Flow cell with a hydrogel coating

Abstract

In an example of the method, a functionalized coating is applied in a recess of a patterned flow cell substrate. The recesses are separated by spacer regions. Primers are grafted onto the functionalized coating to form a grafted functionalized coating in the recess. A hydrogel is applied over at least the grafted functionalized coating.

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of December 17, 2018, an application number of 201880044290.6, and an invention title of "Flow Cell with Hydrogel Coating".

[0002] Cross - Reference to Related Applications

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 609,105, filed December 21, 2017, the contents of which are hereby incorporated by reference in their entirety. Background Art

[0004] Bioarrays are one of the broad tools for detecting and analyzing molecules, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In these applications, the arrays are engineered to include probes for nucleotide sequences present in the genes of humans and other organisms. In certain applications, for example, individual DNA and RNA probes can be attached at positions in a geometric grid (or randomly) on an array support. A test sample, such as from a human or an organism, can be exposed to the grid such that complementary fragments hybridize to the probes at individual sites in the array. The array can then be examined by scanning light at a specific frequency at the sites to identify which fragments are present in the sample by fluorescence at the sites of fragment hybridization.

[0005] Bioarrays can be used for gene sequencing. Generally, gene sequencing involves determining the order of nucleotides or nucleic acids in a length of genetic material, such as a DNA or RNA fragment. Longer and longer base pair sequences are being analyzed, and the resulting sequence information can be used in various bioinformatics methods to logically assemble the fragments together to reliably determine the sequence of a broad length of the genetic material from which the fragments are derived. Automated, computer-based detection methods for characteristic fragments have been developed and are used for genomic mapping, identification of genes and their functions, risk assessment of certain disorders and disease states, etc. In addition to these applications, bioarrays can also be used to detect and evaluate a wide range of molecules, molecular families, genetic expression levels, single nucleotide polymorphisms, and genotyping. Summary of the Invention

[0006] In a first aspect, a method includes applying a functionalized coating in a recess of a patterned flow cell substrate, where the recess is separated by spacer gaps; grafting primers onto the functionalized coating to form a grafted functionalized coating in the recess; and applying a hydrogel onto the grafted functionalized coating.

[0007] In an instance of this first aspect of the method, the hydrogel is selected from poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide), crosslinked polyacrylamide, agarose gel, and crosslinked polyethylene glycol.

[0008] In an example of this first aspect of the method, a hydrogel is disposed on the grafted functionalized coating. In one example, the hydrogel is applied to the functionalized coating in the recesses and on at least some of the interstitial regions. In another example, applying the hydrogel comprises selectively disposing the hydrogel on the grafted functionalized coating in the recesses.

[0009] In an example of this first aspect of the method, prior to applying the functionalized coating, the method further comprises treating the surface of the patterned flow cell substrate to attach functional groups to the surface, thereby forming treated recesses and treated interstitial regions. In this example, applying the functionalized coating in the recesses comprises: applying the functionalized coating in the treated recesses and on the treated interstitial regions; and polishing the functionalized coating from the treated interstitial layer.

[0010] In an example of this first aspect of the method, applying the hydrogel comprises applying an aqueous mixture comprising from about 0.001% to up to about 0.1% (mass / volume) of a hydrogel material. In one example, the hydrogel material is selected from poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide), crosslinked polyacrylamide, agarose gel, and crosslinked polyethylene glycol.

[0011] In an example of this first aspect of the method, the perimeter of the patterned flow cell substrate has a spacer layer bonded thereto, and after applying the hydrogel, the method further comprises bonding a lid to the spacer layer.

[0012] In an example of this first aspect of the method, after applying the functionalized coating and prior to grafting the primer, the method comprises bonding a lid to at least some of the interstitial regions.

[0013] In an example of this first aspect of the method, applying the hydrogel comprises selectively depositing the hydrogel on the grafted functionalized coating. It should be understood that any features of this first aspect of the method may be combined together in any desired manner and / or configuration.

[0014] In a second aspect, a method comprises attaching a silane or silane derivative to the surface of a patterned substrate, the substrate comprising a flow-through channel having recesses defined therein, wherein the recesses are separated by interstitial regions, thereby forming silanized recesses and silanized interstitial regions; applying a functionalized coating in the silanized recesses and on the silanized interstitial regions; polishing the functionalized coating from the silanized interstitial regions; grafting a primer to the functionalized coating in the silanized recesses to form a grafted functionalized coating in the recesses; and applying a hydrogel to the grafted functionalized coating in the recesses.

[0015] In an example of this second aspect, applying the hydrogel comprises applying an aqueous mixture comprising from about 0.001% to up to about 0.1% (mass / volume) of a hydrogel material. In this example, the hydrogel material is selected from poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide), crosslinked polyacrylamide, agarose gel, and crosslinked polyethylene glycol.

[0016] In an example of this second aspect, the spacer layer is bonded to the patterned substrate and defines the perimeter of the flow channel; and after applying the hydrogel, the method further comprises bonding a lid to the spacer layer.

[0017] In an example of this second aspect, after polishing the functionalized coating and prior to grafting primers, the method further comprises bonding a lid to at least some of the gap regions.

[0018] In an example of this second aspect of the method, applying the hydrogel comprises applying the hydrogel onto the grafted functionalized coating in the recess. In one example, the hydrogel is applied onto the functionalized coating in the recess and onto at least some of the gap regions. In another example, applying the hydrogel comprises selectively depositing the hydrogel onto the grafted functionalized coating.

[0019] It should be understood that any features of this second aspect of the method can be combined together in any desired manner. Additionally, it should be understood that any combination of features of this aspect of the method and / or features of the first aspect of the method can be used together, and / or any feature from either or both of these aspects can be combined with any example disclosed herein.

[0020] In another aspect, a flow cell comprises a patterned substrate comprising recesses separated by gap regions; sequencing surface chemistry attached to each recess, the sequencing surface chemistry comprising: a functionalized coating, and primers grafted to the functionalized coating; and a hydrogel on the sequencing surface chemistry and optionally on some of the gap regions.

[0021] In an example of the flow cell, the hydrogel is also on at least some of the gap regions.

[0022] In an example of the flow cell, the functionalized coating is poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide).

[0023] In an example of the flow cell, the hydrogel is selected from poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide), crosslinked polyacrylamide, agarose gel, and crosslinked polyethylene glycol.

[0024] In an example of the flow cell, the hydrogel is not grafted to the surface chemistry.

[0025] In an example of the flow cell, the patterned substrate includes at least one flow channel; a recess is defined in at least one flow channel; and the flow cell further includes a spacer layer attached to other interstitial regions of the patterned substrate such that the spacer layer defines the perimeter of at least one flow channel. In this example, the flow cell may further include a lid attached to the spacer layer.

[0026] It should be understood that any features of this aspect of the flow cell can be combined together in any desired manner. Additionally, it should be understood that any combination of features of this aspect of the flow cell and / or the first and / or second aspects of the method can be used together, and / or any feature from any one aspect can be combined with any one example disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The features of the examples of the present disclosure will become apparent by reference to the following detailed description and the drawings, in which like reference numerals correspond to like but possibly different components. For the sake of brevity, reference numerals or features having a previously described function may be described in combination with or without the other drawings in which they appear.

[0028] Figure 1 is a flowchart illustrating an example of the method disclosed herein;

[0029] Figure 2 is a flowchart illustrating another example of the method disclosed herein;

[0030] Figures 3A to 3G and Figures 3A to 3D 3H and 3I are schematic cross-sectional views depicting corresponding examples of the method disclosed herein;

[0031] Figure 4 is by Figures 3A to 3G and Figures 3A to 3D 3H and 3I are cross-sectional views of an example flow cell formed by the methods shown;

[0032] Figure 5 is a graph showing the percentage of clusters passing through the filter (%PF) and the percentage of depressions / holes occupied by DNA templates (% occupied) for the tiles (1 - 384 on the X-axis) of a comparative flow cell without a hydrogel coating and the tiles (385 - 768 on the X-axis) of an example flow cell including a hydrogel coating;

[0033] Figure 6 is a graph of the percentage of clusters passing through the filter (%PF) relative to the template concentration (pM) for the comparative example flow cell and the example flow cell including a hydrogel coating;

[0034] Figure 7 Graph of the percentage of clusters passing through the filter (%PF) relative to the template concentration (pM) after removal of the replication template for the comparative example flow cell and the example flow cell including the hydrogel coating; and

[0035] Figure 8A and 8B Graph of the mismatch rate of Read 1 (R1) ( Figure 8A ) and Read 2 (R2) ( Figure 8B ) after 150 sequencing cycles for the comparative example flow cell and the example flow cell including the hydrogel coating. DETAILED DESCRIPTION

[0036] Flow cells are commonly used in sequencing operations, analysis, and other biological applications. A patterned flow cell can include a substrate or carrier having depressions defined therein or thereon; and chemical and / or bioactive surface chemistries can be confined within the depressions. For example, the surface chemistries include functionalized coatings and primers. In some sequencing operations, after a primer is immobilized in a depression of a flow cell substrate, a sequencing template (including a portion complementary to the primer) can be introduced into the depression, and the sequencing template can subsequently be amplified to produce identical copies of the sequencing template (a process referred to herein as cluster generation).

[0037] In the examples disclosed herein, a hydrogel (also referred to herein as a hydrogel coating) is directly included on the surface chemistry, i.e., on the functionalized coating and the primer. It has been found that the hydrogel coating can slow down the sequencing template seeding rate during cluster generation. As a result, after one sequencing template is seeded into a depression, there is more time (compared to when no hydrogel is included) to amplify that template into a larger cluster before any subsequent sequencing template has a chance to diffuse through the hydrogel and enter the depression. This increases the number of depressions seeded with a single sequencing template. In other words, this increases monoclonal clustering (i.e., formation of multiple copies of one type of sequencing template) within a particular depression and reduces polyclonal clustering (i.e., formation of multiple copies of multiple types of sequencing templates) within a particular depression. The number of clusters passing through the filter after removal of duplicates can indicate the increased monoclonal clusters. In one example, the net PF% range for the examples including the hydrogel coating disclosed herein is about 2% to about 17% higher than the net PF% of the comparative examples that do not include the hydrogel coating.

