RU2778014C1 - Resin composition and flow cells including this resin composition - Google Patents

Abstract

FIELD: genetic engineering.

SUBSTANCE: invention relates to materials for genetic sequencing. A resin composition is proposed for the production of a flow cell for genetic sequencing, including a curable free-radical resin matrix including polysiloxane substituted with acryloxy- or methacryloxy-groups or acryl-substituted polyhedral oligomeric silsesquioxane, a free-radical photo-initiator, a matrix of epoxide resin, and a photo acid generator. In a cured state, the proposed resin composition differs in weak autofluorescence or absence of autofluorescence in radiation with blue light with an excitation wave length from about 380 nm to about 480 nm, or in radiation with green light with an excitation wave length from about 510 nm to about 560 nm. A flow cell for genetic sequencing and its production method are also proposed.

EFFECT: proposed composition allows for the creation of effective flow cells for genetic sequencing with a reduced level of autofluorescence.

21 cl, 5 dwg, 2 ex

Description

Cross-references to related applications

This application claims the benefit of Provisional Patent Application US 62/701228, filed July 20, 2018, the contents of which are hereby incorporated by reference in their entirety.

Prior Art

A biological array (English biological array, also called a biochip or biological matrix) is one of a huge variety of tools used to detect and analyze molecules, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Arrays for this application are designed to include probes for nucleotide sequences found in the genes of humans and other organisms. For example, in some applications, separate DNA and RNA probes can be attached to small patches created on the array substrate in a geometric grid (or random manner). For example, a test sample derived from a known human or other organism may be applied to the grid such that complementary fragments hybridize to the probes at individual sites in the array. The array can then be examined by scanning the sites at certain frequencies of light, and the fluorescence of the sites to which the fragments are hybridized can determine which fragments are present in the sample.

Biological arrays can be used for genetic sequencing. In general, genetic sequencing involves determining the order of nucleotides or nucleic acids along the length of genetic material, such as a piece of DNA or RNA. Progressive base pair sequences are analyzed and the resulting sequence information can be used in various bioinformatics techniques to logically arrange fragments relative to each other to reliably determine the sequence of the long genetic material from which the fragments were derived. Techniques for automated computerized study of characteristic fragments have been developed, which are used for genome mapping, identification of genes and their functions, risk assessment for certain conditions and diseases, etc. In addition to these applications, biological arrays can be used to detect and study a variety of molecules, families of molecules, gene expression levels, single nucleotide polymorphisms, and to perform genotyping.

The essence of the invention

According to one aspect, the resin composition includes a curable free radical resin matrix, including acrylate and siloxane, and a free radical photoinitiator; however, in the cured state, the resin composition exhibits little or no autofluorescence when irradiated with blue light with an excitation wavelength of about 380 nm to about 480 nm, or when irradiated with green light with an excitation wavelength of about 510 nm to about 560 nm.

In one example, the cured resin composition exhibits low autofluorescence, where low autofluorescence upon irradiation of the cured resin composition with light having an excitation wavelength in the blue range corresponds to a gray value of less than 25,000.

In another example, the cured resin composition exhibits low autofluorescence, where low autofluorescence when the cured resin composition is irradiated with light having an excitation wavelength in the blue range corresponds to a gray value of less than 5000.

According to another example, the cured resin composition is characterized by low autofluorescence, where low autofluorescence when the cured resin composition is irradiated with light having an excitation wavelength in the green range corresponds to a gray value of less than 10,000.

According to another example, the cured resin composition is characterized by low autofluorescence, where low autofluorescence when the cured resin composition is irradiated with light having an excitation wavelength in the green range corresponds to a gray value of less than 2500.

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

In one example, the free radical photoinitiator is 2-ethyl-9,10-dimethoxyanthracene.

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

According to another example, the free radical photoinitiator is a phosphine oxide, where the phosphine oxide is selected from the group consisting of diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, a mixture 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)phenylphosphinate, and combinations of these substances.

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

In one example, the weight percentage of curable free radical resin matrix to free radical photoinitiator is from about 99.8:0.2 to about 90:10.

In one example, the resin composition further includes an epoxy resin matrix and a photoacid generator.

It should be understood that any features of this aspect of the resin composition may be combined with each other in any desired manner and/or in any desired configuration to achieve the beneficial effects discussed herein, which include, for example, relatively weak autofluorescence.

According to another aspect, the flow cell includes a substrate and a cured patterned resin deposited on the substrate, where the cured structured resin includes recesses separated by intermediate sections, and the cured structured resin was obtained from a resin composition, including: a curable free radical resin matrix, including acrylate and siloxane, and a free radical photoinitiator; wherein the cured structured resin exhibits little or no autofluorescence when irradiated with blue light at an excitation wavelength of about 380 nm to about 480 nm, or when irradiated with green light with an excitation wavelength of about 510 nm to about 560 nm.

In one example, the flow cell further includes a recessed polymer coating and a primer grafted onto the polymer coating.

In one example, the cured structured resin exhibits low autofluorescence, where low autofluorescence when the cured structured resin is irradiated with light having an excitation wavelength in the blue range corresponds to a gray value of less than 25,000.

In another example, the cured structured resin exhibits low autofluorescence, where low autofluorescence when the cured structured resin is irradiated with light having an excitation wavelength in the green range corresponds to a gray value of less than 10,000.

In another example, the resin composition further includes an epoxy resin matrix and a photoacid generator.

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

In one 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.

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 mixture 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)phenylphosphinate and combinations of these substances.

In one example, the resin composition further includes a dark quencher or electron acceptor.

In one example, the weight percentage of the amount of curable free radical resin matrix to the amount of free radical photoinitiator in the resin composition is from about 99.8:0.2 to about 90:10.

It should be understood that any features of this aspect of the flow cell may be combined with each other in any desired manner to achieve the beneficial effects discussed herein, including, for example, relatively weak autofluorescence. In addition, it should be understood that any combination of features of this aspect of the flow cell and/or resin composition can be used together, and/or that any features of any or both of these aspects can be combined with any of the examples discussed to achieve the beneficial effects discussed in this work, including, for example, relatively weak autofluorescence.

In another aspect, the method comprises depositing (applying) a resin composition onto a substrate, wherein the resin composition comprises a curable free radical resin matrix comprising an acrylate and a siloxane, and a free radical photoinitiator; nanoimprinting (i.e. pressing a template with nanosized elements into a layer of material) of the deposited resin composition using a working stamp; and curing the deposited resin composition to form a cured structured resin, wherein the cured structured resin exhibits little or no autofluorescence when irradiated with blue light with an excitation wavelength of about 380 nm to about 480 nm, or when irradiated with green light with an excitation wavelength, component from about 510 nm to about 560 nm.

It should be understood that any of the features of this aspect of the method can be combined with each other in any desired manner to achieve the beneficial effects discussed in this work, including, for example, relatively weak autofluorescence. In addition, it should be understood that any combination of features of the method and/or flow cell and/or resin composition can be used together, and/or that any features of any or all of the considered aspects can be combined with any features of the considered examples to achieve beneficial effects, considered in this work, including, for example, relatively weak autofluorescence.

Additionally, it should be understood that any features of any method and/or flow cell and/or resin composition can be combined with each other in any desired manner, and/or can be combined with any of the considered examples to achieve the beneficial effects discussed in this work, including, for example, relatively weak autofluorescence.

Brief description of graphic materials

Features of the exemplary embodiments of the present invention will become more apparent upon closer examination of the figures, in which like reference numerals refer to similar, but not necessarily identical, components. For brevity, numerals or features having the functions previously described may or may not be described in combination with other figures in which they are depicted.

On FIG. 1 is a block diagram showing one example of the method discussed in this paper;

On FIG. 2A-2E are schematic perspective views which together illustrate an example of the method disclosed herein;

On FIG. 2F is a schematic and cross-sectional view along line 2F-2F shown in FIG. 2E;

On FIG. 3A-3F are scanning electron micrographs of exemplary wells formed from various examples of the resin compositions discussed herein;

On FIG. 4 is a graph showing fluorescence intensity (in arbitrary units) versus wavelength (in nanometers (nm)) of the resin obtained in the comparative example, the embodiment of the resin and the optical glass substrate; and

On FIG. 5A and 5B are graphs showing the expected autofluorescence results for the Model Resin Composition Example and the Model Resin Composition Comparative Example.

Information confirming the possibility of carrying out the invention

It is desirable that patterned flow cells used for sequencing have baseline levels of autofluorescence that allow sufficiently high signal-to-noise ratios (SNR for short) to be achieved to allow signal resolution of individual clusters in the process. sequencing. Resin compositions used to form structured flow cells often include photoinitiators (eg, free radical and/or cationic particle generators) that initiate curing upon application of the structure from the composition. In theory, if the photoinitiators used do not absorb light in the visible region of the spectrum, then no fluorescence is observed when irradiated during sequencing with sources of excitatory blue, green, and/or red light. However, based on studies in solution of some combinations of free radical and cationic photoinitiators, it was found that at the excitation wavelength of interest, unwanted autofluorescence can be observed even when, based on the spectral properties of the resin components, absorption should not be observed in the visible region of the spectrum, or only weak absorption.

The resin matrix of the resin composition discussed herein is based on acrylate (eg, the resin includes acrylate and/or methacrylate) and exhibits relatively low levels of fluorescence after curing. In addition, acrylate-based resins cure exclusively according to the free radical mechanism. With this curing mechanism, a free radical photoinitiator can be used without involving other types of photoinitiators such as cationic photoinitiators. This, in turn, can minimize and in some cases even eliminate intramolecular interactions that may otherwise occur between different types of photoinitiators (eg, between certain free radical and cationic photoinitiators in combinations). It is believed that such intramolecular interactions may take place before or during ultraviolet (UV) irradiation, indicating that energy exchange between the combination of photoinitiators and the subsequent formation of excited state complexes (exciplexes) and adducts may contribute to unwanted autofluorescence. It is also believed that due to the absence of the above intramolecular interactions in the resin composition discussed in this work, the considered cured resin composition can generate autofluorescence (when irradiated with light with an excitation wavelength of interest (for example, with an excitation wavelength in blue and/or in green range)), which is about an order of magnitude lower than the autofluorescence of resins that include a combination of a free radical photoinitiator and a cationic photoinitiator.

The resin composition discussed in this paper can also more easily undergo photobleaching, rendering the cured resin composition permanently incapable of autofluorescence (i.e., the amount of autofluorescence that can still develop is less than the detection threshold or does not affect detection methods). when sequencing).

As stated in this paper, examples of the considered cured resin composition are characterized by minimal emission of radiation in the blue and green regions, and are also characterized by very little or no autofluorescence when irradiated with light with an excitation wavelength in the blue and green range. According to the invention, blue light wavelengths are from about 463 nm to about 514 nm, and green light wavelengths are from about 583 nm to about 660 nm. Also according to the invention, the excitation wavelength in the blue range is from about 380 nm to about 480 nm, and the excitation wavelength in the green range is from about 510 nm to about 560 nm. In another example, the blue excitation wavelength is from about 440 nm to about 457 nm, or the green excitation wavelength is from about 519 nm to about 535 nm. According to another example, the excitation wavelength in the blue range is from about 400 nm to about 480 nm.

In some instances, the cured resin composition may be considered non-fluorescent (non-light emitting) when excited with light at a wavelength in the blue range and/or at a wavelength in the green range. No fluorescence or no autofluorescence means that the fluorescence level lies below the detection threshold. According to the invention, no fluorescence or no autofluorescence can occur if the cured resin composition has been subjected to photobleaching. In other cases, the cured resin composition fluoresces (emits light) when excited by light with a wavelength in the blue range and/or a wavelength in the green range. In these examples, the term "weak autofluorescence" may mean that the level of radiation (cured resin irradiated with light with an excitation wavelength in the blue range and/or in the green range) lies above the detection threshold, but is low enough to be considered noise, and noise does not interfere with the identification of cluster signals during sequencing (eg, autofluorescence levels correspond to sufficiently high signal-to-noise ratios (SNRs) to allow signal resolution of individual clusters during sequencing).

