Hydrogel

Abstract

The hydrogel comprises a dendritic core having 2 to 30 arms and a first and a second acrylamide monomer incorporated into each arm. The first acrylamide monomer is (I) [Formula 1] TIFF2023504948000074.tif23128, wherein R1 and R2 are independently selected from alkyl, alkylamino, alkylamido, alkylthio, aryl, glycol, and any substituted versions thereof; and the second acrylamide monomer is (II). [Case 2] TIFF2023504948000075.tif26128, wherein R3 and R4 are independently hydrogen or alkyl; L is a linker comprising a linear chain of 2 to 20 atoms selected from carbon, oxygen, and nitrogen, and optional substituents on the carbon and any nitrogen atoms; and A is an N-substituted amide: (III) [C3] TIFF2023504948000076.tif23128, wherein R5 is hydrogen or alkyl, E is 1 to 4 atoms selected from carbon, oxygen, and nitrogen, and optional substituents on the carbon and any nitrogen atoms, and Z is an optional nitrogen-containing heterocycle.

Description

Technical Field

[0001] (Cross - reference to Related Applications) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 942,52 filed on December 2, 2019 and Dutch Application No. N2024749 filed on January 24, 2020. The entire content of each of these is incorporated herein by reference in its entirety.

Background Art

[0002] Substrates coated with polymers or hydrogels are used in many technical applications. In one example, implantable medical devices can be coated with biocompatible polymers. In another example, wound dressings may be coated with a thin hydrogel layer. In yet another example, substrates coated with polymers or hydrogels can be used for the preparation and / or analysis of biomolecules. Some molecular analyses, such as certain nucleic acid sequencing methods, involve attaching nucleic acid strands to the surface of a substrate coated with a polymer or hydrogel. Introduction

[0003] A dendritic core having 2 to 30 arms, for example 2 to 20 or 2 to 10 arms, and a first acrylamide monomer incorporated into each arm of the dendritic core, having the structure:

Chemical formula

Chemical formula

[0004] The first acrylamide monomer can be N,N-dimethylacrylamide.

[0005] The dendritic core optionally contains a thiocarbonylthio group in each arm. The thiocarbonylthio group can be selected from the group consisting of dithiobenzoate, trithiocarbonate, and dithiocarbamate. The dendritic core can be selected from the group consisting of 3,5-bis(2-dodecylthiocarbonothioylthio-1-oxopropoxy)benzoic acid, 1,1,1-tris[(dodecylthiocarbonothioylthio)-2-methylpropionate]ethane, and pentaerythritol tetrakis[2-(dodecylthiocarbonothioylthio)-2-methylpropionate].

[0006] The dendritic core can contain an atom transfer radical polymerization initiator in each arm. The dendritic core can be selected from the group consisting of bis[2-(2'-bromoisobutyryloxy)ethyl]disulfide, 2-bromoisobutyric anhydride, ethylene bis(2-bromoisobutyrate), pentaerythritol tetrakis(2-bromoisobutyrate), dipentaerythritol hexakis(2-bromoisobutyrate), and 1,1,1-tris(2-bromoisobutyryloxymethyl)ethane.

[0007] The dendritic core may include a multifunctional central molecule and a plurality of atom transfer radical polymerization monofunctional initiators bonded to the multifunctional central molecule. The atom transfer radical polymerization monofunctional initiator may be selected from the group consisting of 2-azidoethyl 2-bromoisobutyrate, poly(ethylene glycol) methyl ether 2-bromoisobutyrate, 2-(2-bromoisobutyryloxy)ethyl methacrylate, dodecyl 2-bromoisobutyrate, 2-hydroxyethyl 2-bromoisobutyrate, 1-(phthalimidomethyl) 2-bromoisobutyrate, and propargyl 2-bromoisobutyrate.

[0008] The dendritic core may include a nitroxide-mediated polymerization initiator in each arm. In certain examples, the dendritic core is selected from the group consisting of 1,3,5-tris((4-(1-((2,2,6,6-tetramethylpiperidin-1-yl)oxy)ethyl)benzyl)oxy)benzene and 1,3,5-tris((3,5-bis((4-(1-((2,2,6,6-tetramethylpiperidin-1-yl)oxy)ethyl)benzyl)oxy)benzyl)oxy)benzene.

[0009] The dendritic core may include a multifunctional central molecule and a plurality of nitroxide-mediated polymerization monofunctional initiators bonded to the multifunctional central molecule. Each of the plurality of nitroxide-mediated polymerization monofunctional initiators

Chemical formula

Chemical formula

[0010] The first acrylamide monomer and the second acrylamide monomer may form a block copolymer, a random copolymer, a statistical copolymer, or an alternating copolymer on each arm of the dendritic core.

[0011] The second acrylamide monomer is, optionally, azidoacetamidopentyl acrylamide.

[0012] It should be understood that any of the features of the hydrogels disclosed herein can be combined together in any desired manner and / or configuration to achieve the advantages described in this disclosure, including, for example, producing a polymeric hydrogel that exhibits suitable sequencing performance even after being exposed to dry storage at room temperature (e.g., from about 18 °C to about 25 °C).

[0013] Also provided is a flow cell comprising a substrate and a multi-arm polymeric hydrogel on the substrate, the multi-arm polymeric hydrogel comprising a dendritic core having 2 to 30 arms, a first acrylamide monomer incorporated into each arm of the dendritic core and having the structure: [Chemical formula] wherein R1 and R2 are independently selected from the group consisting of alkyl, alkylamino, alkylamide, alkylthio, aryl, glycol, and any substituents thereof; a second acrylamide monomer incorporated into each arm of the dendritic core and having the structure: [Chemical formula] wherein R3 is hydrogen or alkyl, R4 is hydrogen or alkyl, L is a linear chain of 2 to 20 atoms selected from the group consisting of carbon, oxygen, and nitrogen, and including optional substituents on the carbon atoms and any nitrogen atoms within the chain, and A has the structure [Chemical formula] an N-substituted amide, wherein R5 is hydrogen or alkyl, E is a linear chain of 1 to 4 atoms selected from the group consisting of carbon, oxygen, and nitrogen, and optional substituents on the carbon atoms and any nitrogen atoms within the chain, and Z is an optional nitrogen-containing heterocyclic ring, a second acrylamide monomer, is also disclosed.

[0014] The substrate optionally includes a plurality of recesses separated by interstitial regions, and the hydrogel is disposed within each of the recesses.

[0015] The flow cell optionally further includes amplification primers grafted to the hydrogel.

[0016] The substrate optionally includes channels, and the hydrogel is optionally disposed within the channels. The flow cell optionally further includes amplification primers grafted to the hydrogel.

[0017] The first acrylamide monomer and the second acrylamide monomer may form a random copolymer on each arm of the dendritic core, or the first acrylamide monomer and the second acrylamide monomer may form a statistical copolymer on each arm of the dendritic core, or the first acrylamide monomer and the second acrylamide monomer may form an alternating copolymer on each arm of the dendritic core, or the first acrylamide monomer and the second acrylamide monomer may form a block copolymer on each arm of the dendritic core.

[0018] The first acrylamide monomer is optionally N,N-dimethylacrylamide.

[0019] The second acrylamide monomer is optionally azidoacetamidopentyl acrylamide.

[0020] It should be understood that any features of the flow cell can be combined together in any desired manner. Further, any combination of features of the flow cell and / or the hydrogel can be used together to achieve the advantages described in this disclosure, including, for example, improved sequencing methods, and / or can be combined with any of the examples disclosed herein.

[0021] A method comprising incorporating a copolymer into each arm of a multi-arm dendritic core component having 2 to 30 arms, the copolymer comprising a first acrylamide monomer and a second acrylamide monomer, the first acrylamide monomer having the structure:

Chemical formula

Chemical formula

Chemical formula

[0022] Incorporating can include polymerizing a mixture of a first acrylamide monomer and a second acrylamide monomer in the presence of a multi-arm component.

[0023] Incorporating can include forming a block copolymer in the presence of a multi-arm component by: i) polymerizing a first block with a first acrylamide monomer in the presence of a multi-arm component to form a modified multi-arm component, and then polymerizing a second block with a second acrylamide monomer in the presence of the modified multi-arm component; or ii) polymerizing a first block with a second acrylamide monomer in the presence of a multi-arm component to form a modified multi-arm component, and then polymerizing a second block with a first acrylamide monomer in the presence of the modified multi-arm component.

[0024] Incorporating can include reversible addition-fragmentation chain transfer polymerization or atom transfer radical polymerization or nitroxide-mediated polymerization. The present disclosure also refers to wound dressing materials and medical devices containing hydrogels.

[0025] It should be understood that any features of the method can be combined together in any desirable manner. Further, any combination of the features of the method and / or the features of the flow cell and / or the hydrogel can be used together to achieve the advantages described in the present disclosure, including, for example, controlling the molecular weight distribution of the hydrogel, and / or can be combined with any of the examples disclosed herein.

[0026] The present disclosure also includes the following items. 1. A dendritic core having 2 to 30 arms, and A first acrylamide monomer incorporated into each arm of the dendritic core, having the structure:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chem.

Chem.

Chem.

Chem.

Chem.

Brief Description of the Drawings

[0027] The features of the embodiments of the present disclosure will become apparent by referring to the following detailed description and the drawings. In the drawings, like reference numerals correspond to components that are similar but not necessarily identical. For the sake of brevity, reference numerals or features having the above-described functions may or may not be described in association with other drawings in which they appear.

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Figure 1A

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DETAILED DESCRIPTION OF THE INVENTION

[0044] Hydrogels are disclosed herein. One example of the hydrogels described herein is a polymeric hydrogel. The hydrogels disclosed herein can be multi-arm polymeric hydrogels. Examples of the hydrogels include a dendrimer core. When preparing the hydrogels disclosed herein, the number of arms of the dendrimer core can define the degree of branching of the polymer and thus provide control over crosslinking. In other words, the crosslinked state of the hydrogel is fixed and restricted depending on the dendrimer core used. Further, any crosslinking between branches can be adjusted by monomer selection. The ability to adjust multiple parameters (e.g., initiator concentration, mobile agent, etc.) allows for more control over the dispersity (e.g., compared to a free radical polymerization process), and thus the resulting product has a relatively narrow molecular weight distribution (e.g., the dispersity is 5 or less, or in some cases 4 or less, or 2.5 or less, or 1.7 or less, or 1.3 or less). Thus, the hydrogels can be consistently produced from one batch to the next. The dispersity referred to in the present invention is M w and M n defined as the ratio of, where M w is the weight average molecular weight and M n is the number average molecular weight of the hydrogel. M w and M n can be determined using gel permeation chromatography.

[0045] Furthermore, examples of the hydrogels exhibit suitable sequencing performance even after being exposed to dry storage at room temperature (e.g., about 18 °C to about 25 °C). For example, unwanted intramolecular and intermolecular interactions of polymer strands during dry storage can adversely affect downstream sequencing performance. In some embodiments, the acrylamide units of the hydrogels disclosed herein include functional groups that can at least reduce hydrogen bonding between polymer strands, thus making it possible to dry store the hydrogels without adversely affecting downstream sequencing performance.

