NL2024528B1 - On-flow cell three-dimensional polymer structures having functionalized surfaces - Google Patents

Abstract

A method for making on-flow cell three-dimensional polymer structures, comprising loading a polymer precursor solution into a flow cell, wherein the polymer precursor solution includes a monomer, 5 a crosslinker, a photoinitiator, and a functionalized polymer, and wherein the flow cell includes at least one channel for receiving the polymer precursor solution, and wherein the at least one channel has an upper interior surface and a lower interior surface, and illuminating the polymer precursor solution through a photomask with light at a wavelength that activates the photoinitiator , wherein the photomask includes a series of aperture formed therein, wherein the photomask has been placed over an exterior 10 surface of the channel, and wherein activation of the photoinitiator polymerizes the polymer precursor solution underneath the apertures in the photomask and forms three-dimensional polymer structures extending from the upper interior surface to the lower interior surface of the at least one channel. 15

Description

ON-FLOW CELL THREE-DIMENSIONAL POLYMER STRUCTURES HAVING FUNCTIONALIZED SURFACES BACKGROUND

[0001] Next-generation sequencing (NGS) 1s a high-throughput sequencing technology capable of sequencing entire genomes in a rapid and cost-effective manner. NGS typically begins with the creation of a sequencing library that includes genomic DNA that has been randomly fragmented, extracted, and purified. NGS processes such as sequencing-by-synthesis can then be utilized for massively parallel sequencing of the entire genomic library. The large amount of data generated by whole genome sequencing can complicate data processing and analysis; therefore, as a workaround, portions of genomes may be enriched using various techniques to focus on genes or other specific targets of interest. However. current methods for library preparation and library enrichment often requires multiple manual operation and reagents transfer that lead to losses of targeted library. Accordingly, automatic systems and processes for mitigating losses associated with current sequencing library preparation and enrichment methods would be beneficial.

SUMMARY

[0002] The following provides a summary of certain examples. This summary is not an extensive overview and is not intended to identify key or critical aspects or elements of the present invention or to delineate its scope. It is to be understood that any respective features/examples of each of the aspects of the disclosure as described herein may be implemented together in any combination to achieve the results as described herein, and that any features/examples from any one or more of these aspects may be implemented together with any of the features of the other aspect(s) as described herein in any combination to achieve the benefits as described herein.

[0003] In accordance with one implementation, a first method for making on-flow cell three- dimensional polymer structures having functionalized surfaces is provided. This method comprises loading a polymer precursor solution into a flow cell, wherein the polymer precursor solution includes a monomer, a crosslinker, a photoinitiator, and a functionalized polymer, and wherein the flow cell includes atleast one channel for receiving the polymer precursor solution, and wherein the at least one channel has an upper interior surface and a lower interior surface; and illuminating the polymer precursor solution through a photomask with light at a wavelength that activates the photoinitiator, wherein the photomask includes a series of apertures formed therein, wherein the photomask has been placed over an exterior surface of the channel, and wherein activation of the photoinitiator polymerizes at least some of the polymer precursor solution underneath the apertures in the photomask and forms three-dimensional polymer structures extending from the upper interior surface to the lower interior surface of the at least one channel.

[0004] The method may further comprise reacting a bi-functional linker, having a first end and a second end, with the functionalized polymer, wherein the first end of the bi-functional linker is chemically or enzymatically attached to the functionalized polymer, and wherein the second end of the bi- functional linker selectively binds predetermined types of molecules. The functionalized polymer may be poly(N-(5- azidoacetamidylpentyl) acrylamide-co-acrylamide (PAZAM) containing azide moieties, the bi-functional linker may be a biotin-PEG-alkyne complex, and the method may further comprise reacting the biotin-PEG-alkvne complex with the azide moieties in the PAZAM using an azide-alkyne click reaction a click reaction. The method may further comprise binding streptavidin to the biotin in the biotin-PEG-alkyne complex. The method may further comprise binding biotinylated capture oligonucleotides to the streptavidin, wherein the biotinylated capture oligonucleotides are specific for targets of interest in a sequencing library. The method may further comprise washing unpolvmerized polymer precursor solution out of the flow cell. The method may further comprise cleaving at least some of the three-dimensional polymer structures from the flow cell using heat, cleaving chemicals, or a combination of heat and cleaving chemicals. The flow cell may have oligonucleotides of predetermined lengths and sequences bound to both the upper and lower interior surfaces of the at least one channel, and wherein the oligonucleotides include primers adapted for nucleic acid amplification. The polymer may be ahydrogel.

[0005] In accordance with another implementation, a second method for making on-flow cell three-dimensional polymer structures having functionalized surfaces is provided. This method comprises loading a hydrogel precursor solution into a flow cell, wherein the hydrogel precursor solution includes a monomer, a crosslinker, a photoinitiator, and PAZAM containing azide moieties, and wherein the flow cell includes at least one channel for receiving the hydrogel precursor solution, and wherein the at least one channel has an upper interior surface and a lower interior surface; placing a photomask over the at least one channel, wherein the photomask includes a series of apertures formed therein; and illuminating the hydrogel precursor solution through the photomask with light at a wavelength that activates the photoinitiator, and wherein activation of the photoinitiator polymerizes at least some of the hydrogel precursor solution underneath the apertures in the photomask and forms three-dimensional hydrogel structures extending from the upper interior surface to the lower interior surface of the at least one channel; reacting a biotin-PEG-alkvne complex with the azide moieties in the PAZAM in the three-dimensional polymer structures using an azide-alkyne click reaction: binding streptavidin to the biotin in the biotin-

PEG-alkyne complex; and binding biotinylated capture oligonucleotides to the streptavidin, wherein the biotinylated capture oligonucleotides are specific for target molecules of interest in a sequencing library.

