Light-operated chemistry for reversible hydrogels and reusable flow cells

Abstract

In some examples, novel photochemically reversible hydrogel particles and nanogel particles are described, the photochemically reversible hydrogel particles and nanogel particles comprising a copolymer chain that comprises a copolymer capable of polymerizing at gt, gt, gt, gt, gt, gt, gt, gt, gt, gt, gt, gt, gt, gt, gt, gt, gt, gt; and at least one reactive olefin or reactive 1, 4-diene end group that respectively undergoes [2 + 2] or [2 + 2 + 2 + 2] photodimerization at a wavelength of 270 nm. In various examples, the photochemically reversible hydrogel includes a copolymer chain that includes at least one-N3,-C = CH, or-CO2H end group to achieve bifunctionality and / or pH responsiveness. For nucleic acid sequencing, amplification primers are transplanted onto photochemically reversible hydrogel particles or nanogel particles that reversibly bind to a surface within a flow cell. After sequencing is completed, the photochemically reversible hydrogel particles or nanogel particles can be removed from the surface of the flow cell by irradiation, so that the flow cell can be reused.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 378,943, filed on October 10, 2022, and entitled “Photo-switchable Chemistry for Reversible Hydrogels and Reusable Flow Cells,” the disclosure of which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present disclosure relates generally to nucleic acid sequencing methods and apparatus, and in particular to photochemically reversible hydrogel particles and nanogel particles that can be used in sequencing-by-synthesis (SBS) methods. Background Art

[0004] Nucleic acid sequencing remains an important tool in many different fields, from ancestry research to law enforcement to medical diagnosis. Today's sequencing methods are fast and cost-effective. Nevertheless, there is still a need to further reduce the cost of gene sequencing, such as by simplifying the individual processing steps in the sequencing method and improving various equipment (such as the flow cells used in synthesis sequencing (SBS)). Summary of the invention

[0005] Provided herein are photochemically reversible hydrogels and photochemically reversible nanogel particles with light-controlled chemistry for use in nucleic acid sequencing systems.

[0006] For example, as provided herein, certain polymer hydrogel particles and nanogel particles with photochemical reversibility can be used to replace the hydrogel coating in a flow cell for synthesis sequencing (SBS). Polymer hydrogel particles and nanogel particles with photochemical reversibility can be used to improve many aspects of the SBS method, such as enabling the sequencing flow cell to be reused after the removal of the hydrogel particles or nanogel particles.

[0007] In various examples provided herein, certain polymer hydrogel particles and nanogel particles having photochemical reversibility can be attached to the surface of a flow cell when exposed to light with a frequency hυ1>270nm, and can be detached from the surface of the flow cell when exposed to light with a frequency hυ2<300nm.

[0008] In various examples provided herein, certain polymer hydrogels and nanogel particles having photochemical reversibility include copolymer chains comprising at least one reactive olefin or reactive 1,4-diene end group capable of undergoing [2+2] or [2+2+2+2] photodimerization, respectively, at a wavelength of hυ1>270 nm.

[0009] In various examples provided herein, nanogel particles with photochemical reversibility are used as a substitute for nanowells in a flow cell, thereby eliminating the need to configure nanowells in a flow cell for SBS. Other examples may include the operation of capturing nanogel particles in nanowells configured in a flow cell.

[0010] In various examples provided herein, sequencing on nanogel particles relative to sequencing on hydrogel surfaces improves the monoclonality of multi-copy clusters of sequencing templates. For example, during sequencing, confining clusters to nanoscale particles can improve signal-to-noise ratios, error rates, and the overall quality and coverage of the genome.

[0011] In various examples, the nanogel particles disclosed herein with photochemical reversibility also exhibit bifunctionality by the presence of at least two types of reactive end groups on the copolymer chains within the nanogel particles. In various examples, the nanogel particles with photochemical reversibility and bifunctionality include copolymer chains, the copolymer chains include at least one reactive olefin or reactive 1,4-diene end group capable of [2+2] or [2+2+2+2] photodimerization at a wavelength of hυ1>270nm, respectively, and the nanogel particles include copolymer chains, the copolymer chains having at least one of an azide end group and a carboxylic acid end group.

[0012] In various examples, the photochemically reversible nanogel particles disclosed herein also exhibit dual responsiveness, namely temperature and pH responsiveness. The temperature responsiveness is partially due to the copolymer chains having poly(N-isopropylacrylamide) unit portions, and the pH responsiveness is partially due to the copolymer chains having carboxylic acid end groups.

[0013] The bifunctionality and dual responsiveness (temperature / pH) characteristics of nanogel particles as provided herein allow the initial attachment of alkyne-functionalized amplification primers to the particles using, for example, the -N3 functionality on the particles, while the pH responsiveness enhances chemical capture on the flow cell surface using bioconjugation techniques. These amplification primer-functionalized nanogel particles have been shown to support on-board particle clustering and SBS sequencing.

[0014] In various examples, the hydrogel polymer includes a copolymer chain further comprising:

[0015] The first repeating unit of formula (I):

[0016] in

[0017] R 1 , R 1’ and R 1”Each of is independently selected from H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl or heterocyclyl;

[0018] X is -O- or -NH-; and

[0019] R 2 is -CH2-C≡CH, or R 2 Has the following structure:

[0020]

[0021] Where R 2’ is -NH2, alkyl, alkoxy, alkenyl, alkynyl or optionally substituted variants thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, aziridine, triazoline or -C≡CH; and

[0022] p is an integer from 1 to 50; and

[0023] The second repeating unit of formula (II),

[0024] in:

[0025] R 3 , R 3’ , R 4 and R 4’ Each of -H, -R 5 、-OR 5 、-CO2R 5 、-C(O)R 5 、-OC(O)R 5 、-C(O)NR 6 R 7 、-NR 6 R 7 or a substructure of formula (III),

[0026]

[0027] R 5 is -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl or heterocyclyl;

[0028] R 6 and R 7 Each of is independently selected from -H and alkyl;

[0029] A is an aryl or thymidyl moiety;

[0030] R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl or their optionally substituted variants, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN,

[0031] -CO2H, -SH, glycidyl, epoxy or two R' groups, when bonded to adjacent atoms on ring A and combined with ring A, form coumarin, anthracenyl, acenaphthenyl, thioguanidine

[0032] a thionaphthene-1,1-dioxide moiety;

[0033] X 1 is a bond, -(CH2) q -, -O- or -NH-;

[0034] L is a -(CH2) q -X 2 -C(=O)- or -(CH2CH2O) q -X 2 -C(=O)- divalent linker;

[0036] X 2 is -O- or -NH-;

[0037] m is an integer from 1 to 9; and

[0038] q is an integer from 0 to 50;

[0039] Wherein at least some of the copolymer chains include at least one reactive olefin or reactive 1,4-diene end group capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm.

[0040] In various examples, at least some of the copolymer chains include at least one N3, -C≡CH, or -CO2H terminal group.

[0041] In various examples, formula (III) includes subgeneric structures of formula (IV):

[0042]

[0043] Where R 8 and R 9 Each of is -H, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl or heterocyclyl; each R 10 and R 11 is independently -H, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl or heterocyclyl, or R 10 is -C(=O)-, R 11is -O-, and R 10 and R 11 are bonded together such that formula (IV) includes a substituted coumarin moiety.

[0044] In various examples, the repeating unit of formula (I) is:

[0045]

[0046] In various examples, the repeating unit of formula (II) is:

[0047]

[0048] and / or

[0049]

[0050] In various examples, the repeating unit of formula (II) is:

[0051]

[0052] and / or

[0053] wherein R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl or optionally substituted variants thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy; and

[0054] m is an integer of 1-9.

[0055] In various examples, the repeating unit of formula (II) is:

[0056] and / or

[0057] wherein q is an integer from 0 to 50.

[0058] In various examples, the repeating unit of formula (II) is

[0059]

[0060] and / or

[0061] wherein R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl or optionally substituted variants thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy; and

[0062] m is an integer of 1-7.

[0063] In various examples, the repeating unit of formula (II) is:

[0064]

[0065] and / or

[0066] wherein R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl or optionally substituted variants thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy; and

[0067] m is an integer of 1-5.

[0068] In various examples, the repeating unit of formula (II) is:

[0069]

[0070] and / or

[0071]

[0072] In various examples, R 9 or R 10 is -CO2H, so that formula (IV) is a cis- or trans-cinnamic acid moiety.

[0073] In various examples, R 9 or R 10 is an aryl group, so that formula (IV) is a cis- or trans-stilbene moiety.

[0074] In various examples, the hydrogel polymer is derived from a monomer mixture comprising: 7-((2-methacryloyloxy)ethoxy)-4-methylcoumarin; N-isopropylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

[0075] In various examples, the hydrogel polymer is derived from a monomer mixture comprising: 7-((2-methacryloyloxy)ethoxy)-4-methylcoumarin; N,N-dimethylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

[0076] In various examples, the hydrogel polymer is derived from a monomer mixture comprising: 7-((2-acrylamido)ethoxy)-4-methylcoumarin; N-isopropylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

[0077] In various examples, the hydrogel polymer is derived from a monomer mixture comprising: 7-((2-acrylamido)ethoxy)-4-methylcoumarin; N,N-dimethylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

[0078] In various examples, the hydrogel polymer is derived from a monomer mixture comprising: 7-(acrylamido)-4-methylcoumarin; N-isopropylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

[0079] In various examples, the hydrogel polymer is derived from a monomer mixture comprising: 7-(acrylamido)-4-methylcoumarin; N,N-dimethylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

[0080] In various examples, the hydrogel polymer is derived from a monomer mixture comprising: 7-(methacrylamido)-4-methylcoumarin; N-isopropylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

[0081] In various examples, the hydrogel polymer is derived from a monomer mixture comprising: 7-(methacrylamido)-4-methylcoumarin; N,N-dimethylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

[0082] In various examples, the hydrogel polymer is derived from a monomer mixture comprising: 7-(acryloyloxy)-4-methylcoumarin; N-isopropylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

[0083] In various examples, the hydrogel polymer is derived from a monomer mixture comprising: 7-(acryloyloxy)-4-methylcoumarin; N,N-dimethylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

[0084] In various examples, the hydrogel polymer is derived from a monomer mixture comprising: 7-(methacryloyloxy)-4-methylcoumarin; N-isopropylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

[0085] In various examples, the hydrogel polymer is derived from a monomer mixture comprising: 7-(methacryloyloxy)-4-methylcoumarin; N,N-dimethylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

[0086] In various examples, the monomer mixture further comprises a multifunctional compound selected from the group consisting of: N,N'-methylenebisacrylamide, N,N'-methylenebismethacrylamide, polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, N-vinyl acrylamide, glycidyl acrylate, divinylbenzene, tetraallylammonium chloride, and mixtures thereof.

[0087] In various examples, the hydrogel polymer is in the form of nanogel particles.

[0088] In various examples, the hydrogel polymer also includes an amplification primer conjugated thereto.

[0089] In various examples, each conjugation between an amplification primer and the hydrogel polymer comprises a click chemistry between a terminal alkyne substituent on the amplification primer and an azide group on the corresponding copolymer chain end, or a click chemistry between a terminal azide substituent on the amplification primer and an alkyne group on the corresponding copolymer chain end.

[0090] In various examples, at least some of the copolymer chains are crosslinked by photodimerization between the reactive olefin or reactive 1,4-diene end groups capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm.

[0091] In various examples, a substrate having a surface includes a hydrogel polymer covalently attached thereto, wherein the hydrogel polymer includes a plurality of copolymer chains further comprising a repeating unit of formula (I) and a repeating unit of formula (II);

[0092]

[0093] in:

[0094] R 1 , R 1’ and R 1” Each of is independently selected from H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl or heterocyclyl;

[0095] X is -O- or -NH-;

[0096] R 2 is -CH2-C≡CH, or R 2 Has the following structure:

[0097] R 2’ is -NH2, alkyl, alkoxy, alkenyl, alkynyl or optionally substituted variants thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, aziridine, triazoline or -C≡CH;

[0098] p is an integer from 1 to 50;

[0099] R 3 , R 3’ , R 4 and R 4’ Each of -H, -R 5 、-OR 5 、-CO2R 5 、-C(O)R 5 、-OC(O)R 5 、-C(O)NR 6 R 7 、-NR 6 R 7 or a substructure of formula (III),

[0100]

[0101] R 5 is -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl or heterocyclyl;

[0102] R 6 and R 7 Each of is independently selected from -H and alkyl;

[0103] A is an aryl or thymidyl moiety;

[0104] R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl or optionally substituted variants thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, or two R' groups that, when bonded to adjacent atoms on ring A and taken together with ring A, form a coumarinyl, anthracenyl, acenaphthenyl, thioguandanyl-1-oxide or thioguandanyl-1,1-dioxide moiety;

[0105] X 1 is a bond, -(CH2) q -, -O- or -NH-;

[0106] L is a -(CH2) q -X 2 -C(=O)- or -(CH2CH2O) q -X 2 -C(=O)- divalent linker;

[0108] X 2 is -O- or -NH-;

[0109] m is an integer from 1 to 9; and

[0110] q is an integer from 0 to 50;

[0111] wherein at least some of the copolymer chains include at least one -N3, -C≡CH or -CO2H end group; and wherein at least some of the copolymer chains include at least one reactive olefin or reactive 1,4-diene end group capable of [2+2] or [2+2+2+2] photodimerization at wavelengths >270 nm, respectively.

[0112] In various examples, the covalent connection between the substrate and the hydrogel polymer includes a photodimerization bond between the reactive olefin or reactive 1,4-diene end group of the copolymer chain capable of [2+2] or [2+2+2+2] photodimerization and a corresponding reactive olefin or reactive 1,4-diene group disposed on the surface of the substrate.

[0113] In various examples, the photodimerization bond includes at least one of a coumarin dimer, an anthracene dimer, a thymidine dimer, a cinnamic acid dimer, a stilbene dimer, an acenaphthene dimer, a 2-methylbenzothiophene-1-oxide dimer, a 2-methylbenzothiophene-1,1-dioxide dimer, or a styrylquinoxaline dimer. In various examples, the hydrogel polymer is in the form of nanogel particles. In various examples, the hydrogel polymer also includes an amplification primer conjugated thereto.

[0114] In various examples, each conjugation between an amplification primer and the hydrogel polymer comprises a click chemistry between a terminal alkyne substituent on the amplification primer and an azide terminal group on a corresponding copolymer chain, or a click chemistry between a terminal azide substituent on the amplification primer and an alkyne terminal group on a corresponding copolymer chain.

[0115] In various examples, at least some of the copolymer chains of the hydrogel polymer are crosslinked by photodimerization between the reactive olefin or reactive 1,4-diene end groups capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm.

[0116] In various examples, the flow cell includes a substrate as described herein above.

[0117] In various examples, a method of synthesizing a hydrogel polymer having cross-linked copolymer chains includes:

[0118] (1) A method comprising: (a) 7-((2-acryloyloxy)ethoxy)-4-methylcoumarin, 7-((2-methacryloyloxy)ethoxy)-4-methylcoumarin, 7-((2-acrylamide)ethoxy)-4-methylcoumarin, 7-((2-methacrylamide)ethoxy)-4-methylcoumarin, 7-((2-acryloxy)aminoethyl)-4-methylcoumarin, 7-((2-methacryloxy)aminoethyl)-4-methylcoumarin, 7-((2-acrylamide)aminoethyl)-4-methylcoumarin or 7-

[0119] an aqueous dispersion of a monomer mixture of ((2-methacrylamido)aminoethyl)-4-methylcoumarin; (b) N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA); (c) N,N-dimethylacrylamide or N-isopropylacrylamide (NiPAM); and (d) acrylic acid (AAc) under conditions suitable for free radical polymerization to form the hydrogel polymer having non-crosslinked copolymer chains, wherein at least some of the copolymer chains include reactive coumarin end groups from monomer (a); and

[0120] (2) crosslinking at least some of the copolymer chains by irradiating the hydrogel polymer with light of wavelength >270 nm, the crosslinks comprising dimers between the reactive coumarin end groups of the copolymer chains.

