Resin composition and flow cell incorporating the resin composition

Abstract

Examples of resin compositions include free radical curable resin matrices containing acrylates and siloxanes, and free radical photoinitiators. When cured, the resin composition has low or no autofluorescence when exposed to a blue excitation wavelength in the range of from about 380 nm to about 480 nm or a green excitation wavelength in the range of from about 510 nm to about 560 nm.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 701,228, filed on July 20, 2018, the contents of which are incorporated herein by reference in their entirety.

[0003] background

[0004] Bioarray is one of the multiple tools for detecting and analyzing molecules, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In these applications, arrays are designed to include probes for nucleotide sequences present in the genes of humans and other organisms. For example, in some applications, independent DNA and RNA probes can be attached to small positions in a geometric grid (or random) on an array support. For example, a test sample from a known person or organism can be exposed to a grid so that complementary fragments are hybridized with probes at a separate site in the array. The array can then be checked by scanning the light of a specific frequency on the site to identify which fragments are present in the sample by the fluorescence of the site hybridized by the fragment.

[0005] Bioarrays can be used for gene sequencing. In general, gene sequencing involves determining the order of nucleotides or nucleic acids in a segment of genetic material (such as a segment of DNA or RNA). Longer and longer base pair sequences are being analyzed, and the resulting sequence information can be used in a variety of bioinformatics methods to logically fit the segments together so as to reliably determine the sequence of a wide range of lengths of genetic material from which the segments are derived. Automated computer-based inspection of characteristic segments has been developed and has been used for genome mapping, identification of genes and their functions, risk assessment of certain conditions and disease states, and the like. In addition to these applications, bioarrays can also be used to detect and evaluate a wide range of molecules, molecular families, gene expression levels, single nucleotide polymorphisms, and genotyping.

[0006] introduction

[0007] In one aspect, the resin composition comprises a free radical curable resin matrix comprising an acrylate and a siloxane; and a free radical photoinitiator; wherein, when cured, the resin composition has low or no autofluorescence when exposed to a blue excitation wavelength in a range from about 380 nm to about 480 nm or a green excitation wavelength in a range from about 510 nm to about 560 nm.

[0008] In an example, the cured resin composition has low autofluorescence, wherein the low autofluorescence corresponds to a grey value of less than 25,000 when the cured resin composition is exposed to a blue excitation wavelength.

[0009] In another example, the cured resin composition has low autofluorescence, wherein the low autofluorescence corresponds to a grayscale value of less than 5,000 when the cured resin composition is exposed to a blue excitation wavelength.

[0010] In yet another example, the cured resin composition has low autofluorescence, wherein the low autofluorescence corresponds to a grayscale value of less than 10,000 when the cured resin composition is exposed to a green excitation wavelength.

[0011] In yet further examples, the cured resin composition has low autofluorescence, wherein the low autofluorescence corresponds to a grayscale value of less than 2,500 when the cured resin composition is exposed to a green excitation wavelength.

[0012] In an example, the free radical curable resin matrix comprises an acrylate selected from the group consisting of 1,3-bis(3-methacryloxypropyl)tetramethyldisiloxane, methacryloxypropyl terminated polydimethylsiloxane, tetramethyltetra[3-acryloxypropyl]cyclotetrasiloxane, methacryl polyhedral oligomeric silsesquioxane, acrylo polyhedral oligomeric silsesquioxane, acryloxypropyl methylsiloxane homopolymer, and combinations thereof.

[0013] In an example, the free radical photoinitiator is 2-ethyl-9,10-dimethoxyanthracene.

[0014] In another example, the free radical photoinitiator is selected from the group consisting of 2,2-dimethoxy-2-phenylacetophenone and 2-ethoxy-2-phenylacetophenone.

[0015] In yet another example, the free radical photoinitiator is a phosphine oxide, wherein the phosphine oxide is selected from the group consisting of diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide, a blend of diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide and 2-hydroxy-2-methylpropiophenone, phenyl bis (2,4,6-trimethylbenzoyl) phosphine oxide, ethyl (2,4,6-trimethylbenzoyl) phenyl phosphite, and combinations thereof.

[0016] In an example, the resin composition further includes a dark quencher or an electron acceptor.

[0017] In an example, the weight % ratio of the free radical curable resin matrix to the free radical photoinitiator ranges from about 99.8:0.2 to about 90:10.

[0018] In an example, the resin composition further includes an epoxy resin matrix; and a photoacid generator.

[0019] It should be understood that any of the features of this aspect of the resin composition may be combined together in any desired manner and / or configuration to achieve the benefits as described in the present disclosure, including, for example, relatively low autofluorescence.

[0020] In another aspect, a flow cell comprises a substrate; and a cured patterned resin on the substrate, the cured patterned resin comprising recesses separated by gap regions, and the cured patterned resin has been formed from a resin composition comprising: a free radical curable resin matrix containing an acrylate and a siloxane; and a free radical photoinitiator; wherein the cured patterned resin has low or no autofluorescence when exposed to a blue excitation wavelength in a range from about 380 nm to about 480 nm or a green excitation wavelength in a range from about 510 nm to about 560 nm.

[0021] In an example, the flow cell further includes a polymer coating in the recess; and a primer grafted to the polymer coating.

[0022] In an example, the cured patterned resin has low autofluorescence, wherein the low autofluorescence corresponds to a grayscale value of less than 25,000 when the cured patterned resin is exposed to a blue excitation wavelength.

[0023] In another example, the cured patterned resin has low autofluorescence, wherein the low autofluorescence corresponds to a grayscale value of less than 10,000 when the cured patterned resin is exposed to a green excitation wavelength.

[0024] In yet another example, the resin composition further includes an epoxy resin matrix; and a photoacid generator.

[0025] In an example, the free radical curable resin matrix comprises an acrylate selected from the group consisting of 1,3-bis(3-methacryloxypropyl)tetramethyldisiloxane, methacryloxypropyl terminated polydimethylsiloxane, tetramethyltetra[3-acryloxypropyl]cyclotetrasiloxane, methacryl polyhedral oligomeric silsesquioxane, acryl polyhedral oligomeric silsesquioxane, acryloxypropyl methylsiloxane homopolymer, and combinations thereof.

[0026] In an example, the free radical photoinitiator is selected from the group consisting of 2-ethyl-9,10-dimethoxyanthracene, 2,2-dimethoxy-2-phenylacetophenone, and 2-ethoxy-2-phenylacetophenone.

[0027] In another example, the free radical photoinitiator is a phosphine oxide selected from the group consisting of diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide, a blend of diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide and 2-hydroxy-2-methylpropiophenone, phenyl bis (2,4,6-trimethylbenzoyl) phosphine oxide, ethyl (2,4,6-trimethylbenzoyl) phenyl phosphite, and combinations thereof.

[0028] In an example, the resin composition further comprises a dark quencher or an electron acceptor.

[0029] In an example, the weight % ratio of the free radical curable resin matrix to the free radical photoinitiator in the resin composition ranges from about 99.8:0.2 to about 90:10.

[0030] It will be appreciated that any features of this aspect of the flow cell may be combined in any desired manner to obtain benefits as described in the present disclosure, including, for example, relatively low autofluorescence. In addition, it will be appreciated that any combination of features of this aspect of the flow cell and / or resin composition may be used together, and / or any features from either or both of these aspects may be combined with any example disclosed herein to obtain benefits as described in the present disclosure, including, for example, relatively low autofluorescence.

[0031] In yet another aspect, a method includes depositing a resin composition on a substrate, the resin composition comprising: a free radical curable resin matrix containing an acrylate and a siloxane and a free radical photoinitiator; the resin composition is deposited using a working stamp nanoimprint; and curing the deposited resin composition to form a cured patterned resin; wherein the cured patterned resin has low or no autofluorescence when exposed to a blue excitation wavelength in a range from about 380 nm to about 480 nm or a green excitation wavelength in a range from about 510 nm to about 560 nm.

[0032] It will be appreciated that any features of this aspect of the method may be combined in any desired manner to obtain benefits as described in the present disclosure, including, for example, relatively low autofluorescence. In addition, it will be appreciated that any combination of features from the method and / or from the flow cell and / or from the resin composition may be used together, and / or any features from any or all of these aspects may be combined with any features of the examples disclosed herein to obtain benefits as described in the present disclosure, including, for example, relatively low autofluorescence.

[0033] Furthermore, it should be understood that any features of any of the methods and / or flow cells and / or resin compositions may be combined together in any desired manner and / or may be combined with any examples disclosed herein to obtain benefits as described in the present disclosure, including, for example, relatively low autofluorescence. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Features of examples of the present disclosure will become apparent by reference to the following detailed description and accompanying drawings, in which like reference numerals correspond to similar, though perhaps not identical, components. For the sake of brevity, reference numerals or features having previously described functions may or may not be described in conjunction with other drawings in which they appear.

[0036] Figure 1 is a flow chart illustrating an embodiment of the method disclosed herein;

[0037] FIG. 2A to FIG. 2E are schematic perspective views that together depict embodiments of the methods disclosed herein;

[0038] Figure 2F is along Figure 2E A schematic cross-sectional view taken along line 2F-2F of ;

[0039] FIG. 3A to FIG. 3F are scanning electron micrograph images of example wells formed using various embodiments of the resin compositions disclosed herein;

[0040] Figure 4 is a graph illustrating fluorescence intensity (in arbitrary units) versus wavelength (in nanometers (nm)) for Comparative Example resin, Example resin, and a blank glass substrate; and

[0041] Figure 5A and Figure 5B is a graph illustrating proposed autofluorescence results of the prophetic example resin composition and the prophetic comparative example resin composition.

[0042] Detailed Description

[0043] It is desirable that the patterned flow cell used in sequencing applications has a base level of autofluorescence so that the signal-to-noise ratio (SNR) is high enough to resolve the signal from a separate cluster during sequencing. The resin composition used in the patterned flow cell typically includes a photoinitiator (e.g., a free radical generator and / or a cation generator) to initiate curing during patterning of the composition. In theory, when the photoinitiator used does not absorb in the visible region, when the blue, green, and / or red light source used during sequencing is excited, no fluorescence should occur. However, based on solution measurements of some free radical photoinitiators and cationic photoinitiators combined, it has been found that undesirable autofluorescence may occur at the excitation wavelength of interest, even when little or no absorption is expected in the visible region based on the spectral properties of the resin component.

[0044] The resin matrix of the resin composition disclosed herein is based on acrylate (for example, the resin includes acrylate and / or methacrylate), and exhibits a relatively low level of fluorescence after curing. In addition, the acrylate-based resin is cured only by a free radical mechanism. With regard to the curing mechanism, a free radical photoinitiator can be used, rather than other types of photoinitiators, such as cationic photoinitiators. This can then be minimized, and in some cases even eliminated, intramolecular interactions that may otherwise occur between different types of photoinitiators (for example, between some free radical photoinitiators and cationic photoinitiator combinations). It is believed that these intramolecular interactions may occur before or during ultraviolet (UV) light exposure, indicating that the energy exchange between the combination of photoinitiators and the subsequent formation of excited state complexes (exciplexes) and adducts may contribute to undesirable autofluorescence. It is also believed that because the resin compositions disclosed herein do not undergo the above-mentioned intramolecular interactions, the cured resin compositions disclosed herein can exhibit autofluorescence (when exposed to excitation wavelengths of interest (e.g., blue and / or green excitation wavelengths)) that is about an order of magnitude lower than resins including a combination of a free radical photoinitiator and a cationic photoinitiator.

[0045] The resin compositions disclosed herein may also be more easily photobleached, which would render the cured resin composition permanently incapable of autofluoresce (ie, any autofluorescence that does occur is below the threshold limit of detection or does not interfere with the sequencing detection process).

