Flow Cell

Abstract

The flow cell comprises a substrate and a copolymer coating comprising copolymer chains, each of which has the formula (I): [Formula 1] TIFF2023545338000047.tif51128 and formula (II): [chemical 2] TIFF2023545338000048.tif30128. In formula (I), R 1 is selected from the group consisting of -H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocycle, or optionally substituted variants thereof; R 2 is an azide, and each (CH2) p may be optionally substituted, and p is an integer from 1 to 50. In formula (II), R 3 , R 3’ , R 4 , R 4’ Each of -H, R 5 , -OR 5 , -C(O)OR 5 , -C(O)R 5 , -OC(O)R 5 , -C(O)NR 6 R 7 , or -NR 6 R 7 and R 5 is -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, heterocycle, or an optionally substituted variant thereof; R 6 and R 7 Each of is —H or alkyl. Some copolymer chains contain at least one alkoxyamine end group.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the interests of U.S. Provisional Patent Application No. 63 / 094,147, filed on 20 October 2020, the entirety of which is incorporated herein by reference. [Background technology]

[0002] Polymer or hydrogel coated substrates are used in many technical applications. For example, implantable medical devices can be coated with a bioinert polymer. In another example, wound dressings may be coated with a thin hydrogel layer. Yet another example shows that polymer or hydrogel coated substrates can be used for the preparation and / or analysis of biomolecules. Some molecular analyses, such as certain nucleic acid sequencing methods, involve the adhesion of nucleic acid strands to a polymer or hydrogel coated surface of a substrate in a flow cell. [Overview of the project]

[0003] An exemplary flow cell disclosed herein comprises a copolymer coating comprising at least one alkoxyamine-terminated group. Another exemplary flow cell disclosed herein comprises a copolymer coating comprising at least one side chain having an alkoxyamine group. The alkoxyamine group provides a copolymer coating that includes additional adhesion sites on the flow cell surface.

[0004] introduction A first aspect disclosed herein is a substrate and a copolymer coating attached to at least a portion of the substrate, wherein the copolymer coating comprises a plurality of copolymer chains, each copolymer chain comprising formula (I):

[0005] [ka] The first repeating unit of the formula, R 1 is selected from the group consisting of -H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocycle, and optionally substituted variants thereof, R 2 is azide, each (CH2) p may be optionally substituted, p is an integer from 1 to 50, the first repeating unit, and the formula (II):

[0006]

Chemical formula

[0007] In an example of the first aspect, the substrate includes a base carrier and a plurality of norbornenesilane molecules attached to the base carrier. In some examples, at least one of the plurality of norbornenesilane molecules attaches to each copolymer chain via at least one alkoxyamine end group.

[0008] In one example of the first embodiment, several R of the copolymer chain 2 However, this is replaced with tetramethylethylenediamine. In some examples, the copolymer coating contains several branched copolymer chains.

[0009] In one example of the first embodiment, R 3’ , R 4 , and R 4’ Each of them is -H, and R 3 However, -C(O)NR 6 R 7 And R 6 and R 7 Each of these is -H. In some examples, R 1 However, it is -H, and p is 5.

[0010] In one example of the first embodiment, the substrate includes recesses separated by interstitial regions, and the copolymer coating is attached to the recesses.

[0011] In one example of the first embodiment, the copolymer coating forms isolated pads on the surface of the substrate, and the interstitial regions separate the isolated pads.

[0012] In some examples of the first embodiment, the first repeating unit and the second repeating unit form a random copolymer, or the first repeating unit and the second repeating unit form a statistical copolymer, or the first repeating unit and the second repeating unit form a block copolymer.

[0013] It should be understood that any of the features of the flow cells disclosed herein can be combined in any desired manner and / or configuration to achieve the advantages described herein, including, for example, improved adhesion of copolymer coatings to the flow cell substrate.

[0014] A second aspect disclosed herein is formula (III):

[0015] [ka] The monomer and formula (IV):

[0016] [ka] The reaction mixture with the monomer is copolymerized, and for formula (III), R 1 However, R is selected from the group consisting of -H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, and optionally substituted variants thereof. 2’ However, they are azide or halogen, and each (CH2) p However, it can be replaced by any choice, p is an integer from 1 to 50, and for equation (IV), R 3 , R 3’ , R 4 , R 4’ Each of these independently corresponds to -H, R 5 , -OR 5 , -C(O)OR 5 , -C(O)R 5 -OC(O)R 5 -C(O)NR 6 R 7 , and -NR 6 R 7 Selected from the group consisting of R 5 R is selected from the group consisting of -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, heterocyclic, and their optionally substituted variants, 6 and R 7The method comprises each of the following: copolymerizing independently selected from the group consisting of -H and alkyl; adding i) a nitroxide-mediated polymerization monomolecule initiator, or ii) a combination of a water-soluble initiator and a nitroxide to the reaction mixture to initiate copolymerization, thereby producing a product mixture with copolymer chains, wherein at least some of the copolymer chains have alkoxyamine terminal groups; depositing the product mixture onto a substrate having surface-bound norbornenesilane molecules; and enabling at least some of the alkoxyamine terminal groups to react with at least some of the surface-bound norbornenesilane molecules, thereby forming a copolymer coating.

[0017] In one example of the second aspect, R 2’ However, the azide is present, and the method further comprises curing the copolymer coating, thereby causing some of the azides to react with some of the other surface-bonded norbornenesilane molecules.

[0018] In one example of the second aspect, R 2’ However, it is an azide, and before copolymerization, the method is formula (V):

[0019] [ka] The process further includes reacting the monomer with NaN3 to produce the monomer of formula (III) and introducing an azide.

[0020] In one example of the second aspect, R 2’ However, the halogen is present, and the method further comprises introducing NaN3 into the product mixture before deposition of the product mixture, and heating the product mixture to replace the halogen with an azide.

[0021] One example of the second embodiment further comprises adding tetramethylethylenediamine to the reaction mixture, wherein at least some of the copolymer chains are branched.

[0022] It should be understood that any feature of this method can be combined together in any desired manner. Furthermore, it should be understood that any combination of the features of this method and / or the flow cell can be used together and / or in combination with any of the examples disclosed herein to achieve the advantages described herein, for example, the adhesion of copolymer coatings to a substrate at room temperature.

[0023] A third aspect disclosed herein is a nitroxide-mediated polymerization monomolecule initiator, or a combination of a water-soluble initiator and a nitroxide, using formula (III):

[0024] [ka] The monomer and formula (IV):

[0025] [ka] The mixture of monomers is added in the presence of a substrate having surface-bonded norbornenesilane molecules, wherein for formula (III), R 1 R is selected from the group consisting of -H, halogens, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, and optionally substituted variants thereof. 2’ However, they are azide or halogen, and each (CH2) p However, it can be replaced by any choice, p is an integer from 1 to 50, and for equation (IV), R 3 , R 3’ , R 4 , R 4’ Each of these independently corresponds to -H, R 5 , -OR 5 , -C(O)OR 5 , -C(O)R 5 -OC(O)R 5 -C(O)NR 6 R 7 , and -NR 6 R 7Selected from the group consisting of R 5 R is selected from the group consisting of -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, heterocyclic, and their optionally substituted variants, 6 and R 7 The method comprises adding a component, each independently selected from the group consisting of -H and alkyl, thereby generating a copolymer coating covalently attached to the norbornenesilane molecule.

[0026] In one example of the third aspect, R 2 However, the azide is present, and the method further comprises curing a copolymer coating on a substrate, thereby causing some of the azides to react with some of the other surface-bonded norbornenesilane molecules.

[0027] In one example of the third aspect, R 2’ However, it is an azide, and before copolymerization, the method is formula (V):

[0028] [ka] The process further includes reacting the monomer with NaN3 to produce the monomer of formula (III) and introducing an azide.

[0029] In one example of the third aspect, R 2’ The halogen is involved, and after copolymerization, the method further comprises introducing NaN3 into the copolymer coating and heating the copolymer coating to replace the halogen with an azide.

[0030] In one example of the third embodiment, the method further comprises curing a copolymer coating on a substrate, thereby in which some of the azides react with some of the other surface-bound norbornenesilane molecules.

[0031] One example of the third embodiment further comprises adding tetramethylethylenediamine to the mixture during copolymerization, such that at least some of the copolymer chains are branched.

[0032] It should be understood that any feature of this method can be combined together in any desired manner. Furthermore, it should be understood that any combination of this method and / or other methods and / or flow cell features can be used together and / or in combination with any of the examples disclosed herein to achieve the advantages described herein, for example, including the adhesion of copolymer coatings to a substrate at room temperature.

[0033] A fourth aspect disclosed herein is formula (III):

[0034] [ka] The reaction mixture of monomer A and other monomers is copolymerized in the presence of i) a nitroxide-mediated polymerization monomolecule initiator, or ii) a combination of a water-soluble initiator and a nitroxide, wherein R 1 However, R is selected from the group consisting of -H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, and optionally substituted variants thereof. 2’ However, it is an Azid, and each (CH2) p However, it can be optionally substituted, and p is an integer from 1 to 50, copolymerization and quenching of polymerization, thereby producing a first product mixture with block copolymer chains, thereby producing a product in which at least some of the block copolymer chains have alkoxyamine terminal groups, and formula (IV):

[0035] [ka] The process involves adding the monomer to the first product mixture to produce a second reaction mixture, wherein R 3 ,R 3’ ,R4 , R 4’ each independently is -H, R 5 , -OR 5 , -C(O)OR 5 , -C(O)R 5 , -OC(O)R 5 , -C(O)NR 6 R 7 , and -NR 6 R 7 selected from the group consisting of, R 5 is selected from the group consisting of -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, heterocycle, and optionally substituted variants thereof, R 6 and R 7 each independently is selected from the group consisting of -H and alkyl, producing, i) polymerizing a second reaction mixture in the presence of a nitroxide-mediated polymerization unimolecular initiator or ii) a combination of a water-soluble initiator and a nitroxide, quenching the polymerization, thereby producing a second product mixture with block copolymer chains, whereby at least some of the block copolymer chains have alkoxyamine end groups, depositing the second product mixture on a substrate having surface-bound norbornenesilane molecules, and reacting at least some of the alkoxyamine end groups with at least some of the surface-bound norbornenesilane molecules.

[0036] It should be understood that any features of this method can be combined together in any desirable manner. Further, any combination of the features of this method and / or other methods and / or flow cells can be used together and / or in combination with any of the examples disclosed herein to achieve the advantages described in this disclosure, including, for example, the attachment of a copolymer coating to a substrate at room temperature.

[0037] The fifth aspect disclosed in this specification is a flow cell, comprising a substrate and a copolymer coating attached to at least a part of the substrate, wherein the copolymer coating contains a plurality of copolymer chains, and each copolymer chain has a first repeating unit of formula (I):

[0038]

Chemical formula

[0039]

Chemical formula

[0040]

Chemical formula

[0041] In one example of the fifth embodiment, at least some of the copolymer chains include at least one alkoxyamine terminal group.