It should be understood that when quantifying autofluorescence, the definition of "weak" or "low" may vary depending on the characteristics of the instrument used to measure autofluorescence and/or the lamps used to generate the excitation radiation. For example, if an Amersham TYPHOON™ (formerly called TYPHOON™ FLA 7000) device (available from GE Healthcare Life Sciences) is used to measure the autofluorescence of a cured resin composition when irradiated with light at an excitation wavelength in the blue range, then weak autofluorescence corresponds to a gray value of less than 25,000. In another example, if an Amersham TYPHOON™ device is used to measure the autofluorescence of a cured resin composition when irradiated with light at an excitation wavelength in the blue range, then weak autofluorescence corresponds to a gray value of less than 5000. In another example, if the autofluorescence measurement of a cured resin composition compositions when irradiated with light with an excitation wavelength in the green range using the Amersham TYPHOON™ device, then weak autofluorescence corresponds to a gray value of less than 10,000. In another example, if for measuring autofluorescence of a cured resin compound When exposed to green excitation wavelengths using an Amersham TYPHOON™ device, low autofluorescence corresponds to a gray value of less than 2500.

Autofluorescence can also be measured with a spectrometer-based instrument. A discussion of examples of spectrometer-based devices is provided below in Example 2. In one example, if a spectrometer-based device is used to measure the autofluorescence of a cured resin composition when irradiated with light with an excitation wavelength in the blue range, then weak autofluorescence corresponds to the intensity of fluorescence (in arbitrary units (PU)) of less than about 400 PU. In another example, if a spectrometer-based device is used to measure the autofluorescence of a cured resin composition when irradiated with light with an excitation wavelength in the green range, then weak autofluorescence corresponds to a fluorescence intensity (in arbitrary units (PU)) of less than about 500 PU.

It should be understood that, unless otherwise indicated, the terms used in the present description have their usual meanings known in the relevant field of technology. Some of the terms used in the present description and their meanings are given below.

The singular forms include the plural unless the context makes it clear otherwise.

The terms "comprising", "comprising" and various forms of these terms are synonymous with each other and have the same broad meaning.

The terms "top", "bottom", "top", "bottom", "on", etc. are used herein to describe the flow cell module and/or the various components of the flow cell. It should be understood that such direction designations do not refer to a specific orientation, but are used to indicate the orientation of components relative to each other. The use of directional designations should not be construed as limiting the examples discussed herein to any particular orientation(s).

или мономер, включающий акриламидную группу, имеющую эту структуру. Акриламид может представлять собой химическое соединение ациламид, имеющее заместитель вместо одного или более атомов водорода (например, метакриламид). Примеры мономеров, включающих акриламидную группу, включают азидоацетамидопентилакриламид:

и N-изопропилакриламид:

Могут быть применены другие акриламидные мономеры."Acrylamide" is a functional group having the structure

or a monomer containing an acrylamide group having this structure. Acrylamide can be an acylamide chemical compound having a substituent in place of one or more hydrogen atoms (eg, methacrylamide). Examples of monomers containing an acrylamide group include azidoacetamidopentyl acrylamide:

and N-isopropylacrylamide:

Other acrylamide monomers may be used.

As used herein, the term "aldehyde" refers to an organic compound containing a functional group having the structure -CHO, which includes a carbonyl center (i.e., a carbon double bonded to oxygen) in which the carbon atom is also bonded to a hydrogen and a group R, such as alkyl, or with another side chain. The aldehyde has the following general structure:

As used herein, the term "alkyl" refers to a fully saturated (ie, free of double or triple bonds) straight or branched hydrocarbon chain. The alkyl group may contain from 1 to 20 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like. For example, the designation "C1-4 alkyl" indicates that the alkyl chain contains from one to four carbon atoms, i.e. the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

Used in the present description, the term "alkenyl" refers to a straight or branched hydrocarbon chain containing one or more double bonds. The alkenyl group may contain from 2 to 20 carbon atoms. Examples of alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like.

As used herein, the term "alkyne" or "alkynyl" refers to a straight or branched hydrocarbon chain containing one or more triple bonds. The alkynyl group may contain from 2 to 20 carbon atoms.

As used herein, the term "aryl" refers to an aromatic ring or ring system (ie, two or more fused rings that share two adjacent carbon atoms) that contain only carbon atoms in the backbone of the ring. If aryl is a ring system, then each ring in the system is an aromatic ring. The aryl group may contain from 6 to 18 carbon atoms. Examples of aryl groups include phenyl, naphthyl, azulenyl and anthracenyl.

), С1-С6 алкила, С2-С6 алкенила, С2-С6 алкинила, С3-С7 карбоциклила, С6-С10 арила, 5-10-членного гетероарила и 5-10-членного гетероциклила, определения которых приведены в настоящей работе.By "amino" functionality is meant a -NR _a R _b group in which R _a and R _b are each independently selected from hydrogen (e.g.,

), C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclyl, C6-C10 aryl, 5-10-membered heteroaryl, and 5-10-membered heterocyclyl, which are defined in this paper.

Used in the present description, the term "attached" means the state of two items that are combined, fastened, glued, connected or attached to each other either directly or indirectly. For example, the nucleic acid can be attached to the polymer coating by a covalent or non-covalent bond. A covalent bond is characterized by the socialization of a pair of electrons by a set of atoms. A non-covalent bond is a physical bond that does not involve the sharing of electron pairs and may include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, and hydrophobic interactions.

The term "azide" or "azido" functional means -N ₃ .

As used herein, the term "bonding region" means a portion located on a substrate to be bonded to another material, which may be, for example, a release layer, a lid, another substrate, etc. or combinations of such materials (for example, a release layer and a lid). The bond formed in the bonding region may be a chemical bond (discussed above) or a mechanical bond (eg via a fastener, etc.).

Used in the present description, the term "carbocyclyl" means a non-aromatic cyclic ring (cycle) or a cyclic system containing only carbon atoms in the main chain of the cyclic system. If the carbocyclyl is a ring system, then two or more rings may be connected to each other in a fused system, bridged, or as a spiro ring system. The carbocyclyls may be of any degree of saturation, provided that at least one ring in the ring system is non-aromatic. Thus, carbocyclyls include cycloalkyls, cycloalkenyls and cycloalkynyls. The carbocyclyl group may contain from 3 to 20 carbon atoms. Examples of carbocyclyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydroindene, bicyclo[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl.

Used in the present description, the term "cycloalkenyl" or "cycloalkene" means a carbocyclic ring (cycle) or a system of cycles containing at least one double bond, and the system of cycles does not contain aromatic cycles. Examples include cyclohexenyl or cyclohexene and norbornenyl or norbornene. Also used in the present description, the term "heterocycloalkenyl" or "heterocycloalkene" means a carbocyclic ring (cycle) or a cycle system containing at least one heteroatom in the main chain of the cycle and containing at least one double bond, and the cycle system does not contain aromatic cycles.

As used herein, the term "cycloalkyl" refers to any monovalent groups derived from cycloalkanes by abstraction of a hydrogen atom from a ring carbon atom (eg, from a cycloalkane). An example of such a group includes 2-methylcyclopropyl.

Used in the present description, the term "cycloalkynyl" or "cycloalkyne" means a carbocyclic ring (cycle) or a system of cycles containing at least one triple bond, and the system of cycles does not contain aromatic cycles. An example is cyclooctyne. Another example is bicyclononin. Also used in the present description, the term "heterocycloalkynyl" or "heterocycloalkyne" means a carbocyclic ring (cycle) or a cycle system containing at least one heteroatom in the main chain of the cycle and containing at least one triple bond, and the cycle system does not contain aromatic cycles.

As used herein, the term "dimple" refers to a discrete (separate) concave feature in a structured resin having an opening on its surface that is at least partially surrounded by an intermediate region(s) of the structured resin. The depressions can have many different shapes of the opening on the surface, examples of which include round, elliptical, square, polygonal, star-shaped (with any number of rays), and so on. The cross section of the depression in a plane perpendicular to the plane of the surface may be curved, square, polygonal, hyperbolic, conical, angled, etc. As examples of a recess, a hole or a groove/track/trough can be mentioned. The recess may also have a more complex structure, including, for example, ledges, steps, etc.

As used herein, the term "flow cell" means a container including a chamber (i.e., a flow channel) in which a reaction can be carried out, an inlet for introducing reagent(s) into the chamber, and an outlet for removing reagent(s) from the cameras. In some examples, the camera allows you to observe the progress of the reaction in the chamber. For example, the camera/flow channel may include one or more transparent surfaces through which an optical examination of arrays, optically labeled molecules, or the like located in the recess can be performed.

As used herein, the term "flow channel" may refer to the area defined by the two connected components into which a liquid sample can be selectively placed. In some examples, the flow channel may be enclosed between the structured resin and the cover, and thus may be in fluid communication with one or more recesses present in the structured resin.

As used herein, the term "heteroaryl" refers to an aromatic ring or ring system (i.e., two or more fused rings sharing two neighboring atoms) that contain one or more heteroatoms, i.e., elements other than carbon, and non-limiting examples of heteroaryls include nitrogen, oxygen and sulfur in the backbone of the ring. If heteroaryl is a ring system, then each ring in the system is aromatic. The heteroaryl group may have 5 to 18 ring members.

Used in the present description, the term "heterocyclyl" means a non-aromatic cyclic ring (cycle) or system of cycles containing at least one heteroatom in the main chain of the cycle. Heterocyclyls can be connected to each other in a fused system, connected by a bridge or in the form of a spirocyclic system. The heterocyclyls may be of any degree of saturation, provided that at least one ring in the ring system is non-aromatic. In a ring system, the heteroatom(s) may be present in a non-aromatic or aromatic ring. The heterocyclyl group may contain from 3 to 20 ring members (ie, the number of atoms that make up the backbone of the ring, including carbon atoms and heteroatoms). In some examples, the heteroatom(s) is O, N, or S.

Used in the present description, the term "hydrazine" or "hydrazinyl" refers to the group-NHNH ₂ .

в которой каждый из R_a и R_b независимо выбран из водорода (например,

), С1-С6 алкила, С2-С6 алкенила, С2-С6 алкинила, С3-С7 карбоциклила, С6-С10 арила, 5-10-членного гетероарила и 5-10-членного гетероциклила, определения которых приведены в настоящей работе.As used herein, the term "hydrazone" or "hydrazonil" refers to the group

in which each of R _a and R _b is independently selected from hydrogen (for example,

), C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclyl, C6-C10 aryl, 5-10-membered heteroaryl, and 5-10-membered heterocyclyl, which are defined in this paper.

As used herein, the term "hydroxy" or "hydroxyl" refers to the -OH group.

Used in the present description, the term "intermediate area" refers to the area located on the surface (eg, structured resin), which separates the recesses. For example, an intermediate section may separate one element of the array (biochip) from another element of the array. Two separated elements can be discrete, i.e. without physical contact with each other. In another example, the intermediate portion may separate the first element part from the second element part. In many instances, the intermediate portion is continuous while the features are discrete, such as in the case of a plurality of wells bounded by an otherwise continuous surface. In other examples, the intermediate sections and elements are discrete, such as in the case of a plurality of grooves separated by respective intermediate sections. The separation provided by the intermediate section may be partial or complete separation. The surface material of the intermediate portions may be different from the surface material of the elements formed in the surface. For example, the elements of the array may have an amount or concentration of polymer coating and primer(s) that exceeds the amount or concentration of these substances in the intermediate regions. In some instances, the polymer coating and primer(s) may be omitted from the intermediate regions.

Used in the present description, the term "nitrile oxide" means the group "R _a C≡N ⁺ O ^- ", in which R _a has the definition discussed in the present description. Examples of the production of nitrile oxide include in situ generation from aldoximes by the action of chloramide-T or by the action of a base on imidoyl chlorides [RC(Cl)=NOH] or by the reaction of hydroxylamine with an aldehyde.

в которой R₁, R₂ и R₃ могут представлять собой любые из групп R_a и R_b, рассмотренных в настоящем описании.As used herein, the term "nitrone" means a group

in which R ₁ , R ₂ and R ₃ may represent any of the groups R _a and R _b discussed in the present description.