[0046] As mentioned herein, examples of cured resin compositions disclosed herein have minimal blue and green emission, and also exhibit very low or no autofluorescence when exposed to blue and green excitation wavelengths. As used herein, the blue emission wavelength includes from about 463nm to about 514nm, and the green emission wavelength includes from about 583nm to about 660nm. Also as used herein, the blue excitation wavelength includes from about 380nm to about 480nm, and the green excitation wavelength includes from about 510nm to about 560nm. In another example, the blue excitation wavelength is in the range of from about 440nm to about 457nm, or the green excitation wavelength is in the range of from about 519nm to about 535nm. In yet another example, the blue excitation wavelength is in the range of from about 400nm to about 480nm.

[0047] In some cases, when exposed to blue excitation wavelength and / or green excitation wavelength, the cured resin composition is described as no fluorescence (light emission). No fluorescence or no autofluorescence means that the fluorescence level is lower than the threshold limit of detection. No fluorescence or no autofluorescence (when the term is defined in this article) may occur when the cured resin composition has been photobleached. In other cases, the cured resin composition fluoresces (emitted light) when exposed to blue excitation wavelength and / or green excitation wavelength. In these cases, the term "low autofluorescence" can mean that (cured resin when exposed to blue excitation wavelength and / or green excitation wavelength) emission level is higher than the threshold limit of detection, but low enough to be considered as noise, and the noise does not interfere with the identification of cluster signals during sequencing (for example, the level of autofluorescence can make the signal-to-noise ratio (SNR) high enough so that the signal from a separate cluster can be resolved during sequencing).

[0048] It will be appreciated that the definition of "low" or "low level" for purposes of quantifying autofluorescence may vary depending on the tool used to measure the autofluorescence and / or the lamp used to provide the excitation radiation. TM (Former TYPHOON TM When a FLA 7000 (available from GE Healthcare Life Sciences) is used to measure the autofluorescence of a cured resin composition when exposed to a blue excitation wavelength, low autofluorescence corresponds to a grayscale value of less than 25,000. For another example, when the Amersham TYPHOON TM When used to measure the autofluorescence of a cured resin composition when exposed to a blue excitation wavelength, low autofluorescence corresponds to a grayscale value of less than 5,000. For yet another example, when Amersham TYPHOON TMWhen used to measure the autofluorescence of a cured resin composition when exposed to a green excitation wavelength, low autofluorescence corresponds to a grayscale value of less than 10,000. For yet another example, when the Amersham TYPHOON TM When used to measure the autofluorescence of a cured resin composition when exposed to a green excitation wavelength, low autofluorescence corresponds to a grayscale value of less than 2,500.

[0049] Autofluorescence can also be measured using a spectrometer-based tool. Discussion of examples of spectrometer-based tools is found in Example 2 below. In an example, when a spectrometer-based tool is used to measure the autofluorescence of a cured resin composition when exposed to a blue excitation wavelength, low autofluorescence corresponds to a fluorescence intensity of less than about 400AU (in arbitrary units (AU)). In another example, when a spectrometer-based tool is used to measure the autofluorescence of a cured resin composition when exposed to a green excitation wavelength, low autofluorescence corresponds to a fluorescence intensity of less than about 500AU (in arbitrary units (AU)).

[0050] It should be understood that the terms used herein will adopt their ordinary meanings in the relevant art unless otherwise specified. Several terms used herein and their meanings are set forth below.

[0051] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0052] The terms comprising, including, containing and various forms of these terms are synonymous with each other and are equally broadly meant.

[0053] The terms top, bottom, lower, upper, on, etc. are used herein to describe a flow cell and / or various components of a flow cell. It should be understood that these directional terms are not meant to imply a particular direction, but are used to indicate the relative direction between components. The use of directional terms should not be construed as limiting the examples disclosed herein to any particular direction.

[0054] Acrylamide is a substance with the structure The monomers containing the acrylamide group include a functional group of or a monomer including an acrylamide group having the structure. Acrylamide may be a compound amide (e.g., methacrylamide) having a substituent replacing one or more hydrogen atoms. Examples of monomers containing the acrylamide group include azidoacetamidopentyl acrylamide: And N-isopropylacrylamide: Other acrylamide monomers may be used.

[0055] As used herein, an "aldehyde" is an organic compound containing a functional group having the structure -CHO, which includes a carbonyl center (i.e., a carbon double-bonded to an oxygen), wherein the carbon atom is also bonded to a hydrogen and an R group such as an alkyl or other side chain. The general structure of an aldehyde is:

[0056] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., does not contain double or triple bonds). The alkyl group may have 1 to 20 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, and the like. As an example, the name "C1-4 alkyl" indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0057] As used herein, "alkenyl" refers to a straight or branched hydrocarbon chain containing one or more double bonds. An alkenyl group can have 2 to 20 carbon atoms. Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl and the like.

[0058] As used herein, "alkyne" or "alkynyl" refers to a straight or branched hydrocarbon chain containing one or more triple bonds. An alkynyl group can have 2 to 20 carbon atoms.

[0059] As used herein, "aryl" refers to an aromatic ring or aromatic ring system containing only carbon in the ring backbone (i.e., two or more fused rings sharing two adjacent carbon atoms). When aryl is a ring system, each ring in the system is aromatic. Aryl groups can have 6 to 18 carbon atoms. Examples of aryl groups include phenyl, naphthyl, azulenyl, and anthracenyl.

[0060] The "amino" functional group refers to -NR a R b Group, where R a and R b are each independently selected from hydrogen (e.g., ), C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, C6-10 aryl, 5-10 membered heteroaryl and 5-10 membered heterocyclyl, as defined herein.

[0061] As used herein, the term "attachment" refers to the state in which two things are joined, fastened, adhered, connected or combined directly or indirectly to each other. For example, nucleic acids can be attached to polymer coatings by covalent bonds or non-covalent bonds. Covalent bonds are characterized by sharing electron pairs between atoms. Non-covalent bonds are physical bonds that do not involve sharing of electron pairs, and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, and hydrophobic interactions.

[0062] An "azide" or "azido" functional group refers to -N3.

[0063] As used herein, "bonding region" refers to an area on a substrate that will be bonded to another material, such as a spacer layer, a cover, another substrate, etc., or a combination thereof (e.g., a spacer layer and a cover). The bond formed at the bonding region can be a chemical bond (as described above), or a mechanical bond (e.g., using fasteners, etc.).

[0064] As used herein, "carbocyclyl" means a non-aromatic ring or non-aromatic ring system containing only carbon atoms in the ring system skeleton. When the carbocyclyl is a ring system, two or more rings can be connected together in a fused, bridged or spiral connection. The carbocyclyl can have any saturation, provided that at least one ring in the ring system is not aromatic. Therefore, the carbocyclyl includes cycloalkyl, cycloalkenyl and cycloalkynyl. The carbocyclyl group can have 3 to 20 carbon atoms. Examples of carbocyclyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicyclo [2.2.2] octyl, adamantyl and spiro [4.4] nonyl.

[0065] As used herein, "cycloalkenyl" or "cycloalkene" means a carbocyclyl ring or carbocyclyl ring system having at least one double bond, wherein no ring in the ring system is aromatic. Examples include cyclohexenyl or cyclohexene and norbornenyl or norbornene. Also as used herein, "heterocycloalkenyl" or "heterocycloalkene" means a carbocyclyl ring or carbocyclyl ring system having at least one double bond, having at least one heteroatom in the ring backbone, wherein no ring in the ring system is aromatic.

[0066] As used herein, "cycloalkyl" means any monovalent group derived from a cycloalkane by removing a hydrogen atom from a ring carbon atom (eg, from a cycloalkane). Examples include 2-methylcyclopropyl.

[0067] As used herein, "cycloalkynyl" or "cycloalkyne" means a carbocyclyl ring or carbocyclyl ring system having at least one triple bond, wherein no ring in the ring system is aromatic. An example is cyclooctyne. Another example is bicyclononyne. Also as used herein, "heterocycloalkynyl" or "heterocycloalkyne" means a carbocyclyl ring or carbocyclyl ring system having at least one triple bond, having at least one heteroatom in the ring backbone, wherein no ring in the ring system is aromatic.

[0068] As used herein, the term "depression" refers to a discrete concave feature in a patterned resin having a surface opening at least partially surrounded by a gap region of the patterned resin. Depression can have any of a variety of shapes at the opening in its surface, including circular, elliptical, square, polygonal, star-shaped (with any number of vertices) etc. as examples. The cross section of the depression intercepted orthogonally to the surface can be curved, square, polygonal, hyperbolic, conical, angular etc. As an example, the depression can be a well or trench (trench) / line / trough (trough). Depression can also have a more complex structure, such as ridges, step features, etc.

[0069] As used herein, the term "flow cell" is intended to refer to a container having a chamber (i.e., a flow channel) in which a reaction can be carried out, an inlet for delivering reagents to the chamber, and an outlet for removing reagents from the chamber. In some instances, the chamber is capable of detecting a reaction occurring in the chamber. For example, the chamber / flow channel may include one or more transparent surfaces, allowing optical detection of an array of recesses, optically labeled molecules, or the like.

[0070] As used herein, a "flow channel" can be a region defined between two bonded components that can selectively receive a liquid sample. In some examples, a flow channel can be defined between a patterned resin and a cover, and can thus be in fluid communication with one or more recesses defined in the patterned resin.

[0071] As used herein, "heteroaryl" refers to an aromatic ring or aromatic ring system (i.e., two or more fused rings sharing two adjacent atoms) containing one or more heteroatoms (i.e., elements other than carbon, including but not limited to nitrogen, oxygen, and sulfur) in the ring backbone. When heteroaryl is a ring system, each ring in the system is aromatic. A heteroaryl group can have 5-18 ring members.

[0072] As used herein, "heterocyclyl" means a non-aromatic ring or non-aromatic ring system containing at least one heteroatom in the ring skeleton. The heterocyclyl can be connected together in a fused, bridged or spirally connected manner. The heterocyclyl can have any degree of saturation, provided that at least one ring in the ring system is not aromatic. In the ring system, heteroatoms can be present in non-aromatic rings or aromatic rings. The heterocyclyl group can have 3 to 20 ring members (that is, the number of atoms that make up the ring skeleton, including carbon atoms and heteroatoms). In some instances, the heteroatom is O, N or S.

[0073] As used herein, the term "hydrazine" or "hydrazine" refers to a -NHNH2 group.

[0074] As used herein, the term "hydrazone" or "hydrazone group" refers to Group, where R a and R b are each independently selected from hydrogen (e.g., ), C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, C6-10 aryl, 5-10 membered heteroaryl and 5-10 membered heterocyclyl, as defined herein.

[0075] As used herein, "hydroxyl" or "hydroxyl" refers to an -OH group.

[0076] As used herein, the term "gap region" refers to a region separating depressions on a surface (e.g., of a patterned resin). For example, a gap region may separate one feature of an array from another feature of an array. Two features separated from each other may be discrete, i.e., lack physical contact with each other. In another example, a gap region may separate a first portion of a feature from a second portion of a feature. In many examples, the gap region is continuous, and the feature is discrete, e.g., as in the case of more than one well defined in an otherwise continuous surface. In other examples, the gap region and the feature are discrete, e.g., as in the case of more than one groove separated by corresponding gap regions. The separation provided by the gap region may be partial or complete separation. The gap region may have a surface material different from the surface material of the feature defined in the surface. For example, the feature of the array may have an amount or concentration of a polymer coating and a primer, the amount or concentration of the polymer coating and the primer exceeding the amount or concentration present at the gap region. In some examples, the polymer coating and the primer may not be present at the gap region.

[0077] As used herein, "nitrile oxide" means "R a C≡N + O - " group, where R aAs defined herein. Examples of the preparation of nitrile oxides include in situ generation from aldoximes by treatment with chloroamide-T or by the action of a base on imidoyl chlorides [RC(Cl)=NOH] or in situ generation from a reaction between hydroxylamine and an aldehyde.

[0078] As used herein, "nitrone" means A group, wherein R1, R2 and R3 can be R as defined herein a Group and R b Any one of the groups.

