RU2829848C1 - Kits and flow cells - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to enzymology of single molecules and can be used to study enzymatic activities of single enzyme molecules using nanotechnology, as well as processes of denaturation of single enzyme molecules. Sequencing kit includes: a flow cell comprising: a substrate; catalytic polymer hydrogel on a substrate; an amplification primer attached to a catalytic polymer hydrogel; cleavage mixture containing a component which initiates cleavage of the blocking group. Catalytic polymer hydrogel includes a deblocking catalyst which accelerates cleavage of 3' OH blocking group of a blocked nucleotide introduced into a flow cell and embedded in a matrix chain attached to an amplification primer. Method of sequencing in a flow cell includes introducing the added mixture into the flow cell; embedding the individual nucleotides into corresponding nascent chains along the template chains; removing the added mixture; inclusion of individual nucleotides using optical imaging; and introducing the splitting mixture.

EFFECT: group of inventions provides improved release kinetics.

15 cl, 3 dwg

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 62/948,605, filed December 16, 2019, the contents of which are incorporated herein by reference in their entirety.

PREREQUISITES FOR THE CREATION OF THE INVENTION

[0002] Various protocols in biological or chemical research involve performing a large number of controlled reactions on local substrate surfaces or within predetermined reaction chambers. The intended reactions can then be observed or detected, and subsequent analysis can help identify or reveal the properties of the chemicals involved in the reaction. In some examples, the controlled reactions generate fluorescence, and thus an optical system can be used to detect them. In other examples, the controlled reactions change charge, conductivity, or some other electrical property, and thus an electronic system can be used to detect them.

INTRODUCTION

[0003] The first aspect of the invention described herein is a kit that includes a flow cell comprising: a support; a catalytic polymer hydrogel on the support, wherein the catalytic polymer hydrogel includes a deblocking catalyst; and an amplification primer attached to the catalytic polymer hydrogel; wherein the catalyst accelerates the cleavage of the 3' OH blocking group of a blocked nucleotide introduced into the flow cell and incorporated into a template strand attached to the amplification primer; and a cleavage mixture including a component that initiates the cleavage of the blocking group.

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

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

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

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

[0008] In one example according to the first aspect, the deblocking catalyst is an enzyme.

[0009] In one example according to the first aspect, the deblocking catalyst is a peptide.

[0010] In one example according to the first aspect, the deblocking catalyst is a DNA enzyme.

[0011] It should be understood that any of the features of the kit described herein can be combined in any desired manner and/or in any desired configuration to achieve the advantages described herein, including, for example, improved deblocking kinetics.

[0012] A second aspect described herein includes introducing an add-on mixture into a flow cell comprising: a support, a catalytic polymer hydrogel on the support, wherein the catalytic polymer hydrogel includes a deblocking catalyst; and template strands attached to the catalytic polymer hydrogel; thereby incorporating individual nucleotides into respective nascent strands along the template strands, wherein the individual nucleotides include: a dye label attached to a base; and a 3' OH blocking group attached to a sugar; removing the add-on mixture; incorporating the individual nucleotides using optical imaging; and introducing a cleavage mixture comprising a component that initiates cleavage of the 3' OH blocking group into the flow cell, whereby the deblocking catalyst accelerates cleavage of the 3' OH blocking group.

[0013] In the example according to the second aspect, the catalyst accelerates the removal of the external 3' OH protecting group of the blocking group, and wherein the reagent in the cleavage mixture removes the internal 3' OH protecting group of the blocking group.

[0014] It should be understood that any features of the method can be combined together in any desired manner. In addition, it should be understood that any combination of features of the method and/or kit can 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, improving deblocking kinetics.

[0015] A third aspect of the invention described herein is a flow cell comprising: a support; a catalytic polymer hydrogel on the support, wherein the catalytic polymer hydrogel comprises a deblocking catalyst selected from the group consisting of a phosphonic acid; a heterocyclic amine; an enzyme; a peptide; a DNA enzyme; a metal 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 according to the third aspect, the catalyst is integrated into a monomer unit of the catalytic polymer hydrogel.

[0017] In one example according to the third aspect, the catalyst is grafted onto the starting polymer hydrogel.

[0018] In one example according to the third aspect, the catalytic polymer hydrogel includes a starting polymer hydrogel, and wherein the flow cell further comprises an oligonucleotide attached to the starting polymer hydrogel, and the deblocking catalyst is attached to a complementary oligonucleotide ligand that hybridizes with the oligonucleotide.

[0019] It should be understood that any of the flow cell elements can be combined in any desired manner. Moreover, it should be understood that any combination of flow cell characteristics and/or kit and/or method characteristics can be used together and/or in combination with any of the examples disclosed herein to achieve advantages such as those described in this disclosure, including, for example, creating a polymer hydrogel that promotes improved deblocking kinetics.

[0020] A fourth aspect of the invention described herein is a flow cell comprising: a support; a polymer hydrogel on the support, wherein the polymer hydrogel includes 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 elements of this flow cell can be combined in any desired manner. It should also be understood that any combination of the characteristics of this flow cell and/or another flow cell and/or the characteristics of the kit and/or method can 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, improving deblocking kinetics.

[0022] A fifth aspect described herein is a method comprising: applying a catalytic polymer hydrogel to a surface of a flow cell substrate, wherein the catalytic polymer hydrogel comprises a deblocking catalyst selected from the group consisting of a phosphonic acid; a heterocyclic amine; an enzyme; a peptide; a DNA enzyme; a metal 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 amplification primers attached to the catalytic polymer hydrogel.

[0023] An example of the fifth aspect further includes forming a catalytic polymer hydrogel including a deblocking catalyst.

[0024] In the example according to the fifth aspect, forming the catalytic polymer hydrogel comprises copolymerizing a first monomer comprising a functional group for grafting a primer with a second monomer comprising a deblocking catalyst.

[0025] In the example according to the fifth aspect, forming the catalytic polymer hydrogel includes: synthesizing a starting polymer hydrogel; and grafting a catalyst onto the starting polymer hydrogel.

[0026] In an example according to the fifth aspect, forming a catalytic polymer hydrogel includes: synthesizing a starting polymer hydrogel; grafting an oligonucleotide onto the starting polymer hydrogel; and hybridizing a complementary oligonucleotide linkage with the oligonucleotide, wherein the catalyst is attached to the complementary oligonucleotide linkage.

[0027] In an example according to a fifth aspect, forming a catalytic polymer hydrogel includes: synthesizing a starting polymer hydrogel; and attaching a metal-ligand complex to the starting polymer hydrogel, wherein the metal of the metal-ligand complex is a catalyst.

[0028] It should be understood that any features of the method can be combined together in any desired manner. It should also be understood that any combination of features of this method and/or another method and/or features of the kit and/or flow cell can 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, improving deblocking kinetics.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0029] Features according to examples of the present invention will become apparent from the following detailed description and drawings, in which like reference numerals correspond to similar, although possibly non-identical, components. For brevity, reference numerals or features having a previously described function may or may not be described with respect to other drawings in which they occur.

[0030] Fig. 1 is a schematic flow chart illustrating a multi-step process for deblocking an exemplary 3' OH blocked nucleotide;

[0031] Fig. 2 is a schematic illustration of an example catalyst attached to an example polymer hydrogel via an example non-covalent bond;

[0032] Fig. 3A shows a top view of an example flow cell.

[0033] Fig. 3B shows an enlarged, partially cut-away view of an example flow channel of a flow cell, including an example of a polymer hydrogel located in the flow channel; and

[0034] Fig. 3C shows an enlarged, partially cut-away view of an example flow channel of a flow cell, including an example of a polymer hydrogel disposed in recesses formed in the flow channel.

DETAILED DESCRIPTION

[0035] Some nucleic acid sequencing techniques utilize a 3' OH blocking group bound to an oxygen atom of a sugar molecule in a nucleotide. The 3' OH blocking group can serve as a reversible terminator that allows only one base to be incorporated in each sequencing cycle. In some cases, the 3' OH blocking group must be removed to initiate the next sequencing cycle. Temporarily stopping the incorporation of nucleotides allows, among other things, for the washout of unincorporated nucleotides with excitation of a detectable label (e.g., a fluorescent dye) attached to the incorporated nucleotides, visualization of the excited detectable labels, etc.

[0036] A catalytic polymer hydrogel is described 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 such that it is not active during the incorporation process. The catalyst may be selected to carry out the reaction that occurs during deblocking (after incorporation and imaging) and thus may accelerate the cleavage of any 3' OH blocking group. In some embodiments, the catalyst may be used for 3' OH blocking groups that are more stable in aqueous solutions but tend to have slower deblocking kinetics. In these cases, the catalyst may help reduce the time of each sequencing cycle, the overall sequence rotation time, and phasing issues that may arise during slow deblocking, to name a few of its advantages.