[0042] It should be understood that any feature of this flow cell can be combined together in any desired manner. Furthermore, it should be understood that any combination of this flow cell and / or other methods and / or features of other flow cells can be used together and / or in combination with any of the examples disclosed herein to achieve the advantages described herein, including, for example, improved adhesion of copolymer coatings to flow cell substrates.

[0043] A sixth aspect disclosed herein is a nitroxide-mediated polymerization monomolecule initiator, or a combination of a water-soluble initiator and a nitroxide, using formula (V):

[0044] [ka] The monomer and formula (IV):

[0045] [ka] The addition is to a mixture with the monomer, where in formula (V), R 1However, the group is selected from -H, halogens, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, and optionally substituted mutants thereof, where the halo is a halogen, and each (CH2) p However, it can be replaced by any choice, p is an integer from 1 to 50, and for equation (IV), R 3 , R 3’ , R 4 , R 4’ Each of these independently corresponds to -H, R 5 , -OR 5 , -C(O)OR 5 , -C(O)R 5 -OC(O)R 5 -C(O)NR 6 R 7 , and -NR 6 R 7 Selected from the group consisting of R 5 R is selected from the group consisting of -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, heterocyclic, and their optionally substituted variants, 6 and R 7 The method comprises each of the following steps: adding a halogen independently selected from the group consisting of -H and alkyl; thereby generating a copolymer; and, after copolymerization, converting some of the halogens to alkoxyamines and some of the halogens to azides.

[0046] In one example of the sixth embodiment, the conversion of some halogens to alkoxyamines and some of the other halogens to azides includes introducing hydroxylamine into the copolymer such that the halogens outnumber the hydroxylamines, heating the copolymer to initiate a reaction in which the hydroxylamines displace some of the halogens, introducing NaN3 into the copolymer, and heating the copolymer to initiate a reaction in which the azides displace some of the halogens.

[0047] In one example of the sixth embodiment, the conversion of some halogens to hydroxyamines and some of the other halogens to azides includes introducing a mixture of hydroxylamine and NaN3 into a copolymer and heating the copolymer to initiate a reaction in which the hydroxylamine displaces some of the halogens and the azide displaces some of the other halogens.

[0048] In one example of the sixth embodiment, the conversion of some halogens to hydroxyamines and some of the other halogens to azides includes deprotonating the hydroxyamines to form alkoxyamine anions, introducing the alkoxyamine anions into the copolymer such that the halogens outnumber the alkoxyamine anions, heating the copolymer to initiate a reaction in which the alkoxyamine anions displace some of the halogens, introducing NaN3 into the copolymer, and heating the copolymer to initiate a reaction in which the azides displace some of the other halogens.

[0049] It should be understood that any feature of this method can be combined together in any desired manner. Furthermore, it should be understood that any combination of this method and / or other methods and / or other flow cell features can be used together and / or in combination with any of the examples disclosed herein to achieve the advantages described herein, including, for example, improved adhesion of copolymer coatings to flow cell substrates. [Brief explanation of the drawing]

[0050] The features of the examples in this disclosure will become apparent from the following detailed description and drawings. In the drawings, similar reference numbers correspond to components that are similar but probably not identical. For brevity, reference numbers or features having the aforementioned functions may or may not be described in relation to other drawings in which they appear. [Figure 1] This is a schematic diagram of the chemical structure of an example copolymer disclosed herein. [Figure 2] This is a schematic diagram of the chemical structure of another example of a copolymer disclosed herein. [Figure 3] This is a schematic diagram of the chemical structure of another example of a copolymer disclosed herein. [Figure 4] This is a schematic diagram of an example of a chemical reaction for forming an example of a copolymer disclosed herein. [Figure 5] This is a schematic diagram of another example of a chemical reaction for forming an example of the copolymer disclosed herein. [Figure 6] This is a schematic diagram of an additional chemical reaction for forming another example of the copolymer disclosed herein. [Figure 7] This is a schematic diagram of a recess in a flow cell surface having a silane-treated surface, and an example of a copolymer coating attached to the silane-treated surface. [Figure 8] A is a top view of the flow cell, and B-D are enlarged and partially cropped views of different exemplary structures within the flow channels of the flow cell. [Modes for carrying out the invention]

[0051] Copolymer coatings are disclosed herein. The copolymer coating comprises copolymer chains, each containing a repeating acrylamide monomer having an azide group. The azide group allows for the adhesion of primers, and therefore the copolymer coating may be suitable for forming a reactive surface in a flow cell.

[0052] During the preparation of several examples of copolymer chains, nitroxide-mediated polymerization monoinitiators (NMP monoinitiators) are used as polymerization initiators and mediators, and optionally as polymerization quenchers. The NMP monoinitiator is thermally separated into two free radicals, one of which is a carbon-centered radical that acts as an initiator for free radical polymerization, and therefore a separate free radical initiator cannot be used. The other free radical is a nitroxide, a stable free radical that controls polymerization by reversibly terminating the polymerization. Thus, at least some of the copolymer chains have an alkoxyamine-terminated group. In the copolymer chain, the term “alkoxyamine-terminated group” refers to the resting species -ONR1R2, where R1 and R2 may be the same or different, independently linear or branched alkyl, or cyclic, and the oxygen atom is attached to the rest of the copolymer chain. The alkoxyamine-terminated group can be thermally activated as described herein to generate two free radicals. When NMP monomolecule initiators are used beyond monomers, they can quench the polymerization reaction, resulting in copolymer chains that, while not all, have alkoxyamine terminal groups.

[0053] In the preparation of several other examples of copolymer chains, water-soluble initiators and nitroxides are used as polymerization initiators and mediators, respectively. The water-soluble initiators initiate free radical polymerization, and the nitroxides control polymerization by reversibly terminating it. Thus, at least some of the copolymer chains have alkoxyamine terminal groups.

[0054] During the preparation of several other examples of copolymer chains, alkoxyamines are introduced into some of the repeating acrylamide monomers instead of azide groups. This introduces alkoxyamines into the side chains along the polymer backbone, which can provide more adhesion sites to the flow cell substrate and improve the copolymer's stability.

[0055] The inventors unexpectedly found that the alkoxyamines in the terminal groups and / or side chains of the copolymers disclosed herein remain intact and active even after the copolymers have been stored for, for example, at least six months. Furthermore, the inventors found that the alkoxyamine terminal groups or alkoxyamines in the side chains provide an adhesion mechanism to silanized carriers, particularly norbornenesilane molecules. This adhesion mechanism can occur simultaneously with azido-norbornene adhesion. The additional adhesion mechanism via alkoxyamine may not require the inclusion of several azido groups for primer adhesion, thereby providing more primers to the surface and thus potentially improving the sequencing metric. Increased alkoxyamine-norbornene adhesion may also enhance the stability of the copolymer.

[0056] definition Unless otherwise specified, terms used herein should be understood to have their common meanings in the relevant technical field. Some terms used herein and their meanings are listed below.

[0057] The singular forms "a," "an," and "the" refer to multiple objects unless explicitly indicated otherwise in the context.

[0058] The terms "comprising," "including," and "containing," and their various forms, are synonymous and equally broad in meaning.

[0059] To describe flow cells and / or the various components of a flow cell, terms such as top, bottom, lower, upper, and on are used herein. It should be understood that these directional terms are not intended to indicate a specific orientation, but rather to specify the relative orientation between components. The use of directional terms is not to be construed as limiting the examples disclosed herein to any particular orientation.

[0060] Any numerical range provided herein includes the last digit unless otherwise specified.

[0061] "Acrylamide" has a structure

[0062] [ka] A monomer having a monomer, or a monomer containing an acrylamide group. An example of a monomer containing an acrylamide group is azidoacetamidopentylacrylamide:

[0063] [ka] and N-isopropylacrylamide:

[0064] [ka] Examples include the following. Other acrylamide monomers may be used. In the exemplary copolymers disclosed herein, the acrylamide monomer is a precursor unit of the first repeating unit of formula (I). In some examples, a different acrylamide monomer is a precursor unit of the second repeating unit of formula (II).

[0065] As used herein, “alkyl” refers to a fully saturated (i.e., non-double or non-triple) linear or branched hydrocarbon chain. Alkyl chains may have 1 to 20 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, and hexyl. For example, the notation “C1-C6 alkyl” indicates that the alkyl chain contains 1 to 6 carbon atoms, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, pentyl, and hexyl. Any alkyl group may be substituted or unsubstituted.

[0066] As used herein, "alkenyl" refers to a straight or branched hydrocarbon chain containing one or more double bonds. An alkenyl group has 2 to 20 carbon atoms. Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, and hexenyl. Any alkenyl may be substituted or unsubstituted.

[0067] The “alkoxy” group refers to the formula -OR, where R is an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or cycloalkynyl as defined herein. Some exemplary alkoxy groups include methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy. Any alkoxy may be substituted or unsubstituted.

[0068] As described above, the terms "alkoxyamine" and "alkoxyamine terminal group" refer to the resting species -ONR1R2, where R1 and R2 may be the same or different, and may independently be a linear or branched alkyl, or a cyclic carbon group. The oxygen atom is attached to the remainder of the copolymer chain in the side chain or to the terminal group. The alkoxyamine terminal group in the copolymer chain can detach to form an unbound free radical, allowing the copolymer to undergo further reactions. The unbound free radical has the following structure:

[0069] [ka] It is a nitroxide-free radical having (wherein R1 and R2 are carbon-based groups such as linear or branched alkyl or cyclic structures).

[0070] As used herein, "alkyne" or "alkynyl" refers to a linear or branched hydrocarbon chain containing one or more triple bonds. An alkynyl group may have 2 to 20 carbon atoms. Any alkyl group may be substituted or unsubstituted.

[0071] The term "aryl" refers to an aromatic ring or ring system (i.e., two or more fused rings sharing two adjacent carbon atoms) containing only carbon atoms in its ring skeleton. If an aryl is a ring system, all rings in the system are aromatic. An aryl group can have 6 to 18 carbon atoms. Examples of aryl groups include phenyl, naphthyl, azlenyl, and anthracenyl. Any aryl may be a heteroaryl having at least one heteroatom, i.e., an element other than carbon (e.g., nitrogen, oxygen, sulfur, etc.), in its ring skeleton. Any aryl group may be substituted or unsubstituted.

[0072] As used herein, the term “attached” refers to a state in which two things are joined, fastened, adhered, connected, or bonded to each other, either directly or indirectly. Some attachments may be covalent or non-covalent. Covalent bonds are characterized by the sharing of electron pairs between atoms. Non-covalent bonds are physical bonds that do not involve the sharing of electron pairs and include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, and hydrophobic interactions.

[0073] The functional group "azide" or "azido" refers to the -N3 group.

[0074] A "block copolymer" is a copolymer formed when two or more monomers cluster together to form a repeating block. Each block should have at least one feature that is not present in adjacent blocks. Specific examples of block copolymers are described further below.