[0038] The methods disclosed herein can be carried out entirely at the wafer level, entirely at the die level, partially at the wafer level, and / or partially at the die level. As an example of carrying out the method partially at the wafer and die levels, a wafer can be used to initiate the method and then diced into small pieces to form several dies, and the method can continue using each die. At least in some instances, the ability to perform open wafer processing enables various metrology / analysis techniques to be used for quality control and characterization. Before being combined to form a flow cell, the patterned and surface-modified wafer / substrate can be exposed to, for example, atomic force microscopy (AFM), scanning electron microscopy (SEM), ellipsometry, goniometry, scattering, and / or fluorescence techniques. Alternatively, the combined flow cell can be exposed to these techniques. At the die level, the method can be carried out on an open die or on an assembled flow cell (with a closed flow channel).

[0039] It should be understood that unless otherwise specified, the terms used herein have their ordinary meanings in the relevant art. Several terms used herein and their meanings are listed below.

[0040] The singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise.

[0041] The terms "comprising", "including", "containing", and various forms of these terms are synonymous with each other and mean equally broad.

[0042] The terms "top", "bottom", "lower", "higher", "upper", etc. are used herein to describe the flow cell and / or the various components of the flow cell. It should be understood that these directional terms do not imply a particular orientation, but are used to specify the relative orientation between components. The use of directional terms should not be construed as limiting the examples disclosed herein to any particular orientation.

[0043] As used herein, "alkyl" refers to a fully saturated straight-chain or branched hydrocarbon chain (i.e., containing no double or triple bonds). An alkyl group can have 1 to 20 carbon atoms. Example alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, etc. For example, the designation "C1-4 alkyl" means that one to four carbon atoms are present in the alkyl chain, i.e., the alkyl chain is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0044] As used herein, "alkenyl" refers to a straight-chain or branched hydrocarbon chain containing one or more double bonds. An alkenyl group can have 2 to 20 carbon atoms. Example alkenyl groups include vinyl, propenyl, butenyl, pentenyl, hexenyl, etc.

[0045] As used herein, "alkyne" or "alkynyl" refers to a straight-chain or branched hydrocarbon chain containing one or more triple bonds. The alkynyl group can have from 2 to 20 carbon atoms.

[0046] As used herein, "aryl" refers to an aromatic ring or ring system containing only carbon in the ring backbone (i.e., two or more fused rings sharing two adjacent carbon rings). When the aryl is a ring system, each ring in the system is aromatic. The aryl group can have from 6 to 18 carbon atoms. Examples of aryl groups include phenyl, naphthyl, azulenyl, and anthracenyl.

[0047] As used herein, the term "attached" refers to the state in which two objects are joined, fixed, adhered, connected, or bonded to each other. The attachment can be mechanical or can be chemical. For example, a nucleic acid can be chemically attached to a functionalized coating by covalent or non-covalent bonds. Covalent bonds are characterized by the sharing of electron pairs between atoms. Non-covalent bonds are physical bonds that do 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.

[0048] The "azide" or "azido" functional group refers to -N3.

[0049] As used herein, "bonding zone" refers to the area on a substrate that bonds to another material, which can be, for example, a spacer layer, a lid, another substrate, etc., or a combination thereof (e.g., a spacer layer and a lid). The bond formed in the bonding zone can be a chemical bond (as described above) or a mechanical bond (e.g., using fasteners, etc.).

[0050] As used herein, "carbocyclyl" represents a non-aromatic ring or ring system containing only carbon atoms in the ring system backbone. When the carbocyclyl is a ring system, two or more rings can be joined together in a fused, bridged, or spiro manner. The carbocyclyl can have any degree of saturation as long as at least one ring in the ring system is non-aromatic. Thus, carbocyclyl includes cycloalkyl, cycloalkenyl, and cycloalkynyl. The carbocyclyl group can have from 3 to 20 carbon atoms. Examples of carbocyclyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicyclo[2.2.2]octyl, adamantyl, and spiro[4.4]naphthyl.

[0051] As used herein, the term "carboxylic acid" or as used herein "carboxyl" refers to -C(O)OH.

[0052] As used herein, the term "subcycloalkyl" represents a fully saturated carbocyclyl ring or ring system attached to the remainder of the molecule through two attachment points.

[0053] As used herein, "cycloalkenyl" or "cycloalkene" refers to a carbocyclic group or ring system having at least one double bond, wherein no ring in the ring system is aromatic. Examples include cyclohexenyl or cyclohexene and norbornenyl or norbornene. Also as used herein, "heterocycloalkenyl" or "heterocycloalkene" refers to a carbocyclic ring or ring system having at least one double bond and having at least one heteroatom in the ring backbone, wherein no ring in the ring system is aromatic.

[0054] As used herein, "cycloalkynyl" or "cycloalkyne" refers to a carbocyclic ring or ring system having at least one triple bond, wherein no ring in the ring system is aromatic. An example is cyclooctyne. Another example is bicyclooctyne. Also as used herein, "heterocycloalkynyl" or "heterocycloalkyne" refers to a carbocyclic ring or ring system having at least one triple bond and having at least one heteroatom in the ring backbone, wherein no ring in the ring system is aromatic.

[0055] As used herein, the term "deposition" refers to any suitable application technique (which may be manual or automated) and results in a change in surface properties. Generally, 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, blade coating, puddle dispensing, cast coating, aerosol printing, inkjet printing, etc.

[0056] As used herein, the term "recess" refers to a discrete recessed feature in a patterned substrate that has a surface opening completely surrounded by a gap region of the surface of the patterned substrate. The recess can have any of a variety of shapes at its opening in the surface, including, for example, circular, elliptical, square, polygonal, star-shaped (having any number of apexes), etc. The cross-section of the recess taken orthogonal to the surface can be curved, square, polygonal, hyperbolic, conical, angular, etc. For example, the recess can be a hole.

[0057] The term "each", when used in connection with a collection of items, is intended to identify individual items in the collection, but does not necessarily refer to every item in the collection. Exceptions may occur if there is an express disclosure or context that clearly dictates otherwise.

[0058] As used herein, the term "flow cell" is intended to denote a container having a chamber (i.e., a flow channel) in which a reaction can occur, an inlet for delivering reagents to the chamber, and an outlet for removing reagents from the chamber. In some examples, the chamber enables detection of the reaction occurring in the chamber. For example, the chamber can include one or more transparent surfaces that allow optical detection of an array, optically labeled molecules, etc. in the chamber.

[0059] As used herein, a "flow channel" can be a region defined between two bonded components that can selectively receive a liquid sample. In some instances, the flow channel can be defined between a patterned substrate and a lid and can thus be in fluid communication with one or more recesses defined in the patterned substrate.

[0060] As used herein, the term "functionalized coating" is intended to denote a liquid- and gas-permeable semi-rigid material. The functionalized coating can be a hydrogel that can swell upon absorption of a liquid and contract upon removal of the liquid by drying. In the examples disclosed herein, the functionalized coating includes azide / azido functional groups that can react with alkyne functional groups. In one example, the functionalized coating is poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide) (PAZAM).

[0061] As used herein, "heteroaryl" refers to an aromatic ring or ring system (e.g., two or more rings sharing two adjacent atoms) containing one or more heteroatoms in the ring skeleton, where the heteroatoms (i.e., elements other than carbon) include, but are not limited to, nitrogen, oxygen, and sulfur. When the heteroaryl is a ring system, each ring in the system is aromatic. Heteroaryl groups can have 5 - 18 ring members.

[0062] As used herein, "heterocyclic group" denotes a non-aromatic ring or ring system containing at least one heteroatom in the ring skeleton. Heterocyclic groups can be joined together in a fused, bridged, or spiro fashion. Heterocyclic groups can have any degree of saturation, provided that at least one ring in the ring system is non-aromatic. In the ring system, heteroatoms can be present in non-aromatic or aromatic rings. Heterocyclic groups can have 3 to 20 ring members (i.e., the number of atoms making up the ring skeleton, including carbon atoms and heteroatoms). In some instances, the heteroatoms are O, N, or S.

[0063] As used herein, the term "hydrazine" or "hydrazino" refers to the -NHNH2 group.

[0064] As used herein, the term "hydrazone" or "hydrazono" refers to a group where R a and R b are as defined herein.

[0065] As used herein, a "hydrogel" refers to a three-dimensional polymer network structure composed of cross-linked polymer chains. The hydrogel is not water-soluble or removable in the liquid to which it is exposed during sequencing.

[0066] As used herein, "hydroxy" or "hydroxyl" refers to the -OH group.

[0067] As used herein, the term "gap region" refers to a region in or on a substrate that separates recesses. For example, a gap region can separate one feature of an array from another feature of the array. Two features that are separated from each other can be discrete, i.e., lacking physical contact with each other. In another example, a gap region can separate a first portion of a feature from a second portion of the feature. In many instances, the gap region is continuous while the features are discrete, such as in the case of multiple holes defined in an otherwise continuous surface. The separation provided by the gap region can be partial or complete. The gap region can have a surface material that is different from the surface material of the features defined in the surface. For example, the amount or concentration of a coating and primer present in the features of an array can exceed the amount and concentration present in the gap region. In some instances, the coating and primer can be absent from the gap region.

[0068] As used herein, "oxynitrile" means "R" a C≡N + O - " group, where R a is defined herein. Examples of the preparation of oxynitriles include in-situ generation from aldoximes by treatment with chloramine-T or by the action of a base on imidoyl chlorides [RC(Cl)=NOH].

[0069] As used herein, "nitrone" means group, where R1, R2, and R3 can be any of the Rs defined herein a and R b as defined herein.

[0070] As used herein, "nucleotide" includes a nitrogenous heterocyclic base, a sugar, and one or more phosphate groups. Nucleotides are the monomeric units of nucleic acid sequences. In RNA, the sugar is ribose, while in DNA, the sugar is deoxyribose, i.e., a sugar lacking the hydroxyl group present at the 2'-position of ribose. The nitrogenous heterocyclic base (i.e., nucleobase) can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G) and their modified derivatives or analogs. 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 the N-1 of pyrimidine or the N-9 of purine.

