Photoswitchable chemistry and reusable flow cells for reversible hydrogels

Abstract

In some embodiments, novel photochemically reversible hydrogels and nanogel particles are described that contain copolymer chains containing at least one reactive alkene or reactive 1,4-diene end group capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm. In various embodiments, the photochemically reversible hydrogels contain at least one -N3, -N4, -N5, -N6, -N7, -N8, -N9, -N10, -N11, -N12, -N13, -N14, -N15, -N16, -N17, -N18, -N19, -N20, -N21, -N22, -N23, -N24, -N35, -N36, -N37, -N38, -N39, -N40, -N41, -N52, -N53, -N54, -N60, -N61, -N72, -N73, -N84, -N95, -N19, -N19, -N19, -N19, -N22, -N23, -N24, -N25, -N26, -N27, -N38, -N39, -N39, -N39, -N39, -N41, -N52, -N18, -N25, -N39, -N38, -N39, -N42, -N53, -N25, -N39, -N39, -N43, -N54, -N26, -N39, -N38, -N39, -N44, -N55, -N56, -N57, -N58, -N61, -N72, -N25, -N39, -N39, -N39, -N45, -N59, -N5 [Equation 1] TIFF2025533701000104.tif5128 or copolymer chains containing -COH end groups. For nucleic acid sequencing, amplification primers are grafted onto photochemically reversible hydrogel or nanogel particles that are reversibly bound to the surface within the flow cell. After sequencing is complete, the photochemically reversible hydrogel or nanogel particles can be removed from the flow cell surface by irradiation, thereby making the flow cell reusable.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 378,943, filed October 10, 2022, entitled "Photo-switchable Chemistry for Reversible Hydrogels and Reusable Flow Cells," the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates generally to nucleic acid sequencing methods and devices, and more particularly to photochemically reversible hydrogel and nanogel particles that can be used in sequencing by synthesis (SBS) methods. [Background technology]

[0003] Nucleic acid sequencing continues to be an important tool in many diverse fields, from ancestry to medical diagnostics to law enforcement. Today's sequencing methods are fast and cost-effective. Nevertheless, there is a continuing need to further reduce the cost of gene sequencing, for example, by streamlining the individual process steps in sequencing methods and improving various devices, such as flow cells, used in sequencing by synthesis. Summary of the Invention

[0004] Provided herein are photochemically reversible hydrogels and photochemically reversible nanogel particles with photoswitching chemistries for nucleic acid sequencing systems.

[0005] For example, as provided herein, certain photochemically reversible polymeric hydrogel and nanogel particles can be used to replace the hydrogel coating in a flow cell for sequencing by synthesis (SBS). The use of photochemically reversible polymeric hydrogel and nanogel particles can improve many aspects of the SBS method, such as making the sequencing flow cell reusable after removing the hydrogel or nanogel particles.

[0006] In various examples provided herein, certain polymeric hydrogel and nanogel particles with photochemical reversibility can be bound to a flow cell surface upon exposure to light with a frequency hν1>270 nm and can be detached from the flow cell surface upon exposure to light with a frequency hν2<300 nm.

[0007] In various examples provided herein, certain polymer hydrogel and nanogel particles with photochemical reversibility comprise copolymer chains containing at least one reactive alkene or reactive 1,4-diene end group capable of [2 + 2] or [2 + 2 + 2 + 2] photodimerization, respectively, at wavelengths hν>270 nm.

[0008] In various examples provided herein, photochemically reversible nanogel particles serve as a substitute for nanowells in a flow cell, thus eliminating the need to configure nanowells in a flow cell for SBS. Other examples may involve trapping nanogel particles within nanowells configured in a flow cell.

[0009] In various examples provided herein, sequencing on nanogel particles versus hydrogel surfaces improves the monoclonality of clustering of multiple copies of sequencing templates. For example, confining clustering to nanoscale particles may improve signal-to-noise ratios, error rates, and overall quality and coverage of genomes during sequencing.

[0010] In various embodiments, the photochemically reversible nanogel particles disclosed herein also exhibit dual functionality due to the presence of at least two types of reactive end groups on the copolymer chains within the nanogel particles. In various embodiments, the photochemically reversible and dual-functional nanogel particles include copolymer chains containing at least one reactive alkene or 1,4-diene end group capable of [2 + 2] or [2 + 2 + 2 + 2] photodimerization, respectively, at wavelengths hν>270 nm, and copolymer chains having at least one of an azide end group and a carboxylic acid end group.

[0011] In various examples, the photochemically reversible nanogel particles disclosed herein also exhibit dual responsiveness, i.e., temperature and pH responsiveness, where the temperature responsiveness is partially due to copolymer chains having a portion of poly(N-isopropylacrylamide) units, and the pH responsiveness is partially due to copolymer chains having carboxylic acid end groups.

[0012] The dual functionality and dual-responsive (temperature / pH) properties of nanogel particles as provided herein enable the initial attachment of alkyne-functionalized amplification primers onto the particles, for example, using the -N3 functional groups on the particles, while the pH responsiveness enhances chemical capture to the flow cell surface using bioconjugation techniques. These amplification primer-functionalized nanogel particles are demonstrated to support on-board particle clustering and SBS sequencing.

[0013] In various embodiments, the hydrogel polymer comprises a copolymer chain, the copolymer chain comprising: Formula (I):

[0014] [ka] A first repeat unit of the formula: R 1 , R 1’ , and R 1”each is independently selected from H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl; X is —O— or —NH—, and R 2 teeth,

[0015]

number

[0016] [ka] and In the formula, R 2’ is -NH2, alkyl, alkoxy, alkenyl, alkynyl, or optionally substituted versions thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, aziridine, triazoline, or

[0017]

number

[0018] [ka] A second repeat unit of the formula: R 3 , R 3’ , R 4 , and R 4’ each independently represents -H, -R 5 , -OR 5 , -CO2R 5 , -C(O)R 5 , -OC(O)R 5 , -C(O)NR 6 R 7 , -NR6 R 7 or formula (III)

[0019] [ka] is selected from the substructures of R 5 is -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl; R 6 and R 7 each is independently selected from —H and alkyl; A is an aryl or thymidinyl moiety; R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl, or optionally substituted versions thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, or two R' groups when attached to adjacent atoms on Ring A and, together with Ring A, form a coumarinyl, anthracenyl, acenaphthylenyl, thianaphthenyl-1-oxide, or thianaphthenyl-1,1-dioxide moiety; X 1 is the bond, -(CH2) q -, -O-, or -NH-; L is the structure -(CH2) q -X 2 -C(=O)- or -(CH2CH2O) q -X 2 a bivalent linker having -C(=O)-, X 2 is —O— or —NH—, m is an integer from 1 to 9, a second repeating unit, wherein q is an integer of 0 to 50; further comprising At least some of the copolymer chains contain at least one reactive alkene or reactive 1,4-diene end group capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm.

[0020] In various embodiments, at least some of the copolymer chains contain at least one N3,

[0021]

number

[0022] In various embodiments, formula (III) is a compound of formula (IV):

[0023] [ka] including the subgeneric structure of In the formula, R 8 and R 9 each is —H, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl, and each R 10 and R 11 are independently -H, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl, or R 10 is -C(=O)- and R 11 is -O and R 10 and R 11 are linked together such that formula (IV) contains a substituted coumarin moiety.

[0024] In various embodiments, the repeat unit of formula (I) is

[0025] [ka] is.

[0026] In various embodiments, the repeat unit of formula (II) is

[0027] [ka] is.

[0028] In various embodiments, the repeat unit of formula (II) is

[0029] [ka] and wherein R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl, or optionally substituted versions thereof, or halogen, -N, -OH, -C(O)H, -NH=NH, -SCN, -COH, -SH, glycidyl, epoxy; m is an integer of 1 to 9.

[0030] In various embodiments, the repeat unit of formula (II) is

[0031] [ka] and In the formula, q is an integer of 0 to 50.

[0032] In various embodiments, the first repeat unit of formula (I) is

[0033] [ka] and wherein R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl, or optionally substituted versions thereof, or halogen, -N, -OH, -C(O)H, -NH=NH, -SCN, -COH, -SH, glycidyl, epoxy; m is an integer of 1 to 7.

[0034] In various embodiments, the repeat unit of formula (II) is

[0035] [ka] and wherein R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl, or optionally substituted versions thereof, or halogen, -N, -OH, -C(O)H, -NH=NH, -SCN, -COH, -SH, glycidyl, epoxy; m is an integer of 1 to 5.

[0036] In various embodiments, the repeat unit of formula (II) is

[0037] [ka] is.

[0038] In various embodiments, R 9 or R 10 is —CO 2 H, whereby formula (IV) is a cis- or trans-cinnamic acid moiety.

[0039] In various embodiments, R 9 or R 10 is aryl, whereby formula (IV) is a cis- or trans-stilbene moiety.

[0040] In various embodiments, the hydrogel polymer is derived from a monomer mixture including 7-((2-methacryloyloxy)ethoxy)-4-methylcoumarin, N-isopropylacrylamide, N-(5-(2-azidoacetamido)pentyl)acrylamide, and acrylic acid.

[0041] In various embodiments, the hydrogel polymer is derived from a monomer mixture including 7-((2-methacryloyloxy)ethoxy)-4-methylcoumarin, N,N-dimethylacrylamide, N-(5-(2-azidoacetamido)pentyl)acrylamide, and acrylic acid.

[0042] In various embodiments, the hydrogel polymer is derived from a monomer mixture including 7-((2-acrylamido)ethoxy)-4-methylcoumarin, N-isopropylacrylamide, N-(5-(2-azidoacetamido)pentyl)acrylamide, and acrylic acid.

[0043] In various embodiments, the hydrogel polymer is derived from a monomer mixture including 7-((2-acrylamido)ethoxy)-4-methylcoumarin, N,N-dimethylacrylamide, N-(5-(2-azidoacetamido)pentyl)acrylamide, and acrylic acid.

[0044] In various embodiments, the hydrogel polymer is derived from a monomer mixture including 7-(acrylamido)-4-methylcoumarin, N-isopropylacrylamide, N-(5-(2-azidoacetamido)pentyl)acrylamide, and acrylic acid.

[0045] In various embodiments, the hydrogel polymer is derived from a monomer mixture including 7-(acrylamido)-4-methylcoumarin, N,N-dimethylacrylamide, N-(5-(2-azidoacetamido)pentyl)acrylamide, and acrylic acid.

[0046] In various embodiments, the hydrogel polymer is derived from a monomer mixture including 7-(methacrylamido)-4-methylcoumarin, N-isopropylacrylamide, N-(5-(2-azidoacetamido)pentyl)acrylamide, and acrylic acid.

[0047] In various embodiments, the hydrogel polymer is derived from a monomer mixture including 7-(methacrylamido)-4-methylcoumarin, N,N-dimethylacrylamide, N-(5-(2-azidoacetamido)pentyl)acrylamide, and acrylic acid.

[0048] In various embodiments, the hydrogel polymer is derived from a monomer mixture including 7-(acryloyloxy)-4-methylcoumarin, N-isopropylacrylamide, N-(5-(2-azidoacetamido)pentyl)acrylamide, and acrylic acid.

[0049] In various embodiments, the hydrogel polymer is derived from a monomer mixture including 7-(acryloyloxy)-4-methylcoumarin, N,N-dimethylacrylamide, N-(5-(2-azidoacetamido)pentyl)acrylamide, and acrylic acid.

[0050] In various embodiments, the hydrogel polymer is derived from a monomer mixture including 7-(methacryloyloxy)-4-methylcoumarin, N-isopropylacrylamide, N-(5-(2-azidoacetamido)pentyl)acrylamide, and acrylic acid.

[0051] In various embodiments, the hydrogel polymer is derived from a monomer mixture including 7-(methacryloyloxy)-4-methylcoumarin, N,N-dimethylacrylamide, N-(5-(2-azidoacetamido)pentyl)acrylamide, and acrylic acid.

[0052] In various embodiments, the monomer mixture comprises: The composition 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, divinyl benzene, tetraallyl ammonium chloride, diallyl dimethyl ammonium chloride, and mixtures thereof.

[0053] In various embodiments, the hydrogel polymer is in the form of nanogel particles.

[0054] In various embodiments, the hydrogel polymer further comprises amplification primers conjugated thereto.

[0055] In various embodiments, each conjugation between an amplification primer and a hydrogel polymer comprises click chemistry between a terminal alkyne substituent on the amplification primer and an azide group at the end of the respective copolymer chain, or click chemistry between a terminal azide substituent on the amplification primer and an alkyne group at the end of the respective copolymer chain.

[0056] In various embodiments, at least some of the copolymer chains are crosslinked by photodimerization between reactive alkene or reactive 1,4-diene end groups capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm.

[0057] In various embodiments, a substrate having a surface includes a hydrogel polymer covalently attached to the surface, the hydrogel polymer including a plurality of copolymer chains, the plurality of copolymer chains including a repeating unit of formula (I) and a repeating unit of formula (II):

[0058] [ka] further comprising During the ceremony, R 1 , R 1’ , and R 1” each is independently selected from H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl; X is —O— or —NH—; R 2 teeth,

[0059]

number

[0060] [ka] and R 2’ is -NH2, alkyl, alkoxy, alkenyl, alkynyl, or optionally substituted versions thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, aziridine, triazoline, or

[0061]

number

[0062] [ka] is selected from the substructures of R 5 is -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl; R 6 and R 7 each is independently selected from —H and alkyl; A is an aryl or thymidinyl moiety; R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl, or optionally substituted versions thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, or two R' groups when attached to adjacent atoms on Ring A and, together with Ring A, form a coumarinyl, anthracenyl, acenaphthylenyl, thianaphthenyl-1-oxide, or thianaphthenyl-1,1-dioxide moiety; X 1 is the bond, -(CH2) q -, -O-, or -NH-; L is the structure -(CH2) q -X 2 -C(=O)- or -(CH2CH2O) q -X 2 a bivalent linker having -C(=O)-, X 2 is —O— or —NH—, m is an integer from 1 to 9, q is an integer from 0 to 50, At least some of the copolymer chains have at least one -N3,

[0063]

number

[0064] In various embodiments, the covalent bond between the substrate and the hydrogel polymer comprises a photodimerization bond between a reactive alkene or reactive 1,4-diene end group of a copolymer chain capable of [2+2] or [2+2+2+2] photodimerization and a corresponding reactive alkene or reactive 1,4-diene group disposed on the substrate surface.