[0046] Definition

[0047] The terms used in this specification are to be understood as taking their ordinary meaning in the relevant technical field, unless otherwise specified. Some of the terms used in this specification and their meanings are described below.

[0048] The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0049] The terms "comprising", "including", "containing", and various forms of these terms are synonymous with each other and are meant to be equally broad.

[0050] To describe the flow cell and / or various components of the flow cell, terms such as "top", "bottom", "lower", "upper", "on", etc. are used in this specification. It should be understood that these terms indicating directions are not meant to indicate a specific orientation, but are used to specify the relative orientation between components. The use of terms indicating directions is not to be construed as limiting the embodiments disclosed in this specification to any specific orientation.

[0051] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). An alkyl group can have 1 to 20 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, etc. By way of example, the notation "C1-C6 alkyl" indicates that there are 1 to 6 carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, isobutyl, sec-butyl, t-butyl, pentyl, and hexyl.

[0052] As used herein, "alkylamino" refers to an alkyl group in which one or more of the hydrogen atoms are substituted with an amino group, and the amino group refers to an -NR a R b group, where R a and R b are each independently selected from C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclic, C6-C10 aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclic.

[0053] As used herein, "alkylamide" refers to an alkyl group in which one or more of the hydrogen atoms are substituted with a C-amide group or an N-amide group. The "C-amide" group refers to a "-C(=O)N(R a R b )" group, where R a and R b are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicycle, aralkyl, or (heteroalicycle)alkyl. The "N-amide" group refers to an "RC(=O)N(R a )-" group, where R and R a are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclic, aralkyl, or (heterocyclic)alkyl. Any alkylamide may be substituted or unsubstituted.

[0054] As used herein, "alkylthio" refers to RS-, where R is alkyl. Alkylthio may be substituted or unsubstituted.

[0055] As used herein, "alkenyl" refers to a straight or branched chain hydrocarbon chain containing one or more double bonds. The alkenyl group may have 2 to 20 carbon atoms. Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like.

[0056] As used herein, "alkyne" or "alkynyl" refers to a straight or branched chain hydrocarbon chain containing one or more triple bonds. The alkynyl group may have 2 to 20 carbon atoms.

[0057] As used herein, "aralkyl" and "aryl(alkyl)" refer to an aryl group bonded as a substituent via a lower alkylene group. The lower alkylene group and the aryl group of the aralkyl may be substituted or unsubstituted. Examples include, but are not limited to, benzyl, 2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl.

[0058] The term "aryl" refers to an aromatic ring or ring system (i.e., two or more fused rings sharing two adjacent carbon atoms) containing only carbon in the ring skeleton. When the aryl is a ring system, all rings within the system are aromatic rings. The aryl group may have 6 to 18 carbon atoms. Examples of aryl groups include phenyl, naphthyl, azulenyl, and anthracenyl. Any aryl may be a heteroaryl having at least one heteroatom, i.e., an element other than carbon (e.g., nitrogen, oxygen, sulfur, etc.) in the ring skeleton.

[0059] As used herein, the term "linked" refers to a state in which two entities are joined, fastened, adhered, connected, or coupled to each other, either directly or indirectly. For example, a nucleic acid can be linked to a functionalized polymer by a covalent or non-covalent bond. A covalent bond is characterized by the sharing of electron pairs between atoms. A non-covalent bond is a physical bond that does not involve the sharing of electron pairs and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, and hydrophobic interactions.

[0060] The "azide" or "azido" functional group refers to -N3.

[0061] A "block copolymer" is a copolymer formed when two or more monomers cluster together to form blocks of repeating units. Each block should have at least one characteristic that is not present in the adjacent blocks. Specific examples of block copolymers are further described below.

[0062] As used herein, "carbocycle" means a non-aromatic cyclic ring or ring system that contains only carbon atoms in the ring system backbone. When the carbocycle is a ring system, two or more rings can be joined together in a fused, bridged, or spiro linkage fashion. The carbocycle can have any degree of saturation, provided that at least one ring within the ring system is not aromatic. Thus, carbocycles include cycloalkyl, cycloalkenyl, and cycloalkynyl. A carbocyclic group can have from 3 to 20 carbon atoms. Examples of carbocyclic rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicyclo[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl. Any of the carbocycles can be a heterocycle having at least one heteroatom in the ring backbone.

[0063] As used herein, "cycloalkyl" refers to a completely saturated (no double or triple bonds) monocyclic or polycyclic hydrocarbon ring system. When composed of two or more rings, the rings can be joined together in a fused manner. A cycloalkyl group can contain 3 to 10 atoms in the ring. In some examples, a cycloalkyl group can contain 3 to 8 atoms in the ring. A cycloalkyl group can be unsubstituted or substituted. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0064] As used herein, "cycloalkenyl" or "cycloalkene" means a carbocyclic ring or ring system having at least one double bond, and none of the rings within the ring system are aromatic. Examples include cyclohexenyl or cyclohexene and norbornenyl or norbornene.

[0065] As used herein, "cycloalkynyl" or "cycloalkyne" means a carbocyclic ring or ring system having at least one triple bond, and none of the rings within the ring system are aromatic. An example is cyclooctyne. Another example is bicyclononyne.

[0066] As used herein, "dendritic core" refers to the center of a hydrogel. The dendritic core is a synthetic polymer with branches and, in some cases, has a tree-like structure. The dendritic core can have any number of arms (branches) from 2 to 30.

[0067] As used herein, the term "deposition" refers to any suitable application technique, which may be manual or automatic and which, in some cases, results in the modification of surface properties. In general, deposition can be carried out using vapor deposition techniques, coating techniques, grafting techniques, etc. Some specific examples include chemical vapor deposition (CVD), spray coating (e.g., ultrasonic spray coating), spin coating, dunk or dip coating, doctor blade coating, puddle dispensing, flow-through coating, aerosol printing, screen printing, microcontact printing, inkjet printing, and the like.

[0068] As used herein, the term "recess" refers to a discontinuous concave feature in a substrate or patterned resin having a surface opening at least partially surrounded by an interstitial region of the substrate or patterned resin. The recess may have any of a variety of shapes for the surface opening, including, for example, circular, elliptical, square, polygonal, star-shaped (having any number of vertices). The cross-section of the recess perpendicular to the surface can be curved, square, polygonal, hyperbolic, conical, angular, etc. By way of example, the recess can be a well or two interconnected wells. The recess may also have a more complex structure, such as a raised portion, a step feature, etc.

[0069] The term "each", when used with reference to a set of items, is intended to identify the individual items within the set, but does not necessarily refer to all of the items within the set. Exceptions can occur where there is an explicit disclosure or where the context clearly dictates otherwise.

[0070] As used herein, the term "flow cell" is intended to mean a container having a chamber (e.g., a flow channel) in which a reaction can occur, an inlet for delivering a reagent to the chamber, and an outlet for removing the reagent from the chamber. In some examples, the chamber enables detection of the reaction occurring within the chamber. For example, the chamber may include one or more permeable surfaces that enable optical detection, such as an array, optically labeled molecules, and the like.

[0071] As used herein, "flow channel" or "channel" can be a region defined between two joined components that can selectively receive a liquid sample. In some examples, the flow channel may be defined between a patterned or unpatterned substrate and a lid, and thus may be in fluid communication with one or more recesses defined within the patterned resin. The flow channel may also be defined between two joined surfaces of a patterned or unpatterned substrate.

[0072] As used herein, "heteroalicyclic" or "heteroalicyclic ring" refers to monocyclic, bicyclic, and tricyclic ring systems having 3, 4, 5, 6, 7, 8, 9, 10, up to 18 members, where the carbon atoms together with 1 to 5 heteroatoms constitute the ring system. However, the heteroalicyclic ring system may optionally contain one or more unsaturated bonds positioned in such a manner that a completely delocalized pi-electron system does not occur throughout all of the rings. The heteroatoms are independently selected from oxygen, sulfur, and nitrogen. The heteroalicyclic ring system may further contain one or more carbonyl or thiocarbonyl functional groups such that its definition includes oxo- and thio-based systems such as lactams, lactones, cyclic imides, cyclic thioimides, and cyclic carbamates. The rings may be joined together in a fused manner. Further, any nitrogen in the heteroalicyclic may be quaternized. The heteroalicyclic or heteroalicyclic group may be unsubstituted or substituted. Examples of such "heteroalicyclic" or "heteroalicyclic" groups include 1,3-dioxin, 1,3-dioxane, 1,4-dioxane, 1,2-dioxolane, 1,3-dioxolane, 1,4-dioxolane, 1,3-oxathiane, 1,4-oxathiin, 1,3-oxathiolane, 1,3-dithiol, 1,3-dithiolane, 1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine, imidazoline, imidazolidine, isoxazoline, isoxazolidine, oxazoline, oxazolidine, oxazolidinone, thiazoline, thiazolidine, morpholine, oxirane, piperidine N-oxide, piperidine, piperazine, pyrrolidine, pyrrolidone, pyrrolidione, 4-piperidone, pyrazoline, pyrazolidine, 2-oxopyrrolidine, tetrahydropyran, 4H-pyran, tetrahydrothiopyran, thiomorpholine, thiomorpholine sulfoxide, thiomorpholine sulfone, and their benzofused analogs (e.g., benzimidazolidinone, tetrahydroquinoline, 3,4-methylenedioxyphenyl).

[0073] As used herein, "heteroalkyl" and "heteroaryl(alkyl)" refer to a heteroaryl group bonded as a substituent via a lower alkylene group. The lower alkylene group and heteroaryl group of heteroalkyl may be substituted or unsubstituted. Examples include 2-thienylalkyl, 3-thienylalkyl, furylalkyl, thienylalkyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl, and their benzo-fused analogs.

[0074] "(Heteroalicyclic)alkyl" refers to a heterocyclic or heteroalicyclic group bonded as a substituent via a lower alkylene group. The lower alkylene and heterocyclic, or heterocyclic ring of (heteroalicyclic)alkyl may be substituted or unsubstituted. Examples include (tetrahydro-2H-pyran-4-yl)methyl, (piperidin-4-yl)ethyl, (piperidin-4-yl)propyl, (tetrahydro-2H-thiopyran-4-yl)methyl, and (1,3-thiazinan-4-yl)methyl, but are not limited thereto.

[0075] As used herein, "hydroxy" or "hydroxyl" refers to the -OH group.

[0076] The term "dicol" refers to the terminal group -(CH2) n OH, where n ranges from 2 to 10. As a specific example, the glycol can be an ethylene glycol terminal group -CH2CH2OH, a propylene glycol terminal group -CH2CH2CH2OH, or a butylene glycol terminal group -CH2CH2CH2CH2OH.

[0077] As used herein, the term "gap region" refers to the region that separates the recesses, e.g., the region of a substrate, a patterned resin, or other support. For example, the gap region can separate one recess of an array from another recess of the array. Two recesses separated from each other may be distinct, i.e., may lack physical contact with each other. In many instances, the gap region is continuous, although the recesses are discontinuous, as in the case of a plurality of recesses defined in a surface that is otherwise continuous. In other instances, the gap regions and features are discontinuous, as in the case of a plurality of grooves separated by respective gap regions. The separation provided by the gap region can be partial or complete. The gap region may have a surface material different from the surface material of the recesses defined in the surface. For example, the recesses may have a polymer and a first primer set therein, and the gap region may have a polymer and a second primer set thereon. In another example, the recesses of the array may have beads therein, while the intervening region does not have beads thereon.