[0071] The term "flow cell substrate" or "substrate" refers to a carrier on which surface chemistry can be added. The term "patterned substrate" refers to a carrier in or on which depressions are defined. The substrate can be a wafer, panel, rectangular sheet, mold, or any other suitable configuration. The substrate is typically rigid and insoluble in aqueous liquids. The substrate can be inert to the chemicals used to modify the depressions. For example, the substrate can be inert to the chemical actions for applying a functionalized coating, attaching a primer to the functionalized coating, applying a hydrogel, etc. Examples of suitable substrates include epoxy siloxanes, polyhedral oligomeric silsesquioxanes (POSS) or their derivatives, glass and modified or functionalized glass, plastics (including acrylic resins, polystyrene, and copolymers of styrene with other materials, polypropylene, polyethylene, polybutene, polyurethane, polytetrafluoroethylene (e.g., from Chemours ), cycloolefin / epoxy-olefin polymers (COP) (e.g., from Zeon ), polyimides, etc.), nylon, ceramics / ceramic oxides, silica, fused silica, or silica-based materials (e.g., including at least 10% silica), aluminosilicates, silicon and modified silicon (e.g., boron-doped p+ silicon), silicon nitride (Si3N4), silicon oxide (SiO2), tantalum pentoxide (TaO5), or other tantalum oxides (TaO x ), hafnium oxide (HaO2), carbon, metals, inorganic glass, etc. The substrate can also be glass or silicon or POSS or their derivatives with a coating of tantalum oxide or another ceramic oxide on its surface.

[0072] As used herein, "plasma ashing" refers to the process of removing organic matter from a substrate by oxygen plasma. The products generated by plasma ashing can be removed by a vacuum pump / system. Plasma ashing can activate the substrate by introducing reactive hydroxyl or carboxyl groups.

[0073] As used herein, "primer" is defined as a single-stranded nucleic acid sequence (e.g., single-stranded DNA or single-stranded RNA) that serves as a starting point for DNA or RNA synthesis. The 5' end of the primer can be modified to allow a coupling reaction with the functionalized coating. The primer length can be any number of base lengths and can include various non-natural nucleotides. In one example, a sequencing primer is a short chain of 20 to 40 bases.

[0074] As used herein, the terms "silane" and "silane derivatives" refer to organic or inorganic compounds containing one or more silicon atoms. Examples of inorganic silane compounds are SiH4 or halogenated SiH4 in which hydrogen is replaced by one or more halogen atoms. Examples of organic silane compounds are X-R B -Si(OR C)3, where X is an organic group such as amino, vinyl, methacrylate, epoxy sulfur, alkyl, alkenyl or alkynyl; R B is a spacer, for example, -(CH2) n -, where n is from 0 to 1000; R C is selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclic group, optionally substituted aryl, optionally substituted 5-10 membered heteroaryl and optionally substituted 5-10 membered heterocyclic group, as defined herein. As used herein, the terms "silane" and "silane derivative" may include mixtures of different silane and / or silane derivative compounds.

[0075] In some instances, the silane or silane derivative includes an unsaturated moiety capable of reacting with a functional group of a functionalized polymer layer. As used herein, the term "unsaturated moiety" refers to a chemical group including olefins, alkynes, cycloolefins, cycloalkynes, heterocycloolefins, heterocycloalkynes or their optionally substituted variants (including at least one double bond or one triple bond). The unsaturated moiety can be monovalent or divalent. When the unsaturated moiety is monovalent, cycloolefins, cycloalkynes, heterocycloolefins and heterocycloalkynes are used interchangeably with cycloalkenyl, cycloalkynyl, heterocycloalkenyl and heterocycloalkynyl, respectively. When the unsaturated moiety is divalent, cycloolefins, cycloalkynes, heterocycloolefins and heterocycloalkynes are used interchangeably with subcycloalkenyl, subcycloalkynyl, subheterocycloalkenyl and subheterocycloalkynyl, respectively.

[0076] The unsaturated moiety can be directly covalently attached to the silicon atom of the silane or silane derivative, or indirectly attached through a linker. Examples of suitable linkers include optionally substituted alkylene groups (e.g., a divalent saturated aliphatic group considered to be derived from an olefin by opening a double bond or from an alkane by removing two hydrogen atoms from different carbon atoms (such as ethylene)), substituted polyethylene glycols, etc.

[0077] As used herein, a "spacer layer" refers to a material that bonds two components together. In some instances, the spacer layer can be a radiation-absorbing material that aids in bonding, or can be in contact with a radiation-absorbing material that aids in bonding.

[0078] As used herein, the term "surface chemistry" refers to chemical and / or bioactive components incorporated into the depressions of a patterned substrate. Examples of surface chemistries disclosed herein include a functionalized polymer layer attached to at least a portion of the substrate surface and / or a primer that links at least a portion of the functionalized polymer layer.

[0079] A "thiol" functional group refers to -SH.

[0080] As used herein, the terms "tetrazine" and "tetrazinyl" refer to a six-membered heteroaryl group containing four nitrogen atoms. The tetrazine can be optionally substituted.

[0081] As used herein, "tetrazole" refers to a five-membered heterocyclic group that includes four nitrogen atoms. The tetrazole can be optionally substituted.

[0082] Figure 1 An example of method 100 is depicted in. Method 100 includes applying a functionalized coating in a recess of a patterned flow cell substrate, where the recesses are separated by gap regions (as shown by reference numeral 102), grafting primers onto the functionalized coating to form a grafted functionalized coating in the recesses (as shown by reference numeral 104), and applying a hydrogel to at least the grafted functionalized coating (as shown by reference numeral 106).

[0083] The patterned flow cell substrate can be any one of a patterned wafer, a patterned mold, or other patterned substrates disclosed herein. Any example of the substrates described herein can be used. The patterned substrate ( Figure 3A and 4 shown as reference numeral 12 in ) includes recesses defined above or within an exposed layer or substrate surface, and gap regions separating adjacent recesses. Various techniques can be used to fabricate the recesses in or on the substrate, including, for example, photolithography, nanoimprinting, stamping techniques, embossing techniques, molding techniques, microetching techniques, printing techniques, etc. As will be understood by those skilled in the art, the technique used will depend on the composition and shape of the substrate. Many different recess layouts can be envisioned, as discussed below with reference to Figure 4

[0084] Although not shown in Figure 1 prior to applying the functionalized coating and grafting the primers (i.e., before adding the surface chemistry), the method can include treating the surface by exposing the patterned substrate to a cleaning process and / or another process for preparing the patterned substrate surface (e.g., the recesses, and in some cases, the adjacent gap regions) for subsequent deposition of the surface chemistry. For example, the method can include treating the surface of the flow cell substrate to attach functional groups to the surface to form treated recesses, and in some cases, form treated gap regions. More detailed examples of the treatment processes (e.g., the cleaning process and the surface preparation process) are discussed below with reference to Figures 3A to 3I

[0085] In Figure 1 the example shown, adding the surface chemistry includes applying a functionalized coating in the recesses (reference numeral 102) and grafting primers onto the functionalized coating (reference numeral 104).

[0086] ​​Examples of functionalized coatings include acrylamide copolymers, such as poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide), PAZAM. PAZAM and some other forms of acrylamide copolymers are represented by formula (I):

[0087]

[0088] wherein:

[0089] R A is selected from azido, optionally substituted amino, optionally substituted alkenyl, optionally substituted hydrazone, optionally substituted hydrazine, carboxyl, hydroxyl, optionally substituted tetrazole, optionally substituted tetrazine, oxynitrile, nitrone and thiol;

[0090] R B is hydrogen or optionally substituted alkyl;

[0091] R C 、R D and R E are independently selected from H and optionally substituted alkyl;

[0092] -(CH2) p - can each be optionally substituted;

[0093] p is an integer in the range of 1 to 50;

[0094] n is an integer in the range of 1 to 50,000; and

[0095] m is an integer in the range of 1 to 100,000.

[0096] Those of ordinary skill in the art will recognize that the arrangement of the recurring "n" and "m" features in formula (I) is representative, and the monomer subunits can be present in the polymer structure in any order (e.g., random, block, patterned, or combinations thereof).

[0097] A specific example of PAZAM is represented by the following formula:

[0098]

[0099] where n is an integer in the range of 1 - 20,000, and m is an integer in the range of 1 - 100,000.

[0100] The molecular weight range of PAZAM can be from about 10 kDa to about 1500 kDa, or in a specific example, can be about 312 kDa.

[0101] In some examples, PAZAM is a linear polymer. In some other examples, PAZAM is a lightly cross-linked polymer.

[0102] In other instances, the functionalized coating can be a variant of formula (I). In one instance, the acrylamide unit can be replaced by N,N-dimethylacrylamide. In this instance, the acrylamide unit in formula (I) can be replaced by , where R D , R E and R F are each H, and R G and R H are each methyl groups (different from H in the case of acrylamide). In this instance, q can be an integer in the range of 1 to 10,000. In another instance, in addition to the acrylamide unit, N,N-dimethylacrylamide can also be used. In this instance, formula (I) can also include (in addition to the recurring "n" and "m" features), where R D , R E and R F are each H, and R G and R H are each methyl groups. In this instance, q can be an integer in the range of 1 to 100,000.

[0103] It should be understood that other functionalized molecules can be used to form the functionalized coating as long as they are functionalized to interact with the patterned substrate and the subsequently applied primer. Other examples of suitable molecules for forming the functionalized coating include those having a colloidal structure such as agarose; or a polymer network structure such as gelatin; or a cross-linked polymer structure such as polyacrylamide polymers and copolymers, silane-free acrylamide (SFA) or the azide form of SFA. Examples of suitable polyacrylamide polymers can be synthesized from acrylamide and acrylic acid or acrylic acid containing vinyl, or from monomers that form [2+2] photocycloaddition reactions.

[0104] Spin coating, or dip or immersion coating, or the functionalized molecules can flow under positive or negative pressure, or another suitable technique can be used to deposit the functionalized molecules (e.g., PAZAM) on the surface of the patterned substrate. The functionalized molecules can be present as a mixture. In one instance, the mixture includes PAZAM in water or in a mixture of ethanol and water.

[0105] After coating, the functionalized molecules can also be exposed to a curing process to form a functionalized coating over the entire patterned substrate (i.e., over the recessed and void areas). In one instance, the curing of the functionalized molecules can be carried out at a temperature in the temperature range from room temperature (e.g., about 25 °C) to about 60 °C for a time from about 5 minutes to about 2 hours.

[0106] To form a functionalized coating in the recesses of the patterned substrate and not on the gap regions, one can use i) an alkaline aqueous slurry having a pH range of from about 7.5 to about 11 and including abrasive particles; or ii) a polishing pad and a solution without abrasive particles to polish off the functionalized coating from the gap regions.

[0107] In this example of method 100, a primer is then grafted onto the functionalized coating retained in the recesses, as shown by reference numeral 104, to form a grafted functionalized coating. Examples of suitable primers include forward amplification primers or reverse amplification primers. Specific examples of suitable primers include P5 or P7 primers, which are used for sequencing on the surface of commercial flow cells sold by Illumina Inc. in MISEQ TM 、MISEQX TM 、NEXTSEQ TM 、NOVASEQ TM 、GENOME ANALYZER TM and other instrument platforms.