[0006] In accordance with still another implementation, a third method for making on-flow cell three-dimensional polymer structures having functionalized surfaces is provided. This method comprises loading a polymer precursor solution into a flow cell. wherein the polymer precursor solution includes a monomer, a crosslinker, a photoinitiator, and a streptavidin-labeled acrylamide monomer, and wherein the flow cell includes at least one channel for receiving the polymer precursor solution, and wherein the at least one channel has an upper interior surface and a lower interior surface, and wherein oligonucleotides of predetermined lengths are bound to both the upper and lower surfaces of the at least one channel; placing a photomask over the at least one channel, wherein the photomask includes a series of apertures formed therein; illuminating the polymer precursor solution through the photomask with light at a wavelength that activates the photoinitiator, and wherein activation of the photoinitiator polymerizes at least some of the polymer precursor solution underneath the apertures in the photomask and forms three- dimensional polymer structures extending from the upper interior surface to the lower interior surface of the at least one channel; selectively binding biotinylated capture oligonucleotides to the streptavidin in the three-dimensional polymer structures, wherein the biotinylated capture oligonucleotides are specific for target molecules of interest in a library and bind thereto; and eluting the bound target molecules and seeding the eluted target molecules on the surfaces of the flow cell having oligonucleotides bound thereto.

[0007] In all implementations of the methods disclosed above, the monomer may be the compound of formula I:

QF en i oN R2 Co wherein each R” is independently hydrogen or (Cs) alkyl

[0008] In all implementations of the methods disclosed above, the crosslinker may be the compound of formula II: 2 Re:

II wherein: each n is independently an integer from 1-6; and each R! is independently a hydrogen or (Cy) alkyl.

[0009] In each of the methods disclosed above, the monomer may be acrylamide, the crosslinker may be N‚N'-Bis(acryloyl)cystamine (BACy), and the photoinitiator may be lithium phenyl-2.4,6- trimethylbenzoylphosphinate (LAP). The hydrogel precursor solution may include polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N.N'-Bis(acryloyl)cvstaminge (BACy), PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA). poly(methy]l methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), polyv(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L- glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate. dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallvlamine, divinyl sulfone, diethyleneglycol diallyl ether, ethyleneglycol diacrylate. polymethyleneglycol diacrylate, polyethyleneglycol diacrylate, trimethylopropoane trimethacrylate, ethoxvlated trimethylol triacrylate, or ethoxylated pentaerythritol tetracrylate, or combinations thereof. The hydrogel precursor solution may include polyethylene glvcol (PEG)-thiol/PEG-acrylate: acrylamide/N.N'-bis(acryloyl)cystamine (BACy): PEG/polypropylene oxide (PPO). or combinations thereof. The photomask may include Mylar™ (polyester film/polyethylene terephthalate) or similar low- cost plastic material and may be laminated to the upper surface of the flow cell. The light source may be an ultraviolet light source. The three-dimensional polymer structures may be cylindrical.

[0010] In still another implementation. a flow cell is disclosed. This flow cell comprises a channel, wherein the channel includes an upper interior surface having primers coated thereon and a lower interior surface having primers coated thereon; and reversible, permeable, three-dimensional polymer structures in the channel from a polymer precursor solution, wherein the three-dimensional polymer structures extend from the upper interior surface of the channel to the lower interior surface of the channel. The flow cell may further comprise a photomask placed over an outer exterior surface of the channel. The three-dimensional polymer structures may include hydrogels. The flow cell, polymer precursor solutions, and photomask may be provided in a kit.

It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being 5 part of the inventive subject matter disclosed herein and may be implemented to achieve the benefits as described herein. Additional features and aspects of the disclosed system, devices, and methods will become apparent to those of ordinary skill in the art upon reading and understanding the following detailed description of the example embodiments. As will be appreciated by the skilled artisan, further implementations are possible without departing from the scope and spirit of what is disclosed herein. Accordingly, the drawings and associated descriptions are to be regarded as illustrative and not restrictive in nature.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The details of one or more implementations are set forth in the accompanying drawings and the description below. Other features, aspects. and advantages will become apparent from the description, the drawings, and the claims, in which:

[0013] FIG. 1A is a perspective view of a flow cell in accordance with one implementation of the disclosed systems and methods;

[0014] FIG. 1B is a top view and close-up top view of the flow cell of FIG. 1A wherein arrays of hydrogel structures have been formed on the flow cell;

[0015] FIG. IC depicts the flow cell of FIG. 1A properly inserted into a cartridge used in sequencing-by-synthesis processes;

[0016] FIG. 2A depicts a first step in an example of the disclosed systems and methods for forming polymer (e.g.. hydrogel) structures on a flow cell such as the flow cell shown in FIG. 1A, wherein a polymer precursor solution has been introduced into a fluidics channel of the flow cell and a prepattemed photomask has been placed over the channel;

[0017] FIG. 2B depicts a second step in an example of the disclosed systems and methods for forming polymer (e.g., hydrogel) structures on a flow cell wherein ultraviolet light is directed into the channel of the flow cell though openings in the photomask for polymerizing the contents of the polymer precursor solution;

[0018] FIG. 2C depicts a plurality of hydrogel structures formed inside the channel of a flow cell wherein the hydrogel structures are cylindrical in shape and are attached to upper and lower internal surface of the channel:

[0019] FIG. 2D depicts an example method for cleaving hydrogel structures formed in the channel of a flow cell by introducing oil containing a cleaving agent into the channel of the flow cell;

[0020] FIG. 2E depicts an example method for removing cleaved hydrogel structures from the channel of a flow cell by washing the channel;

[0021] FIG. 3A depicts a first step in another example of the disclosed systems and methods for forming polymer (e.g., hydrogel) structures on a flow cell wherein a prepatterned photomask is placed on or attached to a flow cell that is then inserted into a cartridge;

[0022] FIG. 3B depicts a second step in another example of the disclosed systems and methods for forming polymer (e.g., hydrogel) structures on a flow cell wherein a polymer precursor solution containing biological cells is loaded into the flow cell of FIG. 3A and the flow cell is then loaded into a device or instrument using an extendable tray.