[0078] As used herein, a "nucleotide" includes a nitrogen-containing heterocyclic base, a sugar, and one or more phosphate groups. Nucleotides are the monomeric units of nucleic acid sequences. In the case of ribonucleic acid (RNA), the sugar is ribose, and in deoxyribonucleic acid (DNA), the sugar is deoxyribose, i.e., a sugar lacking the hydroxyl group present at the 2'-position of ribose. The nitrogen-containing heterocyclic base (i.e., nucleobase) can be either a purine base or a pyrimidine base. Examples of purine bases include adenine (A) and guanine (G), and their modified derivatives or analogs. Examples of pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and their modified derivatives or analogs. The C-1 atom of deoxyribose is bonded to N-1 of pyrimidine or N-9 of purine. Nucleic acid analogs may have a changed phosphate backbone, sugar, or nucleobase. Examples of nucleic acid analogs include universal bases or phosphate-sugar backbone analogs such as, for example, peptide nucleic acid (PNA).

[0079] "Patterned resin" refers to any polymer that may have recesses defined therein. Specific examples of resins and techniques for patterning resins are further described herein.

[0080] As used herein, "primer" is defined as a single-stranded nucleic acid sequence (e.g., single-stranded DNA or single-stranded RNA). Some primers referred to herein as amplification primers function as starting points for template amplification and cluster generation. Other primers referred to herein as sequencing primers function as starting points for DNA or RNA synthesis. The 5'-end of the primer may be modified to enable a coupling reaction with a functional group of the polymer or the bead surface. The length of the primer can be of any number of bases and can include various unnatural nucleotides. In one example, the sequencing primer is a short strand in the range of 10 to 60 bases, or 20 to 40 bases.

[0081] The term "substrate" refers to a structure onto which various components of a flow cell (e.g., hydrogel, primer, etc.) can be added. The substrate can be a wafer, panel, rectangular sheet, die, or any other suitable configuration. The substrate is generally hard and insoluble in aqueous liquids. The substrate may be inert to the chemicals used to modify the recesses or present in the recesses. For example, the substrate can be inert to the chemicals used to form a polymer, e.g., to bind a primer. The substrate may be a single-layer structure or a multi-layer structure (e.g., including a support and a patterned resin on the support). Examples of suitable substrates are further described herein.

[0082] Multi-arm polymer hydrogel

[0083] One example of the hydrogels described in this specification is a multi-arm polymer hydrogel. The multi-arm polymer hydrogel comprises a dendritic core having 2 to 30 arms and a first acrylamide monomer incorporated into each arm of the dendritic core, with the structure: [Chemical Formula] wherein R1 and R2 are independently selected from the group consisting of alkyl, alkylamino, alkylamide, alkylthiol, aryl, glycol, and any substituents thereof; a first acrylamide monomer; and a second acrylamide monomer incorporated into each arm of the dendritic core, with the structure: [Chemical Formula] wherein R3 is hydrogen or alkyl, R4 is hydrogen or alkyl, L is a linear chain of 2 to 20 atoms selected from the group consisting of carbon, oxygen, and nitrogen, and including optional substituents on the carbon atoms and any nitrogen atoms within the chain; a linker; and A is an N-substituted amide having the structure [Chemical Formula] wherein R5 is hydrogen or alkyl, E is a linear chain of 1 to 4 atoms selected from the group consisting of carbon, oxygen, and nitrogen, and including optional substituents on the carbon atoms and any nitrogen atoms within the chain, and Z is an optional nitrogen-containing heterocyclic ring; a second acrylamide monomer, and includes.

[0084] A multi-arm polymer hydrogel can be prepared by incorporating a copolymer into each arm of a multi-arm component having 2 to 30 arms, and the copolymer includes a first acrylamide monomer and a second acrylamide monomer. The incorporation of the acrylamide monomer into the multi-arm component may be statistical, random, alternating, or block. The incorporation of the acrylamide monomer into the multi-arm component can be achieved by various techniques including reversible addition-fragmentation chain transfer (RAFT) polymerization, atom transfer radical polymerization (ATRP), nitroxide mediated radical (NMP) polymerization combined with RAFT or ATRP, NMP with an additional cross-linking step, cobalt mediated polymerization, group transfer polymerization (GTP), ring opening polymerization (ROP), or any other polymerization process that results in the incorporation (statistical, random, alternating, or block) of the acrylamide monomer into the multi-arm structure and each arm, either directly or indirectly. As an example of an indirect process, RAFT polymerization may be performed following NMP.

[0085] Figure 1A shows an example of RAFT polymerization for generating an example of a hydrogel, which in this example is a multi-arm polymer hydrogel 10.

[0086] In the example shown in Figure 1A, the dendritic core 12 includes a central molecule / compound 13 and arms 14 (or branches) extending from the central molecule / compound 13. The dendritic core 12 may be any multi-functional component that enables a controlled polymerization mechanism, which results in a defined arm length in the polymer structure and at least substantially uniform arm lengths among the polymer structures. In one example, the arms of the dendritic core 12 are identical to each other.

[0087] The central molecule / compound 13 of the dendritic core 12 may be any polyfunctional molecule such as a macrocycle (e.g., cyclodextrin, porphyrin, etc.), an extended π-system (e.g., perylene, fullerene, etc.), a metal-ligand complex, a polymer core, etc. Some specific examples of the central molecule / compound 13 of the dendritic core 12 include a phenyl group, benzoic acid, pentaerythritol, a phosphazene group, and the like.

[0088] As described above, the dendritic core 12 includes arms 14 extending from the central molecule / compound 13.

[0089] In one example, the dendritic core 12 contains a thiocarbonylthio group in each arm 12 and is thus a reversible addition-fragmentation chain transfer agent (RAFT agent). This example of the dendritic core 12 may have 2 to 30 arms, and each of these arms contains a thiocarbonylthio group at or near the end of each arm. In some examples, the dendritic core 12 containing a thiocarbonylthio group has 2 arms, 3 arms, 4 arms, 6 arms, or 8 arms.

[0090] Each RAFT agent contains a thiocarbonylthio group (S=C-S) having substituents R and Z that affect the polymerization reaction kinetics and the degree of structural control. As an example, the thiocarbonylthio group in each arm 14 of the dendritic core 12 is dithiobenzoate:

Chemical formula

Chemical formula

Chemical formula

[0091] In the RAFT agent, the R group is a free radical leaving group, and the Z group controls the C=S bond reactivity and affects the rates of radical addition and cleavage.

[0092] In some examples, the dendritic core 12 containing a thiocarbonylthio group in each arm 14 has an R group configuration, and the central molecule 13 is a leaving group during the chain transfer process. Two examples of dendritic cores having the RAFT agent configuration of the R group are as follows: [Chemical formula] (wherein Ph is a phenyl group), and [Chemical formula] In other examples, the dendritic core 12 containing a thiocarbonylthio group in each arm 14 has a Z group configuration. In these examples, the reactive polymer arm 14 is separated from the central molecule / compound 13 during growth, undergoes chain transfer, and reacts again with the central molecule / compound 13. One example of a dendritic core having the RAFT configuration of the Z group is as follows: [Chemical formula]

[0093] In one example, the dendritic core 12 containing a thiocarbonylthio group in each arm 14 is 3,5-bis(2-dodecylthiocarbonothioylthio-1-oxopropoxy)benzoic acid: [Chemical formula] (example of a two-arm dendritic core), 1,1,1-tris[(dodecylthiocarbonothioylthio)-2-methylpropionate]ethane: [Chemical formula] (example of a three-arm dendritic core), and pentaerythritol tetrakis[2-(dodecylthiocarbonothioylthio)-2-methylpropionate]: [Chemical] Selected from the group consisting of (examples of dendritic cores with four arms).

[0094] An example of a dendritic core 12 containing a phosphazene ring as the central molecule / compound 13 is [Chemical] wherein each R is a trithiocarbonyl group. This is an example of a dendritic core 12 containing 30 arms.

[0095] Yet another example of a dendritic core 12 containing a thiocarbonylthio group in each arm 14 can be produced by RAFT polymerization of acrylamide with 3-(((benzylthio)carbonothioyl)thio)propanoic acid using N,N'-methylenebis(acrylamide) (BisAM) as a crosslinking agent, followed by chain extension with different levels of acrylamide.

[0096] In another example, the dendritic core 12 contains an atom transfer radical polymerization (ATRP) initiator in each arm 14. This example of the dendritic core 12 may have 2 to 30 arms, and each of these arms contains an ATRP initiator at or near the end of each arm 14. In some examples, the dendritic core containing an ATRP initiator has 2 arms, 3 arms, 4 arms, 6 arms, or 8 arms.

[0097] In some examples, the dendritic core 12 containing an atom transfer radical polymerization (ATRP) initiator is a multifunctional initiator. In these examples, the dendritic core 12 can be selected from the group consisting of bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide, 2-bromoisobutyric anhydride, ethylene bis(2-bromoisobutyrate), pentaerythritol tetrakis(2-bromoisobutyrate), dipentaerythritol hexakis(2-bromoisobutyrate), and 1,1,1-tris(2-bromoisobutyryloxymethyl)ethane.

[0098] In other examples, a monofunctional initiator is attached to a non-ATRP polyfunctional core molecule to produce a dendritic core 12 containing an atom transfer radical polymerization (ATRP) initiator on each arm. The non-ATRP polyfunctional core molecule can be any example of the polyfunctional core molecules 13 described herein.

[0099] Examples of ATRP monofunctional initiators include 2-azidoethyl 2-bromoisobutyrate, poly(ethylene glycol) methyl ether 2-bromoisobutyrate (of various molecular weights), 2-(2-bromoisobutyryloxy)ethyl methacrylate, dodecyl 2-bromoisobutyrate, 2-hydroxyethyl 2-bromoisobutyrate, 1-(phthalimidomethyl) 2-bromoisobutyrate, propargyl 2-bromoisobutyrate, and the like. These monofunctional initiators can be attached to any example of the core molecules / compounds 13 disclosed herein to form a dendritic core 12 containing an atom transfer radical polymerization (ATRP) initiator on each arm.

[0100] In yet another example, the dendritic core 12 contains a nitroxide (aminooxyl)-mediated polymerization (NMP) initiator on each arm 14. This exemplary dendritic core may have from 2 to 30 arms, and each of these arms contains an NMP initiator at or near the end of each arm 14. In some examples, the dendritic core containing an NMP initiator has 2 arms, 3 arms, 4 arms, 6 arms, or 8 arms.