[0108] Grafting can be accomplished by immersion coating, spray coating, agitation dispensing, or by another suitable method of attaching the primer to the functionalized coating in at least some of the recesses. Each of these exemplary techniques can utilize a primer solution or mixture, which can include a primer, water, buffer, and catalyst.

[0109] Immersion coating can include immersing the patterned substrate (having a functionalized coating in its recesses) into a series of temperature-controlled baths. The baths can also be flow-controlled and / or covered with a nitrogen layer. The baths can include the primer solution or mixture. In the various baths, the primer attaches to the functionalized coating in at least some of the recesses. In one example, the coated and polished patterned substrate is introduced into a first bath including the primer solution or mixture, in which a reaction occurs to attach the primer, and then the patterned substrate is moved to additional baths for washing. The patterned substrate can be moved between the baths using a robotic arm or manually. A drying system can also be used in immersion coating.

[0110] Spray coating can be accomplished by directly spraying the primer solution or mixture onto the coated and polished patterned substrate. The sprayed wafer can be incubated for a time of from about 4 minutes to about 60 minutes at a temperature in the temperature range of from about 0 °C to about 70 °C. After incubation, the primer solution or mixture can be diluted and removed using, for example, a spin coater.

[0111] Mixing and dispensing can be performed according to the pool and spin off method and can thus be accomplished using a spin coater. The primer solution or mixture can be applied (manually or through an automated process) to the coated and polished patterned substrate. The applied primer solution or mixture can be applied to or diffused over the entire surface of the coated and polished patterned substrate. The primer-coated patterned substrate can be incubated for a time of about 2 minutes to about 60 minutes at a temperature in the range of about 0 °C to about 80 °C. After incubation, the primer solution or mixture can be diluted and removed using, for example, a spin coater.

[0112] In one example, after the primer is grafted to the functionalized coating in the recess to form a grafted functionalized coating, this example of method 100 further includes applying a hydrogel to the grafted functionalized coating (as shown by reference numeral 106).

[0113] The hydrogel can be any hydrophilic polymer that is exposed to the flow cell and serves as a sequencing template filter. The deposition of the hydrogel is partially controlled by the polymer concentration in the solution deposited on the flow cell. The hydrogel slows down the diffusion of the sequencing template into the recesses, thus allowing a single sequencing template to seed and cluster in the recesses before another sequencing template can diffuse through the hydrogel. The hydrogel also remains on the flow cell during the sequencing template seeding and during other sequencing steps and is thus not water-soluble or removable in the liquid to which it is exposed during the sequencing process. Some examples of hydrogels include PAZAM (or variants thereof as described herein), crosslinked polyacrylamide, agarose gel, crosslinked polyethylene glycol (PEG), etc. The hydrogel can be other acrylamide-based copolymers, agarose-based copolymers, or PEG-based copolymers. It should be understood that a copolymer based on X (e.g., based on acrylamide, based on agarose, based on PEG, etc.) includes an amount of the X component of about 10% or more of the molecular weight composition. In some examples, the copolymer based on X includes an amount of the X component of about 10%, or about 11%, or about 12%, or about 15%, or about 20%, or about 39%, or a higher percentage of the molecular weight composition. Additionally, the X component can be higher or lower than the given percentage as long as the copolymer functions as a hydrogel. Crosslinked PEG hydrogels can be synthesized by covalent crosslinking of PEG macromonomers with reactive chain ends such as acrylate, methacrylate, allyl ether, maleimide, vinyl sulfone, NHS ester, and vinyl ether groups. Any of the exemplary hydrogels can include hydrophobic or hydrophilic side chains.

[0114] The hydrogel is not grafted with the primer but is coated with the primer.

[0115] In some instances, the hydrogel can be selectively deposited or patterned such that it covers the surface chemistry (in this instance, the functionalized coating and the primer thereon), and such that the bonding regions of the patterned flow cell substrate remain exposed. The bonding regions of the patterned flow cell substrate are typically located on some of the interstitial regions of the patterned flow cell substrate where the lid bonds to the patterned substrate. When the patterned substrate is a wafer, the bonding regions can define the boundaries (e.g., the perimeter) of several flow cells formed by the wafer. When the patterned substrate is a mold, the bonding regions can define the outer boundary (e.g., the perimeter) of one flow cell formed. It should be understood that other portions of the patterned flow cell substrate (which are not part of the bonding regions) can be covered by the hydrogel.

[0116] In this instance of method 100, the selective deposition or patterning of the hydrogel can be accomplished by solution incubation, dip coating, spin coating, spray coating, ultrasonic spray coating, doctor blade coating, aerosol printing, or inkjet printing. A mask can be used to cover the bonding regions of the patterned substrate such that the hydrogel is not applied to the bonding regions. The selective deposition of the hydrogel can be used to deposit the hydrogel on the grafted functionalized coating in the recesses, but not on the interstitial regions.

[0117] In other instances, after forming the functionalized coating, the lid can be bonded to the bonding regions of the patterned flow cell substrate, and a flow through process can be used to apply the primer and the hydrogel.

[0118] Each of the exemplary techniques for applying the hydrogel can utilize an aqueous mixture, which can include water and up to about 0.1% (weight / volume) of the hydrogel material. In some instances, the hydrogel material constitutes 0.1% or less of the aqueous mixture. In other instances, the aqueous mixture contains from about 0.001% to about 0.1% of the hydrogel material, or from about 0.025% to about 0.005% of the hydrogel material. It should be understood that the concentration of the aqueous mixture can vary depending on the flow cell structure (e.g., the size of the flow channels, inlets, and outlets, etc.). For example, when using flow through deposition, the concentration can be selected such that the aqueous mixture can flow through the flow cell without clogging the interfaces, flow channels, etc. Thus, the concentration can also be higher than about 0.1%. The hydrogel material (and the resulting hydrogel coating) can be any of the instances disclosed herein (i.e., PAZAM or its variants, crosslinked polyacrylamide, agarose gel, etc.).

[0119] In some instances, the aqueous mixture can also include additives such as co-solvents, antioxidants, dyes, UV stabilizers, processing aids, etc. These additives can be included in the aqueous mixture in amounts that do not adversely affect the fluidity of the mixture or the film-forming ability of the hydrogel.

[0120] After applying the aqueous mixture, incubation is allowed to form a hydrogel. The time and temperature for solution incubation can be any time and temperature sufficient to form the hydrogel. For example, the temperature range can be from room temperature to about 65 °C, and the time range can be from about 5 minutes to about 1 hour, or longer. In one example, the solution incubation is carried out at a temperature of about 50 °C for about 10 minutes.

[0121] In some cases, the aqueous mixture can be partially dried during hydrogel formation. The partial drying can be accomplished by air exposure, nitrogen exposure, vacuum, heating (e.g., in an oven), or spin coating (i.e., spinning until dry). In an example where heating is used, the temperature can be about 50 °C, and the hydrogel can be maintained at this temperature for about 10 minutes. The hydrogel can also be washed with a dilute buffer.

[0122] Another example of method 200 is depicted in Figure 2 The method 200 includes attaching a silane or silane derivative to the surface of a patterned substrate that includes flow channels having defined recesses therein, where the recesses are separated by gap regions, thereby forming silanized recesses and silanized gap regions (reference numeral 202), applying a functionalized coating to the silanized recesses and the silanized gap regions (reference numeral 204); polishing the functionalized coating from the silanized gap regions (reference numeral 206); grafting primers to the functionalized coating in the silanized recesses to form grafted functionalized coating in the recesses (reference numeral 208); and applying a hydrogel to the grafted functionalized coating in the recesses (reference numeral 210). Reference will be made to Figures 3A to 3E and in conjunction with Figure 3H and 3I In Figures 3A to 3D further examples of the method 200 are described.

[0123] Figure 3A is a cross-sectional view of an example of a patterned substrate 12. The patterned substrate 12 can be a patterned wafer or a patterned mold or any other patterned substrate (e.g., a panel, a rectangular sheet, etc.). Any example of the substrates described herein can be used. The patterned wafer can be used to form several flow cells, and the patterned mold can be used to form a single flow cell. In one example, the substrate can have a diameter in the range of about 2 mm to about 300 mm, or a rectangular sheet or panel having a maximum dimension of up to 10 feet (~3 meters). In one example, the substrate wafer has a diameter in the range of about 200 mm to about 300 mm. In another example, the substrate mold has a width in the range of about 0.1 mm to about 10 mm. Although example dimensions have been provided, it should be understood that substrates having any suitable dimensions can be used.

[0124] The patterned substrate 12 includes recesses defined on or in an exposed layer or surface of the substrate and interstitial regions 16 separating adjacent recesses. In the examples disclosed herein, the recesses are functionalized with surface chemistries (e.g., 20, 22), while the interstitial regions 16 can be used for adhesion but do not have primers present thereon ( Figures 3E - 3G and 22 as shown in 3I).

[0125] A variety of techniques can be used to create recesses in or on the substrate, including, for example, photolithography, nanoimprinting, stamping techniques, embossing techniques, molding techniques, microetching techniques, printing techniques, etc. As will be understood by those skilled in the art, the technique used will depend on the composition and shape of the substrate.

[0126] Many different layouts of recesses can be envisioned, including regular, repeating, and irregular patterns. In one example, the recesses are arranged in a hexagonal grid for close packing and increased density. Other layouts can include, for example, a linear (i.e., rectangular) layout, a triangular layout, etc. In some examples, the layout or pattern can be an x - y format of recesses in rows and columns. In some other examples, the layout or pattern can be a repeating arrangement of recesses and / or interstitial regions 16. In still other examples, the layout or pattern can be a random arrangement of recesses and / or interstitial regions 16. The pattern can include spots, pads, holes, pillars, stripes, swirls, lines, triangles, rectangles, circles, arcs, checks, lattices, diagonals, arrows, squares, and / or reticulations.