[0075] As used herein, "cycloalkyl" refers to a fully saturated (without double or triple bonds) monocyclic or polycyclic hydrocarbon ring system. When composed of two or more rings, the rings may be joined together in a condensation manner. A cycloalkyl group may contain 3 to 10 atoms in the ring. In some examples, a cycloalkyl group may contain 3 to 8 atoms in the ring. Cycloalkyl groups may be unsubstituted or substituted. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0076] As used herein, "cycloalkenyl" or "cycloalkene" means a carbocyclic ring or ring system having at least one double bond, where none of the rings in the ring system are aromatic. Examples include cyclohexenyl or cyclohexene and norbornenyl or norbornene.

[0077] As used herein, "cycloalkynyl" or "cycloalkyne" means a carbocyclic ring or ring system having at least one triple bond, where none of the rings in the ring system are aromatic. An example is cyclooctyne. Another example is biclononine.

[0078] As used herein, the term “deposition” refers to any suitable application technique, whether manual or automated, that results in a modification of surface properties in some cases. Generally, deposition can be carried out using vapor deposition techniques, coating techniques, grafting techniques, etc. Some specific examples include chemical vapor deposition (CVD), spray coating (e.g., ultrasonic spray coating), spin coating, dunk or dip coating, doctor blade coating, puddle dispensing, flow-through coating, aerosol printing, screen printing, microcontact printing, and inkjet printing.

[0079] As used herein, the term “recess” refers to a discontinuous concave feature in a base carrier or multilayer stack having a surface opening at least partially surrounded by the interstitial regions of the layers of the base carrier or multilayer stack. Recesses can take various shapes at the surface opening, such as circular, elliptical, square, polygonal, or star-shaped (with any number of vertices). The cross-section of a recess taken perpendicular to the surface can be curved, square, polygonal, hyperbolic, conical, or angular. For example, a recess may be a well or two interconnected wells. Recesses may also have more complex structures, such as ridges or stepped structures.

[0080] The term "each" is intended to identify individual items within a set of items when used to refer to a set of items, but not necessarily all items within the set. Exceptions may be made if explicit disclosure or context clearly indicates otherwise.

[0081] As used herein, the term “flow cell” is intended to mean a container having a flow channel on which a reaction can take place, an inlet for delivering a reagent into the flow channel, and an outlet for removing the reagent from the flow channel. In some examples, a flow cell corresponds to the detection of a reaction occurring within the flow cell. For example, a flow cell may include one or more transparent surfaces that enable optical detection, such as an array or optically labeled molecules.

[0082] As used herein, “flow channel” or “channel” may be a region defined between two combined components that can selectively receive a liquid sample. In some examples, a flow channel may be defined between a patterned or unpatterned substrate and a lid. In other examples, a flow channel may be defined between two patterned or unpatterned substrates that are joined together. The flow channel is in fluid communication with the surface chemicals of the patterned or unpatterned substrate.

[0083] A "halogen" or "halo" refers to any one of the radioactive stable atoms in the seventh column of the periodic table, such as bromine, chloride, fluorine, and iodine.

[0084] As used herein, “heterocycle” means a non-aromatic ring or ring system containing at least one heteroatom in its ring skeleton. Heterocycles may be joined together integrally by condensation, bridging, or spirobonding. Heterocycles may have any degree of saturation, provided that at least one ring in the ring system is not aromatic. In the ring system, the heteroatom may be present in either a non-aromatic or aromatic ring. Heterocycles may have 3 to 20 ring members (i.e., the number of atoms forming the ring skeleton, including carbon atoms and heteroatoms). In some examples, the heteroatom is O, N, or S.

[0085] As used herein, "hydroxy" or "hydroxyl" refers to the -OH group.

[0086] As used herein, the term “gap region” refers to a region of, for example, a substrate, patterned resin, or other carrier that separates a recess or pad. For example, a gap region can separate one recess in an array from another recess in the array. Two recesses or pads separated from each other may be discontinuous, i.e., they may lack physical contact with each other. In many examples, the gap region is continuous, while the recesses or pads are discontinuous, for example, in the case of multiple recesses or pads defined on a surface that is otherwise continuous. In other examples, the gap region and feature are discontinuous, for example, in the case of multiple elongated recesses (e.g., trenches) separated by each gap region. The separation provided by the gap region may be partial or complete. The gap region may have a surface material different from the surface material of the recess defined on the surface.

[0087] As used herein, “hydroxyalkyl” refers to an alkyl group in which one or more hydrogen atoms are replaced by a hydroxyl group. Exemplary hydroxyalkyl groups include 2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxypropyl, and 2,2-dihydroxyethyl. Any hydroxyalkyl group may be substituted or unsubstituted.

[0088] The term "hydroxylamine" refers to HONR1R2, where R1 and R2 may be the same or different, independently of a linear or branched alkyl or cyclic structure, and the oxygen atom is attached to the rest of the copolymer chain.

[0089] As used herein, “nucleotide” comprises a nitrogen-containing heterocyclic base, a sugar, and one or more phosphate groups. A nucleotide is a monomeric unit of a nucleic acid sequence. In RNA, the sugar is ribose, and in DNA, the sugar is deoxyribose, i.e., a sugar lacking the hydroxyl group at the 2' position of ribose. The nitrogen-containing heterocyclic base (i.e., nucleic acid base) may be a purine base or a pyrimidine base. Examples of purine bases include adenine (A) and guanine (G), and their modified derivatives or analogs. Examples of pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and their modified derivatives or analogs. The C-1 atom of deoxyribose is bonded to N-1 of the pyrimidine or N-9 of the purine. Nucleic acid analogs may have altered phosphate backbone, sugar, or nucleic acid base. Examples of nucleic acid analogs include, for instance, universal base or phosphate-sugar skeleton analogs such as peptide nucleic acid (PNA).

[0090] As used herein, “primer” is defined as a single-stranded nucleic acid sequence (e.g., single-stranded DNA). Some primers, sometimes referred to as capture or amplification primers, function as starting points for template amplification and clustering. Other primers, referred to herein as sequencing primers, function as starting points for DNA synthesis. The 5' end of a primer may be modified to enable a coupling reaction with a polymer functional group (e.g., an azide). The length of a primer can be any number of bases and may contain a variety of non-natural nucleotides. For example, sequencing primers are short chains ranging from 10 to 60 bases, or from 20 to 40 bases.

[0091] The term "substrate" refers to a single-layer base carrier or a multilayer structure into which surface chemicals are introduced.

[0092] "Surface chemical" refers to the copolymers described herein and examples of any primers attached thereto. The surface chemical forms a reactive surface on the substrate.

[0093] Copolymer The copolymer chains disclosed herein comprise at least two distinct repeating units. In some examples, the copolymer chain comprises three distinct repeating units. All of the chains comprise two terminal groups, and in some examples, at least one terminal group is an alkoxyamine terminal group. In other examples, at least some of the repeating units comprise an alkoxyamine in their side chains.

[0094] Each copolymer chain is represented by formula (I):

[0095] [ka] The first repeating unit of the formula, where R 1 R is selected from the group consisting of -H, halogens, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, and optionally substituted variants thereof.2 This is an Azid, and each (CH2) p The first repeating unit is, and equation (II): can be replaced by any choice, where p is an integer from 1 to 50.

[0096] [ka] The second repeating unit of the formula, where R 3 , R 3’ , R 4 , R 4’ Each of these is independently -H, R 5 , -OR 5 , -C(O)OR 5 , -C(O)R 5 -OC(O)R 5 -C(O)NR 6 R 7 , and -NR 6 R 7 Selected from the group consisting of R 5 R is selected from the group consisting of -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, heterocyclic, and their optionally substituted variants, 6 and R 7 Each of these independently comprises a second repeating unit selected from the group consisting of -H and alkyl groups.

[0097] Some exemplary copolymer chains also include a third repeating unit of formula (VI):

[0098] [ka] In the formula, R 1 The group consists of -H, halogens, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, and optionally substituted variants thereof, where halo is a halogen, and each (CH2) p can be replaced by any choice, and p is an integer between 1 and 50.

[0099] In an example of the repeating unit of equation (I), R 1 is -H, and p is 5.

[0100] In an example of the repeating unit of equation (II), R 3’ , R 4 , and R 4’ Each of them is -H, and R 3 is -C(O)NR 6 R 7 And R 6 and R 7 Each of these is -H. Another example of the repeating unit in equation (II) is R 3’ , R 4 , and R 4’ Each of them is -H, and R 3 is -C(O)NR 6 R 7 And R 6 and R 7 Each of these is -CH3. In yet another example of the repeating unit of equation (II), R 3’ , R 4 , and R 4’ Each of them is -H, and R 3 is -C(O)NR 6 R 7 And R 6 is -H, and R 7 is -CH3. In yet another example of the repeating unit of equation (II), R 3’ , R 4 , and R 4’ Each of them is -H, and R 3 is -C(O)NR 6 R 7 And R 6 is -H, and R 7 It is -(CH2)2OH. Another example of the repeating unit of formula (II) is R 3’ , R 4 , and R 4’ Each of them is -H, and R 3 is -C(O)NR 6 R 7 And R 6 It is -CH3, and R 7 It is -(CH2)2OH. In yet another example of the repeating unit of formula (II), R3’ , R 4 , and R 4’ Each of them is -H, and R 3 is -C(O)OR 5 And R 5 This is -H, -CH3, or -(CH2)2OH.

[0101] In an example of the repeating unit of equation (VI), R 1 is -H, halo is bromine, and p is 5.

[0102] In some examples, at least some of the copolymer chains also include a first terminal group selected from the group consisting of hydroxyl, -OSO3, alkyl, and alkoxyamine terminal groups having about 1 to about 12 carbon atoms, and a second terminal group which is an alkoxyamine terminal group. The first terminal group may be controlled through the selection of an initiator, and the second terminal group may be controlled through a mediator that can quench the polymerization reaction. Exemplary initiators and mediators are discussed below.

[0103] Figure 1 shows an example of copolymer 10. In this example of copolymer 10, an example of the repeating unit of formula (I) is shown at reference no. 12, and an example of the repeating unit of formula (II) is shown at reference no. 14. In this particular example, R in the repeating unit 12 of formula (I) 1 is -H, and p in repeating unit 12 of equation (I) is 5. Also, in this particular example, R in repeating unit 14 of equation (II) 3’ , R 4 , and R 4’ Each of these is -H, and R in the repeating unit 14 of equation (II) 3 This is -C(O)NH2. Specific examples of repeating units 12 and 14 are shown, but it should be understood that any example of repeating units 12 and 14 of formulas (I) and (II) may be included in copolymer 10.

[0104] In the example shown in Figure 1, the first terminal group E of copolymer 10 1It is -OSO3, and the alkoxyamine terminal group E of copolymer 10 2 It is -O-NH-C(CH3)2. The first terminal group E 1 A specific example is shown, but terminal base E 1 It should be understood that any example of the alkoxyamine terminal group E may be included in copolymer 10. 2 While specific examples are shown, it should be understood that any alkoxyamine capable of reversibly terminating the copolymer chain during synthesis can be incorporated into copolymer 10.