[0121] In various examples, the crosslinks include about 5 mol % of available reactive coumarin end groups. In various examples, the hydrogel is in the physical form of nanogel particles.

[0122] In various examples, the free radical polymerization includes suspension / precipitation free radical polymerization, which also includes a free radical initiator and a dispersion.

[0123] In various examples, a method for assembling a flow cell capable of sequencing a nucleic acid includes:

[0124] (a) treating the surface of the flow cell with any one of 3-mercaptopropylsilanetriol, 3-mercaptopropyltrimethoxysilane or 3-mercaptopropyltriethoxysilane to form a surface having a plurality of reactive -SH groups tethered to the surface;

[0125] (b) reacting the plurality of -SH groups with an α,β-unsaturated carbonylthiol acceptor to provide reactive olefin or reactive 1,4-diene groups on the surface, the acceptor further comprising a reactive olefin or reactive 1,4-diene moiety covalently tethered thereto capable of [2+2] or [2+2+2+2] photodimerization, respectively, at a wavelength of >270 nm;

[0126] (c) preparing a hydrogel polymer comprising a copolymer chain further comprising a repeating unit of formula (I) and a repeating unit of formula (II);

[0127]

[0128] in:

[0129] R 1 is H, alkyl, alkoxy, alkenyl, alkynyl or optionally substituted variants thereof;

[0130] R 2 is -NH2, alkyl, alkoxy, alkenyl, alkynyl or their optionally substituted variants, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, aziridine, triazoline;

[0131] p is an integer from 1 to 50;

[0132] R 3 , R 3’ , R 4 and R 4’ Each of -H, -R 5 、-OR 5 、-CO2R 5 、-C(O)R 5 、-OC(O)R 5 、-C(O)NR 6 R 7 、-NR 6 R7 or a substructure of formula (III),

[0133]

[0134] R 5 is -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl or heterocyclyl;

[0135] R 6 and R 7 Each of is independently selected from -H and alkyl;

[0136] A is an aryl or thymidyl moiety;

[0137] R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl or optionally substituted variants thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, or two R' groups that, when bonded to adjacent atoms on ring A and taken together with ring A, form a coumarinyl, anthracenyl, acenaphthenyl, thioguandanyl-1-oxide or thioguandanyl-1,1-dioxide moiety;

[0138] X 1 is a bond, -(CH2) q -, -O- or -NH-;

[0139] L is a -(CH2) q -X 2 -C(=O)- or -(CH2CH2O) q -X 2 -C(=O)- divalent linker;

[0140] X 2 is -O- or -NH-;

[0141] m is an integer from 1 to 9; and

[0142] q is an integer from 0 to 50;

[0143] wherein at least some of the copolymer chains include at least one reactive olefin or reactive 1,4-diene end group capable of [2+2] or [2+2+2+2] photodimerization at wavelengths >270 nm, respectively; and wherein at least some of the copolymer chains include at least one

[0144] -N3 or -C≡CH terminal groups; and

[0145] (d) binding the hydrogel polymer to the surface of the flow cell by: (i)

[0146] contacting the surface with the hydrogel polymer, and (ii) irradiating the hydrogel polymer and the surface of the flow cell with incident light having a wavelength >270 nm to form [2+2] or [2+2+2+2] photodimers between the reactive olefin or 1,4-diene groups on the surface and the reactive olefin or 1,4-diene end groups on the corresponding copolymer chains.

[0147] In various examples, the irradiation step in (d) also crosslinks the copolymer chains in the hydrogel polymer via [2+2] or [2+2+2+2] photochemical addition of reactive olefin or reactive 1,4-diene end groups present on the corresponding copolymer chains.

[0148] In various examples, the method further includes: grafting an amplification primer onto the hydrogel polymer before step (d) or after step (d) by performing a click chemistry reaction between a terminal alkyne substituent on the amplification primer and an azide end group on a corresponding copolymer chain, or performing a click chemistry reaction between a terminal azide substituent on the amplification primer and an alkyne end group on a corresponding copolymer chain.

[0149] In various examples, the hydrogel polymer is prepared by: reacting a mixture of (a) 7-((2-acryloyloxy)ethoxy)-4-methylcoumarin, 7-((2-methacryloyloxy)ethoxy)-4-methylcoumarin, 7-((2-acrylamido)ethoxy)-4-methylcoumarin, 7-((2-methacrylamido)ethoxy)-4-methylcoumarin, 7-((2-acryloyloxy)aminoethyl)-4-methylcoumarin, 7-((2-methacryloyloxy)aminoethyl)-4-methylcoumarin, A monomer mixture of (a) N-(5-(2-acrylamido)aminoethyl)-4-methylcoumarin or 7-((2-methylacrylamido)aminoethyl)-4-methylcoumarin; (b) N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA); (c) N,N-dimethylacrylamide or N-isopropylacrylamide (NiPAM); and (d) acrylic acid (AAc) is free radically polymerized to form a copolymer chain, wherein the reactive olefin end group capable of [2+2] photodimerization at a wavelength of >270 nm includes a coumarin group.

[0150] In various examples, the method also includes recovering the flow cell, including reversing dimerization of the hydrogel polymer and binding of the hydrogel polymer to the surface of the flow cell by irradiating the hydrogel polymer and the surface of the flow cell with light having a wavelength <300 nm, thereby removing the hydrogel polymer from the surface of the flow cell.

[0151] In various examples, a method of synthesizing a hydrogel polymer includes:

[0152] A method comprising: (a) N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA); (b)

[0153] A monomer mixture of (a) N,N-dimethylacrylamide or (b) N-isopropylacrylamide (NiPAM); and (c) acrylic acid (AAc) is reacted under free radical polymerization conditions to form a hydrogel polymer comprising copolymer chains having reactive azide end groups; and

[0154] At least a portion of the azide end groups are reacted with N-(but-3-yn-1-yl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide to form a hydrogel polymer having at least some copolymer chains containing tethered 4-methylcoumarin end groups.

[0155] In various examples, the method further includes irradiating the hydrogel polymer with light having a wavelength >270 nm to crosslink at least some of the copolymer chains via coumarin photodimerization.

[0156] In various examples, the reaction is performed inside a flow cell with the monomer mixture in contact with the surface of the flow cell. In various examples, the hydrogel polymer is in the physical form of nanogel particles.

[0157] It should be understood that any corresponding features / examples of each of the aspects of the present disclosure as described herein may be implemented together in any appropriate combination, and any features / examples from any one or more of these aspects may be implemented together with any of the features of the other (multiple) aspects as described herein in any appropriate combination to achieve the benefits as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0158] Figure 1 The use of a photochemically reversible hydrogel in a flow cell for nucleic acid sequencing according to various examples of the present disclosure is schematically illustrated. Figure 1 Part A) illustrates the attachment of a preformed polymer comprising a photocontrollable moiety to a functionalized flow cell surface. Figure 1 Part B) illustrates the in situ formation of a hydrogel by polymerization of a monomer mixture on the surface of a flow cell.

[0159] Figure 2 Photochemically reversible nanogel particles according to various examples of the present disclosure are schematically illustrated attached to a functionalized surface of a flow cell, wherein the nanogel particles designated as RAP include designated functional groups for primer grafting and for photoreversibility of detachment from the surface.

[0160] Figure 3 Schematically illustrates a partial chemical structure of a photochemically reversible hydrogel particle or nanogel particle according to various examples of the present disclosure, which includes a poly(NDMAM-co-AzAPA-co-CAA) copolymer chain with a molar ratio of NDMAM:AzAPA:CAA of 85:10:5. When irradiated with 365nm light, the coumarin groups present as free end groups on the copolymer chain are available for dimerization. The double reaction arrows indicate that the crosslinks are cut when irradiated with 254nm light.

[0161] Figure 4 Two methods for preparing cross-linkable hydrogel particles and nanogel particles according to various examples of the present disclosure are schematically illustrated. In method A, a preformed polymer such as PAZAM is functionalized with N-(but-3-yn-1-yl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide ("alkyne coumarin") under copper-catalyzed Blackpool grafting. In method B, poly(NDMAM-co-AzAPA-co-CAA) copolymer chains are synthesized by free radical polymerization of a monomer mixture including NDMAM, AzAPA and CAA monomers.

[0162] Figure 5A Schematically illustrated are nanogel particles having available azide groups according to various examples of the present disclosure being functionalized with N-(but-3-yn-1-yl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide (“alkyne coumarin”) to form nanogel particles capable of [2+2] dimerization.

[0163] Figure 5B Schematically illustrated are the synthesis of photochemically reversible nanogel particles having poly(CAA-co-NiPAM-co-AzAPA-co-AAc-co-BisAM) copolymer chains by free radical aqueous suspension / polymerization of a mixture of CAA, NiPAM, AzAPA, AAc and BisAM according to various examples of the present disclosure.

[0164] Figure 6 The synthesis of monomer 7-(acrylamido)-4-methylcoumarin (N-(4-methyl-2-oxo-2H-chromen-7-yl)acrylamide) by the reaction of 7-amino-4-methylcoumarin and acryloyl chloride in dichloromethane (DCM) according to various examples of the present disclosure is illustrated.

[0165] Figure 7The functionalization of the flow cell surface according to various examples of the present disclosure is schematically illustrated, wherein the surface is first silanized with 3-mercaptopropyltrimethoxysilane (MPTMS) and then the tethered -SH groups are reacted with N-(2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl)acrylamide ("CAM").

[0166] Figure 8 Schematically illustrates the reactions of P5 / P7 amplification primers with photoreversible motifs according to various examples of the present disclosure. In A), the photoreversible motif is covalently bonded to the 5' end of the primer. In B), photocrosslinking induces primer transplantation using a first wavelength (hυ1), followed by clustering and sequencing, after which ssDNA can be removed by exposure to incident radiation at a second wavelength (hυ2). DETAILED DESCRIPTION

[0167] The detailed description of the examples herein refers to the accompanying drawings, which illustrate examples by illustration and its best mode. Although these examples are described in sufficient detail to enable those skilled in the art to practice this theme, it should be understood that other examples can be implemented and logical, chemical and mechanical changes can be made without departing from the essence and scope of the theme provided herein. Therefore, presenting a detailed description is only for illustration and not limitation. For example, unless otherwise stated, the steps set forth in any method or process description in the method or process description can be performed in any order and are not necessarily limited to the order presented. In addition, any reference to a singular element or step includes plural elements or steps, and any reference to more than one component or step may include a singular element or step. Moreover, any reference to connection, fixing, connection, etc. may include permanent, removable, temporary, partial, complete and / or any other possible connection options. In addition, any reference to contactless (or similar phrases) may also include reduced contact or minimal contact.

[0168] the term

[0169] As used herein, the term "hydrogel" is intended to refer to a polymer comprising crosslinked or crosslinkable copolymer chains. Such polymers or their monomer mixture precursors can be coated on a surface in the form of a continuous layer or in the form of divided areas. In various examples herein, the hydrogel polymer and the nanogel particles can have the same polymer composition.

[0170] As used herein, the term "nanogel particles" is intended to refer to nanoscale polymer particles including optionally crosslinked copolymer chains. For convenience only, the nanogel particles herein can be exemplified as "soccer balls", i.e., substantially spherical in shape, but their structures may not be so simple. Spherical representation allows the reader to understand the concept of accessible functional groups in / on the nanogel particles, because these groups (usually functional end groups on the copolymer chains) can be shown to protrude from the particle surface. However, particle size analysis can be performed such as by light scattering to obtain relevant particle size distribution or Z average values. Therefore, even if the nanogel particles herein may not be completely spherical in shape, with functional groups protruding from the surface, their average size can be determined. Typically, nanogel particles according to the present disclosure have a Z average value of about 50nm to about 500nm. Moreover, at least for simplicity, the narration herein describes the chemical reaction as occurring on the nanogel particles. Although the nanogel particles described herein may be spherical in shape due to the synthetic method including suspension / precipitation polymerization, the present disclosure is not limited in particle shape. Regardless of shape, all nanogel "objects" are within the scope of the present disclosure. Furthermore, it should be understood that a variety of chemical reactions can occur on and in the nanogel particles because the particles include a lightly cross-linked network containing primarily water.

[0171] As used herein, the term "photochemically reversible" is intended to refer to the property or characteristic of a hydrogel polymer or nanogel particle when the hydrogel polymer or nanogel particle includes at least some copolymer chains having at least one reactive olefin or reactive 1,4-diene end group capable of undergoing a photochemically reversible [2+2] or [2+2+2+2] cycloaddition (i.e., photodimerization). Hydrogel polymers and nanogel particles having photochemical reversibility can be reversibly attached to and removed from certain surfaces.

[0172] As used herein, the term "end group" is intended to refer to a substituent at a physical terminal position on the copolymer chain structure of the hydrogel polymer or nanogel particle, including a position at the end of a polyene backbone of the copolymer chain or at the end of a branch appended from the polyene backbone. In particular, the copolymers herein may be characterized as polyenes, but certain reactive end groups of interest (e.g., -coumarin, -4-methylcoumarin, -N3, -CO2H, -C≡CH, etc.) may be bonded to the end of an appendage branched from the polyene backbone, thereby retaining steric accessibility to various chemical reactions.

[0173] As used herein, the term "bifunctionality" is intended to refer to the properties or characteristics of both the hydrogel particles or nanogel particles when the hydrogel particles or nanogel particles include at least some copolymer chains having at least two types of functional group substituents present as copolymer chain end groups, such as (1) at least one reactive olefin or reactive 1,4-diene end group capable of [2+2] or [2+2+2+2] photodimerization at wavelengths >270 nm, respectively, and (2) at least one of a carboxylic acid end group, a -N3 end group, and / or a -C≡CH end group. The functional groups in type (2) can be used for specific binding or conjugation reactions. In the case where there are more than two types of reactive end groups on the copolymer chains, bifunctionality is intended to include "multifunctionality". For example, hydrogel particles and nanogel particles having bifunctionality allow covalent attachment of amplification primers to hydrogel particles or nanogel particles (such as by reacting alkyne-functionalized amplification primers with free -N3 end groups present on copolymer chains of hydrogel polymers or nanogel particles, or vice versa), and binding of bifunctionalized hydrogel particles or nanogel particles to surfaces in a flow cell (such as by reacting free carboxylic acid end groups on copolymer chains of hydrogel particles or nanogel particles with functionalized groups attached to the flow cell surface). In other examples, the presence of copolymer chains having at least one reactive olefin or reactive 1,4-diene end group capable of [2+2] or [2+2+2+2] photodimerization at wavelengths >270 nm, respectively, allows binding of hydrogel particles or nanogel particles to surfaces previously functionalized with reactive olefins or 1,4-dienes, wherein binding to the surface comprises photodimerization between the reactive end groups on the copolymer chains and the olefin or 1,4-diene groups tethered to the surface.

[0174] As used herein, the term "temperature responsiveness" is intended to refer to the properties or characteristics of nanogel particles when the nanogel particles include at least some copolymer chains, which have a portion of a polymer structure that is physically responsive to temperature. More specifically, nanogel particles with temperature responsiveness exhibit contraction when exposed to an increased or decreased temperature, and expansion when exposed to an opposite temperature trend. In various examples, nanogel particles with copolymer chains containing poly(NiPAM) blocks shrink as temperature increases. The temperature responsiveness provides a method for placing nanogel particles into holes (such as nanowells) and then locking them in place only by temperature manipulation. Although temperature responsiveness is present to some extent in hydrogels including at least some copolymer chains, which have a portion of a polymer structure that is physically responsive to temperature, such as poly(NiPAM) blocks in the copolymer chains, the temperature responsiveness of the polymer layer may not be as useful as demonstrated in nanogel particles because the nanogel particles are physically manipulated on the surface.