[0101] In some examples, the dendritic core 12 containing an NMP initiator is a polyfunctional initiator. As an example, the polyfunctional initiator (I) can be

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0102] In other examples, a plurality of monofunctional NMP initiators are attached to a non-NMP polyfunctional core molecule to produce a dendritic core 12 containing NMP initiators on each arm. The non-NMP polyfunctional core molecule can be any example of the polyfunctional core molecules 13 described herein. Examples of NMP monofunctional initiators include

Chemical formula

Chemical formula

Chemical formula

[0103] Although some examples of the dendritic core 12 are described, it should be understood that the structure of the dendritic core 12 depends on the polymerization process used to produce the multi-arm polymer hydrogel 10. For example, a dendritic core 12 containing a thiocarbonylthio group can be used in RAFT polymerization, while a dendritic core 12 containing an ATRP initiator can be used in ATRP, and a dendritic core 12 containing an NMP initiator can be used in NMP. Other dendritic cores 12 can be prepared or obtained and used in other polymerization processes such as ROP.

[0104] In an example of the multi-arm polymer hydrogel 10 disclosed herein, a first acrylamide monomer 16 and a second acrylamide monomer 18 are incorporated into the arms 14 of the dendritic core 12.

[0105] The first acrylamide monomer 16 has the structure: [Chemical formula] wherein R1 and R2 are independently selected from the group consisting of alkyl, alkylamino, alkylamide, alkylthiol, aryl, glycol, and any substituents thereof. R1 and R2 are selected to provide a more hydrophobic backbone to the arm 14. The R1 and R2 groups cannot form hydrogen bonds between polymer strands, which can help improve the dry storage ability of the multi-arm polymer hydrogel 10 without having a harmful effect on downstream sequencing operations. In one example, the first acrylamide monomer 16 is N,N-dimethylacrylamide.

[0106] The second acrylamide monomer 18 has the structure: [Chemical formula] wherein R3 is hydrogen or alkyl, R4 is hydrogen or alkyl, L is a linker comprising a linear chain of 2 to 20 atoms selected from the group consisting of carbon, oxygen, and nitrogen, and optional substituents on the carbon atoms and any nitrogen atoms within the chain, and A is an N-substituted amide having the structure [Chemical formula] wherein R5 is hydrogen or alkyl, E is a linear chain of 1 to 4 atoms selected from the group consisting of carbon, oxygen, and nitrogen, and optional substituents on the carbon atoms and any nitrogen atoms within the chain, and Z is an optional nitrogen-containing heterocyclic ring.

[0107] The azide group of the second acrylamide monomer 18 can participate in the crosslinking of the multi-arm polymer hydrogel 10. For example, the multi-arm polymer hydrogel 10 can be bonded to the surface of the flow cell (see, for example, FIG. 2A), and a primer can be bonded (see, for example, FIGS. 2B and 2C).

[0108] When R3 and / or R4 is alkyl, the number of carbon atoms can range from 1 to 6 or from 1 to 4.

[0109] In the second acrylamide monomer 18, E may be optionally substituted C1-C4 alkylene, and each carbon is optionally substituted with one or more substituents selected from, for example, C1-C4 alkyl, -OH, -OC1-C4 alkyl, or =O. As an example, E may be unsubstituted C1-C4 alkylene, such as CH2, (CH2)2, (CH2)3 or (CH2)4.

[0110] In other examples, E may contain an ether, an ester, or an amide. For example, E may contain -CH2CH2OCH2-, -COCNHCH2-, or -CH2COOCH2-.

[0111] In the second acrylamide monomer 18, L is a linker containing a linear chain which is -C2-C20 alkylene- or a linear heteroalkylene of 3 to 20 atoms, optionally substituted with one or more substituents each selected from the group consisting of -C1-C4 alkyl, -OH, -OC1-C4 alkyl, or =O. L may be a linker having a linear chain which is -C2-C6 alkylene optionally substituted with one or more -C1-C4 alkyl, -OH, -OC1-C4 alkyl, or =O substituents. L may be unsubstituted -C2-C6 alkylene- (also depicted as -((CH2) 2~6 -). For example, L may be unsubstituted -C3-C4 alkylene-, such as -(CH2)3- or -(CH2)4-.

[0112] In another example, L may be a linker comprising a linear chain that is a straight-chain heteroalkylene of 3 to 20 atoms optionally substituted with one or more substituents selected from the group consisting of -C1-C4 alkyl, -OH, -OC1-C4 alkyl, or =O. L may contain one or more ethylene glycol units. L may be -CH2CH2(OCH2CH2) x -OCH2CH2-, where x is from 0 to 10. In one example, x is 1, 2, 3, 4, 5, or 6. L may contain one or more amide groups. For example, L may be -C2-C6 alkyl-NHC(O)-C2-C6 alkyl-, or L may be -(CH2)2-NHC(O)-(CH2)2- or -(CH2)3-NHC(O)-(CH2)2-. L may contain one or more natural or unnatural amino acids. For example, L may contain one or more amino acids selected from the group consisting of glycine, alanine, valine, isoleucine, leucine, lysine, serine, threonine, cysteine, asparagine, or glutamine. In some examples, L may contain one, two, or three amino acid units.

[0113] In the second acrylamide monomer 18, the N-substituted amide A may be bonded to L and Z in two possible configurations. For example, the carbonyl carbon of A may be bonded to L and the amide nitrogen of A may be bonded to Z. Alternatively, the carbonyl carbon of A may be bonded to Z and the amide nitrogen of A may be bonded to L.

[0114] In the second acrylamide monomer 18, Z may include a nitrogen-containing heterocyclic ring having 5 to 10 ring members (5 to 10 atoms), for example, a 5- to 10-membered heterocyclic ring, and the ring members are atoms forming the backbone of the heterocyclic ring. Z may include a monocyclic structure or a condensed structure including two or more ring systems. In the case of a monocyclic structure, Z may include 5 or 6 ring members. For example, Z may be a 5- or 6-membered heterocyclic ring. In the case of a condensed structure, Z may include 9 or 10 ring members. The nitrogen-containing heterocyclic ring may include two or more heteroatoms, for example, one or more additional nitrogen heteroatoms, or one or more oxygen heteroatoms, or one or more sulfur heteroatoms, or any suitable combination of such heteroatoms. The nitrogen-containing heterocyclic ring may be aromatic, for example, pyridinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, indolyl, quinolinyl, quinazolinyl. The nitrogen-containing heterocyclic ring may be aliphatic, for example, cycloalkyl. The aliphatic nitrogen-containing heterocyclic ring may be saturated or may include one or more double bonds without being aromatic. In one example, the aliphatic nitrogen-containing heterocyclic ring may be pyrrolidinyl, pyridinyl, or pyrimidinyl.

[0115] An example of the second acrylamide monomer 18 (as shown in FIG. 1A and not including Z) is azidoacetamidopentyl acrylamide, specifically, N-(5-azidoacetamidylpentyl) acrylamide. Variations of N-(5-azidoacetamidylpentyl) acrylamide may also be used. For example, the alkyl chain -(CH2)- may be in the range of 1 to 20, and / or each of -(CH2)- may be optionally substituted.

[0116] Some other examples of the second acrylamide monomer 18 including Z are

Chemical formula

[0117] In the example shown in FIG. 1A, a mixture of acrylamide monomers 16, 18 is polymerized in the presence of a multi-arm component (e.g., dendritic core 12). In this example, the multi-arm component is a four-arm RAFT agent containing four trithiocarbonate groups, the first acrylamide monomer 16 is N,N-dimethylacrylamide, and the second acrylamide monomer is azidoacetamidopentyl acrylamide 18.

[0118] The mixture of monomers 16, 18 may include water and a co-solvent (e.g., N-methyl-2-pyrrolidone (NMP), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile (MeCN), methanol (MeOH), ethanol (EtOH), isopropyl alcohol (IPA), dioxane, acetone, dimethylacetamide (DMAc), etc.). The mixture may also include a buffer to at least substantially prevent unwanted changes in pH. The pH of the mixture may be acidic (less than 7). Examples of suitable buffers include TRIS (tris(hydroxymethyl)aminomethane or TRIZMA®), bis-tris methane buffer, ADA buffer (zwitterionic buffer), MES (2-(N-morpholino)ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), or another acidic buffer.

[0119] The polymerization reaction can be carried out at a temperature in the range of about 50°C to about 80°C for a time in the range of about 1 hour to about 48 hours. An initiator including an azo initiator such as azobisisobutyronitrile or 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (one commercial example is VA-044 from FujiFilm) may also be included in the mixture.

[0120] In some examples, the process shown in FIG. 1A randomly incorporates acrylamide monomers 16, 18 into each of the arms 14, although other monomer incorporation scenarios (e.g., statistical, alternating, etc.) are possible. Random incorporation can result in some blocks of each monomer 16 and / or 18. Thus, in one example, the first acrylamide monomer 16 and the second acrylamide monomer 18 form a random copolymer in each of the arms 14 of the dendritic core 12. The molar ratio of monomer 16 to monomer 18 can range from about 5:95 to about 1:50, or from about 5:95 to about 50:1.

[0121] In another example, the acrylamide monomers 16, 18 may be incorporated into each of the arms 14 in controlled blocks. In this example, a block copolymer can be formed in the presence of a multi-arm component (e.g., dendritic core 12). One example of this method is to polymerize a first block with a first acrylamide monomer 16 in the presence of a multi-arm component (e.g., dendritic core 12) to form a modified multi-arm component (including the first block in each arm 14), and then polymerize a second block with a second acrylamide monomer 18 in the presence of the modified multi-arm component to form a multi-arm polymer hydrogel 10 (including both blocks in each arm 14). Another example of this method is to polymerize a first block with a second acrylamide monomer 18 in the presence of a multi-arm component (e.g., dendritic core 12) to form a modified multi-arm component (including the first block in each arm 14), and then polymerize a second block with a first acrylamide monomer 16 in the presence of the modified multi-arm component to form a multi-arm polymer hydrogel 10 (including both blocks in each arm 14). In this example, the first acrylamide monomer 16 and the second acrylamide monomer 18 form a block copolymer in each of the arms 14 of the dendritic core 12.

[0122] In still other examples, another block may be added to the block copolymer. This block may include monomer units not utilized in the other blocks. In one example, the resulting block copolymer is a triblock copolymer.

[0123] In still other examples, acrylamide monomers 16, 18 may be incorporated into each of arms 14 statistically, and the sequential distribution of monomer units follows known statistical laws.

[0124] In yet further examples, acrylamide monomers 16, 18 may be incorporated into each of arms 14 such that they alternate along the length.

[0125] In still other examples, proteins and / or nanoparticles and / or other polymers may be conjugated to the ends of each arm 14 of dendritic core 12. These units may be copolymerized with monomer units or introduced after polymerization.

[0126] It should be understood that the arrangement of the "n" and "m" features that occur repeatedly in FIG. 1A is representative, and monomer subunits 16, 18 may be present in any order (randomly, statistically, as alternating units, as a block copolymer). In one example, n is an integer in the range of 1 to 2,500, and m is an integer in the range of 1 to 2,500. In another example, n + m is an integer in the range of 2 to 5,000.

[0127] Any example of the hydrogels disclosed herein that includes multi-arm polymer hydrogel 10 may contain a single crosslink per polymer molecule.

[0128] The molecular weight of any example of the hydrogels disclosed herein that includes multi-arm polymer hydrogel 10 may vary, at least in part, depending on the starting materials and the percentage of conversion. As an example, the molecular weight of multi-arm polymer hydrogel 10 is about 850,000 g / mol.