[0065] In various embodiments, the photodimerization linkage comprises at least one of a coumarin dimer, an anthracene dimer, a thymidine dimer, a cinnamic acid dimer, a stilbene dimer, an acenaphthylene dimer, a 2-methylthianaphthene-1-oxide dimer, a 2-methylthianaphthene-1,1-dioxide dimer, or a styrylquinoxaline dimer. In various embodiments, the hydrogel polymer is in the form of a nanogel particle. In various embodiments, the hydrogel polymer further comprises an amplification primer conjugated thereto.

[0066] In various embodiments, each conjugation between an amplification primer and a hydrogel polymer comprises click chemistry between a terminal alkyne substituent on the amplification primer and an azide end group of the respective copolymer chain, or click chemistry between a terminal azide substituent on the amplification primer and an alkyne end group of the respective copolymer chain.

[0067] In various embodiments, at least some of the copolymer chains of the hydrogel polymer are crosslinked by photodimerization between reactive alkene or reactive 1,4-diene end groups capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm.

[0068] In various embodiments, the flow cell comprises a substrate as described herein above.

[0069] In various embodiments, a method for synthesizing a hydrogel polymer having cross-linked copolymer chains comprises: (1)(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-methacrylamido)aminoethyl)-4-methylcoumarin, or 7-((2-methacrylamido) (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 a hydrogel polymer having non-crosslinked copolymer chains, at least some of which contain reactive coumarin end groups derived from monomer (a); and (2) crosslinking at least some of the copolymer chains by irradiating the hydrogel polymer with light having a wavelength >270 nm, wherein the crosslinks comprise dimers between reactive coumarin end groups of the copolymer chains; Includes.

[0070] In various embodiments, the crosslinks contain about 5 mole % available reactive coumarin end groups. In various embodiments, the hydrogel is the physical form of the nanogel particles.

[0071] In various embodiments, the free radical polymerization comprises a suspension / precipitation free radical polymerization further comprising a free radical initiator and a dispersing agent.

[0072] In various embodiments, a method for constructing a flow cell capable of sequencing nucleic acids includes: (a) treating the surface of a flow cell with one of 3-mercaptopropylsilanetriol, 3-mercaptopropyltrimethoxysilane, or 3-mercaptopropyltriethoxysilane to form a surface having a plurality of reactive -SH groups immobilized thereon; (b) reacting a plurality of -SH groups with an α,β-unsaturated carbonyl thiol-ene acceptor further comprising a reactive alkene or reactive 1,4-diene moiety capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm covalently attached to the α,β-unsaturated carbonyl thiol-ene acceptor to provide reactive alkene or reactive 1,4-diene groups on the surface; (c) a repeating unit of formula (I) and a repeating unit of formula (II)

[0073] [ka] preparing a hydrogel polymer comprising copolymer chains further comprising: During the ceremony, R 1 is H, alkyl, alkoxy, alkenyl, alkynyl, or optionally substituted versions thereof; R 2 is -NH, alkyl, alkoxy, alkenyl, alkynyl, or optionally substituted versions thereof, or halogen, -N, -OH, -C(O)H, -NH=NH, -SCN, -COH, -SH, glycidyl, epoxy, aziridine, triazoline; p is an integer from 1 to 50, R 3 , R 3’ , R 4 , and R 4’ each independently represents -H, -R 5 , -OR 5 , -CO2R 5 , -C(O)R 5 , -OC(O)R 5 , -C(O)NR 6 R 7 , -NR 6 R 7or formula (III)

[0074] [ka] is selected from the substructures of R 5 is -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl; R 6 and R 7 each is independently selected from —H and alkyl; A is an aryl or thymidinyl moiety; R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl, or optionally substituted versions thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, or two R' groups when attached to adjacent atoms on Ring A and, together with Ring A, form a coumarinyl, anthracenyl, acenaphthylenyl, thianaphthenyl-1-oxide, or thianaphthenyl-1,1-dioxide moiety; X 1 is the bond, -(CH2) q -, -O-, or -NH-; L is the structure -(CH2) q -X 2 -C(=O)- or -(CH2CH2O) q -X 2 a bivalent linker having -C(=O)-, X 2 is —O— or —NH—, m is an integer from 1 to 9, q is an integer from 0 to 50, At least some of the copolymer chains contain at least one reactive alkene or reactive 1,4-diene end group capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm, and at least some of the copolymer chains contain at least one -N3 or

[0075]

number

[0076] In various embodiments, the irradiation step in (d) also crosslinks the copolymer chains of the hydrogel polymer by [2+2] or [2+2+2+2] photoaddition of reactive alkene or reactive 1,4-diene end groups present on the respective copolymer chains.

[0077] In various embodiments, the method further comprises grafting the amplification primer onto the hydrogel polymer either 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 terminal group on each copolymer chain, or by performing a click chemistry reaction between a terminal azide substituent on the amplification primer and an alkyne terminal group on each copolymer chain.

[0078] In various embodiments, the hydrogel polymer is (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-methacrylamido)aminoethyl)-4-methylcoumarin, 7-((2-acrylamido)aminoethyl)-4-methylcoumarin, (b) N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA); (c) N,N-dimethylacrylamide or N-isopropylacrylamide (NiPAM); and (d) acrylic acid (AAc) by free radical polymerization of a monomer mixture containing coumarin, (b) methylcoumarin, or 7-((2-methacrylamido)aminoethyl)-4-methylcoumarin, (c) N,N-dimethylacrylamide or N-isopropylacrylamide (NiPAM), and (d) acrylic acid (AAc), thereby forming a copolymer chain, where the reactive alkene end group capable of [2+2] photodimerization at wavelengths >270 nm contains a coumarin group.

[0079] In various embodiments, the method further includes recycling the flow cell, which includes removing the hydrogel polymer from the surface of the flow cell by irradiating the hydrogel polymer and the surface of the flow cell with light having a wavelength of <300 nm to reverse dimerization and binding of the hydrogel polymer to the surface of the flow cell.

[0080] In various embodiments, a method for synthesizing a hydrogel polymer comprises: 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 with reactive azide end groups; and reacting at least a portion of the azide end groups 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 with attached 4-methylcoumarin end groups.

[0081] In various embodiments, the method further comprises irradiating the hydrogel polymer with light having a wavelength >270 nm to crosslink at least some of the copolymer chains by coumarin photodimerization.

[0082] In various embodiments, the reaction occurs in a flow cell where the monomer mixture is in contact with the surface of the flow cell. In various embodiments, the hydrogel polymer is in the physical form of nanogel particles.

[0083] It should be understood that any respective feature / example of each of the aspects of the present disclosure described herein may be implemented together in any suitable combination, and that any feature / example from any one or more of these aspects may be implemented together in any suitable combination with any of the features of the other aspects described herein, to achieve the benefits described herein. [Brief explanation of the drawings]

[0084] [Figure 1]

[0023] Figure 1 shows a schematic diagram of the use of a photochemically reversible hydrogel in a flow cell for nucleic acid sequencing according to various embodiments of the present disclosure. Part A of Figure 1 shows the attachment of a preformed polymer containing a photoswitchable moiety to a functionalized flow cell surface. Part B of Figure 1 shows the in situ formation of a hydrogel by polymerization of a monomer mixture on the flow cell surface. [Figure 2]FIG. 1 is a schematic diagram illustrating photochemically reversible attachment of nanogel particles onto a functionalized surface of a flow cell according to various embodiments of the present disclosure, where the nanogel particles, designated as RAP, contain the indicated functional groups for primer grafting and photoreversibility for detachment from the surface. [Figure 3] FIG. 1A is a schematic diagram illustrating the partial chemical structure of a photochemically reversible hydrogel or nanogel particle comprising poly(NDMAM-co-AzAPA-co-CAA) copolymer chains with a monomer ratio of 85:10:5 NDMAM:AzAPA:CAA, according to various embodiments of the present disclosure. Coumarin groups present as free end groups on the copolymer chains are available for dimerization upon irradiation with 365 nm light. Two reaction arrows indicate crosslink cleavage upon irradiation with 254 nm light. [Figure 4] 1A-1C are schematic diagrams illustrating two approaches for preparing crosslinkable hydrogel and nanogel particles according to various embodiments of the present disclosure. In Approach 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 (an "alkyne coumarin") under copper-catalyzed Blackpool grafting. In Approach B, poly(NDMAM-co-AzAPA-co-CAA) copolymer chains are synthesized by free radical polymerization of a monomer mixture containing NDMAM, AzAPA, and CAA monomers. [Figure 5A] FIG. 1A is a schematic illustrating the functionalization of nanogel particles bearing available azide groups with N-(but-3-yn-1-yl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide ("alkine coumarin") to form nanogel particles capable of [2+2] dimerization, according to various embodiments of the present disclosure. [Figure 5B]FIG. 10 is a schematic illustrating 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 monomers, according to various embodiments of the present disclosure. [Figure 6] FIG. 1 illustrates the synthesis of the monomer 7-(acrylamido)-4-methylcoumarin (N-(4-methyl-2-oxo-2H-chromen-7-yl)acrylamide) by the reaction of 7-amino-4-methylcoumarin with acryloyl chloride in dichloromethane (DCM), according to various embodiments of the present disclosure. [Figure 7] FIG. 1 is a schematic diagram illustrating the functionalization of a flow cell surface according to various embodiments of the present disclosure, where the surface is first silanized with 3-mercaptopropyltrimethoxysilane (MPTMS), followed by reaction of the immobilized —SH groups with N-(2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl)acrylamide (“CAM”). [Figure 8] 1A-1C are schematic diagrams illustrating the reaction of P5 / P7 amplification primers with photoreversible motifs, according to various embodiments of the present disclosure. In A), the photoreversible motif is covalently attached to the 5' end of the primer. In B), photocrosslinking induces primer grafting using a first wavelength (hv1), followed by clustering and sequencing, after which the ssDNA can be removed by exposure to a second wavelength (hv2) of incident radiation. DETAILED DESCRIPTION OF THE INVENTION

[0085] The detailed description of the embodiments herein refers to the accompanying drawings, which illustrate examples by way of example and their best mode. These embodiments are described in sufficient detail to enable those skilled in the art to practice the present subject matter, but it should be understood that other embodiments may be realized, and that logical, chemical, and mechanical changes may be made without departing from the spirit and scope of the subject matter provided herein. Accordingly, the detailed description is presented for purposes of illustration only, and not limitation. For example, unless otherwise stated, the steps recited in any method or process description may be performed in any order and are not necessarily limited to the order presented. Furthermore, any reference to a singular element or step may encompass multiple elements or steps, and any reference to two or more elements or steps may encompass a singular element or step. Furthermore, any reference to attached, fixed, connected, etc. may include permanent, detachable, temporary, partial, complete, and / or any other possible attachment options. Additionally, any reference to no contact (or similar phrases) may encompass reduced or minimal contact.

[0086] term As used herein, the term "hydrogel" is intended to mean a polymer comprising crosslinked or crosslinkable copolymer chains. Such polymers or their monomer mixture precursors can be coated onto a surface either in a continuous layer or in discrete regions. In various examples herein, the hydrogel polymer and nanogel particles can have the same polymer composition.

[0087] As used herein, the term "nanogel particle" is intended to mean a nanoscale polymeric particle comprising optionally crosslinked copolymer chains. For convenience, nanogel particles herein may be depicted as "soccer balls," i.e., substantially spherical, although their structure may not be this simple. The spherical representation allows the reader to grasp the concept of accessible functional groups within / on the nanogel particle, since these groups, typically terminal functional groups on the copolymer chains, may be depicted as protruding from the particle's surface. Nevertheless, particle size analysis, such as by light scattering, can be performed to obtain an associated particle size distribution or Z-average. Thus, nanogel particles herein may not necessarily be perfectly spherical in shape with functional groups protruding from the surface, and their average size can be determined. Typically, nanogel particles according to the present disclosure have a Z-average of about 50 nm to about 500 nm. Similarly, enumerations herein, at least for simplicity, describe chemical reactions occurring on the nanogel particle. While the nanogel particles herein are likely spherical in shape due to the synthesis method, which involves suspension / precipitation polymerization, the present disclosure is not limited with respect to particle shape. All nanogel "objects" are within the scope of the present disclosure, regardless of shape. Furthermore, because the particles comprise a cross-linked network, often containing water, it should be understood that various chemical reactions can occur both on and within the nanogel particles.

[0088] As used herein, the term "photochemically reversible" is intended to mean a property or characteristic of either a hydrogel polymer or nanogel particle when the hydrogel polymer or nanogel particle contains at least some copolymer chains having at least one reactive alkene end group or reactive 1,4-diene end group capable of photochemically reversible [2 + 2] or [2 + 2 + 2 + 2] cycloaddition, i.e., photodimerization. Hydrogel polymers and nanogel particles with photochemical reversibility can be reversibly bound to and removed from certain surfaces.