As used herein, the term "nucleotide" includes a nitrogen-containing heterocyclic base, a sugar, and one or more phosphate groups. Nucleotides are the monomeric units of a nucleic acid sequence. In RNA, the sugar is ribose, and in DNA, the sugar is deoxyribose, i.e. a sugar that does not have a hydroxyl group that is present at the 2' position of the ribose. The nitrogen-containing heterocyclic base (ie, nucleic base) may be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G) and modified derivatives or analogues thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U) and modified derivatives or analogs thereof. The C-1 atom of deoxyribose is linked to the N-1 of the pyrimidine or N-9 of the purine. In a nucleic acid analog, either the phosphate backbone or any sugar or any nucleotide base can be changed. Examples of nucleic acid analogs include, for example, universal bases or sugar-phosphate backbone analogs such as peptide nucleic acids (PNA, abbr.).

As used herein, the term "photoacid generator" (English photoacid generator, abbreviated as PAG) is a molecule that releases protons under the action of radiation. Usually, the photodetachment of a proton from PAG is irreversible.

Used in the present description, the term "photoinitiator" (English photoinitiator, abbreviated as PI) is a molecule that, after absorbing radiation, enters into a photoreaction, forming reactive particles. Photoinitiators will be able to initiate or catalyze chemical reactions that lead to the solubility and/or physical properties of drugs.

Used in the present description, the term "primer" means a single-stranded nucleic acid sequence (for example, single-stranded DNA or single-stranded RNA). Some primers, which may be referred to as amplification primers, serve as the starting point for template amplification and cluster generation. Other primers, which may be referred to as sequencing primers, serve as the starting point for DNA or RNA synthesis. The 5' end of the primer can be modified to allow the addition reaction to take place at the functional group of the polymer coating. The primer may be any number of bases in length, and the primer may include various non-naturally occurring nucleotides. In one example, the sequencing primer is a short chain of 10 to 60 bases or 20 to 40 bases.

Used in the present description, the term "separation layer" refers to a material that binds two components to each other. In some examples, the separating layer may be composed of a material capable of absorbing radiation that promotes bonding, or the layer may be in contact with a material capable of absorbing radiation that promotes bonding.

The functional "thiol" group means -SH.

Used in the present description, the terms "tetrazine" and "tetrazinyl" mean a six-membered heteroaryl group containing four nitrogen atoms. Tetrazine may be optionally substituted.

Used in the present description, the term "tetrazol" means a five-membered heterocyclic group containing four nitrogen atoms. The tetrazole may optionally be substituted.

In the examples discussed herein, the resin complex comprises a curable free radical resin matrix comprising an acrylate and a siloxane and a free radical photoinitiator, wherein the resin composition after curing exhibits no autofluorescence or weak autofluorescence when irradiated with blue light with an excitation wavelength of from about 380 nm to about 480 nm, or when irradiated with green light with an excitation wavelength of from about 510 nm to about 560 nm.

) или функциональную группу "СН₂=С(СН₃)СОО-" (т.е. метакрилат). Акрилаты включают замещенные варианты (например, примером акрилата является метакрилат). Силоксановая группа представляет собой функциональную группу, которая включает соединительный фрагмент Si-O-Si. Молекулярная масса и плотность поперечных связей между молекулами мономера (мономеров) в отверждаемой свободнорадикальной смоляной матрице могут быть различными, при условии, что смоляную композицию можно обрабатывать способами, приводящими к получению оттисков, рассмотренными в настоящей работе. В одном из примеров молекулярная масса мономера (мономеров) в отверждаемой свободнорадикальной смоляной матрицы может составлять от приблизительно 0,5 кДа до приблизительно 5 кДа или от приблизительно 1 кДа до приблизительно 4,5 кДа. Сшивающая способность и, таким образом, плотность поперечных связей, зависит от количества точек сшивки (например, две, три, четыре) в мономере.The curable free radical resin matrix includes an acrylate group and a siloxane group. As used herein, the term "acrylate" refers to the functional group "CH ₂ =CHCOO-" (i.e.

) or a functional group "CH ₂ =C(CH ₃ )COO-" (i.e. methacrylate). Acrylates include substituted variants (for example, methacrylate is an example of an acrylate). The siloxane group is a functional group that includes a Si-O-Si connecting moiety. The molecular weight and cross-linking density between the molecules of the monomer(s) in the curable free-radical resin matrix can be varied, provided that the resin composition can be processed by the imprinting methods discussed in this paper. In one example, the molecular weight of the monomer(s) in the curable free radical resin matrix may be from about 0.5 kDa to about 5 kDa, or from about 1 kDa to about 4.5 kDa. The cross-linking capacity and thus the cross-link density depends on the number of cross-link points (eg two, three, four) in the monomer.

Examples of curable free radical resin matrices are matrices comprising an acrylate selected from the group consisting of:

i) 1,3-bis(3-methacryloxypropyl)tetramethyldisiloxane:

ii) polydimethylsiloxane with terminal methacryloxypropyl groups:

(where n is from 1 to 1000 or is in any range between the specified values, for example, from 1 to 500 or from 1 to 100, or from 2 to 50, from 1 to 10, from 1 to 2, etc. .),

iii) tetramethyltetrakis[3-acryloxypropyl]cyclotetrasiloxane:

iv) methacryl-polyhedral oligomeric silsesquioxane (polyhedral oligomeric silsesquioxane, abbreviated as POSS):

v) acrylic-polyhedral oligomeric silsesquioxane:

vi) acryloxypropyl-methylsiloxane homopolymer:

(where m is from 1 to 1000 or is in any range between the specified values, for example, from 1 to 500, from 30 to 200, from 30 to 100, etc.), and

vii) combinations of the listed substances. If combinations are used, it should be understood that any two or more of the listed curable free radical resin matrices may be co-included in the resin composition, provided that they are all soluble in the solvent included in the composition.

The photoinitiator added to the resin compositions of the present invention can be any free radical generating photoinitiator that exhibits low autofluorescence in the cured resin.

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

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

In other examples, the free radical photoinitiator is 2-ethoxy-2-phenylacetophenone (also called benzoinethyl ether):

In other examples, the free radical photoinitiator is a phosphine oxide. If a phosphine oxide is used, it may be selected from the group consisting of:

i) diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide:

ii) mixtures of diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide and 2-hydroxy-2-methylpropiophenone:

iii) phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide:

iv) ethyl (2,4,6-trimethylbenzoyl)phenyl phosphinate:

v) combinations of the listed substances. If combinations are used, it should be understood that any two or more of the listed free radical photoinitiators may be co-included in the resin composition, provided that they are all soluble in the solvent included in the composition.

The resin composition can be obtained by mixing a curable free radical resin matrix with a photoinitiator. In one example, the weight percentage of curable free radical resin matrix to photoinitiator is from about 99.8:0.2 to 90:10. In another example, the weight percentage of curable free radical resin matrix to photoinitiator is from about 98:2 to 95:5. According to another example, the weight percentage of the amount of curable free radical resin matrix to the amount of photoinitiator is from about 96:4 to 99:1. If smaller amounts of photoinitiator are used, a longer UV curing time may be required to complete the reaction.

It should be understood that the weight percentage of the amount of curable free radical resin matrix to the amount of photoinitiator may be greater or less depending, at least in part, on the type of curable free radical resin matrix component(s) used.

To apply the resin composition, the components considered (free radical curable resin matrix and photoinitiator) can be diluted with a suitable solvent (to achieve the viscosity required for the application technique used), such as propylene glycol monomethyl ether acetate (English propylene glycol monomethyl ether acetate, abbr. PGMEA), toluene, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), etc. In one example, the concentration of the curable free radical resin matrix in the solvent is from about 15 wt. % (mass percent) to about 56 wt. %, and the concentration of the photoinitiator in the solvent is from about 1 wt. % to about 10 wt. %; however, it is believed that the upper end of the range may be higher and depend on the solubility of the curable free radical resin matrix and photoinitiator, respectively, in the chosen solvent. In one example, the solvent is PGMEA. The total concentration (including curable free radical resin matrix and photoinitiator (and, if present, polyacrylate or surfactant)) of the finished resin composition can be from about 16 wt. % to about 66 wt. % The amount of solvent can be from about 34 wt. % to about 34 wt. %.

In some examples, the resin composition may further include a dark quencher or electron acceptor. A dark quencher is a substance that absorbs light energy generated by a fluorophore and dissipates it as heat. This provides a non-radiative path for the relaxation of particles in an excited state. In the considered embodiments, the selected dark quencher must be capable of absorbing the blue and/or green autofluorescence of the cured resin composition. An electron acceptor can also provide fluorescence quenching.

DABCYL азида (где DABCYL=4-(dimethylaminoazo)benzene-4-carboxylic acid, т.е. 4-(диметиламиноазо)бензол-4-карбоновая кислота):

DABSYL азида (где DABSYL=4-(dimethylamino)azobenzene-4'-sulfonyl, т.е. 4-(диметиламино)азобензол-4'-сульфонил):

дисперсного красного 19:

добавки в виде сажевого наполнителя (англ. carbon black pigment, сокр. СВР), гасителя на основе черного красителя (такого как, например, TRUEBLACK® липофусциновый гаситель аутофлуоресценции, 20Х в ДМФА (диметилформамиде)) и комбинаций перечисленным веществ. Примеры подходящих акцепторов электронов могут включать [5,6]-фуллерен-С70:

или фуллерен-С60:

In one example, the dark quencher may include azo dyes. In one additional example, the dark quencher comprises a substituted azobenzene derivative selected from the group consisting of: 4-dimethylaminoazobenzene-4'-carboxylic acid:

Azide DABCYL (where DABCYL=4-(dimethylaminoazo)benzene-4-carboxylic acid, i.e. 4-(dimethylaminoazo)benzene-4-carboxylic acid):

Azide DABSYL (where DABSYL=4-(dimethylamino)azobenzene-4'-sulfonyl, i.e. 4-(dimethylamino)azobenzene-4'-sulfonyl):

dispersed red 19:

additives in the form of a carbon black pigment (CBP), a black dye quencher (such as, for example, TRUEBLACK® lipofuscin autofluorescence quencher, 20X in DMF (dimethylformamide)) and combinations of these substances. Examples of suitable electron acceptors may include [5,6]-fullerene-C70:

or fullerene-C60:

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

Some other examples of the resin composition may further include a polyacrylate or a surfactant. An example of a commercially available polyacrylate is BYK®-350 (available from BYK Additives & Instruments). Any biocompatible surfactant may be used, such as TWEEN® surfactants (e.g., polyethylene sorbitol esters (TWEEN® 80) and polyoxyethylene sorbitol esters (TWEEN® 20) supplied by Uniqema Americas LLC; TRITON™ X-100 ( octylphenol ethoxylate supplied by The Dow Chemical Co.), and polymeric surfactants supplied by BYK Additives and Instruments In one example, the polyacrylate or surfactant may be present in the resin composition in an amount of from about 0.4 wt. % of total solids to about 1.6 wt % of total solids.

In some examples, the resin composition may further include an epoxy resin matrix in combination with a curable free radical resin matrix. Unlike the curable free-radical resin matrix discussed in this paper, epoxy resin matrices contain a two-component curing system in which free radicals generated by the photoinitiator discussed in this paper react with a photoacid generator, which decomposes to form a superacid, which, in turn, in turn, initiates polymerization and/or cross-linking of the epoxy resin matrix component(s). Thus, it should be understood that in the examples that include an epoxy matrix along with a curable free radical resin matrix, the resin composition also includes a photoacid generator. It is believed that in these examples, the anthracene derived Norrish Type II photoinitiator and the acetophenone or phosphine oxide based Norrish Type I photoinitiators discussed in this paper can generate radicals when exposed to ultraviolet A light, which can enhance a two-part curing system containing a photoacid generator. , but not initiate intramolecular interactions that lead to unwanted autofluorescence in the blue and/or green wavelength range of interest. The following are examples of suitable epoxy resin matrices and photoacid generators that can be added to the resin composition discussed in this paper.