[0105] In this exemplary copolymer 10, "n" (the number of repeating units in formula (I)) is in the range of 2 to 50,000, and "m" (the number of repeating units in formula (II)) is in the range of 2 to 100,000. Figure 1 shows the individual units as blocks. However, it should be understood that the incorporation of the individual units may be statistical, random, or blocky, and may depend on the method used to synthesize copolymer 10.

[0106] Referring here to Figure 2, another example of the copolymer is shown at reference no. 10'. In this example, copolymer 10' contains another repeating unit 12'. Repeating unit 12' is the product of a possible side reaction between repeating unit 12 of formula (1) and tetramethylethylenediamine (TeMED). TeMED is a reaction accelerator that can be introduced during copolymerization. As a result of the side reaction, TeMED replaces some of the azide (N3) groups. Thus, in some examples, some of the R3 groups in the copolymer chain are replaced. 2 This is replaced with tetramethylethylenediamine. This reaction reduces the azide content of these copolymer chains but also introduces branching sites. Branching sites can provide places where copolymer chains can branch off from one another. Thus, in some examples, a coating containing copolymer 10' contains several branched copolymer chains. When TeMED is used in the synthesis shown in Figure 5, crosslinking can occur between the two nitrogen atoms on TeMED and the halogen of the intermediate copolymer 16.

[0107] In this particular example, R in repeating units 12 and 12' 1 is -H, and p in repeating units 12 and 12' is 5. Also, in this particular example, R in repeating unit 14 of equation (II) 3’ , R 4 , and R 4’ Each of these is -H, and R in the repeating unit 14 of equation (II) 3 This is -C(O)NH2. Specific examples of repeating units 12, 12', and 14 are shown, but it should be understood that any example of repeating units 12, 14 and repeating unit 12' of formulas (I) and (II) may be included in copolymer 10'.

[0108] In the example shown in Figure 2, the first terminal group E of copolymer 10' 1 This is -OH, and is the alkoxyamine terminal group E of copolymer 10. 2 It is -O-NH-C(CH3)2. The first terminal group E 1 A specific example is shown, but terminal group E 1 It should be understood that any example of can be included in copolymer 10'. alkoxyamine terminal group E 2 While specific examples are shown, it should be understood that any alkoxyamine that can reversibly terminate the copolymer chain during synthesis can be incorporated into copolymer 10'.

[0109] In this exemplary copolymer 10', "n" + "λ" are integers in the range of 2 to 50,000, and "m" are integers in the range of 2 to 100,000. Figure 2 shows the individual units in blocks. However, it should be understood that the incorporation of the individual units can be statistical, random, or blocky, and may depend on the method used to synthesize copolymer 10'.

[0110] Referring now to FIG. 3, yet another example of a copolymer is shown at reference numeral 10''. In this example, copolymer 10'' includes repeating units of formulas (I) and (II), as well as another repeating unit 12'' of formula (VI). Repeating unit 12'' is the product of an alternative reaction that can occur between the monomer used to form repeating unit (I) and a hydroxylamine or alkoxyamine anion added after polymerization. As a result of the alternative reaction, alkoxyamine (-ONR1R2) is present in some of the side chains instead of an azide (N3) group.

[0111] In this particular example, R in repeating units 12, 12'' 1 is -H and p is 5. Also, in this particular example, R in repeating unit 14 3’ , R 4 and R 4’ each is -H, and R in repeating unit 14 3 is -C(O)NH2. Also, in this particular example, R1 and R2 in repeating unit 12'' are any carbon-based substituents. Specific examples of repeating units 12, 12', 14 are shown, but it should be understood that any examples of repeating units 12, 14 of formulas (I) and (II) and repeating unit 12' can be included in copolymer 10'.

[0112] The end groups in this example of copolymer 10'' can be any of the examples of end groups described herein and depend on the initiator and mediator used in the copolymerization method.

[0113] In this exemplary copolymer 10'', "n" + "λ" is an integer in the range of 2 to 50,000 and "m" is an integer in the range of 2 to 100,000. FIG. 3 shows the individual units in blocks. However, it should be understood that the incorporation of the individual units can be statistical, random, or within blocks and can depend on the method used to synthesize copolymer 10''.

[0114] The molecular weight of any example of copolymer 10, 10', 10'' may be in the range of approximately 5 kDa to approximately 1500 kDa or approximately 10 kDa to approximately 1000 kDa, or in certain examples, approximately 500 kDa.

[0115] Method for preparing copolymers Each of the methods for producing examples of copolymers 10, 10', and 10'' disclosed herein involves nitroxide-mediated controlled free radical polymerization. Some of the polymerization processes include an NMP monomolecule initiator mediator to control free radical polymerization with reversible termination. Other examples of polymerization processes include a nitroxide mediator to control free radical polymerization with reversible termination. During synthesis, the copolymer is considered a “living polymer”. The alkoxyamine is retained at one end of the growing chain, and therefore it can be separated to form a free radical (active nitroxide species). Free radical generation can be controlled using temperature. The growing polymer chain can be modified by adding one or more monomer units until the chain is again reversibly terminated by the alkoxyamine.

[0116] The monomers used to produce copolymers 10, 10', and 10'' contain carbon-carbon double bonds that can undergo free radical polymerization.

[0117] In any of the methods described herein, repeating units 12, 12' can be generated using the monomer of formula (III). Formula (III) is,

[0118] [ka] And in the formula, R 1 R is selected from the group consisting of -H, halogens, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, and optionally substituted variants thereof. 2’ These are azides or halogens, each (CH2) pcan be replaced by any choice, and p is an integer between 1 and 50.

[0119] Azide group R 2’ The monomer of formula (III) has a halogen as R 2’ It can be obtained from monomers of formula (III) having as follows. In some examples, the method is to copolymerize the monomer of formula (III) with the monomer of formula (IV) before (R 2’ This may include producing monomers of formula (III) (containing azide). Formula (V):

[0120] [ka] This involves reacting a monomer of formula (for example, where the halo is Br) with excess NaN3 to introduce an azide group in place of the halogen. In one example, NaN3 is present in a 50-fold excess relative to the monomer of formula (V). The monomer of formula (V) can also be used in the synthesis of copolymer 10'' to produce both repeating unit 12 and repeating unit 12''.

[0121] In any of the methods described herein, the monomer of formula (IV) may be used to produce the repeating unit 14 of formula (II). Formula (IV) is

[0122] [ka] And, In the formula, R 3 , R 3’ , R 4 , R 4’ Each of these is independently -H, R 5 , -OR 5 , -C(O)OR 5 , -C(O)R 5 -OC(O)R 5 -C(O)NR 6 R 7 , and -NR 6 R 7 Selected from the group consisting of R 5is selected from the group consisting of -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, heterocycle, and optionally substituted variants thereof, R 6 and R 7 each independently is selected from the group consisting of -H and alkyl.

[0123] In some of the methods described herein, a nitroxide-mediated polymerization unimolecular initiator can be used. As an example, the NMP unimolecular initiator is

[0124] [Chemical formula] (2,2,6,6-tetramethylpiperidinyloxy (2,2,6,6-Tetramethylpiperidinyloxy, TEMPO)),

[0125] [Chemical formula] (di-tert-butyl nitroxide),

[0126] [Chemical formula] (2,2,5-trimethyl-4-phenyl-3-azahexane-3-nitroxide), and

[0127] [Chemical formula] (β-phosphonylated nitroxide (SG1) having a structure selected from the group consisting of, wherein I is

[0128] [Chemical formula] selected from the group consisting of. In these examples, "I" does not represent the element iodine. These NMP unimolecular initiators can be added to initiate polymerization.

[0129] Other examples of the methods described herein involve the use of a combination of potassium persulfate, a water-soluble radical initiator such as 4,4'-azobis(4-cyanovaleric acid), and a nitroxide. Any of the previously listed nitroxides may be used in radical form, i.e., without the "I" group.

[0130] As referred to herein, the incorporation of individual units 12, 14, or 12, 14, 12', or 12, 14, 12'' along the copolymer chain may be statistical, random, or blocky.

[0131] Statistical copolymers (e.g., the continuous distribution of monomer units follows known statistical laws) and random copolymers (e.g., monomers are introduced randomly along the copolymer chain, potentially resulting in several blocks of repeating units 12, or 12 and 12', or 12 and 12'', as well as several blocks of repeating units 14) can be prepared by one-pot synthesis. In one-pot synthesis, monomers of formula (III) are mixed with monomers of formula (IV), and copolymerization is carried out in the presence of an NMP monomolecule initiator or a combination of a water-soluble initiator and a nitroxide under conditions suitable for the monomers used, the initiator or the combination of initiator and mediator, and the desired product (including its molecular weight).

[0132] A schematic example of a one-pot synthesis is shown in Figure 4. In this example, the monomer of formula (III) is R 2’ As such, the monomer of formula (IV) contains an azide group, 3’ , R 4 , and R 4’ As each of them, -H and R 3 It contains C(O)NH2.

[0133] In the one-pot synthesis shown in Figure 4, the reaction mixture is composed of monomer (R) of formula (III). 2’The reaction comprises an azide group (as a base), a monomer of formula (IV), and an NMP monomolecule initiator, or a combination of a water-soluble initiator and a nitroxide. The monomers of formula (III) and (IV) may be present in a suitable weight ratio to each other so that a desirable number of repeating units 12, 14, respectively, are introduced into the resulting copolymer 10. The NMP monomolecule initiator, or the combination of a water-soluble initiator and a nitroxide, may be present in an amount that allows all monomers in the reaction mixture to copolymerize. In one example, the NMP monomolecule initiator, or the combination of a water-soluble initiator and a nitroxide, may constitute about 0.005% to about 5% by weight of the reaction mixture.

[0134] The reaction mixture may also contain water, a solvent, or a combination of water and a solvent. Exemplary solvents include N-methyl-2-pyrrolidone (NMP), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile (MeCN), methanol (MeOH), ethanol (isopropyl alcohol, EtOH), isopropyl alcohol (IPA), dioxane, acetone, dimethylacetamide (DMAc), and the like. The mixture may also contain a buffer to at least substantially prevent undesirable changes in pH. The pH of the reaction mixture may be acidic (<7). Suitable buffering agents include TRIS (tris(hydroxymethyl)aminomethane or TRIZMA®), bis-trismethane buffer, ADA buffer (zwitterionic buffer), MES (2-ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), or other acidic buffering agents.