[0079] As used herein, "nucleotide" includes nitrogenous heterocyclic bases, sugars and one or more phosphate groups. Nucleotide is the monomer unit of a nucleic acid sequence. In RNA, the sugar is ribose, and in DNA, the sugar is deoxyribose, i.e., a sugar lacking the hydroxyl group at the 2' position present in ribose. Nitrogenous heterocyclic bases (i.e., nucleobases) can be purine bases or pyrimidine bases. Purine bases include adenine (A) and guanine (G) and modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T) and uracil (U) and modified derivatives or analogs thereof. The C-1 atom of deoxyribose is bound to the N-1 of pyrimidine or the N-9 of purine. Nucleic acid analogs can have any of the phosphate backbone, sugar or nucleobase that are changed. Examples of nucleic acid analogs include, for example, universal bases or phosphate-sugar backbone analogs, such as peptide nucleic acids (PNA).

[0080] As used herein, a "photoacid generator" (PAG) is a molecule that releases protons when exposed to radiation. The PAG typically undergoes irreversible proton photodissociation.

[0081] As used herein, a "photoinitiator" (PI) is a molecule that undergoes a photoreaction upon absorption of radiation to produce a reactive species. A photoinitiator is capable of initiating or catalyzing a chemical reaction that results in a change in the solubility and / or physical properties of a formulation.

[0082] As used herein, "primer" is defined as a single-stranded nucleic acid sequence (e.g., single-stranded DNA or single-stranded RNA). Some primers, which may be referred to as amplification primers, are used as starting points for template amplification and cluster generation. Other primers, which may be referred to as sequencing primers, are used as starting points for DNA or RNA synthesis. The 5' end of the primer may be modified to allow coupling reactions with the functional groups of the polymer coating. The primer length may be any number of bases long and may include a variety of non-natural nucleotides. In an example, a sequencing primer is a short chain in the range of from 10 to 60 bases or from 20 to 40 bases.

[0083] As used herein, "spacer layer" refers to a material that bonds two components together. In some examples, the spacer layer can be a radiation absorbing material that facilitates bonding, or can be in contact with a radiation absorbing material that facilitates bonding.

[0084] A "thiol" functional group refers to -SH.

[0085] As used herein, the terms "tetrazine" and "tetrazinyl" refer to a six-membered heteroaryl group containing four nitrogen atoms. The tetrazine may be optionally substituted.

[0086] As used herein, "tetrazolyl" refers to a five-membered heterocyclic group containing four nitrogen atoms. Tetrazolyl may be optionally substituted.

[0087] In the examples disclosed herein, the resin composite includes a free radical curable resin matrix containing an acrylate and a siloxane; and a free radical photoinitiator; wherein, when cured, the resin composition has no autofluorescence or has low autofluorescence when exposed to a blue excitation wavelength in a range from about 380 nm to about 480 nm or a green excitation wavelength in a range from about 510 nm to about 560 nm.

[0088] The free radical curable resin matrix contains acrylate groups and siloxane groups. As used herein, the term "acrylate" refers to a "CH2=CHCOO-" functional group (i.e. ) or "CH2=C(CH3)COO-" functional group (i.e., methacrylate). Acrylate includes substituted variations thereof (e.g., methacrylate is an example of an acrylate). Siloxane groups are functional groups that include Si-O-Si bonds. The molecular weight and crosslink density of the monomers of the free radical-curable resin matrix can vary, as long as the resin composition is imprintable via the methods disclosed herein. In an example, the molecular weight of the monomers of the free radical-curable resin matrix can range from about 0.5 kDa to about 5 kDa or from about 1 kDa to about 4.5 kDa. The crosslinking functionality, and therefore the crosslinking density, depends on the number of arms of the monomer (e.g., two, three, four).

[0089] An example of a free radical curable resin matrix is ​​a matrix comprising an acrylate selected from the group consisting of:

[0090] i) 1,3-bis(3-methacryloxypropyl)tetramethyldisiloxane:

[0091]

[0092] ii) Methacryloyloxypropyl terminated polydimethylsiloxane:

[0093]

[0094] (wherein n is in the range from 1 to 1000, or any range therebetween, such as from 1 to 500, or 1 to 100, or 2 to 50, 1 to 10, 1 to 2, etc.),

[0095] iii) Tetramethyltetrakis[3-acryloxypropyl]cyclotetrasiloxane:

[0096]

[0097] iv) Methacryloyl Polyhedral Oligomeric Silsesquioxane (POSS):

[0098]

[0099] v) Acryloyl polyhedral oligomeric silsesquioxane:

[0100]

[0101] vi) Acryloxypropylmethylsiloxane homopolymer:

[0102]

[0103] (wherein m is in the range from 1 to 1000 or any range therebetween, such as 1 to 500, 30 to 200, 30 to 100, etc.), and

[0104] vii) Combinations thereof. When a combination is used, it is to be understood that any two or more of the free radical curable resin matrices listed may be used together in the resin composition, provided they are all soluble in the solvent used in the composition.

[0105] The photoinitiator in the example resin compositions disclosed herein can be any free radical generating photoinitiator that exhibits low autofluorescence in the cured resin.

[0106] In some examples, the free radical photoinitiator is 2-ethyl-9,10-dimethoxyanthracene:

[0107]

[0108] In other examples, the free radical photoinitiator is 2,2-dimethoxy-2-phenylacetophenone:

[0109]

[0110] In yet other examples, the free radical photoinitiator is 2-ethoxy-2-phenylacetophenone (also known as benzoin ethyl ether):

[0111]

[0112] In yet other examples, the free radical photoinitiator is a phosphine oxide. When a phosphine oxide is used, it can be selected from the group consisting of:

[0113] i) Diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide:

[0114]

[0115] ii) A blend of diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide and 2-hydroxy-2-methylpropiophenone:

[0116]

[0117] iii) Phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide:

[0118]

[0119] iv) Ethyl (2,4,6-trimethylbenzoyl) phenyl phosphonite:

[0120] as well as

[0121] v) Combinations thereof. When a combination is used, it is understood that any two or more of the free radical photoinitiators listed may be used together in the resin composition so long as they are all soluble in the solvent used in the composition.

[0122] The resin composition can be prepared by mixing a free radical curable resin matrix with a photoinitiator. In an example, the weight % ratio of the free radical curable resin matrix to the photoinitiator is in the range of from about 99.8:0.2 to 90:10. In another example, the weight % ratio of the free radical curable resin matrix to the photoinitiator is in the range of from about 98:2 to 95:5. In yet another example, the weight % ratio of the free radical curable resin matrix to the photoinitiator is in the range of from about 96:4 to 99:1. When a lower amount of photoinitiator is included, the UV curing time can be increased to allow for complete reaction.

[0123] It will be appreciated that the weight percent ratio of free radical curable resin matrix to photoinitiator may be higher or lower depending, at least in part, on the free radical curable resin matrix component used.

[0124] To deposit the resin composition, these components (free radical curable resin matrix and photoinitiator) can be diluted in a suitable solvent (to obtain the viscosity required for the deposition technology used), such as propylene glycol monomethyl ether acetate (PGMEA), toluene, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), etc. In an example, the concentration of the free radical curable resin matrix in the solvent is in the range of from about 15 weight % (wt%) to about 56 wt%, and the concentration of the photoinitiator in the solvent is in the range of from about 1 wt% to about 10 wt%, although it is believed that the upper limit may be higher depending on the solubility of the free radical curable resin matrix and the photoinitiator in the selected solvent. In an example, the solvent is PGMEA. The total concentration of the final resin composition (including the free radical curable resin matrix and the photoinitiator (and polyacrylate or surfactant, if used)) can be in the range of from about 16 wt% to about 66 wt%. The amount of solvent can be in the range of from about 34 wt% to about 84 wt%.

[0125] In some examples, the resin composition may also include a dark quencher or an electron acceptor. A dark quencher is a substance that absorbs light energy from a fluorophore and dissipates the energy as heat. This provides a non-radiative pathway for relaxation of excited state species. In the examples disclosed herein, the selected dark quencher should be able to absorb blue and / or green autofluorescence from the cured resin composition. Electron acceptors may also induce fluorescence quenching.

[0126] In an example, the dark quencher can include an azo dye. In a further example, the dark quencher includes a substituted azobenzene derivative selected from the group consisting of: 4-dimethylaminoazobenzene-4'-carboxylic acid: Dabcyl Azide:

[0127] Dabsyl Azide: Disperse Red 19: Carbon black pigment (CBP) additives, black dye-based quenchers (such as Lipofuscin autofluorescence quencher, 20X in DMF) and combinations thereof. Examples of suitable electron acceptors may include [5,6]-fullerene-C70: or Fullerene-C60:

[0128] In an example, the dark quencher or the electron acceptor may be present in an amount ranging from about 0.1 wt % to about 10 wt % based on the total weight of the resin composition.

[0129] Some other examples of resin compositions may also include polyacrylates or surfactants. Examples of commercially available polyacrylates are -350 (available from BYK Additives & Instruments). Any biocompatible surfactant may be used, such as Surfactants (e.g., polyethylene sorbitan esters ( 80) and polyoxyethylene sorbitan ester ( 20); TRITON TM X-100 (octylphenol ethoxylate from Dow Chemical Co.) and polymeric surfactants available from BYK Additives and Instruments. In an example, the polyacrylate or surfactant can be present in the resin composition in an amount ranging from about 0.4 wt % total solids to about 1.6 wt % total solids.

[0130] In some examples, the resin composition may also include an epoxy resin matrix combined with a free radical curable resin matrix. Different from the free radical curable resin matrix disclosed herein, the epoxy resin matrix relates to a two-part curing system, wherein the free radicals produced by the photoinitiator disclosed herein react with a photoacid generator, and the photoacid generator decomposes to produce a superacid, which in turn triggers the polymerization and / or crosslinking of the epoxy resin matrix component. In this way, in addition to the free radical curable resin matrix, an epoxy resin (epoxy) is also included in the example, it should be understood that the resin composition also includes a photoacid generator. In these examples, it is believed that the Norrish II type anthracene derivative photoinitiator disclosed herein and the Norrish I type acetophenone or phosphine oxide photoinitiator can produce free radicals in the case of UVA exposure, which can contribute to the two-part curing system in the case of a photoacid generator, without experiencing the intramolecular interaction that causes undesirable autofluorescence in the blue and / or green excitation wavelengths of interest. The example of a suitable epoxy resin matrix and a photoacid generator that can be added to the resin composition disclosed herein will now be described.

[0131] Any epoxy monomer that can crosslink with the free radical curable resin disclosed herein can be used. Examples of suitable epoxy resin matrices include:

[0132] i) epoxy-functionalized polyhedral oligomeric silsesquioxane (POSS) (described further below);

[0133] ii) Trimethylolpropane triglycidyl ether:

[0134]

[0135] iii) Tetrakis(epoxycyclohexylethyl)tetramethylcyclotetrasiloxane:

[0136]

[0137] iv) Copolymer of (epoxycyclohexylethyl)methylsiloxane and dimethylsiloxane:

[0138]

[0139] (wherein the ratio of m:n is in the range from 8:92 to 10:90);

[0140] v) 1,3-Bis[2-(3,4-epoxycyclohexyl)ethyl]tetramethyldisiloxane:

[0141]

[0142] vi) 1,3-Bis(glycidyloxypropyl)tetramethyldisiloxane:

[0143] as well as

[0144] vii) Combinations thereof. When a combination is used, it is understood that any two or more of the listed epoxy resin matrices may be used together in the resin compositions disclosed herein.

[0145] Epoxy-functionalized polyhedral oligomeric silsesquioxanes include polyhedral oligomeric silsesquioxane (POSS) cores functionalized with epoxy groups. As used herein, the term "polyhedral oligomeric silsesquioxane" (POSS) refers to a hybrid intermediate (RSiO) between silicon dioxide (SiO2) and organosilicon (R2SiO2). 1.5 ). An example of POSS may be the POSS described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776-778, which is incorporated by reference in its entirety. The composition is a chemical composition having the formula [RSiO 3 / 2 ] n The organosilicon compound wherein the R groups may be the same or different. The resin composition disclosed herein may contain one or more different cage or core structures as monomer units. In some cases, the structure includes a polyoctahedral cage or core structure. For example, the polyhedral structure may be a T8 structure, such as:

[0146] And by The monomer unit usually has eight functional group arms R1 to R8.

