Dual-functional and temperature-responsive nanogel particles for particle clustering in nucleic acid sequencing systems

Abstract

In some examples, novel nanogel particles with dual functionality, temperature responsiveness, and pH responsiveness are described. For nucleic acid sequencing, amplification primers are grafted to nanogel particles to form primer-grafted nanogel particles, which are captured on a surface within a flow cell. In flow cells used, for example, in SBS nucleic acid sequencing, each primer-grafted nanogel particle functions as a nanowell in the flow cell, thus, in some examples, eliminating the need for a nanowell.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 407,852, filed September 19, 2022, entitled "NANOGEL PARTICLES HAVING DUAL FUNCTIONALITY AND TEMPERATURE RESPONSIVENESS FOR PARTICLE CLUSTERING IN NUCLEIC ACID SEQUENCING SYSTEMS," the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates generally to nucleic acid sequencing methods and devices, and more particularly to functionalized 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 rapid 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 by improving various devices, such as flow cells used in sequencing by synthesis (SBS). Summary of the Invention

[0004] Provided herein are nanogel particles with dual functionality and temperature and / or pH responsiveness for particle clustering in nucleic acid sequencing systems.

[0005] For example, as provided herein, certain polymeric nanogel particles can be used to replace hydrogel coatings in flow cells for sequencing by synthesis (SBS). The nanogel particles can additionally or alternatively be used to improve many aspects of the SBS method.

[0006] In various examples provided herein, 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.

[0007] 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.

[0008] In various embodiments, the nanogel particles disclosed herein exhibit dual functionality due to the presence of at least two types of reactive end groups on the copolymer chains within the nanogel particle. In various embodiments, the nanogel particles disclosed herein exhibit temperature responsiveness, in which the nanogel particle can shrink or swell in response to a change in temperature, and pH responsiveness, in which the nanogel particle can include at least some copolymer chains with carboxylic acid end groups that are more nonionic in nature in certain pH ranges and more anionic in nature in other pH ranges.

[0009] 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.

[0010] In various embodiments of the present disclosure, polymeric nanogel particles are described. The polymeric nanogel particles are represented by formula (I):

[0011] [ka] A first repeat unit of the formula: 1 , R 1’ , and R 1” is independently selected from H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl; X is —O— or —NH—; and R 2 teeth,

[0012]

number

[0013] [ka] wherein R 2’ is -N3 or

[0014]

number

[0015] [ka] A second repeat unit of the formula: 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 , and -NR 6 R 7 wherein R 5 is -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl; R 6 and R 7 each of which further comprises a copolymer chain comprising a second repeat unit independently selected from -H and alkyl, wherein at least some of the copolymer chains comprise at least one carboxylic acid end group, and at least some of the copolymer chains comprise at least one -N3 or

[0016]

number

[0017] In various embodiments, R 1 , R 1’ , and R 1” are H respectively.

[0018] In various embodiments, R 1 and R 1” is H and R 1’ is CH3.

[0019] In various embodiments, the first repeat unit of formula (I) is

[0020] [ka]

[0021] In various embodiments, formula (I) is

[0022] [ka]

[0023] In various embodiments, the second repeat unit of formula (II) is

[0024] [ka] At least one of the following is true:

[0025] In various embodiments, the polymeric nanogel particles are derived from a monomer mixture including N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), N-isopropylacrylamide (NiPAM), acrylic acid (AAc), and N,N'-methylenebisacrylamide (BisAM).

[0026] In various embodiments, the nanogel particles comprise poly(AzAPA-co-NiPAM-co-AAc-co-BisAM) copolymer chains.

[0027] In various embodiments, the polymeric nanogel particles are derived from a monomer mixture including propargyl acrylate (PAG) and / or N-propargyl acrylamide (PAM), N-isopropyl acrylamide (NiPAM), acrylic acid (AAc), and N,N'-methylenebisacrylamide (BisAM).

[0028] In various embodiments, the polymeric nanogel particles comprise poly(PAG-co-NiPAM-co-AAc-co-BisAM) and / or poly(PAM-co-NiPAM-co-AAc-co-BisAM) copolymer chains.

[0029] In various embodiments of the present disclosure, a substrate has a surface comprising a plurality of pH and temperature responsive organic polymer nanogel particles covalently attached to the surface, wherein the organic polymer nanogel particles have a structure represented by Formula (I):

[0030] [ka] A first repeat unit of the formula: 1 , R 1’ , and R 1” is independently selected from H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl; X is —O— or —NH—; and R 2 teeth,

[0031]

number

[0032] [ka] wherein R 2’ is -N3 or

[0033]

number

[0034] [ka] A second repeat unit of the formula: 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 , and -NR 6 R 7 wherein R 5 is -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl; R 6 and R 7 each of which further comprises a second repeat unit independently selected from -H and alkyl, and a plurality of copolymer chains, at least some of which comprise at least one carboxylic acid end group, and at least some of which comprise at least one -N3 or

[0035]

number

[0036] In various embodiments, organic polymeric nanogel particles are formed from a monomer mixture including (a) propargyl acrylate (PAG) and / or N-propargyl acrylamide (PAM), (b) N-isopropyl acrylamide (NiPAM), and (c) acrylic acid (AAc).

[0037] In various embodiments, the organic polymeric nanogel particles have an average size of about 265 nm to about 280 nm.

[0038] In various embodiments, organic polymeric nanogel particles are formed from a monomer mixture including N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), N-isopropylacrylamide (NiPAM), and acrylic acid (AAc).

[0039] In various embodiments, the organic polymeric nanogel particles have an average size of about 225 nm to about 250 nm.

[0040] In various embodiments, the monomer mixture used in the synthesis of the organic polymeric nanogel particles further comprises a multifunctional compound selected from the group consisting of N,N'-methylenebisacrylamide, N,N'-methylenebismethacrylamide, polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, N-vinyl acrylamide, glycidyl acrylate, divinylbenzene, tetraallyl ammonium chloride, diallyl dimethyl ammonium chloride, and mixtures thereof.

[0041] In various examples, the covalent bond between the substrate and the plurality of nanogel particles comprises an amide -NH-C(O)- bond, where the -NH- moiety of each amide bond occurs as an -NH2 group present in the plurality of -NH2 groups on the substrate, and the -C(O)- moiety of each amide bond occurs as a carboxylic acid end group on the respective copolymer chain.

[0042] In various embodiments, the organic polymeric nanogel particles further comprise amplification primers grafted thereon.

[0043] In various embodiments, each graft of an amplification primer onto an organic polymeric nanogel particle comprises a triazine bond formed from a click chemistry reaction between a terminal alkyne substituent on the amplification primer and an azide group at the end of the respective copolymer chain, or a click chemistry reaction between a terminal azide substituent on the amplification primer and an alkyne group at the end of the respective copolymer chain.

