the group consisting of azide / azide, thiol, poly(ethylene glycol), norbornene, tetrazine, amino, hydroxyl, alkynyl, ketone, aldehyde, ester group, alkyl, aryl, alkoxy, and haloalkyl.

[0152] As shown in FIG. 3C, the patterned material 34 includes depressions 36 defined therein and interstitial regions 28 separating adjacent depressions 36. Many different layouts of the depressions 36 are possible, including regular, repeating, and non-regular patterns. In one example, the depressions 36 are arranged in a hexagonal lattice for close packing and increased density. Other layouts may include, for example, linear (rectangular) layouts, triangular layouts, and the like. In some examples, the layout or pattern may be an xy format of depressions 36 in rows and columns. In some other embodiments, the layout or pattern may be a repeating arrangement of depressions 36 and / or interstitial regions 28. In yet other examples, the layout or pattern may be a random arrangement of depressions 36 and / or interstitial regions 28. The patterns may include spots, pads, holes, posts, stripes, swirls, lines, triangles, rectangles, circles, arcs, checks, plaids, diagonals, arrows, squares, and / or cross hatches.

[0153] The layout or pattern of the dimples 36 can be characterized in terms of the density of the dimples 36 (the number of dimples 36) within a defined area. For example, the dimples 36 may be approximately 2 million / mm 2 The density can be, for example, about 100 particles / mm 2 , about 1,000 pieces / mm 2 , about 100,000 pieces / mm 2, about 1 million pieces / mm 2 , about 2 million pieces / mm 2 , about 5 million pieces / mm 2 , about 10 million pieces / mm 2 , about 50 million pieces / mm 2 , or a variety of densities, including densities greater than or less than 100 nm. It should be further understood that the density of the depressions 36 in the patterned material 34 can be between one of the lower limits and one of the upper limits selected from the ranges above. By way of example, a high density array may be characterized as having depressions 36 spaced less than about 100 nm apart, a medium density array may be characterized as having depressions 36 spaced between about 400 nm and about 1 μm apart, and a low density array may be characterized as having depressions 36 spaced greater than about 1 μm apart. Although exemplary densities have been provided, it should be understood that any suitable density may be used. The density of the depressions 36 may depend in part on the depth of the depressions 36. In some cases, it may be desirable for the spacing between the depressions 36 to be even greater than the examples listed herein.

[0154] Additionally or alternatively, the layout or pattern of the recesses 36 can be characterized in terms of an average pitch, or the distance from the center of a recess 36 to the center of an adjacent recess 36 (center-to-center distance), or the distance from the edge of one recess 36 to the edge of an adjacent recess 36 (end-to-end distance). The pattern can be regular such that the coefficient of variation about the average pitch is small, but the pattern can also be irregular, in which case the coefficient of variation can be relatively large. In either case, the average pitch can be, for example, about 50 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 100 μm, or more or less. The average pitch of a particular pattern of recesses 36 can be between one of the lower and upper limits selected from the ranges above. In one example, the recesses 36 have a pitch (center-to-center distance) of about 1.5 μm. Although exemplary average pitch values ​​have been provided, it should be understood that other average pitch values ​​may be used.

[0155] The size of each recess 36 may be characterized by its volume, open area, depth, and / or diameter.

[0156] Each well 36 may have any volume capable of containing fluid. The minimum or maximum volume may be selected to accommodate, for example, the expected throughput (e.g., multiplexing), resolution, nucleotide count, or analyte reactivity for downstream use of the flow cell 22. For example, the volume may be at least about 1×10 -3 μm 3 , at least about 1 x 10 -2 μm 3 , at least about 0.1 μm 3 , at least about 1 μm 3 , at least about 10 μm 3 , at least about 100 μm 3 Alternatively or additionally, the volume may be up to about 1×10 4 μm 3 , up to about 1×10 3 μm 3 , maximum approximately 100μm 3 , maximum approximately 10μm 3 , maximum approximately 1μm 3 , maximum approximately 0.1μm 3 , or even less.