[0175] As used herein, the term "pH responsiveness" is intended to refer to a property or characteristic of a hydrogel particle or nanogel particle when the hydrogel particle or nanogel particle includes at least some copolymer chains having carboxylic acid end groups such that in certain pH ranges, these groups are predominantly -CO2H and in other pH ranges, these groups are predominantly -CO2. In other words, the pH responsive carboxylic acid end groups on at least some of the copolymer chains of the hydrogel particle or nanogel particle impart pH responsiveness to the hydrogel particle or nanogel particle. In various examples, the pH responsiveness allows the hydrogel particle or nanogel particle to undergo pH-driven binding to a functionalized flow cell surface.

[0176] As used herein, the term "dual stimulation (temperature / pH)" is intended to refer to the combination of the properties of temperature responsiveness and pH responsiveness exhibited by certain hydrogel particles and nanogel particles (according to the above definitions). In various examples, the polyNiPAM blocks in the copolymer chains of the hydrogel particles or nanogel particles impart temperature responsiveness (i.e., contraction / swelling) to the hydrogel particles or nanogel particles, while the presence of AAc units in the copolymer chains of the hydrogel particles or nanogel particles contributes to the pH responsiveness of the hydrogel particles or nanogel particles.

[0177] As used herein, the term "suspension / precipitation polymerization" is intended to refer to a free radical suspension polymerization reaction in which a water-soluble monomer and a free radical initiator produce polymer nanogel particles in a dispersed solid phase when a dispersant or a steric stabilizer is used and the reaction mixture is vigorously stirred. Suspension / precipitation polymerization is fully explained in the following academic references: S. Beck et al., Chapter 3, pages 21 to 85, "Polymer Science and Nanotechnology-Fundamentalsand Applications," Elsevier, 2020, https: / / doi.org / 10.1016 / B978-0-12-816806-6.00003-0, the entire contents of which are incorporated herein by reference. In addition, the present disclosure is not limited to this particular polymerization method for synthesizing nanogel particles. For example, emulsion polymerization techniques can be used, and non-aqueous solvents can be used.

[0178] As used herein, the abbreviation "CAA" is intended to refer to the monomer, ethyl 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acrylate (or more simply, "coumarin acrylate"), which has the chemical structure

[0179] As used herein, the abbreviation "CAM" is intended to refer to the monomer N-(2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl)acrylamide (or more simply, "coumarin acrylamide"), which has the chemical structure

[0180] As used herein, the abbreviation "AzAPA" is intended to refer to the monomer N-(5-(2-azidoacetamido)pentyl)acrylamide.

[0181] As used herein, the abbreviation "NiPAM" is intended to refer to the monomer N-isopropylacrylamide.

[0182] As used herein, the abbreviation "NDMAM" is intended to refer to the monomer N,N-dimethylacrylamide.

[0183] As used herein, the abbreviation "BisAM" is intended to refer to the multifunctional monomer N,N'-methylenebisacrylamide.

[0184] As used herein, the abbreviation "PAG" is intended to refer to the monomer propargyl acrylate.

[0185] As used herein, the abbreviation "PAM" is intended to refer to the monomer N-propargyl acrylamide.

[0186] As used herein, the abbreviation "AAc" is intended to refer to the monomeric acrylic acid.

[0187] As used herein, the abbreviation "BrAPA" is intended to refer to the monomer N-(5-(2-bromoacetamido)pentyl)acrylamide, which is used in various examples to form a PAZAM coating on the flow cell (FC) surface.

[0188] As used herein, the term "alkyne coumarin" is intended to refer to the compound and monomer N-(but-3-yn-1-yl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide, which has the chemical structure

[0189] As used herein, the abbreviation "SDS" is intended to refer to the anionic dispersion sodium dodecyl sulfate.

[0190] As used herein, the abbreviation "APS" is intended to refer to the free radical polymerization initiator ammonium persulfate.

[0191] As used herein, the abbreviation "ANA" is intended to refer to hydrogel particles or nanogel particles comprising poly(AzAPA-co-NiPAM-co-AAc-co-BisAM) copolymer chains. ANA hydrogel particles and nanogel particles are characterized by having both carboxylic acid end groups and -N3 end groups on at least some of the copolymer chains.

[0192] As used herein, the abbreviation "PANA" is intended to refer to hydrogel particles and nanogel particles comprising poly(PAG-co-NiPAM-co-AAc-co-BisAM) copolymer chains. PANA hydrogels and particles are characterized by having both carboxylic acid end groups and -C≡CH end groups on at least some of the copolymer chains.

[0193] As used herein, the abbreviation "PANA" is intended to refer to hydrogel particles and nanogel particles comprising poly(PAM-co-NiPAM-co-AAc-co-BisAM) copolymer chains. PANA hydrogels and particles are characterized by having both carboxylic acid end groups and -C≡CH end groups on at least some of the copolymer chains.

[0194] As used herein, the term "flow cell" (and abbreviation "FC") is intended to refer to a container having a chamber (e.g., a flow channel or "swimming lane") in which a reaction may be carried out, an inlet for delivering reagents to the chamber, and an outlet for removing reagents from the chamber. In various examples, the chamber enables detection of the reaction occurring in the chamber. For example, the chamber may include one or more transparent surfaces that allow optical detection of the array, optical marker molecules, etc. in the chamber. In various examples, a polymer material such as nanogel particles or a hydrogel polymer coating may be connected to the surface of the flow cell channel.

[0195] As used herein, the term "covalently linked" or "covalently bonded" is intended to refer to the formation of a chemical bond characterized by the sharing of electron pairs between atoms. For example, a covalently linked polymer coating is intended to refer to a polymer coating that forms a chemical bond with a functionalized surface of a substrate, as opposed to being attached to the surface via other means (e.g., adhesion or electrostatic interactions). It should be understood that a polymer covalently linked to a surface may also be bonded via means other than covalent attachment.

[0196] As used herein, the abbreviation "PAZAM" is intended to refer to a functionalized polymer coating comprising poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide).

[0197] As used herein, the abbreviation "DMTMM" is intended to refer to the compound 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride.

[0198] As used herein, the abbreviation "CuAAC" is intended to refer to copper-catalyzed azide-alkyne cycloaddition click chemistry.

[0199] As used herein, the abbreviation "dz" or "Dz" is intended to refer to the "Z average" reported by the particle size analysis and is considered in the art to be a reliable measure of the average size of the particle size distribution. The Z average can be determined directly by light scattering experiments using a nanoparticle analyzer. See, for example, JC Thomas, "The determination of log normal particle size distributions by dynamic light scattering," J. Colloid Interface Sci., 117 (1), 187-192 (1987).

[0200] As used herein, the abbreviation "SBS" is intended to refer to "synthesis sequencing", a sequencing technique that uses fluorescently labeled nucleotides to sequence a large number of clusters present on the surface of a circulation cell in parallel. In some examples of SBS, during each sequencing cycle, a single labeled dNTP is added to the nucleic acid chain. The nucleotide label serves as a terminator for polymerization, so that after each incorporation of dNTP, the fluorescent dye is imaged to identify the base, and then cleaved to allow the incorporation of the next nucleotide. A further understanding of SBS is disclosed in PCT application publications WO 2018 / 119101 and WO 2020 / 005501 (both belonging to Illumina, Inc.), the disclosures of which are incorporated herein by reference in their entirety.

[0201] As used herein, the term "seeding" is intended to refer to the binding of single-stranded oligonucleotides (ssDNA) to amplification primers covalently attached to nanogel particles. In various examples, seeding includes monoclonal seeding.

[0202] As used herein, the term "particle clustering" is intended to refer to the clustering of multiple copies of one or more sequencing templates of one type (monoclonal) or multiple types (polyclonal) on a single nanogel particle previously grafted with amplification primers and seeded with ssDNA. The term particle clustering is intended to refer to the activity of the nanogel particles and is not to be confused with the physical clustering of the nanogel particles themselves.

[0203] As used herein, the term "suspension clustering" is intended to refer to a method in which nanogel particles previously seeded with ssDNA and clustered are subsequently captured on FC for sequencing.

[0204] As used herein, the term "on-board clustering" is intended to refer to a cluster that has been previously seeded with primer densities compatible with sequencing and subsequently seeded on a FC (e.g., HiSeqX TMIn one embodiment, nanogel particles of appropriate size (e.g., ranging from about 200 nm to about 400 nm) are captured in nanowells of a DNA sequencing platform and then clustered to generate sufficient copies of the template that can be used for sequencing.

[0205] As used herein, the term "Typhoon" is intended to refer to Amersham TM Typhoon TM , a commercially available laser scanner platform from Cytiva Life Sciences for the imaging and quantification of nucleic acids and proteins. When used as an action verb, the term is intended to refer to the use of Amersham TM Typhoon TM Laser scanners perform imaging methods such as fluorescence imaging.

[0206] For additional abbreviations and terminology related to hydrogel coatings in flow cells and the use of these flow cells in SBS, see US 10,919,033 (Illumina, Inc.), the disclosure of which is incorporated herein in its entirety.

[0207] As used herein, any "R" group specified on a chemical structure (such as R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 R, ...

[0208] As used herein, the term "alkyl" is intended to refer to a straight or branched monovalent fully saturated hydrocarbon substituent, which is optionally substituted with one or more functional groups at any position of the substituent. Unless otherwise specified, an alkyl group may contain any number of carbon atoms, such as, for example, C1-C 24 、C1-C 18 、C1-C 10, C1-C8, C1-C6 or C1-C4. Examples of alkyl substituents include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, octadecyl, dodecyl, and the like. The alkyl substituents herein may be substituted, i.e., have one or more substituents attached to the alkyl group or incorporated into the alkyl chain. Substitution within the alkyl substituent chain may include ether, sulfide or imine bonds, i.e., for example, -O-, -S- or -N=, or some other (multiple) intermediate heteroatoms. Examples of substitutions on alkyl substituents include, but are not limited to, -CN, -N3, -NH2, -NHR, -N(R)2, -N(R)3 + 、-NO2、-NH-NH2、-NH-NHR、-NH-NR2、-halogen、-SH、-SR、-S(=O)R、-SO2R、-OPO3 2- 、-PO3 2- , -OH, -OR, -C(=O)R, -OC(=O)R, -CO2R, -NHC(=O)R, -NRC(=O)R, -C(=O)NHR, -C(=O)NR2, alkyl, alkenyl, cycloalkyl, heterocyclyl and aryl, wherein each R is independently selected from hydrogen-H and alkyl moieties, including, for example, C 1-6 Alkyl (e.g., -CH3, -C2H5, -isopropyl, -tert-butyl, etc.), C 1-6 Alkoxy (e.g., -OCH3, -OC2H5), halogenated C 1-6 Alkyl (e.g., -CF3, -CHF2, -CH2F) and halogenated C 1-6 Alkoxy (e.g., -OCF3, -OC2F5).

[0209] As used herein, the term "cycloalkyl" includes any 3-, 4-, 5-, 6-, 7- or 8-membered saturated or unsaturated non-aromatic carbocyclic ring, which is optionally substituted by one or more functional groups at any position on the cyclic substituent. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-, 2- or 5-cyclopentadienyl, cyclohexyl, 1-, 3- or 4-cyclohexenyl, 1-, 2- or 5-(1,3-cyclohexadienyl), 1- or 3-(1,4-cyclohexadienyl), cycloheptyl, 1-, 3-, 4- or 5-cycloheptenyl, cyclooctyl, etc. Examples of substitutions on cycloalkyl substituents include, but are not limited to -CN, -N3, -NH2, -NHR, -N(R)2, -N(R)3 + 、-NO2、-NH-NH2、-NH-NHR、-NH-NR2、-halogen、-SH、-SR、-S(=O)R、-SO2R、-OPO3 2- 、-PO3 2-, -OH, -OR, -C(=O)R, -OC(=O)R, -CO2R, -NHC(=O)R, -NRC(=O)R, -C(=O)NHR, -C(=O)NR2, alkyl, alkenyl, cycloalkyl, heterocyclyl and aryl, wherein each R is independently selected from -H and alkyl moieties, including, for example, C 1-6 Alkyl (e.g., -CH3, -C2H5, -isopropyl, -tert-butyl, etc.), C 1-6 Alkoxy (e.g., -OCH3, -OC2H5), halogenated C 1-6 Alkyl (e.g., -CF3, -CHF2, -CH2F) and halogenated C 1-6 Alkoxy (e.g., -OCF3, -OC2F5).

[0210] As used herein, the term "alkenyl" is intended to refer to a straight or branched monovalent or divalent unsaturated hydrocarbon substituent optionally substituted with one or more functional groups at any position on or within the substituent. 2 If carbon is part of the molecule bearing the alkenyl substituent, the alkenyl substituent may be considered divalent. An illustrative example is methylenecyclohexane, which may be considered cyclohexane substituted with a methylene group (i.e., a divalent alkenyl substituent = CH2). Unless otherwise specified, an alkenyl group may contain any number of carbon atoms, such as, for example, C1-C 24 、C1-C 18 、C1-C 10 , C1-C8 or C1-C6 and any degree of unsaturation. Examples of alkenyl substituents include, but are not limited to, methylene (methylene / methylidine) (=CH2), ethylene / vinyl (-CH=CH2 or =CH-CH3), propylene / propenyl (-CH2-CH=CH2, cis or trans-CH=CH-CH3, =C(CH3)2 or cis or trans=CH-CH2CH3), etc. The alkenyl substituents herein may be substituted, i.e., have one or more substituents attached to the alkenyl group or incorporated into the alkenyl chain. Substitutions within the alkenyl substituent may include ether, sulfide or imine bonds, i.e., for example, -O-, -S- or -N=, or some other (multiple) intermediate heteroatoms. Examples of substitutions on alkenyl substituents include, but are not limited to, -CN, -N3, -NH2, -NHR, -N(R)2, -N(R)3 + 、-NO2、-NH-NH2、-NH-NHR、-NH-NR2、-halogen、-SH、-SR、-S(=O)R、-SO2R、-OPO3 2- 、-PO3 2-, -OH, -OR, -C(=O)R, -OC(=O)R, -CO2R, -NHC(=O)R, -NRC(=O)R, -C(=O)NHR, -C(=O)NR2, alkyl, alkenyl, cycloalkyl, heterocyclyl and aryl, wherein each R is independently selected from an alkyl moiety, including, for example, C 1-6 Alkyl (e.g., -CH3, -C2H5, -isopropyl, -tert-butyl, etc.), C 1-6 Alkoxy (e.g., -OCH3, -OC2H5), halogenated C 1-6 Alkyl (e.g., -CF3, -CHF2, -CH2F) and halogenated C 1-6 Alkoxy (e.g., -OCF3, -OC2F5).

[0211] As used herein, the term "aryl" includes any aromatic ring or fused polycyclic aromatic ring system, such as phenyl, naphthyl, anthracenyl and phenanthrenyl, which is optionally substituted by one or more functional groups at any position on the aromatic substituent. Unsubstituted phenyl substituents may also be represented as -C6H5 or more simply as -Ph. Aromatic heterocyclic and fused heterocyclic aromatic substituents are different and are included in the definition of heterocyclyl substituents set forth below. Examples of substitutions on aryl substituents include, but are not limited to, -CN, -N3, -NH2, -NHR, -N(R)2, -N(R)3 + 、-NO2、-NH-NH2、-NH-NHR、-NH-NR2、-halogen、-SH、-SR、-S(=O)R、-SO2R、-OPO3 2- 、-PO3 2- , -OH, -OR, -C(=O)R, -OC(=O)R, -CO2R, -NHC(=O)R, -NRC(=O)R, -C(=O)NHR, -C(=O)NR2, alkyl, alkenyl, cycloalkyl, heterocyclyl and aryl, wherein each R is independently selected from an alkyl moiety, including, for example, C 1-6 Alkyl (e.g., -CH3, -C2H5, -isopropyl, -tert-butyl, etc.), C 1-6 Alkoxy (e.g., -OCH3, -OC2H5), halogenated C 1-6 Alkyl (e.g., -CF3, -CHF2, -CH2F) and halogenated C 1-6 Alkoxy (e.g., -OCF3, -OC2F5).