[0044] In various embodiments, the first repeat unit of formula (I) is

[0045] [ka]

[0046] In various embodiments, the first repeat unit of formula (I) is

[0047] [ka]

[0048] In various embodiments, the second repeat unit of formula (II) is

[0049] [ka] At least one of the following is true:

[0050] In various embodiments of the present disclosure, a flow cell that can be used in nucleic acid sequencing is described. In various examples, the flow cell includes a substrate having a surface with a plurality of pH- and temperature-responsive organic polymeric nanogel particles covalently attached to the surface, where the organic polymeric nanogel particles include a plurality of copolymer chains having both a first repeating unit of Formula (I) and a second repeating unit of Formula (II), and the organic polymeric nanogel particles further include amplification primers grafted thereon.

[0051] In various embodiments, a method for synthesizing organic polymeric nanogel particles comprises reacting an aqueous dispersion of N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA) monomers, N-isopropylacrylamide (NiPAM) monomers, acrylic acid (AAc) monomers, and N,N'-methylenebisacrylamide (BisAM) monomers in the presence of a dispersing agent and a free radical initiator under conditions suitable for suspension / precipitation free radical polymerization, wherein the polymeric nanogel particles thus synthesized comprise poly(AzAPA-co-NiPAM-co-AAc-co-BisAM) copolymer chains.

[0052] In various embodiments, a method for synthesizing organic polymeric nanogel particles comprises reacting an aqueous dispersion of propargyl acrylate (PAG) monomers, N-isopropylacrylamide (NiPAM) monomers, acrylic acid (AAc) monomers, and N,N'-methylenebisacrylamide (BisAM) monomers in the presence of a dispersing agent and a free radical initiator under conditions suitable for suspension / precipitation free radical polymerization, wherein the polymeric nanogel particles thus synthesized comprise poly(PAG-co-NiPAM-co-AAc-co-BisAM) copolymer chains.

[0053] In various embodiments, a method for synthesizing organic polymeric nanogel particles comprises reacting an aqueous dispersion of propargyl acrylamide (PAM), N-isopropyl acrylamide (NiPAM), acrylic acid (AAc), and N,N'-methylenebisacrylamide (BisAM) monomers in the presence of a dispersing agent and a free radical initiator under conditions suitable for suspension / precipitation free radical polymerization, and the polymeric nanogel particles thus synthesized comprise poly(PAM-co-NiPAM-co-AAc-co-BisAM) copolymer chains.

[0054] In various embodiments, the dispersing agent includes sodium dodecyl sulfate.

[0055] In various embodiments, the free radical initiator includes ammonium persulfate.

[0056] In various embodiments, a method for assembling a flow cell usable for nucleic acid sequencing, the method comprising: (a) preparing a plurality of organic polymer nanogel particles, each nanogel particle comprising a repeating unit of formula (I) and a repeating unit of formula (II):

[0057] [ka] wherein R 1 , R 1’, and R 1” is independently selected from H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl; X is —O— or —NH—; and R 2 teeth,

[0058]

number

[0059] [ka] wherein R 3 , R 3’ , R 4 , 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 , and -NR 6 R 7 and R 5 is -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl; R 6 and R 7 is independently selected from the group consisting of —H and alkyl; 2’ is -N3 or

[0060]

number

[0061]

number

[0062]

number

[0063]

number

[0064] In various embodiments, the preparation of organic polymeric nanogel particles involves reacting an aqueous dispersion of N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), N-isopropylacrylamide (NiPAM), acrylic acid (AAc), and N,N'-methylenebisacrylamide (BisAM) monomers in the presence of a dispersing agent and a free radical initiator under conditions suitable for suspension / precipitation free radical polymerization, wherein the polymeric nanogel particles thus synthesized comprise poly(AzAPA-co-NiPAM-co-AAc-co-BisAM) copolymer chains.

[0065] In various embodiments, the preparation of organic polymeric nanogel particles involves reacting an aqueous dispersion of propargyl acrylate (PAG), N-isopropylacrylamide (NiPAM), acrylic acid (AAc), and N,N'-methylenebisacrylamide (BisAM) monomers in the presence of a dispersing agent and a free radical initiator under conditions suitable for suspension / precipitation free radical polymerization, wherein the polymeric nanogel particles thus synthesized comprise poly(PAG-co-NiPAM-co-AAc-co-BisAM) copolymer chains.

[0066] In various embodiments, the preparation of organic polymeric nanogel particles involves reacting an aqueous dispersion of propargyl acrylamide (PAM), N-isopropyl acrylamide (NiPAM), acrylic acid (AAc), and N,N'-methylenebisacrylamide (BisAM) monomers in the presence of a dispersing agent and a free radical initiator under conditions suitable for suspension / precipitation free radical polymerization, wherein the polymeric nanogel particles thus synthesized comprise poly(PAM-co-NiPAM-co-AAc-co-BisAM) copolymer chains.

[0067] In various embodiments, grafting the amplification primer onto the organic polymer nanogel particles in (b) above further comprises swelling the organic polymer nanogel particles prior to said grafting by reducing the temperature of the amide condensation reaction to less than about 20°C.

[0068] In various embodiments, the attachment of the grafted organic polymer nanogel particles onto the designated area of ​​the surface of the flow cell in (c) above is preceded by physically capturing the grafted organic polymer nanogel particles from the solution of grafted organic polymer nanogel particles onto the designated area of ​​the surface of the flow cell by performing the steps of: (i) shrinking the grafted organic polymer nanogel particles by increasing the temperature of the solution to about 60°C; (ii) positioning the shrunken grafted organic polymer nanogel particles on the designated area of ​​the surface of the flow cell; and (iii) lowering the temperature of the solution to below about 20°C to swell the positioned grafted organic polymer nanogel particles and physically capturing the grafted organic polymer nanogel particles on the designated area of ​​the surface of the flow cell.

[0069] In various embodiments, the designated areas of the surface of the flow cell include nanowells patterned in the substrate.

[0070] 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]

[0071] [Figure 1A] FIG. 10 shows a schematic diagram of an exemplary procedure for preparing ANA nanogel particles containing poly(AzAPA-co-NiPAM-co-AAc-co-BisAM) copolymer chains, synthesized under suspension / precipitation polymerization conditions according to various embodiments of the present disclosure. [Figure 1B] FIG. 1 shows exemplary temperature-dependent shrinkage / swelling properties of exemplary nanogel particles comprising poly(AzAPA-co-NiPAM-co-AAc-co-BisAM) copolymer chains. [Figure 1C]FIG. 10 is a schematic representation of the grafting of alkyne-P5 / P7 amplification primers onto exemplary nanogel particles comprising poly(AzAPA-co-NiPAM-co-AAc-co-BisAM) copolymer chains under CuAAc conditions to form ANA-P5P7 primer-grafted nanogel particles, and a plot showing the temperature-dependent size of exemplary ANA-P5P7-grafted nanogel particles. [Figure 2A] FIG. 10 is a schematic depicting the capture of ANA-P5P7 primer-grafted nanogel particles onto a CMS-treated PAZAM coating in an SBS flow cell in the presence of DMTMM activation. [Figure 2B] FIG. 1 shows exemplary SBS sequencing and SBS metrics thus obtained using ANA-P5P7 primer-grafted nanogel particles in an SBS flow cell. [Figure 2C] FIG. 1 shows exemplary SBS sequencing and SBS metrics thus obtained using ANA-P5P7 primer-grafted nanogel particles in an SBS flow cell. [Figure 2D] FIG. 1 shows exemplary SBS sequencing and SBS metrics thus obtained using ANA-P5P7 primer-grafted nanogel particles in an SBS flow cell. [Figure 3A] FIG. 10 shows a schematic diagram of an exemplary procedure for preparing PANA nanogel particles comprising poly(PAG-co-NiPAM-co-AAc-co-BisAM) copolymer chains, synthesized under suspension / precipitation polymerization conditions according to various embodiments of the present disclosure. [Figure 3B] FIG. 1 shows exemplary temperature-dependent shrinkage / swelling properties of exemplary nanogel particles comprising poly(PAG-co-NiPAM-co-AAc-co-BisAM) copolymer chains. [Figure 3C] FIG. 10 shows schematic diagrams of various exemplary reactions from PANA nanogel particles containing poly(PAG-co-NiPAM-co-AAc-co-BisAM) copolymer chains, including grafting of amplification primers onto nanogel particles and entrapment of nanogel particles on untreated and treated flow cell surfaces. [Figure 4A] using N3-FITC with CuAAC chemistry or using UV-activated click chemistry reaction between HS-PEG12k-FITC and PANA particles.