[0037] During the cleavage of the 3' OH blocking group, a number of reactions can occur. Figure 1 depicts one example of this series of reactions for an example 3' OH blocked nucleotide 10. 3' OH blocked nucleotide 10 includes a heterocyclic base B, a sugar (shown as deoxyribose in Figure 1), 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 shown in Figure 1, a phosphine (PR ₃ , where R is H or (CH ₂ ) _n 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 the hemiaminal ester, and in step 3, the hemiaminal ester is hydrolyzed to form 3' OH. For the deblocking process shown in Fig. 1, any catalyst that accelerates hydrolysis steps 2 and/or 3 shown in Fig. 1 can be incorporated into or placed on the polymer hydrogel, or any catalyst that accelerates the reaction in step 1 shown in Fig. 1 can be incorporated into or placed on the polymer hydrogel. If the catalyst is specifically selected to catalyze step 1 shown in Fig. 1, the catalyst accelerates the removal of the external 3' OH protecting group of the blocking group 12 (e.g., the azide group shown in Fig. 1), and a reagent in the cleavage mixture (e.g., -CH ₂ - shown in Fig. 1) can be used to remove the internal protecting group of the 3' OH blocking group 12.

[0038] Although Fig. 1 illustrates one example of a deblocking process, it should be understood that the catalyst used may depend on the 3' OH blocking group and the reaction(s) used to cleave that 3' OH blocking group.

[0039] Definitions

[0040] It should be understood that the terms used in this document will have their ordinary meaning in the relevant field unless otherwise specified. Below are a number of terms used in this document and their meanings.

[0041] Singular forms include plural references unless the context clearly dictates otherwise.

[0042] The terms “consisting of,” “including,” “containing,” and various forms of these terms are synonymous with each other and have the same meaning.

[0043] The terms "top," "bottom," "below," "above," "on," and the like are used to describe the flow cell and/or various components of the flow cell. It should be understood that these directional terms do not imply a particular orientation, but are used to indicate the relative orientation between components. The use of directional terms should not be interpreted as limiting the examples described herein to any particular orientation(s).

[0044] An "acrylamide monomer" is a monomer with the structure or a monomer containing an acrylamide group with this structure. Examples of a monomer containing an acrylamide group include azidoacetamidopentyl acrylamide: and N-isopropylacrylamide: Other acrylamide monomers may be used, some examples of which are described in this document.

[0045] As used herein, the term "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). An alkyl group may have from 1 to 20 carbon atoms. Typical alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, and the like. By way of example only, "C1-C6 alkyl" indicates that there are from one to six carbon atoms present 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 tert-butyl.

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

[0047] As used herein, the term "alkynyl" refers to a straight or branched hydrocarbon chain containing one or more triple bonds. An alkynyl group may have from 2 to 20 carbon atoms.

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

[0049] As used herein, the term "aryl" refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent carbon atoms) that contains only carbon in the ring backbone. When aryl is a ring system, each ring in the system is aromatic. An aryl group may have from 6 to 18 carbon atoms. Examples of aryl groups include phenyl, naphthyl, azulenyl, and anthracenyl.

[0050] The functional group "amine" or "amino" refers to the group -NR _a R _b , where R _a and R _b are independently selected from hydrogen (e.g., ), 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 of two components being attached, fastened, glued, connected, or linked to each other. For example, a nucleic acid may be attached to a polymer hydrogel via a covalent or non-covalent bond. A covalent bond is characterized by the presence of electron pairs between atoms. A non-covalent bond is a chemical bond that does not involve the sharing of electron pairs, but may include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic or hydrophobic interactions.

[0052] The "azide" functional group or "azido" refers to -N ₃ .

[0053] The terms "block copolymer" and monomer units formed "in a block" refer to a copolymer formed when two or more monomers are grouped together to form blocks of repeating units. Each block must have at least one feature and/or at least one block-specific functional group (e.g., azide for primer attachment, catalyst, etc.) that is(are) absent from adjacent blocks.

[0054] As used herein, the term "catalytic polymer hydrogel" refers to a copolymer in which a catalyst is incorporated into one of the monomer units, or to a parent polymer hydrogel to which a catalyst is attached. As used herein, the term "parent polymer hydrogel" refers to a polymerized hydrogel prior to any reaction/interaction to introduce a catalyst.

[0055] As used herein, the term "carbocyclyl" means a non-aromatic cyclic ring or ring system containing only carbon atoms in the main chain of the ring system. When the carbocyclyl is a ring system, two or more rings may be joined together by condensation, bridge formation, or spiro bonding. Carbocyclyls may have any degree of saturation, provided that at least one ring in the ring system is not aromatic. Thus, carbocyclyls include cycloalkyls, cycloalkenyls, and cycloalkynyls. A carbocyclyl group may contain from 3 to 20 carbon atoms. Examples of carbocyclyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydroindene, bicyclo[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl.

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

[0057] As used herein, the term "cycloalkyl" refers to a fully saturated (no double or triple bonds) mono- or multicyclic hydrocarbon ring system. When the rings are comprised of two or more rings, they may be joined by a fusion mechanism. Cycloalkyl groups may contain from 3 to 10 atoms in the ring(s). In some embodiments, cycloalkyl groups may contain from 3 to 8 atoms in the ring(s). A cycloalkyl group may be substituted or unsubstituted. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0058] As used herein, the term "cycloalkenyl" or "cycloalkene" means a carbocyclyl ring or ring system having at least one double bond, wherein no ring in the ring system is aromatic. Examples include cyclohexenyl or cyclohexene and norbornenyl or norbornene.

[0059] As used herein, the term "cycloalkynyl" or "cycloalkyne" means a carbocyclyl ring or ring system having at least one triple bond, wherein neither ring in the ring system is aromatic. An example is cyclooctyne. Another example is bicyclononine.

[0060] As used herein, the term "deposition" refers to any suitable application technique, which may be manual or automated and in some cases may result in a change in surface properties. Typically, deposition may be accomplished using vapor deposition technologies, coating technologies, grafting technologies, and the like. Some specific examples include chemical vapor deposition (CVD), spray coating (e.g., ultrasonic spraying), spin coating, dip coating, doctor blade coating, inkjet coating, aerosol printing, screen printing, microcontact printing, inkjet printing, and the like.

[0061] As used herein, the term "dimple" refers to a discrete concave feature in a substrate or patterned resin that has an opening in the surface that is at least partially surrounded by a gap(s) of the substrate or patterned resin. Dimples may have any of a variety of shapes at their openings in the surface, including, for example, circular, elliptical, square, polygonal, star-shaped (with any number of vertices), etc. The cross-section of the depression, taken perpendicular to the surface, may be curved, square, polygonal, hyperbolic, conical, angular, etc. For example, the depression may be a dimple or two interconnected dimples. The depression may also have more complex architectures, such as ridges, stepped features, etc.

[0062] As used herein, the term "each" with respect to a collection of elements is intended to refer to an individual element in the collection, but does not necessarily refer to every element in the collection. Exceptions may arise where express disclosure or context clearly indicates otherwise.

[0063] As used herein, the term "flow cell" means a vessel having a chamber (e.g., a flow channel) in which a reaction can occur, an inlet for delivering reagent(s) into the chamber, and an outlet for removing reagent(s) from the chamber. In some examples, the chamber allows for detection of the reaction occurring in the chamber. For example, the chamber may include one or more transparent surfaces that allow for optical detection of chips, optically labeled molecules, and the like.

[0064] As used herein, the term "flow channel" or "channel" may be a region formed between two components that are connected or otherwise attached to each other and that can selectively receive a liquid sample. In some examples, the flow channel may be formed between a patterned or unpatterned substrate and a cover and may thus be in fluid communication with one or more recesses formed in a patterned resin. The flow channel may also be formed between two surfaces of a patterned or unpatterned substrate that are bonded to each other.

[0065] As used herein, the term "heterocyclic amine" refers to an aromatic ring or ring system (i.e., two or more fused rings having two adjacent atoms) containing an amine nitrogen as the one or only heteroatom in the main chain of the ring.

[0066] As used herein, the term "heteroaryl" refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent atoms) that contain one or more heteroatoms, i.e., an element other than carbon, including, but not limited to, nitrogen, oxygen, and sulfur, in the ring backbone. When the heteroaryl is a ring system, each ring in the system is aromatic. A heteroaryl group may contain 5-18 ring members (i.e., the number of atoms that make up the ring backbone).

[0067] As used herein, the term "heterocyclyl" means a non-aromatic cyclic ring or ring system containing at least one heteroatom in the ring backbone. Heterocycles may be joined together by condensation, bridging, or spiro bonding. Heterocyclyls may have any degree of saturation, provided that at least one ring in the ring system is not aromatic. The heteroatom(s) may be present in either the non-aromatic or aromatic ring in the ring system. A heterocyclyl group may contain from 3 to 20 ring members (i.e., the number of atoms that make up the backbone, including carbon atoms and heteroatoms). In some examples, the heteroatom(s) is/are O, N, or S.

[0068] As used herein, the terms "hydrazine" or "hydrazinyl" refer to the group -NHNH ₂ .

[0069] As used herein, the term “hydrazone” or “hydrazonyl” refers to group wherein R _a and R _b are 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.

[0070] As used herein, the terms "hydroxy" or "hydroxyl" refer to the -OH group.

[0071] As used herein, the term "intermediate region" refers to a region, such as a substrate, structured resin, or other support, that separates depressions. For example, an intermediate region may separate one depression of an array from another depression of the array. The two depressions separated from each other may be discrete, i.e., without physical contact with each other. In many examples, the intermediate region is continuous while the depressions are discrete, such as in the case of a plurality of depressions formed on an otherwise continuous surface. In other examples, the intermediate regions and features are discrete, such as in the case of a plurality of grooves separated by corresponding intermediate regions. The separation provided by the intermediate region may be partial or complete. The intermediate regions may have a surface material that is different from the surface material of the depressions formed on the surface. For example, the trench regions may contain a polymer and a primer set, while the intermediate regions may contain neither a polymer nor a primer set.