[0212] As used herein, "heterocycle" is intended to refer to an unsubstituted or optionally substituted, saturated, unsaturated or aromatic carbocyclic ring interrupted by at least one heteroatom selected from oxygen (O), sulfur (S) or nitrogen (N) in its carbocyclic structure. As used herein, the term "heterocyclic radical" is intended to refer to a heterocycle as a substituent group, which is connected to another atom of any C atom or heteroatom present in the heterocycle in the compound. For example, "pyridyl" includes 2-, 3- and 4-pyridyl moieties as substituent groups. The heterocycle can be a monocyclic or fused polycyclic ring in structure. Examples of optional substitutions on heterocyclic substituents include, but are not limited to, oxo, -CN, -N3, -NH2, -NHR, -N(R)2, -N(R)3 + 、-NO2、-NH-NH2、-NH-NHR、-NH-NR2、-halogen、-SH、-SR、-S(=O)R、-SO2R、-OPO3 2- 、-PO3 2- , -OH, -OR, -C(=O)R, -OC(=O)R, -CO2R, -NHC(=O)R, -NRC(=O)R, -C(=O)NHR, -C(=O)NR2, alkyl, alkenyl, cycloalkyl, heterocyclyl and aryl, wherein each R is independently selected from an alkyl moiety, including, for example, C 1-6 Alkyl (e.g., -CH3, -C2H5, -isopropyl, -tert-butyl, etc.), C 1-6 Alkoxy (e.g., -OCH3, -OC2H5), halogenated C 1-6 Alkyl (e.g., -CF3, -CHF2, -CH2F) and halogenated C 1-6 Alkoxy (e.g., -OCF3, -OC2F5).

[0213] Examples of heterocycles include, but are not limited to, azepanyl, aziridinyl, azetidinyl, coumarinyl (2H-chromen-2-one), diazepanyl, dithiadiazinyl, dioxazepanyl, dioxolanyl, dithiazolinyl, furanyl, isoxazolyl, isothiazolyl, imidazolyl, morpholinyl, morpholino, oxetanyl, oxadiazolyl, oxiranyl, oxazinyl, oxazole yl, piperazinyl, pyrazinyl, pyridazinyl, pyrimidinyl, piperidinyl, piperidinyl, pyridinyl, pyranyl, pyrazolyl, pyrrolyl, pyrrolidinyl, thiatriazolyl, tetrazolyl, thiadiazolyl, triazolyl, thiazolyl, thienyl, tetrazinyl, thiadiazinyl, triazinyl, thiazinyl, thiapyranyl, furanisoxazolyl, imidazothiazolyl, thienoisothiazolyl, thienothiazolyl, thienothiazolyl, imidazo thiazolyl, cyclopentadiazolyl, pyrrolopyrrolyl, thienothiphenyl, thiadiazolopyrimidinyl, thiazolothiazolyl, thiazolopyrimidinyl, thiazolopyridinyl, oxazolopyrimidinyl, oxazolopyrimidinyl, benzoxazolyl, benzisothiazolyl, benzothiazolyl, imidazopyrazinyl, purinyl, pyrazolopyrimidinyl, imidazopyridinyl, benzimidazolyl, indazolyl, benzothiothio, benzodioxy Heterocyclopentyl, benzodithio, indolinyl, indolinyl, isoindolinyl, furapyrimidinyl, furapyridinyl, benzofuranyl, isobenzofuranyl, thienopyrimidinyl, thienopyridine, benzothienyl, cyclopentazinyl, cyclopentafuranyl, benzoxazinyl, benzothiazinyl, quinazolinyl, naphthyridinyl, quinolinyl, isoquinolinyl, benzopyranyl, pyridopyridazinyl and pyridopyrimidinyl. Additional examples of heterocyclic systems can be found in A. Katritzky et al., Handbook of Heterocyclic Chemistry, 3rd edition, Elsevier, 2010, which is incorporated herein by reference.

[0214] General Examples

[0215] In various examples of the present disclosure, novel photochemically reversible hydrogel polymers and polymer nanogel particles are described. Various nanogel particles herein exhibit dual functionality by the presence of at least two types of reactive end groups on the copolymer chains within the nanogel particles. For example, the nanogel particles of the present invention may exhibit temperature responsiveness and pH responsiveness, wherein the nanogel particles may shrink or swell in response to temperature changes, and the pH responsiveness assists surface binding reactions. Nanogel particles according to the present disclosure are particularly useful in nucleic acid sequencing methods, particularly in flow cells used in SBS methods.

[0216] In various examples, nanogel particles are prepared by suspension / precipitation free radical polymerization of various monomer types. The nanogel particles herein are described by the synthetic methods used to prepare them (i.e., the monomers and reaction conditions used in the suspension / precipitation free radical polymerization reaction) and also structurally such as by describing certain repeating units present in the copolymer chains of the nanogel particles so prepared and the physical properties. In various examples, the repeating monomer units in the copolymer chains of the nanogel particles may include a portion of a block within a block copolymer.

[0217] In various examples, photochemically reversible hydrogel polymers and nanogel particles include copolymer chains that are crosslinkable and / or at least partially crosslinked. For example, if multifunctional monomers and other monomer types are used in suspension / precipitation free radical polymerization, crosslinking can be expected to occur. As explained herein, crosslinking can also be photochemically initiated, such as dimerizing reactive olefin or diene end groups on the copolymer chains.

[0218] In various examples, where the incorporation of monomers results in temperature or pH responsive nanogel particles, the nanogel particles can be fine-tuned to suit any step in the SBS sequencing protocol, such as library seeding, nanogel particle capture in the nanowells of FC, clustering of particles, and sequencing of particles. In various examples, temperature responsiveness can be incorporated using LCST (lower critical solution temperature) or UCST (upper critical solution temperature).

[0219] Objectives and general considerations

[0220] In various examples, the light-controlled chemistry of photochemically reversible hydrogel particles and nanogel particles provides for a potential sustainable solution by making sequencing flow cells reusable.

[0221] In various examples, photochemically reversible hydrogel particles and nanogel particles are attached to a functionalized FC surface, followed by clustering and sequencing steps. Once completed, the photochemically reversible hydrogel particles and nanogel particles are cut out from the surface and discarded from the FC lane by washing. Fresh solutions of photochemically reversible hydrogel particles and nanogel particles can then be used for the next sequencing step.

[0222] For these purposes, many physical, chemical and light-assisted controllable / reversible pathways have been proposed. Compared with other systems, an advantage of photoreversible chemicals is that no additional chemical or physical stimulation is required. Many photoreversible cross-linking chemicals have been explored, and they can be activated by a wide range of wavelengths. They are classified as short wavelengths (<400nm) and long wavelengths (400nm to 1000nm). However, they are usually activated and deactivated using long wavelengths (600nm to 1000nm). In principle, in order to be compatible with SBS sequencing methods (e.g., Illumina sales), these reversible chemicals should also show zero (or low) absorbance at wavelengths greater than 400nm. By using short wavelengths for photocleavage and photocrosslinking, respectively, the derivatives of coumarin, anthracene, thymine, cinnamic acid and stilbenes are good candidates for photochemically reversible hydrogel particles and nanogel particles in nucleic acid sequencing methods.

[0223] In various examples, the photochemically reversible hydrogel particles and nanogel particles include copolymer chains having reactive olefin or reactive 1,4-diene end groups that can participate in [2+2] or [2+2+2+2] photoaddition reactions, respectively. These photoaddition reactions can be characterized as photodimerization and can be used to reversibly attach hydrogel particles or nanogel particles to functionalized FC surfaces and / or to crosslink copolymer chains.

[0224] In various examples, Table 1 provides a summary of short-wavelength light-controllable chemicals incorporated into the photochemically reversible hydrogel particles and nanogel particles herein.

[0225] Table 1: Light-controlled (short wavelength) chemistry

[0226]

[0227] Monomers for the synthesis of photochemically reversible hydrogel polymers and nanogel particles

[0228] In various examples, the first type of monomers used to synthesize the photochemically reversible hydrogel polymers and polymer nanogel particles in a free radical polymerization reaction include substances having the following structure:

[0229] in:

[0230] R 1 , R 1’ and R 1” Each of which is independently selected from H, halogen, alkyl, alkoxy, alkene

[0231] alkyl, alkynyl, cycloalkyl, aryl, heteroaryl or heterocyclyl;

[0232] X is -O- or -NH-; and

[0233] R2 is -CH2-C≡CH, or R 2 Has the following structure:

[0234]

[0235] Where R 2’ is -NH2, alkyl, alkoxy, alkenyl, alkynyl or their optionally substituted variants, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H,

[0236] -SH, glycidyl, epoxy, aziridine, triazoline, or -C≡CH; and

[0237] p is an integer of 1 to 50.

[0238] Examples of monomers of the first type include, but are not limited to, propargyl acrylate, N-propargyl acrylamide, N-(5-(2-azidoacetamido)pentyl)acrylamide, (2-methacryloyloxy)trimethylammonium chloride, 2-acrylamido-2-methyl-1-propanesulfonic acid, [2-(acryloyloxy)ethyl]trimethylammonium chloride, and 2-hydroxyethyl methacrylate.

[0239] In various examples, the second class of monomers used to synthesize photochemically reversible hydrogel polymers and polymer nanogel particles in a free radical polymerization reaction includes species having the following structures:

[0240] in:

[0241] R 3 , R 3’ , R 4 and R 4’ Each of -H, -R 5 、-OR 5 、-CO2R 5 ,

[0242] -C(O)R 5 、-OC(O)R 5 、-C(O)NR 6 R 7 、-NR 6 R 7 or a substructure of formula (III),

[0243]

[0244] R 5 is -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl or heterocyclyl;

[0245] R6 and R 7 Each of is independently selected from -H and alkyl;

[0246] A is an aryl or thymidyl moiety;

[0247] R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl or their optionally substituted variants, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN,

[0248] -CO2H, -SH, glycidyl, epoxy or two R' groups, when bonded to adjacent atoms on ring A and combined with ring A, form coumarin, anthracenyl, acenaphthenyl, thioguanidine

[0249] a thionaphthene-1,1-dioxide moiety;

[0250] X 1 is a bond, -(CH2) q -, -O- or -NH-;

[0251] L is a -(CH2) q -X 2 -C(=O)- or -(CH2CH2O) q -X 2 -C(=O)- divalent linker;

[0253] X 2 is -O- or -NH-;

[0254] m is an integer from 1 to 9; and

[0255] q is an integer from 0 to 50.

[0256] In various examples of the second type of monomer, formula (III) has the subgeneric structure (IV):

[0257]

[0258] Where R 8 and R 9 Each of is -H, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl or heterocyclyl; each R 10 and R 11 are independently -H, alkyl, cycloalkyl, hydroxyalkyl,

[0259] aryl, heteroaryl or heterocyclic group, or R 10 is -C(=O)-, R 11 is -O-, and R 10 and R 11are bonded together such that formula (IV) includes a substituted coumarin moiety.

[0260] Examples of the second type of monomer include, but are not limited to, acrylic acid, methacrylic acid, acrylamide, methacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N,N-dimethylacrylamide, N,N-dimethylmethacrylamide, N-vinylpyrrolidone, N-vinylpyridine, N-(4-methyl-2-oxo-2H-chromen-7-yl)acrylamide, 4-methyl-2-oxo-2H-chromen-7-yl acrylate, 2-((4-methyl 2-((4-methyl-2-oxo-2H-chromen-7-yl)amino)ethyl)acrylamide, 2-((4-methyl-2-oxo-2H-chromen-7-yl)amino)ethyl)acrylamide, N-(2-((4-methyl-2-oxo-2H-chromen-7-yl)amino)ethyl)acrylamide, N-(but-3-yn-1-yl)-2-(((2-oxo-2H-chromen-7-yl)oxy)ethyl)acrylamide -yl)oxy)methyl)acrylamide, ethyl 2-(5-methyl-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)acrylate, N-(2-(5-methyl-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)ethyl)acrylamide, ethyl 2-(anthracen-2-yloxy)acrylate, N-(2-(anthracen-2-yloxy)ethyl)acrylamide, ethyl 2-(anthracen-2-ylamino)acrylate, N-(2-(anthracen-2-ylamino)acrylate (E)-ethyl 2-((4-(2-(quinoxalin-2-yl)vinyl)phenyl)amino)acrylate, and (E)-N-(2-((4-(2-(quinoxalin-2-yl)vinyl)phenyl)amino)ethyl)acrylamide.

[0261] In various examples, photochemically reversible hydrogel polymers are prepared under various free radical polymerization conditions by reacting at least one first type of monomer and at least one second type of monomer according to the above structure. For an overview of the synthesis methods, see, for example, U. Madduma-Bandarage et al., "Synthetic Hydrogels: Synthesis, Novel Trends, and Applications," J. Appl. Polym. Sci., 2021; 138: e50376, https: / / doi.org / 10.1002 / app.50376 and E. Ahmed, "Hydrogel: Preparation, Characterization, and Applications: A Review," J. Adv. Res., 6 (2), 105-121 (2015), the entire contents of each of which are incorporated herein by reference.

[0262] When these two types of monomers are used in a free radical polymerization reaction, the various photochemically reversible hydrogel polymers obtained therefrom include a copolymer chain having at least one first repeating unit incorporating the first type of monomer and at least one second repeating unit incorporating the second type of monomer.

[0263] In various examples, according to the above structure, photochemically reversible nanogel particles are prepared under suspension / precipitation or emulsion free radical polymerization conditions by reacting at least one first type of monomer and at least one second type of monomer. When these two types of monomers are used in suspension / precipitation or emulsion free radical polymerization, the resulting photochemically reversible nanogel particles include a copolymer chain having at least one first repeating unit incorporating the first type of monomer and at least one second repeating unit incorporating the second type of monomer.

[0264] Multifunctional monomers that may be included in the free radical polymerization reaction to form photochemically reversible hydrogel polymers and nanogel particles having a degree of crosslinking between copolymer chains include, but are not limited to, N,N'-methylenebisacrylamide, N,N'-methylenebismethacrylamide, polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, N-vinyl acrylamide, glycidyl acrylate, divinylbenzene, diallyldimethylammonium chloride, and tetraallylammonium chloride.

[0265] In various examples, photochemically reversible hydrogel polymers and nanogel particles are prepared under free radical polymerization conditions by reacting at least one first type of monomer, at least one second type of monomer, and at least one multifunctional monomer according to the above structures simultaneously. When these two types of monomers and multifunctional monomers are used in free radical polymerization, the resulting photochemically reversible hydrogel polymers and nanogel particles include copolymer chains having at least one first repeating unit incorporated into the first type of monomer and at least one second repeating unit incorporated into the second type of monomer, wherein the copolymer chains have at least a certain degree of crosslinking between the copolymer chains. Additional crosslinking of the copolymer chains and / or binding of the photochemically reversible hydrogel polymer or nanogel polymer to the functionalized surface can be achieved by photochemical dimerization of olefin or diene groups in the copolymer chains or between the copolymer chains and the functionalized surface.

[0266] In various examples, photochemically reversible hydrogel polymers and nanogel particles are prepared under free radical polymerization conditions by reacting at least one first type monomer, at least one second type monomer and a multifunctional monomer N,N'-methylenebismethacrylamide (BisAM) simultaneously according to the above structure.

[0267] In various examples, the photochemically reversible hydrogel polymers and nanogel particles thus prepared under free radical polymerization conditions including each of the two types of monomers as described above and optionally at least one of the multifunctional monomers include copolymer chains comprising at least one reactive olefin or reactive 1,4-diene end group and at least one of a carboxylic acid end group, a -N3 end group and / or a -C≡CH end group capable of undergoing [2+2] or [2+2+2+2] photodimerization, respectively, at a wavelength of >270 nm.