Any epoxy monomer that can form crosslinks with the curable free radical resin of the invention can be used. Examples of suitable epoxy resin matrices include:

i) epoxy-functionalized polyhedral oligomeric silsesquioxane (POSS) (further disclosed below);

ii) Trimethylolpropane triglycidyl ether:

iii) tetrakis(epoxycyclohexylethyl)tetramethylcyclotetrasiloxane:

iv) copolymer of (epoxycyclohexylethyl)methylsiloxane and dimethylsiloxane

(in which the ratio m:n is from 8:92 to 10:90);

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

vi) 1,3-bis(glycidoxypropyl)tetramethyldisiloxane:

vii) combinations of the listed substances. If combinations are used, it should be understood that any two or more of the listed epoxy resin matrices may be co-included in the resin composition of the invention.

которая может быть представлена как:

Такая мономерная единица обычно содержит восемь ответвлений в виде функциональных групп R₁-R₈.The epoxy-functionalized polyhedral oligomeric silsesquioxane includes a polyhedral oligomeric silsesquioxane (POSS) core that is functionalized with epoxy groups. Used in the present description, the term "polyhedral oligomeric silsesquioxane (English polyhedral oligomeric silsesquioxane, abbreviated as POSS)" refers to a chemical composition that is an intermediate hybrid (RSiO _1.5 ) between silicon oxide (SiO ₂ ) and organosilicon material (R ₂ SiO ). An example of POSS is the substance described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776-778, the contents of which are hereby incorporated by reference in their entirety. This composition is an organosilicon compound having the chemical formula [RSiO _3/2 ] _n in which the R groups may be the same or different. The resin composition discussed in this work may include one or more different "cage" or "core" structures as monomeric units. In some instances, the structure includes a "cage" or "core" polyoctahedral structure. For example, the polyhedral structure may be a T ₈ structure such as:

which can be represented as:

Such a monomeric unit usually contains eight branches in the form of functional groups R ₁ -R ₈ .

или может иметь структуру типа клетки, содержащую 12 атомов кремния и 12 групп R, обозначаемую Т₁₂, такую как:

В альтернативном случае материал на основе POSS может включать структуры Т₆, Т₁₄ или Т₁₆ типа клетки. Среднее содержимое клетки может быть уточнено в процессе синтеза и/или регулироваться типом способа очистки, и примеры осуществления изобретения могут включать определенное распределение размеров клетки, образованной мономерной единицей (единицами). Например, любая из структур типа клетки может присутствовать в количестве, составляющем от приблизительно 30% до приблизительно 100% от общего количества применяемых мономерных единиц POSS. Материал на основе POSS может представлять собой смесь структур типа клетки и структур в виде клеток открытого и частично открытого типа. Таким образом, предшественник смолы или смола на основе POSS может включать эпоксидированные материалы POSS, которые могут включать смеси конфигураций силсесквиоксана. Например, любой материал POSS, рассмотренный в настоящей заявке, может быть смесью дискретных клеток POSS и недискретных силсесквиоксановых структур и/или не полностью сконденсированных дискретных структур, таких как полимеры, лестничные полимеры и подобные структуры. Таким образом, частично сконденсированные материалы включают рассмотренные в настоящей заявке эпоксидные группы R, расположенные на некоторых вершинах, образуемых атомами кремния, но при этом к некоторым атомам кремния не будут присоединены замещающие группы R, а, напротив, могут быть присоединены замещающие группы ОН. В некоторых примерах материалы POSS включают смесь различных форм, таких как формы, приведенные ниже:The monomeric unit may have a cell type structure containing 10 silicon atoms and 10 R groups, denoted T ₁₀ , such as:

or may have a cage type structure containing 12 silicon atoms and 12 R groups, denoted T ₁₂ , such as:

Alternatively, the POSS-based material may include T ₆ , T ₁₄ or T ₁₆ cell type structures. The average content of the cell can be refined during synthesis and/or controlled by the type of purification method, and embodiments of the invention may include a certain size distribution of the cells formed by the monomeric unit (units). For example, any of the cell type structures may be present in an amount ranging from about 30% to about 100% of the total POSS monomer units used. The POSS-based material may be a mixture of cage structures and open and partially open cage structures. Thus, the resin precursor or POSS resin may include epoxidized POSS materials, which may include mixtures of silsesquioxane configurations. For example, any POSS material contemplated herein may be a mixture of discrete POSS cells and non-discrete silsesquioxane structures and/or incompletely condensed discrete structures such as polymers, ladder polymers, and the like. Thus, partially fused materials include the epoxy R groups discussed herein located on some of the vertices formed by silicon atoms, but some of the silicon atoms will not have substituent R groups attached, but, on the contrary, substituent OH groups can be attached. In some examples, the POSS materials include a mixture of different shapes, such as the shapes below:

condensed cells

incompletely condensed cells

Content outside the cells

Large and poorly defined structures

In the examples discussed herein, at least one of R ₁ -R ₈ or R ₁₀ or R ₁₂ includes an epoxy group, and thus POSS is referred to as epoxy-POSS (or epoxidized POSS). In some examples, most of the branches, for example, eight, ten or twelve branches or R groups include epoxy groups. In other examples, R ₁ -R ₈ or R ₁₀ or R ₁₂ are the same, and thus each of R ₁ -R ₈ or R ₁₀ or R ₁₂ includes an epoxy group. In other examples, R ₁ -R ₈ or R ₁₀ or R ₁₂ are not the same, and thus at least one of the groups R ₁ -R ₈ or R ₁₀ or R ₁₂ includes an epoxy group, and at least one of the other groups R ₁ -R ₈ or R ₁₀ or R ₁₂ is a non-epoxy functional group, which in some cases is selected from the group consisting of azide/azido group, thiol, polyethylene glycol, norbornene, tetrazine, acrylates and methacrylates or additionally, for example, alkyl, aryl, alkoxy and haloalkyl groups. In some aspects, the non-epoxy functional group is chosen to increase the surface energy of the resin. In such examples, the ratio of epoxy to non-epoxy groups is 7:1 to 1:7, or 9:1 to 1:9, or 11:1 to 1:11. In any instance, the presence of a disubstituted or monosubstituted (terminal) epoxy group(s) allows polymerization of the monomeric units to proceed to form a crosslinked matrix after ultraviolet (UV) radiation and acid initiation. In some aspects, the epoxy-POSS includes terminal epoxy groups. An example of this type of POSS is a glycidyl functionalized POSS having the following structure:

Another example of this type of POSS is epoxycyclohexylethyl functionalized POSS which has the following structure:

One example of an epoxy resin matrix discussed in this paper includes a combination of two epoxy POSS compounds, wherein said combination includes glycidyl functionalized POSS and epoxycyclohexylethyl functionalized POSS.

In the examples discussed in this work, epoxy-POSS can also be a modified epoxy-POSS, which includes a controlled radical polymerization agent (English controlled radical polymerization, abbr. CRP) and / or other target functional group introduced into the resin or structure nuclei or cells in the form of one or more functional groups R ₁ -R ₈ or R ₁₀ or R ₁₂ .

As noted above, if an epoxy resin matrix is used in combination with a curable free radical resin matrix, then the resin composition also includes a photoacid generator. It is believed that any suitable photoacid generator can be used that does not enter into undesirable intramolecular interactions with the free radical photoinitiator discussed in this paper. Examples of suitable photoacid generators may include benzyl, iminoester, conjugated iminoester, spiropyran, tetrylene, two-photon and organometallic PAG photoacid generator systems. Some specific examples of suitable photoacid generators are selected from the group consisting of:

i) N-hydroxynaphthalimide triflate (trifluoromethanesulfonate):

ii) triarylsulfonium hexafluorophosphate mixed salts:

iii) triarylsulfonium hexafluoroantimonate mixed salts:

iv) 1-naphthyldiphenylsulfonium triflate (denoted NDS-TF):

v) (4-phenylthiophenyl)diphenylsulfonium triflate:

vi) bis-(4-methylphenyl)iodonium hexafluorophosphate:

vii) bis(4-tert-butylphenyl)iodonium hexafluorophosphate:

viii) (2-methylphenyl)(2,4,6-trimethylphenyl)iodonium triflate:

xi) bis(2,4,6-trimethylphenyl)iodonium triflate:

x) Bis(4-dodecylphenyl)iodonium hexafluoroantimonate salt:

xi) Combinations of the listed substances. Combinations of photoacid generators can be used provided they are soluble in the chosen solvent.

In one example of a resin composition comprising an epoxy resin matrix and a photoacid generator, the weight percentage of the epoxy resin matrix to the photoacid generator is approximately 99.8:0.2 to 90:10. In another example, the weight percentage of epoxy resin matrix to photoacid generator is about 98:2 to about 95:5. According to another example, the weight percentage of the epoxy resin matrix to the photoacid generator is about 96:4 to 99:1. If smaller amounts of photoacid generator are added, a longer UV curing time may be required to complete the reaction.

It should be understood that the weight percentage of the amount of epoxy matrix to the amount of photoacid generator may be higher or lower depending at least in part on the type of epoxy matrix component(s) used.

Any example of the resin composition discussed in this paper can be used to fabricate the flow cell. An exemplary method 100 for obtaining an example flow cell is shown in FIG. 1. As shown in FIG. 1, method 100 includes depositing (applying) to a substrate a resin composition (reference symbol 102), wherein the resin composition comprises a curable free radical resin matrix comprising an acrylate and a siloxane, and a free radical photoinitiator; nanoimprinting of the deposited resin composition using a working stamp (symbol 104); and curing the precipitated resin composition to form a cured structured resin (reference symbol 106), wherein the cured structured resin exhibits little or no autofluorescence when irradiated with blue light with an excitation wavelength of about 380 nm to about 480 nm, or when irradiated with green light. with an excitation wavelength of about 510 nm to about 560 nm. The resulting flow cell includes a substrate and a cured structured resin deposited on the substrate, where the cured structured resin includes depressions separated by intermediate sections, and the cured structured resin is obtained from the resin composition example discussed in this work.

Although not shown in FIG. 1, examples of method 100 may further include applying a polymer coating to the recesses, and grafting a primer onto the polymer coating. Method 100, including such additional steps, is discussed in more detail below with reference to FIG. 2A-2E.

On FIG. 2A shows substrate 12 and FIG. 2B shows resin composition 14 deposited on substrate 12.

Examples of suitable substrates 12 include epoxysiloxane, glass and modified or functionalized glass, plastics (including acrylic polymers, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, polytetrafluoroethylene (such as TEFLON®, celebrated by Chemours), cyclic olefins/ cycloolefin polymers (COP for short) (such as ZEONOR® supplied by Zeon), polyimides, etc., nylon, ceramic materials/ceramic oxides, silicon oxide, fused silica or silica-based materials, silicate aluminium, silicon and modified silicon (for example, boron-doped p+ silicon), silicon nitride (Si ₃ N ₄ ), silicon oxide (SiO ₂ ), tantalum pentoxide (TaO ₅ ) or other tantalum oxide(s) (TaO _x ), hafnium oxide (HaO ₂ ), carbon, metals, inorganic glasses, or the like The substrate 12 may also be made of glass or silicon and have on the surface the following th coating of titanium oxide or other ceramic oxide.

In one example, the diameter of substrate 12 may be from about 2 mm to about 300 mm, or a rectangular sheet or panel, the longest side of which is up to about 10 feet (~3 meters). In one example, substrate 12 is a multilayer structure that is between about 200 mm and about 300 mm in diameter. In another example, substrate 12 is a cube that is about 0.1 mm to about 10 mm wide. While examples of sizes are provided herein, it should be understood that substrate 12 can be used in any suitable size. In another example, a panel may be used which is a rectangular substrate 12 whose surface area is greater than that of a circular sandwich structure with a diameter of 300 mm.

Resin composition 14 can be any of the examples of resin compositions discussed herein that includes a curable free radical resin matrix comprising an acrylate and a siloxane and a free radical initiator (or an epoxy resin matrix in combination with a curable free radical resin matrix). Resin composition 14 may be applied to substrate 12 using any suitable application technique, which may be manual or automated. For example, the deposition of the resin composition 14 may be performed by vapor deposition, coating methods, grafting, or the like. Some specific examples include chemical vapor deposition (CVD), spray coating (e.g., ultrasonic spray coating), spin coating, dip or dip coating, knife coating, coating distribution, aerosol printing, screen printing, microcontact printing, inkjet printing, or the like. In one example, spin coating is used to deposit the resin composition 14.

