Flow Cell

Abstract

An example flow cell includes a substrate and a cured patterned resin on the substrate. The cured patterned resin has nanorecesses separated by gap regions. Each nanorecess has a maximum opening dimension ranging from about 10 nm to about 1000 nm. The cured patterned resin also includes an interpenetrating polymer network. The interpenetrating polymer network of the cured patterned resin includes an epoxy-based polymer and a (meth)acryloyl-based polymer.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the interests of U.S. Provisional Patent Application No. 63 / 015,259, filed on 24 April 2020, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Nanoimprinting technology enables the economical and efficient production of nanostructures. Nanoimprint lithography utilizes the direct mechanical deformation of a resist material by a stamp containing a nanostructure. The resist material is cured while the stamp is in place, fixing the shape of the nanostructure in the resist material. [Overview of the project]

[0003] introduction In a first embodiment, the flow cell comprises a substrate and a cured patterned resin on the substrate, the cured patterned resin comprising nanorecesses separated by void regions, the nanorecesses each having a maximum opening dimension in the range of about 10 nm to about 1000 nm, and the cured patterned resin comprising an interpenetrating polymer network comprising epoxy polymers and (meth)acryloyl polymers.

[0004] An example of the first embodiment further comprises a grid layer positioned on a substrate and a planar waveguide layer positioned on the grid layer. In one example, the refractive index of the interpenetrating polymer network is in the range of about 1.35 to about 1.52, and the refractive index of the planar waveguide layer is in the range of about 1.6 to about 2.5.

[0005] An example of the first embodiment further includes a hydrogel positioned in each of the nano-recesses, and an amplification primer bound to the hydrogel.

[0006] In one example of the first embodiment, the thickness of the cured patterned resin is in the range of approximately 225 nm to approximately 600 nm.

[0007] In one example of the first aspect, the weight ratio of the epoxy-based polymer to the (meth)acryloyl-based polymer ranges from 25:75 to about 75:25.

[0008] It should be understood that any features of the flow cell disclosed herein can be combined together in any desirable manner and / or configuration, for example, including having an imprint layer with one or more adjusted properties, to achieve the advantages described in this disclosure.

[0009] In the second aspect, the method comprises applying a resin mixture to a substrate, the resin mixture comprising two independently crosslinkable monomers present in the resin mixture in a predetermined weight ratio, wherein the first of the two independently crosslinkable monomers is a cationically polymerizable monomer and the second of the two independently crosslinkable monomers is a free radical polymerizable monomer; imprinting the resin mixture with a working stamp having a plurality of nanofeatures; curing the resin mixture while the working stamp is in a predetermined position; thereby forming an interpenetrating polymer network imprinted in the nano-recesses of the flow cell.

[0010] In one example of the second aspect, the cationically polymerizable monomer is a polyfunctional epoxy monomer and the free radical polymerizable monomer is a polyfunctional (meth)acryloyl monomer. In one example, the polyfunctional epoxy monomer is selected from the group consisting of: i) 2,4,6,8 - tetramethyl - 2,4,6,8 - tetrakis(propyl glycidyl ether) cyclotetrasiloxane:

Chem.

Chem.

[0011] In one example of the second embodiment, the polyfunctional epoxy monomer is poly(propylene glycol) diglycidyl ether: [ka] The polyfunctional (meth)acryloyl monomer is 2,2,3,3,4,4,5,5-octafluoro-1,6-hexanediol diacrylate: [ka] The specified weight ratio is approximately 50:50.

[0012] In one example of the second embodiment, the polyfunctional epoxy monomer is poly(propylene glycol) diglycidyl ether: [ka] The polyfunctional (meth)acryloyl monomer is glycerol 1,3-diglycerolate diacrylate: [ka] The specified weight ratio is in the range of approximately 25:75 to approximately 75:25.

[0013] It should be understood that any feature of this method can be combined together in any desired manner. Furthermore, it should be understood that any combination of features of the method and / or flow cell can be combined with each other and / or with any of the examples disclosed herein to achieve the benefits described herein, including, for example, modifying one or more properties of the imprint layer.

[0014] In a third embodiment, the method comprises: forming a resin mixture by mixing a cationic polymerizable monomer and a free radical polymerizable monomer, wherein the resin mixture is a precursor of an interpenetrating polymer network incorporated into a flow cell; adjusting the weight ratio of the cationic polymerizable monomer to the free radical polymerizable monomer using at least one property imparted to the resin mixture or the interpenetrating polymer network, wherein the property is selected from the group consisting of the refractive index of the interpenetrating polymer network, the absorption of the interpenetrating polymer network, the hardness of the interpenetrating polymer network, the thickness of the interpenetrating polymer network, the hydrophilic / hydrophobic balance of the interpenetrating polymer network, the viscosity of the resin mixture, the surface chemical compatibility between the resin mixture and a work stamp, the surface chemical compatibility of the interpenetrating polymer network, the shrinkage of the interpenetrating polymer network, and combinations thereof; and patterning the resin mixture to form the interpenetrating polymer network.

[0015] In one example of the third embodiment, the cationic polymerizable monomer is an epoxysiloxane monomer, and the free radical polymerizable monomer is a (meth)acryloyl monomer.

[0016] In an example of the third embodiment, the desired property is the surface chemical compatibility between the resin mixture and the work stamp, and the method further comprises selecting the material of the work stamp, wherein the weight ratio of cationic polymerizable monomer to free radical polymerizable monomer is adjusted to be in the range of about 25:75 to about 75:25.

[0017] In one example of the third embodiment, the patterning is performed using nanoimprint lithography.

[0018] It should be understood that any feature of this method can be combined together in any desired manner. Furthermore, it should be understood that any combination of features of the method and / or other methods and / or flow cells can be combined with each other and / or with any of the examples disclosed herein to achieve the benefits described herein, including, for example, modifying one or more properties of the imprint layer. [Brief explanation of the drawing]

[0019] The features of the examples in this disclosure will become clear from the following detailed description and drawings. In the drawings, similar reference numbers correspond to components that are similar but not identical in some cases. For brevity, reference numbers or features having the aforementioned functions may or may not be described in relation to other drawings in which they appear.

[0020] [Figure 1] This is a schematic diagram of an example of a cross-penetrating polymer network.

[0021] [Figure 2] This is a flowchart illustrating an exemplary method for fabricating an example of a mutually penetrating polymer network.

[0022] [Figure 3A] This is a schematic diagram of one example of a method for producing a cured patterned resin. [Figure 3B] This is a schematic diagram of one example of a method for producing a cured patterned resin. [Figure 3C] This is a schematic diagram of one example of a method for producing a cured patterned resin.

[0023] [Figure 4A] This is a top view of an example of a flow cell.

[0024] [Figure 4B]This is an enlarged cross-sectional view along the line 4B-4B in Figure 4A of an example of a flow channel and patterned sequencing surface in a flow cell of the first example.

[0025] [Figure 4C] This is an enlarged cross-sectional view along the 4C-4C line in Figure 4A of an example of a flow channel and patterned sequencing surface in a flow cell of the second example.

[0026] [Figure 5A] This graph shows the refractive index of the resin mixture and the cured resin as a function of the epoxy monomer content in the resin mixture.

[0027] [Figure 5B] This graph shows the thickness of the deposited resin mixture and cured resin in relation to the epoxy monomer content in the resin mixture. [Modes for carrying out the invention]

[0028] Patterning techniques have been used to create individual recesses on the surface of flow cells. These recesses can be functionalized, for example, with capture primers. Within each functionalized recess, monoclonal populations (localized and independent clusters) of amplicons can be generated from each gene oligonucleotide fragment. Higher cluster densities can be obtained when the recesses have small dimensions and narrow pitches (e.g., the distance from the center of one recess to the center of an adjacent or nearest adjacent recess, or from the edge of one recess to the edge of an adjacent or nearest adjacent recess). Higher cluster density means that more bases can be read from a given unit area, thereby increasing the genetic yield from the patterned flow cell.

[0029] Nanoimprint lithography (NIL) is an example of a high-throughput patterning technique that can achieve higher accuracy and lower costs than, for example, patterning techniques using photons or electrons. In NIL, a working stamp is used to create features in a curable resin mixture.

[0030] Some resin mixtures are easily imprintable but do not possess properties suitable for use in flow cells. For example, some curing resins have compositions that react undesirably when exposed to sequencing chemicals. Other resin mixtures are difficult to imprint. For example, resins intended to adhere upon curing may not easily detach from the work stamp. Also, some resins may become more difficult to imprint when the size of the features and / or the spacing between features becomes smaller (e.g., size and / or spacing is less than 500 nm). As an example, some resins can replicate large features, for example, larger than 1 μm, but as the size of the features decreases, defects that clog and / or cover the features may occur.

[0031] Examples of resin mixtures disclosed herein cure to form interpenetrating polymer networks. The formulation of the resin mixture may be modified so that one or more properties of the resin mixture are tailored to a specific application, such as imprinting, and / or so that one or more properties of the resulting interpenetrating polymer network are tailored to a specific application, such as flow cell sequencing. For example, the viscosity of the resin mixture may be adjusted to facilitate processing and / or reflow (the time required to fill all work stamp features with the resin formulation by applying reasonable pressure (e.g., roller load)). For another example, the wettability of the resin mixture may be adjusted to improve the compatibility of the formulation with a specific work stamp material and / or a specific substrate material. For yet another example, the hydrophilic / hydrophobic balance, thickness, refractive index, surface properties (e.g., tackiness, chemical resistance, etc.), shrinkage, and / or hardness of the resulting interpenetrating polymer network may be adjusted to a specific application. As one specific example, the formulation of the resin mixture may be adjusted so that an interpenetrating polymer network with a refractive index suitable for use with a plane waveguide in a flow cell is produced. As another specific example, the resin mixture formulation may be adjusted to produce an interpenetrating polymer network with surface properties that allow the work stamp to peel off cleanly after curing. Other properties of the interpenetrating polymer network, such as autofluorescence, may be adjusted by selecting specific initiators to be included in the resin mixture. Targeting specific properties of the final resin and / or the resulting interpenetrating polymer network may determine the components of the resin mixture and the amounts of each component in the resin mixture.

[0032] In the flow cell applications disclosed herein, the resulting interpenetrating polymer network can also be subjected to polymer bonding and other surface modification processes to introduce desired surface chemistry for sequencing.

[0033] definition

[0034] Unless otherwise specified, terms used herein will be understood to have their common meanings in the relevant art. Some terms used herein and their meanings are listed below.

[0035] As used herein, the singular forms "a," "an," and "the" refer to both the singular and plural forms unless explicitly indicated otherwise in the context.

[0036] As used herein, the term “includes” is synonymous with “contains,” “includes,” or “characterizes,” and is comprehensive or non-exclusive, not excluding any elements or methods or processes not further enumerated.

[0037] The terms “substantially” and “about” as used throughout this disclosure, including the claims, are used to describe and explain small variations, such as those resulting from variations in processing. These terms may refer to variations of ±10% or less from the stated value, for example, ±5% or less from the stated value, ±2% or less from the stated value, ±1% or less from the stated value, ±0.5% or less from the stated value, ±0.2% or less from the stated value, ±0.1% or less from the stated value, and ±0.05% or less from the stated value.

[0038] "Acryloyl" is an enone having the structure H2C=CH-C(=O)-OR (wherein R may be an alkyl chain, a phenyl chain (e.g., bisphenol), a fluorinated carbon chain, an alcohol, a glycol chain, or a siloxane chain (e.g., dimethylsiloxane, cyclosiloxane)). Acryloyl can be monofunctional or polyfunctional molecule or part of a metal complex (e.g., zirconium or hafnium complex). The acryloyl group may also be methacryloyl, having a methyl group instead of a single hydrogen atom in the C=C bond. When the term "(meth)acryloyl" is used, it means that the group may be either acryloyl or methacryloyl. (Meth)acryloyl polymers are homopolymers or copolymers of (meth)acryloyl monomer units.

[0039] A "cationically polymerizable monomer" is a monomer whose polymerization and / or crosslinking is initiated by a cation.