[0147] The monomer unit may have a cage structure containing 10 silicon atoms and 10 R groups, referred to as T 10 , such as: Or it can have a cage structure containing 12 silicon atoms and 12 R groups, known as T 12 , such as: POSS-based materials may optionally include T6, T 14 or T 16 Cage structure. The average cage content can be adjusted during synthesis, and / or controlled by a purification method, and the distribution of the cage size of the monomer unit can be used in the examples disclosed herein. As an example, any cage structure can be present in an amount ranging from about 30% to about 100% of the total POSS monomer units used. The material based on POSS can be a mixture of a cage structure and an open and partially open cage structure. Therefore, a resin precursor or resin based on POSS can include an epoxy POSS material, which can be a mixture of silsesquioxane configurations. For example, any POSS material described herein can be a mixture of discrete POSS cages and non-discrete silsesquioxane structures and / or incompletely condensed discrete structures, such as polymers, ladders and the like. Therefore, the partially condensed material will include epoxy R groups as described herein at some silicon vertices, but some silicon atoms will not be substituted by R groups, but can be substituted by OH groups. In some examples, the POSS material includes a mixture of various forms, such as:

[0148]

[0149] In the examples disclosed herein, R1 to R8 or R 10 or R 12 At least one of the arms or R groups comprises an epoxy group, and thus the POSS is referred to as epoxy POSS. In some instances, a majority of the arms, such as eight, ten, or twelve arms or R groups, comprise an epoxy group. In other instances, R1 to R8 or R 10 or R 12 are the same, and therefore R1 to R8 or R 10 or R 12 Each of R1 to R8 or R 10 or R 12 are not the same, and therefore R1 to R8 or R 10 or R 12 At least one of R1 to R8 or R 10 or R 12At least another of the non-epoxy functional groups is a non-epoxy functional group, in some cases, the non-epoxy functional group is selected from the group consisting of: azide / azido, thiol, poly (ethylene glycol), norbornene, tetrazine, acrylate and methacrylate or additionally, such as alkyl, aryl, alkoxy and haloalkyl groups. In some respects, non-epoxy functional groups are selected to increase the surface energy of the resin. In these other examples, the ratio of epoxy groups to non-epoxy groups is in the range of from 7:1 to 1:7, or 9:1 to 1:9, or 11:1 to 1:11. In any example, disubstituted or monosubstituted (terminal) epoxy groups allow monomer units to be polymerized into a cross-linked matrix when using ultraviolet (UV) light and acid initiation. In some respects, epoxy POSS includes terminal epoxy groups. The example of this type of POSS is the glycidyl POSS with the following structure:

[0150]

[0151] Another example of this type of POSS is an epoxycyclohexylethyl functionalized POSS having the following structure:

[0152]

[0153] One example of an epoxy resin matrix disclosed herein includes a combination of two epoxy POSS compounds, wherein the combination includes glycidyl POSS and epoxycyclohexylethyl functionalized POSS.

[0154] In the examples disclosed herein, the epoxy POSS may also be a modified epoxy POSS, which includes a controlled radical polymerization (CRP) agent and / or as functional groups R1 to R8 or R 10 or R 12 One or more of another functional group of interest is incorporated into the resin or core or cage structure.

[0155] As mentioned above, when the epoxy resin matrix is ​​used in combination with a free radical curable resin matrix, the resin composition also includes a photoacid generator. It is believed that any suitable photoacid generator that will not undergo undesirable intramolecular interactions with the free radical photoinitiator disclosed herein can be used. Examples of suitable photoacid generators can include benzyl, imino esters, conjugated imino esters, spiropyrans, teraylene-based, two-photon and organometallic PAG systems. Some specific examples of suitable photoacid generators are selected from the group consisting of:

[0156] i) N-hydroxynaphthalimide triflate:

[0157]

[0158] ii) Mixed triarylsulfonium hexafluorophosphates:

[0159]

[0160] iii) Mixed triarylsulfonium hexafluoroantimonates:

[0161]

[0162] iv) 1-Naphthyldiphenylsulfonium trifluoromethanesulfonate (NDS-TF):

[0163]

[0164] v) (4-phenylthiophenyl)diphenylsulfonium trifluoromethanesulfonate:

[0165]

[0166] vi) Bis-(4-methylphenyl)iodonium hexafluorophosphate:

[0167]

[0168] vii) Bis(4-tert-butylphenyl)iodonium hexafluorophosphate:

[0169]

[0170] viii) (2-Methylphenyl) (2,4,6-trimethylphenyl) iodonium trifluoromethanesulfonate:

[0171]

[0172] xi) Bis(2,4,6-trimethylphenyl)iodonium trifluoromethanesulfonate:

[0173]

[0174] x) Bis-(4-dodecylphenyl)iodonium hexafluoroantimonate:

[0175] as well as

[0176] xi) Combinations thereof. Combinations of photoacid generators may be used so long as they are soluble in the selected solvent.

[0177] In an example of a resin composition comprising an epoxy resin matrix and a photoacid generator, the weight % ratio of the epoxy resin matrix to the photoacid generator is in a range from about 99.8:0.2 to 90:10. In another example, the weight % ratio of the epoxy resin matrix to the photoacid generator is in a range from about 98:2 to 95:5. In yet another example, the weight % ratio of the epoxy resin matrix to the photoacid generator is in a range from about 96:4 to 99:1. When a lower amount of photoacid generator is included, the UV curing time can be increased to allow for complete reaction.

[0178] It will be appreciated that the weight percent ratio of epoxy matrix to photoacid generator may be higher or lower depending, at least in part, on the epoxy matrix component used.

[0179] Any embodiment of the resin composition disclosed herein can be used to form a flow cell. An embodiment of a method 100 for making an embodiment of a flow cell is Figure 1 As shown in Figure 1 As shown in , method 100 includes depositing a resin composition on a substrate, the resin composition comprising a free radical curable resin matrix containing acrylate and siloxane and a free radical photoinitiator (reference number 102); using a working stamp to nanoimprint the deposited resin composition (reference number 104); and curing the deposited resin composition to form a cured patterned resin, wherein the cured patterned resin has low or no autofluorescence when exposed to a blue excitation wavelength in a range from about 380 nm to about 480 nm or a green excitation wavelength in a range from about 510 nm to about 560 nm (reference number 106). The resulting flow cell includes a substrate and a cured patterned resin on the substrate, the cured patterned resin including recesses separated by gap regions, and the cured patterned resin has been formed by an embodiment of the resin composition disclosed herein.

[0180] Although in Figure 1 Although not shown in the drawings, embodiments of method 100 may further include applying a polymer coating in the recesses, and grafting the primers to the polymer coating. FIG. 2A to FIG. 2E The method 100 including these additional processes is further described.

[0181] Figure 2A A substrate 12 is depicted, and Figure 2B A resin composition 14 deposited on a substrate 12 is depicted.

[0182] Examples of suitable substrates 12 include epoxysilicones, glass and modified or functionalized glass, plastics including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, polytetrafluoroethylene (such as available from Chemours), ), cycloolefins / cycloolefin polymers (COP) (such as from Zeon ), polyimide, etc.), nylon, ceramic / ceramic oxide, silicon dioxide, fused silica or silicon dioxide-based materials, aluminum silicate, silicon and modified silicon (e.g., boron-doped p+ silicon), silicon nitride (Si3N4), silicon oxide (SiO2), tantalum pentoxide (TaO5) or other tantalum oxides (TaO x ), hafnium oxide (HaO2), carbon, metal, inorganic glass or the like. The substrate 12 may also be glass or silicon with a coating of tantalum oxide or another ceramic oxide at the surface.

[0183] In an example, the substrate 12 can have a diameter in the range of from about 2 mm to about 300 mm, or a rectangular sheet or panel with a maximum dimension of up to about 10 feet (~3 meters). In an example, the substrate 12 is a wafer with a diameter in the range of from about 200 mm to about 300 mm. In another example, the substrate 12 is a die with a width in the range of from about 0.1 mm to about 10 mm. Although exemplary dimensions have been provided, it should be understood that a substrate 12 with any suitable size can be used. For another example, a panel that is a rectangular substrate 12 can be used, which has a larger surface area than a 300 mm round wafer.

[0184] Resin composition 14 can be any example of resin composition described herein, which comprises a free radical curable resin matrix containing acrylate and siloxane and a free radical initiator (or an epoxy resin matrix combined with a free radical curable resin matrix). Resin composition 14 can be deposited on substrate 12 using any suitable application technology, which can be manual or automatic. As an example, the deposition of resin composition 14 can be carried out using vapor deposition technology, coating technology, grafting technology or similar technology. Some specific examples include chemical vapor deposition (CVD), spraying (such as ultrasonic spraying), spin coating, soaking or dip coating, scraper coating, puddle dispensing, aerosol printing, screen printing, micro-contact printing, inkjet printing or similar technology. In an example, spin coating is used to deposit resin composition 14.

[0185] After the resin composition 14 is deposited, it can be soft-baked to remove excess solvent. The deposited resin composition 14 is then patterned using any of the patterning techniques mentioned herein. Figure 2BIn the embodiment shown in , nanoimprint lithography is used to pattern the resin composition 14. A nanoimprint lithography mold or working stamp 20 is pressed against a layer of the resin composition 14 to create an impression on the resin composition 14. In other words, the resin composition 14 is recessed or perforated by the protrusions of the working stamp 20. In an example, the resin composition 14 can be cured with the working stamp 20 in place.

[0186] For the resin composition 14 disclosed herein, curing can be accomplished by exposure to actinic radiation, such as ultraviolet (UV) radiation (e.g., metal halide light source, mercury vapor light source, LED light source emitting UV, etc.). Due to the presence of the photoinitiator, curing promotes free radical formation, and these free radicals are used to cure the acrylate portion of the resin composition. In this way, curing promotes polymerization and / or crosslinking of the resin composition 14. As an example, curing may include a single UV exposure stage, or may include multiple stages, including soft baking (e.g., to remove solvents) and then UV exposure. When included, the soft baking may be performed at a lower temperature in the range of from about 50°C to about 150°C for a duration of greater than 0 seconds to about 3 minutes, and may be performed before the working stamp 20 is placed in the resin composition 14. In an example, the soft baking time is in the range of from about 30 seconds to about 2.5 minutes. During the soft baking, one or more chemical processes may occur, which further contribute to reducing autofluorescence. Exemplary chemical processes may include evaporation of some resin composition materials, sublimation of some resin composition materials, polymerization of some resin composition materials, and / or combinations thereof. Some examples may also include hard baking. However, the curing mechanism of the resin composition 14 disclosed herein is so fast that the resin composition 14 can be fully cured without hard baking. If performed, the working stamp 20 is released / separated before hard baking, for example, so that the working stamp 20 is not bonded to the cured resin composition 14. If performed, the duration of hard baking can last from about 5 seconds to about 10 minutes at a temperature in the range of from about 100°C to about 300°C. Hard baking can be performed, for example, to remove residual solvents from the resin composition 14, to further polymerize some resin composition materials (and thus enhance the degree of cure), and / or to further reduce autofluorescence. Examples of devices that can be used for soft baking and / or hard baking include hot plates, ovens, etc.

[0187] After releasing the working stamp 20, topographical features, such as depressions 16, are present in the resin composition 14. Figure 2C, the resin composition 14 having recesses 16 defined therein is referred to as a cured patterned resin 14'. In some examples, the cured patterned resin 14' can be subjected to a further hard bake, for example, to help lock the imprinted topography, and / or to further reduce fluorescence. In some examples, the additional hard bake can be performed at a temperature ranging from about 60°C to about 300°C.

[0188] The chemical composition of the cured patterning resin 14 ′ depends on the one or more free radical curable resin matrices and free radical photoinitiators used in the resin composition 14 .

[0189] like Figure 2C As shown in FIG. 1 , the cured patterned resin 14′ includes recesses 16 defined therein, and gap regions 22 separating adjacent recesses 16. In the embodiments disclosed herein, the recesses 16 become covered with a polymer coating 18 ( Figure 2C-Figure 2D ) and primer 24 ( Figure 2E-2F ) functionalized, while portions of the gap region 22 may be available for binding but will not have a polymer coating 18 or primer 24 thereon.