After the resin composition 14 is deposited, it can be subjected to heat treatment at a low temperature (English softbaked) to remove excess solvent. The deposited resin composition 14 is then cross-linked using any of the cross-linking techniques mentioned. In the example shown in FIG. 2B, nanoimprint lithography is used to structure resin composition 14. The form or working stamp 20 for nanoimprint lithography is pressed against the layer of resin composition 14, receiving an imprint on the resin composition 14. In other words, the resin composition 14 is pressed or pierced by the protrusions of the work die 20. In one example, the resin composition 14 may be cured with the work die 20 pressed against it.

The resin compositions 14 discussed herein can be cured by actinic radiation, such as ultraviolet (UV) radiation (eg, metal halide light sources, mercury vapor light sources, LED UV light sources, etc.). The presence of the photoinitiator during curing causes the formation of radicals, and these radicals cause the acrylate portion of the resin composition to cure. Thus, curing results in polymerization and/or cross-linking of the resin composition 14. For example, curing may include a single UV exposure step, or may include a combination of steps including low temperature heat treatment (e.g., to remove solvent(s)) followed by exposure to UV radiation. If the heat treatment is performed at a low temperature, then it can be carried out at a lower temperature of from about 50°C to about 150°C for a time in excess of 0 seconds to about 3 minutes, and can be carried out before placing the working die 20 to resin composition 14. In one example, the duration of the heat treatment at a low temperature is from about 30 seconds to about 2.5 minutes. During the heat treatment at a low temperature, one or more chemical processes may occur that further contribute to the reduction of autofluorescence. Examples of chemical processes may include evaporation of any resin composition material(s), sublimation of any resin composition material(s), polymerization of any resin composition material(s), and/or combinations of these processes. Some examples may also include heat treatment at an elevated temperature. However, the cure rate of the resin composition 14 discussed in this paper is so fast that the resin composition 14 can be fully cured without heat treatment at an elevated temperature. If heat treatment is performed at an elevated temperature, then the work die 20 is released/removed prior to heat treatment, for example, so that the work die 20 does not adhere to the cured resin composition 14. up to about 10 minutes at a temperature of from about 100°C to about 300°C. Heat treatment at an elevated temperature may be performed, for example, to remove residual solvent(s) from the resin composition 14, to further polymerize any material(s) of the resin composition (and thus increase the degree of curing), and/or to further decrease in autofluorescence. Examples of devices that can be used for low temperature heat treatment and/or high temperature heat treatment include a hot plate, a furnace, and the like.

After the working die 20 is released, topography is formed in the resin composition 14, for example, recesses 16. Shown in FIG. 2C, the resin composition 14 in which the recesses 16 are formed is referred to as the cured structured resin 14'. In some examples, the cured structured resin 14' may be subjected to additional heat treatment at an elevated temperature, for example, to fix the embossed topography and/or to further reduce fluorescence. In some examples, additional heat treatment at elevated temperature can be performed at a temperature of from about 60°C to about 300°C.

The chemical structure of the cured structured resin 14' depends on the type of curable free radical resin matrix or matrices and the free radical photoinitiator(s) added to the resin composition 14.

As shown in FIG. 2C, recesses 16 are made in the cured structured resin 14' and there are intermediate regions 22 separating adjacent recesses 16. In the examples discussed in this work, recesses 16 are functionalized with polymer coating 18 (Fig. 2C-2D) and primers 24 (Fig. 2E -2F), while parts of the intermediate sections 22 can be used for bonding and do not have polymer coating 18 or primer(s) 24.

The invention includes the use of various patterns of recesses 16, which include ordered, repeating and random patterns. In one example, the recesses 16 are arranged in a hexagonal pattern to create a close packing and achieve increased density. Other layouts may include, for example, straight (ie, rectangular) layouts (eg, lines or grooves), triangular layouts, and so on. In some examples, the layout or pattern of recesses 16 may be in x-y format, that is, include rows and columns. In some other examples, the pattern or pattern of recesses 16 and/or intermediate portions 22 may be a repeating arrangement. In other examples, the pattern or pattern of recesses 16 and/or intermediate portions 22 may be randomized. The pattern may include patches, planes, holes, bars, stripes, curves, lines, triangles, rectangles, circles, arches, checkerboard patterns, criss-cross stripes, diagonals, arrows, squares, and/or criss-cross strokes. In one example, as shown in FIG. 2C, recesses 16 are wells.

The pattern or pattern of recesses can be characterized by the density of recesses 16 (ie, the number of recesses 16) in a limited area. For example, recesses 16 may be present at a density of approximately 2 million per mm ² . Various densities can be achieved, including, for example, densities of at least about 100/ ^mm2 , about 1000/ ^mm2 , about 0.1 ppm/ ^mm2 , about 1 ppm/mm2, about ² ppm/mm ² , approximately 5 ppm mm ² , approximately 10 ppm mm ² , approximately 50 ppm mm ² or more. Alternatively or additionally, a density value may be selected not to exceed 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 0, 1 million per mm ² , approximately 1000 per mm ² , approximately 100 per mm ² or less. It should also be understood that the placement density of the recesses 16 in the cured structured resin'14' may be from one of the lower values to one of the upper values selected from the above ranges. In some examples, a high density array can be characterized as an array containing pits 16 separated by a region of less than about 100 nm, a medium density array can be characterized as an array containing pits 16 separated by a region ranging from about 400 nm to about 1 µm. , and a low density array can be characterized as an array containing depressions 16 separated by an area larger than about 1 micron. While examples of densities have been given above, it should be understood that substrates having any suitable densities may be used.

Also or alternatively, the pattern or pattern of recesses 16 may be characterized by a medium pitch, i. e. the distance from the center of a recess 16 to the center of an adjacent recess 16 (center-to-center distance) or the distance from the edge of one recess 16 to the edge of an adjacent recess 16 (edge-to-edge distance). The pattern may be ordered, ie have a small average pitch deviation factor, or the pattern may be disordered, in which case the deviation factor may be relatively large. In any case, the average pitch may 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 additionally, the largest average pitch may be, for example, about 100 µm, about 10 µm, about 5 µm, about 1 µm, about 0.5 µm, about 0.1 µm, or less. The average pitch value of a particular recess pattern 16 may be from one of the lower values to one of the upper values selected from the above ranges. In one example, the pitch between the recesses 16 (distance from center to center) is approximately 1.5 microns. While examples of average pitch values have been given above, it should be understood that other average pitch values may be used.

In the example shown in FIG. 2A-2E, the depressions 16 are pits, and thus there is an array of pits on the surface of the cured structured resin 14'. The wells may be microwells or nanowells. The size of each well may be characterized by volume, well opening area, well depth and/or diameter.

Each well may be of any volume sufficient to hold liquid. The minimum or maximum volume may be selected, for example, to achieve the desired throughput (eg, multiplexity) and resolution, to suit the analyte composition or analyte reactivity expected when using the flow cell as the post-treatment unit. For example, the volume may be at least about 1×10 ^-3 μm ³ , approximately 1×10 ^-2 μm ³ , approximately 0.1 μm ³ , approximately 1 μm ³ , approximately 10 μm ³ , the maximum volume may be approximately 1× 10 ⁴ µm ³ , about 1×10 ³ µm ³ , about 100 µm ³ , about 10 µm ³ , about 1 µm ³ , about 0.1 µm ³ or less. It should be understood that the polymer coating 18 may fill the entire volume of the well or part of the volume of the well.

The area occupied by the opening of each well on the surface can be selected according to criteria similar to those above for well volume. For example, the area occupied by the opening of each well on the surface may be at least about 1×10 ^-3 μm ² , approximately 1×10 ^-2 μm ² , approximately 0.1 μm ² , approximately 1 μm ² , approximately 10 μm ² , about 100 µm ² or more. Alternatively or additionally, the maximum area may be about 1×10 ³ µm ² , about 100 µm ² , about 10 µm ² , about 1 µm ² , about 0.1 µm ² , about 1×10 ^-2 µm ² or less. The area occupied by the opening of each well may be greater than the above values, less than the above values, or be in between.

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 maximum depth may be about 1×10 ³ µm, about 100 µm, about 10 µm, about 1 µm, about 0.1 µm, or less. The depth of each well may be greater than the above values, less than the above values, or in between.

In some cases, the diameter of each well may 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 maximum diameter may be 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 the above values, less than the above values, or be in between.

As indicated between FIG. 2C and FIG. 2D, after the resin composition 14 has been cross-linked and cured, the cured cross-linked resin 14' may be treated to prepare the surface for polymer coating 18.

In one example, the cured structured resin 14' may be subjected to a silanization that adds the silane or silane derivative to the cured structured resin 14'. Silanization involves the introduction of a silane or silane derivative over the entire surface, including depressions 16 (for example, on the bottom surface and along the side walls) and intermediate areas 22.

Any silane or silane derivative can be used for silanization. The choice of silane or silane derivative may depend in part on the type of functionalized molecule that is used to form polymer coating 18 (shown in FIG. 2D), since it may be desirable to form a covalent bond between the silane or silane derivative and polymer coating 18. The method used to attach silane or silane derivative to the cured structured resin 14' may depend on the type of silane or silane derivative used. Below are some examples.

In one example, the silane or silane derivative is (3-aminopropyl)triethoxysilane (English (3-aminopropyl)triethoxysilane, abbreviated APTES) or (3-aminopropyl)trimethoxysilane (English (3-aminopropyl)trimethoxysilane, abbreviated APTMS) (t .e XR ^B -Si(OR ^C ) ₃ where X is an amino group, R ^B is -(CH ₂ ) ₃ -, and ^RC is ethyl or methyl). In this example, the surface of substrate 12 may be pre-treated with (3-aminopropyl)triethoxysilane (APTES) or (3-aminopropyl)trimethoxysilane (APTMS) to form a covalent bond between silicon and one or more oxygen atoms present on the surface (without adhering to any or mechanism, it can be assumed that each silicon atom can bond with one, two or three oxygen atoms). Such a chemically treated surface is subjected to elevated temperatures to form a monolayer of amino groups. The amino groups are then reacted with Sulfo-HSAB (N-hydroxysulfosuccinimidyl-4-azidobenzoate, which means N-hydroxysulfosuccinimidyl-4-azidobenzoate) to form the azido derivative. Activation by UV radiation with energies ranging from ¹ J/cm2 to 30 J/cm2 at 21°C results in the generation ^of active nitrene species that can easily enter into various inclusion reactions with PAZAM (that is, with one example of a functionalized molecule, used to form the polymer coating 18).

Other silanization methods may also be used. Examples of suitable silanization methods include vapor deposition, a method (eg, developed by Yield Engineering Systems, "YES method"), spin coating, or other precipitation methods. Some examples of methods and materials that can be used to silanize the cured structured resin 14', discussed in this work; however, it should be understood that other methods and materials may be used.

In one example, in which a CVD (chemical vapor deposition, abbr. CVD) oven is used to carry out the YES method, the cured structured resin 14' on the substrate 12 is placed in the oven CVD. The chamber can be purged, after which the silanization cycle is started. During the cycles, the silane or silane derivative vessel can be maintained at a suitable temperature (for example, approximately 120 ° C for norbornene silane), a suitable temperature can be maintained in pipelines for supplying vapors of the silane or silane derivative (for example, approximately 125 ° C for norbornene silane), and a suitable temperature (eg, approximately 145° C.) can be maintained in the vacuum lines.

In another example, a silane or silane derivative (eg, liquid norbornene silane) may be deposited within a glass vial and the vial may be placed in a glass vacuum desiccator containing a structured support 12. The desiccator may then be evacuated until a pressure of between about 15 millitor to about 30 millitor (about 2 to 4 Pa), and the desiccator can be placed in an oven at a temperature of from about 60°C to about 125°C. Upon completion of the silanization, the desiccator is removed from the oven, cooled and equilibrated with the atmosphere.