[0040] As used herein, the term “deposition” refers to any suitable application technique, whether manual or automated, that results in modification of surface properties in some cases. Generally, deposition can be carried out using vapor deposition techniques, coating techniques, grafting techniques, etc. Some specific examples include chemical vapor deposition (CVD), spray coating (e.g., ultrasonic spray coating), spin coating, dunk or dip coating, doctor blade coating, puddle dispensing, flow-through coating, aerosol printing, screen printing, microcontact printing, and inkjet printing.

[0041] As used herein, the terms “recess” and “nanorecess” refer to isolated recessed features in patterned resins of interpenetrating polymer networks. Nanorecesses are imprint features transferred from a work stamp during the nanoimprinting process and are therefore negative replicas of the work stamp nanofeatures. Each nanorecess has a maximum opening dimension (e.g., diameter or length depending on the shape) in the range of about 10 nm to about 1000 nm. In some examples, the maximum opening dimension is a diameter or length in the range of about 25 nm to about 750 nm, e.g., about 50 nm to about 500 nm, about 40 nm to about 400 nm, etc. The maximum opening is at least partially surrounded by a void region of the resin. Recesses may have any of the following shapes at the opening on the surface, including, for example, circular, elliptical, square, polygonal, star-shaped (with any number of vertices), etc. The cross-section of a recess perpendicular to the surface may be curved, square, polygonal, hyperbolic, conical, angular, etc.

[0042] The term "each" is intended to identify individual items within a set of items, but not necessarily all items within the set. Exceptions may be made if explicit disclosure or context clearly indicates otherwise.

[0043] The term "epoxy" (also referred to as glycidyl or oxirane group) is used herein, [ka] This refers to epoxy polymers, which are homopolymers or copolymers of epoxy monomer units.

[0044] The term "feature," as used herein, refers to a point or region in a pattern that can be distinguished from other points or regions according to its relative position. Exemplary features include recesses in patterned resin and protrusions on a work stamp. The term "nanofeature" is specifically used to describe features of a work stamp transferred onto a resin mixture. Nanofeatures are part of a pattern and, during imprinting, generate a negative replica of the pattern in the material being imprinted. For example, a nanofeature may be a nanoprotrusion that generates a nanorecess during imprinting. Each nanoprotrusion has a maximum dimension corresponding to the maximum opening dimension of the nanorecess being imprinted, which in some examples ranges from about 10 nm to about 1000 nm.

[0045] As used herein, the term “flow cell” is intended to mean a container having a chamber (e.g., a flow channel) on which a reaction can take place, an inlet for delivering reagents into the chamber, and an outlet for removing reagents from the chamber. In some examples, the chamber allows for the detection of the reaction occurring within the chamber. For example, the chamber / flow channel may include one or more transparent surfaces that allow for optical detection, such as arrays or optically labeled molecules, in nanorecesses.

[0046] A "free radical polymerizable monomer" is a monomer whose polymerization and / or crosslinking is initiated by free radicals.

[0047] As used herein, the term “independently crosslinkable monomers” refers to two or more different monomers that react and crosslink independently without crosslinking each other. Since the two or more different monomers are miscible, their respective polymerization and crosslinking reactions occur in the same phase. This ensures that the crosslinked polymers interlock and are fixed into a mutual penetration network. In contrast, in immiscible monomer systems, phase separation occurs, and the polymerization and crosslinking reactions occur in separate phases. As a result, the crosslinked polymers are physically separated into two phases.

[0048] In the examples disclosed herein, independently crosslinkable monomers can be polymerized and crosslinked simultaneously or sequentially (but in the same phase).

[0049] As used herein, the term “gap region” refers to a region on a surface (e.g., substrate, patterned resin, etc.) that separates features. For example, a gap region can separate one feature in an array from another feature in the array or pattern. The two separated features may be isolated, that is, they may not be in physical contact with each other. In another example, a gap region can separate a first part of a feature from a second part of the feature. In many examples, the gap regions are continuous, while the features are separated, for example, as in the case of multiple nanorecesses defined on another continuous surface. The separation provided by the gap region may be partial or complete. The gap region may have a different surface material than the surface material of the features defined on the surface. For example, features in a flow cell array may have polymer coatings and primers in amounts or concentrations greater than those present in the gap region. In some examples, polymer coatings and primers may not be present in the gap region.

[0050] A "penetrating polymer network" refers to two or more individual polymer networks that are at least partially intertwined with each other but are not crosslinked. These two or more individual polymer networks cannot be separated unless the chemical bonds in one or both of the individual networks are broken, because their polymer chains are mechanically connected through physical twisting and entanglement. Each individual polymer network is crosslinked, but two or more individual polymer networks are not covalently bonded to each other (e.g., not crosslinked). Penetrating polymer networks are produced from independently crosslinkable monomers. These independently crosslinkable monomers are mixed and then polymerized and crosslinked simultaneously or sequentially in the same phase so that the monomers are physically twisted together, thereby creating this polymer network. Penetrating polymer networks are distinguishable from polymer blends formed by physically mixing two (or more) polymers together. Polymers in polymer blends are not twisted together because the two polymers are mixed together after polymerization.

[0051] As used herein, the term “pitch” refers to the spacing between features. In one example, pitch refers to the distance from the center of a feature to the center of an adjacent or nearest neighboring feature. This pitch may also be called the center-to-center spacing. In another example, pitch refers to the distance from the edge of a feature to the edge of an adjacent or nearest neighboring feature. This pitch may also be called the edge-to-edge spacing.

[0052] As used herein, the term “primer” is defined as a single-stranded nucleic acid sequence (e.g., single-stranded DNA). Some primers, sometimes referred to as amplification primers, serve as starting points for template amplification and cluster generation. Other primers, sometimes referred to as sequencing primers, serve as starting points for DNA synthesis. The 5' end of a primer may be modified to allow coupling reactions with functional groups of a polymer coating. The length of a primer can be any number of bases and may contain a variety of non-natural nucleotides. For example, sequencing primers are short chains ranging from 10 to 60 bases, or from 20 to 40 bases.

[0053] Resin mixtures and interpenetrating polymer networks

[0054] As schematically shown in Figure 1, an example of an interpenetrating polymer network 10 includes two or more individual but twisted polymer networks 12 and 14. Polymer networks 12, 14, and thus the interpenetrating polymer network 10, can be formed when a resin mixture of at least two different types of monomers is cured. In the examples disclosed herein, the curing mechanism of one type of monomer is orthogonal to the curing mechanism of another type of monomer, and therefore these components can be mixed without adversely affecting the curing efficiency. The orthogonal curing mechanisms are different and do not interfere with each other.

[0055] One of the polymer networks 12 can be produced through cationic polymerization of cationic polymerizable monomers. Epoxide-containing monomers are examples of cationic polymerizable monomers. In the examples disclosed herein, the epoxide-containing monomer is a polyfunctional epoxy monomer selected from the group consisting of: i) 2,4,6,8-tetramethyl-2,4,6,8-tetrakis(propylglycidyl ether)cyclotetrasiloxane: [ka] ii) Tetrakis(epoxycyclohexylethyl)tetramethylcyclotetrasiloxane: [ka] iii) Diglycidyl ether-terminated poly(dimethylsiloxane): [ka] (In the formula, 4 <n<8); iv) Poly(propylene glycol) diglycidyl ether: [ka] (In the formula, 5 <n<10); v) 3,4-Epoxycyclohexylmethyl 3,4-Epoxycyclohexanecarboxylate: [ka] (Can be used to increase hardness); vi) Brominated bisphenol A diglycidyl ether: [ka] (Can be used for higher refractive indices); vii) Glycidyl terminal-protected poly(bisphenol A-co-epichlorohydrin) [ka] (In the formula, 0 <n<2); viii) Bisphenol A propoxylate diglycidyl ether: [ka] ix) Monophenyl-functionalized tris(epoxy-terminated polydimethylsiloxane): [ka] x) Trimethylolpropane triglycidyl ether: [ka] xi)2,2'-(2,2,3,3,4,4,5,5-octafluorohexane-1,6-diyl)bis(oxirane): [ka] (Can be used to reduce refractive index); xii) 1,3-Bis(3-glycidoxypropyl)tetramethyldisiloxane: [ka] xiii) 1,3-bis[2(3,4-epoxycyclohexa-1-yl)ethyl]tetra-methyldisiloxane: [ka] xiv) Glycidyl polyoctahedral silsesquioxane: [ka] xv) Epoxycyclohexyl polyoctahedral silsesquioxane: [ka] xvi) Tris(4-hydroxyphenyl)methane triglycidyl ether [ka] xvii) 4,4'-methylenebis(N,N-diglycidylaniline) [ka] Any combination of xviii)i) to xvii).

[0056] While several examples have been provided, it should be understood that these examples are non-limiting and any other epoxide-containing monomers that can be crosslinked may be used. For example, any polyhedral oligomeric silsesquioxane (POSS) core functionalized with epoxy groups can be used. As used herein, the term "polyhedral oligomeric silsesquioxane" (POSS) refers to a hybrid intermediate between silica (SiO2) and silicone (R2SiO) (e.g., R2SiO). 1.5 This refers to a chemical composition having the chemical formula [RSiO]. An example of a POSS may be the one described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776-778, which is incorporated herein by reference in its entirety. The composition has the chemical formula [RSiO]. 3 / 2 ] n It is an organosilicon compound having (wherein the formula, the R groups may be the same or different).

[0057] In yet another example, monofunctional epoxy monomers may be used together with polyfunctional epoxy monomers as comonomers to help adjust the overall resin properties.

[0058] Examples of such monofunctional epoxy monomers include the following molecules: i) Glycidyl 2,2,3,3-tetrafluoropropyl ether (useful for reducing the refractive index of resins): [ka] ii) Glycidyl 2,2,3,3,4,4,5,5-octafluoropentyl ether (useful for lowering the refractive index of resins): [ka] iii) (2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,9-heptadecafluorononyl)oxirane (useful for reducing the refractive index of resins): [ka] iv) Any combination of i) to iii).

[0059] While we have provided several examples, please understand that these examples are not limiting and any other monofunctional epoxy monomer can be used.

[0060] The other 14 of the polymer network is generated through free radical polymerization of free radical polymerizable monomers. The free radical polymerization rate is generally much faster than, for example, cation-initiated epoxy ring-opening polymerization. (meth)acryloyl-containing monomers are examples of free radical polymerizable monomers. In the examples disclosed herein, the (meth)acryloyl-containing monomers are polyfunctional (meth)acryloyl monomers selected from the group consisting of: i) 2,2,3,3,4,4,5,5-Octafluoro-1,6-Hexanediol diacrylate: [ka] ii) Pentaerythritol tetraacrylate: [ka] iii) Pentaerythritol triacrylate: [ka] iv) Glycerol 1,3-diglycerolate diacrylate: [ka] v) Poly(ethylene glycol) dimethacrylate: [ka] (In the formula, 8 <n<10); vi) Glycerol dimethacrylate, mixture of isomers: [ka] vii) 3-(acryloyloxy)-2-hydroxypropyl methacrylate: [ka] viii) Ethylene glycol dimethacrylate; [ka] ix) Bis(2-methacryloyl)oxyethyl disulfide [ka] x) Tricyclodecane dimethanol diacrylate [ka] xi) Bisphenol A glycerolate (1 glycerol / phenol) diacrylate [ka] xii) Bisphenol A dimethacrylate: [ka] xiii) Methacrylateoxypropyl-terminated polydimethylsiloxane: [ka] (wherein n is selected to reflect molecular weights of approximately 420 g / mol, 950 g / mol, 4,000 g / mol, 10,000 g / mol, or 25,000 g / mol for the monomer); and xiv) Zirconium bromonolbornane lactone carboxylate triacrylate: [ka] (In the equation, m is in the range of 0 to 4, n is in the range of 0 to 4, and m + n = 4); xv) Zirconium acrylate: [ka] xvi) Hafnium carboxyethyl acrylate: [ka] xvii) Acrylopolyoctahedralsilsesquioxane: [ka] xviii) Methacrylic polyoctahedral silsesquioxane: [ka] xix)2,4,6,8-tetramethyl-2,4,6,8-tetrakis(3-acryloyloxypropyl)cyclotetrasiloxane: [ka] Any combination of xx)i) to xix).