[0127] The layout or pattern can be characterized in terms of the density of recesses (i.e., the number of recesses) in a defined area. For example, the recesses can be present at a density of about 2 million / mm 2 . This density can be adjusted to different densities, including, for example, at least about 100 / mm 2 , about 1,000 / mm 2 , about 100,000 / mm 2 , about 1 million / mm 2 , about 2 million / mm 2 , about 5 million / mm 2 , about 10 million / mm 2 , about 50 million / mm 2 or greater. Alternatively or additionally, the density can be adjusted to not exceed about 50 million / mm 2 , about 10 million / mm 2 , about 5 million / mm 2 , about 2 million / mm 2 , about 1 million / mm 2 , about 100,000 / mm 2 , about 1,000 / mm 2, about 100 / mm 2 or less. It should be further understood that the density of the depressions on the substrate can be between one of the lower limit values and one of the upper limit values selected from the above ranges. For example, a high-density array can be characterized as having depressions separated by less than about 100 nm, a medium-density array can be characterized as having depressions separated by about 400 nm to about 1 μm, and a low-density array can be characterized as having depressions separated by greater than about 1 μm. Although example densities are provided, it should be understood that substrates with any suitable density can be used.

[0128] The layout or pattern can also or alternatively be characterized according to the average spacing, i.e., the spacing from the center of the depression to the center of the adjacent gap region 16 (center-to-center spacing). The pattern can be regular, such that the coefficient of variation of the average spacing is small, or the pattern can be irregular, in which case the coefficient of variation can be relatively large. In either case, the average spacing can be, for example, at least about 10 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 100 μm or greater. Alternatively or additionally, the average spacing can be, for example, at most about 100 μm, about 10 μm, about 5 μm, about 1 μm, about 0.5 μm, about 0.1 μm or less. The average spacing of a particular pattern of a site can be between one of the lower limit values and one of the upper limit values selected from the above ranges. In one example, the depressions have a spacing (center-to-center spacing) of about 1.5 μm. Although example average spacing values have been provided, it should be understood that other average spacing values can be used.

[0129] In Figures 3A to 3I the example shown, the depressions are holes 14', and thus the patterned substrate 12 includes an array of holes 14' on its surface. The holes 14' can be microholes or nanopores. The size of each hole 14' can be characterized by its volume, hole opening area, depth, and / or diameter.

[0130] Each hole 14' can have any volume capable of confining a liquid. For example, a minimum or maximum volume can be selected to accommodate the flux (e.g., multiplicity), resolution, analyte composition, or analyte reactivity expected for uses downstream of the flow cell. For example, the volume can be at least about 1×10 -3 μm 3 , about 1×10 -2 μm 3 , about 0.1 μm 3 , about 1 μm 3 , about 10 μm 3 , about 100 μm 3 or greater. Alternatively or additionally, the volume can be at most about 1×10 4 μm 3 , about 1×103 μm 3 , about 100 μm 3 , about 10 μm 3 , about 1 μm 3 , about 0.1 μm 3 or smaller. It should be understood that the functionalized coating can fill all or part of the volume of the hole 14'. The volume of the coating in a single hole 14' can be greater than, less than, or between the above-specified values.

[0131] The area occupied by each hole opening on the surface can be selected based on criteria similar to those proposed above for the hole volume. For example, the area of each hole opening on the surface can be at least about 1×10 -3 μm 2 , about 1×10 -2 μm 2 , about 0.1 μm 2 , about 1 μm 2 , about 10 μm 2 , about 100 μm 2 or larger. Alternatively or additionally, the area can be at most about 1×10 3 μm 2 , about 100 μm 2 , about 10 μm 2 , about 1 μm 2 , about 0.1 μm 2 , about 1×10 -2 μm 2 or smaller. The area occupied by each hole opening can be greater than, less than, or between the above-specified values.

[0132] The depth of each hole 14' can be at least about 0.1 μm, about 1 μm, about 10 μm, about 100 μm or larger. Alternatively or additionally, the depth can be at most about 1×10 3 μm, about 100 μm, about 10 μm, about 1 μm, about 0.1 μm or smaller. The depth of each hole 14' can be greater than, less than, or between the above-specified values.

[0133] In some cases, the diameter of each hole 14' can be at least about 50 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 10 μm, about 100 μm or larger. Alternatively or additionally, the diameter can be at most about 1×10 3 μm, about 100 μm, about 10 μm, about 1 μm, about 0.5 μm, about 0.1 μm or smaller (e.g., about 50 nm). The diameter of each hole 14' can be greater than, less than, or between the above-specified values.

[0134] The patterned substrate 12 can be exposed to a series of processes to add surface chemistries 20, 22 in the recesses.

[0135] Although not shown, it should be understood that the patterned substrate 12 can be exposed to plasma ashing to clean and activate the surface. For example, the plasma ashing process can remove organic materials and introduce surface hydroxyl groups. Other suitable cleaning processes can be used to clean the substrate, which depends in part on the type of substrate. For example, an oxidizing agent or caustic solution can be used for chemical cleaning.

[0136] Subsequently, the patterned substrate ( Figure 3A shown in) can be exposed to a process for preparing the substrate for deposition of a functionalized polymer to form a functionalized polymer layer ( Figure 3C ). In one example, the patterned substrate 12 can be exposed to silanization, which attaches a silane or silane derivative 18 ( Figure 3B ) to the surface of the patterned wafer. Silanization introduces the silane or silane derivative 18 over the entire surface, including in the recesses (e.g., on the bottom surface and along the sidewalls) and on the gap regions 16. In some aspects, the silane or silane derivative is introduced only selectively into the recesses of the patterned substrate or into micro-locations (which are separated from each other) of an unpatterned substrate.

[0137] Any silane or silane derivative 18 can be used to effect silanization. The choice of silane or silane derivative 18 can depend in part on the functionalized molecules ( Figure 3C shown in) to be used to form the functionalized polymer layer, as it may be desirable to form a covalent bond between the silane or silane derivative 18 and the functionalized polymer layer. The method for attaching the silane or silane derivative 18 to the substrate can vary depending on the silane or silane derivative 18 to be used. Several examples are listed herein.

[0138] In one example, the silane or silane derivative 18 is (3-aminopropyl)triethoxysilane (APTES) or (3-aminopropyl)trimethoxysilane (APTMS) (i.e., X-R B -Si(OR C )3), where X is an amino group, R B - is -(CH2)3-, and R Cis ethyl or methyl. In this example, the substrate surface can be pretreated with (3-aminopropyl)triethoxysilane (APTES) or (3-aminopropyl)trimethoxysilane (APTMS) to covalently link silicon to one or more oxygen atoms on the surface (not intending to be limited by the mechanism, each silicon can bond to one, two, or three oxygen atoms). The surface of such chemical treatment is baked to form an amine monolayer. Subsequently, the amine groups are reacted with sulfo-HSAB to form azide derivatives. Using 1 J / cm 2 to 30 J / cm 2 energy of UV activation generates reactive nitrene species, which can readily undergo various insertion reactions with PAZAM (e.g., functionalized molecules). In some aspects, the silane or silane derivative is selectively applied to the depressions of the patterned substrate or the micro-locations on the unpatterned substrate.

[0139] Other silanization methods can also be used. Examples of suitable silanization methods include vapor deposition, the YES method, spin coating, or other deposition methods. Some examples of methods and materials that can be used to silanize the substrate are described herein, although it will be understood that other methods and materials can be used.

[0140] In an example using a YES CVD furnace, the patterned substrate 12 is placed in the CVD furnace. The chamber can be purged with gas, and then the silanization cycle can be started. During the cycle, the silane or silane derivative container can be maintained at a suitable temperature (e.g., for norbornene silane, about 120 °C), the silane or silane derivative vapor line can be maintained at a suitable temperature (e.g., for norbornene silane, about 125 °C), and the vacuum line can be maintained at a suitable temperature (e.g., about 145 °C).

[0141] In another example, the silane or silane derivative 18 (e.g., liquid norbornene silane) can be deposited inside a glass vial and placed together with the patterned substrate 12 inside a glass vacuum desiccator. Subsequently, the desiccator can be evacuated to a pressure in the range of about 15 mTorr to about 30 mTorr and placed inside a furnace with a temperature range of about 60 °C to about 125 °C. The silanization is allowed to proceed, and then the desiccator is removed from the furnace, cooled, and purged with air.

[0142] Chemical vapor deposition, YES method, and / or vacuum dryer can be used with various silanes or silane derivatives 18, such as those silanes or silane derivatives 18 including the examples of unsaturated moieties disclosed herein. For example, when the silane or silane derivative 18 includes an olefin or cycloolefin unsaturated moiety (such as norbornene, norbornene derivatives (e.g., (hetero)norbornene, including replacement of one of the carbon atoms with oxygen or nitrogen), trans-cyclooctene, trans-cyclooctene derivatives, trans-cyclopentene, trans-cycloheptene, trans-cyclononene, bicyclo[3.3.1]non-1-ene, bicyclo[4.3.1]dec-1(9)-ene, bicyclo[4.2.1]non-1(8)-ene, and bicyclo[4.2.1]non-1-ene), these methods can be used. Any of these cycloolefins can be substituted with, for example, an R group such as hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocycloalkyl, aralkyl, or (heterocycloalkyl)alkyl. Examples of norbornene derivatives include [(5-bicyclo[2.2.1]hept-2-enyl)ethyl]trimethoxysilane. As other examples, when the silane or silane derivative 18 includes an alkyne or cycloalkyne unsaturated moiety, these methods can be used, such as cyclooctyne, cyclooctyne derivatives, or bicyclononyne (e.g., bicyclo[6.1.0]non-4-yne or its derivatives, bicyclo[6.1.0]non-2-yne, or bicyclo[6.1.0]non-3-yne). Any of these cycloalkynes can be substituted with any of the R groups described herein.

[0143] As Figure 3B shown in, the linking of the silane or silane derivative 18 forms a silanized patterned substrate, including silanized depressions and silanized gap regions (which is an example of the processed depressions and processed gap regions).

[0144] Subsequently, the silanized patterned wafer can be exposed to a process of forming a functionalized polymer layer on the silanized depressions and silanized gap regions.

[0145] As described herein, examples of the functionalized polymer layer include PAZAM, or any other molecule functionalized to interact with the patterned substrate 12 and a subsequently applied primer. The functionalized molecules can be present in a mixture. In one example, the mixture includes PAZAM in water or a mixture of ethanol and water. Any suitable technique can be used to form the functionalized polymer layer on the surface of the silanized patterned wafer (e.g., on the silanized depressions and silanized gap regions). Spin coating, or dipping or immersion coating, or the flow of functionalized molecules under positive or negative pressure, or other suitable techniques can be used to deposit the functionalized molecules on the surface of the patterned substrate 12. The resulting layer is shown in Figure 3C in.

[0146] A functionalized polymer layer can be covalently bonded to the silanized recess and the silanized gap region (i.e., 18). The covalent connection of the functionalized polymer layer to the silanized recess helps to maintain the functionalized polymer layer in the recess during the entire lifetime of the final flow cell during various uses. The following are some examples of reactions that can occur between the silane or silane derivative 18 and the functionalized polymer layer.