Vapor deposition, YES process and/or silanization in a vacuum desiccator can be used to treat various silanes or silane derivatives such as silane or silane derivatives containing an unsaturated cycloalkene moiety such as norbornene, a norbornene derivative (e.g. (hetero)norbornene , including an oxygen or nitrogen atom instead of one of the carbon atoms), transcyclooctene, derivatives of transcyclooctene, transcyclopentene, 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 the cycloalkenes mentioned may be substituted, for example, with an R group such as hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. An example of a norbornene derivative includes [(5-bicyclo[2.2.1]hept-2-enyl)ethyl]trimethoxysilane. In other examples, these methods can be applied if the silane or silane derivative includes an unsaturated cycloalkyne moiety, such as cyclooctyne, a cyclooctyne derivative, or bicyclononines (e.g., bicyclo[6.1.0]non-4-yne or derivatives thereof, bicyclo[6 ,1,0]non-2-yne or bicyclo[6,1,0]non-3-yne). These cycloalkynes may be substituted by any of the R groups discussed in this work.

As a result of the addition of the silane or silane derivative, a pre-treated (eg, silanized) cured structured resin 14' is formed, which includes silanized depressions and silanized intermediate regions.

In other examples, the cured structured resin 14' may not be silanized. In contrast, the cured structured resin 14' may be subjected to plasma etching, after which the plasma etched cured structured resin 14' may be directly spin-coated (or otherwise deposited) with the polymer coating 18. In this example, the plasma etching may form substance(s) activating the surface (for example, hydroxyl (C-OH or Si-OH) and/or carboxyl groups), with which the polymer coating 18 can be fixed on the cured structured resin 14'. In these examples, the polymer coating 18 is chosen to be reactive with the surface groups formed during plasma etching.

In other examples, the cured structured resin 14' may include unreacted epoxy groups (for example, if a curable free radical resin matrix is used in conjunction with an epoxy resin matrix), and thus may not be silanized because unreacted epoxy groups may directly react with the amino functional groups of the polymer. coatings 18. In this example, plasma etching can be performed, for example, if it is necessary to clean the surface of possibly present contaminants.

The polymer coating 18 may then be applied to the pre-treated cured structured resin 14' (as indicated between FIGS. 2C and 2C). The polymeric coating 18 may be a semi-rigid polymeric material permeable to liquids and gases, fixed on the cured structured resin 14'.

Poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide, abbreviated as PAZAM) copolymer of acrylamide, such as N-(5-azidoacetamidylpentyl)acrylamide-acrylamide copolymer can be represented by the following structure (I):

where:

R ¹ is H or optionally substituted alkyl;

R ^A is selected from the group consisting of azido, optionally substituted amino, optionally substituted alkenyl, optionally substituted hydrazone, optionally substituted hydrazine, carboxyl, hydroxy, optionally substituted tetrazole, optionally substituted tetrazine, nitrile oxide, nitrone, and thiol;

each of R ⁵ , R ₆ and R ₈ is independently selected from the group consisting of H and optionally substituted alkyl;

each of the -(CH ₂ ) _p - groups may be optionally substituted;

p is an integer from 1 to 50;

n is an integer from 1 to 50000; and

m is an integer ranging from 1 to 100000.

One skilled in the art will appreciate that the placement of the repeating elements "n" and "m" in structure (I) is representative, and the monomeric subunits can be in any order in the polymer structure (e.g., randomly, block-wise). polymer, structured polymer, or combinations thereof).

The molecular weight of PAZAM can be from about 10 kDa to about 1500 kDa, or in one specific example, it can be about 312 kDa.

In some examples, PAZAM is a linear polymer. In some other examples, PAZAM is a crosslinked polymer with a low crosslink density.

В этом примере акриламидная единица в структуре (I) может быть заменена единицей

в которой каждый из R₆, R₇ и R₈ представляет собой Н, и каждый из R₉ и R₁₀ представляет собой метильную группу (а не Н, как в случае акриламида). В этом примере q может представлять собой целое число, составляющее от 1 до 100000. В другом примере наряду с акриламидной единицей может быть применен N,N-диметилакриламид. В этом примере кроме повторяющихся элементов "n" и "m" структура (I) может включать единицу

в которой каждый из R₆, R₇ и R₈ представляет собой Н, и каждый из R₉ и R₁₀ представляет собой метильную группу. В этом примере q может представлять собой целое число, составляющее от 1 до 100000.In other examples, the structure constituting polymer coating 18 may be a variant of structure (I). In one example, the acrylamide unit can be replaced by N,N-dimethylacrylamide

In this example, the acrylamide unit in structure (I) can be replaced by the unit

in which each of R ₆ , R ₇ and R ₈ represents H, and each of R ₉ and R ₁₀ represents a methyl group (and not H, as in the case of acrylamide). In this example, q may be an integer ranging from 1 to 100,000. In another example, N,N-dimethylacrylamide may be used along with the acrylamide unit. In this example, in addition to the repeated elements "n" and "m", the structure (I) can include a unit

in which each of R ₆ , R ₇ and R ₈ represents H, and each of R ₉ and R ₁₀ represents a methyl group. In this example, q may be an integer ranging from 1 to 100000.

In another polymer example, the repeating element "n" in structure (I) can be replaced by a monomer containing a heterocyclic azido group having structure (II):

where R ¹ represents H or C1-C4 alkyl; R _z is H or C1-C4 alkyl; L is a linker (connecting group) comprising a straight chain containing from 2 to 20 atoms selected from the group consisting of carbon, oxygen and nitrogen, and 10 optional substituents on carbon atoms and any nitrogen atoms of the chain; E is a straight chain containing from 1 to 4 atoms selected from the group consisting of carbon, oxygen and nitrogen, and optional substituents on carbon atoms and any nitrogen atoms of the chain; A is an amide substituted at the N atom, where H or C1-C4 alkyl is attached to the N atom; and Z is a nitrogen-containing heterocycle. Examples of Z include rings containing 5 to 10 members as a single ring structure or a fused ring structure.

In another example, the polymer may include a repeating unit corresponding to each of structures (III) and (IV):

where each of R ^1a , R ^2a , R ^1b and R ^2b is independently selected from hydrogen, optionally substituted alkyl or optionally substituted phenyl; each of R ^3a and R ^3b is independently selected from hydrogen, optionally substituted alkyl, optionally substituted phenyl, or optionally substituted C7-C14 aralkyl; and each of L ¹ and L ² is independently selected from an optionally substituted alkylene linker or an optionally substituted heteroalkylene linker.

It should be understood that other functionalized molecules may be used to form the polymer coating 18, provided that they have been functionalized to react with the pre-treated cured structured resin 14' and the subsequently applied primer(s) 24. Other examples of suitable molecules to form a polymer coating 18, include molecules forming a colloidal structure, such as agarose, or a network polymer structure, such as gelatin, or a cross-linked polymer structure, such as polyacrylamide polymers and copolymers, silane free acrylamide (eng. silane free acrylamide, abbreviated SFA) or an azidolized version of SFA. Examples of suitable polyacrylamide polymers can be synthesized from acrylamide and acrylic acid or acrylic acid containing a vinyl group, or from [2+2] photo-cycloaddition monomers. Other examples of molecules suitable for forming polymer coating 18 include mixed copolymers of acrylamides and acrylates. Branched polymers such as star polymers, star polymers or star block copolymers, dendrimers and the like can also be used.

The functionalized molecule (e.g., PAZAM) may be deposited onto the surface of the pre-treated cured structured resin 14' by spin coating or dip or dip coating, or positive or negative pressure distribution of the functionalized molecule, or other suitable technique. The functionalized molecule may be present in the mixture. In one example, the mixture includes PAZAM in water or in a mixture of ethanol and water.

Once coated, the functionalized molecule can also be cured to form a polymeric coating 18 over the entire patterned substrate (i.e., recess(s) 16 and intermediate region(s) 22). In one example, the curing of the functionalized molecule can be carried out at a temperature ranging from room temperature (eg, about 25°C) to about 95°C for a time of from about 1 millisecond to about several days. According to another example, the curing time may be from 10 seconds to at least 24 hours. In another example, the duration may be from about 5 minutes to about 2 hours.

The attachment of the polymeric coating 18 to the pre-treated depressions and intermediate areas may occur through covalent bonds. The covalent bonding of the polymer coating 18 to the silanized or plasma-etched recesses allows the polymer coating 18 to be fixed in the recesses 16 for the life of the finished flow cell in various operations. The following are some examples of reactions that can take place between a silane or silane derivative and polymer coating 18.

If the silane or silane derivative includes norbornene or norbornene derivative as an unsaturated moiety, then norbornene or norbornene derivative can: i) undergo a 1,3-bipolar cycloaddition reaction with the PAZAM azide/azido group; ii) couple with a tetrazine group attached to PAZAM; enter into a cycloaddition reaction with a hydrazone group attached to PAZAM; enter into a photo-click reaction with a tetrazole group attached to PAZAM; or enter into a cycloaddition reaction with a nitrile oxide group attached to PAZAM.

If the silane or silane derivative contains cyclooctyne or a cyclooctyne derivative as an unsaturated moiety, then the cyclooctyne or cyclooctyne derivative can: i) undergo strain-promoted azide-alkyne 1,3-cycloaddition reactions under stress. 3-cycloaddition, abbreviated as SPAAC) with an azide/azido group of PAZAM, or ii) undergo a stress-induced alkine-nitrile oxide cycloaddition reaction with a nitrile oxide group attached to PAZAM.

If the silane or silane derivative includes bicyclononine as an unsaturated moiety, then bicyclononine, under the influence of tension in the bicyclic ring system, can enter into a similar alkyne cycloaddition reaction according to the SPAAC mechanism with azides or nitrile oxides attached to PAZAM.

In order to form the resin coating 18 in the recess(s) 16 but not in the intermediate region(s) 22 of the cured structured resin 14', the polymer coating 18 may be removed from the intermediate regions 22 by grinding. Grinding can be done using a mild chemical slurry (including, for example, an abrasive, buffer, chelating agent, surfactant, and/or dispersant) that can remove polymeric coating 18 from intermediate areas 22 without damaging the underlying cured structured resin 14' and/or underlying substrate 12 located in the areas to be treated. Alternatively, grinding can be done with a solution that does not include abrasive particles. The chemical slurry can be used in a mechanical grinding chemical removal system. In this example, the grinding head(s)/plate(s) or other grinding device(s) is capable of sanding off the resin coating 18 from the intermediate areas 22, leaving the resin coating 18 in the recesses 16 and leaving the underlying cured structured resin 14' for at least essence intact. For example, the grinding head may be a Strasbaugh ViPRR II grinding head. In another example, sanding can be done with a sanding pad and a non-abrasive solution. For example, a sanding pad may be used in combination with a solution that does not contain abrasive particles (ie, a solution that does not include abrasive particles).

On FIG. 2D shows the flow cell preform 10 after applying a layer of polymer 18 to the recesses 16. The flow cell preform 10 may be cleaned. Cleaning may include the use of a water bath and ultrasonic treatment. The temperature of the water bath can be kept relatively low and range from about 22°C to about 30°C. The silanized structured support with a polished finish can also be dried by spin or other suitable technique.

As indicated between the images in Fig. 2D and FIG. 2E, primer 24 is grafted onto polymeric coating 18 in recess(s) 16 to graft. Primer 24 may be any of the forward amplification primers or reverse amplification primers that include an alkyne functional group or another terminated primer (a primer with a different terminal group). Other examples of terminated primers that can be used include tetrazine-terminated primer, azido-terminated primer, amino-terminated primer, epoxy- or glycidyl-terminated primer, thiophosphate-terminated primer, thiol-terminated primer, aldehyde group, a hydrazino-terminated primer, a phosphoramidite-terminated primer, and a triazolinedione-terminated primer. A mixture of primers may also be used. Specific examples of suitable primers include primers P5 or P7, which are available on the surface of commercially available flow cells supplied by Illumina Inc. for sequencing on HISEQ™, HISEQX™, MISEQ™, MISEQDX™, MINISEQ™, NEXTSEQ™, NEXTSEQDX™, NOVASEQ™, ISEQ™, GENOME ANALYZER™ and other instrument platforms.

In one example, grafting can be done by flow coating (e.g., using a temporarily attached cap), dipping coating, spray coating, distribution coating, or any other suitable method by which primer(s) 24 can be attached to the polymer coating. 18. In each of the above examples of techniques, a primer solution or mixture may be used, which may include primer(s), water, buffer, and catalyst.