[0061] While we have provided several examples, please understand that these examples are not limiting, and any other (meth)acryloyl-containing monomers that can be crosslinked may be used.

[0062] In further examples, monofunctional (meth)acryloyl monomers may be used together with polyfunctional (meth)acryloyl monomers as comonomers to help adjust the overall resin properties. Examples of such monofunctional (meth)acryloyl monomers include the following molecules: i) Pentabromobenzyl acrylate: [ka] ii) 2,2,3,3,4,4,5,5,6,6,7,7-Dodecafluoroheptylacrylate: [ka] iii) 2-[(1',1',1'-trifluoro-2'-(trifluoromethyl)-2'-hydroxy)propyl]-3-norbornyl methacrylate: [ka] iv) 2,2,2-trifluoroethyl methacrylate: [ka] v) 2,2,3,3-tetrafluoropropyl methacrylate: [ka] vi)3-(acrylamide)phenylboronic acid [ka] vii) Hexafluoroisopropyl methacrylate: [ka] viii) Pentafluorophenylacrylate: [ka] ix) Pentafluorophenyl methacrylate: [ka] x) 1,1,1,3,3,3-Hexafluoroisopropyl acrylate: [ka] xi) 1,1,1,3,3,3-Hexafluoroisopropyl methacrylate: [ka] xii) Poly(dimethylsiloxane), monomethacrylate-terminated: [ka] (In the formula, n is selected to target any molecular weight between 250 g / mol and 50,000 g / mol); xiii) tert-butyl acrylate: [ka] xiv) tert-butyl methacrylate: [ka] xv)2-carboxyethyl acrylate oligomer: [ka] (wherein n is in the range of 0 to 3); and Any combination of xvi)i) to xv).

[0063] In one example, a cationic polymerizable monomer is a polyfunctional epoxy monomer, and a free-radical polymerizable monomer is a polyfunctional (meth)acryloyl monomer. Polyfunctional monomers contain more cites for polymerization and crosslinking. In another, more specific example, a cationic polymerizable monomer is an epoxysiloxane monomer, and a free-radical polymerizable monomer is a (meth)acryloyl monomer.

[0064] Each curing mechanism discussed (cationic and free radical) can be initiated by exposure to ultraviolet (UV) light. In some examples, alternative UV-induced curing mechanisms may be used, for example, thiol-ene chemicals may be used instead of the radical-initiating monomers disclosed herein, since this type of chemical also targets free radical polymerization. Some exemplary vinyl molecules that associate with thiol-ene chemicals include: i) Vinyl-terminated polydimethylsiloxane: [ka] (wherein n is selected to reflect molecular weights of approximately 800 g / mol, 9,400 g / mol, 28,000 g / mol, 49,500 g / mol, 117,000 g / mol, or 155,000 g / mol for the monomer); ii) Bis(divinyl)-terminated polydimethylsiloxane: [ka] iii) Pentaerythritol tetrakis(3-mercaptopropionate): [ka] iv) 2,2'-thiodiethanethiol: [ka] v) 1,5-pentanedithiol: [ka] vi) Octavinyl polyoctahedral silsesquioxane: [ka] Any combination of vii)i)~vi).

[0065] Referring here to Figure 2, an example of the method is shown at reference no. 100. As illustrated, method 100 involves mixing a cationic polymerizable monomer (which may be a polyfunctional monomer alone or a mixture of at least one polyfunctional monomer and one or more monofunctional or polyfunctional monomers) with a free radical polymerizable monomer (which may be a polyfunctional monomer alone or a mixture of at least one polyfunctional monomer and one or more monofunctional or polyfunctional monomers) to form a resin mixture that is a precursor to an interpenetrating polymer network incorporated into a flow cell (reference no. 102); the refractive index of the interpenetrating polymer network, the absorption of the interpenetrating polymer network, the interpenetration The method includes adjusting the weight ratio of the cationic polymerizable monomer to the free radical polymerizable monomer using at least one property to be imparted to the resin mixture or the interpenetrating polymer network, selected from the group consisting of the hardness of the polymer network, the thickness of the interpenetrating polymer network, the hydrophilic / hydrophobic balance of the interpenetrating polymer network, the viscosity of the resin mixture, the surface chemical compatibility between the resin mixture and the work stamp, the surface chemical compatibility of the interpenetrating polymer network, the shrinkage of the interpenetrating polymer network, and combinations thereof (reference no. 104); and patterning the resin mixture to form the interpenetrating polymer network (reference no. 106).

[0066] Any example of cationic polymerizable monomers and free radical polymerizable monomers disclosed herein may be used.

[0067] Generally, the weight ratio of cationic polymerizable monomers to free radical polymerizable monomer resin mixtures is in the range of about 10:90 (1:9) to about 90:10 (9:1). In the examples disclosed herein, the weight ratios of these monomers may be adjusted to obtain predetermined properties of the resin mixture and / or predetermined properties of the resulting interpenetrating polymer network 10. The predetermined properties of the resin mixture may be viscosity and / or wettability by a working stamp and / or substrate (e.g., a flow cell substrate). The predetermined properties of the resulting interpenetrating polymer network 10 may be hydrophilic / hydrophobic balance, thickness, refractive index, absorption, surface properties (e.g., tackiness, chemical resistance, etc.), shrinkage, and / or hardness. The autofluorescence of the interpenetrating polymer network 10 can also be adjusted or eliminated by selecting a specific initiator for the resin mixture.

[0068] The effect of weight ratio on specific properties depends in part on the properties of the monomer and the monomer used.

[0069] Figures 5A and 5B (both of which will be discussed in detail in the Examples section) show the mixed epoxy monomers: [ka] and one acryloyl monomer: [ka] The effect of the weight ratio of the resin mixture and the resulting interpenetrating polymer network 10 on the refractive index of both (Figure 5A), and on the thickness of the resin mixture layer and the resulting interpenetrating polymer network 10 (Figure 5B) is shown. With this particular monomer combination, the refractive index of the resin mixture and the resulting interpenetrating polymer network 10 can be kept at a desirablely low level when the ratio of the cationic polymerizable monomer (epoxy) to the free radical polymerizable monomer (acryloll) is in the range of about 10:90 (1:9) to about 50:50 (1:1). With this particular monomer combination, the thickness of the resin mixture and the resulting interpenetrating polymer network 10 generally decreases as the epoxy content decreases.

[0070] To achieve desirable surface properties (e.g., tackiness, chemical resistance, etc.) for the interpenetrating polymer network 10, each selected monomer may have the same or similar properties so that these properties are imparted to the network 10. In this example, the weight ratio may be within 10:90 (1:9) to approximately 90:10 (9:1).

[0071] Regarding shrinkage, (meth)acrylo-based monomers shrink significantly more than epoxy-based materials. As a result, a larger amount of epoxy material may be used to limit the shrinkage of the final interpenetrating polymer network 10.

[0072] Regarding hardness, some epoxy monomers are harder than some (meth)acrylo monomers. Therefore, the weight ratio depends on the hardness of the individual monomers and the desired hardness of the final interpenetrating polymer network 10.

[0073] Multiple properties that may affect the weight ratio can also be targeted. For example, it may be desirable to target a low refractive index of the interpenetrating polymer network 10 and good wettability by the work stamp. For a low refractive index, a fluorinated monomer may be selected, but this may adversely affect the wettability of the resin mixture by the work stamp. In this example, the second monomer and its amount may be selected to restore some of the compatibility. The second monomer may be present in an amount of at least 33% by weight.

[0074] To make the resin mixture imprintable (imprintable), the weight ratio of cationic polymerizable monomers to free radical polymerizable monomers may be adjusted. The imprintable resin can conform to the features of the applied work stamp, which, upon curing, can be fixed to the structure of the applied work stamp features, and can be cleanly peeled off from the work stamp after curing. Therefore, reference no. 104 of Method 100 may include adjusting the weight ratio of cationic polymerizable monomers to free radical polymerizable monomers to make the resin mixture imprintable by a work stamp. When adjusting the weight ratio of monomers to make the resin mixture imprintable by a work stamp, the material of the work stamp may be taken into consideration. For example, the material of the work stamp may affect the wettability of the resin mixture to the work stamp, as well as the peelability of the resulting interpenetrating polymer network 10.

[0075] Therefore, monomers and their associated weight ratios may be selected to be compatible with the work stamp, or the work stamp may be selected to be compatible with the monomers and their associated weight ratios. In the latter example, the weight ratios may be adjusted to achieve one or more target properties, and then the chemical properties of the work stamp may be selected to suitably imprint the resin mixture. Furthermore, imprintability may also be improved by other factors such as increased UV exposure (for resins with a higher degree of curing) and / or the addition of leveling agents to the resin mixture.

[0076] In the following examples, the work stamp material is a silicon-based material such as polymerized silicon acrylate or methacrylate. In one example of a resin mixture imprintable with these work stamps, the polyfunctional epoxy monomer is poly(propylene glycol) diglycidyl ether and the polyfunctional acryloyl monomer is 2,2,3,3,4,4,5,5-octafluoro-1,6-hexanediol diacrylate, with a given weight ratio of approximately 50:50 (1:1). In another example of a resin mixture imprintable with these work stamps, the polyfunctional epoxy monomer is poly(propylene glycol) diglycidyl ether and the polyfunctional acryloyl monomer is glycerol 1,3-diglycerolate diacrylate, with a given weight ratio ranging from approximately 25:75 (1:3) to approximately 75:25 (3:1).

[0077] For some (meth)acryloyl monomers, the maximum weight ratio is 1:1, and therefore, other examples contain more epoxy monomer and less (meth)acryloyl monomer. This may be due to the fact that epoxy monomers can wet the working stamp material better than (meth)acryloyl monomers.

[0078] In addition to cationic polymerizable monomers and free radical polymerizable monomers (present in desired weight ratios), examples of resin mixtures disclosed herein may also include cationic photoinitiators and free radical photoinitiators. Each photoinitiator may be used in an amount ranging from about 0.25% to about 10% by weight relative to the monomer it initiates. In other examples, each photoinitiator may be present in the mixture in an amount ranging from about 0.5% to about 9.5% by weight relative to the monomer it initiates, for example, from about 1% to about 5% by weight.

[0079] Cationic photoinitiators can be used to initiate the curing of cationic polymerizable monomers. The cationic photoinitiator may be a system comprising a photoacid generator (which is a cationic compound) and a sensitizer (which assists in the formation of the cationic compound). These two compounds can be used in a 1:1 weight ratio or in several other suitable weight ratios. Each of these two compounds may be present in the amounts provided herein for the photoinitiator. Suitable cationic compounds (photoacid generators) include any of the various known useful materials such as onium salts, certain organometallic complexes, and mixtures thereof. Some specific examples of suitable cationic photoinitiators include N-hydroxynaphthalimide triflate; mixed triarylsulfonium hexafluorophosphate; mixed triarylsulfonium hexafluoroantimonate; 1-naphthyldiphenylsulfonium triflate; 4-phenylthiophenyl)diphenylsulfonium triflate; bis-(4-methylphenyl)iodonium hexafluorophosphate; bis(4-tert-butylphenyl)iodonium hexafluorophosphate; (2-methylphenyl)(2,4,6-trimethylphenyl)iodonium triflate; bis(2,4,6-trimethylphenyl)iodonium triflate; bis-(4-dedecylphenyl)iodonium hexafluoroantimonate; and combinations thereof.

[0080] Free radical photoinitiators can be used to initiate the curing of free radical polymerizable monomers. Suitable free radical photoinitiators include benzoin ethers (e.g., benzoin methyl ether and benzoin isopropyl ether), substituted benzoin ethers (e.g., anisoin methyl ether), substituted acetophenones (e.g., 2,2-diethoxyacetophenone and 2,2-dimethoxy-2-phenylacetophenone), substituted alpha-ketols (e.g., 2-methyl-2-hydroxypropiophenone), and aromatic phosphine oxides (e.g., diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide). Examples include diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide blends with 2-hydroxy-2-methylpropiophenone; phenylbis(2,4,6-,trimethylbenzoyl)phosphine oxide; and ethyl(2,4,6-trimethylbenzoyl)phenylphosphine), aromatic sulfonyl chlorides (e.g., 2-naphthalene-sulfonyl chloride), photoactive oximes (e.g., 1-phenyl-1,2-propanedione-2(O-ethoxycarbonyl)oxime), and mixtures thereof.