number

number

[0072] 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.

[0073] term 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.

[0074] As used herein, the term "dual functionality" refers to nanogel particles having carboxylic acid end groups as well as -N3 or

[0075]

number

[0076] 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 portion of the polymer structure 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, as well as 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.

[0077] As used herein, the term "pH-responsive" means that the nanogel particles contain at least some copolymer chains with carboxylic acid end groups, such that 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 a nanogel particle such that the pH-responsive carboxylic acid end groups on at least some of the copolymer chains of the nanogel particle impart pH-responsiveness to the nanogel particle. In various embodiments, the pH-responsiveness allows for pH-driven binding of the nanogel particle to a functionalized flow cell surface.

[0078] As used herein, the term "dual stimulus (temperature / pH)" is intended to mean the combination of temperature-responsive and pH-responsive properties (as defined above) exhibited by certain nanogel particles. In various examples, the poly-NiPAM blocks in the copolymer chains of the nanogel particles impart temperature-responsiveness to the nanogel particles, while the presence of AAc units in the copolymer chains of the nanogel particles contributes to the pH-responsiveness of the nanogel particles.

[0079] 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.

[0080] As used herein, the acronym "AzAPA" is intended to mean the monomer N-(5-(2-azidoacetamido)pentyl)acrylamide.

[0081] As used herein, the acronym "NiPAM" is intended to mean the monomer N-isopropylacrylamide.

[0082] As used herein, the acronym "BisAM" is intended to mean the polyfunctional monomer N,N'-methylenebisacrylamide.

[0083] As used herein, the acronym "PAG" is intended to mean the monomer propargyl acrylate.

[0084] As used herein, the acronym "PAM" is intended to mean the monomer N-propargyl acrylamide.

[0085] As used herein, the acronym "AAc" is intended to mean the monomer acrylic acid.

[0086] 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.

[0087] As used herein, the acronym "SDS" is intended to mean sodium dodecyl sulfate, an anionic dispersing agent.

[0088] As used herein, the acronym "APS" is intended to mean ammonium persulfate, a free radical polymerization initiator.

[0089] As used herein, the acronym "ANA" is intended to mean nanogel particles comprising poly(AzAPA-co-NiPAM-co-AAc-co-BisAM) copolymer chains. ANA particles are characterized by both carboxylic acid and -N3 end groups on at least some of the copolymer chains.

[0090] As used herein, the acronym "PANA" is intended to mean nanogel particles comprising poly(PAG-co-NiPAM-co-AAc-co-BisAM) copolymer chains. PANA particles contain carboxylic acids and

[0091]

number

[0092] As used herein, the acronym "PANA'" is intended to mean nanogel particles comprising poly(PAM-co-NiPAM-co-AAc-co-BisAM) copolymer chains. PANA' particles contain carboxylic acid groups on at least some of the copolymer chains and

[0093]

number

[0094] 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.

[0095] 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.

[0096] As used herein, the acronym "PAZAM" is intended to mean a functionalized polymer coating comprising poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide).

[0097] 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.

[0098] As used herein, the acronym "CuAAC" is intended to mean copper-catalyzed azide-alkyne cycloaddition click chemistry.

[0099] As used herein, the acronym "dz" or "Dz" (as may be found in various figures herein) 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).

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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 heterocycle. In some instances, the ring so formed can form a bicyclic or tricyclic structure. While trivial names may be used for particular substituents, it should be understood that if the group is a substituent, the group must have some valence available for bonding to another atom (e.g., a "carboxylic acid" substituent is more formally the monovalent substituent -CO or -COH).

[0108] 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. The alkyl substituents herein may be substituted, i.e., have one or more substituents attached to the alkyl group or incorporated within the alkyl chain. Substitutions 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 substitutions on alkyl substituents include, but are not limited to, -CN, -N3, -NH2, -NHR, -N(R)2, -N(R)3. +, -NO2, -NH-NH2, -NH-NHR, -NH-NR2, -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.

[0109] 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., 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.

[0110] 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 10Alkenyl groups may have any number of carbon atoms, such as C1-C8, or C1-C6, and any degree of unsaturation. Examples of alkenyl substituents include, but are not limited to, methylene / methylidyne (=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.

[0111] 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 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., 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.

[0112] 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 1-6 alkoxy (e.g., -OCF3, -OC2F5), and the like.

[0113] Examples of heterocycles include, but are not limited to, azepinyl, aziridinyl, azetyl, azetidinyl, diazepinyl, dithiadiazinyl, dioxazepinyl, dioxolanyl, dithiazolyl, furanyl, isoxazolyl, isothiazolyl, imidazolyl, morpholinyl, morpholino, oxetanyl, oxadiazolyl, oxiranyl, oxazinyl, oxazolyl, piperazinyl, pyrazinyl, and 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, cyclopentapyrazolyl, pyrrolopyryl thienothienyl, thiadiazolopyrimidinyl, thiazolothiazinyl, thiazolopyrimidinyl, thiazolopyridinyl, oxazolopyrimidinyl, oxazolopyridyl, benzoxazolyl, benzisothiazolyl, benzothiazolyl, imidazopyrazinyl, purinyl, pyrazolopyrimidinyl, imidazopyridinyl, benzimidazolyl, indazolyl, benzoxthiolyl, benzodioxolyl, benzodithiolyl, indolizinyl, indolinyl, isoindolinyl, furopyrimidinyl, furopyridyl, benzofuranyl, isobenzofuranyl, thienopyrimidinyl, thienpyridyl, benzothienyl, cyclopentaoxazinyl, cyclopentafuranyl, benzoxazinyl, benzothiazinyl, quinazolinyl, naphthyridinyl, quinolinyl, isoquinolinyl, benzopyranyl, pyridopyridazinyl, and pyridopyrimidinyl. Further examples of heterocyclic ring systems are given in A. Katritzky, et al., Handbook of Heterocyclic Chemistry, 3 rd Ed., Elsevier, 2010, the entire contents of which are incorporated herein by reference.