[0190] Many different layouts of the recesses 16 can be envisioned, including regular, repeating, and irregular patterns. In an example, the recesses 16 are arranged in a hexagonal grid for close packing and improved density. Other layouts may include, for example, a rectilinear (i.e., rectangular) layout (e.g., lines or grooves), a triangular layout, and the like. In some examples, the layout or pattern may be an xy format of the recesses 16 in rows and columns. In some other examples, the layout or pattern may be a repeating arrangement of the recesses 16 and / or gap regions 22. In still other examples, the layout or pattern may be a random arrangement of the recesses 16 and / or gap regions 22. The pattern may include spots, pads, wells, columns, stripes, swirls, lines, triangles, rectangles, circles, arcs, checkerboards, checkers, diagonal lines, arrows, squares, and / or cross-hatching. In an example, the recesses 16 are wells, such as Figure 2C As shown in .

[0191] The layout or pattern may be characterized with respect to the density of the depressions 16 (ie, the number of depressions 16) in a defined area. For example, the depressions 16 may be present in a ratio of 1 / 100 mm. 2 Densities of about 2 million exist. The density can be adjusted to different densities, including for example per mm 2 At least 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 2About 10 million per mm 2 Density of about 50 million or more. Alternatively or additionally, the density may be adjusted to per mm 2 No more than about 50 million per mm 2 About 10 million per mm 2 About 5 million per mm 2 About 2 million per mm 2 About 1 million per mm 2 About 100,000 per mm 2 About 1,000 per mm 2 About 100 or less. It should also be understood that the density of the depressions 16 in the cured patterned resin 14' can be between one of the lower values ​​and one of the higher values ​​selected from the range above. As an example, a high density array can be characterized as having depressions 16 separated by less than about 100 nm, a medium density array can be characterized as having depressions 16 separated by about 400 nm to about 1 μm, and a low density array can be characterized as having depressions 16 separated by more than about 1 μm. Although exemplary densities have been provided, it should be understood that a substrate having any suitable density can be used.

[0192] The layout or pattern of the recesses 16 can also or alternatively be characterized in terms of the average spacing, i.e., the spacing from the center of the recess 16 to the center of the adjacent recess 16 (center-to-center spacing) or the spacing from the edge of one recess 16 to the edge of the adjacent recess 16 (edge-to-edge spacing). The pattern can be regular, so that the coefficient of variation around the average spacing is small, or the pattern can be irregular, in which case the coefficient of variation can be relatively large. In either case, the average spacing can be, for example, at least about 10 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 100 μm, or more. Alternatively or in addition, the average spacing can be, for example, at most about 100 μm, about 10 μm, about 5 μm, about 1 μm, about 0.5 μm, about 0.1 μm, or less. The average spacing of a particular pattern of recesses 16 can be between one of the lower values ​​and one of the higher values ​​selected from the range above. In an example, the depressions 16 have a pitch (center-to-center spacing) of about 1.5 μm. While exemplary average pitch values ​​have been provided, it will be appreciated that other average pitch values ​​may be used.

[0193] exist FIG. 2A to FIG. 2E In the embodiment shown in , the depressions 16 are wells, and thus the cured patterned resin 14' includes an array of wells in its surface. The wells may be micro-wells or nano-wells. The size of each well may be characterized by its volume, well opening area, depth and / or diameter.

[0194] Each well can have any volume capable of confining a liquid. The minimum or maximum volume can be selected, for example, to accommodate the throughput (e.g., multiplexity), resolution, analyte composition, or analyte reactivity expected for downstream use of the flow cell. For example, the volume can be at least about 1×10 -3 μm 3 , about 1×10 -2 μm 3 , about 0.1μm 3 , about 1μm 3 , about 10μm 3 , about 100μm 3 Alternatively or additionally, the volume may be at most about 1×10 4 μm 3 , about 1×10 3 μm 3 , about 100μm 3 , about 10μm 3 , about 1μm 3 , about 0.1μm 3 It will be appreciated that the polymer coating 18 may fill all or part of the volume of the well.

[0195] The area occupied by each well opening on the surface can be selected based on criteria similar to those set forth above with respect to well volume. For example, the area for each well opening on the surface can be at least about 1×10 -3 μm 2 , about 1×10 -2 μm 2 , about 0.1μm 2 , about 1μm 2 , about 10μm 2 , about 100μm 2 Alternatively or additionally, the area may be at most about 1×10 3 μm 2 , about 100μm 3 , about 10μm 2 , about 1μm 2 , about 0.1μm 2 , about 1×10 -2 μm 2 The area occupied by each well opening may be greater than the value specified above, less than the value specified above, or between the values ​​specified above.

[0196] The depth of each well may be at least about 0.1 μm, about 1 μm, about 10 μm, about 100 μm, or more. Alternatively or additionally, the depth may be at most about 1×10 3The depth of each well may be greater than the value specified above, less than the value specified above, or between the values ​​specified above.

[0197] In some cases, the diameter of each well can be at least about 50 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 10 μm, about 100 μm, or more. Alternatively or additionally, the diameter can be at most about 1×10 3 μm, about 100 μm, about 10 μm, about 1 μm, about 0.5 μm, about 0.1 μm, or less (eg, about 50 nm). The diameter of each well may be greater than, less than, or between the values ​​specified above.

[0198] like Figure 2C and Figure 2D As shown, after the resin composition 14 is patterned and cured, the cured patterned resin 14 ′ may be processed to prepare the surface for application of the polymer coating 18 .

[0199] In an example, the cured patterned resin 14' can be exposed to silanization, which attaches the silane or silane derivative to the cured patterned resin 14'. The silanization introduces the silane or silane derivative across the surface, including in the recesses 16 (e.g., on the bottom surface and along the sidewalls) and on the gap regions 22.

[0200] Silanization can be accomplished using any silane or silane derivative. The choice of silane or silane derivative may depend in part on the polymer used to form the polymer coating 18 (in Figure 2D ) as it may be desirable to form a covalent bond between the silane or silane derivative and the polymer coating 18. The method for attaching the silane or silane derivative to the cured patterned resin 14' may vary depending on the silane or silane derivative used. Several examples are described herein.

[0201] In an example, the silane or silane derivative is (3-aminopropyl)triethoxysilane (APTES) or (3-aminopropyl)trimethoxysilane (APTMS) (ie, XR B -Si(OR C )3, wherein X is an amino group, R B is -(CH2)3-, and R Cis ethyl or methyl). In this example, the surface of substrate 12 can be pre-treated with (3-aminopropyl) triethoxysilane (APTES) or (3-aminopropyl) trimethoxysilane (APTMS) to covalently link silicon to one or more oxygen atoms on the surface (without intending to be maintained by the mechanism, each silicon can be bound to one, two or three oxygen atoms). The chemically treated surface is baked to form a monolayer of amine groups. The amine groups then react with sulfo HSAB to form an azido derivative. The amine groups are heated at 21°C with 1 J / cm 2 Up to 30J / cm 2 UV activation with energy of 200 Å produces reactive nitrene species that can readily undergo a variety of insertion reactions with PAZAM (eg, one example of a functionalized molecule used to form polymer coating 18).

[0202] Other silanization methods may also be used. Examples of suitable silanization methods include vapor deposition (e.g., YES method), spin coating, or other deposition methods. Some examples of methods and materials that can be used to silanize the cured patterned resin 14' are described herein, but it should be understood that other methods and materials may be used.

[0203] In an example utilizing a YES CVD oven, the cured patterned resin 14' on the substrate 12 is placed in the CVD oven. The chamber may be vented, and then the silanization cycle begins. During the cycle, the silane or silane derivative container may be maintained at a suitable temperature (e.g., about 120°C for norbornene silane), the silane or silane derivative vapor line is maintained at a suitable temperature (e.g., about 125°C for norbornene silane), and the vacuum line is maintained at a suitable temperature (e.g., about 145°C).

[0204] In another example, silane or a silane derivative (e.g., liquid norbornene silane) can be deposited in a glass bottle and placed in a glass vacuum desiccator with patterned substrate 12. The desiccator can then be evacuated to a pressure in a range from about 15 mTorr to about 30 mTorr and placed in an oven at a temperature in a range from about 60° C. to about 125° C. Silanization is allowed to proceed, and the desiccator is then removed from the oven, cooled, and vented to air.

[0205] Vapor deposition, the YES process and / or the vacuum dryer can be used with a variety of silanes or silane derivatives, such as those that include a cycloolefin unsaturated moiety, such as norbornene, norbornene derivatives (e.g., (hetero)norbornene containing oxygen or nitrogen in place of one of the carbon atoms), trans-cyclooctene, trans-cyclooctene derivatives, trans-cyclopentene, trans-cycloheptene, trans-cyclononene, bicyclo[3.3.1]non-1-ene, bicyclo[4.3.1]dec-1(9)-ene, bicyclo[4.2.1]non-1(8)-ene, and bicyclo[4.2.1]non-1-ene. Any of these cycloolefins can be substituted, for example, with R groups such as hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. Examples of norbornene derivatives include [(5-bicyclo[2.2.1]hept-2-enyl)ethyl]trimethoxysilane. As other examples, these methods can be used when the silane or silane derivative includes a cycloalkyne unsaturated moiety such as cyclooctyne, a cyclooctyne derivative, or a bicyclononyne (e.g., bicyclo[6.1.0]non-4-yne or a derivative thereof, bicyclo[6.1.0]non-2-yne, or bicyclo[6.1.0]non-3-yne). These cycloalkynes can be substituted with any of the R groups described herein.

[0206] The attachment of the silane or silane derivative forms a pre-treated (eg, silanized) cured patterned resin 14' that includes silanized recesses and silanized gap regions.

[0207] In other examples, the cured patterned resin 14' may not be exposed to silanization. Instead, the cured patterned resin 14' may be exposed to plasma ashing, and then the polymer coating 18 may be directly spin-coated (or otherwise deposited) on the plasma-ashed cured patterned resin 14'. In this example, plasma ashing may generate surfactants (e.g., hydroxyl (C-OH or Si-OH) and / or carboxyl groups) that may adhere the polymer coating 18 to the cured patterned resin 14'. In these examples, the polymer coating 18 is selected so that it reacts with the surface groups generated by plasma ashing.

[0208] In still other examples, the cured patterned resin 14' may include unreacted epoxy groups (e.g., when an epoxy resin matrix is ​​used with a free radical curable resin matrix); and therefore may not be exposed to silanization because the unreacted epoxy groups can directly react with the amino functional groups of the polymer coating 18. In this example, for example, if it is desired to clean the surface of potential contaminants, plasma ashing can be performed.

[0209] The polymer coating 18 may then be applied to the pre-treated cured patterned resin 14' (eg Figure 2C and Figure 2D ). The polymer coating 18 may be a semi-rigid polymer material that is permeable to liquids and gases and is connected to the cured patterned resin 14'.

[0210] Examples of polymer coatings 18 include acrylamide copolymers, such as poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide, PAZAM. Some other forms of PAZAM and acrylamide copolymers are represented by the following structure (I):

[0211]

[0212] in:

[0213] R 1 is H or optionally substituted alkyl;

[0214] R A is selected from the group consisting of an azido group, an optionally substituted amino group, an optionally substituted alkenyl group, an optionally substituted hydrazone group, an optionally substituted hydrazine group, a carboxyl group, a hydroxyl group, an optionally substituted tetrazole group, an optionally substituted tetrazine group, a nitrile oxide group, a nitrone group, and a thiol group;

[0215] R 5 , R6 and R8 are each independently selected from the group consisting of H and optionally substituted alkyl;

[0216] -(CH2) p - each of which may be optionally substituted;

[0217] p is an integer in the range of 1 to 50;

[0218] n is an integer in the range of 1 to 50,000; and

[0219] m is an integer in the range of 1 to 100,000.