[0072] As used herein, the term "nitrile oxide" means the group "R _a C≡N ⁺ O ^- " in which the element R _a is defined herein. Examples of the preparation of nitrile oxide include its in situ generation from aldoximes by treatment with chloramide-T or by the action of a base on imidoyl chlorides [RC(Cl)=NOH] or as a result of the reaction between hydroxylamine and an aldehyde.

[0073] In the context of this document, the term "nitron" means a group in which R ₁ , R ₂ and R ₃ may be any of the groups _Ra and R _b defined herein.

[0074] As used herein, the term "nucleotide" includes a nitrogen-containing heterocyclic base, a sugar, and one or more phosphate groups. Nucleotides are the monomeric units of a nucleotide sequence. In RNA, the sugar is ribose, and in DNA, it is deoxyribose, i.e., a sugar that lacks the hydroxyl group present at the 2' position of ribose. The nitrogen-containing heterocyclic base (i.e., the nucleotide base) can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G), and modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and modified derivatives or analogs thereof. The C-1 atom of deoxyribose is bonded to N-1 of pyrimidine or N-9 of purine. A nucleic acid analogue may have any altered phosphate backbone, sugar or nitrogenous base. Examples of nucleic acid analogues include, for example, universal bases or phosphate-sugar backbone analogues such as peptide nucleic acid (PNA).

[0075] The term "pattern resin" refers to any polymer in which recesses can be formed. Specific examples of pattern resin resins and methods will be further described below.

[0076] In the context of this document, the term "phosphonic acid" refers to R-PO ₃ H ₂ .

[0077] As used herein, the term "primer" is defined as a single-stranded nucleotide sequence (e.g., single-stranded DNA or single-stranded RNA). Some primers, referred to herein as amplification primers, serve as a starting point for amplifying templates and generating clusters. Other primers, referred to herein as sequencing primers, serve as a starting point for DNA or RNA synthesis. The 5' end of a primer can be modified to allow a binding reaction with a functional group of a polymer. The length of a primer can be any number of bases and can include a plurality of nucleotides of artificial origin. In one example, a sequencing primer is a short chain having a length in the range of 10 to 60 bases or 20 to 40 bases.

[0078] As used herein, the term "separating layer" refers to a material that binds two components together. In some examples, the separating layer may be a radiation-absorbing material that promotes binding, or may be brought into contact with a radiation-absorbing material that promotes binding.

[0079] The term "support" refers to a structure into which various components of the flow cell (e.g., catalytic polymer hydrogel, primer(s), etc.) can be added. The support can be a plate, a panel, a rectangular sheet, a matrix, or any other suitable configuration. The support is substantially rigid and insoluble in an aqueous liquid. The support can be inert with respect to the chemistry used to modify the recesses or present in the recesses. For example, the support can be inert with respect to the chemistry used to form the catalytic polymer hydrogel, to attach the primer(s), etc. The support can be a single-layer structure or a multi-layer structure (e.g., including a support and a resin patterned on the support). Examples of suitable supports will be described below.

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

[0081] The term "thiol" in reference to the functional group refers to -SH.

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

[0083] As used herein, the term "tetrazole" refers to a five-membered heterocyclic group comprising four nitrogen atoms. The tetrazole may be optionally substituted.

[0084] Catalytic polymer hydrogel

[0085] The polymer backbone in any example of the catalytic polymer hydrogel may be a semi-rigid polymeric material that is permeable to liquids and gases. In some examples, the catalytic polymer hydrogel includes a starting polymer hydrogel to which a catalyst is attached. In these examples, the catalyst is added to the starting polymer hydrogel by a post-polymerization treatment. In other examples, the catalytic polymer hydrogel may be a copolymer in which one of the monomer components is a catalyst.

[0086] An example of a parent polymer hydrogel includes an acrylamide copolymer, such as a copolymer of N-(5-azidoacetamidylpentyl)acrylamide and acrylamide (PAZAM). PAZAM and some other forms of acrylamide copolymer are represented by the following structure (I):

moreover:

R ^A is selected from the group consisting of azido, 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;

each of R ^C , R ^D and R ^E is independently selected from the group consisting of H and optionally substituted alkyl;

each of -(CH ₂ ) _p - may be optionally substituted;

p is an integer in the range 1 to 50;

n is an integer in the range 1 to 50,000; and

m is an integer in the range 1 to 100,000.

[0087] One skilled in the art will appreciate that the configuration of the repeating units n and m in structure (I) is typical, and the monomeric subunits may be present in any order in the polymer structure (e.g., random, block, structured form, or a combination thereof).

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

[0089] In some examples, PAZAM and other forms of acrylamide copolymer are linear polymers. In some other examples, PAZAM and other forms of acrylamide copolymer are weakly crosslinked polymers.

[0090] In other examples, the parent polymer hydrogel may be a variant of structure (I). In one example, the acrylamide unit may be replaced with N,N-dimethylacrylamide ( ). In this example, the acrylamide unit in structure (I) can be replaced where each of R ^D , R ^E and R ^F is H or C1-C6 alkyl, and each of R ^G and R ^H is C1-C6 alkyl (instead of H, as in the case of acrylamide). In this example, q can be an integer in the range from 1 to 100,000. In another example, N,N-dimethylacrylamide can be used in addition to the acrylamide unit. In this example, structure (I) can include in addition to repeating elements n and m, wherein each of R ^D , R ^{E ,} and R ^F is H or C1-C6 alkyl, and each of R ^G and R ^H is C1-C6 alkyl. In this example, q may be an integer in the range of 1 to 100,000.

[0091] As another example of the starting polymer hydrogel, the repeating element n in structure (I) can be replaced by a monomer comprising a heterocyclic azide group having the structure (II):

wherein R ¹ is H or C1-C6 alkyl; R ₂ is H or C1-C6 alkyl; L is a linker comprising a linear chain of 2-20 atoms selected from the group consisting of carbon, oxygen and nitrogen and 10 optional substituents on carbon and any nitrogen atoms in the chain; E is a linear chain containing 1 to 4 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on carbon and any nitrogen atoms in the chain; A is an N-substituted amide in which H or C1-C4 alkyl is attached to N; and Z is a nitrogen-containing heterocycle. Examples of Z include 5-10 ring members present as a single cyclic structure or a fused structure. Some specific examples of Z include pyrrolidinyl, pyridinyl or pyrimidinyl.

[0092] As another example, the starting polymer hydrogel may include a repeating unit of each of structures (III) and (IV):

wherein each of R ^1a , R ^2a , R ^1b and R ^2b is independently selected from hydrogen, optionally substituted alkyl or optionally substituted phenyl; each of R ^3a and R ^3b is independently selected from hydrogen, optionally substituted alkyl, optionally substituted phenyl or optionally substituted C7-C14 aralkyl; and each of L ¹ and L ² is independently selected from an optionally substituted alkylene linker or an optionally substituted heteroalkylene linker.

[0093] It should be understood that other molecules can be used to form the starting polymer hydrogel, provided that they are functionalized with grafting oligonucleotide primers. Other examples of suitable polymer layers include those having a colloidal structure, such as agarose; or a polymeric network structure, such as gelatin; or a cross-linked polymer structure, such as polyacrylamide polymers and copolymers, silane-free acrylamide (SFA) or an azidolized version of SFA. Examples of suitable polyacrylamide polymers can be synthesized from acrylamide and acrylic acid or acrylic acid containing a vinyl group, or from monomers that undergo [2+2] photocycloaddition reactions. Other examples of suitable starting polymer hydrogels include mixed copolymers of acrylamides and acrylates. In the examples described herein, various polymer architectures containing acrylic monomers (e.g., acrylamides, acrylates, etc.) can be used, such as branched polymers, including star polymers, star or star block polymers, dendrimers, etc. For example, monomers (e.g., acrylamide, an acrylamide-containing catalyst, etc.) can be introduced, either randomly or en bloc, into branches (fragments) of a star polymer.

[0094] The starting polymer hydrogel can be formed using any suitable copolymerization process. The catalyst can then be attached to the starting polymer hydrogel to form a catalytic polymer hydrogel. A variety of different post-polymerization methods can be used to attach the catalyst to the starting polymer hydrogel.

[0095] In one example, the catalyst may be grafted onto the parent polymer hydrogel. In this example of the method, forming the catalytic polymer hydrogel includes synthesizing the parent polymer hydrogel (which does not include the catalyst); and grafting the catalyst onto the parent polymer hydrogel. In one example, grafting includes a click reaction, such as a copper-catalyzed click reaction, or using a voltage-promoted click reaction without a catalyst, such as with bicyclo[6.1.0] non-4-yne (BCN). In one example, the click reaction results in the covalent attachment of the catalyst to the parent polymer hydrogel. It should be understood that the reaction that occurs will depend on the chemical composition of the parent 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, alkali catalyst, organic catalyst, enzyme catalyst, peptide catalyst, or DNA enzyme catalyst can be grafted onto the starting polymer hydrogel. Some of the listed catalysts can belong to two of the listed catalyst categories. In some specific examples, the deblocking catalyst is selected from the group consisting of a phosphonic acid; a heterocyclic amine; an enzyme; a peptide; a DNA enzyme; a metal 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.