[0157] The area occupied by each recess opening can be selected based on criteria similar to those discussed above for volume. For example, the area of ​​each recess opening is at least about 1×10 -3 μm 2 , at least about 1 x 10 -2 μm 2 , at least about 0.1 μm 2 , at least about 1 μm 2 , at least about 10 μm 2 , at least about 100 μm 2 Alternatively or additionally, the area may be up to about 1×10 3 μm 2 , maximum approximately 100μm 2 , maximum approximately 10μm 2 , maximum approximately 1μm 2, maximum approximately 0.1μm 2 , up to about 1×10 -2 μm 2 The area occupied by each recess opening may be greater than, less than, or between the values ​​specified above.

[0158] The depth of each depression 36 may be large enough to accommodate a portion of the catalytic polymer hydrogel 16'. In one example, the depth may be at least about 0.1 μm, at least about 0.5 μm, at least about 1 μm, at least about 10 μm, at least about 100 μm, or more. Alternatively or additionally, the depth may be up to about 1×10 3 The depth of each recess 36 may be greater than, less than, or between the values ​​specified above.

[0159] In some examples, the diameter or length and width of each recess 36 can be at least about 50 nm, at least about 0.1 μm, at least about 0.5 μm, at least about 1 μm, at least about 10 μm, at least about 100 μm, or more. Alternatively or additionally, the diameter or length and width can be up to about 1×10 3 The diameter or length and width of each recess 36 may be greater than, less than, or between the values ​​specified above.

[0160] In the example shown in Figure 3C, the catalyzed polymer hydrogel 16' is disposed within each of the depressions 36. The catalyzed polymer hydrogel 16' may be applied as described with reference to Figure 3B such that the catalyzed polymer hydrogel 16' is present within the depressions 36 and not on the surrounding interstitial regions 28.

[0161] In the example shown in Figure 3C, a primer 30 may be grafted to the catalytic polymer hydrogel 16' within each of the recesses 36. The primer 30 may be applied as described with reference to Figure 3B, thus grafting to the catalytic polymer hydrogel 16' and not to the surrounding interstitial regions 28.

[0162] 3A, 3B, or 3C, it is understood that flow cell 22 may also include a lid attached to substrate 24. In one example, the lid may be bonded to at least a portion of substrate 24, such as in a portion of interstitial region 28. The bond formed between the lid and substrate 24 may be a chemical bond or a mechanical bond (e.g., using fasteners, etc.).

[0163] The lid can be any material that is transparent to the excitation light directed toward the substrate 24. By way of example, the lid can be glass (e.g., borosilicate, fused silica, etc.), plastic, etc. A commercially available example of a suitable borosilicate glass is D 263® available from Schott North America, Inc. Commercially available examples of suitable plastic materials, i.e., cycloolefin polymers, are the ZEONOR® products available from Zeon Chemicals LP.

[0164] The lid may be bonded to the substrate 24 using any suitable technique, such as laser bonding, diffusion bonding, anodic bonding, eutectic bonding, plasma activated bonding, glass frit bonding, or other methods known in the art. In one example, a spacer layer may be used to bond the lid to the substrate 24. The spacer layer may be any material that seals at least a portion of the substrate 24 and the lid together. In some examples, the spacer layer may be a radiation absorbing material that aids in bonding the substrate 24 and the lid.

[0165] In other examples, the flow cell 22 may further include an additional patterned or unpatterned substrate 24 attached to the substrate 24 .

[0166] nucleotide

[0167] The nucleotide used in the example flow cell 22 is a 3'OH blocked nucleotide 10 (see FIG. 1). As described herein, the 3'OH blocked nucleotide 10 comprises a nucleotide and a 3'OH blocking group 12 attached to the sugar of the nucleotide. The nucleotide can be any of the examples described herein.

[0168] The 3'OH blocking group 12 may be linked to an oxygen atom of a sugar molecule in a nucleotide. The 3'OH blocking group 12 may be a reversible terminator that allows only a single base to be incorporated in each sequencing cycle. The reversible terminator prevents further base incorporation into the nascent strand complementary to the template polynucleotide strand. This allows for detection and identification of the single incorporated base. The 3'OH blocking group 12 may then be removed to allow further sequencing cycles to be performed on each template polynucleotide strand. The cleavage reaction that removes the 3'OH blocking group 12 is catalyzed by a catalyst 18.