[0089] As used herein, the term "end group" is intended to mean a substituent at a terminal physical position on the copolymer chain structure of a hydrogel polymer or nanogel particle, such as the terminal position of the polyene backbone of the copolymer chain or the terminal position of a branched chain attached from the polyene backbone. In particular, copolymers herein may be characterized as polyenes, but may contain specific reactive end groups of interest (e.g., -coumarin, -4-methylcoumarin, -N3, -CO2H,

[0090]

number

[0091] As used herein, the term "dual-functional" refers to a hydrogel or nanogel particle that contains, for example, (1) at least one reactive alkene or reactive 1,4-diene end group capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm, and (2) a carboxylic acid end group, an -N3 end group, and / or

[0092]

number

[0093] As used herein, the term "temperature-responsive" is intended to refer to a property or characteristic of a nanogel particle when the nanogel particle comprises at least some copolymer chains having a polymeric structural portion that is physically responsive to temperature. More specifically, nanogel particles that are temperature-responsive exhibit shrinkage when exposed to an increase or decrease in temperature, and swelling when exposed to the opposite temperature trend. In various embodiments, nanogel particles having copolymer chains with blocks of poly(NiPAM) shrink with increasing temperature. This temperature-responsiveness provides a method for placing nanogel particles into pores, such as nanowells, and then fixing them in place simply by temperature manipulation. While temperature-responsiveness may be present to some extent in hydrogels that include at least some copolymer chains having a polymeric structural portion 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 a property as has been demonstrated for nanogel particles because the nanogel particles are physically manipulated on the surface.

[0094] As used herein, the term "pH-responsive" refers to a hydrogel or nanogel particle that contains at least some copolymer chains with carboxylic acid end groups, and in certain pH ranges, these groups are predominantly -COH, and in other pH ranges, these groups are predominantly -CO. - "pH-responsive" is intended to mean a property or characteristic of the hydrogel or nanogel particle such that the pH-responsive carboxylic acid end groups on at least some of the copolymer chains of the hydrogel or nanogel particle impart pH-responsiveness to the hydrogel or nanogel particle. In various embodiments, the pH-responsiveness allows for pH-driven binding of the hydrogel or nanogel particle to a functionalized flow cell surface.

[0095] As used herein, the term "dual stimulus (temperature / pH)" is intended to mean the combination of temperature- and pH-responsive properties (as defined above) exhibited by a particular hydrogel or nanogel particle. In various embodiments, the poly-NiPAM blocks in the copolymer chains of the hydrogel or nanogel particle impart temperature-responsiveness (i.e., contraction / swelling) to the hydrogel or nanogel particle, while the presence of AAc units in the copolymer chains of the hydrogel or nanogel particle contributes to the pH-responsiveness of the hydrogel or nanogel particle.

[0096] 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 polymeric nanogel particles as a dispersed solid phase when a dispersant or steric stabilizer is used and the reaction mixture is vigorously stirred. Suspension / precipitation polymerization is described in detail in the academic literature, S. Beck, et al., Chapter 3, pp. 21-85 in "Polymer Science and Nanotechnology—Fundamentals and 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. Furthermore, the present disclosure is not limited to this particular polymerization method for the synthesis of nanogel particles. For example, emulsion polymerization techniques may be employed, and non-aqueous solvents may be used.

[0097] As used herein, the acronym "CAA" refers to the chemical structure

[0098] [ka] By "coumarin acrylate" is meant to mean 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl acrylate (or more simply "coumarin acrylate"), a monomer having the formula:

[0099] As used herein, the acronym "CAM" refers to the chemical structure

[0100] [ka] By this reference, it is intended to mean N-(2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl)acrylamide (or more simply "coumarin acrylamide"), a monomer having the formula:

[0101] As used herein, the acronym "AzAPA" is intended to mean the monomer N-(5-(2-azidoacetamido)pentyl)acrylamide.

[0102] As used herein, the acronym "NiPAM" is intended to mean the monomer N-isopropylacrylamide.

[0103] As used herein, the acronym "NDMAM" is intended to mean the monomer N,N-dimethylacrylamide.

[0104] As used herein, the acronym "BisAM" is intended to mean the polyfunctional monomer N,N'-methylenebisacrylamide.

[0105] As used herein, the acronym "PAG" is intended to mean the monomer propargyl acrylate.

[0106] As used herein, the acronym "PAM" is intended to mean the monomer N-propargyl acrylamide.

[0107] As used herein, the acronym "AAc" is intended to mean the monomer acrylic acid.

[0108] As used herein, the acronym "BraPA" is intended to mean 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.

[0109] As used herein, the term "alkine coumarin" refers to the chemical structure

[0110] [ka] and the monomer N-(but-3-yn-1-yl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide.

[0111] As used herein, the acronym "SDS" is intended to mean sodium dodecyl sulfate, an anionic dispersing agent.

[0112] As used herein, the acronym "APS" is intended to mean ammonium persulfate, a free radical polymerization initiator.

[0113] As used herein, the acronym "ANA" is intended to mean hydrogel or nanogel particles comprising poly(AzAPA-co-NiPAM-co-AAc-co-BisAM) copolymer chains. ANA hydrogels and nanogel particles are characterized by both carboxylic acid and -N3 end groups on at least some of the copolymer chains.

[0114] As used herein, the acronym "PANA" is intended to mean hydrogel and nanogel particles comprising poly(PAG-co-NiPAM-co-AAc-co-BisAM) copolymer chains. PANA hydrogel and nanogel particles contain carboxylic acid and

[0115]

number

[0116] As used herein, the acronym "PANA'" is intended to mean hydrogel and nanogel particles comprising poly(PAM-co-NiPAM-co-AAc-co-BisAM) copolymer chains. PANA' hydrogel and nanogel particles contain carboxylic acid and

[0117]

number

[0118] As used herein, the term "flow cell" (and the acronym "FC") is intended to mean a vessel having a chamber (e.g., a flow channel or "lane") in which a reaction can occur, an inlet for delivering reagents to the chamber, and an outlet for removing reagents from the chamber. In various embodiments, the chamber allows for detection of a reaction occurring within the chamber. For example, the chamber may include one or more transparent surfaces that allow for optical detection of arrays, optically labeled molecules, etc. within the chamber. In various embodiments, polymeric materials, such as nanogel particles or hydrogel polymer coatings, can be attached to surfaces within the flow cell channels.

[0119] As used herein, the terms "covalently attached" or "covalently bonded" are intended to refer to the formation of a chemical bond characterized by the sharing of electron pairs between atoms. For example, a covalently bonded polymer coating is intended to refer to a polymer coating that forms a chemical bond with the functionalized surface of a substrate, as compared to attaching to the surface by other means, such as adhesion or electrostatic interactions. It will be understood that a polymer covalently attached to a surface can be attached by means in addition to covalent bonds.

[0120] As used herein, the acronym "PAZAM" is intended to mean a functionalized polymer coating comprising poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide).

[0121] As used herein, the acronym "DMTMM" is intended to mean the compound 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride.

[0122] As used herein, the acronym "CuAAC" is intended to mean copper-catalyzed azide-alkyne cycloaddition click chemistry.

[0123] As used herein, the acronym "dz" or "Dz" is intended to mean the "Z-average" reported from particle size analysis and is known in the art as a reliable measure of the mean size of a particle size distribution. The Z-average can be ascertained directly from light scattering experiments using a nanoparticle analyzer. See, e.g., J.C. Tomas, "The determination of log normal particle size distributions by dynamic light scattering," J. Colloid Interface Sci., 117(1), 187-192 (1987).

[0124] As used herein, the acronym "SBS" is intended to mean "Sequencing by Synthesis," a sequencing technology that uses fluorescently labeled nucleotides to sequence multiple clusters present on a flow cell surface in parallel. In some embodiments of SBS, a single labeled dNTP is added to a nucleic acid strand during each sequencing cycle. The nucleotide label serves as a terminator for polymerization, whereby after each dNTP incorporation, the fluorescent dye is imaged to identify the base and then enzymatically cleaved to allow incorporation of the next nucleotide. Further understanding of SBS is disclosed in PCT Application Publications WO2018 / 119101 and WO2020 / 005501 (both to Illumina, Inc.), the disclosures of which are incorporated herein by reference in their entireties.

[0125] As used herein, the term "seeding" is intended to mean the attachment of single-stranded oligonucleotides (ssDNA) to amplification primers covalently attached to nanogel particles. In various embodiments, seeding includes monoclonal seeding.

[0126] As used herein, the term "particle clustering" is intended to refer to the clustering of multiple copies of each of one type (monoclonal) or multiple types (polyclonal) of sequencing templates on a single nanogel particle bearing ssDNA pre-grafted and seeded with amplification primers. The term particle clustering is intended to refer to activity on the nanogel particles and should not be confused with the physical clustering of the nanogel particles themselves.

[0127] As used herein, the term "suspension clustering" is intended to mean the process whereby clustered nanogel particles, previously seeded with ssDNA, are subsequently captured on FC for sequencing.

[0128] As used herein, the term "on-board clustering" is intended to mean a process in which nanogel particles of appropriate size (e.g., in the range of about 200 nm to about 400 nm), which have been pre-grafted with a primer density compatible with sequencing and subsequently captured in the nanowells of the FC, are then clustered to generate sufficient copies of the template that can be used for sequencing.

[0129] As used herein, the term "Typhoon" is intended to mean the Amersham™ Typhoon™, a laser scanner platform commercially available from Cytiva Life Sciences for imaging and quantification of nucleic acids and proteins. When used as an action verb, the term is intended to mean performing an imaging method, such as fluorescence imaging, using an Amersham™ Typhoon™ laser scanner.

[0130] For additional acronyms and terminology regarding hydrogel coatings on flow cells and the use of these flow cells in SBS, see U.S. Pat. No. 10,919,033 (Illumina, Inc.), the disclosure of which is incorporated herein in its entirety.

[0131] As used herein, any "R" group designated in a chemical structure, e.g., R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8etc. represent substituents in organic chemistry that can be attached to the indicated atom to which the "R" group is attached. The R group can be substituted or unsubstituted. When two "R" groups are described as being "taken together" to form a ring structure, the R groups and the atoms to which they are attached can form a cycloalkyl, aryl, heteroaryl, or heterocyclic ring. In some instances, the ring so formed can form a bicyclic or tricyclic structure.

[0132] As used herein, the term "alkyl" is intended to mean a linear or branched monovalent fully saturated hydrocarbon substituent, optionally substituted anywhere in the substituent with one or more functional groups. Unless otherwise specified, alkyl groups include, for example, C1-C 24 , C1~C 18 , C1~C 10 The alkyl group may contain any number of carbon atoms, such as 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. Alkyl substituents herein may be substituted, i.e., have one or more substituents attached to the alkyl group or incorporated within the alkyl chain. Substitution within the alkyl substituent chain may include, for example, ether, sulfide, or imine linkages, i.e., O-<-S-, or -N=, or some other intervening heteroatom. Examples of substitution with 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, -halo, -SH, -SR, -S(=O)R, -SO2R, -OPO3 2- , -PO3 2-, -OH, -OR, -C(=O)R, -OC(=O)R, -COR, -NHC(=O)R, -NRC(=O)R, -C(=O)NHR, -C(=O)NR, alkyl, alkenyl, cycloalkyl, heterocyclyl, and aryl, wherein each R above independently represents hydrogen -H and an alkyl moiety, e.g., 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), and the like.

[0133] As used herein, the term "cycloalkyl" includes any 3-, 4-, 5-, 6-, 7-, or 8-membered, saturated or unsaturated, non-aromatic carbocyclic ring, optionally substituted at any position on the ring substituent with one or more functional groups. 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, cyclooctanyl, and the like. Examples of substitution on a cycloalkyl substituent include, but are not limited to, -CN, -N, -NH, -NHR, -N(R), and -N(R). + , -NO2, -NH-NH2, -NH-NHR, -NH-NR2, -halo, -SH, -SR, -S(=O)R, -SO2R, -OPO3 2- , -PO3 2- , -OH, -OR, -C(=O)R, -OC(=O)R, -COR, -NHC(=O)R, -NRC(=O)R, -C(=O)NHR, -C(=O)NR, alkyl, alkenyl, cycloalkyl, heterocyclyl, and aryl, wherein each R above independently represents -H and an alkyl moiety, e.g., C1~6 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), and the like.

[0134] As used herein, the term "alkenyl" is intended to mean a linear or branched, monovalent or divalent unsaturated hydrocarbon substituent, optionally substituted on or anywhere within the substituent with one or more functional groups. 2 When a carbon is part of a molecule bearing an alkenyl substituent, the alkenyl substituent can be considered divalent. An illustrative example is methylenecyclohexane, which can be considered a cyclohexane substituted with a methylene group (i.e., =CH2, which is a divalent alkenyl substituent). Unless otherwise specified, an alkenyl group can be, for example, C1-C 24 , C1~C 18 , C1~C 10 , C1-C8, or C1-C6, and any number of carbon atoms, such as methylidine (=CH2), ethylene / ethenyl (-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), and the like. Alkenyl substituents herein may be substituted, i.e., have one or more substituents appended to the alkenyl group or incorporated within the alkenyl chain. Substitution within an alkenyl substituent may include, for example, an ether, sulfide, or imine bond, i.e., O-, -S-, or -N=, or some other intervening heteroatom. Examples of substitution with an alkenyl substituent include, but are not limited to, -CN, -N3, -NH2, -NHR, -N(R)2, -N(R)3+ , -NO2, -NH-NH2, -NH-NHR, -NH-NR2, -halo, -SH, -SR, -S(=O)R, -SO2R, -OPO3 2- , -PO3 2- , -OH, -OR, -C(=O)R, -OC(=O)R, -COR, -NHC(=O)R, -NRC(=O)R, -C(=O)NHR, -C(=O)NR, alkyl, alkenyl, cycloalkyl, heterocyclyl, and aryl, wherein each R above is independently an alkyl moiety, e.g., 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), and the like.