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

[0098] In other examples, the catalyst is an alkali catalyst selected from the group consisting of an amine, a heterocyclic amine, and combinations thereof. Lewis bases such as thioethers, triazoles, imidazoles, etc., may be particularly suitable elements for catalyzing the azide-phosphine reaction (step 1 of Fig. 1). The base catalyst may catalyze the hydrolysis reaction that occurs during deprotection.

[0099] The organic catalyst is comprised of carbon, hydrogen, sulfur, and/or other non-metallic elements found in organic compounds and increases the rate of the chemical reaction of removing protective groups. 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.

[00100] The enzyme catalyst may be a hydrolase. Examples of suitable hydrolases include phosphatases, esterases (e.g., acetylcholinesterase, lipases, etc.), sequence-specific proteases (e.g., TEV protease or thrombin), and glycosidases (which do not cleave ribose, such as cellulose or amylase). Another suitable hydrolase is carbonic anhydrase. Another enzyme catalyst is a serine protease, which cleaves peptide bonds in proteins and can catalyze the hydrolysis of amides and esters.

[00101] Other catalysts include DNA enzymes, which are also called deoxyribozymes. Examples of DNA enzymes include DNA enzymes that target the hydrolysis of the ester group, including DNA mimetics of enzymes such as esterases. In these examples, the chemical reaction to remove the protecting groups is catalyzed by the DNA enzyme.

[00102] Although several examples have been proposed, it is believed that any available enzyme or engineered enzyme can be used, provided that the enzyme is capable of catalyzing the removal of protecting groups from the 3' OH blocking group.

[00103] Examples of a peptide catalyst include supermolecular β-barrel esterases with a hydrophobic outer fraction and a histidine-rich pore interior, self-assembled peptide catalysts, and other catalytic peptide assemblies. In these examples, the chemical reaction of removing protecting groups is catalyzed by the peptide.

[00104] As another example, the catalyst can be introduced by non-covalent attachment to the surface of the parent polymer hydrogel. Non-covalent attachments can include, for example, hybridization with oligonucleotides grafted onto the surface of the parent polymer hydrogel, hydrogen bond arrays, biotin-streptavidin or other similar bonds, metal-ligand complexation, or the like.

[00105] Fig. 2 schematically depicts one example of non-covalent attachment of a catalyst. In particular, Fig. 2 shows oligonucleotide 14 attached to parent polymer hydrogel 16, and catalyst 18 attached to a complementary oligonucleotide ligament 20 that hybridizes with oligonucleotide 14. Oligonucleotide 14 attached to parent polymer hydrogel 16 can be grafted as described herein with reference to Figs. 3A-3C for amplification primers (e.g., via a click reaction on a terminal azide). Oligonucleotide 14 can have from 5 to 20 nucleotides (shown as N in Fig. 2), and the sequence can be similar to or different from the sequence of amplification primers (described below). The catalyst 18 is attached to a linker 20, which has a sequence complementary to the oligonucleotide 14. The catalysts 18, which can be attached according to the example shown in Fig. 2, include any acid catalysts, base catalysts, organic catalysts (e.g., imidazoles, guanidines, etc.), copper catalysts, enzyme catalysts, peptide catalysts, or DNA enzyme catalysts.

[00106] 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.

[00107] An example of a method for forming a catalytic polymer hydrogel 16' shown in Fig. 2 includes synthesizing a starting polymer hydrogel 16 (which does not include a catalyst 18); grafting an oligonucleotide 14 onto the starting polymer hydrogel 16; and hybridizing a complementary oligonucleotide linkage 20 to the oligonucleotide 14, wherein the catalyst 18 is attached to the complementary oligonucleotide linkage 20.

[00108] Another example of non-covalent attachment of catalyst 18 to the starting polymer hydrogel 16 includes arrays of hydrogen bonds. In this example, catalyst 18 is hydrogen bonded to polymer hydrogel 16. Any pair of hydrogen bonds can be used, where one component of the pair is covalently attached to the starting polymer hydrogel and the other component of the pair is attached to catalyst 18. Examples of pairs of hydrogen bonds include, for example, O⋅H, NH⋅N, and CH⋅N.

[00109] Another example of non-covalent attachment of catalyst 18 to starting polymer hydrogel 16 involves the formation of a metal-ligand complex. In one example, catalyst 18 is a metal of a metal-ligand complex attached to starting polymer hydrogel 16. Catalyst 18 can be any suitable catalytic metal, such as copper, palladium, ruthenium, etc. Catalytic metal complexes with a ligand attached to starting polymer hydrogel 16. Examples of a metal-ligand complex include copper(II) complexes with ligands such as bis(2-pyridylmethyl)amine or pyridine-functionalized cyclodextrin.

[00110] An example of a method for forming this example of a catalytic polymer hydrogel 16' comprises synthesizing a starting polymer hydrogel 16; and attaching a metal-ligand complex to the starting polymer hydrogel 16, wherein the metal of the metal-ligand complex is a catalyst 18.

[00111] Other catalysts 18 that can be attached to the initial polymer hydrogel 16 include photoacid generators. When using a photoacid generator as the catalyst 18, the catalytic activity can be initiated or enhanced by exposure to external stimuli, such as photons, with a wavelength corresponding to a particular photoacid generator.

[00112] The photoacid generator may be attached to the polymer hydrogel 16' via a guest-host chemistry, wherein 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 external stimulation. Examples of host compounds include cucurbiturils and cyclodextrins. An example of a guest photoacid generator catalyst is a blue/visible light photoacid generator. Photoinitiated catalysts (e.g., photoacid generators) may also be used as a guest in guest-host examples.

[00113] The host-guest molecule may be attached to the parent polymer hydrogel 16 using any of the post-polymerization methods (e.g., grafting, non-covalent attachment) described herein.

[00114] Although several examples have been provided, it should be understood that the catalyst 18 attached to the starting polymer hydrogel 16 may depend in part on the chemical composition of the starting polymer hydrogel 16 and the chemical composition of the blocking group 12 included in the nucleotide 10 to be introduced into the catalytic polymer hydrogel 16'.

[00115] In other examples described herein, the starting polymer hydrogel 16 is not used. Instead, the copolymerization product is a catalytic polymer hydrogel 16'. In these examples, the catalyst 18 is integrated into the monomer unit used during the copolymerization.

[00116] In these examples, the catalyst 18 can be a portion of any of the acrylamide monomer units of the starting polymer hydrogel 16 described herein. In one example, R ^A of structure (I) can be a carboxyl group, which is one example of an acid catalyst. In another example, R ₂ of structure (II) can be C1-C6 alkyl, and the catalyst 18 can be incorporated into the C1-C6 alkyl chain as an end group. The catalyst containing the monomer unit can be copolymerized (randomly or in block) with any of the acrylamide monomer units described herein, so long as one of the units includes a functional group for primer grafting. One example of a catalyst containing an acrylamide monomer unit that can be used is shown in structure (V):

where R ⁴ is H or CH ₃ , and X is a functional group that acts as a catalyst. In some examples, the catalyst (X) may be any of the examples of an acid catalyst, a base catalyst, or an organic catalyst described herein. In some examples, a linking group (e.g., an alkyl group, a short polyethylene glycol chain, etc.) may be located between the NH and the catalyst (X).

[00117] Other examples of the catalyst containing a monomer unit that can be used include (meth)acrylic monomers (e.g. acrylic acid, methacrylic acid) with a catalyst (X) attached thereto.

[00118] In one example of the method, forming the catalytic polymer hydrogel 16' includes copolymerizing a first monomer comprising a functional group for grafting a primer (e.g., azide) with a second monomer comprising a catalyst 18. In some examples, a third acrylamide monomer unit can also be used in the copolymerization process. One example of a copolymer formed by this method is shown in structure (VI):

where R ^A , R ^B , R ^C , R ^D , R ^E and p are as described for structure (I), X is as described for structure (V), n and r are independently in the range from 5 mol% to 30 mol%, and m is the remainder of the mol%.

[00119] In one example, the surface concentration of catalyst 18 can be in the range of from about 0.01% to about 50% of the repeating units of polymer hydrogel 16'. These concentrations can exceed practical concentrations of the catalyst solution, which contributes to high efficiency within the examples described herein.

[00120] In other examples, a nucleophile-sensitive functional group may be used as an anchor for catalyst 18 or to generate the catalyst in situ . In some examples, the nucleophile-sensitive functional group is a cyclic sulfonate ester (e.g., a sulfone ring) that can undergo a ring-opening reaction upon exposure to a nucleophile, in some cases in an alkaline (high pH) environment, at pH=9 or higher. An example of a sultone ring-opening reaction is as follows:

.

[00121] In one example, this ring opening reaction can be carried out during deblocking such that the catalyst 18, for example, an acid or a base, is formed in situ . The nucleophile-sensitive functional group can be considered a precursor to the catalyst attached to the catalytic polymer hydrogel 16'.

[00122] In another example, "Nu" is catalyst 18, and the ring-opening reaction creates a covalent bond with the element that was previously a sultone. This reaction can be formed prior to sequencing such that catalyst 18 is present when deblocking is performed.