[0268] Suspension / precipitation and other free radical polymerization

[0269] In various examples, the synthesis of photochemically reversible nanogel particles includes aspects of suspension / precipitation free radical polymerization or emulsion polymerization. In various embodiments, the reaction conditions are aqueous and heated, using selected monomers, dispersions and free radical initiators that promote the suspension of the photochemically reversible nanogel particles that are usually insoluble in water and formed in water.

[0270] In various examples, the suspension / precipitation free radical polymerization reaction is performed at a temperature of about 50° C. to about 90° C. for about 1 hour to about 4 hours.

[0271] In various examples, the dispersions herein include anionic or nonionic dispersions. Exemplary anionic dispersions include sodium dodecyl sulfate (SDS). Nonionic dispersions include, but are not limited to, polyethylene glycol (PEG), sorbitan monooleate (e.g., trade name ), ethoxylated sorbitan monooleate (e.g., trade name ) and acryloyl-terminated PEG.

[0272] In various examples, the free radical initiator includes a water soluble compound.

[0273] In various examples, the free radical initiator includes a peroxide.

[0274] In various examples, the free radical initiator includes sodium persulfate, potassium persulfate, or ammonium persulfate.

[0275] In various examples, the free radical initiator includes ammonium persulfate (APS).

[0276] In various examples, photochemically reversible nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction that combines a dispersed monomer mixture including ethyl 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acrylate (CAA); N,N-dimethylacrylamide (NDMAM); and N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA).

[0277] In various examples, photochemically reversible nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction that combines a dispersed monomer mixture including ethyl 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acrylate (CAA); N,N-dimethylacrylamide (NDMAM); N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA); and the multifunctional monomer N,N'-methylenebisacrylamide (BisAM).

[0278] In various examples, photochemically reversible nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction that combines a dispersed monomer mixture including ethyl 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acrylate (CAA); N,N-dimethylacrylamide (NDMAM); N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA); and acrylic acid (AAc).

[0279] In various examples, photochemically reversible nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction that combines a dispersed monomer mixture including ethyl 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acrylate (CAA); N,N-dimethylacrylamide (NDMAM); N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA); acrylic acid (AAc); and the multifunctional monomer N,N'-methylenebisacrylamide (BisAM).

[0280] In various examples, photochemically reversible nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction that combines a dispersed monomer mixture including ethyl 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acrylate (CAA), N-isopropylacrylamide (NiPAM); N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA); and acrylic acid (AAc).

[0281] In various examples, photochemically reversible nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction that combines a dispersed monomer mixture including ethyl 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acrylate (CAA), N-isopropylacrylamide (NiPAM); N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA); acrylic acid (AAc); and the multifunctional monomer N,N'-methylenebisacrylamide (BisAM).

[0282] In various examples, photochemically reversible nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction that combines a dispersed monomer mixture including N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA) and acrylic acid (AAc) to form a copolymer chain, after which at least some of the available -N3 end groups on the resulting copolymer chain are reacted with N-(but-3-yn-1-yl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide (alkyne coumarin).

[0283] In various examples, photochemically reversible nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction that combines a dispersed monomer mixture including N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), acrylic acid (AAc), and the multifunctional monomer N,N'-methylenebisacrylamide (BisAM) to form a copolymer chain, after which at least some of the available -N3 end groups on the resulting copolymer chain are reacted with N-(but-3-yn-1-yl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide (alkyne coumarin).

[0284] In various examples, photochemically reversible nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction that combines a dispersed monomer mixture including N,N-dimethylacrylamide (NDMAM), N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), and acrylic acid (AAc) to form a copolymer chain, after which at least some of the available -N3 end groups on the resulting copolymer chain are reacted with N-(but-3-yn-1-yl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide (alkyne coumarin).

[0285] In various examples, photochemically reversible nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction that combines a dispersed monomer mixture including N,N-dimethylacrylamide (NDMAM), N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), acrylic acid (AAc), and the multifunctional monomer N,N'-methylenebisacrylamide (BisAM) to form a copolymer chain, after which at least some of the available -N3 end groups on the resulting copolymer chain are reacted with N-(but-3-yn-1-yl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide (alkyne coumarin).

[0286] In various examples, photochemically reversible nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction that combines a dispersed monomer mixture including N-isopropylacrylamide (NiPAM), N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA) and acrylic acid (AAc) to form a copolymer chain, after which at least some of the available -N3 end groups on the resulting copolymer chain are reacted with N-(but-3-yn-1-yl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide (alkyne coumarin).

[0287] In various examples, photochemically reversible nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction that combines a dispersed monomer mixture including N-isopropylacrylamide (NiPAM), N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), acrylic acid (AAc), and the multifunctional monomer N,N'-methylenebisacrylamide (BisAM) to form a copolymer chain, after which at least some of the available -N3 end groups on the resulting copolymer chain are reacted with N-(but-3-yn-1-yl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide (alkyne coumarin).

[0288] Photochemically reversible hydrogel polymers and nanogel particles comprising copolymer chains

[0289] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include copolymer chains having various end groups on at least some of the copolymer chains. In various examples, the photochemically reversible hydrogel polymers and nanogel particles include copolymer chains having at least some degree of crosslinking. In various examples, at least some degree of crosslinking is photochemically obtained by dimerizing certain reactive olefin or 1,4-diene end groups on the copolymer chains.

[0290] In various examples, the photochemically reversible hydrogel polymers and polymer nanogel particles according to the present disclosure include a copolymer chain further comprising a first repeating unit of formula (I):

[0291] in:

[0292] R 1 , R 1’ and R 1” Each of is independently selected from H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl or heterocyclyl;

[0293] X is -O- or -NH-; and

[0294] R 2 is -CH2-C≡CH, or R 2 Has the following structure:

[0295]

[0296] Where R 2’ is -NH2, alkyl, alkoxy, alkenyl, alkynyl or optionally substituted variants thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, aziridine, triazoline or -C≡CH; and

[0297] p is an integer from 1 to 50; and

[0298] The second repeating unit of formula (II),

[0299] in:

[0300] R 3 , R 3’ , R 4 and R 4’ Each of -H, -R 5 、-OR 5 、-CO2R 5 、-C(O)R 5 、-OC(O)R 5、-C(O)NR 6 R 7 、-NR 6 R 7 or a substructure of formula (III),

[0301]

[0302] R 5 is -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl or heterocyclyl;

[0303] R 6 and R 7 Each of is independently selected from -H and alkyl;

[0304] A is an aryl or thymidyl moiety;

[0305] R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl or their optionally substituted variants, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy or two R' groups, when bonded to adjacent atoms on ring A and taken together with ring A, form a coumarinyl, anthracenyl, acenaphthenyl, thiophene

[0306] a thionaphthene-1,1-dioxide moiety;

[0307] X 1 is a bond, -(CH2) q -, -O- or -NH-;

[0308] L is a -(CH2) q -X 2 -C(=O)- or -(CH2CH2O) q -X 2 -C(=O)- divalent linker;

[0310] X 2 is -O- or -NH-;

[0311] m is an integer from 1 to 9; and

[0312] q is an integer from 0 to 50.

[0313] In various examples, R 1 =R 1’ =R 1” =H; X is -O- or -NH-; R 2 It is -CH2-C≡CH or has the following structure:

[0314] Where R 2’ is -N3 or -C≡CH; and p is an integer from 1 to 50.

[0315] In various examples of the second repeating unit of formula (II), formula (III) has a subgeneric structure of formula (IV):

[0316]

[0317] Where R 8 and R 9 Each of is -H, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl or heterocyclyl; each R 10 and R 11 is independently -H, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl or heterocyclyl, or R 10 is -C(=O)-, R 11 is -O-, and R 10 and R 11

[0318] are bonded together such that formula (IV) includes a substituted coumarin moiety.

[0319] In various examples, the photochemically reversible hydrogel polymers and nanogel particles having the above repeating units include copolymer chains having at least one reactive olefin or reactive 1,4-diene end group capable of [2+2] or [2+2+2+2] photodimerization at wavelengths >270 nm, respectively.

[0320] In various examples, the photochemically reversible hydrogel polymers and nanogel particles having the above-described repeating units include copolymer chains having (1) at least one reactive olefin or reactive 1,4-diene end group capable of undergoing [2+2] or [2+2+2+2] photodimerization at wavelengths >270 nm, respectively; and (2) at least one of a carboxylic acid end group, a -N3 end group, and / or a -C≡CH end group.

[0321] In various examples, the photochemically reversible hydrogel polymers and nanogel particles having the above-described repeating units include a copolymer chain having at least one carboxylic acid end group, at least one -N3 end group, and at least one reactive olefin or reactive 1,4-diene end group capable of undergoing [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm.

[0322] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include copolymer chains wherein the first repeating unit of formula (I) is:

[0323] Wherein, as described above, p is an integer from 1 to 50.

[0324] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include copolymer chains wherein the first repeating unit of formula (I) is:

[0325] Wherein, as described above, p is an integer from 1 to 50.

[0326] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include copolymer chains wherein the first repeating unit of formula (I) is:

[0327]

[0328] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include copolymer chains wherein the second repeating unit of formula (II) is:

[0329]

[0330] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include copolymer chains wherein the second repeating unit of formula (II) is:

[0331]

[0332] and / or

[0333] wherein R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl or their optionally substituted variants, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -

[0334] SCN, -CO2H, -SH, glycidyl, epoxy; and

[0335] m is an integer of 1-9.

[0336] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include copolymer chains wherein the second repeating unit of formula (II) is:

[0337] and / or

[0338] wherein q is an integer from 0 to 50.

[0339] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include copolymer chains wherein the second repeating unit of formula (II) is:

[0340]

[0341] and / or

[0342] wherein R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl or their optionally substituted variants, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -

[0343] SCN, -CO2H, -SH, glycidyl, epoxy; and

[0344] m is an integer of 1-7.

[0345] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include copolymer chains wherein the second repeating unit of formula (II) is:

[0346]

[0347] and / or

[0348] wherein R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl or their optionally substituted variants, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -

[0349] SCN, -CO2H, -SH, glycidyl, epoxy; and

[0350] m is an integer of 1-5.

[0351] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include copolymer chains wherein the second repeating unit of formula (II) is:

[0352]

[0353] and / or

[0354] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include poly(NiPAM-co-AzAPA-co-AAc) copolymer chains functionalized with an alkyne coumarin such that at least some of the copolymer chains include unused -N3 end groups, unreacted -CO2H end groups, and at least some N-(2-(1λ 2 ,2,3-triazol-4-yl)ethyl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide appendages are provided to provide reactive olefin end groups capable of [2+2] photodimerization at wavelengths >270 nm.

[0355] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include poly(NiPAM-co-AzAPA-co-AAc-co-BisAM) copolymer chains functionalized with an alkyne coumarin such that at least some of the copolymer chains include unused -N3 end groups, unreacted -CO2H end groups, and at least some N-(2-(1λ 2 ,2,3-triazol-4-yl)ethyl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide appendages are provided to provide reactive olefin end groups capable of [2+2] photodimerization at wavelengths >270 nm.

[0356] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include poly(NDMAM-co-AzAPA-co-AAc) copolymer chains functionalized with an alkyne coumarin such that at least some of the copolymer chains include unused -N3 end groups, unreacted -CO2H end groups, and at least some N-(2-(1λ 2 ,2,3-triazol-4-yl)ethyl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide appendages are provided to provide reactive olefin end groups capable of [2+2] photodimerization at wavelengths >270 nm.

[0357] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include poly(NDMAM-co-AzAPA-co-AAc-co-BisAM) copolymer chains functionalized with an alkyne coumarin such that at least some of the copolymer chains include unused -N3 end groups, unreacted -CO2H end groups, and at least some N-(2-(1λ 2 ,2,3-triazol-4-yl)ethyl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide appendages are provided to provide reactive olefin end groups capable of [2+2] photodimerization at wavelengths >270 nm.

[0358] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include poly(CAA-co-NDMAM-co-AAc) copolymer chains. In various examples, at least some of the copolymer chains include -CO2H end groups and reactive olefin end groups capable of [2+2] photodimerization at wavelengths >270 nm to obtain difunctionality.

[0359] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include poly(CAA-co-NDMAM-co-AAc-co-BisAM) copolymer chains. In various examples, at least some of the copolymer chains include -CO2H end groups and reactive olefin end groups capable of [2+2] photodimerization at wavelengths >270 nm to obtain difunctionality.

[0360] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include poly(CAA-co-NDMAM-co-AzAPA) copolymer chains. In various examples, at least some of the copolymer chains include -N3 end groups and reactive olefin end groups capable of [2+2] photodimerization at wavelengths >270 nm to obtain difunctionality.

[0361] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include poly(CAA-co-NDMAM-co-AzAPA-co-BisAM) copolymer chains. In various examples, at least some of the copolymer chains include -N3 end groups and reactive olefin end groups capable of [2+2] photodimerization at wavelengths >270 nm to obtain difunctionality.

[0362] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include poly(CAA-co-NDMAM-co-AzAPA-co-AAc) copolymer chains. In various examples, at least some of the copolymer chains include -N3 end groups, -CO2H end groups, and reactive olefin end groups capable of [2+2] photodimerization at wavelengths >270 nm to obtain difunctionality.

[0363] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include poly(CAA-co-NDMAM-co-AzAPA-co-AAc-co-BisAM) copolymer chains. In various examples, at least some of the copolymer chains include -N3 end groups, -CO2H end groups, and reactive olefin end groups capable of [2+2] photodimerization at wavelengths >270 nm to obtain difunctionality.

[0364] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include poly(CAA-co-NiPAM-co-AzAPA-co-AAc) copolymer chains. In various examples, at least some of the copolymer chains include -N3 end groups, -CO2H end groups, and reactive olefin end groups capable of [2+2] photodimerization at wavelengths >270 nm to obtain difunctionality.

[0365] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include poly(CAA-co-NiPAM-co-AzAPA-co-AAc-co-BisAM) copolymer chains. In various examples, at least some of the copolymer chains include -N3 end groups, -CO2H end groups, and reactive olefin end groups capable of [2+2] photodimerization at wavelengths >270 nm to obtain difunctionality.

[0366] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include poly(PAG-co-CAA-co-NiPAM-co-AAc) copolymer chains. In various examples, at least some of the copolymer chains include -C≡CH end groups, -CO2H end groups, and reactive olefin end groups capable of [2+2] photodimerization at wavelengths >270 nm to obtain difunctionality.

[0367] In various examples, the photochemically reversible hydrogel polymers and nanogel particles include poly(PAG-co-CAA-co-NiPAM-co-AAc-co-BisAM) copolymer chains. In various examples, at least some of the copolymer chains include -C≡CH end groups, -CO2H end groups, and reactive olefin end groups capable of [2+2] photodimerization at wavelengths >270 nm to obtain difunctionality.

[0368] Grafting amplification primers onto photochemically reversible hydrogel polymers and nanogel particles

[0369] The general examples above show that photochemically reversible hydrogel polymers and nanogel particles can be prepared, wherein the copolymer chains of the photochemically reversible hydrogel polymers and nanogel particles include at least one reactive olefin or 1,4-diene end group capable of [2+2] or [2+2+2+2] photodimerization at wavelengths >270 nm, respectively, and at least some -C≡CH end groups, -CO2H end groups, and / or -N3 end groups. In some examples, the -CO2H end groups of the copolymer chains are used to connect the photochemically reversible hydrogel polymer or nanogel particles to a surface, such as a lane within a FC for SBS. In addition, the -N3 end groups or -C≡CH end groups of the copolymer chains can be used to transplant appropriately functionalized amplification primers (such as P5 / P7) onto the photochemically reversible hydrogel polymer or each nanogel particle in preparation for SBS. In some examples, [2+2] or [2+2+2+2] photodimerization was used to attach photochemically reversible hydrogel polymers or nanogel particles to appropriately functionalized FC surfaces in preparation for SBS.