[0135] As described above, copolymerization is carried out under suitable conditions in the presence of an initiator or an initiator and mediator. For example, the temperature can range from about room temperature (e.g., 18°C ​​to 25°C) to about 150°C, depending on the choice of solvent, monomer, and NMP monomolecule initiator or nitroxide. For example, when water alone is used, the temperature may be 90°C or lower. When a solvent, or a combination of water and a solvent, is used, higher temperatures may be used. Some other exemplary temperature ranges include about 18°C ​​to about 130°C, about 50°C to about 75°C, etc. The time of the polymerization reaction can range from about 5 minutes to about 24 hours, for example, from 1 hour to 10 hours.

[0136] The temperature can be set so that the growing copolymer chain can be continuously added monomer units of formulas (III) and (IV). In one example, an excess of NMP monomolecule initiator or a combination of a water-soluble initiator and a nitroxide can be included in the reaction mixture, and when all the monomers have reacted, the temperature can be lowered, and the nitroxide free radicals quench polymerization and the copolymer chain end group E 2 It adheres as such. In another example, polymerization can be quenched by adding an additional NMP monomolecule initiator or nitroxide to the reaction mixture. Quenching copolymerization produces a product mixture with copolymer chains, at least some of which have alkoxyamine terminal groups E 2 It holds.

[0137] It should be understood that the exemplary method described with reference to Figure 4, although not illustrated, may also include tetramethylethylenediamine to form an example of copolymer 10' shown in Figure 2. This exemplary method involves adding tetramethylethylenediamine to the reaction mixture. TeMED may be added before the start of copolymerization or while copolymerization is taking place. As shown in Figure 2, at least some of the azide groups are replaced with TeMED, which provides branching sites in the copolymer chains 10'. In this exemplary method, at least some of the copolymer chains 10' are branched from one another.

[0138] Another example of one-pot synthesis is schematically shown in Figure 5. In this example, the monomer of formula (III) is R 2’ For example, containing a halogen (specifically Br), the monomer of formula (IV) is R 3’ , R 4 , and R 4’ As each of them, -H and R 3 It contains C(O)NH2.

[0139] In this example of one-pot synthesis, the reaction mixture first contains the monomer of formula (III), R 2’ Except for containing halogens, it may be as described with reference to Figure 4. The copolymerization reaction and quenching may be carried out as described with reference to Figure 4. However, in this example, the product mixture contains an intermediate copolymer 16. This intermediate copolymer 16 contains repeating units 18, which are precursors to repeating units 12 of formula (I) and also contain repeating units 14 of formula (II).

[0140] In this example, NaN3 is added to the intermediate product mixture. The mixture is heated to initiate the reaction that replaces the halogen group with the azide group. In one example, NaN3 is present in a 50-fold excess relative to 18 repeating units to ensure that the halogen group is replaced.

[0141] Another example of one-pot synthesis is schematically shown in Figure 6. In this example, the monomer of formula (V) is used together with the monomer of formula (IV). In this example, the "halo" of formula (V) is Br, and the monomer of formula (IV) is R 3’ , R 4 , and R 4’ -H as each of and R 3 It contains C(O)NH2 as an example.

[0142] In this example, the reaction mixture includes monomers of formula (V), monomers of formula (IV), and an NMP monomolecule initiator, or a combination of a water-soluble initiator and a nitroxide. The monomers of formula (V) and formula (IV) may be present in a suitable weight ratio to each other such that a desirable number of their respective repeating units 12+12', 14 are introduced into the resulting copolymer 10''. The NMP monomolecule initiator, or the combination of a water-soluble initiator and a nitroxide, may be present in an amount that allows all monomers in the reaction mixture to copolymerize. The reaction mixture may also include water, a solvent, or a combination of water and a solvent.

[0143] The copolymerization reaction and quenching may be carried out as described with reference to Figure 4. However, in this example, the product mixture includes an intermediate copolymer 16. This intermediate copolymer 16 includes repeating units 18, which are precursors to repeating units 12 and 12'' respectively, and also includes repeating unit 14 of formula (II). The copolymerization is shown in step 1 of Figure 6.

[0144] Each example of the method shown in Figure 6 involves converting some of the halogens (halo groups shown as Br in Figure 6) to alkoxyamines and, after copolymerization, converting some of the other halogens (halo groups) to azides.

[0145] In one exemplary method, the conversion involves first converting some of the halogens to alkoxyamines (Step 2 in Figure 6), and then converting some of the other halogens to azides (Step 3 in Figure 6). Step 2 may include introducing hydroxylamine into the copolymer such that the halogens outnumber the hydroxylamine, and heating the copolymer to initiate a reaction in which the hydroxylamine displaces some of the halogens. Step 3 may include introducing NaN3 into the copolymer (in this case, including some of the alkoxyamines in the side chains), and heating the copolymer to initiate a reaction in which the azides displace some of the other halogens. The resulting copolymer 10'' comprises repeating units 12, 12'', and 14.

[0146] In another exemplary method, the conversion involves first converting some of the halogens to alkoxyamines (Step 2 in Figure 6), and then converting some of the other halogens to azides (Step 3 in Figure 6). In this example, the hydroxylamine is first deprotonated to form an alkoxyamine anion. - ONR1R2) can be formed. For example, deprotonation may be carried out using a strong base in a polar aprotic solvent, such as sodium hydroxide or potassium tert-butoxide. In another example, the deprotonation reaction may be catalyzed by a base such as 1,8-diazabicycloundeca-7-ene (DBU) or N,N-diisopropylethylamine (DIPEA) in a polar aprotic solvent. In this example, step 2 may include introducing alkoxylamine anions into the copolymer such that the halogens outnumber the alkoxylamine anions, and heating the copolymer to initiate a reaction in which the alkoxylamine anions displace some of the halogens. Step 3 may include introducing NaN3 into the copolymer (here including some of the alkoxyamines in the side chains), and heating the copolymer to initiate a reaction in which the azide displaces some of the other halogens. The resulting copolymer 10'' contains repeating units 12, 12'', and 14.

[0147] In another exemplary method, the conversion simultaneously converts some of the halogens to alkoxyamines and some of the other halogens to azides (step 4 in Figure 6). Step 4 may involve introducing a mixture of hydroxylamine and NaN3 into the copolymer so that the mixture exceeds the halogens. N3 is a stronger nucleophile and is used to complete the reaction, and when mixed, it may result in N3 reacting more than hydroxylamine. The copolymer is heated in the presence of the mixture to initiate the reaction in which the hydroxylamine displaces some of the halogens and the azides displace some of the other halogens. If these reactions occur at different temperatures, the temperature may be increased stepwise to initiate both displacement reactions. For example, a higher temperature may be desirable for the hydroxylamine reaction. The resulting copolymer 10'' contains repeating units 12, 12'', and 14.

[0148] Another example of the method may be performed to produce a block copolymer (e.g., at least one block of repeating units 12 and 14, and at least one block of repeating unit 14).

[0149] One example of this method is i) the monomer (R) of formula (III). 2’ The reaction mixture of a monomer (including an azide as a monomer) and another monomer (e.g., any example represented by formula (IV)) comprises: ii) copolymerizing at a temperature in the range of about 18°C ​​to about 150°C in the presence of an NMP monomolecule initiator or a combination of a water-soluble initiator and a nitroxide; ii) allowing the temperature to drop and optionally removing any unreacted monomers (e.g., evaporation, copolymer precipitation, and washing); iii) introducing the monomer of formula (IV); and iv) increasing the temperature to polymerize the newly added monomer. The process can be repeated to form additional blocks. This process can also be carried out by first polymerizing the monomer of formula (IV) and then polymerizing the monomer of formula (III) with another monomer.

[0150] Another example of this method is i) the monomer (R) of formula (III). 2’ A reaction with a halogen (including a halogen as a component) and another monomer (e.g., any example represented by formula (IV)) comprises copolymerizing at a temperature in the range of about 18°C ​​to about 150°C in the presence of an NMP monomolecule initiator or a combination of a water-soluble initiator and a nitroxide, ii) allowing the temperature to decrease and optionally removing any unreacted monomer, iii) introducing the monomer of formula (IV), and iv) increasing the temperature to polymerize the newly added monomer. The process may be repeated to form additional blocks. In this example, the resulting block copolymer is similar to the intermediate copolymer 16 and contains blocks of repeating units 18. In this example of the method, NaN3, or a mixture of hydroxylamine and NaN3, or hydroxylamine followed by NaN3, or an alkoxylamine anion followed by NaN3 is added to the product mixture. The mixture is heated to initiate a reaction in which the halogen group is replaced by an azide group, or an azide group and an alkoxyamine group. In one example, NaN3 is present in a 50-fold excess relative to the 18 repeating units to ensure that the halogen group is replaced.

[0151] Figures 4, 5, and 6 show the individual units of copolymer 10, 10'' in blocks, respectively. However, it should be understood that the incorporation of the individual units may be statistical, random, or blocky, and may depend on the method used to synthesize copolymer 10, 10', 10''.

[0152] Method for incorporating copolymers onto the surface of a flow cell Examples of copolymers 10, 10', and 10'' disclosed herein can adhere to at least a portion of the flow cell surface. Methods for adhering copolymers 10, 10', and 10'' to the flow cell surface are generally described with reference to Figure 7. While some details regarding the flow cell surface are described with reference to Figure 7, it should be understood that examples of individual flow cell structures are described in more detail with reference to Figures 8A to 8D.

[0153] As shown in Figure 7, the flow cell surface includes a substrate 20. The substrate 20 may include a single-layer base carrier 22 (Figures 8B and 8C) or a multilayer structure 24 (Figures 7 and 8D). The substrate 20 may not be patterned (for example, including a single lane 23 surrounded by gap regions 28 as shown in Figures 8B and 8C), or it may be patterned (for example, including recesses 26 separated by gap regions 28 as shown in Figures 7 and 8D).

[0154] Examples of suitable materials for the single-layer base carrier 22 include epoxysiloxane, glass, modified or functionalized glass, plastics (including acrylic, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, polytetrafluoroethylene (such as Chemors' TEFLON®), cyclic olefin / cycloolefin polymer (COP) (such as Zeon's ZEONOR®), polyimide, etc.), nylon (polyamide), ceramic / ceramic oxide, silica, fused silica, or silica-based materials, aluminum silicate, silicon and modified silicon (e.g., boron-doped p+ silicon), silicon nitride (Si3N4), silicon oxide (SiO2), tantalum pentoxide (Ta2O5) or other tantalum oxides (TaO2O5). x Examples include ), hafnium oxide (HfO2), carbon, metals, and inorganic glass.

[0155] In some examples, a single lane 23 can be the upper surface of the base carrier 22, and the walls of the lane 23 can be defined by a binding material positioned on the base carrier. In other examples, a single lane 23 can be etched onto the upper surface of the base carrier 22.

[0156] An example of the multilayer structure 24 includes a base carrier 22 and at least one other layer 30 thereon. Some examples of the multilayer structure 24 include glass or silicon as the base carrier 22, with tantalum oxide (e.g., tantalum pentoxide or another tantalum oxide (TaO)) on the surface. x)) or a coating layer of another ceramic oxide (e.g., layer 30). Other examples of the multilayer structure 24 include a base carrier 22 (e.g., glass, silicon, tantalum pentoxide, etc.) and a patterned resin as the other layer 30. It should be understood that any material that can be selectively deposited, or deposited and patterned, to form the recesses 26 and gap regions 28 may be used for the patterned resin.