[0135] As used herein, the term "aryl" includes any aromatic ring or fused polycyclic aromatic ring system (e.g., phenyl, naphthyl, anthracenyl, and phenanthrenyl) optionally substituted anywhere on the aromatic substituent with one or more functional groups. An unsubstituted phenyl substituent may be represented as -CH or, more simply, as -Ph. Aromatic heterocycles and fused-ring heteroaromatic substituents are distinct and are included in the definition of heterocyclyl substituents below. Examples of substitution on an aryl substituent include, but are not limited to, -CN, -N, -NH, -NHR, -N(R), -N(R). + , -NO2, -NH-NH2, -NH-NHR, -NH-NR2, -halo, -SH, -SR, -S(=O)R, -SO2R, -OPO3 2- , -PO3 2- , -OH, -OR, -C(=O)R, -OC(=O)R, -COR, -NHC(=O)R, -NRC(=O)R, -C(=O)NHR, -C(=O)NR, alkyl, alkenyl, cycloalkyl, heterocyclyl, and aryl, wherein each R above is independently an alkyl moiety, e.g., C1~6 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), and the like.

[0136] As used herein, "heterocycle" is intended to mean an unsubstituted or optionally substituted, saturated, unsaturated, or aromatic carbocyclic ring whose carbocyclic ring structure is interrupted by at least one heteroatom selected from oxygen (O), sulfur (S), or nitrogen (N). As used herein, the term "heterocyclyl" is intended to mean a heterocycle as a substituent, bonded to another atom of the compound from any C atom or heteroatom present in the heterocycle. For example, "pyridinyl" includes 2-, 3-, and 4-pyridinyl moieties as a substituent. Heterocycles can be monocyclic or fused polycyclic in structure. Examples of optional substitution on aryl substituents include, but are not limited to, -CN, -N, -NH, -NHR, -N(R), -N(R). + , -NO2, -NH-NH2, -NH-NHR, -NH-NR2, -halo, -SH, -SR, -S(=O)R, -SO2R, -OPO3 2- , -PO3 2- , -OH, -OR, -C(=O)R, -OC(=O)R, -COR, -NHC(=O)R, -NRC(=O)R, -C(=O)NHR, -C(=O)NR, alkyl, alkenyl, cycloalkyl, heterocyclyl, and aryl, wherein each R above is independently an alkyl moiety, e.g., 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 1~6alkoxy (e.g., -OCF3, -OC2F5), and the like.

[0137] Examples of heterocycles include, but are not limited to, azepinyl, aziridinyl, azetyl, azetidinyl, coumarinyl (2H-chromen-2-one), diazepinyl, dithiadiazinyl, dioxazepinyl, dioxolanyl, dithiazolyl, furanyl, isoxazolyl, isothiazolyl, imidazolyl, morpholinyl, morpholino, oxetanyl, oxadiazolyl, oxiranyl, oxazinyl, oxazolyl, piperazine, Nyl, pyrazinyl, pyridazinyl, pyrimidinyl, piperidyl, piperidino, pyridyl, pyranyl, pyrazolyl, pyrrolyl, pyrrolidinyl, thiatriazolyl, tetrazolyl, thiadiazolyl, triazolyl, thiazolyl, thienyl, tetrazinyl, thiadiazinyl, triazinyl, thiazinyl, thiopyranyl, furoisoxazolyl, imidazothiazolyl, thienoisothiazolyl, thienothiazolyl, imidazopyrazolyl, cyclopenta Pyrazolyl, pyrrolopyrrolyl, thienothienyl, thiadiazolopyrimidinyl, thiazolothiazinyl, thiazolopyrimidinyl, thiazolopyridinyl, oxazolopyrimidinyl, oxazolopyridyl, benzoxazolyl, benzisothiazolyl, benzothiazolyl, imidazopyrazinyl, purinyl, pyrazolopyrimidinyl, imidazopyridinyl, benzimidazolyl, indazolyl, benzoxthiolyl, benzodioxolyl, benzodi Thiolyl, indolizinyl, indolinyl, isoindolinyl, furopyrimidinyl, furopyridyl, benzofuranyl, isobenzofuranyl, thienopyrimidinyl, thienopyridyl, benzothienyl, cyclopentaoxazinyl, cyclopentafuranyl, benzoxazinyl, benzothiazinyl, quinazolinyl, naphthyridinyl, quinolinyl, isoquinolinyl, benzopyranyl, pyridopyridazinyl, and pyridopyrimidinyl. Further examples of heterocyclic ring systems are described in A. Katritzky, et al., Handbook of Heterocyclic Chemistry, 3 rd Ed., Elsevier, 2010, the entire contents of which are incorporated herein by reference.

[0138] Basic Example Various embodiments of the present disclosure describe novel photochemically reversible hydrogel polymers and polymeric nanogel particles. Various nanogel particles herein exhibit dual functionality due to the presence of at least two types of reactive end groups on the copolymer chains within the nanogel particles. For example, nanogel particles of the present disclosure may exhibit temperature-responsive properties, allowing the nanogel particles to shrink or swell in response to temperature changes, and pH-responsive properties, which aid in surface binding reactions. Nanogel particles according to the present disclosure may be used, inter alia, in nucleic acid sequencing methods, particularly within flow cells used in SBS methods.

[0139] In various embodiments, nanogel particles are prepared by suspension / precipitation free radical polymerization of various monomer types. The nanogel particles herein are described by the synthetic process used to prepare them, i.e., by the monomers and reaction conditions used in the suspension / precipitation free radical polymerization reaction, and also structurally, e.g., by describing the specific repeat units present in the copolymer chains of the nanogel particles thus prepared, along with their physical properties. In various embodiments, the repeat monomer units in the copolymer chains of the nanogel particles can comprise part of a block within a block copolymer.

[0140] In various embodiments, photochemically reversible hydrogel polymers and nanogel particles comprise copolymer chains that are capable of crosslinking and / or are at least partially crosslinked. For example, crosslinking is expected when multifunctional monomers are used in suspension / precipitation free radical polymerizations with other monomer types. As described herein, crosslinking can also be initiated photochemically, for example, by dimerizing reactive alkene or diene end groups on the copolymer chains.

[0141] In various embodiments, by incorporating monomers that result in temperature- or pH-responsive nanogel particles, the nanogel particle size can be fine-tuned to suit any step in the SBS sequencing protocol, such as library seeding, nanogel particle capture in FC nanowells, on-particle clustering, and on-particle sequencing. In various embodiments, temperature responsiveness can be incorporated using LCST (Lower Critical Solution Temperature) or UCST (Upper Critical Solution Temperature).

[0142] Goals and Basic Considerations In various embodiments, photoswitchable chemistries for photochemically reversible hydrogel and nanogel particles offer a potential solution for sustainability by making sequencing flow cells reusable.

[0143] In various embodiments, photochemically reversible hydrogel or nanogel particles are attached to a functionalized FC surface, followed by a clustering and sequencing step. After this is complete, the photochemically reversible hydrogel or nanogel particles are cleaved from the surface and discarded from the FC lane by flushing. A fresh solution of photochemically reversible hydrogel or nanogel particles can then be used for the next sequencing step.

[0144] For these purposes, many physical, chemical, and light-assisted switchable / reversible pathways have been proposed. One advantage of photoreversible chemistry compared to other systems is that no additional chemical or physical stimuli are required. Many photoreversible crosslinking chemistries that can be activated by a wide range of wavelengths have been explored. They are classified as short wavelength (<400 nm) and long wavelength (400-1000 nm). However, they are often activated and deactivated using long wavelengths (600-1000 nm). In principle, these reversible chemistries should also exhibit zero (or low) absorbance at wavelengths longer than 400 nm to be compatible with SBS sequencing methods (e.g., commercially available from Illumina). By using short wavelengths for photocleavage and photocrosslinking, respectively, derivatives of coumarin, anthracene, thymine, cinnamic acid, and stilbene are good candidates for photochemically reversible hydrogels and nanogel particles used in nucleic acid sequencing methods.

[0145] In various embodiments, photochemically reversible hydrogel and nanogel particles contain copolymer chains with either reactive alkene 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 photodimerizations that can be used to reversibly attach hydrogel or nanogel particles to functionalized FC surfaces and / or crosslink copolymer chains.

[0146] In various examples, Table 1 provides a summary of short wavelength photoswitchable chemicals that have been incorporated into the photochemically reversible hydrogel and nanogel particles herein.

[0147] [Table 1]

[0148] Monomers for the synthesis of photochemically reversible hydrogel polymers and nanogel particles In various embodiments, a first type of monomer used in synthesizing photochemically reversible hydrogel polymers and polymeric nanogel particles in a free radical polymerization reaction includes monomers having the structure:

[0149] [ka] and species having the formula: R 1 , R 1’ , and R 1” each is independently selected from H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl; X is —O— or —NH—, and R 2 teeth,

[0150]

number

[0151] [ka] and In the formula, R 2’ is -NH2, alkyl, alkoxy, alkenyl, alkynyl, or optionally substituted versions thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, aziridine, triazoline, or

[0152]

number

[0153] Examples of this first type of monomer 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.

[0154] In various embodiments, a second type of monomer used in synthesizing photochemically reversible hydrogel polymers and polymeric nanogel particles in a free radical polymerization reaction includes monomers having the structure:

[0155] [ka] and species having the formula: R 3 , R 3’ , R 4 , and R 4’ each independently represents -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 formula (III)

[0156] [ka] is selected from the substructures of R 5 is -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl; R 6 and R 7 each is independently selected from —H and alkyl; A is an aryl or thymidinyl moiety; R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl, or optionally substituted versions thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, or two R' groups when attached to adjacent atoms on Ring A and, together with Ring A, form a coumarinyl, anthracenyl, acenaphthylenyl, thianaphthenyl-1-oxide, or thianaphthenyl-1,1-dioxide moiety; X 1 is the bond, -(CH2) q -, -O-, or -NH-; L is the structure -(CH2) q -X 2 -C(=O)- or -(CH2CH2O) q -X 2 a bivalent linker having -C(=O)-, X 2 is —O— or —NH—, m is an integer from 1 to 9, q is an integer of 0 to 50.

[0157] In various embodiments of the second type of monomer, formula (III) can be represented by the subgenus structure (IV):

[0158] [ka] and In the formula, R 8 and R 9 each is —H, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl, and each R 10 and R 11 are independently -H, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl, or R 10 is -C(=O)- and R 11 is -O and R 10 and R 11are linked together such that formula (IV) contains a substituted coumarin moiety.

[0159] Examples of this 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-oxo-2H-chromen-7-yl)oxa ethyl acrylate, N-(2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl)acrylamide, 2-((4-methyl-2-oxo-2H-chromen-7-yl)amino)ethyl acrylate, 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)methyl)acrylamide, 2-(5-methyl-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)ethyl Acrylate, N-(2-(5-methyl-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)ethyl)acrylamide, 2-(anthracen-2-yloxy)ethyl acrylate, N-(2-(anthracen-2-yloxy)ethyl)acrylamide, 2-(anthracen-2-ylamino)ethyl acrylate, N-(2-(anthracen-2-ylamino)ethyl)acrylamide, (E)-2-(4-( 2-(quinoxalin-2-yl)vinyl)phenoxy)ethyl acrylate, (E)-N-(2-(4-(2-(quinoxalin-2-yl)vinyl)phenoxy)ethyl)acrylamide, (E)-2-((4-(2-(quinoxalin-2-yl)vinyl)phenyl)amino)ethyl acrylate, and (E)-N-(2-((4-(2-(quinoxalin-2-yl)vinyl)phenyl)amino)ethyl)acrylamide.

[0160] In various embodiments, photochemically reversible hydrogel polymers are prepared under various free radical polymerization conditions by reacting at least one first type of monomer with at least one second type of monomer according to the structures listed above. For reviews of 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.

[0161] With these two types of monomers used in the free radical polymerization reaction, the resulting photochemically reversible hydrogel polymers contain copolymer chains having at least a first repeat unit incorporating the first type of monomer and at least a second repeat unit incorporating the second type of monomer.

[0162] In various embodiments, photochemically reversible nanogel particles are prepared under suspension / precipitation or emulsion free radical polymerization reaction conditions by reacting at least one first type of monomer with at least one second type of monomer according to the structures listed above. With these two types of monomers used in the suspension / precipitation or emulsion free radical polymerization reaction, the resulting photochemically reversible nanogel particles comprise copolymer chains having at least a first repeat unit incorporating the first type of monomer and at least a second repeat unit incorporating the second type of monomer.

[0163] Multifunctional monomers that can be included in free radical polymerization reactions to form photochemically reversible hydrogel polymers and nanogel particles with some degree of cross-linking 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.

[0164] In various embodiments, photochemically reversible hydrogel polymers and nanogel particles are prepared under free radical polymerization reaction conditions by reacting at least one first type of monomer, at least one second type of monomer (both according to the structures listed above), and at least one multifunctional monomer. With these two types of monomers and the multifunctional monomer used in the free radical polymerization reaction, the resulting photochemically reversible hydrogel polymers and nanogel particles comprise copolymer chains having at least a first repeat unit incorporating a first type of monomer and at least a second repeat unit incorporating a second type of monomer, where the copolymer chains have at least some crosslinking between the copolymer chains. Further crosslinking of the copolymer chains and / or attachment of the photochemically reversible hydrogel polymer or nanogel polymer to a functionalized surface can be achieved by photochemical dimerization of alkene or diene groups within the copolymer chains or between the copolymer chains and the functionalized surface.

[0165] In various embodiments, photochemically reversible hydrogel polymers and nanogel particles are prepared under free radical polymerization reaction conditions by reacting at least one first type of monomer, at least one second type of monomer (both according to the structures listed above), and the multifunctional monomer N,N'-methylenebismethacrylamide (BisAM).

[0166] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles thus prepared under free radical polymerization reaction conditions comprising at least one of each of two types of monomers and optionally a multifunctional monomer as described above, comprise at least a reactive alkene or 1,4-diene capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm, and a carboxylic acid, -N3, or

[0167]

number

[0168] Suspension / Precipitation Radical Polymerization and Other Free Radical Polymerizations In various embodiments, the synthesis of photochemically reversible nanogel particles involves various aspects of suspension / precipitation free radical polymerization or emulsion polymerization. In various embodiments, the reaction conditions are aqueous and heated, and employ selected monomers, a dispersant to promote the suspension in water of the thus-formed, generally water-insoluble, photochemically reversible nanogel particles, and a free radical initiator.

