Patent Description:
Biological arrays may be used for genetic sequencing. In general, genetic sequencing involves determining the order of nucleotides or nucleic acids in a length of genetic material, such as a fragment of DNA or RNA. Increasingly longer sequences of base pairs are being analyzed, and the resulting sequence information may be used in various bioinformatics methods to logically fit fragments together so as to reliably determine the sequence of extensive lengths of genetic material from which the fragments were derived. Automated, computer-based examination of characteristic fragments have been developed, and have been used in genome mapping, identification of genes and their function, evaluation of risks of certain conditions and disease states, and so forth. Beyond these applications, biological arrays may be used for the detection and evaluation of a wide range of molecules, families of molecules, genetic expression levels, single nucleotide polymorphisms, and genotyping. <CIT>, <CIT> and <CIT> disclose novel heteropolymers comprising substituted acrylamide recurring units used for surface functionalization and in DNA sequencing.

The present invention is solely defined in the appended claims.

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

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

The terms top, bottom, lower, upper, on, and related terms, are used herein to describe the flow cell and/or the various components of the flow cell. It is to be understood that these directional terms are not meant to imply a specific orientation but are used to designate relative orientation between components. The use of directional terms should not be interpreted to limit the examples disclosed herein to any specific orientation(s).

As used herein, "acetal group" refers to a functional group with the following connectivity R<NUM>C(OR')<NUM>, where the R groups and R' groups are each organic fragments. Acetal groups include acetals, ketals, hemiacetals, and hemiketals. In an acetal, one R group is H. In some aspects, R' is C<NUM>-<NUM>alkyl, or the two R' groups taken together form a C<NUM>-<NUM>alkylene. An acetal protecting group can be used to protect a hydroxyl group, a <NUM>,<NUM>-diol, or a <NUM>,<NUM>-diol.

An "acrylate group" includes the salts, esters, and conjugate bases of acrylic acid and its derivatives (e.g., methacrylic acid). The acrylate ion has the molecular formula CH<NUM>=CHCOO-.

An "acrylamide monomer" is a monomer with the structure
<CHM>
or a substituted analog thereof (e.g., methacrylamide or N-isopropylacrylamide). An example of a monomer including an acrylamide group and an azido group is azido acetamido pentyl acrylamide:
<CHM>.

As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). The alkyl group may have <NUM> to <NUM> carbon atoms. Example alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like. As an example, the designation "C1-<NUM> alkyl" indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, isobutyl, sec-butyl, and t-butyl.

As used herein, "alkenyl" refers to a straight or branched hydrocarbon chain containing one or more double bonds. The alkenyl group may have <NUM> to <NUM> carbon atoms. Example alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like.

As used herein, "alkyne" or "alkynyl" refers to a straight or branched hydrocarbon chain containing one or more triple bonds. The alkynyl group may have <NUM> to <NUM> carbon atoms.

As used herein, "aryl" refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent carbon atoms) containing only carbon in the ring backbone. When the aryl is a ring system, every ring in the system is aromatic. The aryl group may have <NUM> to <NUM> carbon atoms. Examples of aryl groups include phenyl, naphthyl, azulenyl, and anthracenyl.

As used herein, the term "attached" refers to the state of two things being joined, fastened, adhered, connected, or bound to each other, either covalently or non-covalently (e.g., by hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions and hydrophobic interactions). For example, a nucleic acid can be attached to a functionalized polymer by a covalent or non-covalent bond.

An "azide" or "azido" functional group refers to -N<NUM>.

As used herein, the "bonding region" refers to an area on a substrate that is to be bonded to another material, which may be, as examples, a spacer layer, a lid, another substrate, etc., or combinations thereof (e.g., a spacer layer and a lid). The bond that is formed at the bonding region may be a chemical bond (as described above), or a mechanical bond (e.g., using a fastener, etc.).

A "tert-butyloxycarbonyl group" (Boc) refers to a
<CHM>
group. A "butyloxycarbonyloxy group" refers to a -OCO<NUM>tBu group.

As used herein, "carbocyclyl" means a non-aromatic cyclic ring or ring system containing only carbon atoms in the ring system backbone. When the carbocyclyl is a ring system, two or more rings may be joined together in a fused, bridged or spiro-connected fashion. Carbocyclyls may have any degree of saturation, provided that at least one ring in a ring system is not aromatic. Thus, carbocyclyls include cycloalkyls, cycloalkenyls, and cycloalkynyls. The carbocyclyl group may have <NUM> to <NUM> carbon atoms. Examples of carbocyclyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, <NUM>,<NUM>-dihydro-indene, bicyclo[<NUM>. <NUM>]octanyl, adamantyl, and spiro[ <NUM>]nonanyl.

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

As used herein, "cycloalkylene" means a fully saturated carbocyclyl ring or ring system that is attached to the rest of the molecule via two points of attachment.

As used herein, "cycloalkenyl" or "cycloalkene" means a carbocyclyl ring or ring system having at least one double bond, wherein no ring in the ring system is aromatic. Examples include cyclohexenyl or cyclohexene and norbornenyl or norbornene. Also as used herein, "heterocycloalkenyl" or "heterocycloalkene" means a carbocyclyl ring or ring system with at least one heteroatom in ring backbone, having at least one double bond, wherein no ring in the ring system is aromatic.

As used herein, "cycloalkynyl" or "cycloalkyne" means a carbocyclyl 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 bicyclononyne. Also as used herein, "heterocycloalkynyl" or "heterocycloalkyne" means a carbocyclyl ring or ring system with at least one heteroatom in ring backbone, having at least one triple bond, wherein no ring in the ring system is aromatic.

The term "depositing," as used herein, refers to any suitable application technique, which may be manual or automated, and results in modification of the surface properties. Generally, depositing may be performed using vapor deposition techniques, coating techniques, grafting techniques, or the like. Some specific examples include chemical vapor deposition (CVD), spray coating (e.g., ultrasonic spray coating), spin coating, dunk or dip coating, doctor blade coating, puddle dispensing, flow through coating, aerosol printing, screen printing, microcontact printing, inkjet printing, or the like.

As used herein, the term "depression" refers to a discrete concave feature in a patterned support having a surface opening that is completely surrounded by interstitial region(s) of the patterned support surface. Depressions can have any of a variety of shapes at their opening in a surface including, as examples, round, elliptical, square, polygonal, star shaped (with any number of vertices), etc. The cross-section of a depression taken orthogonally with the surface can be curved, square, polygonal, hyperbolic, conical, angular, etc. As an example, the depression can be a well. Also as used herein, a "functionalized depression" refers to the discrete concave feature where the polymer disclosed herein and primer(s) are attached.

The term "each," when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.

As used herein, the term "flow cell" is intended to mean a vessel having a chamber (i.e., flow channel) where a reaction can be carried out, an inlet for delivering reagent(s) to the chamber, and an outlet for removing reagent(s) from the chamber. In some examples, the chamber enables the detection of a reaction or signal that occurs in the chamber. For example, the chamber can include one or more transparent surfaces allowing for the optical detection of arrays, optically labeled molecules, or the like, in the chamber.

As used herein, a "flow channel" or "flow channel region" may be an area defined between two bonded components, which can selectively receive a liquid sample. In some examples, the flow channel may be defined between a patterned support and a lid, and thus may be in fluid communication with one or more depressions defined in the patterned support. In other examples, the flow channel may be defined between a non-patterned support and a lid.

A "fluorenylmethyloxycarbonyl" group (Fmoc) is a base-labile protecting group having a structure
<CHM>.

As used herein, "heteroaryl" refers to an aromatic ring or ring system (e.g., two or more fused rings that share two adjacent atoms) that contain(s) one or more heteroatoms, that is, an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur, in the ring backbone. When the heteroaryl is a ring system, every ring in the system is aromatic. The heteroaryl group may have <NUM>-<NUM> ring members.

As used herein, "heterocyclyl" means a non-aromatic cyclic ring or ring system containing at least one heteroatom in the ring backbone. Heterocyclyls may be joined together in a fused, bridged, or spiro-connected fashion. Heterocyclyls may have any degree of saturation provided that at least one ring in the ring system is not aromatic. In the ring system, the heteroatom(s) may be present in either a non-aromatic or aromatic ring. The heterocyclyl group may have <NUM> to <NUM> ring members (i.e., the number of atoms making up the ring backbone, including carbon atoms and heteroatoms). In some examples, the heteroatom(s) are O, N, or S.

The term "hydrazine" or "hydrazinyl" as used herein refers to an optionally substituted -NHNH<NUM> group.

As used herein, the term "hydrazone" or "hydrazonyl" as used herein refers to a
<CHM>
group in which Ra and Rb are each independently selected from hydrogen, C1-<NUM> alkyl, C2-<NUM> alkenyl, C2-<NUM> alkynyl, C3-<NUM> carbocyclyl, C6-<NUM> aryl, <NUM>-<NUM> membered heteroaryl, and <NUM>-<NUM> membered heterocyclyl, as defined herein.

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

As used herein, the term "interstitial region" refers to an area in a support or on a surface that separates depressions. For example, an interstitial region can separate one feature of an array from another feature of the array. The two features that are separated from each other can be discrete, i.e., lacking physical contact with each other. In another example, an interstitial region can separate a first portion of a feature from a second portion of a feature. In many examples, the interstitial region is continuous whereas the features are discrete, for example, as is the case for a plurality of wells defined in an otherwise continuous surface. The separation provided by an interstitial region can be partial or full separation. Interstitial regions may have a surface material that differs from the surface material of the features defined in the surface. For example, features of an array can have an amount or concentration of the coating layer and primer(s) that exceeds the amount or concentration present at the interstitial regions. In some examples, the coating layer and primer(s) may not be present at the interstitial regions.

As used herein, a "nucleotide" includes a nitrogen-containing heterocyclic base, a sugar, and one or more phosphate groups. Nucleotides are monomeric units of a nucleic acid sequence. In RNA, the sugar is a ribose, and in DNA, the sugar is a deoxyribose, i.e. a sugar lacking a hydroxyl group that is present at the <NUM>' position in ribose. The nitrogen containing heterocyclic base (i.e., nucleobase) can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G), and modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and modified derivatives or analogs thereof. The C-<NUM> atom of deoxyribose is bonded to N-<NUM> of a pyrimidine or N-<NUM> of a purine.

As used herein, "plasma ashing" refers to a process of removing organic matter from a support by an oxygen plasma. The products that result from plasma ashing may be removed with a vacuum pump/system. Plasma ashing can activate the support by introducing reactive hydroxyl groups.

The "heteropolymer" or "heteropolymer coating layer" referred to herein is intended to mean a large molecule of at least two different repeating subunits (monomers), wherein one of the repeating subunits (monomers) includes a stimuli-responsive functional group.

As used herein, the "primer" is defined as a single stranded nucleic acid sequence (e.g., single strand DNA or single strand RNA) that serves as a starting point for DNA or RNA synthesis. The <NUM>' terminus of the primer may be modified to allow a coupling reaction with the functionalized polymer layer. The primer length can be any number of bases long and can include a variety of non-natural nucleotides. In an example, the primer is a short strand, ranging from <NUM> to <NUM> bases.

As used herein, the terms "silane" and "silane derivative" refer to an organic or inorganic compound containing one or more silicon atoms. An example of an inorganic silane compound is SiH<NUM>, or halogenated SiH<NUM> where hydrogen is replaced by one or more halogen atoms. An example of an organic silane compound is X-RB-Si(ORC)<NUM>, wherein R-Si is an organic linker, and wherein X is a functional group, such as amino, vinyl, methacrylate, epoxy, sulfur, alkyl, alkenyl, or alkynyl; RB is a spacer, for example -(CH<NUM>)n-, wherein n is <NUM> to <NUM>; RC is selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted <NUM>-<NUM> membered heteroaryl, and optionally substituted <NUM>-<NUM> membered heterocyclyl, as defined herein. As used herein, the terms "silane" and "silane derivative" can include mixtures of different silane and/or silane derivative compounds.