[0157] Inorganic oxides are an example of patterned resins. Inorganic oxides can be selectively applied to the base carrier 22 via vapor deposition, aerosol printing, or inkjet printing. Suitable examples of inorganic oxides include tantalum oxide (e.g., Ta2O5), aluminum oxide (e.g., Al2O3), silicon oxide (e.g., SiO2), and hafnium oxide (e.g., HfO2).

[0158] As another example of patterned resins, polymer resins can be applied to a base support 22 and then patterned. Suitable deposition techniques include chemical vapor deposition, dip coating, dunk coating, spin coating, spray coating, droplet distribution, ultrasonic spray coating, doctor blade coating, aerosol printing, screen printing, and microcontact printing. Suitable patterning techniques include photolithography, nanoimprint lithography (NIL), stamping techniques, embossing techniques, molding techniques, and microetching techniques. Some examples of suitable resins include polyhedral oligomeric silsesquioxane resin (POSS) based resins, non-POSS epoxy resins, poly(ethylene glycol) resins, polyether resins (e.g., ring-opening epoxy), acrylic resins, acrylate resins, methacrylate resins, amorphous fluoropolymer resins (e.g., CYTOP® from Bellex), and combinations thereof.

[0159] As used herein, the term “polyhedral oligomer silsesquioxane” refers to a hybrid intermediate between silica (SiO2) and silicone (R2SiO) (e.g., RSiO 1.5 This refers to a chemical composition with the chemical formula [RSiO]. One example of a polyhedral oligomeric silsesquioxane may be the one described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776-778, which is incorporated in whole by reference. Several polyhedral oligomeric silsesquioxanes are commercially available from Hybrid Plastics under the trade name POSS®. In one example, the composition has the chemical formula [RSiO]. 3 / 2 ] n The organosilicon compound having the R group may be the same or different. Examples of R groups in polyhedral oligomer silsesquioxanes include epoxy, azide / azido, thiol, poly(ethylene glycol), norbornene, tetrazine, acrylate, and / or methacrylate, or further, for example, alkyl, aryl, alkoxy, and / or haloalkyl groups.

[0160] For example, a single base carrier 22 (whether used alone or as part of a multilayer structure 24) may be a circular sheet, panel, wafer, die, etc., having a diameter in the range of approximately 2 mm to approximately 300 mm, for example, approximately 200 mm to approximately 300 mm, or a rectangular sheet, panel, wafer, die, etc., having a maximum dimension of up to approximately 10 feet (approximately 3 meters). For example, a die may have a width in the range of approximately 0.1 mm to approximately 10 mm. While exemplary dimensions are provided, it should be understood that a single base carrier 22 of any suitable dimensions may be used.

[0161] In the examples disclosed herein, the substrate 20 may be activated before the copolymers 10, 10', and 10'' are attached thereto. If the material selected for a single base carrier 22 or a layer 30 of a multilayer structure 24 has surface-OH groups, the activation of the substrate 20 is accompanied by silanization. If the material selected for a single base carrier 22 or a layer 30 of a multilayer structure 24 has minimal or no surface-OH groups, the activation of the substrate 20 is accompanied by plasma ashing (to introduce or increase surface-OH groups), followed by silanization.

[0162] In the examples disclosed herein, a silane derivative, specifically norbornenesilane 32, is deposited on the surface of a substrate 20 using vapor deposition, spin coating, or other deposition methods. In one example, the norbornenesilane is [(5-bicyclo[2.2.1]hepta-2-enyl)ethyl]trimethoxysilane. Other alkyne or alkene-terminated silanes may also be used.

[0163] It should be understood that any exposed surface of the substrate 20 (e.g., any exposed surface of a single base carrier 22, or any exposed surface of layer 30) will be silanized. In the examples shown in Figures 7 and 8D, each of the recesses 26 and gap regions 28 is silanized. In the example shown in Figure 8C, the entire single lane 23 and gap region 28 are silanized. In this example, the substrate 20 includes a base carrier 22 and multiple norbornenesilane molecules 32 attached to the base carrier 22. In the example shown in 8B, a mask may be used during silanization. In these examples, only a portion of the single lane 23 (Figure 8B) to which copolymers 10, 10', and 10'' are desired is silanized.

[0164] In Figure 7, norbornenesilane 32 is shown covalently attached to the surface-OH groups of the recess 26, but not to the void region 28. It should be understood that the initial silanization process deposits norbornenesilane 32 onto any exposed surface of the substrate 20 (including any exposed void region 28). Subsequent processing can be used to remove the material (e.g., copolymer 10, 10', 10'', norbornenesilane 32) from the void region 28, at least substantially, as will be described in more detail below.

[0165] In some examples of the method, copolymers 10, 10', and 10'' are prepared according to one of the exemplary methods described herein and then attached to norbornenesilane molecules 32 on the surface of a substrate 20.

[0166] For example, copolymerization of monomers of formulas (III) and (IV), followed by quenching, may be performed (e.g., Figure 4 or Figure 5). In another example, copolymerization of monomers of formulas (V) and (IV), followed by halogen displacement, may be performed (e.g., Figure 6). In yet another example, the method may be performed to produce block versions of copolymers 10, 10', and 10''. Any of these methods produces a product mixture. The product mixture contains copolymer chains 10, 10', and 10'', and at least some of the copolymer chains 10, 10', and 10'' have alkoxyamine terminal groups E 2 and / or containing alkoxyamines in some of the side chains.

[0167] Regarding adhesion to the substrate 20, the method involves depositing the product mixture onto the substrate 20 having surface-bonded norbornenesilane molecules 32, and alkoxyamine terminal group E 2The method includes reacting at least some of the alkoxyamines in the side chains with at least some of the surface-bound norbornenesilane molecules 32. The product mixture may be deposited using any of the deposition techniques disclosed herein. The copolymer chains 10, 10', 10'' in the deposited product mixture may be reacted with the surface-bound norbornenesilane molecules 32 for a desired period and temperature. The temperature may be in the range of about 35°C to about 150°C, and the time may be in the range of about 10 minutes to about 24 hours. In one example, the reaction time is about 30 minutes and the reaction temperature is about 50°C.

[0168] The reaction conditions are: alkoxyamine terminal group E 2 and / or alkoxyamines in at least some of the side chains of copolymer chains 10, 10', 10'' may be activated, separating these groups and forming free radicals. In this example, at least one of the multiple norbornenesilane molecules 32 attaches to each copolymer chain 10, 10', 10'' at its terminal or side chain from which the nitroxide free radical is separated. Thus, “alkoxyamine-mediated attachment” means that the separation of the free radical generates an open terminal or side chain that can react with the norbornenesilane molecule, and the free nitroxide may or may not reattach to the norbornenesilane molecule. More specifically, as shown in Figure 7, the norbornene portion of the surface-bound norbornenesilane molecule 32 covalently attaches to the remaining copolymer chains 10, 10', 10'' from which the nitroxide free radical is separated. Although one attachment mechanism has been demonstrated, it is thought that nitroxide free radicals may be released from the side chains to enable attachment to surface-bound norbornenesilane molecule 32 via other mechanisms. Since the alkoxyamine surface bond does not contain azide groups on copolymer 10, 10', and 10'', these groups remain free for additional surface or primer bonding. Some of the alkoxyamine groups may remain attached to copolymer 10, 10', and 10'' and may not be involved in surface attachment.

[0169] In another example of the method, copolymers 10, 10', and 10'' are prepared according to any of the exemplary methods described herein in the presence of a substrate 20 having surface-bound norbornenesilane molecules 32.

[0170] In one example, monomers of formulas (III) and (IV) can be copolymerized in the presence of an NMP monomolecule initiator, or a combination of a water-soluble initiator and a nitroxide, and a substrate 20 having a surface-bound norbornenesilane molecule 32. This example can be carried out with or without TeMED, and may be carried out as shown in Figure 4. Since copolymerization takes place in the presence of the substrate 20, at least some of the resulting copolymer chains 10, 10', 10'' have i) alkoxyamine terminal groups E 2 ii) including and / or alkoxyamine terminal group E 2 It is attached to norbornenesilane molecule 32 via [a certain mechanism].

[0171] In another example, monomers of formulas (III) and (IV) can be sequentially polymerized in the presence of an NMP initiator and a substrate 20 having a surface-bound norbornenesilane molecule 32. This example includes the process described herein for producing a block copolymer. Since continuous polymerization is carried out in the presence of the substrate 20, at least some of the resulting block copolymer chains 10, 10', 10'' have i) alkoxyamine terminal groups E 2 ii) including and / or alkoxyamine terminal group E 2 It is attached to norbornenesilane molecule 32 via [a certain mechanism].

[0172] In yet another example, the method shown in Figure 5 is performed in the presence of substrate 20. This example involves an alkoxyamine terminal group E 2This results in the covalent attachment of the intermediate copolymer 16 to the norbornenesilane molecule 32 via [a specific method]. In this exemplary method, NaN3 is added to the intermediate copolymer coating, which is heated to initiate a reaction that replaces the halogen group with an azide group. This additional reaction converts the intermediate copolymer coating into a copolymer coating (containing copolymer chains 10, 10' covalently attached to the substrate 20).

[0173] An exemplary method shown in Figure 6 may also be performed, after which the copolymer 10'' may be deposited on the substrate 20, or the method may be performed in the presence of the substrate 20.

[0174] Whether the reaction between copolymer chains 10, 10', 10'' and surface-bound norbornenesilane molecules 32 occurs after or during the formation of copolymer chains 10, 10', 10'', this reaction produces a copolymer coating on any exposed surface of the substrate 20. As described herein, this copolymer coating is covalently attached to the substrate 20 via alkoxyamines. In some examples, the copolymer coating is used as is, and a primer may be attached thereto (described below). In other examples, the copolymer coating is subjected to an additional curing process to increase the strength of surface adhesion. In these other examples, the method further comprises curing the product mixture or copolymer coating on the substrate 20, thereby causing some of the azide groups to react with some of the other surface-bound norbornenesilane molecules 32. Any unreacted norbornenesilane molecules 32 may react with the azide groups of copolymer chains 10, 10', 10'' during the curing process. For example, curing can be performed at temperatures ranging from room temperature (e.g., about 25°C) to about 95°C, over a period of time ranging from about 1 millisecond to about several days.

[0175] Any of the surface adhesion methods disclosed herein results in the formation of a copolymer coating on any exposed surface of a substrate 20 containing norbornenesilane molecules 32.