[0023] FIG. 3C depicts a third step in another example of the disclosed systems and methods for forming polymer (e.g.. hydrogel) structures on a flow cell wherein the flow cell is exposed to ultraviolet light to form an array of hydrogel structures on the flow cell (which are shown in the bright field micrograph), and wherein the flow cell is then washed to remove unpolymerized material and unloaded from the instrument;

[0024] FIG. 4 depicts the formation of hydrogel micropillars on a channel within a MiSeq™ flow cell. wherein individual hydrogel micropillars are visible in the bright field micrograph;

[0025] FIG. 5A depicts an example method for on-flow cell fabrication of hydrogel micropillars, wherein hydrogel precursor solution that includes monomers and photo-initiator is introduced into a flow cell:

[0026] FIG. 5B depicts an example method for on-flow cell fabrication of hydrogel micropillars, wherein a prepatterned photomask is placed on the flow cell of FIG. 5A and illuminated with ultraviolet light

[0027] FIG. 5C depicts an example method for on-flow cell fabrication of hydrogel micropillars, wherein hydrogel micropillars are formed on the flow cell of FIG. 5A. and wherein the hydrogel micropillars are attached to the upper and lower surfaces of one of the channels in the flow cell;

[0028] FIG. 6A depicts an example method for on-flow cell fabrication of functionalized hydrogel structures, wherein a hydrogel precursor solution containing 10% polyacrylamide (PA), crosslinker, and 0.23% PAZAM into which azide moieties have been incorporated is loaded onto a flow cell:

[0029] FIG. 6B depicts an example method for on-flow cell fabrication of functionalized hydrogel structures, wherein a photomask, which includes a plurality of apertures formed therein, is placed on top of the flow cell of FIG. 6A and then exposed to UV light for 10 seconds to co-polymerize the acrylamide and PAZAM and form an array of azide-functionalized hydrogel micropillars in the narrow channel of the flow cell;

[0030] FIG. 6C depicts an example method for on-flow cell fabrication of functionalized hydrogel structures, wherein a biotin-PEG-alkvne complex is clicked onto the azide moicties of the hydrogel micropillars of FIG. 6B;

[0031] FIG. 6D depicts an example method for on-flow cell fabrication of functionalized hydrogel structures, wherein streptavidin labeled with Fluorescein binds the biotin in hydrogel micropillars of FIG. 6C;

[0032] FIG. 6E depicts an example method for on-flow cell fabrication of functionalized hydrogel structures, wherein streptavidin binds biotinylated capture oligonucleotides to enable immobilization of target sequencing library molecules;

[0033] FIG. 7A depicts a 4X bright field micrograph of PA/PAZAM control (no biotin);

[0034] FIG. 7B depicts a 4X bright field micrograph of PA/PAZAM plus Blackpool;

[0035] FIG. 7C depicts a 4X fluorescence micrograph of PA/PAZAM control (no biotin) after a reaction time of five minutes;

[0036] FIG. 7D depicts a 4X fluorescence micrograph of PA/PAZAM plus Blackpool after a reaction time of five minutes;

[0037] FIG. 7E depicts a 4X fluorescence micrograph of PA/PAZAM control (no biotin) after a reaction time of ten minutes at 40°C:

[0038] FIG. 7F depicts a 4X fluorescence micrograph of PA/PAZAM plus Blackpool after a reaction time of ten minutes at 40°C;

[0039] FIG. 8A depicts another example method for on-flow cell fabrication of functionalized hydrogel structures, wherein a hydrogel precursor solution containing 10% polyacrylamide (PA) and

0.25% streptavidin-labeled acrylamide monomer is loaded onto a flow cell;

[0040] FIG. 8B depicts another example method for on-flow cell fabrication of functionalized hydrogel structures, wherein a photomask, which includes a plurality of apertures formed therein, is placed on top of the flow cell of FIG. 8A and then exposed to UV light for 10 seconds to co-polvmerize the acrylamide streptavidin-labeled acrylamide monomers and form an array of streptavidin I5 functionalized hydrogel micropillars in the narrow channel of the flow cell;

[0041] FIG. 8C depicts another example method for on-flow cell fabrication of functionalized hydrogel structures, wherein biotinylated capture oligonucleotides are bound to the streptavidin moieties in the hydrogel micropillars of FIG. 8B, and wherein target library molecules hybridize to the biotinylated capture oligonucleotides and become immobilized on the hydrogel micropillars of FIG. 8B;

[0042] FIG. 8D depicts another example method for on-flow cell fabrication of functionalized hydrogel structures, wherein immobilized target molecules are eluted from the capture oligonucleotides of FIG. 8C and seeded on the wide channel of the flow cell;

[0043] FIG. 9A depicts biotinylated P5 and P7 primers binding to a streptavidin functionalized hydrogel micropillar;

[0044] FIG. 9B depicts the biotinylated P5 and P7 primers of FIG. 9A being incubated with TET-labeled complementary P5’ and P7’ oligonucleotides; {0045] FIG. 9C depicts the TET-labeled complementary P5’ and P7” oligonucleotides of FIG. 9B hybridized to the biotinylated PS and P7 primers;

[0046] FIG. 10A is a bright field micrograph showing hvdrogel micropillars incubated with TET-P5° and TET-P7 in the absence of biotin-P5 and biotin-P7 oligonucleotides;

[0047] FIG. 10B 1s a fluorescence micrograph (488 nm excitation) showing hydrogel micropillars incubated with TET-P5" and TET-P7" in the absence of biotin-P5 and Dbiotin-P7 oligonucleotides, wherein uniform staining of flow cell surface P5 and P7 primers was observed;

[0048] FIG. 10C is a bright field micrograph showing hydrogel micropillars incubated with TET-P5" and TET-P7" after incubation with biotin-P5 and biotin-P7 oligonucleotides;

[0049] FIG. 10D is a fluorescence micrograph (488 nm excitation) showing of hydrogel micropillars incubated with TET-P5’ and TET-P7" after incubation with biotin-P5 and biotin-P7 oligonucleotides, wherein localization of TET staining to the edge of the hydrogel micropillars was observed, indicating the TET-labeled oligonucleotides have hybridized to the streptavidin-bound biotinylated P5 and P7 primers;