[0169] In various embodiments, the suspension / precipitation free radical polymerization reaction is carried out at a temperature of about 50° C. to about 90° C. for about 1 hour to about 4 hours.

[0170] In various embodiments, the dispersant herein includes an anionic or non-ionic dispersant. Exemplary anionic dispersants include sodium dodecyl sulfate (SDS). Non-ionic dispersants include, but are not limited to, polyethylene glycol (PEG), sorbitan monooleate (e.g., under the trade name Span®), ethoxylated sorbitan monooleate (e.g., under the trade name Tween®), and acryloyl-terminated PEG.

[0171] In various embodiments, the free radical initiator comprises a water-soluble compound.

[0172] In various embodiments, the free radical initiator comprises a peroxide.

[0173] In various embodiments, the free radical initiator includes sodium persulfate, potassium persulfate, or ammonium persulfate.

[0174] In various embodiments, the free radical initiator includes ammonium persulfate (APS).

[0175] In various examples, photochemically reversible nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction incorporating a dispersed monomer mixture containing 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl acrylate (CAA), N,N-dimethylacrylamide (NDMAM), and N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA).

[0176] In various examples, photochemically reversible nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction incorporating a dispersed monomer mixture containing 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl acrylate (CAA), N,N-dimethylacrylamide (NDMAM), N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), and the multifunctional monomer N,N'-methylenebisacrylamide (BisAM).

[0177] In various examples, photochemically reversible nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction incorporating a dispersed monomer mixture containing 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl acrylate (CAA), N,N-dimethylacrylamide (NDMAM), N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), and acrylic acid (AAc).

[0178] In various examples, photochemically reversible nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction incorporating a dispersed monomer mixture containing 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl acrylate (CAA), N,N-dimethylacrylamide (NDMAM), N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), acrylic acid (AAc), and the multifunctional monomer N,N'-methylenebisacrylamide (BisAM).

[0179] In various examples, photochemically reversible nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction incorporating a dispersed monomer mixture containing 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl acrylate (CAA), N-isopropylacrylamide (NiPAM), N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), and acrylic acid (AAc).

[0180] In various examples, photochemically reversible nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction incorporating a dispersed monomer mixture containing 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl acrylate (CAA), N-isopropylacrylamide (NiPAM), N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), acrylic acid (AAc), and the multifunctional monomer N,N'-methylenebisacrylamide (BisAM).

[0181] In various examples, photochemically reversible nanogel particles are synthesized by forming copolymer chains in a suspension / precipitation free radical polymerization reaction incorporating a dispersed monomer mixture containing N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA) and acrylic acid (AAc), followed by reacting at least some of the available -N3 end groups on the resulting copolymer chains with N-(but-3-yn-1-yl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide (alkyne coumarin).

[0182] In various embodiments, photochemically reversible nanogel particles are synthesized by suspension / precipitation free radical polymerization incorporating a dispersed monomer mixture containing N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), acrylic acid (AAc), and the multifunctional monomer N,N'-methylenebisacrylamide (BisAM) to form copolymer chains, followed by reaction of at least some of the available -N3 end groups on the resulting copolymer chains with N-(but-3-yn-1-yl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide (alkyne coumarin).

[0183] In various embodiments, photochemically reversible nanogel particles are synthesized by forming copolymer chains in a suspension / precipitation free radical polymerization reaction incorporating a dispersed monomer mixture containing N,N-dimethylacrylamide (NDMAM), N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), and acrylic acid (AAc), followed by reacting at least some of the available -N3 end groups on the resulting copolymer chains with N-(but-3-yn-1-yl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide (alkyne coumarin).

[0184] In various embodiments, photochemically reversible nanogel particles are synthesized by forming copolymer chains in a suspension / precipitation free radical polymerization reaction incorporating a dispersed monomer mixture containing N,N-dimethylacrylamide (NDMAM), N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), acrylic acid (AAc), and the multifunctional monomer N,N'-methylenebisacrylamide (BisAM), followed by reaction of at least some of the available -N3 end groups on the resulting copolymer chains with N-(but-3-yn-1-yl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide (alkyne coumarin).

[0185] In various examples, photochemically reversible nanogel particles are synthesized by forming copolymer chains in a suspension / precipitation free radical polymerization reaction incorporating a dispersed monomer mixture containing N-isopropylacrylamide (NiPAM), N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), and acrylic acid (AAc), followed by reacting at least some of the available -N3 end groups on the resulting copolymer chains with N-(but-3-yn-1-yl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide (alkyne coumarin).

[0186] In various embodiments, photochemically reversible nanogel particles are synthesized by forming copolymer chains in a suspension / precipitation free radical polymerization reaction incorporating a dispersed monomer mixture containing N-isopropylacrylamide (NiPAM), N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), acrylic acid (AAc), and the multifunctional monomer N,N'-methylenebisacrylamide (BisAM), followed by reaction of at least some of the available -N3 end groups on the resulting copolymer chains with N-(but-3-yn-1-yl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide (alkyne coumarin).

[0187] Photochemically reversible hydrogel polymers and nanogel particles containing copolymer chains In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise copolymer chains having various end groups on at least some of the copolymer chains. In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise copolymer chains having at least some cross-linking. In various embodiments, the at least some cross-linking is obtained photochemically by dimerizing certain reactive alkene or 1,4-diene end groups on the copolymer chains.

[0188] In various embodiments, the photochemically reversible hydrogel polymers and polymeric nanogel particles according to the present disclosure comprise copolymer chains, the copolymer chains having the formula (I):

[0189] [ka] A first repeat unit of the formula: R 1 , R 1’ , and R 1” each is independently selected from H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl; X is —O— or —NH—, and R 2 teeth,

[0190]

number

[0191] [ka] and In the formula, R 2’is -NH2, alkyl, alkoxy, alkenyl, alkynyl, or optionally substituted versions thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, aziridine, triazoline, or

[0192]

number

[0193] [ka] A second repeat unit of the formula: R 3 , R 3’ , R 4 , and R 4’ each independently represents -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 formula (III)

[0194] [ka] is selected from the substructures of R 5 is -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl; R 6 and R 7 each is independently selected from —H and alkyl; A is an aryl or thymidinyl moiety; R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl, or optionally substituted versions thereof, or halogen, -N3, -OH, -C(O)H, -NH=NH2, -SCN, -CO2H, -SH, glycidyl, epoxy, or two R' groups when attached to adjacent atoms on Ring A and, together with Ring A, form a coumarinyl, anthracenyl, acenaphthylenyl, thianaphthenyl-1-oxide, or thianaphthenyl-1,1-dioxide moiety; X 1 is the bond, -(CH2) q -, -O-, or -NH-; L is the structure -(CH2) q -X 2 -C(=O)- or -(CH2CH2O) q -X 2 a bivalent linker having -C(=O)-, X 2 is —O— or —NH—, m is an integer from 1 to 9, q is an integer from 0 to 50; and a second repeat unit.

[0195] In various embodiments, R 1 =R 1’ =R 1” ═H, X is —O— or —NH—, and R 2 teeth,

[0196]

number

[0197] [ka]

[0198] wherein R 2’ is -N3 or

[0199]

number

[0200] In various embodiments of the second repeat unit of formula (II), formula (III) can be represented by formula (IV):

[0201] [ka] It has a subgeneric structure of In the formula, R 8 and R 9 each is —H, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl, and each R 10 and R 11 are independently -H, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl, or R 10 is -C(=O)- and R 11 is -O and R 10 and R 11 are linked together such that formula (IV) contains a substituted coumarin moiety.

[0202] In various embodiments, photochemically reversible hydrogel polymers and nanogel particles having the repeating units listed above comprise copolymer chains having at least one reactive alkene or reactive 1,4-diene end group capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm.

[0203] In various embodiments, photochemically reversible hydrogel polymers and nanogel particles having the repeating units listed above comprise (1) at least one reactive alkene or reactive 1,4-diene end group capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm, and (2) a carboxylic acid, -N3, or

[0204]

number

[0205] In various embodiments, photochemically reversible hydrogel polymers and nanogel particles having the repeating units listed above comprise copolymer chains having at least one carboxylic acid end group, at least one -N3 end group, and at least one reactive alkene or reactive 1,4-diene end group capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm.

[0206] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise a first repeat unit of formula (I) comprising:

[0207] [ka] wherein p is an integer from 1 to 50, as above.

[0208] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise a first repeat unit of formula (I) comprising:

[0209] [ka] wherein p is an integer from 1 to 50, as above.

[0210] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise a first repeat unit of formula (I) comprising:

[0211] [ka] The copolymer chain comprises:

[0212] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise a polymer in which the second repeat unit of formula (II) is:

[0213] [ka] The copolymer chain comprises:

[0214] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise a second repeat unit of formula (II) comprising:

[0215] [ka] and a copolymer chain in which wherein R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl, or optionally substituted versions thereof, or halogen, -N, -OH, -C(O)H, -NH=NH, -SCN, -COH, -SH, glycidyl, epoxy; m is an integer of 1 to 9.

[0216] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise a second repeat unit of formula (II) comprising:

[0217] [ka] and a copolymer chain in which In the formula, q is an integer of 0 to 50.

[0218] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise a second repeat unit of formula (II) comprising:

[0219] [ka] and a copolymer chain in which wherein R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl, or optionally substituted versions thereof, or halogen, -N, -OH, -C(O)H, -NH=NH, -SCN, -COH, -SH, glycidyl, epoxy; m is an integer of 1 to 7.

[0220] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise a polymer in which the second repeat unit of formula (II) is:

[0221] [ka] and a copolymer chain in which wherein R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl, or optionally substituted versions thereof, or halogen, -N, -OH, -C(O)H, -NH=NH, -SCN, -COH, -SH, glycidyl, epoxy; m is an integer of 1 to 5.

[0222] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise a polymer in which the second repeat unit of formula (II) is:

[0223] [ka] The copolymer chain comprises:

[0224] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles are characterized in that at least some of the copolymer chains have unused -N3 end groups, unreacted -CO2H end groups, and at least some N-(2-(1λ)) end groups that provide reactive alkene end groups capable of [2+2] photodimerization at wavelengths >270 nm. 2The poly(NiPAM-co-AzAPA-co-AAc) copolymer chains were functionalized with alkyne coumarin to contain a 2,3-triazol-4-yl)ethyl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide adduct.

[0225] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles are characterized in that at least some of the copolymer chains have unused -N3 end groups, unreacted -CO2H end groups, and at least some N-(2-(1λ)) end groups that provide reactive alkene end groups capable of [2+2] photodimerization at wavelengths >270 nm. 2 The poly(NiPAM-co-AzAPA-co-AAc-co-BisAM) copolymer chains were functionalized with alkyne coumarin to contain a 2,3-triazol-4-yl)ethyl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide adduct.

[0226] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles are characterized in that at least some of the copolymer chains have unused -N3 end groups, unreacted -CO2H end groups, and at least some N-(2-(1λ)) end groups that provide reactive alkene end groups capable of [2+2] photodimerization at wavelengths >270 nm. 2 The poly(NDMAM-co-AzAPA-co-AAc) copolymer chains were functionalized with alkyne coumarin to contain a 2,3-triazol-4-yl)ethyl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide adduct.

[0227] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles are characterized in that at least some of the copolymer chains have unused -N3 end groups, unreacted -CO2H end groups, and at least some N-(2-(1λ)) end groups that provide reactive alkene end groups capable of [2+2] photodimerization at wavelengths >270 nm. 2The poly(NDMAM-co-AzAPA-co-AAc-co-BisAM) copolymer chains were functionalized with alkyne coumarin to contain a 2,3-triazol-4-yl)ethyl)-2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)acrylamide adduct.

[0228] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise poly(CAA-co-NDMAM-co-AAc) copolymer chains, at least some of which, due to their dual functionality, contain -COH end groups and reactive alkene end groups capable of [2+2] photodimerization at wavelengths >270 nm.

[0229] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise poly(CAA-co-NiPAM-co-AAc-co-BisAM) copolymer chains, at least some of which, due to their dual functionality, contain -COH end groups and reactive alkene end groups capable of [2+2] photodimerization at wavelengths >270 nm.

[0230] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise poly(CAA-co-NDMAM-co-AzAPA) copolymer chains, at least some of which, for dual functionality, contain -N3 end groups and reactive alkene end groups capable of [2+2] photodimerization at wavelengths >270 nm.

[0231] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise poly(CAA-co-NDMAM-co-AzAPA-co-BisAM) copolymer chains, and in various embodiments, at least some of these copolymer chains contain, for dual functionality, -N3 end groups and reactive alkene end groups capable of [2+2] photodimerization at wavelengths >270 nm.

[0232] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise poly(CAA-co-NDMAM-co-AzAPA-co-AAc) copolymer chains, at least some of which, for dual functionality, comprise -N3 end groups, -CO2H end groups, and reactive alkene end groups capable of [2+2] photodimerization at wavelengths >270 nm.

[0233] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise poly(CAA-co-NDMAM-co-AzAPA-co-AAc-co-BisAM) copolymer chains, at least some of which, for dual functionality, comprise -N3 end groups, -CO2H end groups, and reactive alkene end groups capable of [2+2] photodimerization at wavelengths >270 nm.

[0234] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise poly(CAA-co-NiPAM-co-AzAPA-co-AAc) copolymer chains, at least some of which, for dual functionality, contain -N3 end groups, -CO2H end groups, and reactive alkene end groups capable of [2+2] photodimerization at wavelengths >270 nm.

[0235] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise poly(CAA-co-NiPAM-co-AzAPA-co-AAc-co-BisAM) copolymer chains, at least some of which, for dual functionality, comprise -N3 end groups, -CO2H end groups, and reactive alkene end groups capable of [2+2] photodimerization at wavelengths >270 nm.