[00123] Other examples of a nucleophile-sensitive functional group have the following structure:

(a) Y is SO ₂ and Y' is CH ₂ ; or both (b) Y and Y' are C(O). In other aspects, the nucleophile sensitive functional group is: Suitable nucleophiles include primary alkylamines and alkyl alcohols.

[00124] In some aspects, the monomer comprising a nucleophile-sensitive functional group is:

In specific examples, the monomer is:

[00125] Flow cell

[00126] The polymer hydrogel 16 and the embedded catalyst 18 can be used in a flow cell 22, an example of which is shown in Fig. 3A. The flow cell 22 includes a substrate 24 and a catalytic polymer hydrogel 16' located on the substrate 24.

[00127] The substrate 24 may be a single layer/material. Examples of suitable monolayer substrates include epoxy siloxane, glass, modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, polytetrafluoroethylene (e.g., TEFLON® from Chemours), cyclic olefins/cycloolefin polymers (COPs) (such as ZEONOR® from Zeon), polyimides, etc.), nylon (polyamides), ceramics/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 (HfO ₂ ), carbon, metals, inorganic glasses, or the like. The substrate 24 may also be a multilayer structure. Some examples of a multilayer structure include glass or silicon with a coating layer of tantalum oxide or another ceramic oxide on the surface. Other examples of a multilayer structure include an underlying substrate (e.g., glass or silicon) that has a patterned resin deposited on it. Other examples of a multilayer substrate may include a silicon-on-insulator (SOI) substrate.

[00128] In an example, the substrate 24 may have a diameter in the range of about 2 mm to about 300 mm, or be a rectangular sheet or panel, the largest dimension of which is up to about 10 feet (~3 meters). In an example, the substrate 24 is a plate having a diameter in the range of about 200 mm to about 300 mm. In another example, the substrate 24 is a matrix with a width of about 0.1 mm to about 10 mm. Although exemplary dimensions are given, it should be understood that the substrate 24 of any suitable dimensions can be used. In another example, a panel can be used that is a rectangular support that has a larger surface area than a 300 mm round plate.

[00129] In the example shown in Fig. 3A, the flow cell 22 includes flow channels 26. Although multiple flow channels 26 are shown, it should be understood that any number of channels 26 (e.g., one channel 26, four channels 26, etc.) may be included in the flow cell 22. Each flow channel 26 is a region formed between two connected components (e.g., a substrate 24 and a lid or two substrates 24) that may contain fluids (e.g., those described herein) introduced into and removed from them. Each flow channel 26 may be isolated from each other flow channel 26 such that a fluid introduced into any particular flow channel 26 does not flow into any adjacent flow channel 26. Some examples of fluids introduced into the flow channels 26 may introduce reaction components (e.g., polymerases, sequencing primers, nucleotides, etc.), wash solutions, deblocking agents, etc.

[00130] The flow channel 26 may be formed in the substrate 24 using any suitable method, which depends in part on the material(s) of the substrate 24. In one example, the flow channel 26 is etched in a glass substrate 24. In another example, the flow channel 26 may be formed in a polymer substrate 24 using photolithography, nanoimprint lithography, etc. In yet another example, a separate material (not shown) may be deposited on the substrate 24 such that the separate material forms the walls of the flow channel 26 and the substrate 24 forms the bottom of the flow channel 26.

[00131] In one example, the flow channel 26 has a rectangular configuration. The length and width of the flow channel 26 can be respectively smaller than the length and width of the substrate 24, so that a portion of the surface 24 of the substrate surrounding the flow channel 26 is available for attachment to the cover (not shown). In some cases, the width of each flow channel 26 can 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 track 20 can 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 flow channel 26 can be greater, less than the above values, or can have an intermediate value. In another example, the flow channel 26 has a square shape (e.g., 10 mm × 10 mm).

[00132] The depth of each flow channel 26 may be as little as one monolayer when microcontact, aerosol or inkjet printing is used to apply a separate material defining the walls of the flow channel. In other examples, the depth of each flow channel 26 may be about 1 μm, about 10 μm, about 50 μm, about 100 μm or more. In an example, the depth may be in the range of 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 flow channel 26 may be greater, less or an intermediate value than the above values.

[00133] Various examples of architecture within the flow channels 26 of the flow cell 22 are shown in Fig. 3B and Fig. 3C.

[00134] In the example shown in Fig. 3B, the flow cell 22 includes a single-layer substrate 24A and a flow channel 26 formed in the single-layer substrate 24A. In this example, the catalytic polymer hydrogel 16' is located within the flow channel 26.

[00135] To introduce the catalytic polymer hydrogel 16' into the flow channel 26, a mixture of the catalytic polymer hydrogel 16' can be created and then applied to the substrate 24 (if the flow channel 26 is present). In one example, the catalytic polymer hydrogel 16' can be present in a mixture (e.g., with water or with ethanol and water). The mixture can then be applied to the surfaces of the substrate (including the flow channel(s) 26) using spin coating, or dip coating, or positive or negative pressure flow of material, or another suitable technique. These types of techniques loosely deposit the catalytic polymer hydrogel 16' onto the substrate 24 (e.g., into the flow channel 26 and into the intermediate regions 28). For specific deposition of the catalytic polymer hydrogel 16' in the flow channel 26, and not in the intermediate sections 28, other selective deposition methods can be used (e.g., using a mask, controlled printing technology, etc.).

[00136] In some examples, the surface of the substrate (including the portion that is exposed in the flow channel 26) can be activated and then the mixture (including the catalytic polymer hydrogel 16') can be applied thereto. In one example, a silane or silane derivative (e.g., norbornene silane) can be applied to the surface of the substrate using vapor deposition, spin coating, or other deposition methods. In another example, the surface of the substrate can be plasma ashing to form a surface activating agent(s) (e.g., -OH groups) that can be attached to the catalytic polymer hydrogel 16'.

[00137] Depending on the chemical composition of the catalytic polymer hydrogel 16', the deposited mixture may be subjected to a curing process. In one example, curing may occur at a temperature in the range of room temperature (e.g., about 25 °C) to about 95 °C for a time in the range of about 1 millisecond up to several days.

[00138] Polishing may then be performed to remove the catalytic polymer hydrogel 16' from the intermediate regions 28 around the perimeter of the flow channel(s) 26 while leaving the catalytic polymer hydrogel 16' on the surface in the flow channel(s) 26 at least substantially intact.

[00139] In some examples, the catalytic polymer hydrogel 16' already contains the catalyst 18 attached thereto after deposition, and thus no further processing is performed to introduce the catalyst 18 into the flow channel(s) 26. One example is that the catalyst 18 is part of the monomer unit of the main chain of the polymer hydrogel. Another example of this is the attachment of the catalyst 18 after polymerization. Thus, one example of the method described in the present document includes forming a catalytic polymer hydrogel 16' comprising a catalyst 18; and applying the catalytic polymer hydrogel 16' to the surface of the substrate 24.

[00140] In other examples, the polymer hydrogel after deposition is a starting polymer hydrogel 16 to which the catalyst 18 is not attached. Since the starting polymer hydrogel 16 does not include the catalyst 18, further processing is performed to introduce the catalyst 18 into the starting polymer hydrogel 16 to form a catalytic polymer hydrogel 16' in the flow channel(s) 26. In these examples, the starting polymer hydrogel 16 is deposited in the flow channel(s) 26 and polished, and then any of the post-polymerization attachment methods described herein can be used to introduce the catalyst 18 into the starting polymer hydrogel 16. Since the starting polymer hydrogel 16 is present in the flow channel(s) 26 and not in the intermediate region(s) 28, the catalyst 18 will preferably be attached to the starting polymer hydrogel 16 in the flow channel(s) 26.

[00141] The flow cell 22 further includes an amplification primer 30 attached to the catalytic polymer hydrogel 16'. The following description of the primer attachment concerns the catalytic polymer hydrogel 16'. It should be understood that when introducing the original polymer hydrogel 16 into the flow channel(s) 26, the catalyst 18 can be introduced before or after the amplification primers 30. Thus, the description of the primer attachment is also suitable for the original polymer hydrogel 16.

[00142] To incorporate the amplification primers 30 into the catalytic polymer hydrogel 16' in the flow channel 26, a grafting process can be implemented. In one example, the amplification primers 30 can be immobilized on the catalytic polymer hydrogel 16' by a single-point covalent attachment at or near the 5' end of the primers 30. This attachment leaves i) the adapter-specific region of the primers 30 free for annealing to the sequencing-ready nucleic acid fragment it recognizes and ii) the 3'-hydroxyl group free for primer extension. Any suitable covalent coupling means can be used for this purpose. Examples of primers with suitable end groups include primers with end groups (e.g., which can be attached to an azide surface functional group of the catalytic polymer hydrogel 16'), primers with end groups (e.g., which can be attached to a bromine surface functional group of the catalytic polymer hydrogel 16'), or primers with end groups (e.g., which can be attached to an alkyne surface functional group of the catalytic polymer hydrogel 16').

[00143] Specific examples of suitable primers 30 include primers P5 and P7 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.