[0050] FIG. 11A is a fluorescence micrograph depicting the level of TET-P5S*/TET-P7 oligonucleotides in interstitial spaces between hydrogel micropillars at an incubation time of one minute;

[0051] FIG. 11B is a graph depicting the level of TET-P5'TET-P7’ oligonucleotides in interstitial spaces between hydrogel micropillars at an incubation time of one minute;

[0052] FIG. 11C is a fluorescence micrograph depicting the level of TET-PS/TET-P7 oligonucleotides in interstitial spaces between hydrogel micropillars at an incubation time of five minutes;

[0053] FIG. 11D is a graph depicting the level of TET-P5°/TET-P7 oligonucleotides in mterstitial spaces between hydrogel micropillars at an incubation time of five minutes;

[0054] FIG. 11E is a fluorescence micrograph depicting the level of TET-PS/TET-P7 oligonucleotides in interstitial spaces between hydrogel micropillars at an incubation time of ten minutes;

[0055] FIG. 11F is a graph depicting the level of TET-P5’/TET-P7’ oligonucleotides in interstitial spaces between hydrogel micropillars at an incubation time of ten minutes;

[0056] FIG. 12A depicts hybridizing P7° and PS’ regions of sequencing library molecules to biotinylated P5 and P7 oligonucleotides;

[0057] FIG. 12B depicts capturing sequencing library molecules with streptavidin-functionalized hydrogel pillars, which are attached to the surface of a flow cell;

[0058] FIG. 12C depicts seeding bound sequencing library molecules by incubation at 85°C to denature hybridized biotinylated primers and then ramping the temperature to 20°C to allow hybridization of sequencing library molecules to surface primers;

[0059] FIG. 13A is a bright field micrograph of an untreated flow cell (control);

[0060] FIG. 13B is a fluorescence micrograph (488 nm) of a SY TOX-stained untreated flow cell (control) showing no clusters:

[0061] FIG. 13C is a bright field micrograph of a flow cell having streptavidin micropillars;

[0062] FIG. 13D is a fluorescence micrograph (488 nm) of a SYTOX-stamed flow cell having streptavidin micropillars:

[0063] FIG. 13E is bright field micrograph of the hydrogel micropillar of FIG. 13C;

[0064] FIG. 13F is a fluorescence micrograph (488 nm) of the SYTOX-stained micropillar of FIG. 13D;

[0065] FIG. 14A depicts a flow cell in a cartridge, wherein streptavidin micropillars have been formed m the narrow channel, but not in wide channel of the flow cell;

[0066] FIG. 14B is a micrograph of the wide channel of the flow cell of FIG. 14A stained with SYTOX dye after 24 cycles of bridge amplification;

[0067] FIG. 14C is a micrograph of the narrow channel of the flow cell of FIG. 14A stained with SYTOX dve after 24 cycles of bridge amplification.

[0068] FIG. 15 is a flowchart depicting a first method for making functionalized three- dimensional polymer structures on a flow cell;

[0069] FIG. 16 is a flowchart depicting a second method for making functionalized three- dimensional polymer structures on a flow cell; and

[0070] FIG. 17 is a flowchart depicting a third method for making functionalized three- dimensional polymer structures on a flow cell.

DETAILED DESCRIPTION

[0071] Various implementations of the disclosed systems, devices, and methods are useful for creating reversible three-dimensional polymer (e.g., hydrogel) structures within the fluidics channels on flow cells. These structures may be used for introducing temporary functional surfaces within the flow cell, in addition to pre-existing sequencing surfaces, for multiple applications including, for example, (1) target DNA enrichment; (ii) clustered regularly interspaced short palindromic repeats (CRISPR) screening; and (iii) highly multiplexed screening applications using DNA conjugated antigens.

[0072] As used herein, the term "hydrogel" refers to a substance formed when an organic polymer (natural or synthetic) is cross-linked by way of covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure that entraps water molecules to form a gel. In some versions, the hydrogel may be a biocompatible hydrogel, which refers to a polymer that forms a gel that is not toxic to living cells and allows sufficient diffusion of oxygen and nutrients to entrapped cells to maintain viability. In some versions, the hydrogel polymer includes 60-90% fluid, such as water, and 10-30% polymer, wherein in other versions, the water content of hydrogel is about 70-80%.

[0073] An example flow cell includes a channel comprising a surface across which one or more fluid reagents can be flowed and to which adapted fragments of sequencing libraries can transport and bind. A flow cell includes a solid support having a surface on which libraries bind. In some examples, the surface contains a lawn of capture nucleotides that can bind to adapted fragments of a library. In some examples, the surface is a patterned surface. The term “patterned” when referring to a surface may describe an arrangement (such as an array) of different regions (such as amplification sites) in or on an exposed surface of a solid support. For example, one or more of the regions can be features where one or more amplification and/or capture primers are present. The features can be separated by interstitial regions where primers are not present. In some examples, the flow cell device has a channel height of about 50 um, about 60 um. about 70 um, about 80 um, about 90 um, about 100 um, about 110 um. about 120 um, about 130 um, about 140 um, or about 150 um, or an amount within a range defined by any two of the aforementioned values.

[0074] As shown in FIG. 1A, an example flow cell 100 includes top layer of glass 110 having fluidic holes 112 formed therein; channel defining spacer 120, which includes a plurality of fluidic/sequencing channels 122 formed therein; and bottom laver of glass 130 on which array 150 is formed. Array 150 includes a plurality of individual hydrogel structures 152 formed thereon by the disclosed methods. FIG. 1B depicts assembled flow cell 100 upon which an array 150 of individual three- dimensional hydrogel structures 152 has been fabricated in one of the channels 122 and FIG. IC depicts flow cell 100 having multiple three-dimensional hydrogel structures 152 formed thereon inserted into cartridge 160, which 1s used with a sequencing-by-svnthesis apparatus. Reversible, three-dimensional hydrogel structures having a specific, predetermined geometry may be formed on the flow cell by: (i) introducing a hydrogel precursor solution into a channel of the flow cell; (1) placing a photomask having a specific pattern formed thereon over the channel on the flow cell, either before or after introducing the hydrogel precursor solution into the flow cell; and (iii) exposing the hydrogel precursor solution to light at a predetermined wavelength through the photomask, wherein the illumination of the hydrogel precursor solution polvmerizes the contents thereof and forms three-dimensional structures on the flow cell that correspond to the pattern on the photomask. Once the hydrogel structures have served their purpose, they may be cleaved from the flow cell and washed away without affecting the overall functionality of the flow cell.