[0169] Examples of different 3'OH blocking groups 12 include 3'-ONH2 reversible terminators, 3'-O-allyl reversible terminators (-CH=CHCH2), and 3'-O-azidomethyl reversible terminators (-CH2N3). Other suitable reversible terminators include o-nitrobenzyl ethers, alkyl o-nitrobenzyl carbonates, ester moieties, other allyl moieties, acetals (e.g., tert-butoxy-ethoxy), MOM (-CHOCH3) moieties, 2,4-dinitrobenzenesulfenyl, tetrahydrofuranyl ethers, 3' phosphates, ethers, -F, -H2, -OCH3, -N3, -HCOCH3, and 2-nitrobenzene carbonate. In the case of allyl reversible terminators, the ligands on the surface of the polymer hydrogel 16' can be attached to a palladium (Pd(0)) catalyst, a ruthenium catalyst. In the case of esters and acetals, any of the acid and / or base catalysts described herein can be used.

[0170] The 3'OH blocked nucleotide 10 is a fully functional nucleotide that may also include a detectable label attached to the base B of the nucleotide. The detectable label can be any optically detectable moiety, including luminescent, chemiluminescent, fluorescent, fluorogenic, chromogenic and / or chromogenic moieties. Some examples of suitable optically detectable moieties include fluorescein labels, rhodamine labels, cyanine labels (e.g., Cy3, Cy5, etc.), as well as the Alexa family of fluorescent dyes and other fluorescent and fluorogenic dyes.

[0171] Any suitable linking molecule can be used to attach a detectable label to base B of the nucleotide. The linking molecule may be cleavable and undergo a similar series of reactions to remove the deblocking group 12. In one example, the linking molecule has the formula -((CH2)2O) n -spacer group, in which n is an integer of 2 to 50.

[0172] In some applications, it may be desirable to utilize a different type of detectable label for each of the nucleotides 10, including different bases such as, for example, A, T, G, C (as well as U or I). For example, fluorescent or fluorogenic labels may be selected such that each label absorbs excitation radiation and / or fluoresces at a wavelength that is distinguishable from the other label groups. Such distinguishable analogs provide the ability to simultaneously monitor the presence of different labels in the same reaction mixture. In some examples, one of the four nucleotides in a sequence may contain no label, while the other three nucleotides may contain different labels.

[0173] Sequencing methods

[0174] The example flow cell 22 can be used in ensemble sequencing techniques such as sequencing by synthesis (SBS). In ensemble sequencing, amplification primers 30 can be used to form template polynucleotide strands (not shown) to be sequenced on the flow cell 22. At the start of strand formation of the template polynucleotides, library templates can be prepared from any nucleic acid sample (e.g., DNA or RNA sample). The nucleic acid sample can be fragmented into single-stranded, similarly sized (e.g., less than 1000 bp) DNA or RNA fragments. During preparation, adapters can be added to the ends of these fragments. By shortening cycle amplification, various motifs such as sequencing binding sites, indexes, and regions complementary to the primers 30 in the recesses 36 can be introduced into the adapters. The final library template contains DNA or RNA fragments and adapters at both ends. In some examples, identical adapters are added to fragments from a single nucleic acid sample.

[0175] A plurality of library templates may be introduced into flow cell 22. The plurality of library templates are hybridized to one of two primers 30 immobilized, for example, in flow channel 26 or in a recess 36 of flow channel 26.

[0176] Cluster generation may then be performed. In one example of cluster generation, the library templates are copied from the hybridized primers by 3' extension using a high-fidelity DNA polymerase. The original library templates are denatured, leaving copies immobilized in the channels 26 or in the recesses 36. The immobilized copies may be amplified using isothermal bridge amplification or some other form of amplification. For example, the copied templates loop over and hybridize to adjacent complementary primers 30, and the polymerase copies the copied templates to form a double-stranded bridge, which is denatured to form two single strands. These two strands loop over and hybridize to adjacent complementary primers 30 and are extended again to form two new double-stranded loops. This process is repeated for each template copy by cycles of isothermal denaturation and amplification to create compact clonal clusters. Each cluster of double-stranded bridges is denatured. In one example, the reverse strand is removed by specific base cleavage, leaving the forward template polynucleotide strand. Clustering results in the formation of several template polynucleotide strands in the channel 26 or in each well 36. An example of this clustering is bridge amplification, which is one example of a possible amplification. It should be understood that other amplification techniques, such as the Exclusion Amplification (Examp) workflow (Illumina Inc.), may also be used.