[0147] When the silane or silane derivative 18 includes norbornene or a norbornene derivative as the unsaturated moiety, the norbornene or norbornene derivative can: i) undergo a 1,3-dipolar cycloaddition reaction with the azide / azido group of PAZAM; ii) undergo a coupling reaction with the tetrazine group attached to PAZAM; iii) undergo a cycloaddition reaction with the hydrazone group attached to PAZAM; iv) undergo a photo-click reaction with the tetrazole group attached to PAZAM; or v) undergo a cycloaddition with the nitrile oxide group attached to PAZAM.

[0148] When the silane or silane derivative 18 includes cyclooctyne or a cyclooctyne derivative as the unsaturated moiety, the cyclooctyne or cyclooctyne derivative can: i) undergo a strain-promoted azide-alkyne 1,3-cycloaddition (SPAAC) reaction with the azide / azido of PAZAM; or ii) undergo a strain-promoted alkyne-nitrile oxide cycloaddition reaction with the nitrile oxide group attached to PAZAM.

[0149] When the silane or silane derivative 18 includes bicyclononyne as the unsaturated moiety, due to the strain in the bicyclic ring system, the bicyclononyne can undergo a similar SPAAC alkyne cycloaddition with the azide or nitrile oxide attached to PAZAM.

[0150] Although not shown, it should be understood that in some examples of the method, the patterned substrate 12 may not be exposed to silanization. Instead, the patterned substrate 12 can be exposed to plasma ashing, and subsequently the functionalized polymer layer can be directly spin-coated (or otherwise deposited) on the plasma-ashed patterned substrate 12. In this example, plasma ashing can generate surface activators (e.g., -OH groups), which can adhere the functionalized coating to the patterned substrate 12. In these examples, the functionalized polymer layer is selected such that it reacts with the surface groups generated by plasma ashing.

[0151] After coating, the functionalized molecules can also be exposed to a curing process to form a functionalized polymer layer over the entire patterned substrate (i.e., over the recessed and interstitial regions 16). In one example, the curing of the functionalized molecules can be carried out at a temperature in the range from room temperature (e.g., about 25 °C) to about 95 °C for a time period from about 1 millisecond to about several days. In another example, the time period can range from 10 seconds to at least 24 hours. In yet another example, the time period can range from about 5 minutes to about 2 hours.

[0152] The silanized and coated patterned substrate (shown in Figure 3C ) can be exposed to a cleaning process. This process can utilize a water bath and ultrasound. The water bath can be maintained at a relatively low temperature in the range from about 22 °C to about 45 °C. In another example, the water bath temperature range is from about 25 °C to about 30 °C.

[0153] If desired, the silanized and coated patterned substrate is subsequently exposed to polishing to remove a portion of the functionalized polymer layer from the silanized interstitial regions. The silanized, coated, and polished substrate is shown in Figure 3D . The silane or silane derivative portions adjacent to the interstitial regions 16 may or may not be removed as a result of the polishing. Thus, in Figures 3D to 3I , portions of the silane or silane derivative 18 adjacent to the interstitial regions 16 are shown in phantom because they may be at least partially retained after polishing or may be removed after polishing. When these silanized portions are completely removed, it should be understood that the underlying substrate is exposed.

[0154] The polishing process can be carried out with a mild chemical slurry (including, for example, an abrasive, a buffer, a chelating agent, a surfactant, and / or a dispersant) which can remove a thin functionalized polymer layer from the interstitial regions 16 and, in some cases, at least a portion of the silane or silane derivative 18 without having a detrimental effect on the underlying substrate in those regions. Alternatively, the polishing can be carried out with a solution that does not include abrasive particles.

[0155] The chemical slurry can be used in a chemical mechanical polishing system to polish the surface of the silanized and coated patterned substrate shown in Figure 3C . The polishing head / polishing pad or other polishing tool is capable of polishing the functionalized polymer layer from the interstitial regions 16 while retaining the functionalized polymer layer in the recesses and keeping the underlying substrate at least substantially intact. As an example, the polishing head can be a Strasbaugh ViPRR II polishing head.

[0156] As described above, the polishing can be carried out with a polishing pad and a solution without any abrasive. For example, the polishing pad can be used with a solution that does not include abrasive particles (i.e., a solution that does not contain abrasive particles).

[0157] The polishing removes the functionalized polymer layer of the portion from the gap region 16 (and in some cases, at least part of the silane or silane derivative 18), and retains the functionalized polymer layer of the portion in the silanized recesses, as Figure 3D shown. Also as described above, the gap region 16 can remain silanized after polishing. In other words, the silanized gap region can remain intact after polishing. Or (as shown by the phantom portion of 18), as a result of polishing, the silane or silane derivative 18 can be removed from the gap region 16.

[0158] Although not shown, it should be understood that the silanized, coated, and polished patterned substrate (as Figure 3D shown) can be exposed to a cleaning process. This process can utilize a water bath and ultrasound. The water bath can be maintained at a relatively low temperature in the range of about 22 °C to about 30 °C. The silanized, coated, and polished patterned substrate can also be spin-dried or dried by other suitable techniques.

[0159] Then, the Figure 3D shown silanized, coated, and polished patterned substrate can be exposed to the Figures 3E to 3G shown process, which produces the flow cell 10, or exposed to the Figures 3H to 3I shown process, which produces the flow cell 10'. In Figures 3E to 3G , before bonding the lid 26 to the patterned flow cell substrate, the primer is grafted and the hydrogel 24 is applied. As Figure 3H and 3I shown, the lid 26 is bonded to the patterned flow cell substrate before grafting the substrate and applying the hydrogel 24.

[0160] In Figure 3E , a grafting process is performed to graft the primer to the functionalized polymer layer in the recesses. In this example, the grafting can be accomplished by dip coating, spray coating, stirred dispensing, or by another suitable method of attaching the primer to at least some of the functionalized polymer layers in the recesses. Each of these exemplary techniques can utilize the primer solution or mixture disclosed herein (which can include a primer, water, buffer, and catalyst), and be performed as described herein.

[0161] As Figure 3FAs shown, after grafting primers onto the functionalized coating in the recesses, a hydrogel 24 is formed on the grafted functionalized coating and on at least a portion of the patterned flow cell substrate. In this example, the hydrogel 24 can be formed on the exposed surface of the patterned substrate 12 that is not part of the bonding zone 25. In this example, the hydrogel 24 is selectively deposited or patterned on the gap region 16 between adjacent recesses, but not at the edge / periphery of the patterned substrate 12 where the bonding zone 25 is located. As described herein, an aqueous mixture can be used to accomplish the selective deposition / patterning of the hydrogel 24. After deposition of the aqueous mixture, it can be partially dried to form the hydrogel 24.

[0162] As Figure 3G shown, the lid 26 can then be bonded to the bonding zone 25. When the patterned flow cell substrate is a wafer, different regions of the lid 26 can at least partially define the corresponding flow channels 30 formed using the wafer. When the patterned flow cell substrate is a mold, the lid 26 can define one or more flow channels 30 formed.

[0163] The lid 26 can be any material that is transparent to the excitation light directed at the surface chemistries 20, 22 in the recesses. As an example, the lid 26 can be glass (e.g., borosilicate, fused quartz, etc.), plastic, etc. A commercially available example of a suitable borosilicate glass is available from Schott North America, Inc. A commercially available example of a suitable plastic material (i.e., cycloolefin polymer) is available from Zeon Chemicals L.P. product.

[0164] In some examples, the lid 26 can be integrally formed with the sidewalls 29 corresponding to the shape of the bonding zone 25 and bonded to the bonding zone 25. For example, recesses can be etched in a transparent block to form a substantially flat (e.g., top) portion 27 and sidewalls 29 extending from the substantially flat portion 27. When the etched block is mounted to the bonding zone of the patterned substrate 12, the recesses can become the flow channels 30.

[0165] In other examples, the sidewalls 29 and the lid 26 can be separate components coupled to each other. For example, the lid 26 can be a substantially rectangular block having at least a substantially flat outer surface and at least a substantially flat inner surface that defines a portion (e.g., the top portion) of the flow channel 30 (once bonded to the patterned substrate 12). The block can be mounted (e.g., bonded) to the sidewalls 29 that are bonded to the bonding zone 25 of the patterned flow cell substrate and form the sidewalls of the flow channel 30. In this example, the sidewalls 29 can include any of the materials shown herein for the spacer layer (described below).

[0166] The lid 26 can be bonded to the bonding area 25 of the patterned flow cell substrate using any suitable technique, such as laser bonding, diffusion bonding, anodic bonding, eutectic bonding, plasma-activated bonding, glass frit bonding, or other methods known in the art. In one example, a spacer layer 28 can be used to bond the lid 26 to the bonding area 25. The spacer layer 28 can be any material that seals at least a portion of the gap region 16 (e.g., the bonding area 25) of the patterned substrate 12 and the lid 26 together.

[0167] In one example, the spacer layer 28 can be a radiation-absorbing material that absorbs radiation of a wavelength transmitted by the lid 26 and / or the patterned substrate 12. The absorbed energy in turn forms a bond between the spacer layer 28 and the lid 26 and between the spacer layer 28 and the patterned substrate 12. An example of such a radiation-absorbing material is black (carbon black-containing polyimide) from DuPont (USA), which absorbs at approximately 1064 nm. It should be understood that polyimide can be used without adding carbon black, except that the wavelength must be changed to a wavelength significantly absorbed by the natural polyimide material (e.g., 480 nm). As another example, polyimide CEN JP can be bonded when irradiated with light at 532 nm. When the spacer layer 28 is a radiation-absorbing material, the spacer layer 28 can be located at the interface between the lid 26 and the patterned substrate 12 such that the spacer layer 28 contacts the desired bonding area 25. Compression (e.g., a pressure of approximately 100 PSI) can be applied while laser energy of an appropriate wavelength is applied to the interface (i.e., irradiating the radiation-absorbing material). The laser energy can be applied to the interface from the top and from the bottom to achieve a proper bond.

[0168] In another example, the spacer layer 28 can include a radiation-absorbing material in contact therewith. The radiation-absorbing material can be applied at the interface between the spacer layer 28 and the lid 26 and at the interface between the spacer layer 28 and the patterned flow cell substrate. As an example, the spacer layer 28 can be polyimide, and the separate radiation-absorbing material can be carbon black. In this example, the separate radiation-absorbing material absorbs the laser energy that forms a bond between the spacer layer 28 and the lid 26 and between the spacer layer 28 and the patterned substrate 12. In this example, compression can be applied at the respective interfaces while laser energy of an appropriate wavelength is applied to the interface (i.e., irradiating the radiation-absorbing material).