A "spacer layer," as used herein refers to a material that bonds two components together. In some examples, the spacer layer can be a radiation-absorbing material that aids in bonding or can be put into contact with a radiation-absorbing material that aids in bonding.

A "stimuli-responsive functional group," as used herein, refers to a moiety of atoms and/or bonds within the polymer that can change its state in response to a stimulus. The stimuli-responsive functional group may be pH-responsive, temperature-responsive, saccharide-responsive, nucleophile-responsive, or salt-responsive. Specific examples of each stimuli-responsive functional group will be described further below.

The term flow cell "support" or "substrate" refers to a support or substrate upon which surface chemistry may be added. The term "patterned substrate" refers to a support in which or on which depressions are defined. The term "non-patterned substrate" refers to a substantially planar support. The substrate may also be referred to herein as a "support," "patterned support," or "non-patterned support. " The support may be a wafer, a panel, a rectangular sheet, a die, or any other suitable configuration. The support is generally rigid and is insoluble in an aqueous liquid. The support may be inert to a chemistry that is used to modify the depressions. For example, a support can be inert to chemistry used to form the polymer coating layer, to attach the primer(s) to the polymer coating layer, etc. Examples of suitable supports include epoxy siloxane, glass and modified or functionalized glass, polyhedral oligomeric silsequioxanes (POSS) and derivatives thereof, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, polytetrafluoroethylene (such as TEFLON® from Chemours), cyclic olefins/cyclo-olefin polymers (COP) (such as ZEONOR® from Zeon), polyimides, etc.), nylon, ceramics/ceramic oxides, silica, fused silica, or silica-based materials, aluminum silicate, silicon and modified silicon (e.g., boron doped p+ silicon), silicon nitride (Si<NUM>N<NUM>), silicon oxide (SiO<NUM>), tantalum pentoxide (TaO<NUM>) or other tantalum oxide(s) (TaOx), hafnium oxide (HaO<NUM>), carbon, metals, inorganic glasses, or the like. The support may also be glass or silicon or a silicon-based polymer such as a POSS material, optionally with a coating layer of tantalum oxide or another ceramic oxide at the surface.

The term "surface chemistry," as used herein, refers to chemically and/or biologically active component(s) that are incorporated into the channel of the flow cell. Examples of the surface chemistry disclosed herein include the polymer coating layer attached to at least a portion of a surface of the support and the primer attached to at least a portion of the polymer coating layer.

A "thiol" functional group refers to -SH.

As used herein, the terms "tetrazine" and "tetrazinyl" refer to six-membered heteroaryl group comprising four nitrogen atoms. Tetrazine can be optionally substituted.

"Tetrazole," as used herein, refer to five-membered heterocyclic group including four nitrogen atoms. Tetrazole can be optionally substituted.

Examples of the flow cell disclosed herein include a support, a polymer attached to the support, and a primer grafted to the polymer. Examples of the flow cells are shown in <FIG>, <FIG>, and 3D, and will be described further herein. Various examples of the polymer that is attached to the flow cell support will now be described.

Reference throughout the specification to "one example", "another example", "an example", and so forth, means that a particular element (e.g., feature, structure, and/or characteristic) described in connection with the example is included in at least one example described herein, and may or may not be present in other examples. In addition, it is to be understood that the described elements for any example may be combined in any suitable manner in the various examples unless the context clearly dictates otherwise.

It is to be understood that the ranges provided herein include the stated range and any value or sub-range within the stated range, as if such ranges, values or sub-ranges were explicitly recited herein. For example, a range from about <NUM> to about <NUM> (<NUM>), should be interpreted to include not only the explicitly recited limits of from about <NUM> to about <NUM>, but also to include individual values, such as about <NUM>, about <NUM>, etc., and sub-ranges, such as from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, etc. Furthermore, when "about" and/or "substantially" are/is utilized to describe a value, they are meant to encompass minor variations (up to +/- <NUM>%) from the stated value.

The heteropolymers described herein comprise a stimuli-responsive functional group that is capable of undergoing modification when exposed to a predetermined stimulus, wherein the modification changes the polarity and/or conformation of the heteropolymer. Therefore, inventive examples of the heteropolymer disclosed herein are switchable. The switchable heteropolymers can transition from a starting state to a second, switched state upon exposure to the particular stimulus. Examples of switches include changes to polarity such as hydrophobic to hydrophilic, increasing hydrophilicity, hydrophilic to hydrophobic, increasing hydrophobicity, neutral to charged, charged to neutral, anionic to neutral, cationic to neutral, neutral to anionic, neutral to cationic, and neutral to neutral with increased hydrophilicity. Examples of switches also include conformational changes such as swelling, collapsed state to extended state, extended state to collapsed state (e.g., with antipolyelectrolyte behavior), and coil-globule formation. A given stimuli-responsive group may impart more than one switching effect. In some aspects, the switch is irreversible, and in other examples, the switch is reversible under different chemical or thermal conditions.

The stimuli-responsive functional group exhibits a starting state when the switchable heteropolymer is applied to the flow cell support. In some aspects, the starting state is compatible with the hydrophobic nature of a flow cell support, and this compatibility eases manufacture and handling of the flow cell. For example, the starting state (e.g., hydrophobic) may improve adhesion to and coating uniformity of the heteropolymer to a hydrophobic flow cell support, while the switched state may be more compatible with flow cell uses such as sequencing operations.

Thus, in some aspects, the switched state may be a solution conformation that provides improved performance in some applications, including sequencing operations. Prior to a sequencing operation, the stimuli-responsive polymers disclosed herein may be exposed to the predetermined stimulus. Upon exposure to the stimulus, the switchable heteropolymer changes polarity and/or conformation due to the effects of the predetermined stimulus on the stimuli-responsive functional group. The heteropolymer in the switched state(s) can provide a low-fouling surface that may reduce non-specific adsorption of proteins and that may improve sequencing metrics (e.g., base or <NUM>st cycle intensity, quality scores, error rates, etc.).

One of ordinary skill will recognize that any of the heteropolymers described herein may be random, block, linear, and/or branched copolymers comprising two or more recurring monomer units in any order or configuration, and may be linear, cross-linked, or branched, or a combination thereof.

In some examples of the disclosure, the stimuli-responsive functional group is a pH-responsive functional group. In such aspects, the switchable heteropolymer is a copolymer comprising a plurality of monomers comprising a pH-responsive functional group. The plurality of monomers may each have the same pH-responsive functional group or different pH-responsive functional groups that respond to the same pH condition. In some aspects, the heteropolymer is a copolymer with a plurality of acrylamide monomers. A single type of acrylamide monomer may be used, or two or more different acrylamide monomers may be used.

In some aspects, a pH-responsive functional group is converted to a substituent group with increased or decreased polarity (e.g., increased hydrophilicity or increased hydrophobicity) upon exposure to acidic or basic pH conditions. In some aspects, the pH-responsive functional group is neutral and becomes charged upon exposure to the stimulus. In some aspects, the pH-responsive functional group is charged and becomes neutral upon exposure to the stimulus. In some aspects, the pH-responsive functional group is neutral and is converted to a different, neutral, but more polar, group upon exposure to the stimulus.

In some aspects, a pH-responsive functional group is a hydroxyl with an acid-labile protecting group (switches to a more hydrophilic free hydroxyl upon exposure to acidic/low pH conditions), a hydroxyl with a base-labile protecting group (switches to a more hydrophilic free hydroxyl upon exposure to basic/high pH conditions), an amino with an acid-labile protecting group (switches to a more hydrophilic free amino group upon exposure to acidic/low pH conditions), an amino with a base-labile protecting group (switches to a more hydrophilic free amino group upon exposure to basic/high pH conditions), an amino group (switches to an ammonium ion under acidic/low pH conditions), a carboxylate (-CO<NUM>-) group (switches to a neutral carboxylic acid upon exposure to acidic/low pH conditions, a carboxylic acid group (switches to a charged and more hydrophilic carboxylate upon exposure to basic/high pH conditions), a sulfonate (-SO<NUM>-) group (switches to a neutral sulfonic acid upon exposure to acidic/low pH conditions), or a sulfonic acid group (switches to a charged and more hydrophilic sulfonate upon exposure to basic/high pH conditions).

An exemplary switchable heteropolymer comprises a monomer of the following structure:
<CHM>
where:.

In some aspects, X is -O-Boc, -NHBoc, -NHFmoc, -NH<NUM>, -NHCH<NUM>, or -N(CH<NUM>)<NUM>. In some aspects, X is SO<NUM>H, -SO<NUM>-, -CO<NUM>H, or -CO<NUM>-. In some aspects, Ra and Rb are both methyl. In some aspects, Rz is H or methyl. In some aspects, the monomer has the structure:
<CHM>.

Thus, in some aspects, the pH-responsive functional group is a hydroxyl, <NUM>,<NUM>,-diol, or <NUM>,<NUM>-diol protected as an acetal, hemiacetal, or ketal (switches to a more polar/hydrophilic diol upon exposure to acidic/low pH conditions), a tert-butyloxycarbonylamino group, a <NUM>-fluoren-<NUM>-ylmethoxycarbonylamino group, an amino group, a carboxylate (-CO<NUM>-) group, a carboxylic acid group, a sulfonate (-SO<NUM>-) group, or a sulfonic acid group.

A tert-butoxycarbonylamino group may be in a hydrophobic (or less hydrophilic) state, and when exposed to a low (acidic) pH (e.g., having a pH less than <NUM>), may transition to a hydrophilic state (e.g., an amino group). The tert-butoxycarbonylamino group may be attached to an acrylamide monomer or an acrylate monomer. Examples of the tert-butoxycarbonylamino group containing monomer include N-(tert-butoxycarbonyl-aminoethyl) methacrylamide, N-(tert-butoxycarbonyl-aminopropyl) methacrylamide, and (<NUM>-tert-butoxycarbonyl-amino)ethyl methacrylate.

The <NUM>-fluoren-<NUM>-ylmethoxycarbonylamino group may be in a hydrophobic (or less hydrophilic) state, and when exposed to a low (acidic) pH (e.g., having a pH less than <NUM>), may transition to a hydrophilic state (e.g., an amino group). The <NUM>-fluoren-<NUM>-ylmethoxycarbonylamino group may be attached to an acrylamide monomer, or an acrylate monomer, or a vinyl monomer. An example of the <NUM>-fluoren-<NUM>-ylmethoxycarbonyl group containing monomer includes:
<CHM>.

The amino group may be in a neutral state, and when exposed to low (acidic) pH (e.g., having a pH less than <NUM>), may transition to a charged (and more hydrophilic) state (cationic). For example, the amino groups in the synthesized polymer may be protonated, which leads to cationic charges around the polymer backbone. In the synthesized polymer, the amino group may be attached to an acrylamide monomer, or an acrylate monomer, or a vinyl monomer. Examples of an amino group containing monomer include <NUM>-(dimethylamino)ethyl methacrylate, <NUM>-(N,N-dimethylamino)ethyl acrylate, N-[<NUM>-(N,N-dimethylamino)propyl] acrylamide, N-[<NUM>-(N,N-dimethylamino)ethyl]methacrylamide, and N-[<NUM>-(N,N-dimethylamino)propyl] methacrylamide.