[0129] In other examples, the polymer end groups of the multi-arm polymer hydrogel 10 may be cleaved such that the arms remain capped with suitable end groups. Cleavage can be performed using any suitable process, such as reaction with a peroxide (resulting in alcohol end groups), reaction with an azide, radical-induced end group removal, UV-induced removal, oxidation-induced removal, or any other suitable technique. FIGS. 1B and 1C show two examples where the polymer end groups of the multi-arm polymer hydrogel 10 shown in FIG. 1A are cleaved and the arms are capped with different end groups.

[0130] Flow cell

[0131] The hydrogels disclosed herein may be used in a flow cell 20, an example of which is shown in FIG. 2A. The flow cell 20 includes a substrate 22 and a multi-arm polymer hydrogel 10 on the substrate 22.

[0132] The substrate 22 may be a single layer / material. Examples of suitable single-layer substrates include epoxy siloxane, glass, modified or functionalized glass, plastics (acrylic, polystyrene, and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, polytetrafluoroethylene (e.g., TEFLON® from Chemours), cyclic olefin / cyclo-olefin polymer (COP) (e.g., ZEONOR® from Zeon), polyimide, etc.), nylon (polyamide), ceramic / ceramic oxide, 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) Examples include hafnium oxide (HfO2), carbon, metal, inorganic glass, etc. The substrate 22 may also have a multilayer structure. Some examples of the multilayer structure include glass or silicon, which has a coating layer of tantalum oxide or another ceramic oxide on its surface. Another example of the multilayer structure includes a substrate support (e.g., glass or silicon) having a patterned resin thereon. Still another example of the multilayer substrate includes a silicon-on-insulator (SOI) substrate.

[0133] In one example, the substrate 22 can have a rectangular sheet or panel with a diameter in the range of about 2 mm to about 300 mm, or a maximum dimension of up to about 10 feet (about 3 meters). In one example, the substrate 22 is a wafer having a diameter in the range of about 200 mm to about 300 mm. In another example, the substrate 22 is a die having a width in the range of about 0.1 mm to about 10 mm. Although exemplary dimensions are provided, it should be understood that a substrate 22 having any suitable dimensions can be used. In another example, a panel, which is a rectangular support having a larger surface area than a 300 mm circular wafer, can be used.

[0134] In the example shown in FIG. 2A, the flow cell 20 includes flow channels 24. Although several flow channels 24 are shown, it should be understood that any number of channels 24 can be included in the flow cell 20 (e.g., a single channel 24, four channels 24, etc.). Each flow channel 24 is a region defined between two joined components (e.g., the substrate 22 and a lid, or two substrates 22), and a fluid (e.g., those described herein) can be introduced into and removed from there. Each flow channel 24 may be separated from each other flow channel 24 such that the fluid introduced into any particular flow channel 24 does not flow into any adjacent flow channel 24. Some examples of the fluid introduced into the flow channel 24 can include reaction components (e.g., polymerase, sequencing primer, nucleotides, etc.), a washing solution, a deblocking agent, etc.

[0135] The flow channel 24 can be defined in the substrate 22 using any suitable technique that is partially dependent on the material of the substrate 22. In one example, the flow channel 24 is etched into the glass substrate 22. In another example, the flow channel 24 can be patterned into the resin of the multi-layer substrate 22 using photolithography, nanoimprint lithography, etc. In yet another example, a separate material (not shown) can be applied to the substrate 22 such that the separate material defines the wall portions of the flow channel 24 and the substrate 22 defines the bottom of the flow channel 24.

[0136] In one example, the flow channel 24 has a linear configuration. The length and width of the flow channel 24 may each be less than the length and width of the substrate 22, such that the portions of the substrate surface surrounding the flow channel 24 are available for attachment to a lid (not shown) or another substrate 22. In some cases, the width of each flow channel 24 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 lane 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 24 can be greater than, less than, or between the values shown above. In another example, the flow channel 24 is square (e.g., 10 mm × 10 mm).

[0137] The depth of each flow channel 24 can be on the order of the thickness of a single layer when depositing a separate material that defines the flow channel walls using microcontact, aerosol, or inkjet printing. In other examples, the depth of each flow channel 24 can be about 1 μm, about 10 μm, about 50 μm, about 100 μm, or more. In one example, the depth can range from about 10 μm to about 100 μm. In another example, the depth can range from about 10 μm to about 30 μm. In yet another example, the depth is about 5 μm or less. It should be understood that the depth of each flow channel 24 is greater than, less than, or between the values shown above.

[0138] Different examples of the structure within the flow channel 24 of the flow cell 20 are shown in FIGS. 2B and 2C.

[0139] In the example shown in FIG. 2B, the flow cell 20 includes a single layer substrate 22A and a portion of the flow channel 24 defined in the single layer substrate 22A. In this example, the multi-arm polymer hydrogel 10 is disposed within the flow channel 24.

[0140] To introduce the multi-arm polymer hydrogel 10 (or any example of the hydrogels disclosed herein) into the flow channel 24, a mixture of the multi-arm polymer hydrogel 10 can be produced and then applied to the substrate 22 (wherein the flow channel 24 is defined). In one example, the multi-arm polymer hydrogel 10 can be present in a mixture (e.g., with water or ethanol and water). The mixture can then be applied to the surface of the substrate (including that contained in the flow channel 24) using spin coating, or dipping or dip coating, spray coating, or the flow of the material under positive or negative pressure, or another suitable technique. These types of techniques deposit the catalytic polymer hydrogel 16' overlying (e.g., over the in-channel 26 and the gap region 28) on the substrate 24. Other selective deposition techniques (including, for example, masks, controlled printing techniques, etc.) can be used to specifically deposit the catalytic polymer hydrogel 16' within the flow channel 26 without depositing it over the gap region 28.

[0141] In some examples, the surface of the substrate (including the portion exposed in the flow channel 24) may be activated and then a mixture (including a hydrogel such as the multi-arm polymer hydrogel 10) may be applied. In one example, a silane or silane derivative (e.g., norbornene silane) can be deposited on the surface of the substrate using vapor deposition, spin coating, or other deposition methods. In another example, the surface of the substrate can be exposed to plasma ashing to generate a surface activator (e.g., -OH groups) that can adhere to a hydrogel such as the multi-arm polymer hydrogel 10.

[0142] Depending on the hydrogel used, the applied mixture can be exposed to a curing process. In one example, curing can be performed 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 to about several days. Other suitable curing conditions are possible depending on the material of the hydrogel.

[0143] Next, polishing may be performed to remove the hydrogel, e.g., the multi-arm polymer hydrogel 10, from the gap region 34 around the flow channel 24 while leaving the hydrogel at least substantially intact on the surface within the flow channel 24.

[0144] The flow cell 20 also includes amplification primers 26.

[0145] A grafting process can be performed to graft the amplification primers 26 to the hydrogel within the flow channel 24, e.g., the multi-arm polymer hydrogel 10. In one example, the amplification primer 26 can be immobilized to the hydrogel by a single point covalent bond at or near the 5' end of the primer 26. This bond allows i) the adapter-specific portion of the primer 26 to be free to anneal to its cognate sequenceable nucleic acid fragment and ii) the 3' hydroxyl group to be free for primer extension. For this purpose, any suitable covalent bond can be used. Examples of end primers that can be used include alkyne end primers that can bind to the azide portion of the hydrogel. Specific examples of suitable primers 26 include P5 and P7 primers used on the surface of commercially available flow cells sold by Illumina Inc. for sequencing on HISEQ (trademark), HISEQX (trademark), MISEQ (trademark), MISEQDX (trademark), MINISEQ (trademark), NEXTSEQ (trademark), NEXTSEQ (trademark) DX (trademark), NOVASEQ (trademark), GENOME ANALYZER (trademark), ISEQ (trademark), and other instrument platforms.

[0146] In one example, the grafting can include flow-through deposition (e.g., using a temporarily or permanently attached lid), dunk coating, spray coating, paddle dispensing, or another suitable method of attaching primer 26 to the hydrogel within flow channel 24. Each of these exemplary techniques can utilize a primer solution or mixture, which can include primer 26, water, buffer, and a catalyst. By any of the grafting methods, primer 26 reacts with the reactive groups within flow channel 24 and has no affinity for the surrounding substrate 22. Thus, primer 26 selectively grafts to the hydrogel within flow channel 24.

[0147] In the example shown in FIG. 2C, flow cell 20 includes a multilayer substrate 22B that includes a support 28 and a patterned material 30 disposed on support 28. The patterned material 30 defines recesses 32 separated by gap regions 34.

[0148] In the example shown in FIG. 2C, the patterned material 30 is disposed on support 28. It should be understood that any material that can be selectively deposited or deposited and patterned to form recesses 32 and gap regions 34 can be used for the patterned material 30.

[0149] As an example, an inorganic oxide can be selectively applied to support 28 by evaporation, aerosol printing, or inkjet printing. Examples of suitable inorganic oxides include tantalum oxide (e.g., Ta2O5), aluminum oxide (e.g., Al2O3), silicon oxide (e.g., SiO2), hafnium oxide (e.g., HfO2), and the like.

[0150] As another example, a resin may be applied to the support 28 and then patterned. Suitable deposition techniques include chemical vapor deposition, dip coating, dunk coating, spin coating, spray coating, paddle dispensing, ultrasonic spray coating, doctor blade coating, aerosol printing, screen printing, microcontact printing, and the like. Suitable patterning techniques include photolithography, nanoimprint lithography (NIL), stamping techniques, embossing techniques, molding techniques, microetching techniques, printing techniques, and the like. Some examples of suitable resins include polyhedral oligomeric silsesquioxane (POSS) - based resins, non - POSS epoxy resins, poly(ethylene glycol) resins, polyether resins (e.g., ring - opened epoxy), acrylic resins, acrylate resins, methacrylate resins, amorphous fluoropolymer resins (e.g., CYTOP (registered trademark) manufactured by Bellex), and combinations thereof.

[0151] As used herein, the term "polyhedral oligomeric silsesquioxane" (POSS) refers to a chemical composition that is a hybrid intermediate between silica (SiO2) and silicone (R2SiO) (e.g., RSiO 1.5 ). Examples of POSS can be those described in Kehagias et al., Microelectronic Engineering 86(2009), pp.776 - 778, which is hereby incorporated by reference in its entirety. In one example, the composition has the chemical formula [RSiO 3 / 2 n ​It is an organosilicon compound having, and the R groups may be the same or different. Exemplary R groups of POSS include epoxy, azide / azido, thiol, poly(ethylene glycol), norbornene, tetrazine, acrylate, and / or methacrylate, or further, for example, an alkyl group, an aryl group, an alkoxy group, and / or a haloalkyl group. The resin compositions disclosed herein may include one or more different cage or core structures as monomer units. The polyhedral structure may be a T8 structure as follows, for example, [Chemical formula] and may be, [Chemical formula] and can be represented by. This monomer unit typically has eight arms of functional groups R1 to R8.

[0152] The monomer unit may have a cage structure having ten silicon atoms and ten R groups, such as those referred to as T 10 and, [Chemical formula] or may have a cage structure having twelve silicon atoms and twelve R groups, such as those referred to as T 12 and, [Chemical formula] The POSS-based materials may alternatively include a T6, T 14 , or T 16 cage structure. The average cage content can be adjusted during synthesis and / or controlled by purification methods, and the distribution of the cage sizes of the monomer units can be used in the examples disclosed herein.