[0114] Basic Example Various embodiments of the present disclosure describe novel polymeric nanogel particles. The 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.

[0115] 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.

[0116] In various embodiments, the nanogel particles comprise crosslinked copolymer chains, for example, crosslinking is expected when multifunctional monomers are used in suspension / precipitation free radical polymerization with other monomer types.

[0117] 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).

[0118] Monomers for the synthesis of nanogel particles In various embodiments, the first type of monomer used in synthesizing nanogel particles in a suspension / precipitation free radical polymerization reaction includes monomers having the structure:

[0119] [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,

[0120]

number

[0121] [ka] and In the formula, R 2’ is -N3 or

[0122]

number

[0123] 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.

[0124] In various embodiments, the second type of monomer used in synthesizing nanogel particles in a suspension / precipitation free radical polymerization reaction includes monomers having the structure:

[0125] [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 , and -NR 6 R 7 and R 5 is -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl; R 6 and R 7 Each of is independently selected from —H and alkyl.

[0126] 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, and N-vinylpyridine.

[0127] In various embodiments, nanogel particles are prepared by reacting at least one first type of monomer with at least one second type of monomer according to the structures listed above under suspension / precipitation free radical polymerization reaction conditions. With these two types of monomers used in the suspension / precipitation free radical polymerization reaction, the resulting 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.

[0128] Multifunctional monomers that can be included in the suspension / precipitation polymerization reaction to form 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.

[0129] In various embodiments, nanogel particles are prepared 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 under suspension / precipitation free radical polymerization reaction conditions. With these two types of monomers and the multifunctional monomer used in the suspension / precipitation free radical polymerization reaction, the resulting 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, where the copolymer chains have at least some crosslinking between them.

[0130] In various embodiments, nanogel particles are prepared 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) under suspension / precipitation free radical polymerization reaction conditions.

[0131] In various embodiments, nanogel particles comprising at least one of each of the two types of monomers described above and optionally a multifunctional monomer, thus prepared under suspension / precipitation free radical polymerization conditions, contain at least one carboxylic acid end group (i.e., the monovalent substituent -CO2). - or -CO2H) and at least one -N3 or

[0132]

number

[0133] Suspension / Precipitation Radical Polymerization and Other Free Radical Polymerizations In various embodiments, the synthesis of nanogel particles involves various aspects of suspension / precipitation free radical polymerization or emulsion polymerization. In various embodiments, the reaction conditions are aqueous and heated, employing selected monomers, a dispersant to promote the suspension of the thus-formed, generally water-insoluble nanogel particles in water, and a free radical initiator.

[0134] 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.

[0135] 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.

[0136] In various embodiments, the free radical initiator comprises a water-soluble compound.

[0137] In various embodiments, the free radical initiator comprises a peroxide.

[0138] In various embodiments, the free radical initiator includes sodium persulfate, potassium persulfate, or ammonium persulfate.

[0139] In various embodiments, the free radical initiator includes ammonium persulfate (APS).

[0140] In various examples, nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction incorporating a dispersed monomer mixture containing N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), N-isopropylacrylamide (NiPAM), and acrylic acid (AAc).

[0141] In various examples, nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction incorporating a dispersed monomer mixture containing N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), N-isopropylacrylamide (NiPAM), acrylic acid (AAc), and the multifunctional monomer N,N'-methylenebisacrylamide (BisAM).

[0142] In various examples, nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction incorporating a dispersed monomer mixture containing propargyl acrylate (PAG), N-isopropylacrylamide (NiPAM), and acrylic acid (AAc).

[0143] In various examples, nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction incorporating a dispersed monomer mixture containing propargyl acrylate (PAG), N-isopropylacrylamide (NiPAM), acrylic acid (AAc), and the multifunctional monomer N,N'-methylenebisacrylamide (BisAM).

[0144] In various examples, nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction incorporating a dispersed monomer mixture including propargyl acrylamide (PAM), N-isopropyl acrylamide (NiPAM), and acrylic acid (AAc).

[0145] In various examples, nanogel particles are synthesized in a suspension / precipitation free radical polymerization reaction incorporating a dispersed monomer mixture containing propargyl acrylamide (PAM), N-isopropyl acrylamide (NiPAM), and acrylic acid (AAc), as well as the multifunctional monomer N,N'-methylenebisacrylamide (BisAM).

[0146] Nanogel particles containing copolymer chains In various embodiments, the nanogel particles comprise copolymer chains having various end groups on at least some of the copolymer chains. In various embodiments, the nanogel particles comprise copolymer chains having at least some cross-linking.

[0147] In various embodiments, polymeric nanogel particles according to the present disclosure have the formula (I):

[0148] [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,

[0149]

number

[0150] [ka] and In the formula, R 2’ is -N3 or

[0151]

number

[0152] [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 , and -NR 6 R 7 wherein R 5 is -H, -OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl; R 6 and R 7 each of which independently comprises a copolymer chain further comprising a second repeat unit selected from -H and alkyl.

[0153] In various embodiments, R 1 =R 1’ =R 1” ═H, X is —O— or —NH—, and R 2 teeth,

[0154]

number

[0155] [ka] wherein R 2’ is -N3 or

[0156]

number

[0157] In various embodiments, nanogel particles having the repeating units listed above include copolymer chains having at least one carboxylic acid end group.

[0158] In various embodiments, nanogel particles having the repeating units listed above contain at least one -N3 or

[0159]

number

[0160] In various embodiments, the nanogel particles comprise a first repeat unit of formula (I) comprising:

[0161] [ka] wherein p is an integer from 1 to 50, as above.

[0162] In various embodiments, the nanogel particles comprise a first repeat unit of formula (I) comprising:

[0163] [ka] The copolymer chain comprises:

[0164] In various embodiments, the nanogel particles comprise a second repeat unit of formula (II) comprising:

[0165] [ka] The copolymer chain comprises at least one of:

[0166] In various embodiments, the polymeric nanogel particles comprise poly(AzAPA-co-NiPAM-co-AAc-co-BisAM) copolymer chains, and in various embodiments, at least some of these copolymer chains comprise -N3 and -CO2H end groups for dual functionality.

[0167] In various embodiments, the polymeric nanogel particles comprise poly(PAG-co-NiPAM-co-AAc-co-BisAM) copolymer chains. In various embodiments, at least some of these copolymer chains comprise:

[0168]

number

[0169] In various embodiments, the polymeric nanogel particles include poly(PAM-co-NiPAM-co-AAc-co-BisAM) copolymer chains. In various embodiments, at least some of these copolymer chains are bifunctional.

[0170]

number

[0171] Grafting amplification primers onto nanogel particles The basic embodiment described above is a nanogel particle in which the copolymer chains have at least some -CO2H end groups and at least some -N3 end groups or

[0172]

number

[0173]

number

[0174] In various embodiments, grafting of the amplification primers onto the polymeric 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.