An acetal group, when exposed to low (acidic) pH (e.g., pH less than <NUM>), may transition to a more hydrophilic state than its starting state (a hydroxyl or diol). The acetal group may be attached to azide-functionalized hyaluronic acid (HA-N<NUM>). HA-N<NUM> has limited solubility in organic solvents, and thus may be converted to its tetrabutylammonium salt using acidic ion exchange resin prior to the acetalation reaction. The acetalation reaction may be performed by reacting, at room temperature, <NUM>-methoxypropene and pyridinium p-toluenesulfonate with the HA-N<NUM> salt solubilized in dimethyl sulfoxide (DMSO).

In some aspects, the switchable heteropolymer further comprises an azido-containing acrylamide monomer. In some aspects, the switchable heteropolymer comprises:
<CHM>
and optionally
<CHM>
In some aspects, the switchable heteropolymer comprises the structure:
<CHM>
where each Rz is independently H or C<NUM>-<NUM>alkyl. In some examples, X is -O-Boc, -NHBoc, - NHFmoc, -NH<NUM>, -NHCH<NUM>, or -N(CH<NUM>)<NUM>. In some examples, X is -NHBoc. In some aspects, X is SO<NUM>H, -SO<NUM>-, -CO<NUM>H, or -CO<NUM>-. In some aspects, Ra and Rb are both methyl. In some aspects, each Rz is independently H or methyl.

In some examples of the disclosure, the switchable heteropolymer comprises two pH-responsive acetal functional groups (such as one ketal group and one hemiketal group) and a carboxylic acid group. An exemplary heteropolymer has the following structure:
<CHM>
wherein n ranges from <NUM> to <NUM>. The acid-labile acetal groups protect a plurality of alcohols on the polar dextran backbone of the azido-modified hyaluronic acid (HA-N<NUM>), and change the character of the heteropolymer from hydrophilic to hydrophobic. The heteropolymer in its more hydrophobic starting state may be easier to handle and process, allowing the heteropolymer to coat or adhere to the hydrophobic supports disclosed herein (e.g., norbornene-functionalized glass or POSS substrates) more effectively.

In some examples of the disclosure, the stimuli-responsive functional group is a temperature-responsive functional group. In such aspects, the switchable heteropolymer is a copolymer comprising a plurality of monomers comprising a temperature-responsive functional group. The plurality of monomers may each have the same temperature-responsive functional group or different temperature-responsive functional groups that respond to the same temperature condition. In some aspects, the heteropolymer is a copolymer with a plurality of acrylamide monomers. A single type of acrylamide monomer may be used, or two or more different acrylamide monomers may be used.

A temperature-responsive functional group is one that can be converted to a more or less polar functional group or causes conformational change to the polymer due to a temperature change. For example, a temperature-responsive group includes a heat-sensitive hydroxyl or amino protecting group (such as a Boc or Fmoc group) that is removed upon exposure of the heteropolymer to heat (switching from neutral starting state to neutral with increased hydrophilicity). A variety of Boc and Fmoc-protected monomers may be used including acrylic monomers. In another example, a temperature-responsive functional group can cause the polymer to exist in an extended starting state that is neutral and relatively hydrophilic at room temperature and then switch the heteropolymer to a collapsed state that is neutral and relatively hydrophobic at an elevated temperature (such as above <NUM>). This other example is a coil-globule switch, which may be exhibited, for example, by poly(N-isopropylacrylamide). It is to be understood that the primers grafted to this switchable heteropolymers may alter this behavior slightly. In some aspects, this polymeric material undergoes a thermal coil-to-globule transition. In some aspects, the temperature-responsive functional group is part of a polymer that is an ionizable, thermosensitive gel. In some aspects, the monomer comprising the temperature responsive functional group is an N-substituted acrylamide, such as H<NUM>C=C(H or methyl)-C(O)NRcRd, where Rc is H and Rd is a branched C<NUM>-<NUM>alkyl. In some aspects, the monomer comprising the temperature-responsive functional group is N-isopropylacrylamide, optionally in a block of poly(N-isopropylacrylamide).

In an example, the heteropolymer comprises a temperature-responsive functional group monomer and an acrylamide monomer. In some examples, the acrylamide monomer is selected from the group consisting of an azido acetamido pentyl acrylamide monomer and a combination of an acrylamide monomer and the azido acetamido pentyl acrylamide monomer as shown above. In some aspects, the switchable heteropolymer further comprises an azido-containing acrylamide monomer. In some aspects, the switchable heteropolymer comprises:
<CHM>
and optionally
<CHM>
In some aspects, the switchable heteropolymer comprises the structure:
<CHM>
where each Rz is independently H or C<NUM>-<NUM>alkyl.

In some examples of the invention, the stimuli-responsive functional group is a saccharide-responsive functional group, which is a hydrophilic substituent group that reacts with a diol reagent to form an anionic functional group. In some aspects, the diol is an organic diol, or a sugar, or glucose. In the present invention, the saccharide-responsive functional group comprises a boronic acid, such as an alkyl boronic acid or an aryl boronic acid. The boronic acid functional group may be in a charge neutral starting state (and may also be relatively hydrophobic), and when exposed to a saccharide solution, may transition to a negatively charged (anionic) state (that may also be more hydrophilic than the charge neutral state). Boronic acids have the ability to react with saccharides to form boronate esters that undergo reversible swelling due to an influx of water, which may be desirable during sequencing operations.

The boronic acid functional group is attached to an acrylamide monomer, or an acrylate monomer, or a vinyl monomer. In an example, the monomer comprising the saccharide-responsive functional group has the structure:
<CHM>
An example of the boronic acid group containing monomer includes <NUM>-(acrylamido)phenylboronic acid. In some aspects, the switchable heteropolymer further comprises an acrylamide monomer. In some aspects, the acrylamide monomer is an azido-containing acrylamide monomer. In some aspects, the switchable heteropolymer comprises:
<CHM>
and optionally
<CHM>
In some aspects, the heteropolymer has a structure:
<CHM>
where each Rz is independently H or C<NUM>-<NUM>alkyl.

In some some examples of the disclosure, the stimuli-responsive functional group is a nucleophile-responsive functional group. A nucleophile-responsive functional group is a group that is susceptible to attack by a nucleophile to effect a structural change that confers a change in polarity and/or conformation as described herein. In some aspects, the switchable heteropolymer is a copolymer comprising a plurality of monomers comprising a nucleophile-responsive functional group. The plurality of monomers may each have the same nucleophile-responsive functional group or different nucleophile-responsive functional groups that respond to the same nucleophile. In some aspects, the heteropolymer is a copolymer of the monomer comprising a nucleophile-responsive functional group and one or more acrylamide monomers. A single type of acrylamide monomer may be used, or two or more different acrylamide monomers may be used. In some examples, the nucleophile-responsive functional group is a cyclic sulfonate ester (such as a sultone ring) or a cyclic anhydride (such as succinic anhydride) that can undergo a ring-opening reaction upon exposure to a nucleophile, in some cases under basic (high pH) conditions such as pH <NUM> or greater.

In some aspects, the nucleophile-responsive functional group has the following structure:
<CHM>
where (a) Y is SO<NUM> and Y' is CH<NUM>; or (b) Y and Y' are both C(O). In other aspects, the nucleophile-responsive functional group is:
<CHM>
Suitable nucleophiles include primary alkyl amines and alkyl alcohols. An example of the sultone ring opening group and its ring opening reaction is as follows:
<CHM>
where M is H or a monovalent cation (sodium or potassium cation). The sultone ring opening group may be in a hydrophobic (or less hydrophilic) state, and when exposed to a high (basic) pH, may undergo a ring opening reaction and transition to a (more) hydrophilic state. The functional group after the ring opening reaction may also be anionic, and thus in a charged state.

In some aspects, the monomer comprising the nucleophile-responsive functional group is:
<CHM>
In particular examples, the monomer is:
<CHM>.

In some aspects, the nucleophile-responsive functional group may be attached to an acrylamide monomer, or an acrylate monomer, or a vinyl monomer. In some aspects, the switchable heteropolymer further comprises an acrylamide monomer. In some examples, the acrylamide monomer is an azido-containing acrylamide monomer. In some aspects, the switchable heteropolymer comprises:
<CHM>
and optionally
<CHM>.

In some aspects, the heteropolymer has a structure:
<CHM>
where each Rz is independently H or C<NUM>-<NUM>alkyl.

In some examples of the invention, the stimuli-responsive functional group is a salt-responsive functional group. In some examples, the salt-responsive functional group is a zwitterionic functional group exhibiting antipolyelectrolyte behavior, wherein the zwitterionic functional group switches from a collapsed state to an extended state when exposed to a salt. The salt responsive functional group is a zwitterionic functional group having antipolyelectrolyte behavior. "Antipolyelectrolyte behavior," as used herein, means that the monomer including the zwitterionic functional group switches from a collapsed state to an extended state when exposed to a salt (i.e., the monomer possesses greater solubility in salt water than in pure water). As such, the salt responsive functional group may be in a collapsed state (e.g., where the polymer chains are in a globule), and when exposed to a salt solution, may transition to an extended state (i.e., where the polymer chains are extended). The impact of the local salt counterions changes the conformation of the polymer chain including the salt responsive functional group. In one example, a monomer including the zwitterionic functional group is selected from the group consisting of N-(<NUM>-methacryloyloxy)ethyl-N,N-dimethylammonio propanesulfonate andN-(<NUM>-methacryloylimino)propyl-N,N-dimethylammonio propanesulfonate.

In some examples, the monomer comprising the salt-responsive functional group has the structure:
<CHM>
where A is O or NH and Rz is H or C<NUM>-<NUM>alkyl.

In other examples, salt-responsive functional groups are quaternary ammonium groups such as -NMe<NUM>+. An exemplary monomer is:
<CHM>
where Rz is H or C<NUM>-<NUM>alkyl.

When combined with an anionic counterpart (present in a salt solution), these charged materials may exhibit antipolyelectrolyte behavior. Examples of suitable anionic counterparts include a carboxylate salt, a sulfonate salt, a citrate salt, a phosphate salt, etc..

The salt responsive functional group is attached to an acrylamide monomer, or an acrylate monomer, or a vinyl monomer. In some aspects, the switchable heteropolymer further comprises an acrylamide monomer. In some examples, the acrylamide monomer is an azido-containing acrylamide monomer. In some aspects, the switchable heteropolymer comprises:
<CHM>
and optionally
<CHM>.

In one inventive example, a flow cell comprises a support and a switchable heteropolymer attached to the support, wherein the stimuli-responsive functional group is selected from the group consisting of a saccharide-responsive functional group and a salt-responsive functional group. The stimuli-responsive functional group is capable of undergoing modification when exposed to a predetermined stimulus, wherein the modification changes the polarity and/or conformation of the switchable heteropolymer.

In some aspects, the flow cell support is a patterned substrate including depressions separated by interstitial regions, and wherein the heteropolymer is present within the depressions. In other aspects, the support is a non-patterned substrate having flow channel region and a bonding region, and wherein the heteropolymer is attached to the flow channel region.

In some aspects, the flow cell further comprises a primer grafted to the switchable heteropolymer.

In an example of this aspect of the flow cell, a surface of the support is functionalized with a silane or a silane derivative, and the heteropolymer is attached to the silane or the silane derivative. In some examples, the silane or silane derivative includes an unsaturated moiety that is capable of reacting with a functional group of the functionalized polymer layer. As used herein, the term "unsaturated moiety" refers to a chemical group which includes cycloalkenes, cycloalkynes, heterocycloalkenes, heterocycloalkynes, or optionally substituted variants thereof including at least one double bond or one triple bond. The unsaturated moieties can be mono-valent or di-valent. When the unsaturated moiety is mono-valent, cycloalkene, cycloalkyne, heterocycloalkene, and heterocycloalkyne are used interchangeably with cycloalkenyls, cycloalkynyls, heterocycloalkenyl, and heterocycloalkynyl, respectively. When the unsaturated moiety is di-valent, cycloalkene, cycloalkyne, heterocycloalkene, and heterocycloalkyne are used interchangeably with cycloalkenylene, cycloalkynylene, heterocycloalkenylene, and heterocycloalkynylene, respectively.