[0153] In some examples of POSS disclosed herein, R1 to R8 or R 10 or R 12At least one of them contains epoxy. R1 to R8 or R 10 or R 12 may or may not be the same. Also, in some examples, R1 to R8 or R 10 or R 12 at least one of them contains epoxy, and at least one of the others of R1 to R8 or R 10 or R 12 is a non-epoxy functional group. The non-epoxy functional group may be (a) a reactive group that reacts orthogonally to the epoxy group (i.e., reacts under conditions different from those of the epoxy group), which functions as a handle for binding the resin to an amplification primer, polymer, or polymerizing agent, or (b) a group that regulates the mechanical or functional properties of the resin, such as surface energy adjustment. 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.

[0154] As shown in FIG. 2C, the patterned material 30 includes recesses 32 defined therein and gap regions 34 separating adjacent recesses 32. Many different layouts of the recesses 32, including regular, repetitive, and irregular patterns, can be envisioned. In one example, the recesses 32 are arranged in a hexagonal grid for dense packing and improved density. Other layouts can include, for example, a linear (rectangular) layout, a triangular layout, etc. In some examples, the layout or pattern can be in an x-y format of recesses 32 forming rows and columns. In some other examples, the layout or pattern can be a repetitive arrangement of the recesses 32 and / or the gap regions 34. In yet other examples, the layout or pattern can be a random arrangement of the recesses 32 and / or the gap regions 34. Patterns can include spots, pads, wells, pillars, stripes, vortices, lines, triangles, rectangles, circles, arcs, checks, grid stripes, slashes, arrows, squares, and / or cross-hatching.

[0155] The layout or pattern of the recesses 32 can be characterized in terms of the density (number of recesses 32) of the recesses 32 within a defined area. For example, the recesses 32 can be present at a density of about 2,000,000 per 1 mm 2 The density can be, for example, about 100 per 1 mm 2 about 1,000 per 1 mm 2 about 100,000 per 1 mm 2 about 1,000,000 per 1 mm 2 about 2,000,000 per 1 mm 2 about 5,000,000 per 1 mm 2 about 10,000,000 per 1 mm 2 about 10,000,000 per 1 mm 2It may be adjusted to different densities, including a density of about 50,000,000 or more or less. It should be further understood that the density of the recesses 32 in the patterned material 30 may be between one of the lower values selected from the above range and one of the higher values. By way of example, a high-density array may be characterized by having recesses 32 spaced less than about 100 nm apart, a medium-density array may be characterized by having recesses 32 spaced from about 400 nm to about 1 μm apart, and a low-density array may be characterized by having recesses 32 spaced greater than about 1 μm apart. Although exemplary densities are provided, it should be understood that any suitable density may be used. The density of the recesses 32 may depend in part on the depth of the recesses 32. In some cases, it may be desirable for the spacing between the recesses 32 to be even greater than the examples listed herein.

[0156] The layout or pattern of the recesses 32 may further or alternatively be characterized in terms of an average pitch, or the spacing from the center of one recess 32 to the center of an adjacent recess 32 (center-to-center spacing) or the spacing from the end of one recess 32 to the end of an adjacent recess 32 (end-to-end spacing). The pattern may be regular such that the coefficient of variation of the average pitch is small, or the pattern may be irregular, in which case the coefficient of variation may be relatively large. In either 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 of a particular pattern of recesses 32 may be between one of the lower values selected from the above range and one of the higher values. In one example, the recesses 32 have a pitch (center-to-center spacing) of about 1.5 μm. Although exemplary average pitch values are provided, it should be understood that other average pitch values may be used.

[0157] The size of each recess 32 may be characterized by its volume, opening area, depth, and / or diameter.

[0158] Each recess 32 may have any volume capable of confining a fluid. The minimum or maximum volume can be selected, for example, to accommodate the throughput (e.g., multiplicity), resolution, labeled nucleotides, or reactivity of the analyte expected for use downstream of the flow cell 20. For example, the volume can 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 in addition, the volume can be at most about 1×10 4 μm 3 at most about 1×10 3 μm 3 at most about 100 μm 3 at most about 10 μm 3 at most about 1 μm 3 at most about 0.1 μm 3 or less.

[0159] The area occupied by each recess opening can be selected based on the same criteria as described above for the volume. For example, the area of each recess opening can be at least about 1×10 -3 μm 2 at least 1×10 -2 μm 2 at least about 0.1 μm 2 at least about 1 μm 2 at least about 10 μm 2 at least about 100 μm 2 or more. Alternatively, or in addition, the area can be at most about 1×10 3 μm 2 at most about 100 μm 2 at most about 10 μm 2 at most about 1 μm 2 at most about 0.1 μm 2 at most about 1×10 -2μm 2 or may be less than that. The area occupied by each recess opening may be greater than, less than, or between the values shown above.

[0160] The depth of each recess 32 can be large enough to accommodate a portion of a hydrogel, such as a multi-arm polymer hydrogel 10. In one example, the depth can 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 in addition, the depth can be at most about 1×10 3 μm, at most about 100 μm, at most about 10 μm, or less. In some examples, the depth is about 0.4 μm. The depth of each recess 32 can be greater than, less than, or between the values shown above.

[0161] In some cases, the diameter or length and width of each recess 32 can be at least about 50 nm, at least about 0.1 μm, at least about 0.5 μm, at least about 1 μm, at least about 10 μm, at least about 100 μm, or more. Alternatively, or in addition, the diameter or length and width can be at most about 1×10 3 μm, at most about 100 μm, at most about 10 μm, at most about 1 μm, at most about 0.5 μm, at most about 0.1 μm, or less (e.g., about 50 nm). In some examples, the diameter or length and width are about 0.4 μm. The diameter or length and width of each recess 32 can be greater than, less than, or between the values shown above.

[0162] In the example shown in FIG. 2C, the hydrogel (e.g., multi-arm polymer hydrogel 10) is disposed within each of the recesses 32. The multi-arm polymer hydrogel 10 or any other example of a hydrogel disclosed herein may be applied as described with reference to FIG. 2B such that the hydrogel is present within the recesses 32 and not present on the surrounding gap region 34.

[0163] In the example shown in FIG. 2C, primer 26 may be grafted to the hydrogel within each of the recesses 32. Primer 26 may be applied as described with reference to FIG. 2B, and thus is grafted to the hydrogel and not to the surrounding gap region 34.

[0164] Although not shown in FIGS. 2A, 2B, or 2C, it should be understood that flow cell 20 may include a lid attached to substrate 22. In one example, the lid may be coupled to at least a portion of substrate 22, for example, in a portion of gap region 34. The bond formed between the lid and substrate 22 may be a chemical bond or a mechanical bond (e.g., using fasteners, etc.).

[0165] The lid can be any material that is transparent to the excitation light directed towards substrate 22. By way of example, the lid can be glass (e.g., borosilicate, fused silica, etc.), plastic, etc. A commercially available example of a suitable borosilicate glass is D 263® available from Schott North America, Inc. A commercially available example of a suitable plastic material, i.e., a cycloolefin polymer, is the ZEONOR® product available from Zeon Chemicals L.P.

[0166] The lid can be coupled to substrate 22 using any suitable technique such as laser bonding, diffusion bonding, anodic bonding, eutectic bonding, plasma activation bonding, glass frit bonding, or other methods known in the art. In one example, a spacer layer can be used to couple the lid to substrate 22. The spacer layer can be any material that seals together at least a portion of substrate 22 and the lid. In some examples, the spacer layer can be a radiation absorbing material that aids in the bonding of substrate 22 and the lid.

[0167] In other examples, flow cell 20 may also include an additional patterned or unpatterned substrate 22 attached to substrate 22.

[0168] Sequencing method

[0169] Examples of the flow cell 20 can be used in ensemble sequencing techniques such as sequencing by synthesis (SBS). In ensemble sequencing, the template polynucleotide strand to be sequenced (not shown) can be formed on the flow cell using the primer 26. First, in the formation of the template polynucleotide strand, a library template can be prepared from any nucleic acid sample (e.g., a DNA sample or an RNA sample). The DNA nucleic acid sample may similarly be fragmented into single-stranded DNA fragments of a determined size (e.g., less than 1000 bp). The RNA nucleic acid sample can be used to synthesize complementary DNA (cDNA), which may similarly be fragmented into single-stranded cDNA fragments of a determined size (e.g., less than 1000 bp). During preparation, adapters may be added to the ends of these fragments. By reductive cycle amplification, different motifs such as sequencing binding sites, indexes, and regions complementary to the primer 26 in the recess 32 can be introduced into the adapter. The final library template includes the DNA or cDNA fragment and the adapters at both ends. In some examples, the DNA or cDNA fragments derived from a single nucleic acid sample have the same adapter added thereto. The DNA or cDNA fragment represents a part of the final library template to be sequenced.

[0170] A plurality of library templates may be introduced into the flow cell 20. The plurality of library templates hybridize, for example, to one of the two types of primers 26 immobilized in the flow channel 24 or the recess 32.

[0171] Next, cluster generation can be performed. In an example of cluster generation, the library template is copied from the hybridized primer by 3' extension using a high-fidelity DNA polymerase. The original library template is denatured, leaving copies immobilized within flow channel 24 or recess 32. The immobilized copies can be amplified using isothermal bridge amplification or some other amplification form. For example, the copied template loops over and hybridizes to an adjacent complementary primer 26, and the polymerase copies the copied template to form a double-stranded bridge, which is denatured to form two single-stranded strands. These two strands loop over and hybridize to adjacent complementary primers 26 and extend again to form two new double-stranded loops. The process is repeated for each template copy by cycles of isothermal denaturation and amplification to create a dense clone cluster. Each cluster of double-stranded bridges is denatured. In one example, the reverse strand is removed by specific base cleavage, leaving the forward strand template polynucleotide strand. Clustering results in the formation of several template polynucleotide strands within flow channel 24 or within each recess 32. This example of clustering is bridge amplification and is an example of amplification that can be implemented. It should be understood that other amplification techniques such as the Examp workflow (Illumina Inc.) may also be used.

[0172] A sequencing primer that hybridizes to a complementary sequence on the template polynucleotide strand can be introduced. This sequencing primer prepares the template polynucleotide strand for sequencing.

[0173] To initiate sequencing, an incorporation mix may be added to flow cell 20. In one example, the incorporation mix includes a liquid carrier, a polymerase, and fluorescently labeled nucleotides. The fluorescently labeled nucleotides may include a 3’OH blocking group. When the incorporation mix is introduced into flow cell 20, the fluid enters flow channel 24 and / or recess 32 where the template polynucleotide strands are present.

[0174] The fluorescently labeled nucleotides are added to the sequencing primer in a template-dependent manner (thereby extending the sequencing primer) such that 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 each polymerase into a new strand complementary to the template polynucleotide strand by extending the sequencing primer. In other words, in at least a portion of the template polynucleotide strands throughout flow cell 20, each polymerase extends the sequencing primer hybridized by one of the nucleotides in the incorporation mix.