[0370] In various examples, grafting the amplification primer to the photochemically reversible hydrogel polymer or nanogel particle includes click chemistry between the terminal alkyne substituent on the amplification primer and the -N3 terminal group on the corresponding copolymer chain, or click chemistry between the terminal -N3 substituent on the amplification primer and the -C≡CH terminal group on the corresponding copolymer chain. In other words, in various examples, grafting the amplification primer to the photochemically reversible hydrogel polymer or nanogel particle includes alkyne-azide or azide-alkyne cycloaddition click chemistry, thereby covalently linking the primer to the nanogel particle through the triazine moiety.

[0371] In other aspects, thiol-functionalized primers can be grafted onto a photochemically reversible hydrogel polymer or nanogel particle comprising copolymer chains, wherein at least some of the copolymer chains include a -C≡CH terminal group.

[0372] In various examples and as described above, the choice of monomers used to synthesize the photochemically reversible hydrogel polymer or nanogel particle determines whether the resulting copolymer chain includes -N3 or -C≡CH terminal groups in addition to the olefin or 1,4-diene and optional -CO2H terminal groups. For transplantation purposes, complementary functional groups are selected for the functionalized amplification primers to facilitate click chemistry.

[0373] The P5 and P7 amplification primers used in this article were used on the surface of a commercial flow cell sold by Illumina Inc. for use in HiSeq TM 、MiSeq TM 、NextSeq TM and Genome Analyzer TM Sequencing was performed on the platform. The P5 / P7 amplification primers used for transplantation are fully described in U.S. Pat. No. 9,982,250 and U.S. Publication No. 2011 / 0059865, the disclosures of which are incorporated herein by reference in their entirety.

[0374] In various examples, functionalized amplification primers for grafting onto photochemically reversible hydrogel polymers or nanogel particles include, but are not limited to, alkyne-P5 / P7 primers, N3-P5 / P7 primers, and thiol-P5 / P7 primers.

[0375] In various examples, grafting of alkyne-P5 / P7 primers onto a photochemically reversible hydrogel polymer or nanogel particle comprising a copolymer chain having -N3 terminal groups comprises CuAAC grafting, thereby producing a P5 / P7 grafted photochemically reversible hydrogel polymer or nanogel particle.

[0376] In various examples, CUAAC-catalyzed click chemistry involving N3-P5 / P7 or thiol-P5 / P7 primers is performed at a temperature of about 40° C. to about 80° C. for about 1 hour to about 5 hours.

[0377] In various examples, grafting of N3-P5 / P7 or thiol-P5 / P7 primers onto a photochemically reversible hydrogel polymer or nanogel particle comprising a copolymer chain having -C≡CH terminal groups comprises CuAAC grafting, thereby producing a P5 / P7 grafted photochemically reversible hydrogel polymer or nanogel particle.

[0378] Photochemically reversible capture of primers in nanogel particles for SBS

[0379] In various examples and as part of the SBS method, primer-grafted photochemically reversible nanogel particles are captured in a HiSeq RT-PCR system such as from Illumina, Inc. TM On the surface of the flow cell (FC) of the FC. The primer-grafted photochemically reversible nanogel particles can be captured in nanowells patterned in the coating on the FC surface, or directly attached to the coating on the surface without nanowells. In various examples, each primer-grafted nanogel particle can act as a nanowell, and thus can serve as a substitute for the nanowell.

[0380] In various examples, primer-grafted photochemically reversible nanogel particles were captured into the nanowells of FC by:

[0381] (a) Bioconjugation technique, using the reaction between the free carboxylate end groups present on the copolymer chains of the DMTMM-activated primer-grafted photochemically reversible nanogel particles and the available -NH2 groups on the surface of the previously silylated FC to form an amide bond, or

[0382] (b) CuAAC click chemistry, reacting any remaining -C≡CH end groups still present on the copolymer chains of the primer-grafted photochemically reversible nanogel particles (i.e., after grafting) with reactive -N3 groups present on the standard PAZAM-coated and polished FC,

[0383] to form a triazine bond.

[0384] In various examples related to (a) above, silanization of the FC surface can be accomplished using any suitable silane or silane derivative. The method used to attach the silane or silane derivative to the substrate can vary depending on the silane or silane derivative used.

[0385] In various examples, the silane or silane derivative is 3-mercaptopropylsilanetriol, 3-aminopropyltriethoxysilane (APTES), or 3-aminopropyltrimethoxysilane (APTMS) (ie, having the general structure X-RB —Si(OR C )3 silane, wherein X is an amino group, R B is —(CH2)3—, and R C In this example, the FC surface can be pre-treated with APTES or APTMS to covalently attach silicon to one or more oxygen atoms on the surface. The chemically treated surface is optionally baked to form a monolayer of amine groups.

[0386] In various examples, the multiple -NH2 groups present on the FC surface then react with the carboxylate end groups present on the corresponding copolymer chains of the primer-grafted photochemically reversible nanogel particles. This procedure takes advantage of the bifunctionality of the nanogel particles, since the -CO2H end groups are only used to bind the nanogel particles to the FC surface, while the -N3 or -C≡CH end groups were previously used only to graft the functionalized primers to the nanogel particles.

[0387] In various examples related to (b) above, PAZAM coatings on FC surfaces were prepared using bulk N-(5-(2-bromoacetamido)pentyl)acrylamide (BrAPA) as a monomer for the polymer hydrogel coating, followed by conversion of the bromine groups to -N3 groups.

[0388] In various examples, PAZAM can be deposited on the surface of the patterned FC surface by spin coating, dipping, dip coating, or flow of PAZAM under positive or negative pressure, or another suitable technique. PAZAM can be present in a mixture. In one example, the mixture includes water containing PAZAM or a mixture of ethanol and water containing PAZAM.

[0389] After coating, the functionalized molecules may also be exposed to a curing process to form a functionalized coating over the entire patterned substrate (i.e., over the recessed portions and gap regions). In an example, curing the functionalized molecules may be performed at a temperature in the range of room temperature (e.g., about 25° C.) to about 60° C. for a time in the range of about 5 minutes to about 2 hours.

[0390] To form a PAZAM coating in the nanowells but not on the interstitial regions of the patterned substrate, the PAZAM coating can be polished away from the interstitial regions using (a) an alkaline aqueous slurry having a pH ranging from about 7.5 to about 11 and including abrasive particles or (b) a polishing pad and a solution without abrasive particles.

[0391] To capture the photochemically reversible nanogel particles onto the PAZAM-coated FC surface, the PAZAM coating with reactive -N3 groups can react with any remaining -C≡CH end groups present on the copolymer chains of the photochemically reversible nanogel particles under conditions of CuAAC click chemistry. For any additional details, see the '033 patent (Illumina) cited above and incorporated herein.

[0392] In an alternative example, the order of the different steps of primer transplantation and particle capture can be reversed. Thus, photochemically reversible nanogel particles with dual functionality can be captured on a silanized FC surface or a PAZAM-coated FC surface by amide formation or click chemistry, and then the captured particles are subsequently exposed to appropriately functionalized amplification primers (e.g., alkyne-P5 / P7 or N3-P5 / P7) to attach the amplification primers to the captured nanogel particles.

[0393] In an alternative example, the photochemically reversible nanogel particles can be reversibly attached to an appropriately functionalized FC surface by [2+2] or [2+2+2+2] photodimerization. For example, the FC surface can be silanized with 3-mercaptopropylsilanetriol, 3-mercaptopropyltrimethoxysilane, or 3-mercaptopropyltriethoxysilane to tether multiple -SH groups to the FC surface. Then, in a thiol-ene (1,4-addition) reaction, a compound having both acrylate functionality and a reactive olefin or reactive 1,4-diene moiety (e.g., N-(2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl)acrylamide) is reacted with the tethered -SH groups, thereby converting the tethered -SH functionality into multiple tethered reactive olefin or 1,4-diene groups. These tethered reactive olefin or 1,4-diene groups can then be used for [2+2] or [2+2+2+2] photodimerization using photochemically reversible nanogel particles having copolymer chains containing reactive olefin or 1,4-diene end groups, preferably upon exposure to incident radiation of >270 nm. An important feature of this polymer attachment method is that it is reversible, such as upon exposure to incident radiation of <300 nm wavelength.

[0394] Seeding, clustering and SBS sequencing

[0395] In various examples, clustering includes suspended clustering or on-board clustering. On-board clustering can be used for proof of concept because suspended clustering avoids the need to pattern the coated FC surface, and each photochemically reversible nanogel particle captured on the FC surface acts as its own nanowell. In suspended clustering, seeded ssDNA can be clustered on the surface of the photochemically reversible nanogel particles. Clustering on photochemically reversible nanogel particles depends on making sufficiently accessible primers transplanted onto the photochemically reversible nanogel particles.

[0396] In various examples, the temperature responsiveness of photochemically reversible nanogel particles with primer grafts having poly(NiPAM) blocks within the copolymer chains allows temperature-controlled manipulation of seeding, amplification, and sequencing by:

[0397] (a) promote temperature-controlled shrinkage during inoculation to reduce the probability of multiple inoculation events,

[0398] and thus enhance monoclonality;

[0399] (b) promoting temperature-controlled expansion during clustering to increase primer accessibility and facilitate diffusion of materials into the photochemically reversible nanogel particles, resulting in an increase in the number of chains per cluster / particle and improved fluorescence; and / or

[0400] (c) Promoting temperature-controlled shrinkage or expansion to improve SBS incorporation and cleavage steps.

[0401] In various examples, the FC with captured primer-grafted photochemically reversible nanogel particles is then used for various sequencing methods or techniques, including SBS, cycle array sequencing, ligation sequencing, pyrophosphate sequencing, etc. Using any of these techniques, since the sequencing primers are only present on the photochemically reversible nanogel particles, amplification will be confined to each particle. In addition, since amplification is confined to the particle surface, there is more time to amplify one sequencing template into a larger cluster.

[0402] In various examples, SBS can be used in a system such as HISEQ TM HISEQX TM MISEQ TM 、NOVASEQ TM or NEXTSEQ TMSequencer system (Illumina, Inc.) system is run. In SBS, monitoring nucleic acid primers along the extension of nucleic acid template (i.e., sequencing template) to determine the sequence of nucleotides in the template. The basic chemical process can be polymerization (e.g., catalyzed by polymerase) or connection (e.g., catalyzed by ligase). In various SBS methods based on polymerase, fluorescently labeled nucleotides are added to primers to extend the primers in a template-dependent manner so that the order and type of nucleotides added to the primers can be used to determine the sequence of the template. For example, in order to start the first SBS cycle, one or more labeled nucleotides, DNA polymerases, etc. can be delivered to / delivered by the flow channel in the FC, and the flow channel accommodates the primer array on the nanogel particles. The nanogel particles of the primer transplantation that causes the incorporation of labeled nucleotides in primer extension can be detected by imaging events. During the imaging event, the illumination system provides excitation light to the nanogel particles.

[0403] In various examples, the nucleotide may also include a reversible termination property that terminates further primer extension once the nucleotide is added to the primer. For example, a nucleotide analog having a reversible terminator portion may be added to the primer so that subsequent extension does not occur until a deblocking agent is delivered to remove the portion. Thus, for examples using reversible termination, a deblocking agent may be delivered to the flow channel before or after detection.

[0404] Flushing (ie, washing) may be performed between the various fluid delivery steps.The SBS cycle may then be repeated n times to extend the primer by n nucleotides, thereby detecting a sequence of length n.

[0405] Photochemically reversible ligation of hydrogel polymers for SBS

[0406] In various examples, two different methods can be used to form a photochemically reversible hydrogel layer on the FC surface for SBS sequencing:

[0407] (1) providing a mixture of monomers and cross-linker compounds to the FC surface and irradiating the mixture of monomers and cross-linker compounds with a wavelength of >270 nm to form a hydrogel, cross-link at least some of the copolymer chains, and effectively solidify the hydrogel to the FC surface; or

[0408] (2) providing a photochemically reversible hydrogel polymer according to the present disclosure to an appropriately functionalized FC and irradiating the FC surface to bind the hydrogel polymer to the functionalized FC surface via [2+2] or [2+2+2+2] photodimerization.

[0409] For the method (1) referred to as "internal crosslinking of FC", the monomer mixture is in accordance with the present disclosure and is thus a mixture of at least a first type of monomer and at least a second type of monomer as set forth herein. The crosslinker compound added to the monomer mixture may be a multifunctional monomer such as N,N'-methylenebisacrylamide, N,N'-methylenebismethacrylamide, polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, N-vinyl acrylamide, glycidyl acrylate, divinylbenzene or tetraallylammonium chloride or other known substances for crosslinking.

[0410] For method (2), the FC surface is functionalized by silanization with 3-mercaptopropylsilanetriol, 3-mercaptopropyltrimethoxysilane, or 3-mercaptopropyltriethoxysilane, thereby tethering multiple -SH groups to the FC surface. Then, a compound having both acrylate functionality and a reactive olefin or reactive 1,4-diene moiety (e.g., N-(2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl)acrylamide) is reacted with the tethered -SH groups in a thiol-ene (1,4-addition) reaction, thereby converting the tethered -SH functionality into multiple tethered reactive olefin or 1,4-diene groups. Subsequently, these tethered reactive olefin or 1,4-diene groups can be used for [2+2] or [2+2+2+2] photodimerization using a photochemically reversible hydrogel having copolymer chains containing reactive olefin or 1,4-diene end groups. Preferably, the wavelength of the incident radiation used to attach the hydrogel to the functionalized surface is >270 nm.

[0411] Photochemically reversible separation of hydrogel polymers and nanogel particles to reuse FC previously used for SBS

[0412] In various examples, both photochemically reversible hydrogels and photochemically reversible nanogel particles can be removed from FC surfaces. Previously, the removal of hydrogel particles or nanogel particles from FC surfaces required the use of harsh chemicals. According to the present disclosure, photochemically reversible hydrogels and photochemically reversible nanogel particles can be easily removed from FC surfaces without the use of harsh chemicals, simply by irradiating the surface with radiation of a wavelength <300nm. Irradiation cuts the [2+2] or [2+2+2+2] dimers, releasing the photochemically reversible hydrogel or photochemically reversible nanogel particles from the FC surface. Further cleaning of the FC surface (such as removal of tethered silane groups) only requires mild conditions, such as a mild acid rinse.

[0413] In various examples, the sequencing / reuse workflow can be described as follows:

[0414] (1) FC surfaces can be functionalized with photocrosslinking motifs.

[0415] (2) Photochemically reversible hydrogel particles or nanogel particles containing complementary photocrosslinking motifs can be washed by FC. The hydrogel particles or nanogel particles include amplification primers grafted thereon. Alternatively, nanogel particles clustered in suspension containing photocrosslinking motifs can also be used.

[0416] (3) FC is irradiated with a wavelength of λ1, which induces photo-crosslinking of hydrogel particles or nanogel particles to the FC surface.

[0417] (4) Once captured on the FC surface, clustering and sequencing are performed.

[0418] (5) After all nucleic acid sequencing steps are completed, the FC is subjected to a second irradiation including a second wavelength λ2. This wavelength induces photocleavage of the cross-linking motif.

[0419] (6) After photocleavage is complete, the FC is rinsed with a wash solution to remove the sequenced material.

[0420] (7) The FC can then be reused.

[0421] Long-wavelength photochemically controllable chemistry

[0422] In various examples, the photochemically reversible hydrogel polymers and polymer nanogel particles may employ long wavelength (650 nm to 1100 nm) photochemically controllable chemistry rather than short wavelength photochemically controllable chemistry.