[0169] When the patterned flow substrate is a wafer, the spacer layer 28 and the sidewalls 29 (of or connected to the lid 26) can physically separate one flow channel 30 from an adjacent flow channel 30 and can be located at the outer periphery of the wafer. When the patterned substrate 12 is a mold and the formed flow cell 10 includes a single flow channel 30 or lane, the spacer layer 28 and the sidewalls 29 (of or connected to the lid 26) can be located at the outer periphery of the mold to define the flow channel 30 and seal the flow cell 10. When the patterned substrate 12 is a mold and the formed flow cell 10 includes a plurality of separate flow channels 30 (e.g., eight or four flow channels / lanes), the spacer layer 28 and the sidewalls 29 (of or connected to the lid 26) can physically separate one flow channel / lane 30 from an adjacent flow channel / lane 30 and can be located at the outer periphery of the mold. However, it should be understood that, according to embodiments, the spacer layer 28 and the sidewalls 29 can be located in any desired region.

[0170] When the patterned substrate 12 is a mold, the assembly of the flow cell 10 can include the bonding of the lid 26. When the patterned substrate is a wafer, the assembly of the flow cell 10 can include additional processing after the bonding of the lid 26, such as cutting. In one example, the lid 26 can be bonded to the patterned wafer, and cutting is performed to form a single flow cell 10. As mentioned herein, on the wafer, the sidewalls 29 can physically separate one flow channel 30 from an adjacent flow channel 30, and thus the cutting can be performed through at least some of the sidewalls 29 such that each individual flow cell 10 includes a desired number of flow channels 30, each flow channel having a portion of the original sidewall 29 around its perimeter. In another example, the patterned wafer can be cut to form an un-lidded mold, which can have a corresponding lid 26 bonded thereto to form a single flow cell 10.

[0171] In Figure 3G the example shown, the lid 26 includes a top portion 27 integrally formed with the sidewalls 29. The sidewalls 29 are bonded to the bonding region 25 of the patterned substrate 12 through the spacer layer 28.

[0172] In summary, the lid 26 and the patterned flow cell substrate define the flow channel 30, which is selectively in fluid communication with the recess. The flow channel 30 can be used, for example, to selectively introduce reaction components or reactants into the hydrogel 24 and the underlying surface chemistries 20, 22 in order to initiate a specified reaction in / at the recess.

[0173] Examples of the flow cell 10 are shown in Figure 3G .

[0174] Now refer to Figure 3H and 3I, Another instance of method 200 includes bonding lid 26 to the patterned flow cell substrate before grafting primers and applying hydrogel 24.

[0175] As Figure 3H shown in, a functionalized coating (e.g., deposited and polished) has been applied as Figure 3D in and with reference to Figure 1 described. At least some of the polished gap regions 16 may define a bonding region 25, and lid 26 may be bonded to bonding region 25. Lid 26 may be of any material and may have any configuration described herein. Lid 26 may be bonded to bonding region 25 by any technique described herein.

[0176] In Figure 3H the instance shown, lid 26 includes a top portion 27 integrally formed with sidewalls 29. Sidewalls 29 are bonded to bonding region 25 of the patterned substrate 12 by spacer layer 28. After lid 26 is bonded, a flow channel 30 is formed between lid 26 and the patterned substrate 12. Flow channel 30 can be used to selectively introduce various fluids into flow cell 10’ ( Figure 3I ).

[0177] In this instance of method 200, primers are subsequently grafted onto the functionalized coating in the recesses, as Figure 3I shown in. Any primer described herein can be used. In this instance, grafting can be accomplished by a flow-through method. In the flow-through method, a primer solution or mixture described herein can be introduced into flow channel 30 through a corresponding inlet (not shown), can be maintained in flow channel 30 for a sufficient time (i.e., incubation time) for the primers to attach to the functionalized coating in one or more recesses, and can subsequently be removed through a corresponding outlet (not shown). After primer attachment, other fluids can be directed through flow channel 30 to wash the now-functionalized recesses and flow channel 30.

[0178] After primers are grafted onto the functionalized coating in the recesses, this instance of method 200 further includes forming a hydrogel on the grafted functionalized coating and on at least some of the gap regions 16 (e.g., those regions between the recesses).

[0179] In this example, a hydrogel coating can be deposited via a flow-through process. During the flow-through process, an aqueous mixture (including water and a hydrogel material) can be introduced through a corresponding inlet into flow channels 30 of a flow cell and maintained therein. Sufficient aqueous mixture can be introduced to cover the grafted functionalized coating and any exposed surfaces of the patterned flow cell substrate within the flow channels 30. This solution incubation forms the hydrogel coating 24. In some examples, while the mixture is in the flow channels 30, the flow channels 30 can be exposed to a drying process where air, nitrogen, or vacuum is flushed through the inlet for a set amount of time to partially dry the surface chemistry 20, 22 and any exposed portions of the substrate (e.g., some of the interstitial regions 16) with the hydrogel coating 24 thereon. In this example, the hydrogel coating 24 can be any of the examples disclosed herein.

[0180] Examples of flow cells 10” formed by methods 100, 200 disclosed herein are shown in Figure 4 FIG. The flow cell 10” includes a patterned substrate 12, which can be a mold that has been exposed to the processes of methods 100, 200, or a wafer that has been exposed to the processes of methods 100, 200 and cut.

[0181] Generally, the patterned substrate 12 includes depressions separated by interstitial regions 16 and surface chemistries 20, 22 located within the depressions. The surface chemistries include functionalized coatings and primers. Although not shown, it should be understood that the depressions can also have surface preparation or treatment chemistries (e.g., silanes or silane derivatives) located between the substrate and the functionalized coating. This same surface preparation or treatment chemistry can also be located on the interstitial regions 16.

[0182] The flow cell 10” further includes a lid 26 bonded to the bonding region 25 of the patterned substrate 12, where the lid 26 at least partially defines flow channels 30A, 30B, etc. that are selectively in fluid communication with the depressions. In the Figure 4 example shown in FIG., the lid 26 includes a top portion 27 connected to a plurality of sidewalls 29, and these components 27, 29 define a portion of each of the six flow channels 30A, 30B, 30C, 30D, 30E, 30F. The corresponding sidewalls 29 separate one flow channel 30A, 30B, 30C, 30D, 30E, 30F from each adjacent flow channel 30A, 30B, 30C, 30D, 30E, 30F, and each flow channel 30A, 30B, 30C, 30D, 30E, 30F is selectively in fluid communication with a corresponding set of depressions.

[0183] Although not shown, the lid 26 or the patterned substrate 12 may include inlets and outlets that are fluidly coupled to other ports (not shown) for directing fluid into the respective flow channels 30A, 30B, 30C, 30D, 30E, 30F (e.g., from a reagent cartridge or other fluid storage system) and out of the flow channels (e.g., to a waste removal system).

[0184] The hydrogel / hydrogel coating 24 covers the surface chemistries 20, 22 in the recesses, as well as at least a portion of the patterned substrate 12 (e.g., those interstitial regions 16 that are not also bonding regions 25). In the exemplary flow cell 10”, the hydrogel / hydrogel coating 24 is formed as described herein. Thus, the hydrogel / hydrogel coating 24 can be any of the examples disclosed herein (i.e., PAZAM, crosslinked polyacrylamide, agarose gel, etc.).

[0185] Although not shown, it should be understood that some examples of the flow cells 10, 10', 10” can be directly fixed to a detection device (not shown) by one or more fixing mechanisms (e.g., adhesion, bonding, fixtures, etc.) and thus be in physical contact therewith. The detection device can include a CMOS device (which includes multiple stacked layers, including, for example, a silicon layer, a dielectric layer, a metal-dielectric layer, a metal layer, etc.) and optical components. The optical components can be arranged such that the optical sensor of the detection device is at least substantially aligned with and thus operatively associated with a single optical waveguide of the detection device and the surface chemistries 20, 22 within a single recess or within the flow channels 30 of the flow cell.

[0186] Also although not shown, it should be understood that instead of bonding to the lid 26, a functionalized substrate (having surface chemistries 20, 22 and a hydrogel / hydrogel coating 24 thereon) can be bonded to another functionalized substrate having surface chemistries 20, 22 and a hydrogel / hydrogel coating 24 thereon. The two functionalized surfaces can face each other and can have a flow channel defined therebetween. A spacer layer and a suitable bonding method can be used to bond the two functionalized substrates together.

[0187] The flow cells 10, 10', 10” disclosed herein can be used in a variety of sequencing methods or techniques, including techniques commonly referred to as sequencing by synthesis (SBS), cyclic array sequencing, ligation sequencing, pyrosequencing, etc. Using any of these techniques and in examples using a patterned substrate, amplification will be confined to the functionalized recesses (i.e., the recesses having surface chemistries 20, 22 thereon) due to the presence of the functionalized polymer layer and attached sequencing primers in the functionalized recesses and not on the interstitial regions 16. Additionally, due to the presence of the hydrogel 24, there is more time (compared to when no hydrogel is included) to amplify a single sequencing template into a larger cluster, which increases the population of recesses on the entire patterned flow cell substrate that are seeded with a single sequencing template.

[0188] As an example, it can be performed on systems such as HISEQ from Illumina, Inc. (San Diego, CA), TM HISEQ X, TM MISEQ, TM NOVASEQ, TM or NEXTSEQ TM sequencer systems and the like. In SBS, the extension of a nucleic acid primer (e.g., a primer) along a nucleic acid template (i.e., a sequencing template) is monitored to determine the nucleotide sequence in the template. The underlying chemical process can be polymerization (e.g., catalyzed by a polymerase) or ligation (e.g., catalyzed by a ligase). In a particular polymerase-based SBS method, fluorescently labeled nucleotides are added to the primer in a template-dependent manner (thereby extending the primer), such that the detection of the order and type of nucleotides added to the primer can be used to determine the sequence of the template. For example, to initiate the first SBS cycle, one or more labeled nucleotides, DNA polymerase, etc. can be delivered into the flow channel 30 accommodating the primer array coated with the hydrogel 24 or through the flow channel 30, etc. Functionalized depressions (i.e., depressions having surface chemistries 20, 22 thereon) where primer extension results in the incorporation of labeled nucleotides can be detected by imaging events. During the imaging event, an illumination system (not shown) can provide excitation light to the functionalized depressions (i.e., depressions having chemical surface chemistries thereon).