[0175]

number

[0176] In other embodiments, the thiol-functionalized primer has at least some of the copolymer chains

[0177]

number

[0178] In various embodiments, as detailed above, the selection of monomers used in the synthesis of nanogel particles is determined so that the resulting copolymer chains are -N3 or

[0179]

number

[0180] 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 onto nanogel particles 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.

[0181] In various embodiments, functionalized amplification primers for grafting onto nanogel particles include, but are not limited to, alkyne-P5 / P7 primers, N3-P5 / P7 primers, and thiol-P5 / P7 primers.

[0182] In various examples, grafting of an alkyne-P5 / P7 primer onto nanogel particles comprising copolymer chains with -N3 end groups involves CuAAC grafting, resulting in P5 / P7 grafted nanogel particles, such as ANA-P5P7 grafted nanogel particles.

[0183] 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.

[0184] In various embodiments,

[0185]

number

[0186] Entrapment of primer-grafted nanogel particles for SBS In various embodiments, and as part of an SBS method, primer-grafted nanogel particles, such as, for example, ANA-P5P7, PANA-P5P7, or PANA'-P5P7 primer-grafted nanogel particles, are captured on the surface of a flow cell (FC), such as, for example, a HiSeq™ FC manufactured by Illumina, Inc. The primer-grafted nanogel particles can be captured within nanowells patterned in a coating on the FC surface, or can be directly attached to a coating on a surface where no nanowells are present. In various embodiments, each primer-grafted nanogel particle can serve as a nanowell and, therefore, can function as a surrogate for a nanowell.

[0187] In various embodiments, the primer-grafted nanogel particles are (a) a bioconjugation technique using DMTMM to activate the reaction between the free carboxylic acid end groups present on the copolymer chains of the primer-grafted nanogel particles and the available -NH2 groups on the pre-silanized FC surface to form amide bonds; or (b) any residual comonomers still present on the copolymer chains of the primer-grafted nanogel particles (i.e., after grafting)

[0188]

number

[0189] 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.

[0190] In various embodiments, the silane or silane derivative is 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.

[0191] In various embodiments, the -NH groups present on the FC surface are then reacted with carboxylic acid end groups present on the corresponding copolymer chains in the primer-grafted 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

[0192]

number

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] To capture nanogel particles on the PAZAM-coated FC surface, the PAZAM coating with reactive -N3 groups was added under conditions for CuAAC click chemistry to remove any remaining copolymer chains present on the nanogel particles.

[0198]

number

[0199] In an alternative embodiment, the order of the separate steps of primer grafting and particle capture can be reversed. Thus, dual-functional 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.

[0200] Seeding, clustering, and SBS sequencing In various embodiments, clustering includes either in-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, as each nanogel particle captured on the FC surface acts as its own nanowell. In in-suspension clustering, seeded ssDNA can be clustered on the surface of nanogel particles. Clustering on nanogel particles relies on having sufficiently accessible primers grafted onto the nanogel particles.

[0201] In various examples, the temperature responsiveness of primer-grafted nanogel particles having blocks of poly(NiPAM) in the copolymer chain is (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 polymeric 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.

[0202] In various embodiments, the FC with captured primer-grafted 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 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 a larger cluster.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] 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]

[0207] Referring now to FIG. 1A, an exemplary procedure for preparing amide-based dual-responsive nanogel particles is schematically illustrated in accordance with various embodiments of the present disclosure. As shown in the figure, the monomers N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), N-isopropylacrylamide (NiPAM), acrylic acid (AAc), and N,N'-methylenebisacrylamide (BisAM) are reacted under aqueous suspension / precipitation polymerization conditions to form nanogel particles having poly(AzAPA-co-NiPAM-co-AAc-co-BisAM) or "ANA" copolymer chains. In various embodiments, these particles comprise copolymer chains in which at least some of the copolymer chains contain at least one carboxylic acid end group and at least some of the copolymer chains contain at least one -N3 end group.

[0208] In various embodiments, the carboxylic acid end groups can be used to attach the nanogel particles to a surface or to conjugate specific groups to the nanogel particles, and similarly, the -N3 end groups can be used to attach the nanogel particles to a surface or to conjugate specific groups to the nanogel particles. In various embodiments, the nanogel particles are attached to the flow cell surface via free carboxylic acid end groups and / or free -N3 end groups.

[0209] The reaction scheme of FIG. 1A involves a suspension / precipitation polymerization reaction using SDS as a dispersing agent and APS as a free radical polymerization initiator, which is carried out in water at, for example, 70° C. for 4 hours.

[0210] FIG. 1B illustrates a plot showing the size of exemplary nanogel particles as a function of polymerization reaction temperature for exemplary nanogel particles produced according to the reaction scheme of FIG. 1A. Generally, the aqueous suspension / precipitation polymerization reaction shown in FIG. 1A has proven to be well-controlled, allowing for the achievement of desired nanogel particle sizes with narrow nanoparticle polydispersities. For example, in sodium phosphate buffer (pH 7.4), the sizes range from 280 nm when the reaction is carried out at 20°C to 265 nm when the reaction is carried out at 60°C. As shown in the figure, the Z-average particle size remains relatively constant at approximately 280 nm when the suspension / precipitation polymerization reaction is carried out below approximately 40°C. As shown in the plot, above 40°C (the temperature at which polyNiPAM becomes hydrophobic), the size of the nanogel particles decreases with each increase above approximately 40°C. The vertical bars in the plot of FIG. 1B represent error bars.

[0211] Figure 1C shows the grafting of a functionalized amplification primer onto nanogel particles containing poly(AzAPA-co-NiPAM-co-AAc-co-BisAM) or "ANA" copolymer chains. In this example, the free -N3 end groups present on at least some of the copolymer chains are used in click chemistry grafting of an alkyne-functionalized amplification primer to the free -N3 end groups, resulting in the formation of a triazine bond between the particle and the primer. The alkyne-functionalized amplification primer used here is a functionalized P5 / P7 primer, which is discussed at least fully in U.S. Pat. Nos. 9,815,916 and 10,266,891 (Illumina, Inc.), both of which are incorporated herein by reference in their entireties.

[0212] Continuing with Figure 1C, nanogel particles bearing ANA copolymer chains with free -N3 end groups, as prepared according to the reaction scheme described in Figure 1A, were reacted in suspension with alkyne-functionalized P5 / P7 amplification primers at 60 °C for 3 hours using copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry to form P5 / P7-grafted nanogel particles, labeled "ANA-P5P7" in Figure 1C. In the ANA-P5P7 nanogel particles, the free -N3 end groups reacted with the alkyne-functionalized primers to form triazine bonds linking the amplification primers to the particles.

[0213] The plot shown below the CuAAc grafting reaction in Figure 1C shows the temperature-dependent size of the ANA-P5P7-grafted nanogel particles. As shown in the figure, the ANA-P5P7-grafted nanogel particles shrink with increasing temperature.