[0081] The resin mixture may also contain a solvent so that it can be deposited onto a substrate for imprinting and curing. The resin mixture may be diluted with a solvent to achieve the desired viscosity in the deposition technique used and / or to achieve a target thickness of at least substantially uniform resin layer. Examples of suitable solvents include, for example, propylene glycol monomethyl ether acetate (PGMEA), toluene, dimethyl sulfoxide (DMSO), and tetrahydrofuran (THF). In one example, the total concentration of monomers in the solvent is in the range of about 15% by weight (wt%) to about 56% by weight (when monomers are present in the desired weight ratio), and the total concentration of photoinitiators in the solvent is in the range of about 1% by weight to about 10% by weight, although the upper limits may be higher depending on the solubility of the monomers and photoinitiators in the selected solvent.

[0082] Referring here to Figures 3A to 3C, an example of a method for producing a patterned interpenetrating polymer network 10 is shown. This method includes applying a resin mixture to a substrate, wherein the resin mixture contains two independently crosslinkable monomers present in the resin mixture 16 in a predetermined weight ratio, the first of the two independently crosslinkable monomers being a cationic polymerizable monomer and the second of the two independently crosslinkable monomers being a free radical polymerizable monomer; imprinting the resin mixture 16 with a work stamp 20 having a plurality of nanofeatures 22; curing the resin mixture 16 while the work stamp 20 is in a predetermined position; and thereby forming an interpenetrating polymer network (10) imprinted in the nanorecesses of a flow cell.

[0083] Figure 3A shows the application of the resin mixture 16 to the substrate 18. Any example of the resin mixture 16 described herein may be used. The substrate 18 used may depend on the type of flow cell to be formed.

[0084] In some examples, the substrate is epoxysiloxane, glass and modified or functionalized glass, plastics (acrylic, polystyrene, and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, polytetrafluoroethylene (e.g., TEFLON® from Chemors), cyclic olefin / cycloolefin polymer (COP) (e.g., ZEONOR® from Zeon), polyimide, etc.), nylon, ceramic / ceramic oxide, silica, fused silica, or silica-based materials, aluminum silicate, silicon and modified silicon (e.g., boron-doped p+ silicon), silicon nitride (Si3N4), silicon oxide (SiO2), tantalum pentoxide (Ta2O5), or other tantalum oxides (TaO2O5). x ), hafnium oxide (HaO2), carbon, metal, inorganic glass, etc. may be used.

[0085] The substrate may also have a multilayer structure. Some examples of multilayer structures include glass or silicon having a coating layer of tantalum oxide or another metal oxide on its surface. Another example of a multilayer structure is a base support (e.g., glass or silicon) having a patterned resin on it. Yet another example of a multilayer substrate is a silicon-on-insulator (SOI) substrate. Another example of a multilayer substrate includes a planar waveguide. As one example, a glass base may support a grating layer (laser-coupled grating) and a planar waveguide layer.

[0086] It is desirable that the lattice layer has a refractive index in the range of about 1.35 to about 1.55, and the lattice layer can be produced using a resin having a suitable refractive index. In one example, a fluorinated monomer may be included in the resin to achieve a refractive index in the range of about 1.35 to about 1.4. In another example, a slightly fluorinated or siloxane-based monomer may be included in the resin to achieve a refractive index in the range of about 1.4 to about 1.45. Still otherwise, the content of the siloxane-based monomer in the resin may be reduced to achieve a refractive index in the range of about 1.45 to about 1.5.

[0087] It is desirable that the planar waveguide layer has a refractive index greater than that of the patterned interpenetrating polymer network 10. In some examples, it is desirable for the planar waveguide to have a refractive index within the range of about 1.6 to about 2.6, and a resin with a suitable refractive index can be used to generate the planar waveguide layer. A highly transparent polymer having a refractive index in the range of about 1.6 to about 1.65 can be used for the planar waveguide and can be deposited using nanoimprint lithography. A highly transparent monomer having a high polarization rate and / or bromide content and having a refractive index in the range of about 1.65 to about 1.8 can be used for the planar waveguide and can be deposited using nanoimprint lithography. A highly transparent metal oxide having a refractive index greater than 1.8 can be used for the planar waveguide and can be deposited using sputtering. As examples of metal oxides, the following may be used: zinc oxide (ZnO) having a refractive index of about 2.00; tantalum pentoxide (TaO5) having a refractive index of about 2.3; zirconium dioxide (ZrO2) having a refractive index of about 2.14; or titanium dioxide (TiO2) having a refractive index of about 2.64.

[0088] In any of the examples disclosed herein, the substrate can have a diameter in the range of about 2 mm to about 300 mm, or a rectangular sheet or panel having a maximum dimension of up to about 10 feet (≈3 meters). In one example, the substrate is a wafer having a diameter in the range of about 200 mm to about 300 mm. In another example, the substrate is a die having a width in the range of about 0.1 mm to about 10 mm. Although exemplary dimensions have been provided, it should be understood that substrates having any suitable dimensions can be used. In another example, a panel, which is a rectangular support having a larger surface area than a 300 mm round wafer, can be used.

[0089] The application of the resin mixture 16 to the substrate 18 can be carried out using any of the deposition techniques described herein. After depositing the resin mixture 16, it may be soft baked to remove excess solvent.

[0090] Figure 3B shows an imprint of the resin mixture 16 by the working stamp 20 having a plurality of nanofeatures 22. The pattern of the nanofeatures 22 of the working stamp 20 is a negative replica of the desired features (e.g., nano recesses 24 (see FIG. 3C)) formed in the cured patterned resin 28. The size and shape of the nanofeatures 22 depend on the desired size and shape at the nano recesses 24.

[0091] Press the working stamp 20 onto the resin mixture 16 to create an imprint on / into the resin mixture. As shown in FIG. 3B, the working stamp 20 is held in a predetermined position during curing.

[0092] Curing can be performed by exposing to actinic rays such as ultraviolet rays (UV) using, for example, a metal halide light source, a mercury vapor light source, a UV-emitting LED light source, etc. Due to the presence of each photoinitiator, curing promotes the formation of cations and radicals, and these cations and radicals are used to cure the epoxy and (meth)acryloyl moieties of the resin mixture 16, respectively. Thus, curing promotes the separate polymerization and / or crosslinking of the monomers in the resin mixture 16.

[0093] Curing may include a single UV exposure step or may include multiple steps including a soft bake (e.g., to remove solvents) followed by UV exposure. If included, the soft bake may be performed at a lower temperature in the range of about 50 °C to about 150 °C for more than 0 seconds to about 3 minutes and may be performed before placing the working stamp 20 on the resin mixture 16. In one example, the soft bake time is in the range of about 30 seconds to about 2.5 minutes.

[0094] Some multi-stage curing processes may also include hard baking. However, the curing mechanism of the resin mixture 16 disclosed herein is so fast that the resin mixture 16 can reach maximum curing without hard baking. If implemented, for example, the work stamp 20 is peeled / released before hard baking so that it does not bond to the cured patterning resin 28. If implemented, the duration of hard baking may be about 5 seconds to about 10 minutes at a temperature in the range of about 60°C to about 300°C. Hard baking may be performed, for example, to remove residual solvent, to further polymerize some of the material of the resin mixture 16 (thereby increasing the degree of curing and reaching an acceptable layer hardness), and / or to assist in fixing in the imprinted topography. Examples of equipment that can be used for soft baking and / or hard baking include hot plates, ovens, etc.

[0095] As shown in Figure 3C, the work stamp 20 is peeled off after curing. Curing forms a cured patterned resin 28. The chemical structure of the cured patterned resin 28 is an interpenetrating polymer network 10. The chemical structure of the interpenetrating polymer network 10 depends on the composition of the resin mixture 16. In one example, the cured patterned resin 28 of the interpenetrating polymer network 10 may have a weight ratio of epoxy polymer to (meth)acryloyl polymer in the range of 25:75 to about 75:25. In one example, the thickest part of the cured patterned resin is in the range of about 225 nm to about 600 nm.

[0096] As shown in Figure 3C, the cured patterned resin 28 includes nano-recesses 24 defined therein and gap regions 26 separating adjacent nano-recesses 24.

[0097] The nanorecess 24 is a negative replica of the nanofeature 22 of the work stamp. Examples of resin mixtures disclosed herein can be formulated so that their nanofeature 22 is successfully imprinted on the work stamp used to produce the nanorecess 24 disclosed herein.

[0098] Many different layouts of the nano-recesses 24 can be envisioned, including regular, repetitive, and irregular patterns. In one example, the nano-recesses 24 are arranged in a hexagonal lattice for close packing and improved density. Other layouts can include, for example, a linear (i.e., rectangular) layout (e.g., lines or grooves), a triangular layout, and the like. In some examples, the layout or pattern can be an x-y configuration of the nano-recesses 24 forming rows and columns. In some other examples, the layout or pattern can be a repetitive arrangement of the nano-recesses 24 and / or the gap regions 26. In still other examples, the layout or pattern can be a random arrangement of the nano-recesses 24 and / or the gap regions 26.

[0099] The layout or pattern can be characterized in terms of the density of the nano-recesses 24 within a defined area (i.e., the number of nano-recesses 24). For example, the nano-recesses 24 can be present at a density of about 200 million per mm 2 . The density can be adjusted to various densities, including, for example, approximately about 100 per mm 2 , about 1,000 per mm 2 , about 100,000 per mm 2 , about 1 million per mm 2 , about 2 million per mm 2 , about 5 million per mm 2 , about 10 million per mm 2 , about 50 million per mm 2 . It should be further understood that the density of the nano-recesses 24 in the cured patterned resin 28 can be between one of the lower values selected from the above ranges and one of the higher values. As an example, a high-density array can be characterized as having nano-recesses 24 spaced less than about 100 nm apart, a medium-density array can be characterized as having nano-recesses 24 spaced about 400 nm to about 1 μm apart, and a low-density array can be characterized as having nano-recesses 24 spaced more than about 1 μm apart. Although examples of density have been provided, it should be understood that a substrate having any suitable density can be used.

[0100] The layout or pattern of the nanorecesses 24 can also be characterized in terms of average pitch, either further or alternatively. The pattern may be regular such that the coefficient of variation around the average pitch is small, or the pattern may be irregular, in which case the coefficient of variation may be relatively large. In either case, the average pitch may be, for example, approximately 50 nm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, or 100 μm. The average pitch of a particular pattern of nanorecesses 24 may be between one of the lower values ​​and one of the higher values ​​selected from the above range. In one example, the nanorecesses 24 have a pitch (center-to-center spacing) of approximately 1.5 μm. While examples of average pitch values ​​are provided, it should be understood that other average pitch values ​​may also be used.

[0101] The size of each nanorecess 24 can be characterized by its volume, well opening area, depth, and / or diameter.

[0102] Each nanorecess 24 may have an arbitrary volume capable of confining at least some fluid introduced into the flow cell (reference number 30 in Figure 4A). The minimum or maximum volume may be selected to correspond, for example, to throughput (e.g., multiplicity), resolution, nucleotides, or the reactivity of the analyte expected to be used downstream of the flow cell 30. For example, the volume may be approximately about 1 × 10⁻⁶. -3 μm 3 , at least about 1 × 10 -2 μm 3 at least about 0.1 μm 3 , at least about 1 μm 3 , at least about 10 μm 3 , at least about 100 μm 3 That's fine.

[0103] The area occupied by the opening of each nanorecess may be selected based on the same criteria as those described above for volume. For example, the area of ​​the opening of each nanorecess is at least approximately 1 × 10⁻⁶. -3 μm 2 , at least about 1 × 10-2 μm 2 at least about 0.1 μm 2 , at least about 1 μm 2 , at least about 10 μm 2 , at least about 100 μm 2 , or more. Alternatively or further, the area may be at most about 1 × 10 3 μm 2 up to approximately 100 μm 2 up to approximately 10 μm 2 up to approximately 1 μm 2 up to approximately 0.1 μm 2 Up to approximately 1 x 10 -2 μm 2 or less. The area occupied by the opening of each recess may be greater than, less than, or somewhere between the above values.