[00144] In one example, grafting may include flow-through application (e.g., using a temporarily bonded or permanently bonded cap), soak coating, spray coating, tamping, or other suitable method in which the primer(s) 30 are attached to the catalytic polymer hydrogel 16' in the flow channel 26. Each of these example techniques may utilize a primer solution or a primer mixture that may include the primer(s) 30, water, a buffer, and a catalyst. In any of the grafting methods, the primers 30 react with the reactive groups of the catalytic polymer hydrogel 16' in the flow channel 26 and have no affinity for the surrounding substrate 24. Thus, the primers 30 are selectively grafted onto the catalytic polymer hydrogel 16' in the flow channel 26.

[00145] In the example shown in Fig. 3C, the flow cell 22 includes a multilayer substrate 24B that includes a support 32 and a patterned material 34 located on the support 32. The patterned material 34 defines recesses 36 separated by intermediate portions 28. The recesses 36 are located within each of the flow channels 26.

[00146] In the example shown in Fig. 3C, the patterned material 34 is located on the support 32. It should be understood that for the material with the pattern 34, any material can be used that can be selectively deposited or deposited and applied in the form of a pattern to form depressions 36 and intermediate regions 28.

[00147] In one example of the patterned material 34, the inorganic oxide can 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., Ta ₂ O ₅ ), aluminum oxide (e.g., Al ₂ O ₃ ), silicon oxide (e.g., SiO ₂ ), hafnium oxide (e.g., HfO ₂ ), etc.

[00148] As another example of a patterned material 34, a resin can be applied to the support 32 and then a pattern can be applied. Suitable application techniques include chemical vapor deposition, dip coating, overcoating, spin coating, spray coating, blade coating, ultrasonic spray coating, doctor blade coating, spray printing, screen printing, microcontact printing, etc. Suitable patterning technologies include photolithography, nanoimprint lithography (NIL), stamping techniques, embossing techniques, casting techniques, microetching techniques, printing techniques, etc. Some examples of suitable resins include polyhedral oligomer silsesquioxane resin (POSS), POSS-free epoxy resin, polyethylene glycol resin, polyester resin (e.g., ring-opening epoxy resins), acrylic resin, acrylate resin, methacrylate resin, amorphous fluoropolymer resin (e.g., CYTOP® from Bellex), and combinations thereof.

[00149] As used herein, the term "polyhedral oligomeric silsesquioxane" (POSS) refers to a chemical composition that is a hybrid intermediate (e.g., RSiO _1.5 ) between a compound of silica (SiO ₂ ) and silicone (R ₂ SiO). An example of a POSS is described in Kehagias et al., Microelectronics Engineering 86 (2009), pp. 776-778, which is incorporated herein by reference in its entirety. In one example, the composition is an organosilicon compound with the chemical formula [RSiO _3/2 ] _n , where the R groups may be the same or different. Examples of R groups for POSS include epoxy, azide/azido, thiol, polyethyleneglycol, norbornene, tetrazine, acrylates and/or methacrylates, or further, for example, alkyl, aryl, alkoxy and/or haloalkyl groups. The resin composition described herein may comprise one or more different framework or core structures as monomer units. The polyhedral structure may be a _T8 structure such as: and is presented as: This monomeric unit typically has eight functional group arms R ₁ through R ₈ .

[00150] The monomer unit may have a framework structure with 10 silicon atoms and 10 R groups, referred to as _T10 , such as: or it may have a framework structure with 12 silicon atoms and 12 R groups, referred to as T ₁₂ , such as: The POSS-based material may alternatively comprise _T6 , _T14 , or _T16 framework structures. The average framework content may be adjusted during synthesis and/or controlled by purification methods, and in the examples described herein, the framework size distribution of the monomer units may be used.

[00151] In some examples of POSS described herein, at least one of _R1 - _R8 , or _R10 , or _R12 comprises an epoxy group. _R1 - _R8 , or _R10 , or _R12 may be the same or may not be the same, and in some examples, at least one of _R1 - _R8 , or _R10 , or _R12 comprises an epoxy functional group, and at least one other of _R1 - _R8 , or _R10 , or _R12 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 conditions different from the epoxy group) that serves as a handle for linking the resin to an amplification primer, polymer, or polymerization agent; or (b) a group that controls the mechanical or functional properties of the resin, such as adjusting the surface energy. In some examples, the non-epoxy functional group is selected from the group consisting of azide/azido, thiol, poly(ethylene glycol), norbornene, tetrazine, amino, hydroxyl, alkynyl, ketone, aldehyde, ester group, alkyl, aryl, alkoxy, and haloalkyl.

[00152] As shown in Fig. 3C, the patterned material 34 includes depressions 36 formed therein and intermediate portions 28 separating adjacent depressions 36. A variety of different patterns of depressions 36 can be provided, including regular, repeating, and irregular patterns. In one example, the depressions 36 are arranged in a hexagonal grid for dense packing and increased density. Other arrangement patterns may include, for example, rectilinear (rectangular) arrangement patterns, triangular arrangement patterns, and the like. In some examples, the pattern or design may be in an x-y format of depressions 36 that are arranged in rows and columns. In some other examples, the pattern or design may be a repeating arrangement of depressions 36 and/or intervening areas 28. In still other examples, the pattern or design may be a random arrangement of depressions 36 and/or intervening areas 28. The pattern may include spots, lands, wells, columns, stripes, swirls, lines, triangles, rectangles, circles, arcs, checkers, slabs, diagonals, arrows, squares, and/or cross hatching.

[00153] The pattern or design of the depressions 36 can be characterized by the criterion of the density of the depressions 36 (the number of depressions 36) in a certain area. For example, the depressions 36 can have a density of about 2 million per mm ² . The density can be adjusted to various densities, including, for example, a density of about 100 per mm ² , about 1000 per mm ² , about 0.1 million per mm ² , about 1 million per mm ² , about 2 million per mm ² , about 5 million per mm ² , about 10 million per mm ² , about 50 million per mm ² , or more or less. In addition, it should be understood that the density of the depressions 36 in the patterned material 34 can be between one of the lower values and one of the upper values selected from the above-mentioned ranges. For example, a high density array may be characterized by having pits 36 separated by less than about 100 nm, a medium density array may be characterized by having pits 36 separated by from about 400 nm to about 1 μm, and a low density array may be characterized by having pits 36 separated by more than about 1 μm. Although examples of densities are provided, it should be understood that any suitable density values can be used. The density of the pits 36 may depend in part on the depth of the pits 36. In some cases, it may be desirable for the distance between the pits 36 to be even greater than in the examples listed herein.

[00154] The arrangement or pattern of the recesses 36 may also or alternatively be characterized in terms of an average pitch or distance from the center of the recess 36 to the center of an adjacent recess 36 (center-to-center distance) or from the edge of one recess 36 to the edge of an adjacent recess 36 (edge-to-edge distance). The pattern may be regular, such that the coefficient of variation around the average pitch is small, or the pattern may be irregular, in which case the coefficient of variation may be relatively large. In any case, the average pitch may 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 for a particular arrangement of the recesses 36 may be in a range from one of the lower values to one of the upper values selected from the ranges given above. In one example, the recesses 36 have a pitch (distance between centers) of about 1.5 µm. Although examples of average pitch values are provided, it should be understood that other average pitch values may be used.

[00155] The size of each recess 36 may be characterized by its volume, opening area, depth and/or diameter.

[00156] Each well 36 may have any volume capable of holding a fluid. The minimum or maximum volume may be selected, for example, to match the throughput (e.g., multiplexity), resolution, nucleotides 10, or analyte reactivity expected in subsequent applications of the flow cell 22. For example, the volume may be at least about 1×10 ^-3 μm ³ , at least about 1×10 ^-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 or more. Alternatively or additionally, the volume may be no more than about 1×10 ^{4 μm3} , no more than about 1×10 ^{3 μm3} , no more than about 100 ^μm3 , no more than about 10 ^μm3 , no more than about 1 ^μm3 , no more than about 0.1 ^μm3 , or less.

[00157] The area occupied by each recess opening may be selected based on criteria similar to the criteria set forth above for the volume. For example, the area of each recess opening may be at least about 1× ^{10-3 μm2} , at least about 1× ^{10-2 μm2} , at least about 0.1 ^μm2 , at least about 1 ^μm2 , at least about 10 ^μm2 , at least about 100 ^μm2 or more. Alternatively or additionally, the area may be no more than about 1× ^{103 μm2} , no more than about 100 ^μm2 , no more than about 10 ^μm2 , no more than about 1 ^μm2 , no more than about 0.1 ^μm2 , no more than about 1× ^{10-2 μm2} or less. The area occupied by each recess opening may be greater than the values specified, less than the values specified, or be in between the values specified above.

[00158] The depth of each recess 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 no more than about 1×10 ³ μm, no more than about 100 μm, no more than about 10 μm, or less. In some examples, the depth is about 0.4 μm. The depth of each recess 36 may be greater than, less than, or between the values specified above.

[00159] In some cases, the diameter or the length and width of each depression 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. μm or more. Alternatively or additionally, the diameter or the length and width can be no more than about 1×10 ³ μm, no more than about 100 μm, no more than about 10 μm, no more than about 1 μm, no more than about 0.5 μm, no more than about 0.1 μm or less (e.g., about 50 nm). In some examples, the diameter or the length and width are about 0.4 μm. The diameter or the length and width of each depression 36 can be greater than, less than, or between the values specified above.