[0236] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise poly(PAG-co-CAA-co-NiPAM-co-AAc) copolymer chains. In various embodiments, at least some of these copolymer chains, due to their dual functionality,

[0237]

number

[0238] In various embodiments, the photochemically reversible hydrogel polymers and nanogel particles comprise poly(PAG-co-CAA-co-NiPAM-co-AAc-co-BisAM) copolymer chains. In various embodiments, at least some of these copolymer chains, due to their dual functionality,

[0239]

number

[0240] Grafting amplification primers onto photochemically reversible hydrogel polymers and nanogel particles The above basic embodiment relates to a photochemically reversible hydrogel polymer and nanogel particle in which the copolymer chains contain at least some reactive alkene or 1,4-diene end groups capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm, and at least some

[0241]

number

[0242]

number

[0243] In various embodiments, grafting of amplification primers onto photochemically reversible hydrogel polymers and nanogel particles is achieved by click chemistry between a terminal alkyne substituent on the amplification primer and the -N3 terminal group of the respective copolymer chain, or by click chemistry between a terminal -N3 substituent on the amplification primer and the -N3 terminal group of the respective copolymer chain.

[0244]

number

[0245] In other embodiments, the thiol-functionalized primer has at least some of the copolymer chains

[0246]

number

[0247] In various examples, and as detailed above, the selection of monomers used in the synthesis of photochemically reversible hydrogel polymers or nanogel particles is determined so that the resulting copolymer chains contain -N or -N-alkene end groups, in addition to alkene or 1,4-diene end groups and optional -COH end groups.

[0248]

number

[0249] The P5 and P7 amplification primers for use herein are used on the surface of commercially available flow cells sold by Illumina Inc. for sequencing on the HiSeq™, MiSeq™, NextSeq™, and Genome Analyzer™ platforms. The P5 / P7 amplification primers for grafting are fully described in U.S. Pat. No. 9,982,250 and U.S. Patent Application Publication No. 2011 / 0059865, the disclosures of which are incorporated herein by reference in their entireties.

[0250] In various embodiments, 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.

[0251] In various embodiments, grafting of an alkyne-P5 / P7 primer onto a photochemically reversible hydrogel polymer or nanogel particle comprising copolymer chains with -N3 end groups involves CuAAC grafting, resulting in a P5 / P7-grafted photochemically reversible hydrogel polymer or nanogel particle.

[0252] In various embodiments, CUAAC-catalyzed click chemistry involving N3-P5 / P7 or thiol-P5 / P7 primers is carried out at a temperature of about 40° C. to about 80° C. for about 1 hour to about 5 hours.

[0253] In various embodiments,

[0254]

number

[0255] Trapping primer-grafted photochemically reversible nanogel particles for SBS In various embodiments, and as part of the SBS method, primer-grafted photochemically reversible nanogel particles are captured on the surface of a flow cell (FC), such as, for example, a HiSeq™ FC from Illumina, Inc. Primer-grafted photochemically reversible nanogel particles can be captured within nanowells patterned in a coating on the FC surface, or directly attached to a coating on a surface where no nanowells exist. In various embodiments, each primer-grafted nanogel particle can serve as a nanowell and thus function as a surrogate for a nanowell.

[0256] In various embodiments, the primer-grafted photochemically reversible nanogel particles are (a) a bioconjugation technique using DMTMM to activate the reaction between the free carboxylate end groups present on the copolymer chains of primer-grafted photochemically reversible nanogel particles and the available -NH2 groups on the pre-silanized FC surface to form amide bonds; or (b) Any residual copolymer chains still present on the primer-grafted photochemically reversible nanogel particles (i.e., after grafting).

[0257]

number

[0258] In various embodiments regarding (a) above, silanization of the FC surface can be accomplished using any suitable silane or silane derivative. The method used to bond the silane or silane derivative to the substrate can vary depending on the silane or silane derivative used.

[0259] In various embodiments, the silane or silane derivative is 3-mercaptopropylsilanetriol, 3-aminopropyltriethoxysilane (APTES), or 3-aminopropyltrimethoxysilane (APTMS) (i.e., the general structure XR B -Si(OR C ) 3, where X is amino and R B is -(CH2)3- and R C(wherein methyl is ethyl or methyl). In this example, the FC surface can be pretreated with APTES or APTMS to covalently bond silicon to one or more oxygen atoms on the surface. This chemically treated surface is optionally baked to form an amine-group monolayer.

[0260] In various embodiments, the -NH groups present on the FC surface are then reacted with carboxylate end groups present on the corresponding copolymer chains in the primer-grafted photochemically reversible nanogel particles. This procedure allows the -COH end groups to be used only to attach the nanogel particles to the FC surface, while the -N or

[0261]

number

[0262] In various examples related to (b) above, the PAZAM coating on the FC surface is prepared by using N-(5-(2-bromoacetamido)pentyl)acrylamide (BraPA) as a monomer for the polymeric hydrogel coating, followed by conversion of the bromine group to an -N3 group.

[0263] In various embodiments, PAZAM can be coated onto the surface of the patterned FC surface by spin coating, dipping, dip coating, or flowing PAZAM under positive or negative pressure, or another suitable technique. PAZAM can be present in a mixture. In one embodiment, the mixture includes PAZAM in water or a mixture of ethanol and water.

[0264] After coating, the functionalizing molecule can be subjected to a curing process to form a functionalized coating layer over the patterned substrate (i.e., in the recessed and interstitial areas). In one example, curing of the functionalizing molecule can be carried out at a temperature ranging from room temperature (e.g., about 25°C) to about 60°C for a time ranging from about 5 minutes to about 2 hours.

[0265] To form a PAZAM coating layer in the nanowells but not on the gap regions of the patterned substrate, the PAZAM coating layer can be polished away from the gap regions using either (a) a basic aqueous slurry having a pH in the range of about 7.5 to about 11 and containing abrasive particles, or (b) a polishing pad and a solution without abrasive particles.

[0266] To capture photochemically reversible nanogel particles on the PAZAM-coated FC surface, a PAZAM coating with reactive -N3 groups was applied under the conditions for CuAAC click chemistry to remove any remaining copolymer chains of the photochemically reversible nanogel particles.

[0267]

number

[0268] In an alternative embodiment, the order of the separate steps of primer grafting and particle capture can be reversed. Thus, dual-functional photochemically reversible nanogel particles can be captured on silanized or PAZAM-coated FC surfaces by amide formation or click chemistry, and the captured particles are then subsequently exposed to appropriately functionalized amplification primers (e.g., alkyne-P5 / P7 or N3-P5 / P7) to add the amplification primers to the captured nanogel particles.

[0269] In an alternative example, photochemically reversible nanogel particles can be reversibly attached to an appropriately functionalized FC surface by either [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 anchor multiple -SH groups to the FC surface. A compound bearing both acrylate functional groups and either a reactive alkene or reactive 1,4-diene moiety (e.g., N-(2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl)acrylamide) then reacts with the anchored -SH groups in a thiol-ene (1,4-addition) reaction, thereby converting the anchored -SH functional groups into multiple anchored reactive alkene or 1,4-diene groups. These immobilized reactive alkene or 1,4-diene groups are then available for [2 + 2] or [2 + 2 + 2 + 2] photodimerization with photochemically reversible nanogel particles bearing copolymer chains with reactive alkene or 1,4-diene end groups, respectively, upon exposure to incident radiation, preferably at wavelengths >270 nm. A key feature of this method of polymer attachment is that it is reversible, such as upon exposure to incident radiation at wavelengths <300 nm.

[0270] Seeding, clustering, and SBS sequencing In various embodiments, clustering includes either suspension clustering or on-board clustering. Suspension clustering avoids the need to pattern the coated FC surface, and on-board clustering can be used for proof-of-concept purposes because each photochemically reversible nanogel particle captured on the FC surface acts as a nanowell itself. In suspension clustering, seeded ssDNA can be clustered on the surface of photochemically reversible nanogel particles. Clustering on photochemically reversible nanogel particles relies on having sufficiently accessible primers grafted onto the photochemically reversible nanogel particles.

[0271] In various examples, the temperature-responsiveness of primer-grafted photochemically reversible nanogel particles having blocks of poly(NiPAM) in the copolymer chains was (a) Facilitating temperature-controlled contraction during seeding to reduce the probability of multiple seeding events and consequently enhance monoclonality; (b) promoting temperature-controlled swelling during clustering to increase primer accessibility and facilitate diffusion of materials into the photochemically reversible nanogel particles, resulting in an increased number of chains per cluster / particle and improved fluorescence thereof; and / or (c) Promoting temperature-controlled shrinkage or swelling to improve the SBS steps of incorporation and cleavage, thereby enabling temperature-controlled operations of seeding, amplification, and sequencing.

[0272] In various embodiments, the FC with captured primer-grafted photochemically reversible nanogel particles is then used in various sequencing approaches or techniques, such as SBS, cyclic array sequencing, sequencing-by-ligation, and pyrosequencing. With any of these techniques, the sequencing primers are present only on the photochemically reversible nanogel particles, so amplification is limited to each particle. Furthermore, because amplification is limited to the particle surface, there is more time to amplify a single sequencing template into larger clusters.

[0273] In various embodiments, SBS can be performed on a system such as the HISEQ™, HISEQX™, MISEQ™, NOVASEQ™, or NEXTSEQ™ sequencer system (Illumina, Inc.). In SBS, the extension of a nucleic acid primer (e.g., a sequencing primer) along a nucleic acid template (e.g., a sequencing template) is monitored to identify the sequence of nucleotides on the template. The underlying chemical process can be polymerization (e.g., catalyzed by a polymerase enzyme) or ligation (e.g., catalyzed by a ligase enzyme). In various polymerase-based SBS processes, fluorescently labeled nucleotides are added to the primer in a template-dependent manner to extend the primer, so that detection of the order and type of nucleotides added to the primer can be used to identify the sequence of the template. For example, to initiate the first SBS cycle, one or more labeled nucleotides, DNA polymerase, etc., can be delivered through a flow channel of an FC containing an array of primers on nanogel particles. When primer extension results in the incorporation of a labeled nucleotide, the primer-grafted nanogel particle can be detected by an imaging event during which an illumination system provides excitation light to the nanogel particle.

[0274] In various embodiments, the nucleotide can further comprise a reversible termination feature that stops further primer extension once the nucleotide is added to the primer. For example, a nucleotide analog with a reversible terminator moiety can be added to the primer such that further extension cannot occur until a deblocking agent is delivered to remove the moiety. Thus, in embodiments using reversible termination, a deblocking agent can be delivered to the flow channel before or after detection.

[0275] Flushing (i.e., washing) can be performed between the various fluid delivery steps. The SBS cycle is then repeated n times to extend the primer by n nucleotides, thereby allowing detection of a sequence of length n.

[0276] Photochemically reversible hydrogel polymer conjugation for SBS In various embodiments, two separate methods can be used to form a photochemically reversible hydrogel layer on the surface of a FC used for SBS sequencing. (1) providing a mixture of a monomer and a crosslinker compound on the FC surface and irradiating the mixture of the monomer and the crosslinker compound with a wavelength of >270 nm to form a hydrogel, crosslinking at least some of the copolymer chains and effectively curing the hydrogel on the FC surface; or (2) A photochemically reversible hydrogel polymer according to the present disclosure is provided to an appropriately functionalized FC and irradiated onto the FC surface, causing the hydrogel polymer to bind to the functionalized FC surface via [2+2] or [2+2+2+2] photodimerization.

[0277] In method (1), referred to as "in-FC crosslinking," the monomer mixture is in accordance with the present disclosure and is therefore a mixture of at least a first type of monomer and at least a second type of monomer described herein. The crosslinker compound added to the monomer mixture can be a multifunctional monomer, such as N,N'-methylenebisacrylamide, N,N'-methylenebismethacrylamide, polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, N-vinyl acrylamide, glycidyl acrylate, divinyl benzene, or tetraallyl ammonium chloride, or other known materials used in crosslinking.

[0278] In method (2), the FC surface is functionalized by silanization with 3-mercaptopropylsilanetriol, 3-mercaptopropyltrimethoxysilane, or 3-mercaptopropyltriethoxysilane to anchor multiple -SH groups to the FC surface. A compound bearing both acrylate functional groups and either a reactive alkene or reactive 1,4-diene moiety (e.g., N-(2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl)acrylamide) then reacts with the anchored -SH groups in a thiol-ene (1,4-addition) reaction, thereby converting the anchored -SH functional groups into multiple anchored reactive alkene or 1,4-diene groups. These immobilized reactive alkene or 1,4-diene groups are then available for [2 + 2] or [2 + 2 + 2 + 2] photodimerization with photochemically reversible hydrogels bearing copolymer chains with reactive alkene or 1,4-diene end groups, respectively. Preferably, the incident radiation used to attach the hydrogel to the functionalized surface has a wavelength >270 nm.

[0279] Photochemically reversible release of hydrogel polymers and nanogel particles for reuse of FC previously used for SBS In various embodiments, both photochemically reversible hydrogels and photochemically reversible nanogel particles can be removed from FC surfaces. Previously, removal of hydrogels 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 are easily removed from FC surfaces without the use of harsh chemicals, simply by irradiating the surface with radiation having a wavelength of <300 nm. The irradiation cleaves the [2 + 2] or [2 + 2 + 2 + 2] dimers, liberating the photochemically reversible hydrogels or photochemically reversible nanogel particles from the FC surface. Further cleaning of the FC surface, such as to remove immobilized silane groups, requires only mild conditions, such as rinsing with a mild acid.

[0280] In various embodiments, the sequencing / reuse workflow can be described as follows: (1) The FC surface can be functionalized with photocrosslinking motifs. (2) Photochemically reversible hydrogel or nanogel particles containing complementary photocrosslinking motifs can be washed through FC. The hydrogel or nanogel particles have amplification primers grafted onto them. Alternatively, in-suspension clustered nanogel particles containing photocrosslinking motifs can be used. (3) The FC is irradiated with light of wavelength λ1, which causes photocrosslinking of hydrogel or nanogel particles to the FC surface. (4) After capture on the FC surface, clustering and sequencing are performed. (5) Upon completion of all nucleic acid sequencing steps, the FC is subjected to a second irradiation comprising a second wavelength λ2, which causes photocleavage of the cross-linked motifs. (6) Upon completion of photocleavage, the FC is washed with a washing solution to remove the sequenced material. (7) The FC can then be reused.