Dip coating may involve immersing the flow cell preform 10 (shown in FIG. 2D) in a series of temperature controlled baths. The baths may also have flow control and/or be under a nitrogen atmosphere. Baths may contain a primer solution or mixture. When passing through various baths, the primer(s) 24 are attached to the functional group(s) to which the primer can be attached, present on the polymer coating 18 located in at least some recess(s) 16. the cells are introduced into the first bath containing the primer solution or mixture in which the addition reaction of the primer(s) 24 takes place, after which the preform 10 is introduced into the following washing baths. Transfer from one bath to another can be done with an automatic manipulator or manually. In dip coating, a drying system can also be used.

Spray coating can be performed by spraying the primer solution or mixture directly onto the flow cell blank 10 . The spray-coated multilayer structure can be incubated for a period of time from about 4 minutes to about 60 minutes at a temperature of from about 0°C to about 70°C. After incubation, the primer solution or mixture can be diluted and removed using, for example, a spin coater.

Coating by spreading the solution can be performed by a method including pouring and spinning, and thus can be done with a spin coater. The primer solution or mixture can be applied (manually or automatically) to the flow cell preform 10 . The applied primer solution or mixture can be applied to or spread over the entire surface of the flow cell preform 10 . The primer-coated flow cell preform 10 may be incubated for about 2 minutes to about 60 minutes at a temperature of about 0°C to about 80°C. After incubation, the primer solution or mixture can be diluted and removed using, for example, a spin coater.

On FIG. 2F shows an example of a flow cell 10' after primer grafting. While a single type of primer 24 is shown, it should be understood that two or more different primers 24 may be attached.

On FIG. 2E and 2F show examples of a flow cell 10' without a lid attached to it. Although the lid is not shown, the flow cells 10' may have a lid attached to at least a portion of the intermediate portion 22. The lid may be attached before or after primer 24 grafting. If the lid is attached before primer 24 grafting, it should be understood that the flow method can be used for grafting. In a flow-through method, the primer solution or mixture may be introduced into the flow channel(s) (delimited by the lid and intermediate portion 22) through the appropriate inlet(s) (not shown), the solution or mixture may be held in the flow channel(s) for sufficient time (i.e., incubation period) for primer(s) 24 to attach to polymer coating 18 in one or more recesses 16, after which it can be removed from the appropriate outlet(s) (not shown). After primer 24 is attached, additional fluid(s) may be passed through the flow channel(s) to flush the already functionalized recesses and flow channel(s).

A cover may be placed on the intermediate portion 22 in such a way that it defines a single flow channel or a plurality of hydraulically separated flow channels.

The lid may be any material that is transparent to the excitation radiation directed at the recess(s) 16. For example, the lid may be made of glass (e.g., Corning Eagle XG (CEXG), borosilicate glass, fused silica, etc.). ), plastics or similar material. An example of a commercially available suitable borosilicate glass is D 263® available from Schott North America, Inc. Commercially available examples of suitable plastic materials, namely cycloolefin polymers, are the ZEONOR® products supplied by Zeon Chemicals L.P.

In some examples, the lid may be integral with the side wall(s) that conform to the shape of the part of the intermediate portion 22 to which it will be attached. For example, a groove can be etched into the transparent block, resulting in a substantially flat (eg, top) portion and sidewall(s) extending beyond the substantially flat portion. By placing the etched grooved block on the intermediate portion 22, the groove can become a flow channel.

In other examples, the side wall(s) and lid may be separate components connected to each other. For example, the cover may be a substantially rectangular block having at least a substantially flat outer surface and at least a substantially flat inner surface that defines a portion (e.g., top) of the flow channel (after being connected to a portion of the intermediate section 22) . The block may be mounted on (eg, connected to) a side wall(s) which is connected to a portion of the intermediate section 22 and forms the side wall(s) of the flow channel. In this example, the side wall(s) may include any of the materials that are suitable for making the separating layer (described below) according to the present invention.

The lid may be attached using any suitable technique such as laser welding, diffusion bonding, anode welding, eutectic alloy soldering, plasma activated bonding, glass crystal soldering, or other methods known in the art. In one example, a separating layer may be used to connect the lid to a portion of the intermediate portion 22. The separating layer may be any material that can seal at least some of the intermediate portions 22 to the lid.

In one example, the separating layer may be made of a material that absorbs radiation at a wavelength that the cover and/or the cured structured resin 14' transmits. In turn, the absorbed energy is used to form a bond between the release layer and the lid and between the release layer and the cured structured resin 14'. One example of a material that absorbs radiation is black KAPTON® (polyimide containing carbon black) supplied by DuPont (USA), the absorption band of which is at approximately 1064 nm. It should be understood that carbon black-free polyimide can be used, but the wavelength must be chosen at which there is significant energy absorption by the natural polyimide material (eg, at 480 nm). In another example, a CEN JP polyimide can be used for the connection, which is irradiated with light at a wavelength of 532 nm. If the separating layer is made of a radiation absorbing material, then the separating layer may be positioned at the interface between the cover and part of the intermediate portion 22 such that the separating layer is in contact with the target bonding area. A compressive force (eg, a pressure of approximately 100 PSI (corresponding to approximately 6.20 x 10 ⁵ Pa)) can be applied while exposing the interface to laser energy at a suitable wavelength (i.e., irradiating a material that absorbs radiation). To create a suitable connection, the laser energy can act on the interface both from above and below.

In another example, the separating layer may be in contact with a radiation absorbing material. The radiation absorbing material may be applied to the interface between the separating layer and the cover, as well as to the interface between the separating layer and part of the intermediate section 22. For example, the separating layer may be composed of polyimide, and the separate radiation absorbing material may be carbon black. In this example, a separate radiation absorbing material absorbs the laser energy that is expended in forming bonds between the separating layer and the cover and between the separating layer and part of the intermediate section 22. In this example, a compressive force can be applied to the respective interfaces and simultaneously to the interfaces laser energy of a suitable wavelength can be applied (i.e., simultaneous irradiation of the material absorbing the radiation).

The flow cells 10' discussed in this work can be used to implement many different sequencing techniques or methods, including techniques often referred to as sequencing-by-synthesis (English sequencing-by-synthesis, abbreviated as SBS), cycle sequencing of arrays, sequencing by ligation, pyrosequencing etc. Because the polymer coating 18 and the attached primer(s) 24 are in the recesses 16 and not in the intermediate regions 22, any of these techniques will localize amplification only to the recesses.

By way of example, a sequencing-by-synthesis (SBS) reaction can be carried out on a system such as the HISEQ™, HISEQX™, MISEQ™, MISEQDX™, MINISEQ™, ISEQ™, NOVASEQ™, NEXTSEQDX™ or NEXTSEQ™ sequencing systems, supplied by Illumina, Inc. (San Diego, CA). When conducting SBS to determine the sequence of nucleotides in the matrix, the extension (extension) of the nucleic acid primer (eg, sequencing primer) along the nucleic acid matrix (ie, sequencing template) is monitored. The chemical process underlying this process may be polymerization (eg catalyzed by a polymerase enzyme) or ligation (eg catalyzed by a ligase enzyme). In one particular SBS method using a polymerase, fluorescently labeled nucleotides are attached to a sequencing primer (thus extending the sequencing primer) according to the arrangement of the nucleotides in the template, and determining the order and type of nucleotides attached to the sequencing primer can be used to matrix sequence definitions.

Prior to sequencing, amplification primers 24 may be exposed to a sequencing library that is amplified by any suitable method, such as cluster generation.

In one example of cluster generation, library fragments are copied from hybridized primers 24 by 3' extension with high-fidelity DNA polymerase. The original library fragments are denatured, leaving immobilized copies. Isothermal bridge amplification can be used to amplify immobilized copies. For example, the copied templates are folded to hybridize with the adjacent complementary primer 24, and the polymerase copies the copied templates, resulting in the formation of double-stranded (double-stranded) bridges, which are denatured to form single-stranded strands. These two strands fold and hybridize with adjacent complementary primers 24 and re-strand to form two new double-stranded loops. The process is repeated on each copy of the template, carrying out cycles of isothermal denaturation and amplification with the formation of dense clonal clusters. Each cluster of double-stranded bridges undergoes denaturation. In one example, the reverse strand is removed by specific base cleavage, leaving forward polynucleotide template chains. It should be understood that the formation of clusters leads to the formation of several matrix chains.

To initiate the first round of SBS, one or more labeled nucleotides, DNA polymerase, etc. can be directed into/through the flow channel, etc., in which the completion of the sequencing primer leads to the insertion of the labeled nucleotide into template chains. Such embedding can be detected by the act of visualization. During the act of rendering, an illumination system (not shown) may direct excitation radiation to the functionalized recesses. During the rendering event, any radiation (if any) emitted by the cured structured resin 14' as a result of exposure to blue and/or green excitation wavelength light may be i) unsuitable for detection because its intensity is below the detection threshold, or ii) due to the weak autofluorescence of the cured structured resin 14' detectable as noise. Thus, the cured structured resin 14' discussed in this paper is essentially invisible to the detector.

In some examples, the nucleotides may further have the property of a reversible terminator that terminates subsequent primer extension after the addition of the nucleotide to the sequencing primer. For example, a nucleotide analog containing a reversible terminator fragment can be added to a sequencing primer, such that subsequent extension cannot occur until a deblocking agent is added that can remove the fragment. Thus, in those examples where reversible terminators are used, into the flow channel, etc. a deblocking reagent may be sent (before or after detection).

Flushing(s) may be performed between the various fluid supply steps. The SBS cycle can be repeated n times to extend the sequencing primer by n nucleotides, whereby a sequence of length n can be determined.

Although the SBS method has been discussed in detail, it should be understood that the flow cells discussed in this work can be applied in other sequencing protocols, for genotyping, or in other chemical and/or biological assays. Paired-end sequencing can detect genomic rearrangements and repetitive element sequences, as well as gene fusion and new transcripts. In another example, the flow cells discussed in this paper can be used to generate a library on cells.

Although the example shown in FIG. 1 and 2A-2F illustrate the use of the resin composition for making a flow cell, it should be understood that the resin composition may be used for other applications that require low autofluorescence intensities. In one example, the resin composition 14, 14' can be used in any SBS technique with optical detection. In other examples, the resin composition 14, 14' can be used to make planar waveguides, complementary metal-oxide-semiconductor (CMOS for short), etc.

To further illustrate the present invention, two examples and a model example are given below. It should be understood that these examples and the model example are provided to illustrate and do not limit the scope of the present invention.

Description of exemplary embodiments of the invention

Example 1

Six examples of resin compositions were prepared. Each example included a different, free-radical curable resin matrix that included 1,3-bis(3-methacryloxypropyl)tetramethyldisiloxane, methacryloxypropyl-terminated polydimethylsiloxane, tetramethyl-tetrakis[3-acryloxypropyl]cyclotetrasiloxane, methacryl-polyhedral oligomeric silsesquioxane, acrylic- a polyhedral oligomeric silsesquioxane; and an acryloxypropyl-methylsiloxane homopolymer. Each of these curable free radical resin matrices was mixed with a photoinitiator which was bis(2,4,6-trimethylbenzoyl)-phenylphosphine oxide (commercially available from IGM Resins under the name OMNIRAD™ 2022).

The curable free radical resin matrices and photoinitiator were mixed with a PGMEA (propylene glycol monomethyl ether acetate) solvent containing a monomer concentration of from about 15 wt. % to about 66 wt. % and a photoinitiator concentration of approximately 1 wt. %.

Each of the resulting resin compositions was coated by spin coating at 2200 (rpm) rpm. for approximately 1 minute on a glass substrate. After precipitation, the resin compositions were heat treated at a low temperature for about 2 minutes at about 130° C. to remove the PGMEA. The deposited resin compositions were then imprinted by nanoimprint lithography. The work stamp was pressed into the applied composition, and then the resin was irradiated to cure with UV light from a UV LED lamp for about 20 seconds.