[0104] The depth of each nanorecess 24 may be large enough to accommodate a polymer hydrogel (see Figure 4B, described below). In one example, the depth may be at least about 0.1 μm, at least about 0.5 μm, at least about 1 μm, at least about 10 μm, at least about 100 μm, or more. Alternatively or further, the depth may be up to about 1 × 10⁻⁶ 3 The depth can be μm, up to approximately 100 μm, up to approximately 10 μm, or less. In some examples, the depth is approximately 0.4 μm. The depth of each recess 24 may be greater than, less than, or in between the values ​​specified above.

[0105] In some cases, the diameter or length and width of each nanorecess 24 may be in the range of about 10 nm to about 1000 nm. For example, the diameter or length and width of each nanorecess 24 may be about 50 nm, about 0.1 μm, about 0.5 μm, or about 1 μm. Some resins may be imprinted with larger recesses having, for example, a diameter or length and width of about 10 μm, about 100 μm, or more. In some examples of nanorecess 24, the diameter or length and width is about 0.4 μm.

[0106] Flow Cell

[0107] As described above, a substrate 18 having a patterned curing resin 28 thereon can be incorporated into a flow cell 30. An example of a flow cell 30 is shown in Figure 4A. As will be discussed with reference to Figure 4B, some examples of flow cells 30A include two opposing sequencing surfaces 32, 34. In other examples, a flow cell 30B comprises one sequencing surface 32 supported by the substrate 18 and an opposing lid 36 attached to the substrate 18.

[0108] The flow cell 30 includes flow channels 38. While several flow channels 38 are shown in Figure 4A, it should be understood that any number of channels 38 can be included in the flow cell 30 (e.g., one channel, four channels, etc.). In some examples disclosed herein (Figure 4B), each flow channel 38 is a region defined between two opposing sequencing surfaces 32, 34. In other examples disclosed herein (Figure 4C), each flow channel 38 is a region defined between one sequencing surface (e.g., 32) and an opposing lid 36. Fluid can be introduced into and removed from the flow channels 38. Each flow channel 38 may be separated within the flow cell 30 so that fluid introduced into any particular flow channel 38 does not flow into any adjacent flow channel 38.

[0109] In one example, the flow channel 38 has a rectangular configuration. The length and width of the flow channel 38 may be smaller than the length and width of the substrate 18, respectively, so that a portion (e.g., surface 40) of the cured patterned resin 28 (on the substrate 18) surrounding the flow channel 38 is available for attachment to another substrate 18 or lid 36. In some cases, the width of each flow channel 38 may be approximately 1 mm, 2.5 mm, 5 mm, 7 mm, or 10 mm. The width and / or length of each flow channel 38 may be greater than, less than, or in between the values ​​specified above. In another example, the flow channel 38 is square (e.g., 10 mm × 10 mm).

[0110] The depth of each flow channel 38 may be as thin as the thickness of a few monolayers, for example, when depositing a separate material that defines the walls of the flow channel using microcontact, aerosol, or inkjet printing. In other examples, the depth of each flow channel 38 may be about 1 μm, about 10 μm, about 50 μm, about 100 μm, or more. In one example, the depth may be in the range from about 10 μm to about 100 μm. In another example, the depth is about 5 μm or less. It should be understood that the depth of each flow channel 38 may be greater than, less than, or between the values specified above. The depth of the flow channel 38 may also vary along the length and width of the flow cell 30, for example, when using the patterned sequencing surfaces 32, 34.

[0111] FIG. 4B shows a cross-sectional view of a flow cell 30A including the patterned opposing sequencing surfaces 32, 34. In one example, each of these surfaces 32, 34 may be prepared in the nano-recesses 24, 24' of the cured patterned resins 28, 28' on the substrates 18, 18'. The substrates 18, 18' may be attached to each other (e.g., through the surfaces 40, 40') to form an example of the flow cell 30A. Any suitable bonding material 42 such as an adhesive, a radiation-absorbing material that aids in bonding, etc. can be used to bond the substrates 40, 40' to each other.

[0112] The sequencing surfaces 32, 34 include the polymer hydrogels 44, 44' and the amplification primers 46, 46' that are bound to the polymer hydrogels 44, 44'.

[0113] Examples of the polymer hydrogels 44, 44' include acrylamide copolymers such as poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide, PAZAM. PAZAM and several other forms of acrylamide copolymers are represented by the following structure (I):

Chemical formula

[0114] Those skilled in the art will recognize that the arrangement of n and m repeating features in structure (I) is typical, and that monomer subunits can exist in any order within the polymer structure (e.g., random, blocky, patterned, or a combination thereof).

[0115] The molecular weight of PAZAM and other forms of acrylamide copolymers may range from about 5 kDa to about 1500 kDa or from about 10 kDa to about 1000 kDa, or in certain cases, it may be about 312 kDa.

[0116] In some cases, PAZAM and other forms of acrylamide copolymers are linear polymers. In other cases, PAZAM and other forms of acrylamide copolymers are lightly crosslinked polymers.

[0117] In other examples, polymer hydrogels 44, 44' can be variations of structure (I). In one example, the acrylamide unit is N,N-dimethylacrylamide. [ka] It can be replaced by: In this example, the acrylamide unit of structure (I) is [ka] It can be replaced by, in the formula, R D , R E , and R F Each is either H or C1-C6 alkyl, and R G and R H Each of these is a C1-C6 alkyl group (not H as in the case of acrylamide). In this example, q may be an integer in the range of 1 to 100,000. In another example, in addition to the acrylamide unit, N,N-dimethylacrylamide may be used. In this example, structure (I) has repeating "n" and "m" features in addition [ka] It may include, in the formula, R D , R E , and R F Each is either H or C1-C6 alkyl, and R G and R H Each of these is a C1-C6 alkyl group. In this example, q may be an integer in the range of 1 to 100,000.

[0118] As yet another example, polymer hydrogels 44, 44' may contain repeating units of each structure (III) and (IV), [ka] In the formula, R 1a , R 2a , R 1b and R 2b Each of these is independently selected from hydrogen, optionally a substituted alkyl group, or optionally a substituted phenyl group, and R 3a and R 3bEach of these is independently selected from hydrogen, optionally substituted alkyl, optionally substituted phenyl, or optionally substituted C7-C14 aralkyl, L 1 and L 2 Each of these is selected independently of an optional substituted alkylene linker or an optional substituted heteroalkylene linker.

[0119] Furthermore, it should be understood that other molecules may be used to form polymeric hydrogels 44, 44', insofar as they are functionalized to graft oligonucleotide primers 46, 46'. Other examples of suitable polymer layers include colloidal structures such as agarose, polymer mesh structures such as gelatin, or crosslinked polymer structures such as polyacrylamide polymers and copolymers, silane-free acrylamide (SFA), or azide-degraded versions of SFA. Examples of suitable polyacrylamide polymers can be synthesized from acrylamide and acrylic acid or acrylic acid containing vinyl groups, or from monomers that form a [2+2] photoaddition cycloaddition reaction product. Yet another example of a suitable polymeric hydrogel 44, 44' is a mixed copolymer of acrylamide and acrylate. In the examples disclosed herein, a variety of polymer structures containing acrylic monomers (e.g., acrylamide, acrylate, etc.) may be used, such as star-shaped polymers, star-shaped or star-shaped block polymers, and branched polymers including dendrimers. For example, monomers (such as acrylamide) can be incorporated into the branches (arms) of a star-shaped polymer, either randomly or in blocks.

[0120] To introduce polymer hydrogels 44, 44' into nano-recesses 24, 24', a mixture of polymer hydrogels 44, 44' may be prepared and then applied to the cured patterned resins 28, 28'. In one example, the polymer hydrogels 44, 44' may be present in a mixture (e.g., a mixture with water, or a mixture of ethanol and water). The mixture may then be applied to the respective cured patterned resins 28, 28' (including the nano-recesses 24, 24') using spin coating, immersion or dip coating, or material flow under positive or negative pressure, or another preferred technique. These types of techniques deposit the polymer hydrogels 44, 44' onto the cured patterned resins 28, 28' (e.g., onto the nano-recesses 24, 24', as well as the adjacent void regions 26, 26' and surfaces 40, 40'). It is also possible to use other selective deposition techniques (e.g., masks, controlled printing techniques, etc.) to specifically deposit polymer hydrogels 44, 44' within the nano-recesses 24, 24' while preventing deposition on the interstitial regions 26, 26' and surfaces 40, 40'.

[0121] In some examples, the cured patterned resin surface (including nanorecesses 24, 24') may be activated, and then a mixture (including polymer hydrogels 44, 44') may be applied thereto. In one example, a silane or silane derivative (e.g., norbornenesilane) may be deposited on the cured patterned resin surface using vapor deposition, spin coating, or other deposition methods. In another example, the cured patterned resin surface may be exposed to plasma ashing to generate surface activators (e.g., -OH groups) that can adhere to the polymer hydrogels 44, 44'.

[0122] Depending on the chemical properties of the polymer hydrogels 44 and 44', the applied mixture may be subjected to a curing process. In one example, curing can be carried out at a temperature ranging from room temperature (e.g., about 25°C) to about 95°C over a period of time ranging from about 1 millisecond to about several days.

[0123] Polishing may be performed to leave the polymer hydrogel 44, 44' on the surface of the nano-recesses 24, 24' at least substantially intact, and to remove the polymer hydrogel 44, 44' from the gap regions 26, 26' surrounding the nano-recesses 24, 24'.

[0124] The sequencing surfaces 32, 34 also contain amplification primers 46, 46' bound to the polymer hydrogels 44, 44'.

[0125] A grafting process may be performed to graft the amplification primers 46, 46' into the polymer hydrogels 44, 44' in the nanorecesses 24, 24'. In one example, the amplification primers 46, 46' can be immobilized onto the polymer hydrogels 44, 44' by a single covalent bond at or near the 5' end of the primers 46, 46'. This bond leaves i) an adapter-specific portion of the primers 46, 46' that is free to anneal to sequencing-ready nucleic acid fragments of its cognate, and ii) a 3' hydroxyl group that is free to extend the primer. Any suitable covalent bond can be used for this purpose. Examples of terminal primers that can be used include alkyne-terminated primers (e.g., that can bind to the azide surface portion of the polymer hydrogels 44, 44') or azide-terminated primers (e.g., that can bind to the alkyne surface portion of the polymer hydrogels 44, 44').

[0126] Specific examples of suitable primers 46, 46' include P5 and P7 primers used on the surface of flow cells commercially available from Illumina Inc. for sequencing on HISEQ®, HISEQX®, MISEQ®, MiNISeq®, NextSeq®, NovaSeq®, Genome Analyzer®, ISEQ®, and other instrument platforms. Both P5 and P7 primers can be grafted onto polymer hydrogels 44, 44', respectively.

[0127] In one example, grafting may involve flow-through deposition (e.g., using a temporarily bonded lid), dunk coating, spray coating, puddle dispensing, or another preferred method for attaching primers 46, 46' to polymer hydrogels 44, 44'. Each of these exemplary techniques may utilize a primer solution or mixture that may include primers 46, 46', water, buffer, and catalyst. Using any of the grafting methods, primers 46, 46' react with the reactive groups of polymer hydrogels 44, 44' in the nanorecesses 24, 24' and have no affinity for the surrounding cured patterned resin 28, 28'. Thus, primers 46, 46' selectively graft onto polymer hydrogels 44, 44'.

[0128] As shown in Figure 4B, the substrates 18, 18' are bonded to each other via cured patterned resins 28, 28' such that the sequencing surfaces 32, 34 face each other with flow channels 38 defined between them.

[0129] The cured patterned resins 28, 28' can be bonded to each other in part or all of the gap regions 26, 26' (surrounding surfaces 40, 40', etc.). The bond formed may be a chemical bond or a mechanical bond (e.g., using fasteners).