[0177] A sequencing primer may be introduced that hybridizes to a complementary sequence on the template polynucleotide strand, which allows the template polynucleotide strand to be sequenced.

[0178] To initiate sequencing, an incorporation mixture may be added to the flow cell 22. In one example, the incorporation mixture includes a liquid carrier, a polymerase, and a 3'OH blocked nucleotide 10. It should be understood that in some examples, the incorporation mixture is selected so as not to activate the catalyst 18, since it is not desirable to initiate cleavage of the blocking group 12 prior to incorporation and imaging. In a guest-host example, the incorporation mixture may include a guest catalyst (e.g., a metal) in a non-activated state. The guest catalyst may be bound (e.g., via a ligand) to the initial polymer hydrogel 16' and then stimulated by orthogonal means (e.g., exposure to a particular wavelength) during deblocking to convert the guest catalyst to an activated form.

[0179] When the incorporation mixture is introduced into flow cell 22, the fluid enters channels 26 and / or recesses 36 (where the template polynucleotide strands reside).

[0180] The 3'OH blocking nucleotides 10 are added to the sequencing primer (thereby extending the sequencing primer) in a template-dependent manner so that detection of the order and type of nucleotides added to the sequencing primer can be used to determine the sequence of the template. More specifically, one of the nucleotides is incorporated by the respective polymerase into the nascent strand complementary to the template polynucleotide strand as the sequencing primer is extended. In other words, in at least some of the template polynucleotide strands across the flow cell 22, each polymerase extends the hybridized sequencing primer by one of the nucleotides in the incorporation mixture.

[0181] In this exemplary method, after incorporation of a nucleotide base into the nascent strand, the incorporation mixture, including any unincorporated 3'OH blocked nucleotides 10, can be removed from the flow cell 22. This can be accomplished using a wash solution (e.g., a buffer).

[0182] As referred to herein, the 3'OH blocked nucleotide 10 contains a reversible termination feature (e.g., 3'OH blocking group 12) that terminates further primer extension once the nucleotide is added to the sequencing primer. Without further incorporation, the most recently incorporated nucleotide 10 can be detected by an imaging event. During the imaging event, an illumination system (not shown) may provide excitation light to the channel 26 and / or well 36.

[0183] A cleavage mixture may then be introduced into the flow cell 22. In the examples disclosed herein, the cleavage mixture is capable of i) removing the 3'OH blocking group 12 from the incorporated nucleotides and ii) cleaving any detectable label from the incorporated nucleotides. The catalyst present on the polymer hydrogel 16' can accelerate the reaction that occurs during removal of the 3'OH blocking group. Removal of the 3'OH blocking group 12 allows for the execution of a subsequent sequencing cycle, and accelerating this reaction with the catalyst 18 can make the entire sequencing process more efficient.

[0184] Some examples of catalysts accelerate intermediate steps during the deblocking reaction. Thus, the catalyst performs its function at the time of incorporation of the cleavage mixture and at the initiation of the deblocking reaction. When a photoacid generator is used as the catalyst 18, additional light exposure may be used to initiate catalytic activity.

[0185] Examples of 3'OH blocking groups and suitable deblocking agents / components in the cleavage mixture include ester moieties that can be removed by base hydrolysis; allyl moieties that can be removed with Nal, chlorotrimethylsilane and Na2S2O3 or Hg(II) in acetone / water; azidomethyls that can be cleaved with phosphines such as tris(2-carboxyethyl)phosphine (TCEP) or tri(hydroxypropyl)phosphine (THP); acetals such as tert-butoxy-ethoxys that can be cleaved under acidic conditions; MOM (-CHOCH3) moieties that can be cleaved with LiBF4 and CH3CN / H2O; 2,4-dinitrobenzenesulfenyl that can be cleaved with nucleophiles such as thiophenol and thiosulfate; tetrahydrofuranyl ethers that can be cleaved with Ag(I) or Hg(II); and 3' phosphates that can be cleaved by phosphatase enzymes (e.g., polynucleotide kinase).