Scanning electron microscope images were taken of each of the cured structured resins. On FIG. 3A-3F respectively depict cured structured resins prepared from 1,3-bis(3-methacryloxypropyl)tetramethyldisiloxane, methacryloxypropyl-terminated polydimethylsiloxane, tetramethyl-tetrakis[3-acryloxypropyl]cyclotetrasiloxane, methacryl-polyhedral oligomeric silsesquioxane, acrylic a polyhedral oligomeric silsesquioxane; and an acryloxypropyl-methylsiloxane homopolymer. These images show that the resin compositions discussed in this paper can be nanoprinted to form depressions suitable for flow cells or suitable for other applications.

Example 2

Was prepared an example of a resin composition comprising approximately 17 wt. % acrylic-polyhedral oligomeric silsesquioxane (acrylo-POSS - as curable free radical resin matrix), approximately 1 wt. % bis(2,4,6-trimethylbenzoyl)-phenylphosphine oxide (as free radical photoinitiator) and propylene glycol monomethyl ether acetate (PGMEA - as solvent). The solvent in the composition was the addition to 100% (approximately 82 wt.%).

Was prepared comparative resin composition comprising approximately 4 wt. % functionalized glycidyl group POSS and approximately 13 wt. % epoxycyclohexylethyl functionalized POSS (as epoxy resin matrix), approximately 1.2 wt. % in total photoinitiator/photoacid generator thioxanth-9-one (ITX) combination (present at about 0.34 wt %)/TEGO® PC 1467 (Evonik Industries) (present at about 0.85 wt %) ), approximately 1.4 wt. % polyacrylate (BYK®-350) and solvent (PGMEA). The solvent in the composition was the addition to 100% (approximately 80 wt.%).

Both the Comparative Resin Composition Example and the Resin Composition Example were applied to a glass substrate by centrifugation at 2200 (rpm) rpm. for approximately 1 minute.

After applying the Example and Comparative Resin Compositions, the resin compositions of Example and Comparative were heat treated at a low temperature for about 2 minutes at about 120° C. to remove PGMEA. The resins were then cured under UV light from a UV LED lamp for approximately 20 seconds. After UV curing of the comparative example resin, the resin was heat treated at an elevated temperature of 250°C for about 10 minutes. The resin of the example was not subjected to heat treatment at an elevated temperature.

Then, the cured example and the cured comparative example were irradiated with excitation light using a spectrometer-based device. The excitation light source of this device was a laser with a wavelength of 445 nm (blue region of the spectrum). The laser spot size was approximately 200 µm. The laser beam was passed through a filter to cut longer wavelength lines and then passed through the samples. Transparent samples (of the cured example and the cured comparative example) were taken for measurements. In line with the laser beam on the other side of the sample, an Ocean Optics spectrometer was placed, connected with an optical fiber. The energy of the incident laser beam can be adjusted by adjusting the drive current or by adding attenuating filters in the path of the laser beam. To obtain the data presented in the graph of Fig. 4, a laser power of 3 mW was used.

As seen from the graph shown in Fig. 4, the fluorescence intensity (in arbitrary units (PU)) of the comparative example at the blue excitation wavelength is 2500 PU, and at the green excitation wavelength, it is from about 2000 PU to about 2400 PU. In contrast, the fluorescence intensity of the example at excitation wavelengths in the blue and green ranges is generally less than about 500 PU and appears to be slightly higher than the fluorescence intensity of the optical glass substrate.

Model example

The purpose of this model example is to compare two compositions in a hypothetical example.

An example resin composition includes 1,3-bis(3-methacryloxypropyl)tetramethyldisiloxane (as the curable free radical resin matrix) and bis(2,4,6-trimethylbenzoyl)-phenylphosphine oxide (as the free radical photoinitiator) in PGMEA.

Comparative resin composition includes glycidyl functionalized POSS and epoxycyclohexylethyl functionalized POSS (as epoxy resin matrix), a photoinitiator/photoacid generator combination comprising thioxanth-9-one (ITX) and TEGO® PC 1467 (Evonik Industries) in PGMEA.

The Comparative Example composition was applied to the first two lanes (1 and 2) of a glass or silicon made flow cell and the example composition was applied to the last two lanes (4 and 5) of a glass or silicon made flow cell.

After applying the resin compositions of the Example and Comparative Example, the resins were cured.

Both the cured example and the cured comparative example, respectively, were irradiated with excitation light at a wavelength in the blue range and a wavelength in the green range using an Amersham TYPHOON™ device for measuring autofluorescence. It is believed that the autofluorescence intensity of the comparative example will be much higher than the autofluorescence intensity of the example. On FIG. 5A and 5B are graphs depicting the predicted autofluorescence results of the cured example and the cured comparative example when irradiated with blue excitation wavelength (FIG. 5A) and green excitation wavelength (FIG. 5B), respectively. In these graphs, the gray values represent autofluorescence and the distance expressed in centimeters represents the distance at which the lanes are located in the flow cell.

This model example compares a hypothetical acrylate-based composition with a hypothetical epoxy-based composition and presents the estimated autofluorescence intensities for the hypothetical compositions, which show that the combination of a curable free radical resin matrix comprising acrylate and siloxane and a free radical photoinitiator can produce a weaker autofluorescence.

Additional Notes

It should be understood that all combinations of the above features and additional features discussed in more detail below (provided that these features are not mutually exclusive) form part of the subject matter of the invention discussed in this work. In particular, all combinations of the disclosed subjects of the invention, obvious after reading the proposed description, form part of the subject matter of the invention discussed in this work. It should also be understood that the terminology used in the present description, which can also be used in any document included in the present description by reference, should have the meaning that is most consistent with the specific features discussed in this work.

Reference in the present description to "one example", "another example", "example", etc. means that a specific element (eg, element, structure and/or characteristic) disclosed in the description of this example is included in at least one example discussed in this work, and may or may not be present in other examples. In addition, it should be understood that, unless otherwise clear from the context, the discussed elements of any example can be combined in various examples in any suitable way.

The ranges given herein are to be understood to include the indicated ranges and any values or sub-ranges within the stated range, as if such values or sub-ranges were clearly indicated. For example, it should be understood that the range of about 380 nm to about 480 nm includes not only the explicitly mentioned ranges of about 380 nm to about 480 nm, but also includes individual values such as about 408 nm, about 445.5 nm, etc., and subranges such as from about 425 nm to about 475 nm, etc. In addition, if the modifiers "approximately" and/or "essentially" are used to describe a value, then they constitute small variations (up to +/-10%) from the indicated value.

Although some of the examples have been described in detail, it should be understood that the discussed examples can be modified. Thus, the above description is not limiting.

Claims (43)

1. Resin composition for obtaining a flow cell for genetic sequencing, including: a curable free radical resin matrix comprising an acryloxy- or methacryloxy-substituted polysiloxane or an acrylic-substituted polyhedral oligomeric silsesquioxane; and free radical photoinitiator; epoxy resin matrix; and photoacid generator; wherein, in the cured state, the resin composition exhibits little or no autofluorescence when irradiated with blue light with an excitation wavelength of about 380 nm to about 480 nm, or when irradiated with green light with an excitation wavelength of between about 510 nm and about 560 nm . 2. The resin composition of claim 1, wherein the cured resin composition exhibits low autofluorescence, and wherein low autofluorescence when the cured resin composition is irradiated with light having an excitation wavelength in the blue range corresponds to a gray value of less than 25,000. 3. The resin composition of claim 1, wherein the cured resin composition exhibits low autofluorescence, and wherein low autofluorescence when the cured resin composition is irradiated with light having an excitation wavelength in the blue range corresponds to a gray value of less than 5000. 4. The resin composition of claim 1, wherein the cured resin composition exhibits low autofluorescence, and wherein low autofluorescence when the cured resin composition is irradiated with light having an excitation wavelength in the green range corresponds to a gray value of less than 10,000. 5. The resin composition of claim 1, wherein the cured resin composition exhibits low autofluorescence, and wherein low autofluorescence when the cured resin composition is irradiated with light having an excitation wavelength in the green range corresponds to a gray value of less than 2500. 6. The resin composition of claim 1, wherein the curable free radical resin matrix comprises an acrylate selected from the group consisting of 1,3-bis(3-methacryloxypropyl)tetramethyldisiloxane, methacryloxypropyl-terminated polydimethylsiloxane, tetramethyl-tetrakis[3-acryloxypropyl] cyclotetrasiloxane, methacryl-polyhedral oligomeric silsesquioxane, acrylic-polyhedral oligomeric silsesquioxane, acryloxypropyl-methylsiloxane homopolymer, and combinations thereof. 7. The resin composition of claim 1 wherein the free radical photoinitiator is 2-ethyl-9,10-dimethoxyanthracene. 8. The resin composition of claim 1 wherein the free radical photoinitiator is selected from the group consisting of 2,2-dimethoxy-2-phenylacetophenone and 2-ethoxy-2-phenylacetophenone. 9. The resin composition of claim 1 wherein the free radical photoinitiator is a phosphine oxide and the phosphine oxide is selected from the group consisting of diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, a mixture 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)phenylphosphinate and combinations of these substances. 10. The resin composition of claim 1 further comprising a dark quencher or electron acceptor. 11. The resin composition of claim 1, wherein the weight percentage of curable free radical resin matrix to free radical photoinitiator is from about 99.8:0.2 to about 90:10. 12. A flow cell for genetic sequencing, comprising: substrate; and a cured structured resin deposited on a glass substrate, wherein the cured structured resin includes recesses separated by intermediate portions, and the cured structured resin was obtained from a resin composition comprising: a curable free radical resin matrix comprising an acryloxy- or methacryloxy-substituted polysiloxane or an acrylic-substituted polyhedral oligomeric silsesquioxane; and free radical photoinitiator; epoxy resin matrix; and photoacid generator; wherein the cured structured resin exhibits little or no autofluorescence when irradiated with blue light at an excitation wavelength of about 380 nm to about 480 nm, or when irradiated with green light with an excitation wavelength of about 510 nm to about 560 nm. 13. The flow cell according to claim 12, further comprising: a polymer coating located in the recesses; and primer grafted onto the polymer coating. 14. The flow cell of claim 12, wherein the cured structured resin exhibits low autofluorescence, and wherein low autofluorescence when the cured structured resin is irradiated with light having an excitation wavelength in the blue range corresponds to a gray value of less than 25,000. 15. The flow cell of claim 12, wherein the cured structured resin exhibits low autofluorescence, and wherein low autofluorescence when the cured structured resin is irradiated with light having an excitation wavelength in the green range corresponds to a gray value of less than 10,000. 16. The flow cell of claim 12 wherein the curable free radical resin matrix comprises an acrylate selected from the group consisting of 1,3-bis(3-methacryloxypropyl)tetramethyldisiloxane, methacryloxypropyl-terminated polydimethylsiloxane, tetramethyl-tetrakis[3-acryloxypropyl] cyclotetrasiloxane, methacryl-polyhedral oligomeric silsesquioxane, acrylic-polyhedral oligomeric silsesquioxane, acryloxypropyl-methylsiloxane homopolymer, and combinations thereof. 17. The flow cell of claim 12 wherein 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. 18. The flow cell of claim 12 wherein the free radical photoinitiator is a phosphine oxide selected from the group consisting of diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, a mixture 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)phenylphosphinate and combinations of these substances. 19. The flow cell of claim 12, wherein the resin composition further comprises a dark quencher or electron acceptor. 20. The flow cell of claim 12, wherein the weight percent ratio of curable free radical resin matrix to free radical photoinitiator in the resin composition is from about 99.8:0.2 to about 90:10. 21. A method for obtaining a flow cell, including: deposition on a glass substrate of a resin composition, where the resin composition includes: a curable free radical resin matrix comprising an acryloxy- or methacryloxy-substituted polysiloxane or an acrylic-substituted polyhedral oligomeric silsesquioxane; and free radical photoinitiator; epoxy resin matrix; and photoacid generator; nanoimprinting of the deposited resin composition using a working stamp; and curing the precipitated resin composition to form a cured structured resin; wherein the cured structured resin exhibits little or no autofluorescence when irradiated with blue light at an excitation wavelength of about 380 nm to about 480 nm, or when irradiated with green light with an excitation wavelength of about 510 nm to about 560 nm.

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