[0130] The surfaces 40, 40' can be bonded to each other using any suitable technique, such as laser bonding, diffusion bonding, anodic bonding, eutectic bonding, plasma-activated bonding, glass frit bonding, or other methods known in the art. In one example, the surfaces 40, 40' can be bonded using a spacer layer (e.g., material 42). The spacer layer may be any material 42 that seals together at least some portions of the surfaces 40, 40'. In some examples, the spacer layer may be a radiation-absorbing material that aids in bonding.

[0131] In the example shown in Figure 4C, the flow cell 30B comprises a planar waveguide stack 48 positioned between the substrate 18 and the cured patterned resin 28. In other examples, the planar waveguide stack 48 may be similar to the example shown in Figure 4B, and therefore may comprise two opposing planar waveguide stacks 48, one of which relates to the respective sequencing surfaces 32, 34. The planar waveguide stack 48 may comprise a grid layer 50 positioned on the substrate 18 and a planar waveguide layer 52 positioned on the grid layer 50. Any example of the grid layer 50 and the planar waveguide layer 52 disclosed herein may be used. In one example, the refractive index of the interpenetrating polymer network of the cured patterned resin 28 is in the range of about 1.34 to about 1.50, and the refractive index of the planar waveguide layer 52 is in the range of about 1.6 to about 2.5. These refractive indices are carefully controlled to: i) obtain light propagation through the planar waveguide layer 52 through total internal reflection at the boundary with the lattice layer 50; ii) adjust the penetration depth of evanescent waves into the patterned resin 28 (and specifically reach the bottom of the nanorecesses 24); and iii) efficiently guide the light into the planar waveguide layer 52 after impacting the lattice layer 50. It is also desirable to prevent the cured patterned resin 28 from becoming an output coupler. This can be achieved by matching the refractive index of the imaging buffer (introduced, for example, in the nanorecesses 24, 24') with the refractive index of the cured patterned resin 28.

[0132] The sequencing surface 32 (including polymer hydrogel 44 and amplification primer 46) is formed in the nano-recesses 24 of the cured patterned resin 28.

[0133] In this example, the lid 36 is bonded to the surrounding surface 40 of the cured patterned resin 28, as described with reference to Figure 4B.

[0134] Sequencing method

[0135] Flow cells 30, 30A, and 30B, examples of which can be used in ensemble sequencing techniques such as synthesis-based sequencing (SBS), can be used in ensemble sequencing. In ensemble sequencing, the template polynucleotide chain to be sequenced (not shown) can be formed on flow cells 30, 30A, and 30B using primers 46, 46'. At the beginning of template polynucleotide chain formation, a library template can be prepared from any nucleic acid sample (e.g., a DNA or RNA sample). The nucleic acid sample can be fragmented into single-stranded DNA or RNA fragments of similar size (e.g., less than 1000 bp). During preparation, adapters can be added to the ends of these fragments. Through reduced-cycle amplification, various motifs such as sequencing binding sites, indices, and regions complementary to primers 46, 46' in the nanorecesses 24, 24' can be introduced into the adapters. The final library template contains the DNA or RNA fragments and adapters at both ends. In some examples, fragments from a single nucleic acid sample have the same adapters added to the fragments.

[0136] Multiple library templates may be introduced into flow cells 30, 30A, and 30B. The multiple library templates may, for example, hybridize to one of two types of primers 46, 46' immobilized within nanorecesses 24, 24'.

[0137] Next, cluster generation can be performed. In one example of cluster generation, the library template is copied from hybridized primers by 3′ extension using high-fidelity DNA polymerase. The original library template is denatured, leaving a copy immobilized in the nanorecesses 24, 24'. Isothermal bridging amplification or some other form of amplification can be used to amplify the immobilized copy. For example, the copied template loops over and hybridizes to adjacent complementary primers 46, 46', and the polymerase copies the copied template to form a double-stranded bridge, which is denatured to form two single strands. These two strands loop over and hybridize to adjacent complementary primers 46, 46', are extended again to form two new double-stranded loops. This process is repeated for each template copy by isothermal denaturation and amplification cycles to create dense clonal clusters. Each cluster of double-stranded bridges is denatured. In one example, the reverse strand is removed by specific base cleavage, leaving a forward template polynucleotide chain. As a result of clustering, several template polynucleotide chains are formed at each nanorecess 24, 24'. An example of this clustering is bridge amplification, which is one example of amplification that can be performed. It should be understood that other amplification techniques, such as the Exclusion Amplification (Examp) workflow (Illumina Inc.), may be used.

[0138] Sequencing primers that hybridize to complementary sequences on a template polynucleotide chain can be introduced. These sequencing primers prepare the template polynucleotide chain for sequencing.

[0139] To initiate sequencing, the embedded mix may be added to flow cells 30, 30A, and 30B. In one example, the embedded mix contains a liquid carrier, polymerase, and nucleotides with the 3'OH group blocked. Once the embedded mix is ​​introduced into flow cells 30, 30A, and 30B, the fluid enters the flow channel 38 and the nanorecesses 24, 24' (where the template polynucleotide chain is located).

[0140] 3'OH-blocked nucleotides are added to the sequencing primers in a template-dependent manner (thus extending the sequencing primers) so that the template sequence can be determined by detecting the order and type of nucleotides attached to the sequencing primers. More specifically, one of the nucleotides is incorporated by each polymerase into a nascent strand that extends the sequencing primer and is complementary to the template polynucleotide chain. In other words, in at least a portion of the template polynucleotide chain spanning flow cells 30, 30A, and 30B, each polymerase extends the hybridized sequencing primer by one of the nucleotides in the incorporation mix.

[0141] In this exemplary method, after the nucleotide bases have been incorporated into the nascent chain, the incorporation mix containing any unincorporated 3'OH-blocked nucleotides can be removed from flow cells 30, 30A, and 30B. This can be achieved using a washing solution (e.g., a buffer).

[0142] 3'OH-blocked nucleotides contain a reversible termination property (e.g., a 3'OH blocking group) that terminates further primer extension after the nucleotide is attached to the sequencing primer. The most recently incorporated nucleotide can be detected through the imaging event without further incorporation. During the imaging event, an illumination system (not shown) can provide excitation light to the flow channel 38 and / or nanorecess 24, 24'. When flow cell 30B is used, waveguide-based illumination can be used.

[0143] Next, the cleavage mix may be introduced into flow cells 30, 30A, and 30B. In the examples disclosed herein, the cleavage mix can i) remove 3'OH blocking groups from the incorporated nucleotides, and ii) cleave any detectable labels from the incorporated nucleotides. Removal of the 3'OH blocking groups allows subsequent sequencing cycles to be performed.

[0144] Examples of 3'OH blocking groups and suitable deblocking agents / components in the cleavage mix may include: ester moieties that can be removed by base hydrolysis; allyl moieties that can be removed with Na, chlorotrimethylsilane and Na2S2O3 or with Hg(II) in acetone / water; azidomethyl moieties that can be cleaved with phosphines such as tris(2-carboxyethyl)phosphine (TCEP) or tri(hydroxypropyl)phosphine (THP); acetals such as tert-butoxy-ethoxy that can be cleaved under acidic conditions; MOM(-CH2OCH3) moieties that can be cleaved with LiBF4 and CH3CN / H2O; 2,4-dinitrobenzenesulfene that can be cleaved with nucleophiles such as thiophenol and thiosulfate; tetrahydrofuranyl ethers that can be cleaved with Ag(I) or Hg(II); and 3' phosphates that can be cleaved by phosphatase enzymes (e.g., polynucleotide kinases).

[0145] Washing(s) may be performed between various fluid delivery steps. The SBS cycle is then repeated n times to extend the sequencing primer by n nucleotides, thereby allowing detection of a sequence of length n. In some examples, paired-end sequencing may be used, in which the forward strand is sequenced and removed, and then the reverse strand is constructed and sequenced.

[0146] While SBS has been described in detail, it should be understood that the flow cells 30, 30A, and 30B described herein can be used with other sequencing protocols for genotyping or other chemical and / or biological applications. In some cases, the flow cell primers may be selected to enable simultaneous paired-end sequencing, where both the forward and reverse chains are present on the polymer hydrogel 44, 44', allowing simultaneous base calling of each read. Sequential and simultaneous paired-end sequencing facilitates the detection of genomic rearrangements and repeating sequence elements, as well as gene fusions and novel transcripts. In another example, the flow cells 30, 30A, and 30B disclosed herein can be used to construct on-flow cell libraries.

[0147] Examples are provided herein to further illustrate the present disclosure. These examples are provided for illustrative purposes only and should not be construed as limiting the scope of the present disclosure. Non-limiting examples

[0148] [Examples]

[0149] Two control resins (6 and 7) were prepared. By combining control resins 6 and 7 in appropriate ratios, five different resin mixtures (1-5) were prepared. In control resin 7, and by extension, in each resin mixture 1-5, the epoxy monomer system was [ka] (25% by weight), [ka] It contained a 1:3 mixture (75% by weight). In control resin 6, and by extension, in each of these resin mixtures, the acryloyl monomer system is [ka] It included.

[0150] Table 1 shows the monomer content of the mixture and the control. [Table 1] Control resin 6 contained 4% by weight (relative to monomer) of 2,2-dimethoxy-2-phenylacetophenone as a free radical photoinitiator, and control resin 7 contained 4% by weight (relative to monomer) of bis(4-methylphenyl)iodonium hexafluorophosphate as a cationic photoinitiator and 4% by weight (relative to monomer) of isopropyl-9H-thioxan-9-one as a sensitizer. The control resins were diluted with propylene glycol monomethyl ether acetate to a solids content of 8%. The control resins were then mixed in appropriate ratios to obtain resin mixtures 1 to 5. Each mixture and the control resin was spin-coated onto a silicon wafer. The refractive index and thickness of each mixture and the control resin were measured before curing.

[0151] Each mixture and control resin was cured using UV exposure. After curing, the refractive index and thickness of each cured resin were measured.

[0152] The refractive index results are shown in Figure 5A, and the thickness results are shown in Figure 5B. The results in Figure 5A show that by changing the monomer ratio in the mixture, the refractive index of the resin layer decreases from 1.51 (100% epoxy - control resin 7) to 1.49 (100% acrylo - control resin 6). The results in Figure 5B show that a thinner resin layer was obtained by decreasing the epoxy monomer ratio in the mixture.

[0153] [Examples]

[0154] Similar to Example 1, several different resin mixtures were prepared. The monomer content of the mixtures is shown in Table 2.

[0155] Table 2 uses the following acronyms. 2,4,6,8-Tetramethyl-2,4,6,8-tetrakis(propylglycidyl ether)cyclotetrasiloxane:G-D4 Tetrakis(epoxycyclohexylethyl)tetramethylcyclotetrasiloxane:EC-D4 Diglycidyl ether-terminated poly(dimethylsiloxane):G-PDMS Poly(propylene glycol) diglycidyl ether: PPGGE 2,2,3,3,4,4,5,5-Octafluoro-1,6-Hexanediol diacrylate:BFA Pentaerythritol tetraacrylate: PETA Glycerol 1,3-diglycerolate diacrylate: GDA [Table 2]

[0156] Each resin mixture was filtered and spin-coated onto a silicon wafer. Imprint testing was performed by pressing either a silicon acrylate work stamp (Si-WS) or a fluorinated work stamp (F-WS) onto the coated resin mixture, followed by curing.

[0157] The resin mixtures and cured resins were tested for coating properties (uniform wetting of the substrate), wetting by work stamp (efficient filling of WS features), effective curing, imprintability by work stamp (ease of peeling of WS and visual quality of diffraction pattern), refractive index (of the cured resin), and / or thickness (of the cured resin). Coating properties mean that the resin mixture formed a substantially uniform film on the substrate. Imprintability means that the features of the work stamp were successfully transferred to the cured resin without adhesion, feature size reduction, etc. Table 3 shows the resin, work stamp type, and results. Where two work stamp types and one result are provided, this indicates that the results were the same for both work stamps. Where two work stamps and two results are provided, this indicates the results for each work stamp. For example, if the work stamp types are Si-WS and F-WS, and the imprint results are good and bad, then Si-WS was successfully imprinted, and F-WS was not successfully imprinted. [Table 3]

[0158] In resin mixtures 8 and 9 (with different ratios of PPGGE and BFA), a uniform (+) film was coated, and UV curing was efficient. In the case of resin mixture 8, wetting of the work stamp was efficient, and imprintability was achieved. In the case of resin mixture 9, imprintability can be achieved by adding a leveling agent.