[0176] Therefore, in some examples, gap regions 28 adjacent to a single lane 23 (Figure 8C) or gap regions 28 adjacent to a recess 26 (Figures 7 and 8D) may be coated with a copolymer coating. In some cases, it may be desirable for the gap regions 28 to be free of copolymer coating so that these surfaces can be used for bonding and / or adhere within the single lane 23 or recess 26 but not on the gap regions themselves. In these examples, any copolymer coating covering the gap regions 28 may be removed, for example, by a polishing process. The polishing process may be carried out using a chemical slurry (e.g., containing abrasives, buffers, chelating agents, surfactants, and / or dispersants) that can remove the copolymer coating from the gap regions 28 without adversely affecting the substrate 20 (e.g., base carrier 22 or layer 30) beneath those regions 28. Alternatively, polishing may be carried out with a solution that does not contain abrasive particles. The chemical slurry may be used in a chemical mechanical polishing system to polish the surface of the gap regions 28. The polishing head / pad or other polishing tool can polish any copolymer coating that may be present on the gap region 28 while leaving the copolymer coating at least substantially intact in a single lane 23 or recess 26. As an example, the polishing head may be a Strasbaugh ViPRR II polishing head.

[0177] In the example shown in Figure 8C, the copolymer coating 10B remains in a single lane 23 after polishing. In the example shown in Figure 8D, the copolymer coating 10C remains in each of the recesses 26 after polishing.

[0178] In other examples, the copolymer coating selectively adheres to the substrate 20, and therefore no removal process such as polishing is used. For example, if norbornenesilane molecules are deposited on predetermined areas of the substrate 20 using a masking technique (as described above with reference to Figure 8B), the copolymer coating adheres to those predetermined areas but not to any other areas of the substrate 20 that are not silanized. Figure 8A shows an example in which the copolymer coating 10A forms isolated pads on the surface of a single lane 23.

[0179] Flow Cell An exemplary top view of a flow cell 34 is shown in Figure 8A. Examples of different copolymer coatings 10A, 10B, and 10C that can be formed using the methods disclosed herein are shown in Figures 8B, 8C, and 8D, respectively.

[0180] The flow cell 34 may include two of the substrates 20 bonded together, or one substrate 20 bonded to the lid. The substrate 20 includes one or more reactive surfaces, including, for example, copolymer coatings 10A, 10B, 10C and primers 36, 38.

[0181] A flow channel 40 is defined between the bonded substrates 20, or between one substrate 20 and the lid. Thus, the flow channel 40 is in fluid communication with the reactive surface of the substrate 20.

[0182] The example shown in Figure 8A includes eight flow channels 40. Although eight flow channels 40 are shown, it should be understood that any number of flow channels 40 can be contained in the flow cell 34 (e.g., a single flow channel 40, four flow channels 40, etc.). Each flow channel 40 may be isolated so that the fluid introduced into any flow channel 40 does not flow into any adjacent flow channel 40. Some examples of fluids introduced into the flow channels 40 may include reaction components (e.g., DNA samples, polymerase, sequencing primers, nucleotides, etc.), washing solutions, deblocking agents, etc.

[0183] In one example, the flow channel 40 has a linear configuration.

[0184] The substrate 20, or the substrate 20 and the lid, may be bonded together using a bonding material not shown. Any suitable bonding material may be used, such as an adhesive or a radiation-absorbing material to aid bonding. The depth of the flow cell 40 may depend in part on the thickness of the bonding material. The depth of the flow channel 40 may be approximately the same as the thickness of a single layer when the bonding material is deposited using microcontact, aerosol, or inkjet printing. In other examples, the depth of the flow channel 40 may be about 1 μm, about 10 μm, about 50 μm, about 100 μm, or more. In one example, the depth may be in the range of about 10 μm to about 100 μm. In another example, the depth may be in the range of about 10 μm to about 30 μm. In yet another example, the depth is about 5 μm or less. It should be understood that the depth of the flow channel 40 may be greater than or less than the values ​​specified above, or in between.

[0185] Figures 8B, 8C, and 8D show examples of structures within the substrate 20 and flow channel 40. As shown in Figure 8B, the structure may include copolymer coating 10A in the form of isolated pads (separated by gap regions 28') on the surface of the monolayer base carrier 22 / substrate 20. As shown in Figure 8C, the structure may include copolymer coating 10B in the form of a coating layer on the entire surface of a single lane 23 defined within the monolayer base carrier 22 / substrate 20. As shown in Figure 8D, the structure may include copolymer coating 10C in the form of individual layers located in each of the recesses 26 and not located in the gap regions 28.

[0186] Many different layouts of the isolated pad copolymer coatings 10A and recesses 26 can be envisioned, including regular, repeating, and irregular patterns. In one example, the isolated pad copolymer coatings 10A or recesses 26 are arranged in a hexagonal grid for close packing and improved density. Other layouts may include, for example, linear (rectangular) layouts, triangular layouts, and so on. In some examples, the layout or pattern may be in an xy format consisting of rows and columns. In some other examples, the layout or pattern may be a repeating arrangement of the isolated pad copolymer coatings 10A or recesses 26. In yet another example, the layout or pattern may be a random arrangement.

[0187] The layout or pattern can be characterized in terms of the density (number) of isolated pad copolymer coatings 10A or recesses 26 within a given area. For example, the number of isolated pad copolymer coatings 10A or recesses 26 may be approximately 2 million / mm². 2 It can exist at a density of 1 mm. 2 Approximately 100,1mm 2 Approximately 1,000 per unit, 1 mm 2 Approximately 100,000 per unit, 1 mm 2 Approximately 1 million per unit, 1 mm 2 Approximately 2 million per unit, 1 mm 2 Approximately 5 million per unit, 1 mm 2 Approximately 10 million per unit, 1 mm2 It can be adjusted to different densities, including densities of approximately 50 million or less per unit. It should be further understood that the density may be between one of the lower and upper limits selected from the above range, or other densities (outside the given range) may be used.

[0188] The layout or pattern of the isolated pad copolymer coatings 10A or recesses 26 may also be characterized, or alternatively, with respect to the average pitch, or the distance (edge-to-edge distance) from the center of one isolated pad copolymer coating 10A or recess 26 to the center of an adjacent isolated pad copolymer coating 10A or recess 26, or from the right edge of one isolated pad copolymer coating 10A or recess 26 to an adjacent isolated pad copolymer coating 10A or recess 26. The pattern may be regular such that the coefficient of variation around the average pitch is small, or the pattern may be irregular, in which case the coefficient of variation may be relatively large. In either case, the average pitch may be, for example, approximately about 50 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, or about 100 μm. The average pitch of a particular pattern may be between one of the lower values ​​and one of the higher values ​​selected from the above range. In one example, the recess 20 has a pitch (center-to-center distance) of approximately 1.5 μm. An example of an average pitch value is provided, but it should be understood that other average pitch values ​​may also be used.

[0189] The size of each recess 26 may be characterized by its volume, opening area, depth, and / or diameter or length and width. For example, the volume may be approximately 1 × 10⁻⁶. -3 μm 3 ~about 100μm 3 It extends to a range of, for example, approximately 1 × 10 -2 μm 3 , about 0.1μm 3 , about 1μm 3 , about 10μm 3 , or more or less. In another example, the opening area is approximately 1 × 10⁻⁶3 μm 2 ~about 100μm 2 It extends to a range of, for example, approximately 1 × 10 -2 μm 2 , about 0.1μm 2 , about 1μm 2 , at least about 10 μm 2 , or more or less. In yet another example, the depth ranges from about 0.1 μm to about 100 μm, for example, about 0.5 μm, about 1 μm, about 10 μm, or more or less. In yet another example, the depth ranges from about 0.1 μm to about 100 μm, for example, about 0.5 μm, about 1 μm, about 10 μm, or more or less. In yet another example, the diameter or length and width ranges from about 0.1 μm to about 100 μm, for example, about 0.5 μm, about 1 μm, about 10 μm, or more or less.

[0190] The size of each isolated pad copolymer coating 10A may be characterized by its top surface area, height, and / or diameter or length and width. For example, the top surface area may be approximately 1 × 10⁻⁶ 3 μm 2 ~about 100μm 2 It extends to a range of, for example, approximately 1 × 10 -2 μm 2 , about 0.1μm 2 , about 1μm 2 , at least about 10 μm 2 , or more or less. In yet another example, the height ranges from about 0.1 μm to about 100 μm, for example, about 0.5 μm, about 1 μm, about 10 μm, or more or less. In yet another example, the diameter or length and width ranges from about 0.1 μm to about 100 μm, for example, about 0.5 μm, about 1 μm, about 10 μm, or more or less.

[0191] The copolymer coatings 10A, 10B, and 10C may comprise any of the exemplary copolymer chains 10, 10', and 10'' disclosed herein, and may be formed and adhered to the substrate 20 using any of the methods disclosed herein.

[0192] Flowcell 34 also includes primers 36 and 38.

[0193] The primers 36 and 38 can be grafted onto copolymer coatings 10A, 10B, and 10C by a grafting process. In one example, the primers 36 and 38 can be immobilized onto copolymer coatings 10A, 10B, and 10C by a single-point covalent attachment at or near the 5' end of the primers 36 and 38. This attachment allows i) the adapter-specific portion of the primers 36 and 38 to freely anneal to its homologous sequenceable nucleic acid fragment, and ii) the 3' hydroxyl group to freely extend the primer. In the examples disclosed herein, the covalent attachment may occur at any unreacted azide / azide group of the copolymer chains 10 and 10' in copolymer coatings 10A, 10B, and 10C. Examples of terminal primers that can be used include alkyne terminal primers that can bind to the azide portion of copolymer coatings 10A, 10B, and 10C. Specific examples of suitable primers 36, 38 include P5 and P7 primers used on the surface of commercially available flow cells sold by Illumina Inc. for sequencing on HISEQ®, HISEQX®, MISEQ®, MISEQDX®, MINISEQ®, NEXTSEQ®, NEXTSEQ®DX®, NOVASEQ®, GENOME ANALYZER®, ISEQ®, and other instrument platforms.

[0194] In one example, the grafting may be carried out by flow-through deposition (e.g., using a temporarily or permanently bonded lid, or a permanently bonded second substrate 20), dunk coating, spray coating, puddle dispensing, or by another preferred method of attaching primers 36, 38 to copolymer coatings 10A, 10B, 10C in the flow channel 40. Each of these examples of techniques may utilize a primer solution or mixture, which may comprise primers 36, 38, water, buffer, and catalyst. By any of the grafting methods, primers 36, 38 react with the reactive groups of copolymer coatings 10A, 10B, 10C in the flow channel 40 and have no affinity for the surrounding substrate 20 (e.g., interstitial regions 28, 28'). Thus, primers 36, 38 selectively graft onto copolymer coatings 10A, 10B, 10 in the flow channel 40.