[0214] In various embodiments, this temperature-controllable shrinkage and swelling of nanogel particles, such as ANA-P5P7 shown in Figure 1C, is a significant advantage. This feature can be used to increase the efficiency of various biochemical processes occurring during seeding, amplification, and nucleic acid sequencing. For example, promoting particle shrinkage during seeding can reduce the probability of multiple seeding events, resulting in increased monoclonality. Furthermore, promoting particle swelling during clustering can increase primer accessibility and facilitate polymer diffusion, resulting in an increased number of nucleic acid strands per particle cluster and subsequent improved fluorescent signal. Furthermore, SBS steps, such as incorporation or cleavage, can also be improved by this observed timed swelling / shrinkage of amplification primer-grafted nanogel particles.

[0215] 2A-2D show examples of chemically facilitated capture chemistry of ANA-P5P7 particles into the nanowells of a FC and SBS sequencing metrics of particle-captured FCs.

[0216] Figure 2A shows the bioconjugation technique using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) used to attach ANA-P5P7 particles to nanowells, e.g., by reacting the free carboxylic acid end groups on the copolymer chains of the ANA-P5P7 particles with the amine groups provided on the cleavage-mixing (CMS) nanowells. The reaction was carried out in PBS buffer (pH 7.4). The particles were then captured onto a standard PAZAM-coated, polished HiSeqX™ FC (PN15059930) using a "cBot" (an automated fluidics device from Illumina used to prepare HiSeq X flow cells for cluster generation; see https: / / www.illumina.com / products / by-type / accessory-products / cbot.html). The capture process was optimized by testing different flush times and / or particle suspension concentrations (wt%).

[0217] Figure 2B (left image) shows a Cal Fluor Red (CFR) fluorescence image of FCs with ANA-P5P7 particles captured on their surface, imaged at 580 nm with a Typhoon™. P5 / P7 complementary primers conjugated with CFR dyes were hybridized on each lane. Lanes 1 and 8 are control lanes in which the original PAZAM-coated lanes were grafted with P5 / P7 primers. As seen in Figure 2B (left image), capture was successfully achieved, and good intensity of the hybridized CFRs was imaged with a Typhoon™. This experiment demonstrated that ANA-P5P7 nanogel particles can accommodate surface primer densities compatible with clustering and sequencing. The FCs were then clustered and sequenced using an Illumina HiSeqX™ instrument. An analysis image of the FC intensity after sequencing is shown in Figure 2B (right image).

[0218] The particle concentration and number of flushes of the particle solution are crucial for the intensity, as measured by CFR-QC or C1*Cycle 1 intensity using a sequencer. Three different particle concentrations, 0.08, 0.02, and 0.005 wt%, were examined with 5 or 10 flushes per lane. Both CFR and C1 intensities were found to increase with higher particle concentrations and flush numbers.

[0219] 2C and 2D show the sequencing metrics where the average value of the best tile and the average value of all tiles are considered, respectively.

[0220] The target values ​​for the SBS set of successful metrics, including %PF, %alignment, %≥Q30, and %occupancy, were 60, 100, 70, and 90%, respectively. In all experiments, three different particle concentrations, namely, 0.08, 0.02, and 0.005 wt%, were examined with five or ten flushes of each lane, as shown in Figure 2B (under "Chemistry"). The average values ​​of all metrics compared to the control lane and the target values ​​demonstrate the success of SBS sequencing after 36 cycles. More specifically, the average metric values ​​of the best tile (Figure 2C) clearly demonstrate that these values ​​were highly achieved. In particular, for a particle concentration of 0.08 wt% and five flushes of the lane, the %PF, %alignment, %≥Q30, and %occupancy were 58.2, 99.4, 95.1, and 90.5%, respectively. Figure 2D is a bar graph showing each of the metrics for only the best tiles. Thus, the results obtained demonstrate the ability of ANA-P5P7 nanogel particles to support successful SBS sequencing.

[0221] As shown in Figures 3A-3C, amide-based dual-responsive nanogel particles can also be synthesized by reacting propargyl acrylate (PAG) or propargyl acrylamide (PAM), N-isopropylacrylamide (NiPAM), acrylic acid (AAc), and N,N'-methylenebisacrylamide (BisAM) monomers under aqueous suspension / precipitation polymerization conditions to form nanogel particles with poly(PAG-co-NiPAM-co-AAc-co-BisAM) "PANA" or poly(PAM-co-NiPAM-co-AAc-co-BisAM) "PANA'" copolymer chains. In various embodiments, these particles have at least some of the copolymer chains containing at least one carboxylic acid end group and at least some of the copolymer chains containing at least one carboxylic acid end group.

[0222]

number

[0223]

number

[0224]

number

[0225] In the synthesis example shown in Figure 3A, propargyl acrylate (PAG), N-isopropylacrylamide (NiPAM), acrylic acid (AAc), and N,N'-methylenebisacrylamide (BisAM) monomers were reacted under aqueous suspension / precipitation polymerization conditions in the presence of SDS and APS at 70°C for 4 hours to form nanogel particles with poly(PAG-co-NiPAM-co-AAc-co-BisAM) or PANA copolymer chains. At least some of the polymer chains contained carboxylic acid end groups and at least some of the copolymer chains contained carboxylic acid end groups.

[0226]

number

[0227] As shown in the plot in Figure 3B, the resulting PANA nanogel particles exhibited temperature-dependent shrinkage / swelling (measured using light scattering in phosphate buffer at pH 7.4) ranging from a swollen size of approximately 250 nm at temperatures below approximately 30°C to a shrunken size of approximately 225 nm at temperatures of approximately 70°C. Furthermore, above approximately 40°C, the temperature at which poly(NiPAM) present as a block in the block copolymer chain becomes hydrophobic, there was a more significant decrease in particle size with increasing temperature.

[0228] Figure 3C shows a schematic of the various reactions performed on PANA nanogel particles prepared according to Figure 3A. Specifically, PANA nanogel particles were grafted with N3-P5 / P7 primers in a copper-catalyzed click chemistry (CuAAC) reaction at 60 °C for 3 h. The primer-grafted PANA particles were purified and isolated by TFF. The primer density thus obtained appeared to meet the SBS requirements based on the CFR QC. Alternatively, grafting can be achieved by using thiol-P5 / P7 primers in a UV-activated thiol-alkyne click chemistry reaction, as shown in Figure 3C.

[0229] Figure 4A shows the nanogel particles present on the

[0230]

number

[0231] Figure 4B shows a schematic of the capture of PANA particles.

[0232]

number

[0233] Figure 5A shows the Blackpool grafting of N3-P5 / P7 primers via “reverse” CuAAC chemistry onto alkyne-PANA particles, resulting in PANA-P5P7 primer-grafted nanogel particles.

[0234] Figure 5B shows the capture of PANA-P5P7 particles using either of two different chemistries. FC images were captured using a Typhoon at 580 nm via a CFR QC. Lanes 1 and 8 were control lanes with standard primers grafted onto a standard PAZAM surface. Lanes 5-7 had PANA-P5P7 particles captured at different flash factors using CuAAC chemistry. Lanes 2-4 had PANA-P5P7 particles captured at different flash factors using DMTMM chemistry. For lanes 2-4, the grafting and capture were successful, as there was significant intensity of 40-60% compared to the intensity of the control lanes. This has been shown in previous studies to be sufficient to maintain adequate cluster growth. Unfortunately, for lanes 5-7, the available chemistries likely lacked the required chemistries.