[0281] Long-wavelength photochemically switchable chemicals In various embodiments, photochemically reversible hydrogel polymers and polymer nanogel particles may employ long wavelength (650-1100 nm) photochemically switchable chemistries rather than short wavelength photochemically switchable chemistries.

[0282] In various embodiments, photochemically reversible hydrogel polymers and polymer nanogel particles can include copolymer chains having azobenzene moieties capable of photoisomerization between E and Z isomers according to the following scheme:

[0283] [ka]

[0284] In various embodiments, photoisomerization between the E and Z isomers can be used to shift copolymer chains away from the surface, for example, by electrostatic changes that affect monolayer bonding. In various embodiments, the R" substituent on the azobenzene moiety can include an oleyl amide or ester, where the oleyl chain provides van der Waals interactions that support the self-assembled monolayer (SAM). The substituent R" can be the remainder of a copolymer chain of a hydrogel polymer or polymeric nanogel particle. In various embodiments, R" and R" can be attached to the same or different polymer chains.

[0285] In various embodiments, photochemically reversible hydrogel polymers and polymer nanogel particles can include copolymer chains having spiropyran moieties capable of photoisomerization between a ring-open merocyanine isomer and a ring-closed spiropyran isomer according to the following scheme:

[0286] [ka]

[0287] In various embodiments, photochromism in the above schemes can be used to attach / detach hydrogel polymers or polymeric nanogel particles to / from surfaces, for example, by altering the electrostatic interaction between the polymer and the surface. In various embodiments, at least one of Ra, Rb, or Rc can be the remaining portion of a copolymer chain of the hydrogel polymer or polymeric nanogel particle. In alternative embodiments, one portion of the merocyanine can be present in one set of copolymer chains and another portion can be present in a second set of copolymer chains, such that the photochromism that cyclizes the merocyanine connects the copolymer chains together or connects the copolymer chains to the functionalized surface of the substrate.

[0288] In various embodiments, photochemically reversible hydrogel polymers and polymer nanogel particles can comprise copolymer chains with conjugated bis-thiophene ethylene moieties capable of reversible 2+2+2 cycloaddition according to the following scheme:

[0289] [ka]

[0290] In various embodiments, the reversible photocycloaddition in the above scheme can be used to attach / detach hydrogel polymers or polymeric nanogel particles to / from a surface, for example, by changing the electrostatic interaction between the polymer and the surface. In various embodiments, at least one of R, R, or R can be a remaining portion of a copolymer chain of a hydrogel polymer or polymeric nanogel particle. In alternative embodiments, one thiophene moiety can be present in one set of copolymer chains and another thiophene can be present in a second set of copolymer chains, such that photocycloaddition connects the copolymer chains together or connects the copolymer chains to a functionalized surface of a substrate.

[0291] In various embodiments, photochemically reversible hydrogel polymers and polymer nanogel particles can include copolymer chains having hemithioindigo moieties capable of photoisomerization between E and Z isomers according to the following scheme:

[0292] [ka]

[0293] In various embodiments, photoisomerization between the E and Z isomers can be used to shift copolymer chains away from the surface, for example, by electrostatic changes that affect monolayer bonding. In various embodiments, the Ra, Rb, and Rc substituents on the hemithioindigo moiety can include oleoyl amides or esters, in which case the oleoyl chains provide van der Waals interactions that support the self-assembled monolayer (SAM). The Ra, Rb, and Rc substituents can be the remainder of copolymer chains in hydrogel polymers or polymeric nanogel particles. Alternatively, two of Ra, Rb, and Rc can be cyclized as part of the same or different polymer chains.

[0294] In various embodiments, photochemically reversible hydrogel polymers and polymer nanogel particles can include copolymer chains having donor-acceptor Stenhouse adducts capable of reversible cycloaddition according to the following scheme:

[0295] [ka]

[0296] In various embodiments, reversible photocycloaddition in the above scheme can be used to attach / detach hydrogel polymers or polymeric nanogel particles to / from surfaces, for example, by altering steric or electrostatic interactions between the polymer and the surface. In various embodiments, at least one of Ra, Rb, or Rc can be a remaining portion of a copolymer chain of the hydrogel polymer or polymeric nanogel particle.

[0297] In various examples, photochemically reversible hydrogel polymers and polymer nanogel particles can include copolymer chains having aryl-substituted bisimidazoles capable of photochromism toward imidazole radical species according to the following scheme:

[0298] [ka]

[0299] In various embodiments, the reversible reactions in the above schemes can be used to attach / detach hydrogel polymers or polymeric nanogel particles to / from surfaces or to crosslink copolymer chains. In various embodiments, aryl substituents can be attached to copolymer chains of hydrogel polymers or polymeric nanogel particles.

[0300] To further illustrate the present disclosure, the following examples are provided, which are provided for illustrative purposes and should not be construed as limiting the scope of the disclosure in any way. [Example]

[0301] Referring now to Figure 1, a photochemically reversible hydrogel is reversibly attached to an appropriately functionalized surface of a flow cell used for nucleic acid sequencing and then removed after sequencing is completed. In this way, the photochemically reversible hydrogel can make the FC reusable.

[0302] The top portion of Figure 1 shows that upon stimulus 1 (hν1), photochemically reversible hydrogels according to various embodiments of the present disclosure bind to an appropriately functionalized FC surface via multiple photoaddition reactions, e.g., [2+2] photodimerization. The FC surface contains multiple immobilized reactive alkene or 1,4-diene groups such that binding of the hydrogel to the surface is achieved by photoaddition. Preferably, the radiation (hν1) used to bind the hydrogel to the functionalized surface is radiation with a wavelength >270 nm. The exact wavelength used for hydrogel binding depends on whether an alkene or 1,4-diene is used and the electron density of the alkene or 1,4-diene functional group, e.g., whether a specific electron-withdrawing or electron-donating group is attached to the alkene or 1,4-diene moiety. In certain embodiments, both 4-methylcoumarin substituents on the hydrogel copolymer chain and 4-methylcoumarin substituents immobilized on the FC surface participate in the photodimerization that binds the hydrogel to the FC surface.

[0303] The top part of Figure 1 shows the removal of the hydrogel from the FC surface, resulting in the reuse of the FC. As shown in the figure, stimulus 2 (hν2) causes cleavage of the dimer or [2 + 2 + 2 + 2] adduct, allowing the detached hydrogel to be easily washed away under mild conditions.

[0304] The lower part of Figure 1 illustrates two methods for achieving reversible attachment of a hydrogel to a FC surface. Method A) is as described above: a preformed hydrogel polymer is applied to the functionalized FC surface and irradiated to dimerize the alkene moieties or form a [2 + 2 + 2 + 2] cycloadduct, thereby attaching the hydrogel to the functionalized FC surface. In Method B), a monomer mixture is applied to the FC surface, and polymerization is initiated at the surface. This is referred to as "in-FC crosslinking." This example demonstrates the use of at least a monomer M1 (e.g., AzAPA) with an azide functionality and at least a monomer M2 (e.g., CAA) with a reactive alkene or 1,4-diene functionality. A crosslinking compound, such as BisAM, is also added to the mixture prior to irradiation. In this way, in situ polymerization forms a hydrogel polymer on the FC surface.

[0305] Figure 2 provides an example of patterned reversible nanogel particles on FC, referred to as the "RAP approach." "R" represents reversible and responsive chemistry, such as that provided by the presence of reactive alkene or reactive 1,4-diene end groups on the copolymer chains of the nanogel particles. "A" represents an alternative polymer, either synthetic or naturally derived. "P" represents -N3 or -N4, which can participate in click chemistry grafting of appropriately functionalized amplification primers.

[0306]

number

[0307] In the example shown in Figure 3, photochemically reversible hydrogel or nanogel particles with 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 demonstrates the photodimerization of the 4-methylcoumarin end groups of the copolymer chains of the hydrogel or nanogel particles.

[0308] Figure 4 shows a synthetic route to photochemically reversible hydrogel or nanogel particles. In Method A, PAZAM or other polymers containing copolymer chains with -N3 end groups are reacted with alkyne coumarin under copper catalysis to form photochemically reversible hydrogel or nanogel particles with anchored coumarin substituents. In Method A, the photochemically reversible hydrogel or nanogel particles ultimately, but not initially, have reactive alkene end groups on the copolymer chains after the reaction, linking the coumarin tether to the hydrogel or nanogel particle via a triazine linker.

[0309] In exemplary Method B shown in Figure 4, photochemically reversible hydrogel 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 carried out at 70 °C under an inert atmosphere. The resulting photochemically reversible hydrogel or nanogel particles contain poly(CAA-co-NDMAM-co-AzAPA) copolymer chains and, as a result, possess dual functionality due to the presence of both reactive alkene and -N3 end groups on at least some of the copolymer chains. In the absence of -CO2H end groups (no AAc was used for this copolymer), the attachment of the photochemically reversible hydrogel or nanogel particles to FC is via photodimerization.

[0310] In the example shown in Figure 5A, photochemically reversible nanogel particles are prepared by reacting nanogel particles containing -N3 terminal groups on at least some of the copolymer chains with an alkyne coumarin. In this way, reactive coumarin groups are immobilized on preformed nanogel particles to create photochemically reversible nanogel particles. While exemplified by nanogel particles, the same process can be used to immobilize coumarin groups on hydrogel polymers containing -N3 terminal groups on at least some of their copolymer chains.

[0311] In the example shown in Figure 5B, photochemically reversible nanogel particles are prepared directly 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 the free radical initiator, and sodium dodecyl sulfate (SDS) is used as the dispersing agent. The resulting photochemically reversible nanogel particles shown comprise poly(CAA-co-NiPAM-co-AzAPA-co-AAc-co-BisAM) copolymer chains, where at least some of the copolymer chains have coumarin, -N3, and -CO2H end groups.

[0312] In the example shown in Figure 6, N-(4-methyl-2-oxo-2H-chromen-7-yl)acrylamide (7-(acrylamido)-4-methylcoumarin) is prepared by reacting 7-amino-4-methylcoumarin with acryloyl chloride in dichloromethane (DCM) at 0°C to ambient temperature. Although not exemplified, 4-methyl-2-oxo-2H-chromen-7-yl acrylate can be similarly prepared starting from 7-hydroxy-4-methylcoumarin.

[0313] In the example shown in Figure 7, a FC surface is functionalized by sequential treatment with 3-mercaptopropylmethoxysilane and N-(2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl)acrylamide. The resulting FC surface is characterized as having immobilized coumarin groups (or more precisely, immobilized 4-methylcoumarin groups). Photochemically reversible hydrogel or nanogel particles are then provided to this functionalized surface in solution, followed by irradiation of the surface to attach the hydrogel or nanogel particles to the functionalized surface via photodimerization of the coumarin groups.

[0314] In the example shown in Figure 8, P5 / P7 amplification primers are functionalized at the 5' end with a photoreversible motif, such as a reactive alkene or a reactive 1,4-diene-containing substituent. This example replaces grafting of the functionalized P5 / P7 amplification primer to hydrogel or nanogel particles via alkyne-N3 or N3-alkyne click chemistry and is photochemically reversible. As shown in b), the PAZAM hydrogel on the FC surface can also contain a photoreversible motif, resulting in the functionalized P5 / P7 amplification primer being reversibly attached to the hydrogel layer by photodimerization. After the nucleic acid sequencing experiment is completed, irradiation at <300 nm cleaves the primer, and simple washing restores the hydrogel-coated surface.

[0315] In the detailed description, references to "various embodiments," "one embodiment," "one embodiment," etc., indicate that the described embodiment may include a particular feature, structure, or characteristic, but not all embodiments necessarily include that particular feature, structure, or characteristic. Moreover, such phrases do not necessarily refer to the same embodiment. Furthermore, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is believed to be within the knowledge of one skilled in the art to affect such feature, structure, or characteristic in connection with other embodiments, whether or not explicitly described. After reading the description, it will become apparent to one skilled in the art how to implement the present disclosure in alternative embodiments.

[0316] Benefits, other advantages, and solutions to problems have been described herein with reference to specific embodiments. However, the benefits, advantages, solutions to problems, and any elements that may cause or enhance any benefit, advantage, or solution should not be construed as key, necessary, or essential features or elements of the present disclosure. Accordingly, the scope of the present disclosure is not limited by anything other than the appended claims, and references to elements in the singular do not mean "one and only one," unless explicitly stated, but rather "one or more." Furthermore, when phrases like "at least one of A, B, and C" or "at least one of A, B, or C" are used in the claims or specification, this phrase is intended to mean that only A may be present in an embodiment, only B may be present in an embodiment, only C may be present in an embodiment, or that any combination of elements A, B, and C, e.g., A and B, A and C, B and C, or A and B and C, may be present in a single embodiment.

[0317] All structural, chemical, and functional equivalents to the elements of the various embodiments described above that are known to those of skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the present claims. Moreover, it is not necessary for an apparatus or apparatus component, or a method of using an apparatus, to address each and every problem sought to be solved by the present disclosure, for it to be encompassed by the present claims. Furthermore, no element, component, or method step in the present disclosure is intended to be made available to the public, regardless of whether that element, component, or method step is expressly recited in a claim. No claim element is intended to invoke 35 U.S.C. § 112(f) unless the element is expressly recited using the phrase "means for." As used herein, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a chemical, chemical composition, process, method, article, or apparatus that includes a list of elements does not include only those elements, but may include other elements that are not expressly listed or that are inherent to such chemical, chemical composition, process, method, article, or apparatus.