[0075] The hydrogel precursor solution may include monomer solutions that can be photopolvmerized by activation of a photoinitiator. An example of one such a system includes at least one type of monomer, a reversible or cleavable crosslinker, and a photoinitiator. In one version, the monomer 1s acrylamide, the reversible crosslinker is N‚N'-Bis(acryloyl)cystamine (BAC), and the photoinitiator is lithium phenyl-2 4,6-trimethylbenzoylphosphinate (LAP), which is activated by ultraviolet (UV) light at a predetermined wavelength.

[0076] In other versions, the precursor solution may include polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N'-bis(acryloylicystamine, PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N- isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), polylysme. agar, agarose. alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, dicthvleneglycol diallyl ether, ethyleneglycol diacrylate, polymethyleneglycol diacrylate, polyethyleneglycol diacrylate, trimethylopropoane trimethacrvlate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetracrylate, or combinations or mixtures thereof. In other versions, the monomer may include PEG-thiol/PEG-acrylate, acrvlamide/N,N'-bis(acrvloyl)cystamine (BACy), or PEG/PPO.

[0077] In implementations of the disclosed methods, the monomer may be the compound of formula I: 3 ie 0 Re

I wherein each R” is independently hydrogen or (C1) alkyl.

[0078] In implementations of the disclosed methods that include a crosslinker, the crosslinker may be the compound of formula II: 5 8 | In wherein: cach n is independently an integer from 1-6: and each R' is independently hydrogen or (C4) alkyl.

[0079] A reversible or cleavable crosslinker is capable of reversibly crosslinking the polymer chains within the hydrogel. In some versions, a crosslinker can be cleaved, thereby unlinking the polymer chains, by the presence of a reducing agent; by elevated temperature; by an electric field; or by exposing the hydrogel structures to a wavelength of light that cleaves a photo-cleavable crosslinker that crosslinks polymer of the hydrogel. In some versions, the reducing agent may include phosphine compounds, water soluble phosphines, nitrogen containing phosphines and salts and derivatives thereof, dithioerythritol (DTE), dithiothreitol (DTT) (cis and trans isomers, respectively, of 2,3-dihydroxy-1,4-dithiolbutane), 2- mercaptoethanol or B-mercaptoethanol (BME), 2-mercaptoethanol or ammoethanethiol, glutathione, thioglycolate or thioglycolic acid, 2,3-dimercaptopropanol, tris(2-carboxyethyl)phosphine (TCEP), tristhydroxymethy phosphine (THP), or P-[tris(hydroxymethyl)phosphine] propionic acid (THPP). In some versions, the crosslinker is cleaved by increasing the temperature to greater than about 50°C, about 55°C, about 60°C, about 63°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 95°C, or about 100°C. In some versions, the reducing agent is activated by ultraviolet light.

[0080] Other suitable photoinitiators include biocompatible photoinitiators for radical polymerization that do not damage nucleic such as, for example, a diazosulfonate initiator; monoacylphosphineoxide (MAPO}) salts such as, for example, NaUTPO and LiOTPO; and bisacylphosphineoxide (BAPO) salts such as, for example, BAPOOONa and BAPODOI.

[0081] In some examples, crosslinking the polymer chains of the hydrogel structure forms a hydrogel matrix having pores (i.e. a porous hydrogel matrix). In some versions, the size of the pores in the hydrogel structures are regulatable or tunable and may be formulated to encapsulate sufficiently large genetic material, such as cells or nucleic acids of greater than about 300 base pairs, but to allow smaller materials, such as reagents, or smaller sized nucleic acids of less than about 50 base pairs, such as primers, to pass through the pores, thereby passing in and out of the hydrogel structures. The hydrogels can have any pore size having a diameter sufficient to allow diffusion of reagents through the structure while retaining the encapsulated nucleic acid molecules. The term "pore size” can also refer to an average diameter or an average effective diameter of a cross-section of the pores, based on the measurements of a plurality of pores. The effective diameter of a cross-section that is not circular equals the diameter of a circular cross-section that has the same cross-sectional area as that of the non-circular cross-section. In some examples, the hydrogel structure can be swollen when the hvdrogel is hydrated. The sizes of the pores can then change depending on the water content in the hydrogel of the hydrogel structure. In some examples, the pores have a diameter of from about 10 nm to about 100 nm.

[0082] In some examples, the pore size of the hydrogel structures is tuned by varying the ratio of the concentration of polymer to the concentration of crosslinker. In some examples, the ratio of polymer to crosslinker 1s about 30:1, about 25:1. about 20:1, about 19:1, about 18:1, about 17:1. about 16:1, about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1. about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:30, or about any one of these rations, or a ratio within a range defined by any two of the aforementioned ratios.