The unsaturated moiety can be covalently attached either directly to the silicon atoms of the silane or silane derivative, or indirectly attached via linkers. Examples of suitable linkers include optionally substituted alkylenes (e.g., bivalent saturated aliphatic radicals (such as ethylene) regarded as being derived from an alkene by opening of the double bond or from an alkane by removal of two hydrogen atoms from different carbon atoms), substituted polyethylene glycols, or the like.

The heteropolymers disclosed herein are made up of at least two different monomers. One of the monomers comprises a stimuli-responsive functional group. In some aspects, the other of the monomers includes an attachment group that may be reacted with the flow cell support and/or the primer to attach the heteropolymer thereto. This other attachment group may also be capable of attaching to the support, or the other monomer may include a second (different) attachment group that is capable of attaching to the support. It is to be understood that the polymers disclosed herein may also include one or more other monomers that do not interfere with the respective functions of the stimuli-responsive functional group and the attachment group.

In any examples of the polymer disclosed herein, the attachment group is selected from the group consisting of azido, amino, alkenyl (including cycloalkenyl or heterocycloalkenyl groups), alkynyl (including cycloalkynyl or heterocycloalkynyl groups), aldehyde, hydrazone, hydrazine, carboxyl, hydroxy, tetrazole, tetrazine, and thiol.

The attachment group may be capable of reacting with a functional group attached to the <NUM>' end of the primer. For example, a bicyclo[<NUM>. <NUM>] non-<NUM>-yne (BCN) terminated primer may be captured by an azide attachment group of the polymer via strain-promoted catalyst free click chemistry. For another example, an alkyne terminated primer may be captured by an azide attachment group of the polymer via copper catalyzed click chemistry. For still another example, a norbornene terminated primer may undergo a catalyst-free ring strain promoted click reaction with a tetrazine attachment group of the polymer. It is to be understood that other coupling chemistries may be used to attach the primer to the attachment group, including, for example, Staudinger ligations, strain-promoted reactions, and photo-click cycloadditions.

Other examples of terminated primers that may be used include a tetrazine terminated primer, an azido terminated primer, an amino terminated primer, an epoxy or glycidyl terminated primer, a thiophosphate terminated primer, a thiol terminated primer, an aldehyde terminated primer, a hydrazine terminated primer, and a triazolinedione terminated primer.

In an example, the attachment group is attached to an acrylamide monomer. One example of the monomer that includes the attachment group is azido acetamido pentyl acrylamide.

In an example of the method, applying the polymer coating layer to a flow cell involves flow through deposition, chemical vapor deposition, dip coating, dunk coating, spin coating, spray coating, puddle dispensing, ultrasonic spray coating, doctor blade coating, aerosol printing, screen printing, microcontact printing, or inkjet printing.

In an example of the method, the flow cell support is a patterned flow cell support including depressions separated by interstitial regions, and the method further comprises: prior to applying the polymer coating layer, attaching a silane or a silane derivative to a surface of the patterned flow cell support, thereby forming silanized depressions and silanized interstitial regions; applying the polymer coating layer in the silanized depressions and on the silanized interstitial regions; and removing (e.g., polishing) the polymer coating layer from the silanized interstitial regions.

In an example of the method, exposing the polymer coating layer to the predetermined stimulus involves one of: heating the polymer coating layer; exposing the polymer coating layer to a solution of a predetermined pH; exposing the polymer coating layer to a nucleophile; exposing the polymer coating layer to a solution including a saccharide; or exposing the polymer coating layer to a salt solution.

In yet a further aspect, a method comprises exposing a polymer coating layer on at least a portion of a flow cell support to a predetermined stimulus, thereby causing a stimuli-responsive functional group of the polymer coating layer to switch i) from a current state to a more hydrophilic state than the current state, or ii) from a neutral state to a charged state, or iii) from a collapsed state to an extended state; and performing a sequencing operation using the flow cell support when the polymer coating layer is in the more hydrophilic state, the charged state, or the extended state.

In some aspects is a method for making the flow cells. The method includes applying a switchable heteropolymer to at least a portion of a flow cell support.

The addition of the polymer (polymer coating layer) and the primer (i.e., surface chemistry) to a patterned substrate will be described in reference to <FIG>, and in <FIG> in combination with <FIG>, and the addition of the surface chemistry to the non-patterned substrate will be described in reference to <FIG>.

<FIG> is a cross-sectional view of an example of the patterned support <NUM>. The patterned support <NUM> may be a patterned wafer or a patterned die or any other patterned support (e.g., panel, rectangular sheet, etc.). Any example of the support <NUM> described herein may be used. The patterned wafer may be used to form several flow cells, and the patterned die may be used to form a single flow cell. In an example, the support may have a diameter ranging from about <NUM> to about <NUM>, or a rectangular sheet or panel having its largest dimension up to about <NUM> feet (~ <NUM> meters). In an example, the support wafer has a diameter ranging from about <NUM> to about <NUM>. In another example, the support die has a width ranging from about <NUM> to about <NUM>. While example dimensions have been provided, it is to be understood that supports/substrates with any suitable dimensions may be used. For another example, a panel may be used that is a rectangular support, which has a greater surface area than a <NUM> round wafer.

The patterned support <NUM> includes depressions <NUM> defined on or in an exposed layer or surface of the support <NUM>, and interstitial regions <NUM> separating adjacent depressions <NUM>. In the examples disclosed herein, the depressions <NUM> become functionalized with surface chemistry (e.g., <NUM>, <NUM>), while the interstitial regions <NUM> may be used for bonding but will not have primer(s) (shown in <FIG> and <FIG>) present thereon.

The depressions <NUM> may be fabricated in or on the support <NUM> using a variety of techniques, including, for example, photolithography, nanoimprint lithography, stamping techniques, embossing techniques, molding techniques, microetching techniques, printing techniques, etc. As will be appreciated by those in the art, the technique used will depend on the composition and shape of the support <NUM>.

Many different layouts of the depressions <NUM> may be envisaged, including regular, repeating, and non-regular patterns. In an example, the depressions <NUM> are disposed in a hexagonal grid for close packing and improved density. Other layouts may include, for example, rectilinear (i.e., rectangular) layouts, triangular layouts, and so forth. In some examples, the layout or pattern can be an x-y format of depressions <NUM> that are in rows and columns. In some other examples, the layout or pattern can be a repeating arrangement of depressions <NUM> and/or interstitial regions <NUM>. In still other examples, the layout or pattern can be a random arrangement of depressions <NUM> and/or interstitial regions <NUM>. The pattern may include spots, pads, wells, posts, stripes, swirls, lines, triangles, rectangles, circles, arcs, checks, plaids, diagonals, arrows, squares, and/or cross-hatches.

The layout or pattern may be characterized with respect to the density of the depressions <NUM> (i.e., number of depressions <NUM>) in a defined area. For example, the depressions <NUM> may be present at a density of approximately <NUM> million per mm<NUM>. The density may be tuned to different densities including, for example, a density of at least about <NUM> per mm<NUM>, about <NUM>,<NUM> per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, or more. Alternatively or additionally, the density may be tuned to be no more than about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM> million per mm<NUM>, about <NUM>,<NUM> per mm<NUM>, about <NUM> per mm<NUM>, or less. It is to be further understood that the density of depressions <NUM> on the support <NUM> can be between one of the lower values and one of the upper values selected from the ranges above. As examples, a high-density array may be characterized as having depressions <NUM> separated by less than about <NUM>, a medium density array may be characterized as having depressions <NUM> separated by about <NUM> to about <NUM>, and a low density array may be characterized as having depressions <NUM> separated by greater than about <NUM>. While example densities have been provided, it is to be understood that substrates with any suitable densities may be used.

The layout or pattern may also or alternatively be characterized in terms of the average pitch, i.e., the spacing from the center of the depression <NUM> to the center of an adjacent interstitial region <NUM> (center-to-center spacing). The pattern can be regular, such that the coefficient of variation around the average pitch is small, or the pattern can be non-regular in which case the coefficient of variation can be relatively large. In either case, the average pitch can be, for example, at least about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or more. Alternatively or additionally, the average pitch can be, for example, at most about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or less. The average pitch for a particular pattern of sites <NUM> can be between one of the lower values and one of the upper values selected from the ranges above. In an example, the depressions <NUM> have a pitch (center-to-center spacing) of about <NUM>. While example average pitch values have been provided, it is to be understood that other average pitch values may be used.

In the example shown in <FIG>, the depressions <NUM> are wells <NUM>', and thus the patterned support <NUM> includes an array of wells <NUM>' in a surface thereof. The wells <NUM>' may be micro wells or nanowells. The size of each well <NUM>' may be characterized by its volume, well opening area, depth, and/or diameter.

Each well <NUM>' can have any volume that is capable of confining a liquid. The minimum or maximum volume can be selected, for example, to accommodate the throughput (e.g., multiplexity), resolution, analyte composition, or analyte reactivity expected for downstream uses of the flow cell. For example, the volume can be at least about <NUM>×<NUM>-<NUM> µm<NUM>, about <NUM>×<NUM>-<NUM> µm<NUM> , about <NUM><NUM>, about <NUM><NUM>, about <NUM><NUM>, about <NUM><NUM>, or more. Alternatively or additionally, the volume can be at most about <NUM>×<NUM><NUM> µm<NUM>, about <NUM>×<NUM><NUM> µm<NUM>, about <NUM><NUM>, about <NUM><NUM>, about <NUM><NUM>, about <NUM><NUM>, or less. It is to be understood that the functionalized coating layer can fill all or part of the volume of a well <NUM>'. The volume of the coating layer in an individual well <NUM>' can be greater than, less than or between the values specified above.

The area occupied by each well opening on a surface can be selected based upon similar criteria as those set forth above for well volume. For example, the area for each well opening on a surface can be at least about <NUM>×<NUM>-<NUM> µm<NUM>, about <NUM>×<NUM>-<NUM> µm<NUM>, about <NUM><NUM>, about <NUM><NUM>, about <NUM><NUM>, about <NUM><NUM>, or more. Alternatively or additionally, the area can be at most about <NUM>×<NUM><NUM> µm<NUM>, about <NUM><NUM>, about <NUM><NUM>, about <NUM><NUM>, about <NUM><NUM>, about <NUM>×<NUM>-<NUM> µm<NUM>, or less. The area occupied by each well opening can be greater than, less than or between the values specified above.

The depth of each well <NUM>' can be at least about <NUM>, about <NUM>, about <NUM>, about <NUM>, or more. Alternatively or additionally, the depth can be at most about <NUM>×<NUM><NUM> µm, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or less. The depth of each well <NUM>' can be greater than, less than or between the values specified above.

In some instances, the diameter of each well <NUM>' can be at least about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or more. Alternatively or additionally, the diameter can be at most about <NUM>×<NUM><NUM> µm, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or less (e.g., about <NUM>). The diameter of each well <NUM>' can be greater than, less than or between the values specified above.

The patterned support <NUM> may be exposed to a series of processes in order to add the surface chemistry <NUM>, <NUM> in the depression(s) <NUM>.

While not shown, it is to be understood that the patterned support <NUM> may be exposed to a plasma ashing in order to clean and activate the surface. For example, the plasma ashing process may remove organic material and introduce surface hydroxyl groups. Other suitable cleaning processes may be used to clean the support <NUM>, depending, in part, on the type of support <NUM>. For example, chemical cleaning may be performed with oxidizing agents or caustic solutions.