[0220] One of ordinary skill in the art will recognize that the arrangement of the repeating "n" and "m" features in structure (I) is representative and that the monomer subunits may be present in the polymer structure in any order (e.g., random, blocky, patterned, or a combination thereof).

[0221] The molecular weight of PAZAM may range from about 10 kDa to about 1500 kDa, or in a particular example may be about 312 kDa.

[0222] In some instances, PAZAM is a linear polymer. In some other instances, PAZAM is a lightly cross-linked polymer.

[0223] In other examples, the polymer coating 18 can be a variation of structure (I). In one example, the acrylamide unit can be N,N-dimethylacrylamide. In this example, the acrylamide unit in structure (I) can be replaced by substituted, wherein R6, R7 and R8 are each H, and R9 and R 10 Each is a methyl group (rather than H as in the case of acrylamide). In this example, q can be an integer in the range of 1 to 100,000. In another example, in addition to the acrylamide units, N,N-dimethylacrylamide can be used. In this example, in addition to the repeating "n" and "m" features, structure (I) can include wherein R6, R7 and R8 are each H, and R9 and R 10 Each is a methyl group. In this example, q may be an integer in the range of 1 to 100,000.

[0224] As another exemplary polymer, the repeating "n" features in structure (I) may be replaced with a monomer comprising a heterocyclic azido group having structure (II):

[0225]

[0226] Where R 1 R2 is H or C1-C4 alkyl; L is a linker comprising a straight chain having 2 to 20 atoms selected from the group consisting of carbon, oxygen and nitrogen and 10 optional substituents on the carbon and any nitrogen atoms in the chain; E is a straight chain comprising 1 to 4 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents on the carbon and any nitrogen atoms in the chain; A is an N-substituted amide in which H or C1-C4 alkyl is attached to N; and Z is a nitrogen-containing heterocycle. Examples of Z include 5 to 10 ring members present in a monocyclic structure or a condensed structure.

[0227] As yet another example, the polymer may comprise repeating units of each of Structure (III) and Structure (IV):

[0228]

[0229] Where R 1a , R 2a , R 1b and R 2b Each of R is independently selected from hydrogen, optionally substituted alkyl or optionally substituted phenyl; each R 3a and R 3bis independently selected from hydrogen, optionally substituted alkyl, optionally substituted phenyl or optionally substituted C7-14 aralkyl; and each L 1 and L 2 are independently selected from an optionally substituted alkylene linker or an optionally substituted heteroalkylene linker.

[0230] It should be understood that other functionalized molecules can be used to form the polymer coating 18, as long as they are functionalized to interact with the pre-treated cured patterned resin 14' and the primer 24 applied subsequently. Other examples of suitable molecules for forming the polymer coating 18 include those with colloidal structures, such as agarose; or polymer mesh structures, such as gelatin; or cross-linked polymer structures, such as polyacrylamide polymers and copolymers, silane-free acrylamide (SFA) or azid forms 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] photocycloaddition reaction. Still other examples of suitable molecules for forming the polymer coating 18 include mixed copolymers of acrylamide and acrylates. Branched polymers such as star polymers, star or star block polymers, dendrimers and the like can also be used.

[0231] Functionalized molecules (e.g., PAZAM) can be deposited on the surface of the pre-treated cured patterned resin 14' using spin coating, or dipping or dip coating, or flow of functionalized molecules under positive or negative pressure, or another suitable technique. The functionalized molecules can be present in a mixture. In an example, the mixture includes PAZAM in water or in a mixture of ethanol and water.

[0232] After being coated, the functionalized molecules may also be exposed to a curing process to form a polymer coating 18 across the entire patterned substrate (i.e., in the recesses 16 and on the gap regions 22). In an example, curing the functionalized molecules may be performed at a temperature ranging from room temperature (e.g., about 25° C.) to about 95° C. for a time ranging from about 1 millisecond to about several days. In another example, the time may be in the range of from 10 seconds to at least 24 hours. In yet another example, the time may be in the range of from about 5 minutes to about 2 hours.

[0233] Attaching the polymer coating 18 to the pre-treated recesses and interstitial regions can be by covalent bonding. Covalently linking the polymer coating 18 to the silanized or plasma ashed recesses is helpful in maintaining the polymer coating 18 in the recesses 16 during multiple uses throughout the life of the final flow cell. The following are some examples of reactions that can occur between silanes or silane derivatives and the polymer coating 18.

[0234] When the silane or silane derivative includes norbornene or a norbornene derivative as an unsaturated moiety, the norbornene or norbornene derivative can: i) undergo a 1,3-dipolar cycloaddition reaction with an azide / azido group of PAZAM; ii) undergo a coupling reaction with a tetrazine group attached to PAZAM; undergo a cycloaddition reaction with a hydrazone group attached to PAZAM; undergo a photoclick reaction with a tetrazole group attached to PAZAM; or undergo a cycloaddition with a nitrile oxide group attached to PAZAM.

[0235] When the silane or silane derivative includes cyclooctyne or a cyclooctyne derivative as the unsaturated moiety, the cyclooctyne or cyclooctyne derivative can: i) undergo a strain-promoted azide-alkyne 1,3-cycloaddition (SPAAC) reaction with the azide / azide group of PAZAM, or ii) undergo a strain-promoted alkyne-nitrile oxide cycloaddition reaction with the nitrile oxide group attached to PAZAM.

[0236] When the silane or silane derivative includes a bicyclononyne as the unsaturated moiety, the bicyclononyne can undergo similar SPAAC alkyne cycloaddition with an azide or nitrile oxide attached to a PAZAM due to the strain in the bicyclic ring system.

[0237] In order to form the polymer coating 18 in the recess 16 instead of on the gap region 22 of the cured patterned resin 14', the polymer coating 18 can be polished away from the gap region 22. The polishing process can be performed with a mild chemical slurry (including, for example, an abrasive, a buffer, a chelating agent, a surfactant and / or a dispersant) that removes the polymer coating 18 from the gap region 22 without adversely affecting the cured patterned resin 14' and / or the substrate 12 below these regions. Alternatively, the polishing can be performed with a solution that does not contain abrasive particles. The chemical slurry can be used in a chemical mechanical polishing system. In this example, the polishing head / pad or other polishing tool is capable of polishing the polymer coating 18 from the gap region 22 while leaving the polymer coating 18 in the recess 16 and leaving the cured patterned resin 14' below at least substantially intact. As an example, the polishing head can be a Strasbaugh ViPRR II polishing head. In another example, the polishing can be performed with a polishing pad and a solution that does not contain any abrasive. For example, the polishing pad can be used with a solution that does not contain abrasive particles (eg, a solution that does not include abrasive particles).

[0238] Figure 2DThe flow cell precursor 10 is depicted after the polymer layer 18 has been applied to the recess 16. The flow cell precursor 10 can be exposed to a cleaning process. The process can utilize a water bath and sonication. The water bath can be maintained at a relatively low temperature in the range of from about 22° C. to about 30° C. The silanized, coated, and polished patterned substrate can also be spin dried, or dried via another suitable technique.

[0239] like Figure 2D and Figure 2E As shown in the figure, a grafting process is performed so that primer 24 is grafted to the polymer coating 18 in the recess 16. Primer 24 can be any forward amplification primer or reverse amplification primer containing an alkyne functional group, or another end-capped primer. Other examples of end-capped primers that can be used include tetrazine-capped primers, azide-capped primers, amino-capped primers, epoxy- or glycidyl-capped primers, thiophosphate-capped primers, thiol-capped primers, aldehyde-capped primers, hydrazine-capped primers, phosphoramidite-capped primers, and triazolinedione-capped primers. A mixture of primers can also be used. Specific examples of suitable primers include P5 primers and / or P7 primers, which are used on the surface of commercial flow cells sold by Illumina Inc. for use in HISEQ TM HISEQX TM MISEQ TM MISEQDX TM MINISEQ TM 、NEXTSEQ TM 、NEXTSEQDX TM 、NOVASEQ TM 、ISEQ TM 、GENOME ANALYZER TM and other instrument platforms.

[0240] In examples, grafting can be accomplished by flow-through deposition (e.g., using a temporarily bonded cap), dip coating, spray coating, puddle dispensing, or by another suitable method of attaching the primer 24 to the polymer coating 18. Each of these exemplary techniques can utilize a primer solution or mixture, which can include primer, water, buffer, and a catalyst.

[0241] Dip coating may involve applying the flow cell precursor 10 (in Figure 2D16) is immersed in a series of temperature controlled baths. The baths may also be flow controlled and / or covered with nitrogen blankets. The baths may contain primer solutions or mixtures. Throughout the multiple baths, primers 24 will attach to the functional groups of the grafted primers of the polymer coating 18 in at least some of the recesses 16. In an example, the flow cell precursor 10 will be introduced into a first bath containing a primer solution or mixture, in which a reaction occurs to attach the primer 24, and then moved to another bath for washing. Movement from bath to bath may involve a robotic arm, or may be performed manually. A drying system may also be used in immersion coating.

[0242] Spray coating can be accomplished by spraying the primer solution or mixture directly onto the flow cell precursor 10. The spray coated wafer can be incubated at a temperature ranging from about 0° C. to about 70° C. for a time ranging from about 4 minutes to about 60 minutes. After incubation, the primer solution or mixture can be diluted and removed using, for example, a spin coater.

[0243] Puddle distribution can be carried out according to the pool and spin-out method, and can therefore be completed with a spin coater. Primer solution or mixture can be applied to the flow cell precursor 10 (manually or via an automatic process). The applied primer solution or mixture can be applied to the entire surface of the flow cell precursor 10 or spread across the entire surface. The primer-coated flow cell precursor 10 can be incubated at a temperature in the range of from about 0° C. to about 80° C. for a time in the range of from about 2 minutes to about 60 minutes. After incubation, the primer solution or mixture can be diluted and removed using, for example, a spin coater.

[0244] Figure 2F The figure shows an embodiment of a flow cell 10' after primer grafting.While a single type of primer 24 is shown, it will be appreciated that two or more different primers 24 may be attached.

[0245] Figure 2E and Figure 2F The embodiment shown in is an embodiment of the flow cell 10 ' without the cover bonded thereto. Although not shown, the flow cell 10 ' can have a cover bonded to at least a portion of the gap region 22. The cover can be bonded before or after the primer 24 is grafted. When capping is performed before the primer 24 is grafted, it should be understood that the flow-through process can be used for grafting. In the flow-through process, a primer solution or mixture can be introduced into the flow channel (defined between the cover and the gap region 22) through a corresponding input port (not shown), and a time (i.e., incubation period) of continuing to be sufficient to attach the primer 24 to the polymer coating 18 in one or more recesses 16 can be maintained in the flow channel, and then it can be removed from the corresponding output port (not shown). After the primer 24 is attached, additional fluid can be directed through the flow channel to wash the recess and flow channel now functionalized.

[0246] The cover may be positioned over gap region 22 such that it defines a single flow channel or a plurality of fluidly separated flow channels.

[0247] The cover can be any material that is transparent to the excitation light directed toward the recess 16. As an example, the cover can be glass (e.g., Corning Eagle XG (CEXG), borosilicate, fused silica, etc.), plastic, or the like. A commercially available example of a suitable borosilicate glass is D A commercially available example of a suitable plastic material, ie, a cycloolefin polymer, is available from Zeon Chemicals LP product.

[0248] In some examples, the cover can be integrally formed with sidewalls that correspond to the shape of the portion of gap region 22 to which it will be coupled. For example, a recess can be etched into the transparent block to form a generally planar (e.g., top) portion and sidewalls extending from the generally planar portion. When the etched block is mounted to gap region 22, the recess can become a flow channel.

[0249] In other examples, the sidewall and the cover can be separate components coupled to each other. For example, the cover can be a generally rectangular block having at least a generally planar outer surface and at least a generally planar inner surface, the block defining a portion (e.g., a top portion) of the flow channel (once bonded to a portion of the gap region 22). The block can be mounted to (e.g., bonded to) the sidewall, which is bonded to a portion of the gap region 22 and forms a sidewall of the flow channel. In this example, the sidewall can include any material described herein with respect to the spacer layer (described below).