[0175] This nucleotide incorporation can be detected by an imaging event. During imaging, an illumination system (not shown) may provide excitation light to flow channel 24 and / or recess 32.

[0176] In some examples, the nucleotide can further include a reversible termination property (e.g., a 3’OH blocking group) that terminates further primer extension when the nucleotide is added to a sequencing primer. For example, a nucleotide analog having a reversible terminator moiety can be added to the sequencing primer to prevent subsequent extension until a deblocking agent is delivered and that moiety is removed. Thus, in examples using reversible termination, after detection occurs, a deblocking reagent can be delivered to flow cell 20.

[0177] Washing may be performed between various fluid delivery steps. The SBS cycle can then be repeated n times to extend the sequencing primer by only n nucleotides, thereby detecting an array of length n.

[0178] Although SBS has been described in detail, it should be understood that flow cell 20 described herein can be utilized with other sequencing protocols for genotyping or other chemical and / or biological applications. In some cases, the primers of the flow cell may be selected to enable simultaneous paired-end sequencing where both the forward and reverse strands are present on the hydrogel, allowing for simultaneous base calling of each read. Sequential and simultaneous paired-end sequencing can facilitate genome rearrangement and repetitive element detection, as well as gene fusion and novel transcript detection. In another example, flow cell 10 disclosed herein can be used for on-cell library generation.

[0179] To further illustrate the disclosure, examples are provided herein. It should be understood that these examples are provided for illustrative purposes and are not to be construed as limiting the scope of the disclosure. Non-limiting examples

[0180] Example 1

[0181] Examples of multi-arm polymer hydrogels were prepared using RAFT polymerization according to the scheme shown in Figure 1.

[0182] The comparative example polymer hydrogel (poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide)) was prepared by copolymerizing acrylamide and N-(5-azidoacetamidopentyl)acrylamide using free radical synthesis.

[0183] The dispersities of the multi-arm polymer hydrogels of the examples and the polymer hydrogels of the comparative examples were calculated. The results are shown in Figure 3. The median of the polymer hydrogels of the examples was about 1.9, and the median of the polymer hydrogels of the comparative examples was about 3.3. As shown, the dispersity of the multi-arm polymer hydrogels of the examples was much lower than that of the polymer hydrogels of the comparative examples. Therefore, the multi-arm polymer hydrogels of the examples had a narrower molecular weight distribution than the polymer hydrogels of the comparative examples.

[0184] Example 2

[0185] The multi-arm polymer hydrogels of the examples and the polymer hydrogels of the comparative examples of Example 1 were coated in flow channels on their respective glass (specifically, fused silica) slides, and 0.1 μM to 50 μM oligonucleotide primers were grafted onto each of the polymer layers. The flow cells were stored at 60 °C for 20 days.

[0186] After storage, 300 sequencing cycles were performed in each of the channels using the PhiX library.

[0187] The collected sequencing data included phasing (percentage, shown in A of Figure 4), quality score (percentage exceeding Q30, shown in B of Figure 4), and error rate (percentage) (shown in C of Figure 4). Phasing is the rate at which single molecules within a cluster become out of sync with each other. Thus, a lower phasing percentage is more desirable. Q30 is equivalent to a probability of an incorrect base call of 1 in 1000. This means that the base call accuracy (i.e., the probability of a correct base call) is 99.9%. A low base call accuracy of 99% (Q20) means that the probability of an incorrect base call is 1 in 100, which means that for every 100 base pair sequencing reads, there is a high likelihood of containing 1 error. When the sequencing quality reaches Q30, substantially all of the reads are complete and have zero errors and ambiguities. As shown in A, B, and C of Figure 4, the multi-arm polymer hydrogels of the examples functioned better than the comparative examples with respect to phasing, Q30, and error rate. The phasing results of the multi-arm polymer hydrogels of the examples remained below 0.19% even when stored for longer periods such as 14 days and 20 days. In contrast, the phasing results of the multi-arm polymer hydrogels of the comparative examples increased to about 0.26% at 14 days and to about 0.39% at 20 days. The Q30 results of the multi-arm polymer hydrogels of the examples remained above 85% even when stored for longer periods. In contrast, the Q30 results of the multi-arm polymer hydrogels of the comparative examples decreased to about 70% at 14 days and to nearly 40% at 20 days. The error rate results of the multi-arm polymer hydrogels of the examples remained below 2% even when stored for longer periods. In contrast, the error rate results of the multi-arm polymer hydrogels of the comparative examples decreased to about 2.5% at 14 days and to nearly 14% at 20 days. All of these results indicate that the multi-arm polymer hydrogels of the examples are more resistant to irreversible changes as a result of dry staging than the polymer hydrogels of the comparative examples.

[0188] Furthermore, the multi-arm polymer hydrogel structure can also minimize the interaction between the multi-arm polymer hydrogel and DNA during clustering and / or sequencing, which can contribute to improved sequencing performance / metrics.

[0189] Example 3

[0190] The multi-arm polymer hydrogel of the example of Example 1 and the polymer hydrogel of the comparative example were each coated in the recesses of four glass (specifically, fused silica) flow channels (lanes) of two differently patterned flow cells, and 0.1 μM to 50 μM oligonucleotide primers were grafted onto the polymers in the recesses.

[0191] Over 300 sequencing cycles were performed in each of the flow channels using the PhiX library.

[0192] The collected sequencing data included a quality score (percentage exceeding Q30, shown in Figure 5A) and prephasing (percentage, shown in Figure 5B). As shown in Figure 5A, the quality index decreased more slowly for the multi-arm polymer hydrogels of the examples than for the polymer hydrogels of the comparative examples. In particular, for a large number of cycles, a better sequencing run was obtained. The Q30 results of the multi-arm polymer hydrogels of the examples remained above 55% for all cycles and above 85% over approximately 200 cycles. In contrast, the Q30 results of the multi-arm polymer hydrogels of the comparative examples decreased below 80% at approximately 175 cycles and then dropped below 55% at approximately 240 cycles. As shown in Figure 5B, prephasing was significantly reduced for the multi-arm polymer hydrogels of the examples compared to the polymer hydrogels of the comparative examples, resulting in a better sequencing run. The average prephasing result of the multi-arm polymer hydrogels of the examples across four lanes was approximately 0.11%, while the average prephasing result of the multi-arm polymer hydrogels of the comparative examples across four lanes was approximately 0.17%.

[0193] Example 4

[0194] The multi-arm polymer hydrogels of the examples and the polymer hydrogels of the comparative examples of Example 1 were each coated in the recesses of the resin layer of the multilayer substrate, and 0.1 μM to 50 μM of oligonucleotide primers were grafted onto each of the polymer layers.

[0195] Using a human library with a 1% PhiX library, 151 sequencing cycles were performed between Read 1 (R1) and Read 2 (R2) in each of the flow channels.

[0196] The collected sequencing data included an error rate (percentage, shown in Figure 6A for R1 and Figure 6B for R2). As shown in Figures 6A and 6B, the error rate of the multi-arm polymer hydrogel of the examples was significantly reduced compared to the polymer hydrogel of the comparative example between each lead. The average error rate of the flow cell with the multi-arm polymer hydrogel of the examples was 0.65, while the average error rate of the flow cell of the comparative example with the polymer hydrogel of the comparative example was 0.93.

[0197] Example 5

[0198] The multi-arm polymer hydrogel of the example of Example 1 and the polymer hydrogel of the comparative example were respectively coated on the recesses of four glass (specifically, fused silica) flow channels (lanes) of two different patterned flow cells, and oligonucleotide primers of 0.1 μM to 50 μM were grafted onto each of the polymer layers.

[0199] Using a human library with a 1% PhiX library, 151 sequencing cycles were performed in each of the flow channels.

[0200] The collected sequencing data included the intensity of the first cycle (C1), passing filter (PF%) (percentage), fading (%), pre-fading (%), Q30, and error rate. The passing filter (PF) is an indicator used to explain the clusters passing the pure threshold value and is used for further processing and analysis of the sequencing data. Higher passing filter % results indicate an increase in the yield of the unique clusters used in the sequencing data. Reproducible data was observed across the lanes of the flow cell. The sequencing data of one of each lane of the flow cell is shown in Table 1.

Table 1

[0201] As shown in Table 1, the sequencing results of the multi-arm polymer hydrogels of the examples were better than those of the polymer hydrogels of the comparative examples (e.g., PF%, Q30, error rate), or comparable to those of the polymer hydrogels of the comparative examples (e.g., C1 intensity, fading, and pre-fading). The average C1 intensity of all lanes is shown in FIG. 7A. The average C1 intensity of the polymer hydrogels of the examples was about 275, while the average C1 intensity of the polymer hydrogels of the comparative examples was about 250. These results indicate that the intensity of the polymer hydrogels of the examples was as good as or even better than that of the polymer hydrogels of the comparative examples. The average error rate of all lanes is shown in FIG. 7B. The average error rate of the polymer hydrogels of the examples was about 1.5 times lower than the average error rate of the polymer hydrogels of the comparative examples. Therefore, across all lanes, the multi-arm polymer hydrogels of the examples had better performance with respect to C1 intensity and error rate than the polymer hydrogels of the comparative examples.

[0202] Example 6

[0203] The multi-arm polymer hydrogels of the examples of Example 1 and the polymer hydrogels of the comparative examples were also used in this example. Each of the hydrogels was coated in the recesses of the four glass (specifically, fused silica) flow channels (lanes) of two patterned flow cells, respectively, and 0.1 μM to 50 μM oligonucleotide primers were grafted onto each of the polymer layers.

[0204] Using a human library with a 1% PhiX library, multiple sequencing cycles were performed in each of the flow channels.

[0205] For one lane of the flow cell of the example and one lane of the flow cell of the comparative example, duplicate read data was collected over the sequencing cycle. A sequencing read can be determined to be a duplicate if both the forward and reverse reads have the same starting position. It is desirable that the percentage of duplicates be lower. The duplicate read results are shown in FIG. 8A. As shown, the flow cell of the example containing the multi-arm polymer hydrogel of the example showed fewer duplicate reads for the sequencing run. Specifically, the percentage of duplicate reads for the flow cell of the example was in the range of about 2.5% to about 12%. In contrast, the flow cell of the comparative example had a much higher percentage of duplicate reads in the range of about 10% to about 24%.

[0206] Also, for one lane of the flow cell of the example and one lane of the flow cell of the comparative example, pad hopping data was collected over the sequencing cycle. Pad hopping refers to the process by which some adjacent recesses are amplified from the same template array due to the template "hopping" into adjacent recesses during cluster generation. It is desirable that the percentage of pad hopping be lower. The pad hopping results are shown in FIG. 8B. As shown, the flow cell of the example containing the multi-arm polymer hydrogel of the example showed little or no pad hopping (e.g., less than about 1%) for the sequencing run. In contrast, the comparative flow cell containing the polymer hydrogel of the comparative example exhibited much higher pad hopping in the range of about 1% to about 27%.

[0207] The results of all examples show that the multi-arm polymer hydrogel can be used in various different flow cell structures, can improve sequencing assays, and can also improve dry storage stability (e.g., the sequencing performance is not adversely affected even after a period of dry storage).