The patterned support <NUM> (shown in <FIG>) may then be exposed to a process that will prepare the surface for deposition of the stimuli-responsive polymer disclosed herein to form the polymer coating layer <NUM> (<FIG>). In an example, the patterned support <NUM> may be exposed to silanization, which attaches a silane or the silane derivative <NUM> (<FIG>) to the patterned support surface. Silanization introduces the silane or the silane derivative <NUM> across the surface, including in the depression <NUM>, <NUM>' (e.g., on the bottom surface and along the side walls) and on the interstitial regions <NUM>. In some aspects, the silane or silane derivative is selectively introduced only to the depressions of a patterned substrate or to micro-locations (which are isolated from each other) of a non-patterned substrate.

Silanization may be accomplished using any silane or silane derivative <NUM>. The selection of the silane or silane derivative <NUM> may depend, in part, upon the polymer that is to be used to form the polymer coating layer <NUM> (shown in <FIG>), as it may be desirable to form a covalent bond between the silane or silane derivative <NUM> and the polymer coating layer <NUM>. The method used to attach the silane or silane derivative <NUM> to the support <NUM> may vary depending upon the silane or silane derivative <NUM> that is being used. Several examples are set forth herein.

In an example, the silane or silane derivative <NUM> is (<NUM>-aminopropyl)triethoxysilane (APTES) or (<NUM>-aminopropyl)trimethoxysilane (APTMS) (i.e., X-RB-Si(ORC)<NUM>, wherein X is amino, RB is -(CH<NUM>)<NUM>-, and RC is ethyl or methyl). In this example, the support <NUM> surface may be pre-treated with the (<NUM>-aminopropyl)triethoxysilane (APTES) or (<NUM>-aminopropyl)trimethoxysilane (APTMS) to covalently link silicon to one or more oxygen atoms on the surface (without intending to be held by mechanism, each silicon may bond to one, two or three oxygen atoms). This chemically treated surface is baked to form an amine group monolayer. The amine groups are then reacted with Sulfo-HSAB to form an azido derivative. UV activation at <NUM> with <NUM> J/cm<NUM> to <NUM> J/cm<NUM> of energy generates an active nitrene species, which can readily undergo a variety of insertion reactions with the polymers disclosed herein.

Other silanization methods may also be used. Examples of suitable silanization methods include vapor deposition, a YES method, spin coating, or other deposition methods. Some examples of methods and materials that may be used to silanize the support <NUM> are described herein, although it is to be understood that other methods and materials may be used.

In an example utilizing the YES CVD oven, the patterned support <NUM> is placed in the CVD oven. The chamber may be vented and then the silanization cycle started. During cycling, the silane or silane derivative vessel may be maintained at a suitable temperature (e.g., about <NUM> for norbornene silane), the silane or silane derivative vapor lines be maintained at a suitable temperature (e.g., about <NUM> for norbornene silane), and the vacuum lines be maintained at a suitable temperature (e.g., about <NUM>).

In another example, the silane or silane derivative <NUM> (e.g., liquid norbornene silane) may be deposited inside a glass vial and placed inside a glass vacuum desiccator with a patterned support <NUM>. The desiccator can then be evacuated to a pressure ranging from about <NUM> mTorr to about <NUM> mTorr, and placed inside an oven at a temperature ranging from about <NUM> to about <NUM>. Silanization is allowed to proceed, and then the desiccator is removed from the oven, cooled and vented in air.

Vapor deposition, the YES method and/or the vacuum desiccator may be used with a variety of silane or silane derivative <NUM>, such as those silane or silane derivatives <NUM> including examples of the unsaturated moieties disclosed herein. As examples, these methods may be used when the silane or silane derivative <NUM> includes a cycloalkene unsaturated moiety, such as norbornene, a norbornene derivative (e.g., a (hetero)norbomene including an oxygen or nitrogen in place of one of the carbon atoms), transcyclooctene, transcyclooctene derivatives, transcyclopentene, transcycloheptene, trans-cyclononene, bicyclo[<NUM>. <NUM>]non-<NUM>-ene, bicyclo[<NUM>. <NUM>]dec-<NUM> (<NUM>)-ene, bicyclo [<NUM>. <NUM>]non-<NUM>(<NUM>)-ene, and bicyclo[<NUM>. <NUM>]non-<NUM>-ene. Any of these cycloalkenes can be substituted, for example, with an R group, such as hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. An example of the norbornene derivative includes [(<NUM>-bicyclo[<NUM>. <NUM>]hept-<NUM>-enyl)ethyl]trimethoxysilane. As other examples, these methods may be used when the silane or silane derivative <NUM> includes an alkyne or cycloalkyne unsaturated moiety, such as cyclooctyne, a cyclooctyne derivative, or bicyclononynes (e.g., bicyclo[<NUM>. <NUM>]non-<NUM>-yne or derivatives thereof, bicyclo[<NUM>. <NUM>]non-<NUM>-yne, or bicyclo[<NUM>. <NUM>]non-<NUM>-yne). These cycloalkynes can be substituted with any of the R groups described herein.

As shown in <FIG>, the attachment of the silane or silane derivative <NUM> forms a silanized patterned support, including silanized depressions and silanized interstitial regions (which are one example of the treated depressions and treated interstitial regions).

The silanized patterned support may then be exposed to a process that will form the polymer coating layer <NUM> on the silanized depressions and silanized interstitial regions.

Prior to applying the polymer coating layer <NUM>, some examples of the method may involve synthesizing a heteropolymer that is to be deposited to form the polymer coating layer <NUM>. The synthesizing may involve copolymerizing a stimuli-responsive functional group containing monomer with a monomer selected from the group consisting of azido acetamido pentyl acrylamide monomer and a combination of an acrylamide monomer and the azido acetamido pentyl acrylamide monomer. Any of the pH-responsive, temperature-responsive, saccharide-responsive, nucleophile-responsive, or salt-responsive monomers described herein may be used to form the heteropolymers. Several approaches could be used to make the polymer materials disclosed herein. As a few examples, the polymerization method used may be free radical polymerization, controlled radical polymerization, a non-radical method, or another suitable method.

As described herein, examples of the polymer coating layer <NUM> include any of the stimuli-responsive polymers disclosed herein which include any example of the stimuli-responsive functional group and any example of the attachment group. The stimuli-responsive polymer may be present in or incorporated into a mixture. In an example, the mixture includes the stimuli-responsive polymer in an ethanol and water mixture. The polymer coating layer <NUM> may be formed on the surface of the silanized patterned support <NUM> (i.e., onto the silanized depressions and the silanized interstitial regions) using any suitable technique. The stimuli-responsive polymer may be deposited on the surface of the patterned support <NUM> using chemical vapor deposition (CVD), or dipping or dip coating, dunk coating, spin coating, spray coating or ultrasonic spray coating, puddle dispensing, doctor blade coating, aerosol printing, screen printing, microcontact printing, or inkjet printing, or via other suitable techniques. The polymer coating layer <NUM> is shown in <FIG>.

Dunk coating may involve submerging the patterned and silanized support into a series of temperature controlled baths. The baths may also be flow controlled and/or covered with a nitrogen blanket. The baths may include the polymer mixture. Throughout the various baths, the stimuli-responsive polymer will attach to form the polymer coating layer <NUM> in the silanized depression(s) and on the interstitial regions. In an example, the patterned and silanized support will be introduced into a first bath including the polymer mixture where a reaction takes place to attach the polymer, and then the patterned, silanized, and polymer coated support will be moved to additional baths for washing. The patterned support may be moved from bath to bath with a robotic arm or manually. A drying system may also be used in dunk coating.

Spray coating may be accomplished by spraying the polymer mixture directly onto the patterned and silanized support. The spray coated support may be incubated for a time sufficient to attach the polymer. After incubation, any unattached polymer mixture may be diluted and removed using, for example, a spin coater or by sonication in a bath or dunk tank described herein.

Puddle dispensing may be performed according to a pool and spin off method, and thus may be accomplished with a spin coater. The polymer mixture may be applied (manually or via an automated process) to the patterned and silanized support. The applied polymer mixture may be applied to or spread across the entire surface of the patterned and silanized support. The polymer coated patterned support may be incubated for a time a time sufficient to attach the polymer. After incubation, any unattached polymer mixture may be diluted and removed using, for example, the spin coater or by sonication in a bath or dunk tank described herein.

The attachment of the polymer coating layer <NUM> to the silanized depressions and silanized interstitial regions (i.e., <NUM>) may be through covalent bonding. The covalent linking of the polymer coating layer <NUM> to the silanized depressions is helpful for maintaining the polymer coating layer <NUM> in the depressions <NUM>, <NUM>' throughout the lifetime of the ultimately formed flow cell during a variety of uses. The following are some examples of reactions that can take place between the silane or silane derivative <NUM> and the polymer coating layer <NUM>.

When the silane or silane derivative <NUM> includes norbornene or a norbornene derivative as an unsaturated moiety, the norbornene or a norbornene derivative can: i) undergo a <NUM>,<NUM>-dipolar cycloaddition reaction with an azide/azido group of the stimuli-responsive polymer; ii) undergo a coupling reaction with a tetrazine group attached to the stimuli-responsive polymer; iii) undergo a cycloaddition reaction with a hydrazone group attached to the stimuli-responsive polymer; iv) undergo a photo-click reaction with a tetrazole group attached to the stimuli-responsive polymer; or v) undergo a cycloaddition with a nitrile oxide group attached to the stimuli-responsive polymer.

When the silane or silane derivative <NUM> includes cyclooctyne or a cyclooctyne derivative as the unsaturated moiety, the cyclooctyne or cyclooctyne derivative can: i) undergo a strain-promoted azide-alkyne <NUM>,<NUM>-cycloaddition (SPAAC) reaction with an azide/azido of the stimuli-responsive polymer, or ii) undergo a strain-promoted alkyne-nitrile oxide cycloaddition reaction with a nitrile oxide group attached to the stimuli-responsive polymer.

When the silane or silane derivative <NUM> includes a bicyclononyne as the unsaturated moiety, the bicyclononyne can undergo similar SPAAC alkyne cycloaddition with azides or nitrile oxides attached to the stimuli-responsive polymer due to the strain in the bicyclic ring system.

While not shown, it is to be understood that in some examples of the method, the patterned support <NUM> may not be exposed to silanization. Rather, the patterned support <NUM> may be exposed to plasma ashing, and then the polymer coating layer <NUM> may be directly spin coated (or otherwise deposited) on the plasma ashed patterned support <NUM>. In this example, plasma ashing may generate surface-activating agent(s) (e.g., -OH groups, as a hydroxyl or carboxyl) that can adhere the polymer coating layer <NUM> to the patterned support <NUM>. In these examples, the other functional group of the polymer coating layer <NUM> may be selected so that it reacts with the surface groups generated by plasma ashing. For example, the other functional group of the polymer coating layer <NUM> may be an N-hydroxysuccinimide ester (NHS ester).

After being coated, the stimuli-responsive polymer may also be exposed to a curing process to form the polymer coating layer <NUM> across the entire patterned substrate (i.e., on depression(s) <NUM> and interstitial region(s) <NUM>). In an example, curing the stimuli-responsive polymer may take place at a temperature ranging from room temperature (e.g., about <NUM>) to about <NUM> for a time ranging from about <NUM> minutes to about <NUM> hours.

The silanized and coated patterned substrate (shown in <FIG>) may be exposed to a cleaning process. This process may utilize a water bath and sonication. The water bath may be maintained at a relatively low temperature ranging from about <NUM> to about <NUM>. In another example the water bath temperature ranges from about <NUM> to about <NUM>.