[0423] In various examples, the photochemically reversible hydrogel polymers and polymer nanogel particles may include copolymer chains having azobenzene moieties capable of photoisomerization between E and Z isomers according to the following scheme:

[0424]

[0425] In various examples, photoisomerization between the E isomer and the Z isomer can be used to move the copolymer chain off the surface, such as by affecting electrostatic changes in monolayer binding. In various embodiments, the R" substituent on the azobenzene portion can include an oleamide or ester, wherein the oleamide chain provides van der Waals interactions that support a self-assembled monolayer (SAM). The substituent Ra can be the remainder of the copolymer chain of the hydrogel polymer or polymer nanogel particle. In various examples, Ra and R" can be attached in the same or different polymer chains.

[0426] In various examples, the photochemically reversible hydrogel polymers and polymer nanogel particles can include copolymer chains having a spiropyran moiety capable of photoisomerizing between open merocyanine and closed spiropyran isomers according to the following scheme:

[0427]

[0428] In various examples, the photochromicity in the above schemes can be used to attach / detach a hydrogel polymer or polymer nanogel particle to / from a surface, such as by changing the electrostatic interaction between the polymer and the surface. In various examples, at least one of Ra, Rb, or Rc can be the remainder of a copolymer chain of the hydrogel polymer or polymer nanogel particle. In alternative embodiments, one portion of the merocyanine can be in one set of copolymer chains and another portion in a second set of copolymer chains, such that the photochromicity of the cyclized merocyanine attaches the copolymer chains together or attaches the copolymer chains to a functionalized surface of a substrate.

[0429] In various examples, the photochemically reversible hydrogel polymers and polymer nanogel particles may include copolymer chains having conjugated bithienylethylene moieties capable of undergoing a reversible 2+2+2 cycloaddition according to the following scheme:

[0430]

[0431] In various examples, the reversible photocycloaddition in the above scheme can be used to attach / detach a hydrogel polymer or polymer nanogel particle to / from a surface, such as by changing the electrostatic interaction between the polymer and the surface. In various examples, at least one of Ra, Rb, or Rc can be the remainder of a copolymer chain of a hydrogel polymer or polymer nanogel particle. In alternative embodiments, one thiophene moiety can be in one set of copolymer chains and another thiophene moiety in a second set of copolymer chains, such that the photocycloaddition attaches the copolymer chains together or attaches the copolymer chains to a functionalized surface of a substrate.

[0432] In various examples, the photochemically reversible hydrogel polymers and polymer nanogel particles may include copolymer chains having a hemithioindigo moiety capable of photoisomerizing between an E isomer and a Z isomer according to the following scheme:

[0433]

[0434] In various examples, photoisomerization between the E and Z isomers can be used to move the copolymer chain off the surface, such as by affecting electrostatic changes in monolayer binding. In various embodiments, the Ra, Rb, Rc substituents on the hemithioindigo moiety may include oleamide or esters, wherein the oleamide chain provides van der Waals interactions that support the self-assembled monolayer (SAM). The substituents Ra, Rb, Rc can be the remainder of the copolymer chain of the hydrogel polymer or polymer nanogel particle. Or two of Ra, Rb, Rc can be cyclized as part of the same or different polymer chains.

[0435] In various examples, the photochemically reversible hydrogel polymers and polymer nanogel particles can include copolymer chains having donor-acceptor Stenhouse adducts capable of undergoing a reversible cycloaddition according to the following scheme:

[0436]

[0437] In various examples, the reversible photocycloaddition in the above scheme can be used to attach / detach a hydrogel polymer or polymer nanogel particle to / from a surface, such as by changing the steric or electrostatic interaction between the polymer and the surface. In various examples, at least one of Ra, Rb, or Rc can be the remainder of a copolymer chain of a hydrogel polymer or polymer nanogel particle.

[0438] In various examples, the photochemically reversible hydrogel polymers and polymer nanogel particles may include copolymer chains having aryl-substituted bisimidazoles capable of photochromizing to imidazolium free radical species according to the following scheme:

[0439]

[0440] In various examples, the reversible reactions in the above schemes can be used to attach / detach hydrogel polymers or polymer nanogel particles to / from a surface or to crosslink copolymer chains. In various examples, aryl substituents can be attached to copolymer chains of hydrogel polymers or polymer nanogel particles.

[0441] To further illustrate the present disclosure, examples are provided below. The examples are provided for illustration and should not be construed as limiting the scope of the present disclosure in any way.

[0442] Example

[0443] Reference now Figure 1 , the photochemically reversible hydrogel is reversibly attached to an appropriately functionalized surface in a flow cell for nucleic acid sequencing and then removed after sequencing is complete. In this way, the photochemically reversible hydrogel enables the reuse of FC.

[0444] Figure 1The top portion illustrates that in the case of stimulus 1 (hυ1), a photochemically reversible hydrogel according to various examples of the present disclosure is connected to an appropriately functionalized FC surface by multiple photoaddition reactions (e.g., [2+2] photodimerization). The FC surface includes multiple tethered reactive olefin or 1,4-diene groups, such that the connection of the hydrogel to the surface is achieved by photoaddition. Preferably, the wavelength of the radiation (hυ1) used to connect the hydrogel to the functionalized surface is >270nm. The exact wavelength used for hydrogel connection depends on whether olefins or 1,4-dienes are used, and the electron density of the olefin or 1,4-diene functional groups, such as in the case where certain electron-withdrawing or electron-donating groups are bonded to the olefin or 1,4-diene moieties. In some examples, 4-methylcoumarin substituents that are both on the hydrogel copolymer chain and tethered to the FC surface participate in photodimerization to connect the hydrogel to the FC surface.

[0445] Figure 1 The top portion of also illustrates the removal of the hydrogel from the FC surface, allowing the FC to be reused. As illustrated, stimulation 2 (hυ2) causes cleavage of the dimer or [2+2+2+2] adduct, allowing the isolated hydrogel to be easily washed off under mild conditions.

[0446] Figure 1 The lower part of the diagram shows two methods for achieving reversible bonding of the hydrogel to the FC surface. Method A) is as described above - a preformed hydrogel polymer is provided to a functionalized FC surface and irradiated to dimerize the olefin moiety or produce a [2+2+2+2] cycloaddition adduct, bonding the hydrogel to the functionalized FC surface. In method B), a monomer mixture is provided to the FC surface and polymerization is initiated on the surface. This is referred to as "intra-FC cross-linking". This example shows the use of at least one monomer M1 with azide functionality (e.g., AzAPA), and at least one monomer M2 with reactive olefin or 1,4-diene functionality (e.g., CAA). A cross-linking compound, such as BisAM, is also added to the mixture prior to irradiation. In this way, in situ polymerization forms the hydrogel polymer on the FC surface.

[0447] Figure 2 An example of reversibly patterning nanogel particles on FC is provided, referred to as the "RAP method" "R" represents reversible and responsive chemistry, such as provided by the presence of reactive olefin or reactive 1,4-diene end groups on the copolymer chains of the nanogel particles. "A" represents an alternative polymer of synthetic or natural origin. "P" represents a primer-containing monomer having a primer-grafting functional group, such as a -N3 or -C≡CH group, which can participate in click chemistry grafting of appropriately functionalized amplification primers. Figure 2 In the illustrated example, photochemically reversible nanogel particles (such as RAP particles) are patterned on a functionalized FC surface using a photomask.

[0448] exist Figure 3 In the illustrated example, photochemically reversible hydrogel particles or nanogel particles having poly(CAA-co-NDMAM-co-AzAPA) copolymer chains can be reversibly crosslinked and cleaved by exposure to wavelengths of 365 nm and 254 nm, respectively. This example illustrates the photodimerization of 4-methylcoumarin end groups in the copolymer chains of the hydrogel particles or nanogel particles.

[0449] Figure 4 Synthetic routes for photochemically reversible hydrogel particles or nanogel particles are exemplified. In method A, PAZAM or other polymers including copolymer chains with -N3 end groups are reacted with alkyne coumarin under copper catalysis to form photochemically reversible hydrogel particles or nanogel particles with tethered coumarin substituents. In method A, the photochemically reversible hydrogel particles or nanogel particles do not start out as such, but ultimately have reactive olefin end groups on the copolymer chains after the reaction of attaching the coumarin tether to the hydrogel particles or nanogel particles via a triazine linker.

[0450] exist Figure 4 In the illustrated exemplary method B, photochemically reversible hydrogel particles or nanogel particles are directly synthesized by free radical polymerization of a monomer mixture consisting of CAA, NDMAM and AzAPA. The free radical initiator here is potassium persulfate (KPS), and the reaction is performed under an inert atmosphere at 70° C. The resulting photochemically reversible hydrogel particles or nanogel particles include poly(CAA-co-NDMAM-co-AzAPA) copolymer chains, and therefore have dual functionality by the presence of both reactive olefin end groups and -N3 end groups on at least some of the copolymer chains. In the absence of -CO2H end groups (AAc is not used in the copolymer), the photochemically reversible hydrogel particles or nanogel particles are connected to FC by photodimerization.

[0451] exist Figure 5A In the illustrated example, photochemical nanogel particles are prepared by reacting nanogel particles including -N3 end groups on at least some of the copolymer chains with an alkyne coumarin. In this manner, reactive coumarin groups are tethered to previously formed nanogel particles to produce photochemically reversible nanogel particles. Although exemplified with nanogel particles, the same method can be used to tether coumarin groups to hydrogel polymers including -N3 end groups on at least some of their copolymer chains.

[0452] exist Figure 5BIn the illustrated example, photochemically reversible nanogel particles are directly prepared from a monomer mixture of CAA, NiPAM, AzAPA, AAc and BisAM under aqueous suspension / precipitation free radical polymerization conditions. In this example, ammonium persulfate (APS) is used as a free radical initiator, and sodium dodecyl sulfate (SDS) is used as a dispersant. The resulting photochemically reversible nanogel particles illustrated include poly(CAA-co-NiPAM-co-AzAPA-co-AAc-co-BisAM) copolymer chains, wherein at least some of the copolymer chains have coumarin end groups, -N3 end groups and -CO2H end groups.

[0453] exist Figure 6 In the illustrated example, N-(4-methyl-2-oxo-2H-chromen-7-yl)acrylamide (7-(acrylamido-4-methylcoumarin) is prepared by reacting 7-amino-4-methylcoumarin and acryloyl chloride in dichloromethane (DCM) at 0° C. to ambient temperature. Although not illustrated, 4-methyl-2-oxo-2H-chromen-7-yl acrylate can be similarly prepared starting from 7-hydroxy-4-methylcoumarin.

[0454] exist Figure 7 In the example shown, the FC surface is functionalized by sequential treatment with 3-mercaptopropyltrimethoxysilane and N-(2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl)acrylamide. The resulting FC surface is characterized by having tethered coumarin groups (or more accurately, tethered 4-methylcoumarin groups). Photochemically reversible hydrogel particles or nanogel particles are then provided to the functionalized surface in solution, and the surface is then irradiated to bind the hydrogel particles or multiple nanogel particles to the functionalized surface through photodimerization of the coumarin groups.

[0455] exist Figure 8 In the illustrated example, the P5 / P7 amplification primer is functionalized at the 5' end with a photoreversible motif (such as a substituent comprising a reactive olefin or a reactive 1,4-diene). This example replaces the transplantation of the functionalized P5 / P7 amplification primer to the hydrogel particle or nanogel particle by alkyne-N3 or N3-alkyne click chemistry and is reversible by photochemistry. As illustrated in b), the PAZAM hydrogel on the FC surface may also include a photoreversible motif so that the functionalized P5 / P7 amplification primer is reversibly attached to the hydrogel layer by photodimerization. Once the nucleic acid sequencing experiment is completed, irradiation at <300nm will cut off the primer, and a simple wash will restore the hydrogel-coated surface.

[0456] In the detailed description, references to "various embodiments," "one embodiment," "embodiment," etc. indicate that the described embodiments may include certain features, structures, or characteristics, but not every embodiment necessarily includes the certain features, structures, or characteristics. In addition, such phrases do not necessarily refer to the same embodiment. In addition, when a certain feature, structure, or characteristic is described in conjunction with an embodiment, whether or not explicitly described, it is considered to be within the knowledge of those skilled in the art to affect such feature, structure, or characteristic in conjunction with other embodiments. After reading this specification, it will be clear to those skilled in the relevant art how to implement the disclosure in the alternative embodiments.

[0457] Benefits, other advantages and solutions to problems have been described with respect to specific embodiments. However, the benefits, advantages, solutions to problems, and any elements that may cause any benefit, advantage or solution to occur or become more apparent should not be interpreted as key, essential or necessary features or elements of the present disclosure. Therefore, the scope of the present disclosure is limited only by the appended claims, wherein, unless explicitly stated, elements in the singular form are not intended to mean "one and only one", but "one or more". In addition, when a phrase similar to "at least one of A, B and C" or "at least one of A, B or C" is used in a claim or specification, the phrase is intended to be interpreted as meaning that A may exist alone in an example, B may exist alone in an example, C may exist alone in an example, or any combination of elements A, B and C may exist in a single example; for example, A and B, A and C, B and C, or A and B and C.

[0458] All structural equivalents, chemical equivalents, and functional equivalents of the above-mentioned various examples of elements known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be covered by the present claims. In addition, the device or the component of the device or the method of using the device does not have to solve each problem that the present disclosure seeks to solve, because it is covered by the present claims. In addition, the elements, components, or method steps in the present disclosure are not intended to be dedicated to the public, regardless of whether the element, component, or method step is explicitly stated in the claims. Claim elements are not intended to cite 35 U.S.C. 112 (f), unless the element is explicitly stated using the phrase "member for...". As used herein, the term "comprises", "comprising" or any other variant thereof is intended to cover non-exclusive inclusions, so that the chemistry, chemical composition, process, method, article, or device including a series of elements does not only include those elements, but may include other elements that are not clearly listed or inherent to such chemistry, chemical composition, process, method, article, or device.

Claims

1. A hydrogel polymer, comprising a copolymer chain, wherein the copolymer chain further comprises: The first repeating unit of formula (I): in: R 1 , R 1’ and R 1” Each of is independently selected from H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl or heterocyclyl; X is -O- or -NH-; and R 2 is -CH2-C≡CH, or R 2 Has the following structure: Where R 2’ is -NH2, alkyl, alkoxy, alkenyl, alkynyl or optionally substituted variants thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, aziridine, triazoline or -C≡CH; and p is an integer from 1 to 50; and The second repeating unit of formula (II), in: R 3 , R 3’ , R 4 and R 4’ Each of -H, -R 5 、-OR 5 、-CO2R 5 、-C(O)R 5 、-OC(O)R 5 、-C(O)NR 6 R 7 、-NR 6 R 7 or a substructure of formula (III), R 5 is -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl or heterocyclyl; R 6 and R 7 Each of is independently selected from -H and alkyl; A is an aryl or thymidyl moiety; R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl or optionally substituted variants thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, or two R' groups that, when bonded to adjacent atoms on ring A and taken together with ring A, form a coumarinyl, anthracenyl, acenaphthenyl, thioguandanyl-1-oxide or thioguandanyl-1,1-dioxide moiety; X 1 is a bond, -(CH2) q -, -O- or -NH-; L is a -(CH2) q -X 2 -C(=O)- or -(CH2CH2O) q -X 2 -C(=O)- divalent linker; X 2 is -O- or -NH-; m is an integer from 1 to 9; and q is an integer from 0 to 50; wherein at least some of the copolymer chains include at least one reactive olefin or reactive 1,4-diene end group capable of undergoing [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm.

2. The hydrogel polymer of claim 1, wherein at least some of the copolymer chains comprise at least one N3, -C≡CH or -CO2H terminal group.

3. The hydrogel polymer according to any one of claims 1 or 2, wherein formula (III) includes a sub-structure of formula (IV): Where R 8 and R 9 Each of is -H, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl or heterocyclyl; each R 10 and R 11 is independently -H, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl or heterocyclyl, or R 10 is -C(=O)-, R 11 is -O-, and R 10 and R 11 are bonded together such that formula (IV) includes a substituted coumarin moiety.