[0186] Washing may be performed between the various fluid delivery steps. A sequence of length n can then be detected by repeating the SBS cycle n times to extend the sequencing primer by n nucleotides. In some examples, paired-end sequencing can be used, in which the forward strand is sequenced and removed, and then the reverse strand is constructed and sequenced.

[0187] Although SBS has been described in detail, it should be understood that the flow cell 22 described herein may be utilized in other sequencing procedures for genotyping, or in other chemical and / or biological applications. In some examples, the primers of the flow cell may be selected to allow simultaneous paired-end sequencing where both the forward and reverse strands are present on the catalytic polymer hydrogel 16', allowing simultaneous base calling of each read. Sequential and simultaneous paired-end sequencing may facilitate detection of genomic rearrangements and repeated sequence elements, as well as detection of gene fusions and novel transcripts. In another example, the flow cell 10 disclosed herein may be used for on-flow cell library generation.

[0188] kit

[0189] Any of the examples of flow cells 22 described herein may be part of a kit. Thus, any of the examples of polymer hydrogels 16' disclosed herein may be part of a kit. Some examples of kits include a flow cell 22 including a substrate 24, a catalytic polymer hydrogel 16' on the substrate 24, the catalytic polymer hydrogel 16' including a catalyst 18, an amplification primer 30 attached to the catalytic polymer hydrogel 16', the catalyst 18 being introduced into the flow cell 22 and accelerating cleavage of the blocking group 12 of the 3'OH blocked nucleotide 10 incorporated into the template strand attached to the amplification primer 30, and a cleavage mixture including components to initiate cleavage of the blocking group 12.

[0190] Additional Notes

[0191] It should be understood that all combinations of the foregoing concepts and further concepts discussed in more detail below (provided that such concepts are not mutually inconsistent) are contemplated as part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as part of the inventive subject matter disclosed herein. It should also be recognized that the terms expressly used in this specification and which may also appear in any disclosures incorporated by reference should be accorded the meaning most consistent with the particular concepts disclosed herein.

[0192] References throughout this specification to "one example," "another example," "an example," etc. mean that a particular element (e.g., a feature, structure, and / or characteristic) described in connection with an example is included in at least one example described herein, and may or may not be present in other examples. Moreover, unless the context clearly dictates otherwise, it should be understood that the described elements with respect to any example may be combined in any suitable manner in the various examples.

[0193] Ranges provided herein should be understood to include the stated range and any value or subrange within the stated range as if such value or subrange were expressly recited. For example, a range of about 2 mm to about 300 mm should be interpreted to include not only the explicitly recited limits of about 2 mm to about 300 mm, but also individual values ​​such as about 40 mm, about 250.5 mm, and subranges such as about 25 mm to about 175 mm. Additionally, when "about" and / or "substantially" are used to describe values, they are meant to encompass slight variations (up to + / - 10%) from the stated values.

[0194] Although several examples have been described in detail, it should be understood that the disclosed examples may be modified. Thus, the above description should be considered as non-limiting.

Claims

1. A kit comprising: A flow cell comprising: A substrate; a catalyzed polymer hydrogel on said substrate, said catalyzed polymer hydrogel comprising a deblocking catalyst; an amplification primer attached to the catalytic polymer hydrogel; Including, a flow cell, wherein the catalyst is introduced into the flow cell and accelerates cleavage of a blocking group of a 3'OH blocked nucleotide incorporated into a template strand attached to the amplification primer; a cleavage mixture comprising components that initiate cleavage of the blocking group; A kit comprising:

2. 2. The kit of claim 1, wherein the deblocking catalyst is selected from the group consisting of an acid catalyst, a base catalyst, an enzyme, a peptide, a DNAzyme, an organic catalyst, and combinations thereof.

3. The kit of claim 1 , wherein the deblocking catalyst is a metal of a metal-ligand complex attached to the polymer hydrogel.

4. 2. The kit of claim 1, wherein the deblocking catalyst is an acid catalyst selected from the group consisting of carboxylic acids, phosphonic acids, sulfonic acids, and combinations thereof.

5. 2. The kit of claim 1, wherein the deblocking catalyst is a base catalyst selected from the group consisting of amines, heterocyclic amines, and combinations thereof.