[0083] FIGS. 2A-2E depict example method 200 for fabrication and subsequent removal of three-dimensional hydrogel structures on flow cell 210. Flow cell 210 includes upper internal surface 212 and lower internal surface 214 which together define flow cell channel 216. Pre-pattemed photomask 218 has been laminated or otherwise attached to the upper surface of flow cell 210. FIG. 2A depicts mtroducing hydrogel precursor solution 230 containing: (i) monomer (¢.g., acrylamide), (ii) crosslinker (e.g., BAC), and (iii) photo-initiator (¢.g., LAP) into flow cell 210. FIG. 2B depicts exposing hydrogel precursor solution 230 to UV light at a predetermined wavelength through pre-patterned photomask 218, which has multiple apertures 200 formed therein. Exposing hydrogel precursor solution 230 to UV light activates the photoinitiator (LAP), thereby generating radicals that lead to controlled polymerization of the monomer (acrylamide) into hydrogel structures 232 containing disulfide bonds. FIG. 2C depicts the formation of hydrogel features 232, which are anchored to top and bottom surfaces 212 and 214 of channel(s) 216 of flow cell 210, which is adapted to be inserted into cartridge 260. FIG. 2C includes bright field micrograph 250 showing cvlindrical hydrogel structure 232 (100-150 um in diameter) having dense gel walls with a less dense core. FIG. 2D depicts cleaving hydrogel features 232 from flow cell 210 using heat or a combination of heat and chemical cleavage of the crosslinker. For example, incubating hydrogel structures 232 with a reducing agent, such as an oil containmg DTT, cleaves the structures by reducing the disulfide bonds in the hydrogel crosslinker to thiols, thereby permitting the hydrogel to be washed out of flow cell 210 as shown in FIG. 2E. The surfaces of flow cell 210 remain functional after the cleaved hydrogel structures have been washed out of the flow cell, ie, removing the hydrogel structures from flow cell 210 does not affect the functionality of sequencing primers that have been bound to the flow cell prior to fabrication and subsequent removal of the hydrogel gel features.

[0084] Fabrication of hydrogel structures such as those previously described can be accomplished in both a factory environment and in a laboratory environment. However, known hydrogel fabrication techniques typically mvolve the use of expensive and unwieldy equipment such as, for example, a photomask aligner with a collimated UV light source and a chrome mask. Accordingly. to facilitate the fabrication of hydrogel structures on flow cells directly by consumers of sequencing products. a relatively small-scale, low-cost instrument for on-flow cell hydrogel fabrication is provided. By way of example, a generic implementation of this instrument includes: (i) a collimated LED UV light source such as, for example, Thor Labs model M385LP1-C1; (in) a housing that is adapted to receive a flow cell (and flow cell cartridge) therein and that supports and properly positions the light source relative to the flow cell; (iii) a prepatterned MylarTM photomask that is adapted to be laminate adhered on the upper surface of a particular flow cell; and (iv) a shielding enclosure for containing the light source and housing. An opening in the shielding enclosure allows the flow cell to be inserted into the housing for UV illumination of the flow cell through the prepatterned photomask. The housing may include a movable or adjustable stage apparatus for replicating patterns along the length and width of a flow cell if the illumination zone of the housing is smaller than the area of on the flow cell that is to be photopatterned. In addition to operating as a wide-field illuminator, different versions of the disclosed instrument also perform various reagent exchanges and provide thermal control for facilitating automated library preparation. As described in greater detail below. certain implementations of the disclosed instrument operate as stand-alone library preparation devices that output a ready to cluster or ready to sequence library.

[0085] FIGS. 3A-3C depict an example implementation of the disclosed system and method for fabricating hydrogel structures on a flow cell, wherein the hydrogel structures contain a sample to be sequenced or otherwise analyzed. In this implementation, the disclosed instrument is automated, and the housing includes a processor that executes various programs residing thereon for illuminating the flow cell and for performing reagent exchange and other functions in an automated manner. As shown in FIG. 3A, a customer (or other user) orders flow cell 310 onto which photomask 318, having a region that includes a customer-specified pattern formed therein, has been laminated to form assembly 320. The patterned region of photomask 318 is placed over and aligned with channel(s) 312 on flow cell 310. Flow cell 310 is then inserted into an appropriate flow cell cartridge 360. As shown in FIG. 3B, the customer then mixes a sample of interest (¢.g., biological cells or genomic DNA) with a hydrogel precursor solution that includes, for example, a monomer, a cross linker, and a photoinitiator, and loads the solution onto flow cell 310. As shown in FIG. 3C, assembly 320 and cartridge 360 are then loaded into housing 370 on which a UV light source has been mounted using moveable tray 372. Based on the layout or geometric pattern of photomask 318, the customer chooses an appropriate illumination program and exposes flow cell 310 to UV light for polymerizing the solution and patterning the desired hydrogel structures on flow cell 310. FIG. 3C includes a bright field micrograph of hydrogel pillars fabricated on a flow cell using the disclosed system and method. Flow cell 310 is then washed to remove any unpolvmerized solution and excess sample and photomask 318 can be removed from flow cell 310. Flow cell 310 can then be placed into a sequencer or fluid handler for automated downstream processing such as lvsis, tagmentation, bridge amplification, clustering, etc.

[0086] Several alternate implementations are provided regarding the assembly of the photomask and the flow cell. In one implementation. a user first inserts a flow cell into the housing and then inserts the photomask, which is separate from the flow cell (e.g., the photomask is not laminated to the flow cell). Because various photomask patterns and designs are possible, a user may select different photomasks based on required pitch or on specific applications or specific uses for the flow cell. In this and other implementations, the housing of the instrument is adapted to receive a variety of different flow cells including HiSeq™, NextSeq™, NovaSeq™. MiniSeq™, and MiSeq™ flow cells available from [lumina. In another implementation, the flow cell is provided pre-assembled with the photomask already applied to the exterior surface of the flow cell. Depending on the resolution required, the photomask can be either printed on the flow cell using screen-printing or laminated to the surface of the flow cell using an opaque adhesive film patterned to create structures on the flow cell. The photomask may be peeled off of the flow cell after it has been used, if desired. In another implementation, the photomask may be fabricated from aluminum or another metal deposited inside a fluidic channel, during a microfabrication process used to create the flow cell. The photomask may then be etched away with a high pH buffer after creation of hydrogel structures on the flow cell is complete.

Functionalization of On-Flow Cell Hydrogel Structures

[0087] Various implementations of the disclosed systems, devices, and methods may be used to create reversible hydrogel structures within the fluidics channels on flow cells that may be used for introducing temporary functional surfaces within the flow cell, in addition to pre-existing sequencing surfaces. These temporary functional surfaces may be used for multiple applications including, for example, (i) target DNA enrichment; (ii) clustered regularly interspaced short palindromic repeats (CRISPR) screening; and (iii) highly multiplexed screening applications using DNA conjugated antigens. Using methods disclosed herein (including those discussed above) on-flow cell hydrogel micro-pillars decorated with streptavidin moieties are fabricated. As discussed in greater detail below, biotinylated capture oligonucleotides bind to streptavidin and immobilize target library molecules to the hydrogel structures for on-flow cell library enrichment. Similarly, proteins and oligonucleotides can be attached to the hydrogel pillars using biotin-streptavidin linkage for enabling of variety of other screening processes such as CRISPR screening. Disclosed implementations provide a much greater surface area for binding IO reactions due to the porous nature of hydrogels and permit screening of the entire volume of a flow cell for binding events, rather than just the surface, thereby resulting in higher binding capacity and reaction rates.