[0235]

number

[0236] In these practical examples, particle capture of primer graft particles (PANA-P5P7) was successful only at 60°C in deionized water (pH 6.5). This is because the particles shrink most at 60°C in deionized water (average particle size d z This is thought to be due to the particle size (up to 175 nm) at 30 °C. In comparison, the particle size is approximately 240 nm at 30 °C. Furthermore, the size of PANA particles varies from 220 nm to 250 nm when measured in phosphate buffer (pH = 7.4). In deionized water (pH 6.5), the size varies from 175 nm to 225 nm. This indicates the pH-responsiveness of the particles.

[0237] Figure 5C shows the C1 intensity heat map of FC compared to the CFR QC fluorescence intensity shown in Figure 5B. In Figure 5C, the C1 intensity heat map of FC confirms the CFR QC fluorescence intensity observed and shown in Figure 5B. Lanes 2-4, in which PANA-P5P7 particles were captured at different flash factors using DMTMM chemistry, show that these lanes were successfully sequenced. Target values ​​for the SBS set of success metrics, including %PF, %alignment, %≥Q30, and %occupancy, were 60, 100, 70, and 90%, respectively.

[0238] Figure 5D shows the resulting SBS intensities and metrics. Preliminary sequencing data demonstrate the suitability of the generated material as a support for SBS. According to Figure 5D, adequate %PF, %alignment, %≥Q30, and %occupancy metrics were observed in lane 2 for the best tile: 40.9, 98.9, 70.2, and 70.5%, respectively. Furthermore, without optimization, the data are comparable to the baseline for several metrics.

[0239] Further Concepts FIG. 6A details additional exemplary concepts within the scope of the present disclosure.

[0240] As shown in Figure 6A, thiol-alkyne reactions on PANA particles using the following procedure are potential methods for protein conjugation as well as for future use in proteomics. (a) Grafting thiol-terminated peptides onto PANA particles via thiol-alkyne click chemistry; (b) selecting peptides for low affinity / high specificity peptide-protein interactions; and (c) Formation of various protein coronas to facilitate specific binding events.

[0241] FIG. 6B shows the synthesis of propargyl acrylamide (PAM), which is suitable for use in various concepts of FIG. 6A.

[0242]

number

[0243] 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 be apparent to one skilled in the art how to implement the present disclosure in alternative embodiments.

[0244] 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.

[0245] 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 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 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.

[0246] Further comments While various illustrative examples have been described above, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the invention. It is intended that the appended claims cover all such changes and modifications that fall within the true spirit and scope of the invention.

[0247] 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.

Claims

1. Polymeric nanogel particles, Formula (I) 【number】 The first repeat unit of (I) is １ , R １’ , and R １” are each 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 the structure: 【number】 wherein R ２’ Ha-N ３ or [Equation 2] and p is an integer from 1 to 50; and Formula (II) 【number】 The second repeat unit of (II) is 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 , and -NR ６ R ７ wherein R ５ is —H, —OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl, and R ６ and R ７ each of which further comprises a copolymer chain comprising said second repeat unit, independently selected from —H and alkyl; At least some of the copolymer chains contain at least one carboxylic acid end group and at least some of the copolymer chains contain at least one -N ３ or [Equation 3] Polymeric nanogel particles containing end groups.

2. R １ , R １’ , and R １” The polymeric nanogel particle according to claim 1 , wherein

3. R １ and R １” is H and R １’ is CH ３ The polymeric nanogel particle according to claim 1,

4. The first repeat unit of formula (I) is 【Chemistry 4】 The polymeric nanogel particle according to claim 1,

5. The first repeat unit of formula (I) is 【Chemistry 5】 The polymeric nanogel particle according to claim 1,

6. The second repeat unit of formula (II) is 【Chemistry 6】 The polymeric nanogel particle according to any one of claims 1 to 5, wherein the polymeric nanogel particle is at least one of the following:

7. 2. The polymeric nanogel particles of claim 1, derived from a monomer mixture comprising N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), N-isopropylacrylamide (NiPAM), acrylic acid (AAc), and N,N'-methylenebisacrylamide (BisAM).

8. The polymeric nanogel particles of claim 7, comprising poly(AzAPA-co-NiPAM-co-AAc-co-BisAM) copolymer chains.

9. 2. The polymeric nanogel particles of claim 1, which are derived from a monomer mixture comprising propargyl acrylate (PAG) and / or N-propargyl acrylamide (PAM), N-isopropyl acrylamide (NiPAM), acrylic acid (AAc), and N,N'-methylenebisacrylamide (BisAM).

10. The polymeric nanogel particles of claim 9, comprising poly(PAG-co-NiPAM-co-AAc-co-BisAM) and / or poly(PAM-co-NiPAM-co-AAc-co-BisAM) copolymer chains.

11. 1. A substrate having a surface comprising a plurality of pH and temperature responsive organic polymer nanogel particles covalently bound to the surface, wherein the organic polymer nanogel particles comprise: Formula (I) 【number】 The first repeat unit of (I) is １ , R １’ , and R １” are each independently selected from H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl; X is —O— or —NH—; and R ２ teeth, [Equation 4] or the structure: 【number】 wherein R ２’ Ha-N ３ or [Equation 5] and p is an integer from 1 to 50; and Formula (II) 【number】 The second repeat unit of (II) is 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 , and -NR ６ R ７ wherein R ５ is —H, —OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl, and R ６ and R ７ each of which further comprises a plurality of copolymer chains independently comprising said second repeat unit selected from -H and alkyl; At least some of the copolymer chains contain at least one carboxylic acid end group and at least some of the copolymer chains contain at least one -N ３ or [Equation 6] A substrate comprising an end group.

12. 12. The substrate of claim 11, wherein the organic polymeric nanogel particles are formed from a monomer mixture comprising: (a) propargyl acrylate (PAG) and / or N-propargyl acrylamide (PAM), (b) N-isopropyl acrylamide (NiPAM), and (c) acrylic acid (AAc).

13. The substrate of claim 12, wherein the organic polymeric nanogel particles have an average size of about 265 nm to about 280 nm.

14. 12. The substrate of claim 11, wherein the organic polymeric nanogel particles are formed from a monomer mixture comprising N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), N-isopropylacrylamide (NiPAM), and acrylic acid (AAc).

15. The substrate of claim 14, wherein the organic polymeric nanogel particles have an average size of about 225 nm to about 250 nm.

16. 16. The substrate of any one of claims 12 to 15, 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, diallyl dimethyl ammonium chloride, and mixtures thereof.

17. The covalent bond between the substrate and the plurality of nanogel particles comprises an amide -NH-C(O)- bond, and the -NH- moiety of each amide bond is connected to a plurality of -NH ２ -NH present in the group ２ 12. The substrate of claim 11, wherein the -C(O)- moiety of each amide linkage occurs as a carboxylic acid end group on the respective copolymer chain.

18. The substrate of claim 11 , wherein the organic polymeric nanogel particles further comprise amplification primers grafted thereon.