Claims

1. 1. A hydrogel polymer comprising copolymer chains, the copolymer chains comprising: Formula (I): 【number】 A first repeat unit of the formula: R １ , R １’ , and R １” each is independently selected from H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl; X is —O— or —NH—, and R ２ teeth, [Equation 1] or R ２ Here is the structure: 【number】 and In the formula, R ２’ is -NH ２ , alkyl, alkoxy, alkenyl, alkynyl, or optionally substituted versions thereof, or halogen, —N ３ , -OH, -C(O)H, -NH=NH ２ , -SCN, -CO ２ H, —SH, glycidyl, epoxy, aziridine, triazoline, or [Equation 2] and a first repeat unit, wherein p is an integer from 1 to 50; Formula (II) 【number】 A second repeat unit of the formula: R ３ , R ３’ , R ４ , and R ４’ each independently represents —H, —R ５ , -OR ５ , -CO ２ R ５ , -C(O)R ５ , -OC(O)R ５ , —C(O)NR ６ R ７ , -NR ６ R ７ or formula (III) 【number】 is selected from the substructures of R ５ is —H, —OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl; R ６ and R ７ each is independently selected from —H and alkyl; A is an aryl or thymidinyl moiety; R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl, or optionally substituted versions thereof, or halogen, -N ３ , -OH, -C(O)H, -NH=NH ２ , -SCN, -CO ２ H, —SH, glycidyl, epoxy, or two R′ groups when the two R′ groups are attached to adjacent atoms on ring A and together with ring A form a coumarinyl, anthracenyl, acenaphthylenyl, thianaphthenyl-1-oxide, or thianaphthenyl-1,1-dioxide moiety; X １ is a bond, -(CH ２ ) ｑ -, -O-, or -NH-; L is a group having the structure -(CH ２ ) ｑ -X ２ -C(=O)- or -(CH ２ CH ２ O) ｑ -X ２ a bivalent linker having -C(=O)-; X ２ is —O— or —NH—, m is an integer from 1 to 9, q is an integer from 0 to 50; and further comprising A hydrogel polymer, wherein at least some of said copolymer chains contain at least one reactive alkene or reactive 1,4-diene end group capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm.

2. At least some of the copolymer chains have at least one N ３ , [Equation 3] or -CO ２ 10. The hydrogel polymer of claim 1, comprising H end groups.

3. Formula (III) may be converted to formula (IV): 【Chemistry 5】 including the subgeneric structure of In the formula, R ８ and R ９ each is —H, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl, and each R １０ and R １１ is independently —H, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl, or R １０ is —C(═O)—, and R １１ is -O, and R １０ and R １１ are linked together such that formula (IV) comprises a substituted coumarin moiety.

4. The repeating unit of formula (I) is 【Chemistry 6】 The hydrogel polymer of any one of claims 1 to 3, wherein

5. The repeating unit of formula (II) 【Chemistry 7】 The hydrogel polymer of any one of claims 1 to 4, wherein

6. The repeating unit of formula (II) 【Chemistry 8】 and wherein R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl, or optionally substituted versions thereof, or halogen, -N ３ , -OH, -C(O)H, -NH=NH ２ , -SCN, -CO ２ H, —SH, glycidyl, epoxy, The hydrogel polymer of any one of claims 1 to 4, wherein m is an integer from 1 to 9.

7. The repeating unit of formula (II) 【Chemistry 9】 and 5. The hydrogel polymer of claim 1, wherein q is an integer from 0 to 50.

8. The repeating unit of formula (II) 【Chemistry 10】 and wherein R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl, or optionally substituted versions thereof, or halogen, -N ３ , -OH, -C(O)H, -NH=NH ２ , -SCN, -CO ２ H, —SH, glycidyl, epoxy, The hydrogel polymer of any one of claims 1 to 4, wherein m is an integer from 1 to 7.

9. The repeating unit of formula (II) 【Chemistry 11】 and wherein R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl, or optionally substituted versions thereof, or halogen, -N ３ , -OH, -C(O)H, -NH=NH ２ , -SCN, -CO ２ H, —SH, glycidyl, epoxy, The hydrogel polymer of any one of claims 1 to 4, wherein m is an integer from 1 to 5.

10. The repeating unit of formula (II) 【Chemistry 12】 The hydrogel polymer of any one of claims 1 to 4, wherein

11. R ９ or R １０ Either of these is -CO ２ 4. The hydrogel polymer of claim 3, wherein formula (IV) is H, whereby formula (IV) is a cis- or trans-cinnamic acid moiety.

12. R ９ or R １０ The hydrogel polymer of claim 3, wherein is aryl, whereby formula (IV) is a cis- or trans-stilbene moiety.

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

14. 10. The hydrogel polymer of claim 1, 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. 10. The hydrogel polymer of claim 1, derived from a monomer mixture comprising 7-((2-acrylamido)ethoxy)-4-methylcoumarin, N-isopropylacrylamide, N-(5-(2-azidoacetamido)pentyl)acrylamide, and acrylic acid.

16. 10. The hydrogel polymer of claim 1, derived from a monomer mixture comprising 7-((2-acrylamido)ethoxy)-4-methylcoumarin, N,N-dimethylacrylamide, N-(5-(2-azidoacetamido)pentyl)acrylamide, and acrylic acid.

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

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

19. 10. The hydrogel polymer of claim 1, derived from a monomer mixture comprising 7-(methacrylamido)-4-methylcoumarin, N-isopropylacrylamide, N-(5-(2-azidoacetamido)pentyl)acrylamide, and acrylic acid.

20. 10. The hydrogel polymer of claim 1, derived from a monomer mixture comprising 7-(methacrylamido)-4-methylcoumarin, N,N-dimethylacrylamide, N-(5-(2-azidoacetamido)pentyl)acrylamide, and acrylic acid.

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

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

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

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

25. 25. The hydrogel polymer of 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, divinyl benzene, tetraallyl ammonium 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. 28. The hydrogel polymer of claim 27, 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 group at the end of each copolymer chain, or click chemistry between a terminal azide substituent on the amplification primer and an alkyne group at the end of each copolymer chain.

29. 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 alkene or reactive 1,4-diene end groups capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm.

30. 1. A substrate having a surface comprising a hydrogel polymer covalently bonded to the surface, wherein the hydrogel polymer comprises a plurality of copolymer chains, the plurality of copolymer chains comprising a repeating unit of formula (I) and a repeating unit of formula (II): 【number】 further comprising During the ceremony, R １ , R １’ , and R １” each is independently selected from H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl; X is —O— or —NH—; R ２ teeth, [Equation 4] or R ２ Here is the structure: 【number】 and R ２’ is -NH ２ , alkyl, alkoxy, alkenyl, alkynyl, or optionally substituted versions thereof, or halogen, —N ３ , -OH, -C(O)H, -NH=NH ２ , -SCN, -CO ２ H, —SH, glycidyl, epoxy, aziridine, triazoline, or [Equation 5] and p is an integer from 1 to 50, R ３ , R ３’ , R ４ , and R ４’ each independently represents —H, —R ５ , -OR ５ , -CO ２ R ５ , -C(O)R ５ , -OC(O)R ５ , —C(O)NR ６ R ７ , -NR ６ R ７ or formula (III) 【number】 is selected from the substructures of R ５ is —H, —OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl; R ６ and R ７ each is independently selected from —H and alkyl; A is an aryl or thymidinyl moiety; R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl, or optionally substituted versions thereof, or halogen, -N ３ , -OH, -C(O)H, -NH=NH ２ , -SCN, -CO ２ H, —SH, glycidyl, epoxy, or two R′ groups when the two R′ groups are attached to adjacent atoms on ring A and together with ring A form a coumarinyl, anthracenyl, acenaphthylenyl, thianaphthenyl-1-oxide, or thianaphthenyl-1,1-dioxide moiety; X １ is a bond, -(CH ２ ) ｑ -, -O-, or -NH-; L is a group having the structure -(CH ２ ) ｑ -X ２ -C(=O)- or -(CH ２ CH ２ O) ｑ -X ２ a bivalent linker having -C(=O)-; X ２ is —O— or —NH—, m is an integer from 1 to 9, q is an integer from 0 to 50, At least some of the copolymer chains have at least one -N ３ , [Equation 6] or -CO ２ containing an H terminal group, A hydrogel polymer, wherein at least some of said copolymer chains contain at least one reactive alkene or reactive 1,4-diene end group capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm.

31. 31. The substrate of claim 30, wherein the covalent bond between the substrate and the hydrogel polymer comprises a photodimerization bond between the reactive alkene 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 alkene or reactive 1,4-diene group disposed on the substrate surface.

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

33. The 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. 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 end group of the respective copolymer chain, or click chemistry between a terminal azide substituent on the amplification primer and an alkyne end group of the respective copolymer chain.

36. 36. The substrate of any one of claims 30 to 35, wherein at least some of the copolymer chains of the hydrogel polymer are crosslinked by photodimerization between the reactive alkene 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. 1. A method of synthesizing a hydrogel polymer having crosslinked copolymer chains, the method comprising: (1) (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-methacrylamido)aminoethyl)-4-methylcoumarin, or 7-((2-methacrylamido)aminoethyl)-4-methylcoumarin reacting an aqueous dispersion of a monomer mixture comprising (a) N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), (b) N,N-dimethylacrylamide or N-isopropylacrylamide (NiPAM), and (d) acrylic acid (AAc) under conditions suitable for free radical polymerization to form a hydrogel polymer having non-crosslinked copolymer chains, at least some of the copolymer chains containing reactive coumarin end groups derived from monomer (a); (2) cross-linking at least some of the copolymer chains by irradiating the hydrogel polymer with light having a wavelength >270 nm, wherein the cross-links comprise dimers between the reactive coumarin end groups of the copolymer chains; A method comprising:

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

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

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

42. 1. A method of assembling a flow cell capable of sequencing nucleic acids, said method comprising: (a) treating the surface of the flow cell with one of 3-mercaptopropylsilanetriol, 3-mercaptopropyltrimethoxysilane, or 3-mercaptopropyltriethoxysilane to form the surface having a plurality of reactive -SH groups immobilized thereon; (b) reacting the plurality of -SH groups with the α,β-unsaturated carbonyl thiol-ene acceptor, which further comprises a reactive alkene or reactive 1,4-diene moiety capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm, covalently attached to the α,β-unsaturated carbonyl thiol-ene acceptor to provide reactive alkene or reactive 1,4-diene groups on the surface; (c) The hydrogel polymer comprises a repeating unit of formula (I) and a repeating unit of formula (II) 【number】 preparing a hydrogel polymer comprising copolymer chains further comprising: During the ceremony, R １ is H, alkyl, alkoxy, alkenyl, alkynyl, or optionally substituted versions thereof; R ２ is -NH ２ , alkyl, alkoxy, alkenyl, alkynyl, or optionally substituted versions thereof, or halogen, —N ３ , -OH, -C(O)H, -NH=NH ２ , -SCN, -CO ２ H, —SH, glycidyl, epoxy, aziridine, triazoline, p is an integer from 1 to 50, R ３ , R ３’ , R ４ , and R ４’ each independently represents —H, —R ５ , -OR ５ , -CO ２ R ５ , -C(O)R ５ , -OC(O)R ５ , —C(O)NR ６ R ７ , -NR ６ R ７ or formula (III) 【number】 is selected from the substructures of R ５ is —H, —OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl; R ６ and R ７ each is independently selected from —H and alkyl; A is an aryl or thymidinyl moiety; R' is -H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl, or optionally substituted versions thereof, or halogen, -N ３ , -OH, -C(O)H, -NH=NH ２ , -SCN, -CO ２ H, —SH, glycidyl, epoxy, or two R′ groups when the two R′ groups are attached to adjacent atoms on ring A and together with ring A form a coumarinyl, anthracenyl, acenaphthylenyl, thianaphthenyl-1-oxide, or thianaphthenyl-1,1-dioxide moiety; X １ is a bond, -(CH ２ ) ｑ -, -O-, or -NH-; L is a group having the structure -(CH ２ ) ｑ -X ２ -C(=O)- or -(CH ２ CH ２ O) ｑ -X ２ a bivalent linker having -C(=O)-; X ２ is —O— or —NH—, m is an integer from 1 to 9, q is an integer from 0 to 50; at least some of the copolymer chains contain at least one reactive alkene or reactive 1,4-diene end group capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm; At least some of the copolymer chains have at least one -N ３ or [Equation 7] including a terminal group; (d) attaching the hydrogel polymer onto 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 having a wavelength >270 nm to form a [2+2] or [2+2+2+2] photodimer between the reactive alkene or 1,4-diene groups on the surface and the reactive alkene or 1,4-diene end groups on the respective copolymer chains. A method comprising:

43. 43. The method of claim 42, wherein the irradiating step in (d) also crosslinks copolymer chains of the hydrogel polymer by [2+2] or [2+2+2+2] photoaddition of reactive alkene or reactive 1,4-diene end groups present on the respective copolymer chains.

44. 44. The method of claim 42 or 43, further comprising grafting an amplification primer onto the hydrogel polymer either 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 of each copolymer chain, or a click chemistry reaction between a terminal azide substituent on the amplification primer and an alkyne end group of each copolymer chain.

45. The hydrogel polymer is (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-methacrylamido)aminoethyl)-4-methylcoumarin, or 7-((2-methacrylamido)aminoethyl)-4-methylcoumarin.

45. The method of any one of claims 42 to 44, wherein the reactive alkene end groups capable of [2+2] photodimerization at wavelengths >270 nm comprise coumarin groups, prepared by free radical polymerization of a monomer mixture comprising (a) N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), (b) N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), (c) N,N-dimethylacrylamide or N-isopropylacrylamide (NiPAM), and (d) acrylic acid (AAc), thereby forming copolymer chains.

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

47. 1. A method of synthesizing a hydrogel polymer, said 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 with reactive azide end groups; reacting at least a portion of the azide end groups 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 with anchored 4-methylcoumarin end groups; A method comprising:

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

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

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

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