[0189] In some examples, the nucleotides can further include reversible termination properties that terminate further primer extension once the nucleotides are added to the primer. For example, nucleotide analogs having reversible terminator moieties can be added to the primer such that subsequent extension cannot occur until a deblocking agent is delivered to remove the moiety. Thus, for examples using reversible termination, the deblocking agent can be delivered to the flow channel 30, etc. (before or after detection).

[0190] Washing can be performed between the respective fluid delivery steps. Then the SBS cycle can be repeated n times to extend the primer by n nucleotides, thereby detecting a sequence of length n.

[0191] Although SBS has been described in detail, it should be understood that the flow cells 10, 10', 10'' described herein can be used with other sequencing experimental protocols for genotyping or for other chemical and / or biological applications.

[0192] To further illustrate the present invention, examples are given herein. It should be understood that these examples are provided for illustrative purposes and should not be construed as limiting the scope of the disclosure.

[0193] Non - restrictive working examples

[0194] Example 1

[0195] A flow cell including 8 flow channels / lanes defined on a patterned fused silica substrate is used, where each lane includes 96 blocks (which correspond to the imaging area), and where each block is in fluid communication with a plurality of pores. A PAZAM layer is formed in each pore, and 1 - μm primers are grafted onto the PAZAM layer.

[0196] Lanes 1 - 4, and thus blocks 1 to 384, are comparative example lanes and blocks. Thus, in these lanes and blocks, a hydrogel layer is not applied on the PAZAM layer or the primers.

[0197] Lanes 5 - 8, and thus blocks 385 - 768, are example lanes and blocks. Thus, a hydrogel layer is applied on the PAZAM layer and the primers in these lanes and blocks. The hydrogel coating is another PAZAM layer applied by a flow - through method. A 0.025% PAZAM solution in water is introduced into lanes 5 - 8, heated to 60 °C, and maintained at this temperature for about 10 minutes.

[0198] All lanes are washed with a dilution buffer.

[0199] Sequencing cycles are performed in each of lanes 1 - 8. A Phi X sequencing template solution with a concentration of 150 pM is used.

[0200] Figure 5 The percentage of clusters passing through the filter (% passing filter (%PF)) and the percentage of pores occupied by the DNA sequencing template (% occupied) are shown. % passing filter (%PF) is a metric used to describe the clusters passing through the chastity threshold and is used for further processing and analysis of sequencing data. A higher % passing filter results in an increased yield of unique clusters for sequencing data.

[0201] Figure 5 The data in [description of the relevant context] show that when using the hydrogel, the % passing filter increases (by about 5% to about 10%) (comparing the data of blocks 1 to 384 (without hydrogel) with the data of blocks 385 to 768 (with hydrogel)).

[0202] The difference between % occupied and % passing filter is a rough estimate of polyclonal clusters. The difference between % occupied and %PF for example blocks 385 to 768 is much smaller than the difference between % occupied and %PF for comparative blocks 1 to 384, indicating that the PAZAM hydrogel - coated blocks / lanes have far fewer polyclonal clusters compared to the comparative uncoated blocks / lanes.

[0203] Generally speaking,Figure 5 The data in Figure 5 indicate that the presence of the hydrogel coating helps improve the purity of the main components / clusters in the pores of monoclonal and polyclonal clusters, which will also increase the sequencing yield and data quality.

[0204] Example 2

[0205] Two flow cells are used, each including 8 flow channels / lanes defined on a patterned fused silica substrate, where each lane includes 96 blocks (and imaging regions), and where each block is in fluid communication with a plurality of pores. A PAZAM layer is formed in each pore, and 1 μm primers are grafted onto the PAZAM layer.

[0206] In the comparison flow cell, no hydrogel coating is applied to the PAZAM layer or primers in any of the lanes and blocks.

[0207] In the example flow cell, a hydrogel coating is applied to the PAZAM layer and primers in each lane and block. The hydrogel coating is another PAZAM layer applied by a flow-through method. A mixture / solution of PAZAM in water is introduced into lanes 1-8 of the example flow cell, heated to 60 °C, and maintained at that temperature for about 10 minutes.

[0208] All lanes in the comparison flow cell and the example flow cell are washed with a dilute buffer.

[0209] Sequencing cycles are performed in each of lanes 1-8 of each comparison flow cell and example flow cell. 151 cycles are sequenced in Read 1, and another 151 cycles are sequenced in Read 2. Sequencing metrics are derived from the block centers to eliminate edge effects. Different sequencing template solutions with different concentrations in the range from 100 pM to 800 pM are used in each lane. More specifically, lane 1 of each comparison and example flow cell is exposed to a 100 pM sequencing template solution; lane 2 of each comparison and example flow cell is exposed to a 200 pM sequencing template solution; lane 3 of each comparison and example flow cell is exposed to a 300 pM sequencing template solution; lane 4 of each comparison and example flow cell is exposed to a 400 pM sequencing template solution; lane 5 of each comparison and example flow cell is exposed to a 500 pM sequencing template solution; lane 6 of each comparison and example flow cell is exposed to a 600 pM sequencing template solution; lane 7 of each comparison and example flow cell is exposed to a 700 pM sequencing template solution; and lane 8 of each comparison and example flow cell is exposed to an 800 pM sequencing template solution.

[0210] Figure 6Shows the percentage of clusters passing through the filter (% Passing Filter (%PF)) for different lanes of the comparative and example flow cells. As shown, the %PF is more consistent over a wider concentration range for the example flow cell lanes including the hydrogel compared to the comparative flow cell lanes with the hydrogel.

[0211] Based on whether the reads align to the exact same genomic location, duplicate templates are removed bioinformatically from the example flow cell lanes and the comparative flow cell lanes. The net %PF after duplicate removal is as Figure 7 shown. Overall, using sequencing templates in the concentration range of 300 pM to 800 pM, a higher yield (an increase in yield from about 2% to about 17%) can be obtained using the hydrogel-coated flow cell when compared to the comparative flow cell.

[0212] For the comparative flow cell, the maximum %PF after duplicate removal is 76.13% in the lane exposed to the 300 pM sequencing template solution. For the example flow cell, the maximum %PF after duplicate removal is 83.42% in the lane exposed to the 600 pM sequencing template solution. This represents an increase of 9.6% in monoclonal clusters.

[0213] Figure 8A and 8B illustrates the read 1 and read 2 mismatch rates (MMR) of the comparative and example flow cells after 150 sequencing cycles. Similar mismatch rates on the comparative and example flow cells indicate that the hydrogel coating does not adversely affect the sequencing operation.

[0214] Other Notes

[0215] It should be understood that all combinations of the foregoing concepts and other concepts discussed in more detail below (assuming such concepts are not mutually inconsistent) are considered to be part of the inventive subject matter disclosed herein. In particular, all combinations of the claimed subject matter appearing at the end of this disclosure are considered to be part of the inventive subject matter disclosed herein. It should also be understood that the terms explicitly employed herein, and which may also appear in any disclosure incorporated by reference, should be accorded a meaning most consistent with the particular concepts disclosed herein.

[0216] References to "one example", "another example", "example", etc. throughout the specification mean that a particular element (e.g., a feature, structure, and / or characteristic) described in connection with that example is included in at least one example described herein and may or may not be present in other examples. Additionally, it should be understood that, unless the context clearly dictates otherwise, the elements described for any example may be combined in any suitable manner in any example.

[0217] It should be understood that the ranges provided herein include the stated range and any value or sub-range within the stated range. For example, a range from 1 to 50,000 should be interpreted to include not only the explicitly recited limits of 1 to 50,000, but also individual values such as approximately 708, approximately 945, approximately 3500, etc., and sub-ranges such as, for example, from approximately 825 to approximately 29,000, from approximately 95 to approximately 40,000, etc. Further, when the terms "about" and / or "substantially" are used to describe a value, they are intended to encompass minor variations from the stated value (up to + / - 10%).

[0218] Although several examples have been described in detail, it should be understood that the disclosed examples may be modified. Accordingly, the foregoing description should be considered non-limiting.

Claims

1. A flow cell, comprising a patterned substrate including recesses separated by interstitial regions and including bonding regions; sequencing surface chemistries attached to each of the recesses, the sequencing surface chemistries including: a functionalized coating; and primers grafted to the functionalized coating; a non-grafted hydrogel on at least the sequencing surface chemistries, the non-grafted hydrogel selected from crosslinked polyacrylamide, agarose gel, and crosslinked polyethylene glycol; a lid attached to the patterned substrate at the bonding regions; and a flow channel defined between the patterned substrate and the lid; wherein the non-grafted hydrogel is also on at least some of the interstitial regions; wherein the functionalized coating is poly(N-(5-azidoacetamido pentyl) acrylamide-co-acrylamide).

2. A method of preparing a flow cell, comprising: applying a functionalized coating in recesses of a patterned flow cell substrate, wherein the recesses are separated by interstitial regions and wherein the patterned flow cell substrate includes bonding regions; grafting primers to the functionalized coating to form a grafted functionalized coating in the recesses; applying a non-grafted hydrogel on at least the grafted functionalized coating, the non-grafted hydrogel selected from crosslinked polyacrylamide, agarose gel, and crosslinked polyethylene glycol; bonding a lid to the patterned flow cell substrate at the bonding regions, thereby defining a flow channel between the patterned flow cell substrate and the lid; wherein the non-grafted hydrogel is disposed on the grafted functionalized coating and on some of the interstitial regions; wherein prior to applying the functionalized coating, the method further includes treating a surface of the patterned flow cell substrate to attach functional groups to the surface, thereby forming treated recesses and treated interstitial regions.

3. The method as defined in claim 2, wherein applying the functionalized coating in the recesses includes: applying the functionalized coating in the treated recesses and on the treated interstitial regions; and polishing the functionalized coating from the treated interstitial regions.

4. The method as defined in claim 2, wherein applying the non-grafted hydrogel includes applying an aqueous mixture comprising from 0.001% to up to 0.1% mass / volume of hydrogel material.

5. The method as defined in claim 2, wherein the bonding regions of the patterned flow cell substrate have an intervening layer bonded thereto, and wherein bonding the lid to the patterned flow cell substrate after applying the non-grafted hydrogel includes bonding the lid to the intervening layer.

6. The method as defined in claim 2, wherein applying the non-grafted hydrogel includes selectively depositing the non-grafted hydrogel on the grafted functionalized coating.

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