[0250] The cover can be bonded using any suitable technique, such as laser bonding, diffusion bonding, anodic bonding, eutectic bonding, plasma activated bonding, frit bonding, or other methods known in the art. In an example, a spacer layer can be used to bond the cover to portions of the gap region 22. The spacer layer can be any material that seals at least some of the gap region 22 and the cover together.

[0251] In one example, the spacer layer can be a radiation absorbing material that absorbs radiation at wavelengths transmitted by the cover and / or the cured patterned resin 14'. The absorbed energy in turn forms a bond between the spacer layer and the cover and between the spacer layer and the cured patterned resin 14'. An example of such a radiation absorbing material is a black 1064 nm from DuPont (USA) that absorbs at about 1064 nm. (Polyimide containing carbon black). It should be understood that the polyimide can be used without the addition of carbon black, except that the wavelength must be changed to a wavelength that is significantly absorbed by the native polyimide material (e.g., 480nm). As another example, the polyimide CEN JP can be bonded when irradiated with 532nm light. When the spacer layer is a radiation absorbing material, the spacer layer can be positioned at the interface between the cover and the portion of the gap area 22 so that the spacer layer contacts the desired bonding area. Compression (e.g., a pressure of about 100PSI) can be applied while laser energy of a suitable wavelength is applied to the interface (i.e., the radiation absorbing material is irradiated). The laser energy can be applied to the interface from both the top and the bottom to achieve suitable bonding.

[0252] In another example, the spacer layer can include a radiation absorbing material in contact therewith. The radiation absorbing material can be applied at the interface between the spacer layer and the cover and at the interface between the spacer layer and the portion of the gap region 22. As an example, the spacer layer can be a polyimide and the separate radiation absorbing material can be carbon black. In this example, the separate radiation absorbing material absorbs laser energy that forms a bond between the spacer layer and the cover and between the spacer layer and the portion of the gap region 22. In this example, compression can be applied at the corresponding interfaces while laser energy at a suitable wavelength is applied to the interface (i.e., the radiation absorbing material is irradiated).

[0253] The flow cell 10' disclosed herein can be used in a variety of sequencing methods or techniques, including techniques generally referred to as sequencing by synthesis (SBS), cycle array sequencing, ligation sequencing, pyrosequencing, etc. With any of these techniques, since the polymer coating 18 and attached primers 24 are present in the recesses 16 rather than on the interstitial regions 22, amplification will be confined to the recesses.

[0254] As an example, a sequencing-by-synthesis (SBS) reaction can be performed on a system such as the HISEQ TM HISEQX TM MISEQ TM MISEQDX TM MINISEQ TM 、ISEQ TM 、NOVASEQ TM 、NEXTSEQDX TM or NEXTSEQ TMThe SBS is run on a sequencer system. In SBS, the extension of a nucleic acid primer (e.g., a sequencing primer) along a nucleic acid template (i.e., a sequencing template) is monitored to determine the sequence of the nucleotides in the template. The underlying chemical process can be polymerization (e.g., catalyzed by a polymerase) or ligation (e.g., catalyzed by a ligase). In a specific polymerase-based SBS process, fluorescently labeled nucleotides are added to the sequencing primer in a template-dependent manner (thereby extending the sequencing primer), so that the detection of the order and type of nucleotides added to the sequencing primer can be used to determine the sequence of the template.

[0255] Prior to sequencing, amplification primers 24 may be exposed to a sequencing library, which is amplified using any suitable method, such as cluster generation.

[0256] In an example of cluster generation, a high-fidelity DNA polymerase is used to copy library fragments from hybridized primers 24 by 3' extension. The original library fragments are denatured to fix the copies. Isothermal bridge amplification can be used to amplify fixed copies. For example, the copied template is looped on adjacent complementary primers 24 and hybridized with adjacent complementary primers 24, and the copied template is denatured to form a double-stranded bridge by polymerase copy, and the double-stranded bridge is denatured to form two single strands. These two chains are looped on adjacent complementary primers 24 and hybridized with adjacent complementary primers 24, and are extended again to form two new double-stranded loops. The process is repeated on each template copy through a cycle of isothermal denaturation and amplification to produce dense clone clusters. Each cluster of double-stranded bridges is denatured. In an example, the reverse strand is removed by specific base cleavage, leaving a forward template polynucleotide chain. It should be understood that clustering leads to the formation of several template chains.

[0257] To initiate the first SBS cycle, one or more labeled nucleotides, DNA polymerases, etc. can be delivered into / through a flow channel, etc., in which sequencing primer extension causes the labeled nucleotides to be incorporated into the template strand. This incorporation can be detected by an imaging event. During the imaging event, an illumination system (not shown) can provide excitation light to the functionalized recesses. During the imaging event, any emission (if any) from the cured patterned resin 14' due to exposure to blue and / or green excitation wavelengths may be i) undetected because they are below the threshold limit of detection, or ii) distinguished as noise due to the low autofluorescence of the cured patterned resin 14'. In this way, the cured patterned resin 14' disclosed herein is substantially invisible to the detector.

[0258] In some examples, after the nucleotide has been added to the sequencing primer, the nucleotide may also include a reversible termination property that further terminates primer extension. For example, a nucleotide analog having a reversible terminator portion may be added to the sequencing primer such that subsequent extension cannot occur until a deblocking agent is delivered to remove the portion. Thus, for examples using reversible termination, a deblocking agent may be delivered to a flow channel, etc. (before or after detection occurs).

[0259] Washing may occur between the multiple fluid transfer steps.The SBS cycle may then be repeated n times to extend the sequencing primer by n nucleotides, thereby detecting a sequence of length n.

[0260] Although SBS has been described in detail, it should be understood that the flow cell described herein can be used for genotyping with other sequencing schemes, or in other chemical and / or biological applications. Paired-end sequencing is beneficial for detecting genomic rearrangements and repetitive sequence elements, as well as gene fusions and new transcripts. In another example, the flow cell disclosed herein can be used for on-cell library generation.

[0261] Although in Figure 1 and FIG. 2A to FIG. 2F The embodiment diagrams described in the drawings illustrate the use of the resin composition in the formation of a flow cell, but it should be understood that the resin composition can be used in other applications where low autofluorescence is required. As an example, the resin composition 14, 14' can be used in any optical-based SBS technology. As other examples, the resin composition 14, 14' can be used in planar waveguides, complementary metal oxide semiconductors (CMOS), etc.

[0262] To further illustrate the present disclosure, two examples and one prophetic example are given herein. It should be understood that these examples and prophetic examples are provided for illustrative purposes and should not be construed as limiting the scope of the present disclosure. Example

[0263] Example 1

[0264] Six examples of resin compositions were prepared. Each example included a different free radical curable resin matrix including 1,3-bis(3-methacryloxypropyl)tetramethyldisiloxane, methacryloxypropyl terminated polydimethylsiloxane, tetramethyltetra[3-acryloxypropyl]cyclotetrasiloxane, methacryl polyhedral oligomeric silsesquioxane, acryl polyhedral oligomeric silsesquioxane, and acryloxypropylmethylsiloxane homopolymer. Each of these free radical curable resin matrices was reacted with a photoinitiator, namely bis(2,4,6-trimethylbenzoyl)-phenylphosphine oxide (available from IGM Resins as OMNIRAD TM 2022 commercially available) mixed.

[0265] A free radical curable resin matrix and a photoinitiator are mixed with a PGMEA solvent, wherein the concentration of the monomers ranges from about 15 wt % to about 66 wt %, and the concentration of the photoinitiator is about 1 wt %.

[0266] Each of these resin compositions is deposited on a glass substrate by spin coating at 2200 (rev / min) rpm for about 1 minute. After deposition, the resin composition is exposed to soft baking at about 130 ° C for about 2 minutes to remove PGMEA. The deposited resin composition is then imprinted using nanoimprint lithography. The working stamp is pressed into the deposited composition, and the resin is exposed to UV curing with an LED UV lamp for about 20 seconds.

[0267] SEM images of each cured patterned resin were obtained. FIG. 3A to FIG. 3F , the cured patterned resin is formed by 1,3-bis(3-methacryloxypropyl)tetramethyldisiloxane, methacryloxypropyl-terminated polydimethylsiloxane, tetramethyltetra[3-acryloxypropyl]cyclotetrasiloxane, methacryl polyhedral oligomeric silsesquioxane, acryl polyhedral oligomeric silsesquioxane, and acryloxypropylmethylsiloxane homopolymer. These images demonstrate that the resin composition disclosed herein can be nano-imprinted to form suitable recesses for flow cells or other suitable applications.

[0268] Example 2

[0269] An example resin composition was prepared, which contained about 17 wt% of acryl polyhedral oligomeric silsesquioxane (acryl-POSS-as a free radical curable resin matrix), about 1 wt% of bis(2,4,6-trimethylbenzoyl)-phenylphosphine oxide (as a free radical photoinitiator) and propylene glycol monomethyl ether acetate (PGMEA-a solvent). The solvent constituted the balance of the composition (about 82 wt%).

[0270] A comparative resin composition was prepared comprising about 4 wt% of glycidyl functionalized POSS and about 13 wt% of epoxycyclohexylethyl functionalized POSS (as the epoxy resin matrix), a total of about 1.2 wt% of thioxanthen-9-one (ITX) (present at about 0.34 wt%) / A photoinitiator / photoacid generator combination of PC 1467 (Evonik Industries) (present at about 0.85 wt%), about 1.4 wt% of a polyacrylate ( -350) and solvent (PGMEA). The solvent constitutes the balance of the composition (about 80 wt%).

[0271] Each of the comparative example resin composition and the example resin composition was deposited on a glass substrate by spin coating at 2200 (revolutions / minute) rpm for about 1 minute.

[0272] After depositing the Example resin composition and the Comparative Example resin composition, the Example resin composition and the Comparative Example resin composition were exposed to a soft bake at about 120°C for about 2 minutes to remove PGMEA. The resin was then UV cured with an LED UV lamp for about 20 seconds. After UV curing, the Comparative Example resin was hard baked at 250°C for about 10 minutes. The Example resin was not hard baked.

[0273] The cured examples and cured comparative examples were then exposed to an excitation wavelength using a spectrometer based tool. The tool excitation source was a 445nm (blue) laser. The laser spot size was about 200μm. The laser beam passed through a filter to filter out some of the higher wavelength lines and then passed through the sample. A transparent sample (of the cured examples and cured comparative examples) was used for the measurement. In line with the laser beam on the other side of the sample was a fiber optic coupled Ocean Optics spectrometer. The incident laser power can be adjusted by adjusting the drive current or adding an attenuation filter in front of the laser beam. For Figure 4 For the measurements shown in the figure, a laser power of 3 mW was used.

[0274] As from Figure 4 As can be seen from the graph, the fluorescence intensity (in arbitrary units (AU)) of the comparative example in the blue excitation wavelength is as high as 2,500 AU, and the fluorescence intensity in the green excitation wavelength is in the range from about 2,000 AU to about 2,400 AU. In contrast, the fluorescence intensity of the example in the blue and green excitation wavelengths is generally less than about 500 AU, and does not show a fluorescence intensity significantly higher than that of the blank glass substrate.

[0275] Prophecy Example

[0276] The intent of this prophetic example is to compare two formulations in a hypothetical example.

[0277] Example The resin composition comprises 1,3-bis(3-methacryloxypropyl)tetramethyldisiloxane (as free radical curable resin matrix) and bis(2,4,6-trimethylbenzoyl)-phenylphosphine oxide (as free radical photoinitiator) in PGMEA.

[0278] The comparative resin compositions comprised glycidyl-functionalized POSS and epoxycyclohexylethyl-functionalized POSS in PGMEA (as epoxy resin matrix), thioxanthen-9-one (ITX) and A photoinitiator / photoacid generator combination of PC 1467 (Evonik Industries).

[0279] The comparative example composition was deposited in the first two lanes (1 and 2) of the glass or silicon flow cell, and the example composition was deposited in the last two lanes (4 and 5) of the glass or silicon flow cell.