4. The hydrogel polymer according to any one of claims 1 to 3, wherein the repeating unit of formula (I) is:

5. The hydrogel polymer according to any one of claims 1 to 4, wherein the repeating unit of formula (II) is:

6. The hydrogel polymer according to any one of claims 1 to 4, wherein the repeating unit of formula (II) is: and / or wherein R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl or optionally substituted variants thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy; and m is an integer of 1-9.

7. The hydrogel polymer according to any one of claims 1 to 4, wherein the repeating unit of formula (II) is: and / or wherein q is an integer from 0 to 50.

8. The hydrogel polymer according to any one of claims 1 to 4, wherein the repeating unit of formula (II) is: and / or wherein R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl or optionally substituted variants thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy; and m is an integer of 1-7.

9. The hydrogel polymer according to any one of claims 1 to 4, wherein the repeating unit of formula (II) is: wherein R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl or optionally substituted variants thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy; and m is an integer of 1-5.

10. The hydrogel polymer according to any one of claims 1 to 4, wherein the repeating unit of formula (II) is: and / or 11. The hydrogel polymer according to claim 3, wherein R 9 or R 10 is -CO2H, so that formula (IV) is a cis- or trans-cinnamic acid moiety.

12. The hydrogel polymer according to claim 3, wherein R 9 or R 10 is an aryl group, so that formula (IV) is a cis- or trans-stilbene moiety.

13. The hydrogel polymer of claim 1, wherein the hydrogel polymer is derived from a monomer mixture comprising: 7-((2-methacryloyloxy)ethoxy)-4-methylcoumarin; N-isopropylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

14. The hydrogel polymer of claim 1, wherein the hydrogel polymer is derived from a monomer mixture comprising: 7-((2-methacryloyloxy)ethoxy)-4-methylcoumarin; N,N-dimethylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

15. The hydrogel polymer according to claim 1, wherein the hydrogel polymer is derived from a monomer mixture, wherein the monomer mixture comprises: 7-((2-acrylamido)ethoxy)-4-methylcoumarin; N-isopropylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic.

16. The hydrogel polymer according to claim 1, wherein the hydrogel polymer is derived from a monomer mixture, wherein the monomer mixture comprises: 7-((2-acrylamido)ethoxy)-4-methylcoumarin; N,N-dimethylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic.

17. The hydrogel polymer of claim 1, wherein the hydrogel polymer is derived from a monomer mixture comprising: 7-(acrylamido)-4-methylcoumarin; N-isopropylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

18. The hydrogel polymer of claim 1, wherein the hydrogel polymer is derived from a monomer mixture comprising: 7-(acrylamido)-4-methylcoumarin; N,N-dimethylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

19. The hydrogel polymer of claim 1, wherein the hydrogel polymer is derived from a monomer mixture, wherein the monomer mixture comprises: 7-(methacrylamide)-4-methylcoumarin; N-isopropylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

20. The hydrogel polymer of claim 1, wherein the hydrogel polymer is derived from a monomer mixture, wherein the monomer mixture comprises: 7-(methacrylamido)-4-methylcoumarin; N,N-dimethylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

21. The hydrogel polymer of claim 1, wherein the hydrogel polymer is derived from a monomer mixture comprising: 7-(acryloyloxy)-4-methylcoumarin; N-isopropylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

22. The hydrogel polymer of claim 1, wherein the hydrogel polymer is derived from a monomer mixture comprising: 7-(acryloyloxy)-4-methylcoumarin; N,N-dimethylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

23. The hydrogel polymer of claim 1, wherein the hydrogel polymer is derived from a monomer mixture, the monomer mixture comprising: 7-(methacryloyloxy)-4-methylcoumarin; N-isopropylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

24. The hydrogel polymer of claim 1, wherein the hydrogel polymer is derived from a monomer mixture, the monomer mixture comprising: 7-(methacryloyloxy)-4-methylcoumarin; N,N-dimethylacrylamide; N-(5-(2-azidoacetamido)pentyl)acrylamide; and acrylic acid.

25. The hydrogel polymer according to any one of claims 13 to 24, wherein the monomer mixture further comprises a multifunctional compound selected from the group consisting of N,N'-methylenebisacrylamide, N,N'-methylenebismethacrylamide, polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, N-vinyl acrylamide, glycidyl acrylate, divinylbenzene, tetraallylammonium chloride, and mixtures thereof.

26. The hydrogel polymer of any one of claims 1 to 25, wherein the hydrogel polymer is in the form of nanogel particles.

27. The hydrogel polymer of any one of claims 1 to 26, wherein the hydrogel polymer further comprises an amplification primer conjugated thereto.

28. The hydrogel polymer of claim 27, wherein each conjugation between an amplification primer and the hydrogel polymer comprises a click chemistry between a terminal alkyne substituent on the amplification primer and an azide group on a corresponding copolymer chain end, or a click chemistry between a terminal azide substituent on the amplification primer and an alkyne group on a corresponding copolymer chain end.

29. The hydrogel polymer of any one of claims 1 to 28, wherein at least some of the copolymer chains are crosslinked by photodimerization between the reactive olefin or reactive 1,4-diene end groups capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm.

30. A substrate having a surface comprising a hydrogel polymer covalently attached thereto, wherein the hydrogel polymer comprises a plurality of copolymer chains further comprising a repeating unit of formula (I) and a repeating unit of formula (II); in: R 1 , R 1’ and R 1” Each of is independently selected from H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl or heterocyclyl; X is -O- or -NH-; R 2 is -CH2-C≡CH, or R 2 Has the following structure: R 2’ is -NH2, alkyl, alkoxy, alkenyl, alkynyl or optionally substituted variants thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, aziridine, triazoline or -C≡CH; p is an integer from 1 to 50; R 3 , R 3’ , R 4 and R 4’ Each of -H, -R 5 、-OR 5 、-CO2R 5 、-C(O)R 5 、-OC(O)R 5 、-C(O)NR 6 R 7 、-NR 6 R 7 or a substructure of formula (III), R 5 is -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl or heterocyclyl; R 6 and R 7 Each of is independently selected from -H and alkyl; A is an aryl or thymidyl moiety; R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl or optionally substituted variants thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, or two R' groups that, when bonded to adjacent atoms on ring A and taken together with ring A, form a coumarinyl, anthracenyl, acenaphthenyl, thioguandanyl-1-oxide or thioguandanyl-1,1-dioxide moiety; X 1 is a bond, -(CH2) q -, -O- or -NH-; L is a -(CH2) q -X 2 -C(=O)- or -(CH2CH2O) q -X 2 -C(=O)- divalent linker; X 2 is -O- or -NH-; m is an integer from 1 to 9; and q is an integer from 0 to 50; wherein at least some of the copolymer chains include at least one -N3, -C≡CH or -CO2H terminal group; and wherein at least some of the copolymer chains include at least one reactive olefin or reactive 1,4-diene end group capable of undergoing [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm.

31. The substrate of claim 30, wherein the covalent connection between the substrate and the hydrogel polymer comprises a photodimerization bond between the reactive olefin or reactive 1,4-diene end groups of the copolymer chains capable of [2+2] or [2+2+2+2] photodimerization and the corresponding reactive olefin or reactive 1,4-diene groups disposed on the surface of the substrate.

32. The substrate of claim 31, wherein the photodimerization bond comprises at least one of a coumarin dimer, an anthracene dimer, a thymidine dimer, a cinnamic acid dimer, a stilbene dimer, an acenaphthene dimer, a 2-methylbenzothiophene-1-oxide dimer, a 2-methylbenzothiophene-1,1-dioxide dimer, or a styrylquinoxaline dimer.

33. A substrate according to any one of claims 30 to 32, wherein the hydrogel polymer is in the form of nanogel particles.

34. The substrate of any one of claims 30 to 33, wherein the hydrogel polymer further comprises an amplification primer conjugated thereto.

35. The substrate of claim 34, wherein each conjugation between an amplification primer and the hydrogel polymer comprises click chemistry between a terminal alkyne substituent on the amplification primer and an azide terminal group on a corresponding copolymer chain, or click chemistry between a terminal azide substituent on the amplification primer and an alkyne terminal group on a corresponding copolymer chain.

36. The substrate of any one of claims 30 to 35, wherein at least some of the copolymer chains of the hydrogel polymer are cross-linked by photodimerization between the reactive olefin or reactive 1,4-diene end groups capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm.

37. A flow cell comprising a substrate according to any one of claims 30 to 36.

38. A method for synthesizing a hydrogel polymer having cross-linked copolymer chains, the method comprising: (1) A mixture containing (a) 7-((2-acryloyloxy)ethoxy)-4-methylcoumarin, 7-((2-methacryloyloxy)ethoxy)-4-methylcoumarin, 7-((2-acrylamide)ethoxy)-4-methylcoumarin, 7-((2-methacrylamide)ethoxy)-4-methylcoumarin, 7-((2-acryloyloxy)aminoethyl)-4-methylcoumarin, 7-((2-methacryloxy)aminoethyl)-4-methyl An aqueous dispersion of a monomer mixture of coumarin, 7-((2-acrylamido)aminoethyl)-4-methylcoumarin or 7-((2-methacrylamido)aminoethyl)-4-methylcoumarin; (b) N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA); (c) N,N-dimethylacrylamide or N-isopropylacrylamide (NiPAM); and (d) acrylic acid (AAc) is reacted under conditions suitable for free radical polymerization, to form the hydrogel polymer having non-crosslinked copolymer chains, wherein at least some of the copolymer chains include reactive coumarin end groups from monomer (a); and (2) crosslinking at least some of the copolymer chains by irradiating the hydrogel polymer with light of wavelength >270 nm, the crosslinks comprising dimers between the reactive coumarin end groups of the copolymer chains.

39. The method of claim 38, wherein the crosslinks comprise about 5 mole % of available reactive coumarin end groups.

40. The method of claim 38 or 39, wherein the hydrogel is in the physical form of nanogel particles.

41. The method of claim 40, wherein the free radical polymerization comprises suspension / precipitation free radical polymerization further comprising a free radical initiator and a dispersant.

42. A method for assembling a flow cell capable of sequencing nucleic acids, the method comprising: (a) treating the surface of the flow cell with any one of 3-mercaptopropylsilanetriol, 3-mercaptopropyltrimethoxysilane or 3-mercaptopropyltriethoxysilane to form a surface having a plurality of reactive -SH groups tethered to the surface; (b) reacting the plurality of -SH groups with an α,β-unsaturated carbonylthiol acceptor to provide reactive olefin or reactive 1,4-diene groups on the surface, the acceptor further comprising a reactive olefin or reactive 1,4-diene moiety covalently tethered thereto capable of [2+2] or [2+2+2+2] photodimerization, respectively, at a wavelength of >270 nm; (c) preparing a hydrogel polymer comprising a copolymer chain further comprising a repeating unit of formula (I) and a repeating unit of formula (II); in: R 1 is H, alkyl, alkoxy, alkenyl, alkynyl or optionally substituted variants thereof; R 2 is -NH2, alkyl, alkoxy, alkenyl, alkynyl or their optionally substituted variants, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, aziridine, triazoline; p is an integer from 1 to 50; R 3 , R 3’ , R 4 and R 4’ Each of -H, -R 5 、-OR 5 、-CO2R 5 、-C(O)R 5 、-OC(O)R 5 、-C(O)NR 6 R 7 、-NR 6 R 7 or a substructure of formula (III), R 5 is -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl or heterocyclyl; R 6 and R 7 Each of is independently selected from -H and alkyl; A is an aryl or thymidyl moiety; R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl or optionally substituted variants thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, or two R' groups that, when bonded to adjacent atoms on ring A and taken together with ring A, form a coumarinyl, anthracenyl, acenaphthenyl, thioguandanyl-1-oxide or thioguandanyl-1,1-dioxide moiety; X 1 is a bond, -(CH2) q -, -O- or -NH-; L is a -(CH2) q -X 2 -C(=O)- or -(CH2CH2O) q -X 2 -C(=O)- divalent linker; X 2 is -O- or -NH-; m is an integer from 1 to 9; and q is an integer from 0 to 50; wherein at least some of the copolymer chains include at least one reactive olefin or reactive 1,4-diene end group capable of [2+2] or [2+2+2+2] photodimerization at wavelengths >270 nm, respectively; and wherein at least some of the copolymer chains include at least one -N3 or -C≡CH terminal group; as well as (d) binding the hydrogel polymer to the surface of the flow cell by: (i) contacting the surface with the hydrogel polymer, and (ii) irradiating the hydrogel polymer and the surface of the flow cell with incident light of wavelength >270 nm to form [2+2] or [2+2+2+2] photodimers between the reactive olefin or 1,4-diene groups on the surface and the reactive olefin or 1,4-diene end groups on the corresponding copolymer chains.

43. The method of claim 42, wherein the irradiation step in (d) further crosslinks the copolymer chains in the hydrogel polymer via [2+2] or [2+2+2+2] photochemical addition of reactive olefins or reactive 1,4-diene end groups present on the corresponding copolymer chains.

44. The method according to claim 42 or 43, further comprising: Prior to step (d) or after step (d), an amplification primer is grafted onto the hydrogel polymer by performing a click chemistry reaction between a terminal alkyne substituent on the amplification primer and an azide terminal group on a corresponding copolymer chain, or performing a click chemistry reaction between a terminal azide substituent on the amplification primer and an alkyne terminal group on a corresponding copolymer chain.

45. The method of any one of claims 42 to 44, wherein the hydrogel polymer is prepared by reacting a hydrogel polymer comprising (a) 7-((2-acryloyloxy)ethoxy)-4-methylcoumarin, 7-((2-methacryloyloxy)ethoxy)-4-methylcoumarin, 7-((2-acrylamido)ethoxy)-4-methylcoumarin, 7-((2-methacrylamido)ethoxy)-4-methylcoumarin, 7-((2-acryloyloxy)aminoethyl)-4-methylcoumarin, 7-((2-methacryloyloxy)aminoethyl)-4-methylcoumarin, A monomer mixture of (a) methylcoumarin, (b) 7-((2-acrylamido)aminoethyl)-4-methylcoumarin or (c) N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA); (d) N,N-dimethylacrylamide or N-isopropylacrylamide (NiPAM); and (d) acrylic acid (AAc) is free radically polymerized to form a copolymer chain, wherein the reactive olefin end groups capable of [2+2] photodimerization at a wavelength of >270 nm include coumarin groups.

46. ​​The method according to any one of claims 42 to 45, further comprising recovering the flow cell, comprising reversing dimerization of the hydrogel polymer and binding of the hydrogel polymer to the surface of the flow cell by irradiating the hydrogel polymer and the surface of the flow cell with light having a wavelength <300 nm, thereby removing the hydrogel polymer from the surface of the flow cell.

47. A method for synthesizing a hydrogel polymer, the method comprising: reacting a monomer mixture comprising (a) N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA); (b) N,N-dimethylacrylamide or N-isopropylacrylamide (NiPAM); and (c) acrylic acid (AAc) under free radical polymerization conditions to form a hydrogel polymer comprising copolymer chains having reactive azide end groups; and At least a portion of the azide end groups are reacted with N-(but-3-yn-1-yl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide to form a hydrogel polymer having at least some copolymer chains containing tethered 4-methylcoumarin end groups.

48. The method of claim 47, further comprising irradiating the hydrogel polymer with light having a wavelength >270 nm to crosslink at least some of the copolymer chains via coumarin photodimerization.

49. The method of claim 47 or 48, wherein the reaction is performed inside a flow cell with the monomer mixture in contact with the surface of the flow cell.

50. The method of claim 47 or 48, wherein the hydrogel polymer is in the physical form of nanogel particles.

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