[0159] In resin mixtures 10-13 (with different ratios of EC-D4 and BFA), a moderately uniform (+ / -) film was coated. Curing was not always efficient, for example, when there was too much acrylole monomer (90%) or too much epoxy monomer (75%). For this combination, the EC-D4:BFA ratio should be in the range of approximately 50:50 to approximately 25:75. In addition, in resin mixtures 10-13, efficient wetting of the work stamp was achieved, but the peeling of the work stamp was undesirable, so the pattern could not be successfully transferred from the stamp to the resin. Since EC-D4 is a very viscous compound, it may be desirable to reduce its amount (as shown in resin mixtures 11 and 12). Furthermore, the peeling of the work stamp can be improved by adding a leveling agent.

[0160] In resin mixtures 14-16 (with different ratios of EC-D4 / G-D4 mixture and BFA), a moderately (+ / -) uniform film coating was achieved. Curing was efficient in all of these examples. Furthermore, while efficient wetting of the work stamp was achieved in resin mixtures 14-16, the peeling of the work stamp was undesirable, preventing successful transfer of the pattern from the stamp to the resin. The film thickness increased with increasing G-D4 content. Additionally, the peeling of the work stamp can be improved by adding a leveling agent.

[0161] In resin mixtures 17-19 (with different ratios of G-D4 and GDA), a uniform (+) film was coated. In all of these examples, curing was efficient. Furthermore, while efficient wetting of the work stamp was achieved in resin mixtures 17-19, the peeling of the work stamp was undesirable, preventing successful transfer of the pattern from the stamp to the resin. Adding a leveling agent can improve the peeling of the work stamp.

[0162] In the resin mixture 20 (G-PDMS and GDA), a moderately (+ / -) uniform film coating was achieved, and curing was efficient. Furthermore, efficient wetting of the work stamp was achieved with resin mixture 20, but successful transfer of the pattern from the stamp to the resin was not possible. The PDMS material was soft, and the imprintability could be improved by increasing the amount of GDA in some cases. Additionally, the addition of a leveling agent could improve the release of the work stamp.

[0163] In resin mixtures 21-23 (with different ratios of G-PDMS and PETA), a uniform (+) film was coated. Curing was efficient. Furthermore, while efficient wetting of the work stamp was achieved in resin mixtures 21-23, successful transfer of the pattern from the stamp to the resin was not possible. The PDMS material was soft, and the imprintability could be improved by increasing the amount of PETA in some cases. Additionally, the addition of a leveling agent could improve the release of the work stamp.

[0164] In resin mixtures 24-26 (with different ratios of PPGGE and GDA), a uniform (+) film was coated. UV curing was efficient. For resin mixtures 24-26, wetting of the working stamp was efficient, and imprintability was achieved when using Si-WS. However, imprint transfer could not be achieved with F-WS. As the amount of epoxy (PPGGE) increased, the thickness of the cured resin also increased, so the effect on viscosity could also be observed in these examples. In these resin mixtures, the effect of acrylole monomer on refractive index (GDA, RI=1.446 and PPGGE, RI=1.457) could also be observed.

[0165] In resin mixtures 27-29 (with different ratios of PPGGE and PETA), a uniform (+) film was coated. UV curing was efficient. In the case of resin mixtures 27-29, wetting of the work stamp was efficient, but the peeling of the work stamp was undesirable, so imprintability was not achieved. For the mixtures in these examples, the peeling of the work stamp can be improved by adding a leveling agent. As the amount of acryloyl (PETA) increases, the thickness of the cured resin also increases, so the effect on viscosity can also be observed in these examples.

[0166] The results of this embodiment demonstrate how different monomers and different amounts of monomers in the resin mixture can be varied to adjust one or more properties of the resin mixture and / or the resulting interpenetrating network. Furthermore, the results for resin mixtures 24-26 demonstrate how monomers and their weight ratios can be selected to produce resin mixtures that can be imprinted using a specific work stamp.

[0167] Additional information

[0168] It should be understood that all combinations of the aforementioned concepts and further concepts, which will be discussed in more detail below, are intended to be part of the subject matter of the inventions disclosed herein (insofar as such concepts do not contradict each other). Specifically, all combinations of claimed subject matter appearing at the end of this disclosure are intended to be part of the subject matter of the inventions disclosed herein. It should also be understood that terms used expressly herein and that may appear in any disclosure incorporated by reference should be given meanings that most coincide with the specific concepts disclosed herein.

[0169] Throughout this specification, references to "one example," "another example," or "another example" mean that certain elements (e.g., features, structures, and / or characteristics) described in relation to an example are included in at least one example described herein, and may or may not be present in other examples. Furthermore, unless explicitly indicated otherwise in the context, it should be understood that elements described in relation to any example may be combined in any preferred manner in various examples.

[0170] It should be understood that the ranges provided herein include the indicated range and any values ​​or subranges within that indicated range, as if such values ​​or subranges were explicitly enumerated. For example, the range of approximately 225 mm to approximately 600 mm should be interpreted to include not only the explicitly enumerated limit of approximately 225 mm to approximately 600 mm, but also individual values ​​such as approximately 358 mm, approximately 375.5 mm, and subranges such as approximately 355 mm to approximately 395 mm, approximately 350 mm to approximately 575 mm, and so on. Furthermore, where “approximately” and / or “substantially” are used to describe a value, they mean to include a small variation (up to ±10%) from the stated value.

[0171] While several embodiments have been described in detail, it should be understood that the disclosed examples can be modified. Therefore, the above description should be considered non-limiting.

[0172] As used herein and in the claims, the terms “comprising” and its variations mean that the specified features, processes, or integers are included. These terms should not be construed as excluding the presence of other features, processes, or components.

[0173] The present invention may also, in a broader sense, consist of any and all combinations of two or more parts, elements, processes, examples, and / or features that are referred to or specified herein individually or collectively. In particular, one or more features in any of the embodiments described herein may be combined with one or more features from any other embodiments described herein.

[0174] Any feature disclosed in any one or more public documents referenced herein in combination with this disclosure may be subject to protection.

[0175] While specific exemplary embodiments of the present invention have been described, the scope of the appended claims is not intended to be limited to these embodiments only. The claims should be interpreted as literally, purposefully, and / or encompassing equivalents. Typical features Typical features are described in the following clauses, which may be used individually or in any combination with one or more features disclosed in the text and / or drawings of this specification. 1. A flow cell comprising a substrate and a cured patterned resin on the substrate, The cured patterned resin, A flow cell comprising nanorecesses separated by void regions, wherein each nanorecess has a maximum opening dimension in the range of approximately 10 nm to approximately 1000 nm, and the cured patterned resin comprises an interpenetrating polymer network containing epoxy polymers and (meth)acryloyl polymers. 2. A flow cell as described in Clause 1, A grid layer positioned on the substrate, A planar waveguide layer positioned on the grid layer, Flowcell, which is equipped with even more features. 3. A flow cell as described in Clause 1 or 2, The refractive index of the interpenetrating polymer network is in the range of approximately 1.35 to approximately 1.52. A flow cell in which the refractive index of the planar waveguide layer is in the range of approximately 1.6 to approximately 2.5. 4. A flow cell as described in any one of clauses 1 to 3, A hydrogel positioned in each of the nano-recesses, The amplification primer bound to the hydrogel, A flow cell that further includes the following. 5. A flow cell according to any one of clauses 1 to 4, wherein the thickness of the cured patterned resin is in the range of approximately 225 nm to approximately 600 nm. 6. A flow cell according to any one of clauses 1 to 5, wherein the weight ratio of the epoxy polymer to the (meth)acryloyl polymer is in the range of 25:75 to approximately 75:25. 7. A method, The application of a resin mixture to a substrate, wherein the resin mixture contains two independently crosslinkable monomers present in a predetermined weight ratio, the first of the two independently crosslinkable monomers being a cationic polymerizable monomer, and the second of the two independently crosslinkable monomers being a free radical polymerizable monomer. Imprinting the resin mixture with a work stamp having multiple nanofeatures, The process involves curing the resin mixture while the work stamp is in a predetermined position, thereby forming an interpenetrating polymer network imprinted in the nano-recesses of the flow cell, Methods that include... 8. The method according to Clause 7, wherein the cationic polymerizable monomer is a polyfunctional epoxy monomer and the free radical polymerizable monomer is a polyfunctional (meth)acryloyl monomer. 9. The method according to Clause 7 or 8, wherein the polyfunctional epoxy monomer is selected from the group consisting of the following: i) 2,4,6,8-tetramethyl-2,4,6,8-tetrakis(propylglycidyl ether)cyclotetrasiloxane: [ka] ii) Tetrakis(epoxycyclohexylethyl)tetramethylcyclotetrasiloxane: [ka] iii) Diglycidyl ether-terminated poly(dimethylsiloxane): [ka] (In the formula, 4 <n<8); iv) Poly(propylene glycol) diglycidyl ether: [ka] (In the formula, 5 <n<10); v) 3,4-Epoxycyclohexylmethyl 3,4-Epoxycyclohexanecarboxylate: [ka] vi) Brominated bisphenol A diglycidyl ether: [ka] vii) Glycidyl-terminated poly(bisphenol A-co-epichlorohydrin): [ka] (In the formula, 0 <n<2); viii) Bisphenol A propoxylate diglycidyl ether: [ka] ix) Monophenyl-functionalized tris(epoxy-terminated polydimethylsiloxane): [ka] x) Trimethylolpropane triglycidyl ether: [ka] xi)2,2'-(2,2,3,3,4,4,5,5-octafluorohexane-1,6-diyl)bis(oxirane): [ka] xii) 1,3-Bis(3-glycidoxypropyl)tetramethyldisiloxane: [ka] xiii) 1,3-bis[2(3,4-epoxycyclohexa-1-yl)ethyl]tetra-methyldisiloxane: [ka] xiv) Glycidyl polyoctahedral silsesquioxane: [ka] xv) Epoxycyclohexyl polyoctahedral silsesquioxane: [ka] xvi) Tris(4-hydroxyphenyl)methane triglycidyl ether: [ka] xvii) 4,4'-methylenebis(N,N-diglycidylaniline): [ka] Any combination of xviii)i) to xvii). 10. The method according to clauses 7 to 9, wherein the polyfunctional (meth)acryloyl monomer is selected from the group consisting of the following: i) 2,2,3,3,4,4,5,5-Octafluoro-1,6-Hexanediol diacrylate: [ka] ii) Pentaerythritol tetraacrylate: [ka] iii) Pentaerythritol triacrylate: [ka] iv) Glycerol 1,3-diglycerolate diacrylate: [ka] v) Poly(ethylene glycol) dimethacrylate: [ka] (In the formula, 8 <n<10); vi) Glycerol dimethacrylate, mixture of isomers: [ka] vii) 3-(acryloyloxy)-2-hydroxypropyl methacrylate: [ka] viii) Ethylene glycol dimethacrylate: [ka] ix) Bis(2-methacryloyl)oxyethyl disulfide: [ka] x) Tricyclodecanedimethanol diacrylate: [ka] xi) Bisphenol A glycerolate (1 glycerol / phenol) diacrylate: [ka] xii) Bisphenol A dimethacrylate: [ka] xiii) Methacrylateoxypropyl-terminated polydimethylsiloxane: [ka] (wherein n is selected to reflect molecular weights of approximately 420 g / mol, 950 g / mol, 4,000 g / mol, 10,000 g / mol, or 25,000 g / mol for the monomer); xiv) Zirconium bromonolbornane lactone carboxylate triacrylate: [ka] (In the equation, m is in the range of 0 to 4, n is in the range of 0 to 4, and m + n = 4); xv) Zirconium acrylate: [ka] xvi) Hafnium carboxyethyl acrylate: [ka] xvii) Acrylopolyoctahedralsilsesquioxane: [ka] xviii) Methacrylic polyoctahedral silsesquioxane: [ka] xix)2,4,6,8-tetramethyl-2,4,6,8-tetrakis(3-acryloyloxypropyl)cyclotetrasiloxane: [ka] Any combination of xx)i) to xix). 11. Methods described in Articles 7-10, The polyfunctional epoxy monomer is poly(propylene glycol) diglycidyl ether: [ka] The polyfunctional (meth)acryloyl monomer is 2,2,3,3,4,4,5,5-octafluoro-1,6-hexanediol diacrylate: [ka] A method wherein the predetermined weight ratio is approximately 50:50. 12. A method described in any one of the paragraphs 7 to 11, The polyfunctional epoxy monomer is poly(propylene glycol) diglycidyl ether: [ka] The polyfunctional (meth)acryloyl monomer is glycerol 1,3-diglycerolate diacrylate: [ka] A method wherein the predetermined weight ratio is in the range of approximately 25:75 to approximately 75:25. 13. A method, The process involves mixing a cationic polymerizable monomer and a free radical polymerizable monomer to form a resin mixture, wherein the resin mixture is a precursor to an interpenetrating polymer network incorporated into a flow cell. Adjusting the weight ratio of the cationic polymerizable monomer to the free radical polymerizable monomer using at least one property imparted to the resin mixture or the interpenetrating polymer network, wherein the property is selected from the group consisting of the refractive index of the interpenetrating polymer network, the absorption of the interpenetrating polymer network, the hardness of the interpenetrating polymer network, the thickness of the interpenetrating polymer network, the hydrophilic / hydrophobic balance of the interpenetrating polymer network, the viscosity of the resin mixture, the surface chemical compatibility between the resin mixture and the work stamp, the surface chemical compatibility of the interpenetrating polymer network, the shrinkage of the interpenetrating polymer network, and combinations thereof. The resin mixture is patterned to form the interpenetrating polymer network, Methods that include... 14. The method according to Clause 13, wherein the cationic polymerizable monomer is an epoxysiloxane monomer and the free radical polymerizable monomer is a (meth)acryloyl monomer. 15. Methods described in Article 13 or 14, The desired characteristic is the surface chemical compatibility between the resin mixture and the work stamp. The method further includes selecting the material for the work stamp, A method in which the weight ratio of the cationic polymerizable monomer to the free radical polymerizable monomer is adjusted to be within the range of approximately 25:75 to approximately 75:25. 16. The method described in any one of the clauses 13 to 15, wherein the patterning is accompanied by nanoimprint lithography.