The silanized and coated patterned support is then exposed to polishing, if needed, to remove portion(s) of the polymer coating layer <NUM> from the silanized interstitial regions. The silanized, coated, and polished patterned substrate is shown in <FIG>. The portions of the silane or silane derivative <NUM> that are adjacent to the interstitial regions <NUM> may or may not be removed as a result of polishing. As such, in <FIG>, the portions of the silane or silane derivative <NUM> that are adjacent to the interstitial regions <NUM> are shown in phantom, as they may at least partially remain after polishing or they may be removed after polishing. When these silanized portions are completely removed, it is to be understood that the underlying support <NUM> is exposed.

The polishing process may be performed with a gentle chemical slurry (including, e.g., an abrasive, a buffer, a chelating agent, a surfactant, and/or a dispersant) which can remove the thin polymer coating layer <NUM>, and in some instances, at least part of the silane or silane derivative <NUM>, from the interstitial regions <NUM> without deleteriously affecting the underlying support <NUM> at those regions. Alternatively, polishing may be performed with a solution that does not include the abrasive particles.

The chemical slurry may be used in a chemical mechanical polishing system to polish the surface of the silanized and coated patterned support shown in <FIG>. The polishing head(s)/pad(s) or other polishing tool(s) is/are capable of polishing the polymer coating layer <NUM> from the interstitial regions <NUM> while leaving the polymer coating layer <NUM> in the depressions <NUM>, <NUM>' and leaving the underlying support <NUM> at least substantially intact. As an example, the polishing head may be a Strasbaugh ViPRR II polishing head.

As mentioned above, polishing may be performed with a polishing pad and a solution without any abrasive. For example, the polish pad may be utilized with a solution free of the abrasive particle (i.e., a solution that does not include abrasive particles).

Polishing removes portion(s) of the polymer coating layer <NUM> (and in some instances at least part of the silane or silane derivative <NUM>) from the interstitial regions <NUM> and leaves portion(s) of the polymer coating layer <NUM> in the silanized depressions, as shown in <FIG>. Also as mentioned above, the interstitial region(s) <NUM> may remain silanized after polishing is complete. In other words, the silanized interstitial regions may remain intact after the polishing. Alternatively (as indicated by the phantom portions of <NUM>), the silane or silane derivative <NUM> may be removed from the interstitial region(s) <NUM> as a result of polishing.

While not shown, it is to be understood that the silanized, coated, and polished patterned support (shown in <FIG>) may be exposed to a cleaning process. This process may utilize a water bath and sonication. The water bath may be maintained at a relatively low temperature ranging from about <NUM> to about <NUM>. The silanized, coated, and polished patterned substrate may also be spin dried, or dried via another suitable technique.

The silanized, coated, and polished patterned support shown in <FIG> may then be exposed to the processes shown in <FIG>, which generate the flow cell <NUM>, or to the processes shown in <FIG>, which generate the flow cell <NUM>'. In <FIG>, the primers <NUM> are grafted before the lid <NUM> is bonded to the patterned flow cell support <NUM>. In <FIG>, the lid <NUM> is bonded to the patterned flow cell support <NUM> before the primers <NUM> are grafted.

In <FIG>, a grafting process is performed in order to graft the primer <NUM> to the polymer coating layer <NUM> in the depression(s) <NUM>, <NUM>'. The primer <NUM> may be any forward amplification primer or reverse amplification primer that includes the alkyne functional group. Specific examples of suitable primers include P5 and/or P7 primers, which are used on the surface of commercial flow cells sold by Illumina, Inc. , for sequencing on HISEQ™, HISEQX™, MISEQ™, MISEQDX™, MINISEQ™, NEXTSEQ™, NEXTSEQDX™, NOVASEQ™, GENOME ANALYZER™, ISEQ™, and other instrument platforms.

In this example, grafting may be accomplished by flow through deposition (e.g., using a temporarily bound lid), dunk coating, spray coating, puddle dispensing, or by another suitable method that will attach the primer(s) <NUM> to the functionalized polymer layer <NUM> in at least some of the depressions <NUM>, <NUM>'. Each of these example techniques may utilize a primer solution or mixture, which may include the primer(s), water, a buffer, and a catalyst, and may be performed as described herein.

Dunk coating may involve submerging the patterned support (having the polymer coating layer <NUM> in the depression(s) <NUM> thereof) into a series of temperature controlled baths. The baths may also be flow controlled and/or covered with a nitrogen blanket. The baths may include the primer solution or mixture. Throughout the various baths, the primer(s) <NUM> will attach to the attachment group(s) of the polymer coating layer <NUM> in at least some of the depression(s) <NUM>. In an example, the coated and polished patterned support will be introduced into a first bath including the primer solution or mixture where a reaction takes place to attach the primer(s), and then the patterned substrate will be moved to additional baths for washing. The patterned substrate may be moved from bath to bath with a robotic arm or manually. A drying system may also be used in dunk coating.

Spray coating may be accomplished by spraying the primer solution or mixture directly onto the coated and polished patterned support. The spray coated wafer may be incubated for a time ranging from about <NUM> minutes to about <NUM> minutes at a temperature ranging from about <NUM> to about <NUM>. After incubation, the primer solution or mixture may be diluted and removed using, for example, a spin coater.

Puddle dispensing may be performed according to a pool and spin off method, and thus may be accomplished with a spin coater. The primer solution or mixture may be applied (manually or via an automated process) to the coated and polished patterned support. The applied primer solution or mixture may be applied to or spread across the entire surface of the coated and polished patterned support. The primer coated patterned substrate may be incubated for a time ranging from about <NUM> minutes to about <NUM> minutes at a temperature ranging from about <NUM> to about <NUM>. After incubation, the primer solution or mixture may be diluted and removed using, for example, the spin coater.

As depicted in <FIG>, the lid <NUM> may then be bonded to a bonding region <NUM> of the support <NUM>. When the patterned flow cell support <NUM> is a wafer, different areas of the lid <NUM> may at least partially define respective flow channels <NUM> that are being formed using the wafer. When the patterned flow cell support <NUM> is a die, the lid <NUM> may define the one or more flow channels <NUM> that is/are being formed.

The lid <NUM> may be any material that is transparent to an excitation light that is directed toward the surface chemistry <NUM>, <NUM> in the depression(s) <NUM>. As examples, the lid <NUM> may be glass (e.g., borosilicate, fused silica, etc.), plastic, or the like. A commercially available example of a suitable borosilicate glass is D <NUM>®, available from Schott North America, Inc. Commercially available examples of suitable plastic materials, namely cyclo olefin polymers, are the ZEONOR® products available from Zeon Chemicals L.

In some examples, the lid <NUM> may be integrally formed with sidewall(s) <NUM> that correspond with the shape of the bonding region <NUM>, and that will be bonded to the bonding region <NUM>. For example, a recess may be etched into a transparent block to form a substantially planar (e.g., top) portion <NUM> and sidewall(s) <NUM> extending from the substantially planar portion <NUM>. When the etched block is mounted to the bonding region of the patterned substrate <NUM>, the recess may become the flow channel <NUM>.

In other examples, the sidewall(s) <NUM> and the lid <NUM> may be separate components that are coupled to each other. For example, the lid <NUM> may be a substantially rectangular block having an at least substantially planar exterior surface and an at least substantially planar interior surface that defines a portion (e.g., a top portion) of the flow channel <NUM> (once bonded to the patterned support <NUM>). The block may be mounted onto (e.g., bonded to) the sidewall(s) <NUM>, which are bonded to the bonding region <NUM> of the patterned flow cell substrate <NUM> and form sidewall(s) of the flow channel <NUM>. In this example, the sidewall(s) <NUM> may include any of the materials set forth herein for the spacer layer (described below).

The lid <NUM> may be bonded to the bonding region <NUM> of the patterned flow cell support <NUM> using any suitable technique, such as laser bonding, diffusion bonding, anodic bonding, eutectic bonding, plasma activation bonding, glass frit bonding, or others methods known in the art. In an example, a spacer layer <NUM> may be used to bond the lid <NUM> to the bonding region <NUM>. The spacer layer <NUM> may be any material that will seal at least some of the interstitial regions <NUM> (e.g., the bonding region <NUM>) of the patterned substrate <NUM> and the lid <NUM> together.

In one example, the spacer layer <NUM> may be a radiation-absorbing material that absorbs radiation at a wavelength that is transmitted by the lid <NUM> and/or the patterned support <NUM>. The absorbed energy, in turn, forms the bond between the spacer layer <NUM> and the lid <NUM> and between the spacer layer <NUM> and the patterned substrate <NUM>. An example of this radiation-absorbing material is black KAPTON® (polyimide containing carbon black) from DuPont (USA), which absorbs at about <NUM>. It is to be understood that polyimide could be used without the addition of carbon black, except that the wavelength would have to be altered to one that is significantly absorbed by the natural polyimide material (e.g., <NUM>). As another example, polyimide CEN JP can be bonded when irradiated with light at <NUM>. When the spacer layer <NUM> is the radiation-absorbing material, the spacer layer <NUM> may be positioned at an interface between the lid <NUM> and the patterned support <NUM> so that the spacer layer <NUM> contacts the desired bonding region <NUM>. Compression may be applied (e.g., approximately <NUM> PSI of pressure) while laser energy at a suitable wavelength is applied to the interface (i.e., the radiation-absorbing material is irradiated). The laser energy may be applied to the interface both from the top and from the bottom in order to achieve suitable bonding.

In another example, the spacer layer <NUM> may include a radiation-absorbing material in contact therewith. The radiation-absorbing material may be applied at the interface between the spacer layer <NUM> and the lid <NUM> as well as at the interface between the spacer layer <NUM> and the patterned flow cell support <NUM>. As an example, the spacer layer <NUM> may be polyimide and the separate radiation-absorbing material may be carbon black. In this example, the separate radiation-absorbing material absorbs the laser energy that forms the bonds between the spacer layer <NUM> and the lid <NUM> and between the spacer layer <NUM> and the patterned support <NUM>. In this example, compression may be applied at the respective interfaces while laser energy at a suitable wavelength is applied to the interfaces (i.e., the radiation-absorbing material is irradiated).

When the patterned flow support <NUM> is a wafer, the spacer layer <NUM> and sidewalls <NUM> (of or connected to the lid <NUM>) may physically separate one flow channel <NUM> from an adjacent flow channel <NUM> and may be located at the periphery of the wafers. When the patterned support <NUM> is a die and the flow cell <NUM> that is being formed is to include a single flow channel <NUM> or lane, the spacer layer <NUM> and sidewalls <NUM> (of or connected to the lid <NUM>) may be located at the periphery of the die to define the flow channel <NUM> and seal the flow cell <NUM>. When the patterned support <NUM> is a die and the flow cell <NUM> that is being formed is to include multiple isolated flow channels <NUM> (e.g., eight or four flow channels/lanes), the spacer layer <NUM> and sidewalls <NUM> (of or connected to the lid <NUM>) may physically separate one flow channel/lane <NUM> from an adjacent flow channel/lane <NUM> and may be located at the periphery of the die. It is to be understood, however, that the spacer layer <NUM> and sidewalls <NUM> may be located in any desired region depending on the implementation.

When the patterned support <NUM> is a die, assembling the flow cell <NUM> may involve the bonding of the lid <NUM>. When the patterned support <NUM> is a wafer, assembling the flow cell <NUM> may involve additional processing, such as dicing, after the lid <NUM> is bonded. In one example, the lid <NUM> may be bonded to the patterned wafer, and dicing forms individual flow cells <NUM>. As mentioned herein, on a wafer, the sidewalls <NUM> may physically separate one flow channel <NUM> from an adjacent flow channel <NUM>, and thus dicing may take place through at least some of the sidewalls <NUM>, so that each individual flow cell <NUM> includes a desirable number of flow channels <NUM>, each of which has a portion of the original sidewall <NUM> surrounding its periphery. In another example, the patterned wafer may be diced to form non-lidded dies, which can have respective lids <NUM> bonded thereto to form individual flow cells <NUM>.