[0195] Sequencing method An example of flow cell 34 can be used in ensemble sequencing techniques, such as sequencing by synthesis (SBS). In ensemble sequencing, a template polynucleotide chain (not shown) to be sequenced can be formed on the reactive surface of flow cell 34 using primers 36, 38. At the beginning of template polynucleotide chain formation, a library template can be prepared from any nucleic acid sample (e.g., a DNA sample or an RNA sample). The nucleic acid sample can be fragmented into similarly sized (e.g., <1000 bp), single-stranded DNA or RNA fragments, which are then converted into complementary DNA (cDNA) fragments. During preparation, adapters can be added to the ends of these fragments. By reductive cycle amplification, different motifs such as sequencing binding sites, indices, and regions complementary to primers 36, 38 can be introduced into the adapters. The final library template contains DNA or cDNA fragments with adapters at both ends. In some examples, fragments from a single nucleic acid sample have the same adapters added to the fragments.

[0196] Multiple library templates can be introduced into the flow cell 34. The multiple library templates are hybridized, for example, to one of two types of primers 36, 38 immobilized on copolymer coatings 10A, 10B, and 10C.

[0197] Next, cluster generation can be performed. In one example of cluster generation, the library template is copied from hybridized primers by 3' extension using high-fidelity DNA polymerase. The original library template is denatured, leaving an immobilized copy on copolymer coatings 10A, 10B, and 10C. Isothermal bridging amplification or some other form of amplification can be used to amplify the immobilized copy. For example, the copied template loops over and hybridizes to an adjacent complementary primer 38 or 36, and the polymerase copies the copied template to form a double-stranded bridge structure, which then denatures to form two single strands. These two strands loop over and hybridize to an adjacent complementary primer 36 or 38, and are extended again to form two new double-stranded loops. This process is repeated for each template copy by isothermal denaturation and amplification cycles to create dense clonal clusters. Each cluster of double-stranded bridge structures is denatured. In one example, the reverse strand is removed by specific base cleavage, leaving a forward template polynucleotide chain. Clustering results in the formation of several template polynucleotide chains across the copolymer coatings 10A, 10B, and 10C. An example of this clustering is bridge amplification, which is one example of amplification that can be performed. It should be understood that other amplification techniques, such as the Exclusion Amplification (Examp) workflow (Illumina Inc.), may be used.

[0198] Sequencing primers that hybridize to complementary sequences on a template polynucleotide chain can be introduced. These sequencing primers prepare the template polynucleotide chain for sequencing.

[0199] To initiate sequencing, a composite mixture can be added to the flow cell 34. In one example, the composite mixture includes a liquid carrier, polymerase, and a fluorescently labeled nucleotide. The fluorescently labeled nucleotide may contain a 3'OH blocking group. Once the composite mixture is introduced into the flow cell 34, the fluid enters the flow channel 40 and comes into contact with the reactive surface.

[0200] Fluorescently labeled nucleotides are added to sequencing primers in a template-dependent manner (thus extending the sequencing primers), and the detection of the order and type of nucleotides added to the sequencing primers can be used to sequence the template. More specifically, one of the nucleotides is incorporated by its respective polymerase into a nascent strand complementary to the template polynucleotide chain that extends the sequencing primer. In other words, in at least some of the template polynucleotide chains throughout the flow cell 34, each polymerase extends the hybridized sequencing primer by one of the nucleotides in the incorporation mixture.

[0201] Nucleotide incorporation can be detected through imaging events. During imaging events, an illumination system (not shown) may provide excitation light to the reactive surfaces of the flow channel 40 and / or flow cell 34.

[0202] In some examples, the nucleotide may further include a reversible termination property (e.g., a 3'OH blocking group) that terminates further primer extension once the nucleotide is added to the sequencing primer. For example, a nucleotide analog with a reversible terminal portion is added to the sequencing primer, and as a result, no further extension can occur until a deblocking agent is delivered and that portion is removed. Therefore, in examples using reversible termination, the deblocking reagent can be delivered to flow cell 34 after detection has been performed.

[0203] Washing may be performed between various fluid delivery steps. The SBS cycle is then repeated n times to extend the sequencing primers with n nucleotides, thereby allowing for the detection of a sequence of length n.

[0204] While SBS is described in detail, it should be understood that the flow cell 34 described herein may be used in conjunction with other sequencing protocols for genotyping or other chemical and / or biological applications. In some cases, the flow cell primers may be selected to enable simultaneous paired-end sequencing, where both the forward and reverse strands are present on copolymer coatings 10A, 10B, and 10C, allowing simultaneous base calling of each read. Sequential and simultaneous paired-end sequencing facilitates the detection of genomic rearrangements and repeating sequence elements, as well as gene fusions and novel transcripts. In another example, the flow cell 34 disclosed herein may be used for on-cell library generation.

[0205] Additional information It should be understood that all combinations of the aforementioned concepts and further concepts, which are discussed in more detail below, are intended to be part of the subject matter of the inventions disclosed herein (provided that such concepts do not contradict each other). Specifically, all combinations of claimed subject matter appearing at the end of this disclosure are intended to be part of the subject matter of the inventions disclosed herein. It should also be understood that terms used expressly herein and that may appear in any disclosure incorporated by reference should be given meanings that most coincide with the specific concepts disclosed herein.

[0206] Throughout this specification, references to "an example," "another example," or "a certain example" mean that certain elements (e.g., features, structures, and / or characteristics) described in relation to an example are included in at least one example described herein, and may or may not be present in other examples. Furthermore, unless explicitly indicated otherwise in the context, it should be understood that elements described in relation to any example may be combined in any preferred manner in various examples.

[0207] It should be understood that the ranges provided herein include the indicated range and any values ​​or subranges within that indicated range, as if such values ​​or subranges were explicitly enumerated. For example, the range of approximately 200 mm to approximately 300 mm should be interpreted to include not only the explicitly enumerated limits of approximately 200 mm to approximately 300 mm, but also individual values ​​such as approximately 240 mm, approximately 250.5 mm, and subranges such as approximately 225 mm to approximately 275 mm. Furthermore, where “approximately” and / or “substantially” are used to describe a value, they mean to include slight variations (up to ±10%) from the indicated value.

[0208] While several embodiments have been described in detail, it should be understood that the disclosed examples can be modified. Therefore, the above description should be considered non-limiting. This disclosure includes the following aspects. (Aspect 1) It is a flow cell, Substrate and A copolymer coating attached to at least a portion of the substrate, wherein the copolymer coating comprises a plurality of copolymer chains, and each copolymer chain is Equation (I):

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Claims

1. It is a flow cell, Substrate and A copolymer coating attached to at least a portion of the substrate, wherein the copolymer coating comprises a plurality of copolymer chains, and each copolymer chain is Equation (I): 【Chemistry 1】 The first repeating unit of the formula, R １ However, it is selected from the group consisting of -H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, and optionally substituted variants thereof. R ２ However, it is Azid, Each (CH ２ ) ｐ However, it can be replaced by choice. The first repeating unit is an integer from 1 to 50, Formula (II): 【Chemistry 2】 The second repeating unit of the formula, R ３ 、 R ３’ 、 R ４ 、 R ４’ each of which is independently -H, R ５ 、 -OR ５ 、 -C(O)OR ５ 、 -C(O)R ５ 、 -OC(O)R ５ 、 -C(O)NR ６ R ７ 、 and -NR ６ R ７ selected from the group consisting of, R ５ However, it is selected from the group consisting of -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, heterocyclic, and their optionally substituted variants. R ６ and R ７ Each of these independently comprises a second repeating unit selected from the group consisting of -H and alkyl groups, Equation (VI): 【Transformation 3】 The third repeating unit of the formula, R １ However, it is selected from the group consisting of -H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, and optionally substituted variants thereof. R １ and R ２ However, they are independently selected from carbon-based substituents, Each (CH ２ ) ｐ However, it can be replaced by choice. A flow cell comprising a copolymer coating, which comprises a third repeating unit, where p is an integer from 1 to 50.

2. The flow cell according to claim 1, wherein at least some of the copolymer chains include at least one alkoxyamine terminal group.

3. R ３’ , R ４ , and R ４’ Each of them is -H, R ３ However, -C(O)NR ６ R ７ And, R ６ and R ７ The flow cell according to claim 1, wherein each of the is -H.

4. R １ However, it is -H, The flow cell according to claim 3, wherein p is 5.

5. The substrate includes recesses separated by gap regions, The flow cell according to any one of claims 1 to 4, wherein the copolymer coating is attached to the recess.

6. The copolymer coating forms isolated pads on the surface of the substrate. The flow cell according to any one of claims 1 to 5, wherein the gap region separates the isolated pad.

7. It is a method, i) A monomolecule initiator for nitroxide-mediated polymerization, or ii) a combination of a water-soluble initiator and a nitroxide, using formula (V): 【Chemistry 4】 The monomer and formula (IV): 【Transformation 5】 Adding to a mixture with the monomer, in the formula, Regarding equation (V), R １ However, it is selected from the group consisting of -H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, and optionally substituted variants thereof. Halo is a halogen, Each (CH ２ ) ｐ However, it can be replaced by choice. p is an integer between 1 and 50. Regarding equation (IV), R ３ , R ３’ , R ４ , R ４’ Each of these independently is -H, R ５ , -OR ５ , -C(O)OR ５ , -C(O)R ５ , -OC(O)R ５ , -C(O)NR ６ R ７ , and -NR ６ R ７ Selected from the group consisting of, R ５ However, it is selected from the group consisting of -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, heterocyclic, and their optionally substituted variants. R ６ and R ７ Each of these is independently selected from the group consisting of -H and alkyl, and is added. This produces copolymers, A method comprising converting some of the halogens to alkoxyamines and some of the halogens to azides after copolymerization.

8. Converting some of the halogens to alkoxyamines and some of the halogens to azides, Introducing hydroxylamine into the copolymer such that the halogen is present in a molar amount greater than that of the hydroxylamine, The copolymer is heated to initiate a reaction in which the hydroxylamine displaces some of the halogens, NaN ３ Introducing into the copolymer, The method according to claim 7, comprising heating the copolymer to initiate a reaction in which the azide displaces some of the other halogens.

9. Converting some of the halogens to alkoxyamines and some of the halogens to azides, Hydroxylamine and NaN ３ Introducing a mixture of the above into the copolymer, The method according to claim 7, comprising heating the copolymer to initiate a reaction in which the hydroxylamine displaces some of the halogens and the azide displaces some of the other halogens.

10. Converting some of the halogens to alkoxyamines and some of the halogens to azides, Deprotonation of hydroxylamine to form an alkoxyamine anion, The alkoxyamine anion is introduced into the copolymer such that the halogen is present in a molar amount greater than the alkoxyamine anion. The copolymer is heated to initiate a reaction in which the alkoxyamine anion displaces some of the halogens, NaN ３ Introducing into the copolymer, The method according to claim 7, comprising heating the copolymer to initiate a reaction in which the azide displaces some of the other halogens.

11. R ３’ , R ４ , and R ４’ Each of them is -H, R ３ However, -C(O)NR ６ R ７ And, R ６ and R ７ The method according to any one of claims 7 to 10, wherein each of is -H.

12. R １ However, it is -H, The method according to claim 11, wherein p is 5.

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