19. 19. The substrate of claim 18, wherein each graft of an amplification primer onto an organic polymeric nanogel particle comprises a triazine bond formed from a click chemistry reaction between a terminal alkyne substituent on the amplification primer and an azide group at the end of the respective copolymer chain, or a click chemistry reaction between a terminal azide substituent on the amplification primer and an alkyne group at the end of the respective copolymer chain.

20. The first repeat unit of formula (I) is 【Chemistry 10】 The substrate of claim 11 , wherein

21. The first repeat unit of formula (I) is 【Chemistry 11】 The substrate of claim 11 , wherein

22. The second repeat unit of formula (II) is 【Chemistry 12】 22. The substrate according to claim 20 or 21, wherein the substrate is at least one of:

23. 12. A flow cell comprising the substrate of claim 11, wherein the organic polymeric nanogel particles further comprise amplification primers grafted thereon.

24. 1. A method for synthesizing organic polymeric nanogel particles, the method comprising reacting an aqueous dispersion of N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), N-isopropylacrylamide (NiPAM), acrylic acid (AAc), and N,N'-methylenebisacrylamide (BisAM) monomers in the presence of a dispersing agent and a free radical initiator under conditions suitable for suspension / precipitation free radical polymerization, wherein the polymeric nanogel particles so synthesized comprise poly(AzAPA-co-NiPAM-co-AAc-co-BisAM) copolymer chains.

25. 1. A method for synthesizing organic polymeric nanogel particles, the method comprising reacting an aqueous dispersion of propargyl acrylate (PAG), N-isopropylacrylamide (NiPAM), acrylic acid (AAc), and N,N'-methylenebisacrylamide (BisAM) monomers in the presence of a dispersing agent and a free radical initiator under conditions suitable for suspension / precipitation free radical polymerization, wherein the polymeric nanogel particles so synthesized comprise poly(PAG-co-NiPAM-co-AAc-co-BisAM) copolymer chains.

26. 1. A method for synthesizing organic polymeric nanogel particles, the method comprising reacting an aqueous dispersion of propargyl acrylamide (PAM), N-isopropyl acrylamide (NiPAM), acrylic acid (AAc), and N,N'-methylenebisacrylamide (BisAM) monomers in the presence of a dispersing agent and a free radical initiator under conditions suitable for suspension / precipitation free radical polymerization, wherein the polymeric nanogel particles so synthesized comprise poly(PAM-co-NiPAM-co-AAc-co-BisAM) copolymer chains.

27. The method of any one of claims 24 to 26, wherein the dispersing agent comprises sodium dodecyl sulfate.

28. 28. The method of any one of claims 24 to 27, wherein the free radical initiator comprises ammonium persulfate.

29. 1. A method of assembling a flow cell that can be used in sequencing nucleic acids, said method comprising: (a) preparing a plurality of organic polymer nanogel particles, each nanogel particle comprising a repeating unit of formula (I) and a repeating unit of formula (II) 【number】 wherein R １ , R １’ , and R １” are each independently selected from H, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl; X is —O— or —NH—; and R ２ teeth, [Equation 7] or the structure: 【number】 and In 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 , and -NR ６ R ７ and R ５ is —H, —OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, or heterocyclyl, and R ６ and R ７ is independently selected from the group consisting of —H and alkyl; ２’ is -N ３ or [Equation 8] and p is an integer from 1 to 50, and at least some of the copolymer chains contain at least one carboxylic acid end group, and at least some of the copolymer chains contain at least one -N ３ or [Equation 9] including a terminal group; (b) on an alkyne-functionalized amplification primer [Equation 10] groups and -N on each copolymer chain ３ by performing a click chemistry reaction between the terminal group -N on the azide-functionalized amplification primer ３ groups on each copolymer chain [0011] grafting amplification primers onto the organic polymer nanogel particles by performing a click chemistry reaction between the end groups; (c) attaching the primer-grafted organic polymer nanogel particles onto designated areas of the surface of the flow cell via amide -NH-C(O)- bonds by performing a temperature and pH controlled amide condensation reaction, wherein the -NH- moiety of each amide bond is connected to multiple -NH- bonds on the designated areas of the surface. ２ -NH present in the group ２ and the —C(O)— moiety of each amide linkage occurs as a carboxylic acid end group on the respective copolymer chain.

30. 30. The method of claim 29, wherein the step of preparing the organic polymeric nanogel particles comprises reacting an aqueous dispersion of N-(5-(2-azidoacetamido)pentyl)acrylamide (AzAPA), N-isopropylacrylamide (NiPAM), acrylic acid (AAc), and N,N'-methylenebisacrylamide (BisAM) monomers in the presence of a dispersing agent and a free radical initiator under conditions suitable for suspension / precipitation free radical polymerization, and the polymeric nanogel particles so synthesized comprise poly(AzAPA-co-NiPAM-co-AAc-co-BisAM) copolymer chains.

31. 30. The method of claim 29, wherein the step of preparing the organic polymeric nanogel particles comprises reacting an aqueous dispersion of propargyl acrylate (PAG), N-isopropylacrylamide (NiPAM), acrylic acid (AAc), and N,N'-methylenebisacrylamide (BisAM) monomers in the presence of a dispersing agent and a free radical initiator under conditions suitable for suspension / precipitation free radical polymerization, and wherein the polymeric nanogel particles so synthesized comprise poly(PAG-co-NiPAM-co-AAc-co-BisAM) copolymer chains.

32. 30. The method of claim 29, wherein the step of preparing the organic polymeric nanogel particles comprises reacting an aqueous dispersion of propargyl acrylamide (PAM), N-isopropyl acrylamide (NiPAM), acrylic acid (AAc), and N,N'-methylenebisacrylamide (BisAM) monomers in the presence of a dispersing agent and a free radical initiator under conditions suitable for suspension / precipitation free radical polymerization, and the polymeric nanogel particles so synthesized comprise poly(PAM-co-NiPAM-co-AAc-co-BisAM) copolymer chains.

33. 33. The method of any one of claims 29 to 32, wherein the grafting of amplification primers onto the organic polymeric nanogel particles in (b) further comprises swelling the organic polymeric nanogel particles prior to the grafting by reducing the temperature of the amide condensation reaction to below about 20°C.

34. 34. The method of claim 29, wherein the bonding of the grafted organic polymer nanogel particles onto the designated area of ​​the surface of the flow cell in (c) is preceded by physically capturing the grafted organic polymer nanogel particles from the solution onto the designated area of ​​the surface of the flow cell by performing the following steps: (i) shrinking the grafted organic polymer nanogel particles by increasing the temperature of the solution of the grafted organic polymer nanogel particles to about 60°C; (ii) positioning the shrunken grafted organic polymer nanogel particles onto the designated area of ​​the surface of the flow cell; and (iii) reducing the temperature of the solution to below about 20°C to swell the positioned grafted organic polymer nanogel particles, thereby physically capturing the grafted organic polymer nanogel particles onto the designated area of ​​the surface of the flow cell.

35. 35. The method of any one of claims 29 to 34, wherein the designated area of ​​the surface of the flow cell comprises a nanowell patterned in a substrate.

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