In the example flow cell <NUM> shown in <FIG>, the lid <NUM> includes the top portion <NUM> integrally formed with sidewall(s) <NUM>. The sidewall(s) <NUM> are bonded to the bonding region <NUM> of the patterned substrate <NUM> through the spacer layer <NUM>.

Together, the lid <NUM> and the patterned flow cell substrate <NUM> define the flow channel <NUM>, which is in selective fluid communication with the depressions <NUM>, <NUM>'. The flow channel <NUM> may serve to, for example, selectively introduce reaction components or reactants to the surface chemistry <NUM>, <NUM> in order initiate designated reactions in/at the depressions <NUM>, <NUM>'.

Prior to performing a sequencing operation, the flow cell <NUM> may be exposed to the predetermined stimulus of the polymer coating layer <NUM> in order to transition the polymer coating layer <NUM> from a current state to a more hydrophilic state (e.g., from the hydrophobic state to the hydrophilic state), from the neutral state to the charged state, and/or from the collapsed state to the expanded state. The predetermined stimulus used will depend upon the polymer coating layer <NUM> and the stimuli-responsive functional group that it includes. Exposing the polymer coating layer <NUM> to the predetermined stimulus may involve heating the polymer coating layer <NUM>; exposing the polymer coating layer <NUM> to a solution of a predetermined pH; exposing the polymer coating layer <NUM> to a solution including a saccharide; exposing the polymer coating layer <NUM> to a nucleophile; or exposing the polymer coating layer <NUM> to a salt solution. When the lid <NUM> is attached, exposing the polymer coating layer <NUM> to any of the solutions may be accomplished by a flow through process. For example, a basic or acidic solution, a saccharide solution (e.g., glucose), or a salt solution may be introduced into the flow cell channel <NUM> through an input port, allowed to incubate for a time sufficient for the desired property change to take place, and then removed from the channel <NUM> through an output port. In an example, the incubation time may be from seconds to several minutes. When the stimuli-responsive functional group is thermo-responsive, the entire flow cell <NUM> could be heated, or a heated solution may be exposed to the polymer coating layer <NUM> using the flow through process.

The predetermined stimulus will render the polymer coating layer <NUM> more compatible with the conditions of the subsequently performed sequencing operation.

Examples of the solutions of the predetermined pH may include basic solutions, such as <NUM> NaOH, TRIS-HCL buffer, or a carbonate buffer, or acidic solutions, such as citrate buffer (pH <NUM>) or <NUM>-(N-morpholino)ethanesulfonic acid (MES) buffer. An example of a saccharide solution includes a glucose solution having a concentration ranging from about <NUM> to about <NUM>. Examples of the salt solution include saline-sodium citrate buffer and phosphate buffered saline (PBS) buffer.

Referring now to <FIG>, another example of the method includes bonding the lid <NUM> to the patterned flow cell support <NUM> before the primers <NUM> are grafted.

As shown in <FIG>, the polymer coating layer <NUM> has been applied (e.g., deposited and polished) as described in <FIG>. At least some of the polished interstitial regions <NUM> may define the bonding region <NUM>, and the lid <NUM> may be bonded to the bonding region <NUM>. The lid <NUM> may be any of the materials and may have any of the configurations described herein. The lid <NUM> may be bonded to the bonding region <NUM> via any of the techniques described herein.

In the example shown in <FIG>, the lid <NUM> includes a top portion <NUM> integrally formed with sidewall(s) <NUM>. The sidewall(s) <NUM> are bonded to the bonding region <NUM> of the patterned substrate <NUM> through the spacer layer <NUM>. After the lid <NUM> is bonded, the flow channel <NUM> is formed between the lid <NUM> and the patterned substrate <NUM>. The flow channel <NUM> may serve to selectively introduce various fluids to the flow cell <NUM>' (<FIG>).

In this example, the primer <NUM> is then grafted to the polymer coating layer <NUM> in the depression(s) <NUM>, as shown in <FIG>. Any of the primers <NUM> described herein may be used. In this example, grafting may be accomplished by a flow through process. In the flow through process, the primer solution or mixture described herein may be introduced into the flow channel(s) <NUM> through respective input port(s) (not shown), may be maintained in the flow channel(s) <NUM> for a time sufficient (i.e., an incubation period) for the primer <NUM> to attach to the polymer coating layer <NUM> in one or more of the depressions <NUM>, and then may be removed from respective output port(s) (not shown). After primer <NUM> attachment, the additional fluid(s) may be directed through the flow channel(s) <NUM> to wash the now functionalized depressions and the flow channel(s) <NUM>.

Prior to performing a sequencing operation, the flow cell <NUM>' may be exposed to the predetermined stimulus of the polymer coating layer <NUM> in order to transition the polymer coating layer <NUM> from the current (e.g., more hydrophobic) state to the more hydrophilic state than the current state, the neutral state to the charged state, and/or the collapsed state to the expanded state. The predetermined stimulus used will depend upon the polymer coating layer <NUM> and the stimuli-responsive functional group that it includes. Exposing the polymer coating layer <NUM> to the predetermined stimulus may involve heating the polymer coating layer <NUM>; exposing the polymer coating layer <NUM> to a solution of a predetermined pH; exposing the polymer coating layer <NUM> to a solution including a saccharide; exposing the polymer coating layer <NUM> to a nucleophile; or exposing the polymer coating layer <NUM> to a salt solution. When the lid <NUM> is attached, exposing the polymer coating layer <NUM> to any of the solutions may be accomplished by a flow through process as previously described herein. When the stimuli-responsive functional group is thermo-responsive, the entire flow cell <NUM>' could be heated, or a heated solution may be exposed to the polymer coating layer <NUM> using the flow through process.

In other examples, exposing the polymer coating layer <NUM> to the predetermined stimulus may take place prior to primer <NUM> grafting. In examples in which predetermined stimulus exposure takes place prior to primer <NUM> grafting at <FIG>, techniques other than the flow through process, such as dip or dunk coating may be used. For example, the silanized, coated, and polished patterned support shown in <FIG> may be dipped into a basic or acidic solution, a saccharide solution (e.g., glucose), or a salt solution for a time sufficient for the desired property change to take place. For another example, the silanized, coated, and polished patterned support shown in <FIG> may be heated to a desired temperature to initiate the state transition. In examples in which predetermined stimulus exposure takes place prior to primer <NUM> grafting at <FIG>, the flow through process may be used for such exposure. For another example, the silanized, coated, and polished patterned support having the lid <NUM> attached thereto, as shown in <FIG>, may be heated to a desired temperature to initiate the state transition. Heating may be performed in the presence of water or a buffer.

As mentioned above, the surface chemistry <NUM>, <NUM> may also be added to a non-patterned support, and this example will be described in reference to <FIG>. With a non-patterned support <NUM>', a continuous surface would include the same surface chemistry <NUM>, <NUM> that is found in the wells <NUM>' of <FIG>, and <FIG>. Any of the supports disclosed herein may be used as the non-patterned substrate <NUM>', except the non-patterned substrate <NUM>' does not include depressions <NUM> or interstitial regions <NUM>. In this example method, the lid <NUM> (shown in <FIG>) is bonded to the non-patterned substrate <NUM>' at the outset to form the flow channel(s) <NUM>. The lid <NUM> may be any of the materials and in any of the configurations described herein. The lid <NUM> may also be bonded to the non-patterned substrate <NUM>' via any of the techniques described herein.

In the example shown in <FIG>, the lid <NUM> includes a top portion <NUM> integrally formed with sidewall(s) <NUM>. The sidewall(s) <NUM> are bonded to a bonding region <NUM> of the non-patterned substrate <NUM>' through the spacer layer <NUM>. The bonding region <NUM> may be at a periphery of the non-patterned substrate <NUM>', or at any areas where it is desirable to form a boundary of a flow channel <NUM>. In other examples, the spacer layer <NUM> may form the sidewall(s) and may be attached to an at least substantially planar lid <NUM>.

Together, the lid <NUM> (including the sidewall(s) <NUM>) and the non-patterned substrate <NUM>' define the flow channel <NUM>. The flow channel <NUM> may serve to, for example, selectively introduce fluids in order to form the surface chemistry <NUM>, <NUM> and to selectively introduce reaction components or reactants to the surface chemistry <NUM>, <NUM> in order to initiate a state transition of the polymer coating layer <NUM> and/or to initiate other designated reactions within the flow channel <NUM>.

Prior to forming the polymer coating layer <NUM> (shown in <FIG>), the method may involve exposing the non-patterned substrate <NUM>' (via a flow through process) to a cleaning process and/or to another process (e.g., silanization) that prepares the exposed surface of the non-patterned substrate <NUM>' for the subsequent deposition of the stimuli-responsive polymer.

Silanization of the non-patterned substrate <NUM>' is shown in <FIG>. In this example, silanization attaches the silane or the silane derivative <NUM> to the exposed portions of the non-patterned wafer surface <NUM>' that are present in the flow channel <NUM>.

Silanization may be accomplished using any silane or silane derivative <NUM>. The selection of the silane or silane derivative <NUM> may depend, in part, upon the stimuli-responsive polymer that is to be used to form the polymer coating layer <NUM> (shown in <FIG>), as it may be desirable to form a covalent bond between the silane or silane derivative <NUM> and the polymer coating layer <NUM>. The method used to attach the silane or silane derivative <NUM> to the substrate <NUM>' may be a flow through process.

As shown in <FIG>, in this example, the polymer coating layer <NUM> is then formed on the silane or silane derivative <NUM>, or on other chemistry that has been deposited to prepare the exposed surface of the non-patterned substrate <NUM>' within the flow channel <NUM>.

Any of the stimuli-responsive polymers described herein may be used, and combinations of the stimuli-responsive polymers may be used together. In an example, the polymer coating layer formation may be accomplished by a flow through process. In the flow through process, the stimuli-responsive polymer(s) may be introduced into the flow channel(s) <NUM> through respective input port(s) and may or may not be cured. The polymer coating layer <NUM> will form on the exposed surface of the non-patterned substrate <NUM>' and polishing does not take place.

As shown in <FIG>, the primer <NUM> is grafted to the polymer coating layer <NUM> in the flow channel <NUM>. In this example, grafting may be accomplished by a flow through process. In the flow through process, a primer solution or mixture may be introduced into the flow channel(s) <NUM> through respective input port(s), may be maintained in the flow channel(s) for a time sufficient (i.e., an incubation period) for the primer <NUM> to attach to the attachment group of the polymer coating layer <NUM>. The remaining primer solution or mixture may then be removed from respective output port(s). After primer attachment, the additional fluid(s) may be directed through the flow channel(s) to wash the now functionalized flow channel(s) <NUM>. The resulting flow cell <NUM>" in this example is shown in <FIG>.

Prior to performing a sequencing operation, the flow cell <NUM>" may be exposed to the predetermined stimulus of the polymer coating layer <NUM> in order to transition the polymer coating layer <NUM> from a current state to a more hydrophilic state (e.g., from the hydrophobic state to the hydrophilic state, from a hydrophilic state to a more hydrophilic state), the neutral state to the charged state, and/or the collapsed state to the expanded state. The predetermined stimulus used will depend upon the polymer coating layer <NUM> and the stimuli-responsive functional group that it includes. Exposing the polymer coating layer <NUM> to the predetermined stimulus may involve heating the polymer coating layer <NUM>; exposing the polymer coating layer <NUM> to a solution of a predetermined pH; exposing the polymer coating layer <NUM> to a solution including a saccharide; exposing the polymer coating layer <NUM> to a nucleophile; or exposing the polymer coating layer <NUM> to a salt solution. Because the lid <NUM> is attached, exposing the polymer coating layer <NUM> to any of the solutions may be accomplished by a flow through process as previously described herein. When the stimuli-responsive functional group is thermo-responsive, the entire flow cell <NUM>" could be heated, or a heated solution may be exposed to the polymer coating layer <NUM> using the flow through process.