[00160] In the example shown in Fig. 3C, the catalytic polymer hydrogel 16' is located within each of the recesses 36. The catalytic polymer hydrogel 16' can be applied as described with reference to Fig. 3B so that the catalytic polymer hydrogel 16' is located in the recesses 36 and is not located on the surrounding intermediate regions 28.

[00161] In the example shown in Fig. 3C, primers 30 may be grafted onto the catalytic polymer hydrogel 16' in each of the recesses 36. Primers 30 may be applied as described with reference to Fig. 3B and will thus be grafted onto the catalytic polymer hydrogel 16' and not onto the surrounding interstitial regions 28.

[00162] Although not shown in Fig. 3A, Fig. 3B or Fig. 3C, it should be understood that the flow cell 22 may also include a lid attached to the substrate 24. In one example, the lid may be connected to at least a portion of the substrate 24, such as at some intermediate regions 28. The bond formed between the lid and the substrate 24 may be a chemical bond or a mechanical bond (e.g., using a fastener, etc.).

[00163] The lid may be made of any material that is transparent to excitation light directed toward substrate 24. As examples, the lid may be glass (e.g., borosilicate glass, fused silica, etc.), plastic, or the like. A commercially available example of a suitable borosilicate glass is D 263® from Schott North America, Inc. Commercially available examples of suitable plastic materials, namely cycloolefin polymers, are ZEONOR® products from Zeon Chemicals L.P.

[00164] The lid may be connected to the substrate 24 using any suitable method, such as laser soldering, diffusion soldering, anodic soldering, eutectic alloy soldering, plasma activation soldering, glass-ceramic soldering, or other methods known in the art. In one example, a separator layer may be used when attaching the lid to the substrate 24. The separator layer may be made of any material that will hermetically connect at least a portion of the substrate 24 and the lid. In some examples, the separator layer may be a radiation absorbing material that facilitates bonding of the substrate 24 to the lid.

[00165] In other examples, the flow cell 22 may also include an additional substrate 24, patterned or unpatterned, attached to the substrate 24.

[00166] Nucleotides

[00167] The nucleotides used in the examples of the flow cell 22 are 3' OH blocked nucleotide 10 (see FIG. 1). As described herein, the 3' OH blocked nucleotide 10 includes 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.

[00168] The 3' OH Blocking group 12 can be linked to an oxygen atom of a sugar molecule in a nucleotide. The 3' OH Blocking group 12 can serve as a reversible terminator that allows only one base to be incorporated in each sequencing cycle. The reversible terminator stops the incorporation of additional bases into the nascent strand that is complementary to the template polynucleotide strand. This allows for the detection and identification of a single incorporated base. The 3' OH Blocking group 12 can then be removed, allowing for additional sequencing cycles to be performed for each template polynucleotide strand. The cleavage reaction to remove the 3' OH Blocking group 12 is catalyzed by catalyst 18.

[00169] Examples of various 3' OH blocking groups 12 include a 3'-ONH ₂ reversible terminator, a 3'- O -allyl (-CH=CHCH ₂ ) reversible terminator, and a 3'- O -azidomethyl (-CH ₂ N ₃ ) reversible terminator. Other suitable reversible terminators include o-nitrobenzyl ethers, alkyl o-nitrobenzyl carbonate, ester functionalities, other allyl functionalities, acetals (e.g., tert-butoxyethoxy), MOM (-CH ₂ OCH ₃ ) functionalities, 2,4-dinitrobenzene sulfenyl, tetrahydrofuranyl ether, 3' phosphate, ethers, -F, -H ₂ , -OCH ₃ , -N ₃ , -HCOCH _{3 ,} and 2-nitrobenzene carbonate. For allylic reversible terminators, the ligand on the surface of the polymer hydrogel 16' can bind palladium (Pd(0)) and ruthenium catalysts. With esters and acetals, any of the acidic and/or basic catalysts described herein can be used.

[00170] 3' OH Blocked nucleotide 10 is a fully functional nucleotide that may also include a detectable label attached to the B base of the nucleotide. The detectable label may be any optically detectable component, including luminescent, chemiluminescent, fluorescent, fluorogenic, chromophore and/or chromogenic functional groups. Some examples of suitable optically detectable functional groups include fluorescein, rhodamine, cyanine labels (e.g., Cy3, Cy5, etc.) and the Alexa family of fluorescent dyes and other fluorescent and fluorogenic dyes).

[00171] Any suitable linking molecule can be used to attach a detectable label to the B base of the nucleotide. The linking molecule is cleavable and can undergo a series of reactions similar to those that occur to remove the deblocking group 12. In one example, the linking molecule is a spacer group of the formula -((CH ₂ ) ₂ O) _n -, where n is an integer from 2 to 50.

[00172] In some applications, it may be desirable to use different types of detectable labels for each of the nucleotides 10 that include a different base, such as A, T, G, C (as well as U or I). For example, fluorescent or fluorogenic labels can be selected such that each of such labels absorbs excitation radiation and/or emits fluorescence at a wavelength that differs from the wavelengths of other groups of labels. Such distinguishable analogs allow the presence of different labels to be monitored simultaneously in the same reaction mixture. In some examples, one of the four nucleotides that represent the sequences may not include labels, while the other three nucleotides contain different labels.

[00173] Sequencing Methods

[00174] Examples of flow cell 22 can be used in an ensemble sequencing method, such as sequencing by synthesis (SBS). In ensemble sequencing, a template polynucleotide chain (not shown) to be sequenced can be formed on flow cell 22 using amplification primers 30. At the beginning of the formation of the template polynucleotide chain, library templates can be obtained from any nucleic acid sample (e.g., a DNA sample or an RNA sample). The nucleic acid sample can be fragmented into single-stranded DNA or RNA fragments of similar size (e.g., <1000 bp). Adaptors can be added to the ends of these fragments during preparation. By means of reduced cycle amplification, various motifs such as binding sites, indices and sequencing regions that are complementary to primers 30 in wells 36 can be introduced into the adapters. The final library templates include DNA or RNA fragments and adapters at both ends. In some examples, the same adapters are added to fragments from a single nucleic acid sample.

[00175] A plurality of library templates may be introduced into the flow cell 22. Templates from the plurality of libraries are hybridized, for example, with one of two types of primers 30 immobilized in the flow channel(s) 26 or in the recesses 36 in the flow channel(s) 26.

[00176] Next, cluster creation can be performed. In one example of cluster creation, library templates are copied from hybridized primers by 3' extension using high-fidelity DNA polymerase. The original library templates are denatured, leaving copies immobilized in flow channel 26 or in wells 36. Isothermal bridge amplification or some other form of amplification can be used to amplify the immobilized copies. For example, the copied templates form a loop and hybridize to an adjacent complementary primer 30, and the polymerase copies the copied templates to form double-stranded bridges that are denatured to form two single-stranded strands. These two strands form a loop and hybridize to adjacent complementary primers 30, and are then extended again to form two new double-stranded loops. The process is repeated on each copy of the template in cycles of isothermal denaturation and amplification to produce dense clonal clusters. Each cluster of double-stranded bridges is denatured. In one example, the reverse strand is removed by base-specific cleavage, leaving forward-directed template polynucleotide strands. Clustering results in the formation of multiple template polynucleotide strands in flow channel 26 or in each well 36. This example of clustering is a bridge amplification and is one example of the amplification that can be performed. It should be understood that other amplification techniques can be used, such as the size-exclusion amplification (Examp) workflow (Illumina Inc.).

[00177] A sequencing primer may be introduced that hybridizes to a complementary sequence on the template polynucleotide strand. This sequencing primer makes the template polynucleotide strand ready for sequencing.

[00178] To initiate sequencing, an addition mixture may be added to the flow cell 22. In one example, the addition mixture includes a liquid carrier, a polymerase, and 3' OH blocked nucleotides 10. It should be understood that in some examples, the addition mixture is selected such that it does not activate the catalyst 18, since it is undesirable to initiate the cleavage of the blocking group 12 before inclusion and imaging. In a host-guest example, the addition mixture may include a guest catalyst (e.g., a metal) in an unactivated state. The guest catalyst may bind to the parent polymer hydrogel 16' (e.g., via a ligand) and then, upon deblocking, may be orthogonally stimulated (e.g., by exposure to a certain wavelength) to convert the guest catalyst to an activated form.

[00179] When the added mixture is introduced into the flow cell 22, the liquid enters the flow channel 26 and/or the recesses 36 (where the template polynucleotide chains are present).

[00180] 3' OH Blocked nucleotides 10 are added to the sequencing primer (thereby extending the sequencing primer) in a template-dependent manner, such that determination 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 an appropriate polymerase into the nascent strand that extends the sequencing primer and is complementary to the strand of the template polynucleotide. In other words, in at least some strands of the template polynucleotides through the flow cell 22, the appropriate polymerases extend the hybridized sequencing primer with one of the nucleotides in the addition mixture.

[00181] In this example of the method, after incorporation of the nucleotide base into the nascent chain, it is possible to remove the addition mixture, which includes any unincorporated 3' OH blocked nucleotides 10, from the flow cell 22. This can be achieved using a wash solution (e.g., a buffer).

[00182] As described herein, the 3' OH blocked nucleotides 10 include a reversible termination feature (e.g., a 3' OH blocking group 12) that stops further extension of the primer after the nucleotide has been added to the sequencing primer. Without further incorporation, the last incorporated nucleotides 10 can be detected using an imaging event. During the imaging event, the illumination system (not shown) can apply excitation light to the flow channel 26 and/or the wells 36.