6. The kit of claim 1 , wherein the deblocking catalyst is an enzyme.

7. The kit of claim 1 , wherein the deblocking catalyst is a peptide.

8. The kit of claim 1 , wherein the deblocking catalyst is a DNAzyme.

9. 1. A method comprising: Incorporating the mixture A substrate; a catalyzed polymer hydrogel on said substrate, said catalyzed polymer hydrogel comprising a deblocking catalyst; a template strand attached to the catalytic polymer hydrogel; and incorporating individual nucleotides into each nascent strand along the template strand by introducing the individual nucleotides into a flow cell comprising: a dye label attached to the base; a 3'OH blocking group attached to the sugar; and removing the incorporated mixture; and Optically imaging the incorporation of individual nucleotides; and introducing into the flow cell a cleavage mixture comprising a component that initiates cleavage of the 3'OH blocking group, whereby the deblocking catalyst accelerates cleavage of the 3'OH blocking group; A method comprising:

10. 10. The method of claim 9, wherein the catalyst accelerates the removal of the outer protecting group of the 3'OH blocking group and a reagent in the cleavage mixture removes the inner protecting group of the 3'OH blocking group.

11. A flow cell comprising: A substrate; A catalyzed polymer hydrogel on the substrate, Phosphonic acid; Heterocyclic amines; enzyme; peptide; DNAzymes; The metal of the metal-ligand complex; An organic catalyst selected from the group consisting of urea, thiourea, imidazole, guanidine, 1,8-diazabicyclo(5.4.0)-undec-7-ene, and combinations thereof; and Photoacid generator a catalytic polymer hydrogel comprising a deblocking catalyst selected from the group consisting of: an amplification primer attached to the catalytic polymer hydrogel; A flow cell comprising:

12. The flow cell of claim 11 , wherein the catalyst is incorporated into a monomer unit of the catalytic polymer hydrogel.

13. The flow cell of claim 11 , wherein the catalyst is grafted to an initial polymer hydrogel.

14. 12. The flow cell of claim 11, wherein the catalytic polymer hydrogel comprises an initial polymer hydrogel, the flow cell further comprises an oligonucleotide attached to the initial polymer hydrogel, and the deblocking catalyst is attached to a complementary oligonucleotide tether hybridized to the oligonucleotide.

15. A flow cell comprising: A substrate; a polymer hydrogel on the substrate, the polymer hydrogel comprising a first member of a hydrogen bonding pair; a deblocking catalyst attached to the polymer hydrogel via a second member of the hydrogen bonding pair; an amplification primer attached to the catalytic polymer hydrogel; A flow cell comprising:

16. 1. A method comprising: applying a catalytic polymer hydrogel to a surface of a flow cell substrate, said catalytic polymer hydrogel comprising: Phosphonic acid; Heterocyclic amines; enzyme; peptide; DNAzymes; The metal of the metal-ligand complex; An organic catalyst selected from the group consisting of urea, thiourea, imidazole, guanidine, 1,8-diazabicyclo(5.4.0)-undec-7-ene, and combinations thereof; and Photoacid generator and comprising a deblocking catalyst selected from the group consisting of: attaching an amplification primer to the catalytic polymer hydrogel; A method comprising:

17. 17. The method of claim 16, further comprising forming the catalytic polymer hydrogel containing the unblocking catalyst.

18. 20. The method of claim 17, wherein forming the catalytic polymer hydrogel comprises copolymerizing a first monomer comprising a primer-grafted functional group with a second monomer comprising the unblocking catalyst.

19. forming the catalytic polymer hydrogel comprises Synthesizing an initial polymer hydrogel; grafting said catalyst onto said initial polymer hydrogel; 20. The method of claim 17, comprising:

20. forming the catalytic polymer hydrogel comprises Synthesizing an initial polymer hydrogel; grafting oligonucleotides onto said initial polymer hydrogel; hybridizing a complementary oligonucleotide tether to said oligonucleotide, said catalyst being attached to said complementary oligonucleotide tether; 20. The method of claim 17, comprising:

21. forming the catalytic polymer hydrogel comprises Synthesizing an initial polymer hydrogel; attaching a metal-ligand complex to the initial polymer hydrogel, the metal of the metal-ligand complex being the catalyst; 20. The method of claim 17, comprising:

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