[0280] After depositing the Example resin composition and the Comparative Example resin composition, the resin was cured.

[0281] Using Amersham TYPHOON TM Autofluorescence Tool Each of the cured Examples and cured Comparative Examples was exposed to a blue excitation wavelength and a green excitation wavelength, respectively. It is believed that the autofluorescence results of the Comparative Examples will be much higher than the Examples. Figure 5A and Figure 5B is a graph showing the cured examples and the cured comparative examples after exposure to blue excitation wavelengths ( Figure 5A ) and green excitation wavelength ( Figure 5B ). In these figures, the grayscale value represents the autofluorescence and the distance in cm at which the lane is located on the flow cell.

[0282] This prophetic example compares a hypothetical acrylate-based formulation and a hypothetical epoxy-based formulation and presents the autofluorescence results for each hypothetical formulation proposed to illustrate that a combination of a free radical-curable resin matrix comprising acrylates and siloxanes and a free radical photoinitiator can result in lower autofluorescence.

[0283] Additional Notes

[0284] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in more detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of the claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It should also be appreciated that terms used expressly herein that may also appear in any disclosure incorporated by reference should conform to the meaning most consistent with the particular concepts disclosed herein.

[0285] References throughout the specification to "one example," "another example," "an example," etc. mean that a particular element (e.g., features, structures, and / or characteristics) described in conjunction with the example is included in at least one example described herein, and may or may not be present in other examples. In addition, it should be understood that the elements described for any example may be combined in any suitable manner in multiple examples, unless the context clearly dictates otherwise.

[0286] It should be understood that the ranges provided herein include the stated ranges and any values ​​or sub-ranges within the stated ranges, as if such values ​​or sub-ranges were explicitly listed herein. For example, a range from about 380 nm to about 480 nm should be interpreted as including not only the explicitly listed limits from about 380 nm to about 480 nm, but also individual values, such as about 408 nm, about 445.5 nm, etc. and sub-ranges, such as from about 425 nm to about 475 nm, etc. In addition, when "about" and / or "substantially" are used to describe a value, they are meant to cover minor variations (up to + / - 10%) of the stated value.

[0287] While several examples have been described in detail, it should be understood that the disclosed examples may be modified. Therefore, the foregoing description should be considered non-limiting.

Claims

1. A resin composition comprising: a free radical curable resin matrix comprising acrylate groups and siloxane groups, wherein the free radical curable resin matrix comprises acrylate groups selected from the group consisting of 1,3-bis(3-methacryloxypropyl)tetramethyldisiloxane, methacryloxypropyl terminated polydimethylsiloxane, tetramethyltetra[3-acryloxypropyl]cyclotetrasiloxane, methacryl polyhedral oligomeric silsesquioxane, acryl polyhedral oligomeric silsesquioxane, acryloxypropyl methylsiloxane homopolymer, and combinations thereof; A free radical photoinitiator, wherein the free radical photoinitiator is phenyl bis (2,4,6-trimethylbenzoyl) phosphine oxide; An epoxy resin matrix, wherein the epoxy resin matrix is ​​selected from: i) epoxy-functionalized polyhedral oligomeric silsesquioxane POSS; ii) Trimethylolpropane triglycidyl ether: iii) Tetrakis(epoxycyclohexylethyl)tetramethylcyclotetrasiloxane: iv) Copolymer of (epoxycyclohexylethyl)methylsiloxane and dimethylsiloxane: wherein the ratio of m:n is in the range of from 8:92 to 10:90; v) 1,3-Bis[2-(3,4-epoxycyclohexyl)ethyl]tetramethyldisiloxane: vi) 1,3-Bis(glycidyloxypropyl)tetramethyldisiloxane: and vii) combinations thereof; and A photoacid generator, wherein the photoacid generator is selected from: i) N-Hydroxynaphthalimide trifluoromethanesulfonate: ii) Mixed triarylsulfonium hexafluorophosphates: iii) Mixed triarylsulfonium hexafluoroantimonates: iv) 1-naphthyldiphenylsulfonium trifluoromethanesulfonate NDS-TF: v) (4-phenylthiophenyl)diphenylsulfonium trifluoromethanesulfonate: vi) Bis-(4-methylphenyl)iodonium hexafluorophosphate: vii) Bis(4-tert-butylphenyl)iodonium hexafluorophosphate: viii) (2-Methylphenyl) (2,4,6-trimethylphenyl) iodonium trifluoromethanesulfonate: xi) Bis(2,4,6-trimethylphenyl)iodonium trifluoromethanesulfonate: x) Bis-(4-dodecylphenyl)iodonium hexafluoroantimonate: xi) combinations thereof; in, When cured, the resin composition has low or no autofluorescence when exposed to a blue excitation wavelength in a range from 380 nm to 480 nm or a green excitation wavelength in a range from 510 nm to 560 nm.

2. The resin composition of claim 1, wherein the cured resin composition has low autofluorescence, and wherein the low autofluorescence corresponds to a grayscale value of less than 25,000 when the cured resin composition is exposed to the blue excitation wavelength.

3. The resin composition of claim 1, wherein the cured resin composition has low autofluorescence, and wherein the low autofluorescence corresponds to a grayscale value of less than 5,000 when the cured resin composition is exposed to the blue excitation wavelength.

4. The resin composition of claim 1, wherein the cured resin composition has low autofluorescence, and wherein the low autofluorescence corresponds to a grayscale value of less than 10,000 when the cured resin composition is exposed to the green excitation wavelength.

5. The resin composition of claim 1, wherein the cured resin composition has low autofluorescence, and wherein the low autofluorescence corresponds to a grayscale value of less than 2,500 when the cured resin composition is exposed to the green excitation wavelength. The resin composition according to claim 1 , further comprising a dark quencher or an electron acceptor.

7. The resin composition of claim 1, wherein the weight % ratio of the free radical curable resin matrix to the free radical photoinitiator ranges from 99.8:0.2 to 90:

10.

8. A flow cell comprising: substrate; as well as a cured patterned resin on the substrate, the cured patterned resin comprising recesses separated by gap regions, and the cured patterned resin having been formed from a resin composition comprising: a free radical curable resin matrix comprising acrylate groups and siloxane groups, wherein the free radical curable resin matrix comprises acrylate groups selected from the group consisting of 1,3-bis(3-methacryloxypropyl)tetramethyldisiloxane, methacryloxypropyl terminated polydimethylsiloxane, tetramethyltetra[3-acryloxypropyl]cyclotetrasiloxane, methacryl polyhedral oligomeric silsesquioxane, acryl polyhedral oligomeric silsesquioxane, acryloxypropyl methylsiloxane homopolymer, and combinations thereof; A free radical photoinitiator, wherein the free radical photoinitiator is phenyl bis (2,4,6-trimethylbenzoyl) phosphine oxide; An epoxy resin matrix, wherein the epoxy resin matrix is ​​selected from: i) epoxy-functionalized polyhedral oligomeric silsesquioxane POSS; ii) Trimethylolpropane triglycidyl ether: iii) Tetrakis(epoxycyclohexylethyl)tetramethylcyclotetrasiloxane: iv) Copolymer of (epoxycyclohexylethyl)methylsiloxane and dimethylsiloxane: wherein the ratio of m:n is in the range of from 8:92 to 10:90; v) 1,3-Bis[2-(3,4-epoxycyclohexyl)ethyl]tetramethyldisiloxane: vi) 1,3-Bis(glycidyloxypropyl)tetramethyldisiloxane: vii) combinations thereof; and A photoacid generator, wherein the photoacid generator is selected from: i) N-Hydroxynaphthalimide trifluoromethanesulfonate: ii) Mixed triarylsulfonium hexafluorophosphates: iii) Mixed triarylsulfonium hexafluoroantimonates: iv) 1-naphthyldiphenylsulfonium trifluoromethanesulfonate NDS-TF: v) (4-phenylthiophenyl)diphenylsulfonium trifluoromethanesulfonate: vi) Bis-(4-methylphenyl)iodonium hexafluorophosphate: vii) Bis(4-tert-butylphenyl)iodonium hexafluorophosphate: viii) (2-Methylphenyl) (2,4,6-trimethylphenyl) iodonium trifluoromethanesulfonate: xi) Bis(2,4,6-trimethylphenyl)iodonium trifluoromethanesulfonate: x) Bis-(4-dodecylphenyl)iodonium hexafluoroantimonate: xi) combinations thereof; The cured patterned resin has low or no autofluorescence when exposed to a blue excitation wavelength in a range from 380 nm to 480 nm or a green excitation wavelength in a range from 510 nm to 560 nm.

9. The flow cell according to claim 8, further comprising: a polymer coating in said depression; and A primer is grafted to the polymer coating.

10. The flow cell of claim 8, wherein the cured patterned resin has low autofluorescence, and wherein the low autofluorescence corresponds to a grayscale value of less than 25,000 when the cured patterned resin is exposed to the blue excitation wavelength.

11. The flow cell of claim 8, wherein the cured patterned resin has low autofluorescence, and wherein the low autofluorescence corresponds to a grayscale value of less than 10,000 when the cured patterned resin is exposed to the green excitation wavelength. 12 . The flow cell according to claim 8 , wherein the resin composition further comprises a dark quencher or an electron acceptor.

13. The flow cell of claim 8, wherein the weight % ratio of the free radical curable resin matrix to the free radical photoinitiator in the resin composition ranges from 99.8:0.2 to 90:

10.

14. A method for manufacturing a flow cell, comprising: A resin composition is deposited on a substrate, the resin composition comprising: a free radical curable resin matrix comprising acrylate groups and siloxane groups, wherein the free radical curable resin matrix comprises acrylate groups selected from the group consisting of 1,3-bis(3-methacryloxypropyl)tetramethyldisiloxane, methacryloxypropyl terminated polydimethylsiloxane, tetramethyltetra[3-acryloxypropyl]cyclotetrasiloxane, methacryl polyhedral oligomeric silsesquioxane, acryl polyhedral oligomeric silsesquioxane, acryloxypropyl methylsiloxane homopolymer, and combinations thereof; A free radical photoinitiator, wherein the free radical photoinitiator is phenyl bis (2,4,6-trimethylbenzoyl) phosphine oxide; An epoxy resin matrix, wherein the epoxy resin matrix is ​​selected from: i) epoxy-functionalized polyhedral oligomeric silsesquioxane POSS; ii) Trimethylolpropane triglycidyl ether: iii) Tetrakis(epoxycyclohexylethyl)tetramethylcyclotetrasiloxane: iv) Copolymer of (epoxycyclohexylethyl)methylsiloxane and dimethylsiloxane: wherein the ratio of m:n is in the range of from 8:92 to 10:90; v) 1,3-Bis[2-(3,4-epoxycyclohexyl)ethyl]tetramethyldisiloxane: vi) 1,3-Bis(glycidyloxypropyl)tetramethyldisiloxane: vii) combinations thereof; and A photoacid generator, wherein the photoacid generator is selected from: i) N-Hydroxynaphthalimide trifluoromethanesulfonate: ii) Mixed triarylsulfonium hexafluorophosphates: iii) Mixed triarylsulfonium hexafluoroantimonates: iv) 1-naphthyldiphenylsulfonium trifluoromethanesulfonate NDS-TF: v) (4-phenylthiophenyl)diphenylsulfonium trifluoromethanesulfonate: vi) Bis-(4-methylphenyl)iodonium hexafluorophosphate: vii) Bis(4-tert-butylphenyl)iodonium hexafluorophosphate: viii) (2-Methylphenyl) (2,4,6-trimethylphenyl) iodonium trifluoromethanesulfonate: xi) Bis(2,4,6-trimethylphenyl)iodonium trifluoromethanesulfonate: x) Bis-(4-dodecylphenyl)iodonium hexafluoroantimonate: xi) combinations thereof; A resin composition deposited by nanoimprinting using a working stamp; and curing the deposited resin composition to form a cured patterned resin; The cured patterned resin has low or no autofluorescence when exposed to a blue excitation wavelength in a range from 380 nm to 480 nm or a green excitation wavelength in a range from 510 nm to 560 nm.

Images