[00183] A cleavage mixture can then be introduced into the flow cell 22. In the examples described herein, the cleavage mixture is capable of i) removing the 3' OH blocking group 12 from the incorporated nucleotides and ii) cleaving detectable labels from the incorporated nucleotides. A catalyst present on the polymer hydrogel 16' can accelerate the reaction that occurs during the removal of the 3' OH blocking group. Removal of the 3' OH blocking group 12 allows for a subsequent sequencing cycle to be performed, and acceleration of this reaction using the catalyst 18 can improve the efficiency of the overall sequencing process.

[00184] Some examples of the catalyst accelerate intermediate steps during the deblocking reaction. Thus, after the mixture is turned on for cleavage and the deblocking reaction is initiated, the catalyst performs its function. When using a photoacid generator as the catalyst 18, additional optical exposure can be used to initiate the catalytic activity.

[00185] Examples of 3' OH blocking groups and suitable deblocking agents/components in the cleavage mixture may include ester functionalities which can be removed by base hydrolysis; allyl functionalities which can be removed with Nal, _{chlorotrimethylsilane and Na2S2O3 or Hg} (II) in acetone/water; azidomethyl which can be cleaved with phosphines such as tris(2-carboxyethyl)phosphine (TCEP) or tri(hydroxypropyl)phosphine (THP); acetals such as tert-butoxyethoxy which can be cleaved in acidic media; MOM ( _{-CH2OCH3 )} functionalities which can be cleaved with _LiBF4 and _CH3CN / _H2O ; 2,4-dinitrobenzenesulfenyl, which can be cleaved by nucleophiles such as thiophenol and thiosulfate; tetrahydrofuranyl ether, which can be cleaved by Ag(I) or Hg(II); and 3' phosphate, which can be cleaved by phosphatase enzymes (e.g., polynucleotide kinase).

[00186] Wash(s) may occur between the various fluid feed steps. The SBS cycle may then be repeated n times to extend the sequencing primer by n nucleotides and detect a sequence of length n. In some examples, paired-end sequencing may be used, in which the forward strands are sequenced and removed, and then the reverse strands are constructed and sequenced.

[00187] Although the SBS method has been described in detail, it should be understood that the flow cells 22 described herein may be used with another sequencing protocol, for genotyping, or in other chemical and/or biological applications. In some cases, the flow cell primers may be selected to provide for simultaneous paired-end sequencing, in which both forward and reverse strands are present on the catalytic polymer hydrogel 16', allowing each read to be recognized simultaneously. Sequential and simultaneous paired-end sequencing facilitates the detection of genomic rearrangements and repetitive sequence elements, as well as gene fusions and novel transcripts. In another example, the flow cells 10 disclosed herein may be used to generate a flow cell library.

[00188] Sets

[00189] Any example of a flow cell 22 described herein may be part of a kit. Thus, any examples of a polymer hydrogel 16' described herein may be part of a kit. Some examples of a kit include a flow cell 22 that includes a support 24; a catalytic polymer hydrogel 16' on the support 24, wherein the catalytic polymer hydrogel 16' includes a catalyst 18; and an amplification primer 30 attached to the catalytic polymer hydrogel 16'; wherein the catalyst 18 is designed to accelerate the cleavage of the blocking group 12 3' OH of the blocked nucleotide 10 introduced into the flow cell 22 and incorporated into a template strand attached to the amplification primer 30. and a cleavage mixture comprising a component that initiates the cleavage of the blocking group 12.

[00190] Additional Notes

[00191] It should be understood that all combinations of the above concepts and additional concepts described in more detail below (provided that such concepts are not mutually contradictory) are considered to be part of the subject matter described herein. In particular, all combinations of claimed subject matter appearing at the end of this description are considered to be part of the subject matter described herein. It should also be understood that terminology explicitly used herein, which may also appear in any description incorporated herein by reference, is to have the meaning most consistent with the particular concepts described herein.

[00192] Reference throughout the specification to "one example," "another example," "an example," and so forth, means 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. Furthermore, it should be understood that the described elements of any example may be combined in any suitable manner with different examples, unless the context clearly indicates otherwise.

[00193] It should be understood that the ranges provided herein include the stated range and any value or sub-range within the stated range, as if such values or sub-ranges were expressly stated. For example, a range from about 2 mm to about 300 mm should be interpreted as including not only the expressly stated limits from about 2 mm to about 300 mm, but also including individual values, such as about 40 mm, about 250.5 mm, etc., and sub-ranges, such as from about 25 mm to about 175 mm, etc. Moreover, when the terms "about" and/or "substantially" are used to describe a value, they cover minor deviations (up to ±10%) from the stated value.

[00194] Although several examples are described in detail, it should be understood that the examples described may be changed. Therefore, the above description should be considered as not limiting.

Claims (62)

1. Sequencing kit including: a flow cell containing: backing; a catalytic polymer hydrogel on a support, wherein the catalytic polymer hydrogel includes a deblocking catalyst selected from the group consisting of a phosphonic acid; a heterocyclic amine; an enzyme; a peptide; a DNA enzyme; 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 amplification primer attached to a catalytic polymer hydrogel; wherein the catalyst accelerates the cleavage of the 3' OH blocking group of the blocked nucleotide introduced into the flow cell and incorporated into the template strand attached to the amplification primer; and a cleavage mixture comprising a component that initiates cleavage of the blocking group. 2. The kit according to item 1, characterized in that if the deblocking catalyst is a metal of a metal-ligand complex, then it is attached to the polymer hydrogel. 3. A method for flow cell sequencing, comprising: introducing the added mixture into a flow cell, including: backing; a catalytic polymer hydrogel on a support, wherein the catalytic polymer hydrogel includes a deblocking catalyst selected from the group consisting of a phosphonic acid; a heterocyclic amine; an enzyme; a peptide; a DNA enzyme; 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 matrix chains attached to a catalytic polymer hydrogel; with the implementation by this of the incorporation of individual nucleotides into the corresponding nascent chains along the template chains, wherein the individual nucleotides include: a dye label attached to the base; and 3' OH blocking group attached to the sugar; removal of the added mixture; incorporation of individual nucleotides using optical imaging; and introducing a cleavage mixture comprising a component that initiates cleavage of the 3' OH blocking group into the flow cell, whereby the deblocking catalyst accelerates cleavage of the 3' OH blocking group. 4. The method according to claim 3, characterized in that the catalyst accelerates the removal of the external protective group 3' OH blocking group, and wherein the reagent in the cleavage mixture removes the internal protective group 3' OH blocking group. 5. A flow cell for sequencing, containing: backing; a catalytic polymer hydrogel on a support, wherein the catalytic polymer hydrogel comprises a deblocking catalyst selected from the group consisting of: phosphonic acid; heterocyclic amine; enzyme; peptide; DNA enzyme; 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 amplification primer attached to a catalytic polymer hydrogel. 6. A flow cell according to claim 5, wherein the catalyst is embedded in the monomer unit of the catalytic polymer hydrogel. 7. A flow cell according to item 5, in which the catalyst is grafted onto the initial polymer hydrogel. 8. The flow cell of claim 5, wherein the catalytic polymer hydrogel comprises a starting polymer hydrogel, and the flow cell further comprises an oligonucleotide attached to the starting polymer hydrogel, and the deblocking catalyst is attached to a complementary oligonucleotide ligand that is hybridized with the oligonucleotide. 9. A flow cell according to item 5, in which: the polymer hydrogel includes the first element of the pair forming a hydrogen bond; the deblocking catalyst is attached to the polymer hydrogel via a second member of the pair forming a hydrogen bond; and The amplification primer is attached to a catalytic polymer hydrogel. 10. A method for creating a flow cell for sequencing with a catalytic polymer hydrogel on the surface of a substrate, comprising: applying a catalytic polymer hydrogel to the surface of a flow cell substrate, wherein the catalytic polymer hydrogel comprises a deblocking catalyst selected from the group consisting of: phosphonic acid; heterocyclic amine; enzyme; peptide; DNA enzyme; 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 attachment of amplification primers to a catalytic polymer hydrogel. 11. The method of claim 10, further comprising forming a catalytic polymer hydrogel comprising a deblocking catalyst. 12. The method according to claim 11, characterized in that the formation of the catalytic polymer hydrogel includes copolymerization of the first monomer, including a functional group for grafting a primer, with a second monomer, including a deblocking catalyst. 13. The method according to item 11, characterized in that the formation of the catalytic polymer hydrogel includes: synthesis of the initial polymer hydrogel; and grafting the catalyst onto the original polymer hydrogel. 14. The method according to item 11, characterized in that the formation of the catalytic polymer hydrogel includes: synthesis of the initial polymer hydrogel; grafting the oligonucleotide onto the original polymer hydrogel; and hybridization of a complementary oligonucleotide linkage with an oligonucleotide, wherein the catalyst is attached to the complementary oligonucleotide linkage. 15. The method according to item 11, characterized in that the formation of the catalytic polymer hydrogel includes: synthesis of the initial polymer hydrogel; and adding a metal-ligand complex to the original polymer hydrogel, wherein the metal of the metal-ligand complex is a catalyst.

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