[0088] An example implementation is shown in FIG. 4, which depicts the formation of hydrogel micropillars on a channel within a MiSeq™ flow cell using the previously described method for on-flow fabrication of hydrogel microstructures. In FIG. 4, flow cell 400 is shown inserted into cartridge 460. An array 450 of individual hydrogel micropillars 452 have been formed within channel 422 and are visible in the bright field micrograph at the bottom of FIG.4. In an example implementation, hydrogel micropillars 452 are created by co-polvmerization of acrylamide monomer and N‚N'-bis(acryloyl)cystamine crosslinker. Control of spatial patterning is accomplished using photo-initiator lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) to initiate the polymerization reaction by directing ultraviolet light through a photomask that has been positioned on flow cell 400 above channel 422. FIGS. 5A-5C depict on-flow cell fabrication of hydrogel micropillars 452, wherein hydrogel precursor solution that includes acrylamide and crosslinker monomers and photo-initiator is introduced into flow cell 400 (FIG. 5A) and then exposed to UV light through photomask 418 (FIG. 5B) which has been pre-patterned with desired features (e.g., apertures having a particular geometry) to form hydrogel pillars (FIG. 5C). In FIGS. 5A-5C flow cell 400 includes narrow channel 422 in which hydrogel micropillars 452 are formed and wide channel 424. Hydrogel micropillars 452 are attached to both upper surface 412 and lower surface 414 of narrow channel 422.

[0089] Flow cell 400 is provided with two types of oligonucleotides (e.g., P5 and P7), referred to as surface primers or sequencing primers, bound to the upper and lower surfaces of the flow cell. The sequences of these surface primers are complimentary to library adapters, and the fragments of a DNA library are captured by these oligonucleotides. As used herein, P5 and P7 refer to a universal P5 or P7 sequence or PS or P7 primer for capture and/or amplification purposes. A P5 sequence comprises a sequence defined by SEQ ID NO: | (AATGATACGGCGACCACCGA) and a P7 sequence comprises a sequence defined by SEQ ID NO: 2 (CAAGCAGAAGACGGCATACGA). EXAMPLE 1: PAZAM-Conjugated Biotin

[0090] In the example implementation shown in FIGS. 6A-6E. functionalized reversible hydrogel structures were formed within a channel on a flow cell bv utilizing poly(N-(5- azidoacetamidylpentyl) acrylamide-co-acrylamide) (PAZAM) into which azide moieties have been incorporated; and the azide-alkyne click reaction. The azide-alkyne click reaction involves the copper- catalyzed reaction of an azide with and alkyne to form a 5-membered heteroatom ring: a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The azide-alkyne click reaction may be photomitiated using Cu (II) and a photoinitiator system such as a Type II photoinitiator system, ¢.g., camphorquinone., which can use blue light at 470 nm as an excitation source.

[0091] In this example implementation, hydrogel micropillars are first fabricated within a MiSeq™ flow cell (or any other suitable flow cell) using UV-mediated co-polymerization of acrylamide, crosslinker, and PAZAM into which azide moieties have been incorporated. A biotin-polyethylene glycol (PEG)-alkyne complex is then clicked onto the azide moieties of the PAZAM and used to bind streptavidin. The multiple binding sites of streptavidin then allow the immobilized streptavidin to immobilize oligo capture probes, which in turn hybridize to sequencing library molecules of interest that are introduced into the flow cell. Any non-hybridized library fragments are washed out of the flow cell. Bound sequencing library fragments are then eluted from hydrogel micropillars located in the narrow channel (non-sequencing area) of the flow cell and seeded in the wide channel (sequencing area) of the flow cell in preparation for amplification, clustering, and sequencing-by-synthesis or sequencing by another method.

[0092] FIGS. 6A-6E depict an example implementation of a method for fabricating functionalized hydrogel structures on a flow cell. In FIG. GA, a hydrogel precursor solution containing 10% polyacrylamide (PA), crosslinker, and 0.25% PAZAM into which azide moieties have been incorporated is loaded onto flow cell 400. In FIG. 6A, flow cell 400 is a MiSeq™ flow cell having both a narrow channel 422 and a wide channel 424. In FIG. 6B. photomask 418, which includes a plurality of 200 um apertures formed therein, is placed on top of flow cell 400, which is then exposed to UV light for 10 seconds to co-polymerize the acrylamide and PAZAM and form an array 450 of azide-functionalized hydrogel micropillars 452 in narrow channel 422. In FIG. 6C, 5 pM biotin-PEG-alkyne is clicked onto the azide moieties during Blackpool incubation. In FIG. 6D, streptavidin labeled with Fluorescein (1:500)

binds the biotin in hydrogel micropillars 452. In FIG. 6E, the streptavidin binds biotinylated capture oligonucleotides to enable immobilization of target sequencing library molecules that have been tagged during library preparation with sequences complementary to the sequences of the capture oligonucleotides.