[0208] Addendum

[0209] It is to be understood that all combinations of the foregoing concepts and additional concepts, to be discussed in more detail below, are intended, subject to such concepts not being mutually inconsistent, to be part of the subject matter of the invention disclosed herein. Specifically, all combinations of the claimed subject matter appearing at the end of this disclosure are intended to be part of the subject matter of the invention disclosed herein. It should also be understood that any terms used explicitly herein and that may also appear in any disclosure incorporated by reference should be given a meaning that most closely matches the particular concepts disclosed herein.

[0210] References throughout this specification to "one example," "another example," "an example," etc., mean that a particular element (e.g., feature, structure, and / or characteristic) described in connection with the example is included in at least one example described herein, and may or may not be present in other examples. Further, it is to be understood that elements described in connection with any example can be combined in any suitable manner in various examples, unless the context clearly dictates otherwise.

[0211] The ranges provided herein are to be understood to include the recited range and any value or sub-range within the recited range as if such value or sub-range were expressly recited. For example, a range of about 200 mm to about 300 mm is to be interpreted to include not only the explicitly recited limits of about 200 mm to about 300 mm, but also individual values such as about 240 mm, about 250.5 mm, etc., and sub-ranges such as about 225 mm to about 275 mm. Further, when "about" and / or "substantially" are used to describe a value, they are meant to encompass a slight variation (up to ±10%) from the recited value.

[0212] Although several embodiments have been described in detail, it is to be understood that the disclosed examples may be modified. Accordingly, the foregoing description is to be considered non-limiting.

Claims

1. A dendritic core having 2 to 30 arms, and a first acrylamide monomer incorporated into each arm of the dendritic core, of formula (1): 【Chemical Formula 1】 [In the above formula (1), R １ and R ２ are each independently selected from the group consisting of alkyl, alkylamino, alkylamide, alkylthio, aryl, glycol, and any substituents thereof] a first acrylamide monomer having a structure represented by a second acrylamide monomer incorporated into each arm of the dendritic core, of formula (2): [Chemical 2] [In the above formula (2), R ３ is hydrogen or alkyl, R ４ is hydrogen or alkyl, L is a linker comprising a linear chain of 2 to 20 atoms, each atom independently selected from the group consisting of carbon, oxygen, and nitrogen, and optional substituents on any carbon atom and any nitrogen atom within the chain, A is an N-substituted amide having a structure represented by formula (3): 【Chemical Formula 3】 {In the formula (3), R ５ is hydrogen or alkyl} E is a linear chain of 1 to 4 atoms, each atom independently selected from the group consisting of carbon, oxygen, and nitrogen, and optional substituents on any carbon atom and any nitrogen atom within the chain, Z is an optional nitrogen-containing heterocyclic ring] a second acrylamide monomer having a structure represented by A hydrogel comprising.

2. The hydrogel according to claim 1, wherein the first acrylamide monomer is N,N-dimethylacrylamide.

3. The hydrogel according to claim 1 or 2, wherein the dendritic core contains a thiocarbonylthio group in each arm.

4. The hydrogel according to claim 3, wherein the thiocarbonylthio group is selected from the group consisting of dithiobenzoate, trithiocarbonate, and dithiocarbamate.

5. The hydrogel according to claim 3, wherein the dendritic core is selected from the group consisting of 3,5-bis(2-dodecylthiocarbonothioylthio-1-oxopropoxy)benzoic acid, 1,1,1-tris[(dodecylthiocarbonothioylthio)-2-methylpropionate]ethane, and pentaerythritol tetrakis[2-(dodecylthiocarbonothioylthio)-2-methylpropionate].

6. The hydrogel according to any one of claims 1 to 5, wherein the dendritic core contains an atom transfer radical polymerization initiator in each arm.

7. The hydrogel according to claim 6, wherein the dendritic core is selected from the group consisting of bis[2-(2'-bromoisobutyryloxy)ethyl]disulfide, 2-bromoisobutyric anhydride, ethylene bis(2-bromoisobutyrate), pentaerythritol tetrakis(2-bromoisobutyrate), dipentaerythritol hexakis(2-bromoisobutyrate), and 1,1,1-tris(2-bromoisobutyryloxymethyl)ethane.

8. The dendritic core is a polyfunctional central molecule and a plurality of atom transfer radical polymerization monofunctional initiators bonded to the polyfunctional central molecule, and the hydrogel according to any one of claims 1 to 7.

9. The hydrogel according to claim 8, wherein the atom transfer radical polymerization monofunctional initiator is selected from the group consisting of 2-azidoethyl 2-bromoisobutyrate, poly(ethylene glycol) methyl ether 2-bromoisobutyrate, 2-(2-bromoisobutyryloxy)ethyl methacrylate, dodecyl 2-bromoisobutyrate, 2-hydroxyethyl 2-bromoisobutyrate, 1-(phthalimidomethyl)2-bromoisobutyrate, and propargyl 2-bromoisobutyrate.

10. The hydrogel according to any one of claims 1 to 9, wherein the dendritic core contains a nitroxide-mediated polymerization initiator in each arm.

11. The hydrogel according to claim 10, wherein the dendritic core is selected from the group consisting of 1,3,5-tris((4-(1-((2,2,6,6-tetramethylpiperidin-1-yl)oxy)ethyl)benzyl)oxy)benzene and 1,3,5-tris((3,5-bis((4-(1-((2,2,6,6-tetramethylpiperidin-1-yl)oxy)ethyl)benzyl)oxy)benzyl)oxy)benzene.

12. The dendritic core is a polyfunctional central molecule and a plurality of nitroxide-mediated polymerization monofunctional initiators bonded to the polyfunctional central molecule, and the hydrogel according to any one of claims 1 to 11.

13. Each of the plurality of nitroxide-mediated polymerization monofunctional initiators 【Chemical Formula 4】 has a structure selected from the group consisting of wherein I is [Chemical Formula 5] selected from the group consisting of The hydrogel according to claim 12. The hydrogel according to any one of claims 1 to 13, wherein the first acrylamide monomer and the second acrylamide monomer form a block copolymer, a random copolymer, a statistical copolymer, or an alternating copolymer on each arm of the dendritic core.

15. The hydrogel according to any one of claims 1 to 14, wherein the second acrylamide monomer is azidoacetamidopentyl acrylamide.

16. Structure (10): [Chemical Formula 6] Wherein, [Chemical Formula 7] [wherein, m and n are independently integers from 1 to 2500] Or structure: 【Chemical 8】 Wherein, 【Chemical Formula 9】 [wherein, m and n are independently integers from 1 to 2500] Or structure: 【Chemical 10】 Wherein, 【Chemical 11】 [wherein, m and n are independently integers from 1 to 2500] The hydrogel according to any one of claims 1 to 15, having

17. The hydrogel according to any one of claims 1 to 16, having a dispersity of less than 2.

5.

18. The hydrogel according to any one of claims 1 to 15, wherein the dendritic core has a polyfunctional central molecule selected from the group consisting of a phenyl group, benzoic acid, pentaerythritol, and a phosphazene group.

19. The hydrogel according to any one of claims 1 to 15, wherein the dendritic core has two arms, three arms, four arms, six arms, or eight arms.

20. A flow cell comprising a substrate and the hydrogel according to any one of claims 1 to 15 on the substrate.

21. The flow cell according to claim 20, wherein the substrate includes a plurality of recesses separated by a gap region, and the hydrogel is disposed inside each of the recesses.

22. The flow cell according to claim 20 or 21, further comprising an amplification primer grafted to the polymer hydrogel.

23. The flow cell according to any one of claims 20 to 22, wherein the substrate includes a channel, and the hydrogel is disposed in the channel.

24.

25. The first acrylamide monomer and the second acrylamide monomer form a random copolymer on each arm of the dendritic core, or The first acrylamide monomer and the second acrylamide monomer form a statistical copolymer on each arm of the dendritic core, or The first acrylamide monomer and the second acrylamide monomer form an alternating copolymer on each arm of the dendritic core, or The first acrylamide monomer and the second acrylamide monomer form a block copolymer on each arm of the dendritic core, the flow cell according to any one of claims 20 to 23.

25. A method comprising incorporating a copolymer into each arm of a multi-arm dendritic core component having 2 to 30 arms, the copolymer comprising a first acrylamide monomer and a second acrylamide monomer, The first acrylamide monomer has the formula (4): 【Chemical Formula 12】 [In the formula (4), R １ and R ２ are each independently selected from the group consisting of alkyl, alkylamino, alkylamide, alkylthio, aryl, glycol, and any substituents thereof] has a structure represented by The second acrylamide monomer has the formula (5): 【Chemical 13】 [In the formula (5), R ３ is hydrogen or alkyl, R ４ is hydrogen or alkyl, and L is a linker comprising a linear chain of 2 to 20 atoms, each atom independently selected from the group consisting of carbon, oxygen, and nitrogen, and optional substituents on any carbon atom and any nitrogen atom in the chain, A is an N-substituted amide having a structure represented by formula (6): 【Chemical 14】 {In the formula (6), R ５ is hydrogen or alkyl} E is a linear chain of 1 to 4 atoms, each atom independently selected from the group consisting of carbon, oxygen, and nitrogen, and optional substituents on any carbon atom and any nitrogen atom in the chain, Z is an optional nitrogen-containing heterocyclic ring]] A method having a structure represented by.

26. The incorporating comprises polymerizing a mixture of the first acrylamide monomer and the second acrylamide monomer in the presence of the multi-arm component, the method according to claim 25.

27. The incorporating is i) polymerizing a first block with the first acrylamide monomer in the presence of the multi-arm component to form a modified multi-arm component, then polymerizing a second block with the second acrylamide monomer in the presence of the modified multi-arm component, or ii) polymerizing a first block with the second acrylamide monomer in the presence of the multi-arm component to form a modified multi-arm component, then polymerizing a second block with the first acrylamide monomer in the presence of the modified multi-arm component, The method according to claim 25 or 26, comprising forming a block copolymer in the presence of the multi-arm component.

28. The method according to any one of claims 25 to 27, wherein the incorporating comprises reversible addition-fragmentation chain transfer polymerization or atom transfer radical polymerization or nitroxide-mediated polymerization.

29. The multi-arm component comprises a multifunctional central molecule, and each of its arms comprises an initiator selected from the group consisting of a thiocarbonylthio group, or an atom transfer radical polymerization (ATR) initiator and a nitroxide-mediated polymerization monofunctional initiator. The method according to any one of claims 26 to 28.

30. The second acrylamide monomer comprises an azide group, and the method comprises binding the multi-arm polymer hydrogel to the surface of the substrate by reaction of the azide groups of the formed multi-arm polymer hydrogel, binding a primer to the arms of the multi-arm polymer hydrogel by reaction of the azide groups of the formed multi-arm polymer hydrogel, and crosslinking the formed multi-arm polymer hydrogel (10) by reaction of the azide groups of the formed multi-arm polymer hydrogel. The method according to any one of claims 26 to 29, further comprising at least one of the above.

31. A medical device coated with the hydrogel according to any one of claims 1 to 15.

32. A wound dressing material coated with the hydrogel according to any one of claims 1 to 15.

33. A substrate coated with the hydrogel according to any one of claims 1 to 15 for use in sequencing analysis.

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