The predetermined stimulus will render the polymer coating layer <NUM> more compatible with the conditions of the subsequently performed sequencing operation. A sequencing operation is the process of determining the order nucleotides in a sample of DNA or RNA. In an example, the sequencing operation is sequencing by synthesis, which involves imaging a fluorescently labeled reversible terminator as a nucleotide is added to a template strand, and then cleaving the fluorescently labeled reversible terminator to allow for incorporation of the next base.

In other examples using the flow cell <NUM>", exposing the polymer coating layer <NUM> to the predetermined stimulus may take place prior to primer <NUM> grafting. Again, because the lid <NUM> is attached prior to application of the polymer coating layer <NUM>, the flow through process may be used for the predetermined stimulus exposure. For another example, the silanized and coated non-patterned support, as shown in <FIG>, may be heated to a desired temperature to initiate the state transition. Heating may be accomplished in an aqueous environment (e.g., water or a buffer).

While not shown, it is to be understood that the patterned support <NUM> or non-patterned support <NUM>' may include inlet and outlet ports that are to fluidically engage other ports (not shown) for directing fluid(s) into the respective flow channels (e.g., from a reagent cartridge or other fluid storage system) and out of the flow channel (e.g., to a waste removal system).

Also while not shown, it is to be understood that instead of being bonded to a lid <NUM>, a functionalized support (with surface chemistry, <NUM>, <NUM> thereon) may be bonded to another functionalized substrate with surface chemistry, <NUM>, <NUM> thereon. The two functionalized surfaces can face each other and can have a flow channel defined therebetween. A spacer layer and suitable bonding method may be used to bond two of the functionalized substrates together.

The flow cells <NUM>, <NUM>', <NUM>" disclosed herein may be used in a variety of sequencing approaches or technologies, including techniques often referred to as sequencing-by-synthesis (SBS), cyclic-array sequencing, sequencing-by-ligation, pyrosequencing, and so forth. With any of these techniques and in examples using a patterned support <NUM>, since the polymer coating layer <NUM> and attached primer(s) <NUM> are present in the functionalized depressions (i.e., <NUM>, <NUM>' with surface chemistry <NUM>, <NUM> thereon) and not on the interstitial regions <NUM>, amplification will be confined to the functionalized depressions. Sequencing generally involves hybridizing a nucleic acid template to the flow cell, amplifying the nucleic acid template, and detecting a signal when a nucleotide or an oligonucleotide associates with the amplified nucleic acid template.

As one example, a sequencing by synthesis (SBS) reaction may be run on a system such as the HISEQ™, HISEQX™, MISEQ™, MISEQDX™, MINISEQ™, NOVASEQ™, NEXTSEQDX™, or NEXTSEQ™ sequencer systems from Illumina (San Diego, CA).

An SBS sequencing operation generally includes introducing a nucleic acid library template to the flow cell support <NUM>, whereby the nucleic acid library template hybridizes to a primer <NUM> attached to the polymer coating layer <NUM>; generating a nucleic acid template strand from the hybridized nucleic acid library template; introducing a sequencing primer that is complementary to an adapter of the nucleic acid template strand; introducing fluorescently labeled nucleotides and a polymerase to the flow cell support <NUM>, whereby one of the fluorescently labeled nucleotides is incorporated to extend the sequencing primer along the nucleic acid template strand; and detecting a fluorescent signal from the incorporated one of the fluorescently labeled nucleotides.

In SBS, a plurality of nucleic acid library templates may be introduced to the flow cell <NUM>, <NUM>', <NUM>". Multiple nucleic acid library templates are hybridized, for example, to one of two types of primers <NUM> immobilized on the flow cell <NUM>, <NUM>', <NUM>". Cluster generation may then be performed. In one example of cluster generation, the nucleic acid library templates are copied from the hybridized primers <NUM> by <NUM>' extension using a high-fidelity DNA polymerase. The original nucleic acid library templates are denatured, leaving the copies immobilized where primers <NUM> are located. Isothermal bridge amplification may be used to amplify the immobilized copies. For example, the copied templates loop over to hybridize to an adjacent, complementary primer <NUM>, and a polymerase copies the copied templates to form double stranded bridges, which are denatured to form two single stranded strands. These two strands loop over and hybridize to adjacent, complementary primers <NUM> and are extended again to form two new double stranded loops. The process is repeated on each template copy by cycles of isothermal denaturation and amplification to create dense clonal clusters. Each cluster of double stranded bridges is denatured. In an example, the reverse strand is removed by specific base cleavage, leaving forward template strands.

The <NUM>' end of the templates and any primers <NUM> can be blocked to prevent unwanted priming. The sequencing primer can be introduced to the flow cell <NUM>, <NUM>', <NUM>". Because the sequencing primer is complementary to an adapter of the nucleic acid template strand, it will hybridize to the adapter (e.g., a read <NUM> sequencing primer of the template).

Extension of a nucleic acid primer (e.g., the sequencing primer) along the nucleic acid template (e.g., the forward template polynucleotide strand) is monitored to determine the sequence of nucleotides in the template. The underlying chemical process can be polymerization (e.g., catalyzed by a polymerase enzyme) or ligation (e.g., catalyzed by a ligase enzyme). In a particular polymerase-based SBS process, fluorescently labeled nucleotides are added to the template (thereby extending the sequencing primer) in a template dependent fashion such that detection of the order and type of nucleotides added to the sequencing primer can be used to determine the sequence of the template. For example, to initiate a first SBS cycle, one or more labeled nucleotides, DNA polymerase, etc., may be delivered into/through the flow channel <NUM>, etc. that houses an array of primers <NUM> having template strands attached thereto. Sequencing primer extension causes a labeled nucleotide to be incorporated, and this incorporation can be detected through an imaging event. During an imaging event, an illumination system (not shown) may provide an excitation light to the flow cell <NUM>, <NUM>', <NUM>".

In some examples, the nucleotides can further include a reversible termination property that terminates further sequencing primer extension once a nucleotide has been added. For example, a nucleotide analog having a reversible terminator moiety can be added to the sequencing primer along the template strand such that subsequent extension cannot occur until a deblocking agent is delivered to remove the moiety. Thus, for examples that use reversible termination, a deblocking reagent can be delivered to the flow channel <NUM>, etc. (before or after detection occurs).

Wash(es) may take place between the various fluid delivery steps. The SBS cycle can then be repeated n times to extend the sequencing primer by n nucleotides, thereby detecting a sequence of length n.

While SBS has been described in detail, it is to be understood that the flow cells <NUM>, <NUM>', <NUM>" described herein may be utilized with other sequencing protocols, such as flowcell-based library preparation, for genotyping, or in other chemical and/or biological applications.

To further illustrate the present disclosure, example and prophetic examples are given herein. It is to be understood that these examples are provided for illustrative purposes and are not to be construed as limiting the scope of the disclosure.

A mixture of BrAPA (N-(<NUM>-bromoacetamidylpentyl) acrylamide), sodium azide, and dimethylformamide (DMF) was placed in a DrySyn bath and the solution heated under a nitrogen atmosphere with stirring for <NUM> at <NUM> to form AzAPA (N-(<NUM>-azidoacetamidylpentyl) acrylamide). Acrylamide and <NUM>-(acrylamido)phenylboronic acid were dissolved in deionized water. The prepared AzAPA solution was then added to the acrylamide/<NUM>-(acrylamido)phenylboronic acid solution and mixed thoroughly before being filtered through a <NUM> filter. The filtered solution was then transferred to a <NUM> round-bottomed flask equipped with a stirrer bar and nitrogen was bubbled through the mixture for <NUM>. Whilst degassing the acrylamide/AzAPA premix, the required quantity of potassium persulfate in deionized water was prepared and was then transferred to the mixture of monomers. The mixture was then treated with the co-initiator TEMED (Tetramethylethylenediamine). The solution was stirred under nitrogen at <NUM> for <NUM>. At the end of the polymerization, the nitrogen gas line was removed to expose the reaction flask to air. The crude mixture was then added slowly to <NUM>-propanol. The crude polymer was then isolated by filtration.

A mixture of the acrylate derivative and sultone is reacted to form the sulfonate-derivatized acrylate monomer. The monomer is converted to the heteropolymer as described in Example <NUM>.

The sultone-derivatized acrylate monomer is converted to the heteropolymer as described in Example <NUM>.

The succinic anhydride-derivatized acrylate monomer is converted to the heteropolymer as described in Example <NUM>.

The heteropolymer shown was prepared from the appropriate monomers as described in Example <NUM>. This heteropolymer may have improved dry storage robustness.

Four heteropolymers were respectively coated on the surface of the channels of four single-channel, non-patterned, flow cells using a flow through process.

Control: poly(N-(<NUM>-azidoacetamidylpentyl)acrylamide-co-acrylamide), also known as PAZAM) Test <NUM>: Zwitterionic switchable heteropolymer of Example <NUM> (according to the invention).

Test <NUM>: Anionic switchable heteropolymer of Example <NUM> (not according to the invention).

Test <NUM>: Saccharide switchable heteropolymer of Example <NUM> (according to the invention). <NUM>-<NUM> primers were grafted on each of the polymer layers in the separate flowcells.

Prior to sequencing, the Example <NUM> polymer was exposed to a solution of glucose, which transitioned the Example 1polymer from its neutral and relatively hydrophobic state, to its negatively charged and more hydrophilic state. In this example, the Example <NUM> polymer and the Example <NUM> polymer were not switched.

More than <NUM> sequencing cycles were performed in each of the channels using a PhiX library. Read <NUM> corresponds with cycles <NUM>-<NUM> and Read <NUM> corresponds with cycles <NUM>-<NUM>. The sequencing data collected included error rate (percentage) (shown in <FIG>) and quality score (percentage greater than Q30) (shown in <FIG>). Q30 is equivalent to the probability of an incorrect base call <NUM> in <NUM> times. This means that the base call accuracy (i.e., the probability of a correct base call) is <NUM>%. A lower base call accuracy of <NUM>% (Q20) will have an incorrect base call probability of <NUM> in <NUM>, meaning that every <NUM> base pair sequencing read will likely contain an error. When sequencing quality reaches Q30, virtually all of the reads will be perfect, having zero errors and ambiguities. As shown in <FIG>, each of the Example polymers performed similarly to the comparative/control example. These results indicate that the Example1, <NUM> and <NUM> polymers are capable of supporting a sequencing-by-synthesis technique with the performance being approximately matched to the performance of the control example. It is believed that similar results may be obtained with all types of sequencing libraries.

It should be appreciated that all combinations of the concepts described herein and in the appended claims (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein.

Claim 1:
A switchable heteropolymer, comprising:
an acrylamide monomer comprising an attachment group to react with a functional group attached to a primer; and
a monomer comprising a stimuli-responsive functional group, wherein the monomer comprising the stimuli-responsive functional group is selected from the group consisting of
an acrylamide, acrylate, or vinyl monomer including a terminal saccharide-responsive functional group, wherein the terminal saccharide-responsive functional group comprises a boronic acid group; and
an acrylamide, acrylate, or vinyl monomer including a terminal salt-responsive functional group, wherein the salt-responsive functional group is a zwitterionic functional group exhibiting antipolyelectrolyte behavior; and further wherein the term switchable heteropolymer is defined as a heteropolymer which can transition from a starting state to a second, switched state upon exposure to a stimulus.