Claims

1. A flow cell comprising a substrate and a cured patterned resin on the substrate, The cured patterned resin, A flow cell comprising nanorecesses separated by void regions, each of which has a maximum opening dimension in the range of about 10 nm to about 1000 nm, wherein the cured patterned resin comprises interpenetrating polymer networks comprising an epoxy polymer and a (meth)acryloyl polymer, the interpenetrating polymer networks comprising two or more individual but twisted polymer networks of the epoxy polymer and the (meth)acryloyl polymer, the interpenetrating polymer networks having a three-dimensional network structure in which the two or more polymer networks are mechanically connected through the interpenetration and entanglement of the epoxy polymer and the (meth)acryloyl polymer of the two or more polymer networks, and the weight ratio of the epoxy polymer to the (meth)acryloyl polymer is in the range of about 25:75 to about 75:

25.

2. A flow cell according to claim 1, A grid layer positioned on the substrate, A planar waveguide layer positioned on the aforementioned grid layer, Flowcell, which is equipped with even more features.

3. A flow cell according to claim 2, The refractive index of the aforementioned interpenetrating polymer network is in the range of approximately 1.35 to approximately 1.

52. A flow cell in which the refractive index of the planar waveguide layer is in the range of approximately 1.6 to approximately 2.

5.

4. A flow cell according to claim 1, A hydrogel positioned in each of the aforementioned nano-recesses, The amplification primer bound to the hydrogel, A flow cell that further includes the following.

5. The flow cell according to claim 1, wherein the thickness of the cured patterned resin is in the range of about 225 nm to about 600 nm.

6. It is a method, The method involves applying a resin mixture to a substrate, wherein the resin mixture contains two independently crosslinkable monomers present in a predetermined weight ratio, the first monomer of the two independently crosslinkable monomers is a cationic polymerizable monomer, and the second monomer of the two independently crosslinkable monomers is a free radical polymerizable monomer. The resin mixture is imprinted with a work stamp having multiple nanofeatures, The process involves curing the resin mixture while the work stamp is in a predetermined position, thereby forming an interpenetrating polymer network imprinted in the nano-recesses of the flow cell, Includes, The cationic polymerizable monomer is a polyfunctional epoxy monomer, and the free radical polymerizable monomer is a polyfunctional (meth)acryloyl monomer. The method wherein the interpenetrating polymer network comprises an epoxy polymer and a (meth)acryloyl monomer, the interpenetrating polymer network comprises two or more individual but twisted polymer networks of the epoxy polymer and the (meth)acryloyl polymer, the interpenetrating polymer network has a three-dimensional network structure in which the two or more polymer networks are mechanically connected through the entanglement of the epoxy polymer and the (meth)acryloyl polymer of the two or more polymer networks so as to penetrate each other, the weight ratio of the epoxy polymer to the (meth)acryloyl polymer is in the range of about 25:75 to about 75:25, and the predetermined weight ratio is in the range of about 25:75 to about 75:

25.

7. The method according to claim 6, wherein the polyfunctional epoxy monomer is selected from the group consisting of the following: i) 2,4,6,8-tetramethyl-2,4,6,8-tetrakis(propylglycidyl ether)cyclotetrasiloxane: 【Chemistry 1】 ii) Tetrakis(epoxycyclohexylethyl)tetramethylcyclotetrasiloxane: 【Chemistry 2】 iii) Diglycidyl ether-terminated poly(dimethylsiloxane): 【Transformation 3】 (In the formula, 4<n<8); iv) Poly(propylene glycol) diglycidyl ether: 【Chemistry 4】 (in the formula, 5<n<10); v) 3,4-Epoxycyclohexylmethyl 3,4-Epoxycyclohexanecarboxylate: 【Transformation 5】 vi) Brominated bisphenol A diglycidyl ether: 【Transformation 6】 vii) Glycidyl-terminated poly(bisphenol A-co-epichlorohydrin): 【Transformation 7】 (In the formula, 0<n<2); viiii) Bisphenol A propoxylate diglycidyl ether: 【Transformation 8】 ix) Monophenyl-functionalized tris(epoxy-terminated polydimethylsiloxane): 【Chemistry 9】 x) Trimethylolpropane triglycidyl ether: 【Chemistry 10】 xi) 2,2'-(2,2,3,3,4,4,5,5-octafluorohexane-1,6-diyl)bis(oxirane): 【Chemistry 11】 xi) 1,3-bis(3-glycidoxypropyl)tetramethyldisiloxane: 【Chemistry 12】 xiiii) 1,3-bis[2(3,4-epoxycyclohexa-1-yl)ethyl]tetra-methyldisiloxane: 【Chemistry 13】 xiv) Glycidyl polyoctahedral silsesquioxane: 【Chemistry 14】 xv) Epoxycyclohexyl polyoctahedral silsesquioxane: 【Chemistry 15】 xvi) Tris(4-hydroxyphenyl)methane triglycidyl ether: 【Chemistry 16】 xvii) 4,4'-methylenebis(N,N-diglycidylaniline): 【Chemistry 17】 Any combination of xviiii)i) to xvii).

8. The method according to claim 6, wherein the polyfunctional (meth)acryloyl monomer is selected from the group consisting of the following: i) 2,2,3,3,4,4,5,5-Octafluoro-1,6-hexanediol diacrylate: [Chemistry 18] ii) Pentaerythritol tetraacrylate: 【Chemistry 19】 iii) Pentaerythritol triacrylate: 【Chemistry 20】 iv) Glycerol 1,3-diglycerolate diacrylate: 【Chemistry 21】 v) Poly(ethylene glycol) dimethacrylate: 【Chemistry 22】 (In the formula, 8<n<10); vi) Glycerol dimethacrylate, mixture of isomers: 【Chemistry 23】 vii) 3-(acryloyloxy)-2-hydroxypropyl methacrylate: 【Chemistry 24】 viiii) Ethylene glycol dimethacrylate: 【Chemistry 25】 ix) Bis(2-methacryloyl)oxyethyl disulfide: 【Chemistry 26】 x) Tricyclodecane dimethanol diacrylate: 【Chemistry 27】 xi) Bisphenol A glycerolate (1 glycerol / phenol) diacrylate: 【Chemistry 28】 xi) Bisphenol A dimethacrylate: 【Chemistry 29】 xiiii) Methacrylateoxypropyl-terminated polydimethylsiloxane: 【Transformation 30】 (wherein n is selected to reflect molecular weights of approximately 420 g / mol, 950 g / mol, 4,000 g / mol, 10,000 g / mol, or 25,000 g / mol for the monomer); xiv) Zirconium bromonolbornane lactone carboxylate triacrylate: 【Chemistry 31】 (In the formula, m is in the range of 0 to 4, n is in the range of 0 to 4, and m + n = 4); xv) Zirconium acrylate: 【Chemistry 32】 xvi) Hafnium carboxyethyl acrylate: 【Transformation 33】 xvii) Acrylopolyoctahedralsilsesquioxane: 【Transformation 34】 xviiii) Methacrylic polyoctahedral silsesquioxane: 【Chemistry 35】 xix) 2,4,6,8-tetramethyl-2,4,6,8-tetrakis(3-acryloyloxypropyl)cyclotetrasiloxane: 【Transformation 36】 Any combination of xx)i) to xix).

9. The method according to claim 6, The aforementioned polyfunctional epoxy monomer is poly(propylene glycol) diglycidyl ether: 【Chemistry 37】 The polyfunctional (meth)acryloyl monomer is 2,2,3,3,4,4,5,5-octafluoro-1,6-hexanediol diacrylate: 【Transformation 38】 A method wherein the predetermined weight ratio is approximately 50:

50.

10. The method according to claim 6, The aforementioned polyfunctional epoxy monomer is poly(propylene glycol) diglycidyl ether: 【Chemistry 39】 The polyfunctional (meth)acryloyl monomer is glycerol 1,3-diglycerolate diacrylate: 【Chemistry 40】 ,method.

11. It is a method, The process involves mixing a cationic polymerizable monomer and a free radical polymerizable monomer to form a resin mixture, wherein the resin mixture is a precursor to an interpenetrating polymer network incorporated into a flow cell. Adjusting the weight ratio of the cationic polymerizable monomer to the free radical polymerizable monomer using at least one property imparted to the resin mixture or the interpenetrating polymer network, wherein the property is selected from the group consisting of the refractive index of the interpenetrating polymer network, the absorption of the interpenetrating polymer network, the hardness of the interpenetrating polymer network, the thickness of the interpenetrating polymer network, the hydrophilic / hydrophobic balance of the interpenetrating polymer network, the viscosity of the resin mixture, the surface chemical compatibility between the resin mixture and the work stamp, the surface chemical compatibility of the interpenetrating polymer network, the shrinkage of the interpenetrating polymer network, and combinations thereof. The resin mixture is patterned to form the interpenetrating polymer network, Includes, The cationic polymerizable monomer is an epoxysiloxane monomer, and the free radical polymerizable monomer is a (meth)acryloyl monomer. A method wherein the interpenetrating polymer network comprises an epoxy polymer and a (meth)acryloyl monomer, the interpenetrating polymer network comprises two or more individual but twisted polymer networks of the epoxy polymer and the (meth)acryloyl polymer, the interpenetrating polymer network has a three-dimensional network structure in which the two or more polymer networks are mechanically connected by intertwining between the epoxy polymer and the (meth)acryloyl polymer of the two or more polymer networks, the weight ratio of the epoxy polymer to the (meth)acryloyl polymer is in the range of about 25:75 to about 75:25, and the weight ratio of the cationic polymerizable monomer to the free radical polymerizable monomer is adjusted to be in the range of about 25:75 to about 75:

25.

12. The method according to claim 11, The desired characteristic is the surface chemical compatibility between the resin mixture and the work stamp. A method further comprising selecting the material for the work stamp.

13. The method according to claim 11, wherein the patterning is performed using nanoimprint lithography.

Images