[0093] FIGS. 7A-7F are a series of bright field and fluorescence micrographs (488 nm excitation) depicting fluorescein-streptavidin staining of biotinylated hydrogel micropillars. FIG. 7A is 4X bright field micrograph of PA/PAZAM control (no biotin). FIG. 7B is a 4X bright field micrograph of PA/PAZAM plus Blackpool. FIG. 7C is a 4X fluorescence micrograph of PA/PAZAM control (no biotin) after a reaction time of five minutes. FIG. 7D is a 4X fluorescence micrograph of PA/PAZAM plus Blackpool after a reaction time of five minutes. FIG. 7E is a 4X fluorescence micrograph of PA/PAZAM control (no biotin) after a reaction time of ten minutes at 40°C. FIG. 7F is a 4X fluorescence micrograph of PA/PAZAM plus Blackpool after a reaction time of ten minutes at 40°C. FIGS. 7D and 7F demonstrate that fluorescein-labeled streptavidin binds biotin-conjugated hydrogel micropillars. In the control experiment (FIGS. 7C and 7E). polyacrylamide/PAZAM micropillars without biotin did not bind fluorescein-labeled streptavidin. These Figures clearly demonstrate that the biotin-conjugated (functionalized) hydrogel micropillars effectively bind targets of interest, ie. fluorescein-labeled streptavidin, in this particular example. EXAMPLE 2: Streptavidin-Acrylamide Co-polymer

[0094] In the example implementation shown in FIGS. 8A-8D, functionalized reversible hydrogel structures are formed within a channel on a flow cell by photopolymerization of acrylamide monomer, crosslinker, and streptavidin-labeled acrylamide monomer. Streptavidin functionalities of the hydrogel bind biotinylated capture oligonucleotides, which in turn hybridize to sequence library molecules of interest that are introduced into the flow cell. non-hybridized library fragments are washed out of the flow cell. Bound sequencing library fragments are then eluted from hydrogel micropillars located in the narrow channel (non-sequencing area) of the flow cell and seeded in the wide channel (sequencing area) of the flow cell in preparation for amplification, clustering, and sequencing-by- synthesis or sequencing by another method.

[0095] In FIG. 8A. a hydrogel precursor solution containing 10% polvacrvlamide (PA) and

0.25% streptavidin-labeled acrylamide monomer is loaded onto flow cell 400. In FIG. 8A, flow cell 400 is a MiSeq™ flow cell having both a narrow channel 422 and a wide channel 424. In FIG. 8B, photomask 418, which includes a plurality of 200 um apertures formed therein, is placed on top of flow cell 400,

which is then exposed to UV light for 10 seconds to co-polymerize the acrylamide streptavidin-labeled acrylamide monomers and form an array 450 of streptavidin functionalized hydrogel micropillars 452 in narrow channel 422. In FIG. 8C, biotinylated capture oligonucleotides are bound to the streptavidin moieties in the hydrogel structures, and target library molecules hybridize to the biotinylated capture oligonucleotide and become immobilized on the hydrogel micropillars. In FIG. 8D, the immobilized target molecules are eluted from the capture oligonucleotides and seeded on wide channel 424 of flow cell 400 for amplification, clustering, and sequencing-by-synthesis or sequencing by another method.

[0096] With reference to FIGS. 9A-9C, streptavidin on the surface of hydrogel pillars 452 can be detected by incubation with biotinylated primers P5 and P7. FIG. 9A depicts biotinylated P5 and P7 primers (508 and 506 respectively) binding to streptavidin functionalized hydrogel micropillar 452. FIG. 9B depicts the biotinylated P5 and P7 primers (508 and 506 respectively) being incubated with TET- labeled complementary P5’ and P7’ oligonucleotides (509 and 507 respectively). FIG. 9C depicts the TET-labeled complementary PS’ and P7” oligonucleotides (509 and 507 respectively) hybridized to the biotinylated P5 and P7 primers (508 and 506 respectively). Control hydrogel micropillars fabricated without streptavidin do not show staining with TET-labeled primers while streptavidin-containing pillars demonstrate staining with TET-labeled primers.

[0097] FIG. 10A is a bright field micrograph showing hydrogel micropillars 452 incubated with TET-P5’ and TET-P7’ in the absence of biotin-P5 and biotin-P7 oligonucleotides. FIG. 10B is a fluorescence micrograph (488 nm excitation) showing hydrogel micropillars 452 incubated with TET-P5" and TET-P7” in the absence of biotin-P5 and biotin-P7 oligonucleotides, wherein uniform staining of flow cell surface PS and P7 primers was observed. FIG. 10C is a bright field micrograph showing hydrogel micropillars 452 incubated with TET-PS° and TET-P7" after incubation with biotin-P5 and biotin-P7 oligonucleotides. FIG. 10D is a fluorescence micrograph (488 nm excitation) showing of hydrogel micropillars 452 incubated with TET-P5° and TET-P7" after incubation with biotin-P5 and biotin-P7 oligonucleotides, wherein localization of TET staining to the edge of the hydrogel micropillars was observed, indicating the TET-labeled oligonucleotides have hybridized to the streptavidin-bound biotinylated P5 and P7 primers.

[0098] When incubating TET-P5’/TET-P7° oligonucleotides with streptavidin containing micropillars that were previously incubated with biotn-P5 and biotin-P7 oligonucleotides, a depletion of TET-labeled primers was observed in the interstitial space between the micropillars on the flow cell as fluorescence increased at the surface of the micropillars and penetrated the hydrogel. FIG. 11A is a fluorescence micrograph depicting the level of TET-P5/TET-P7" oligonucleotides in interstitial spaces between hydrogel micropillars at an incubation time of one minute and FIG. 11B is a graph depicting the level of TET-P5’/TET-P7° oligonucleotides in interstitial spaces between hydrogel micropillars at an incubation time of one minute, with distance shown on the X-axis and level shown on the Y-axis. FIG. 11C is a fluorescence micrograph depicting the level of TET-PS5"/TET-P7° oligonucleotides in interstitial spaces between hydrogel micropillars at an incubation time of five minutes and FIG. 11D is a graph depicting the level of TET-P3/TET-P7’ oligonucleotides in interstitial spaces between hydrogel micropillars at an incubation time of five minutes, with distance shown on the X-axis and level shown on the Y-axis. FIG. 11E is a fluorescence micrograph depicting the level of TET-PS’/TET-P7 oligonucleotides m interstitial spaces between hydrogel micropillars at an incubation time of ten minutes and FIG. 11F is a graph depicting the level of TET-P5’/TET-P7° oligonucleotides in interstitial spaces between hydrogel micropillars at an incubation time of ten minutes, with distance shown on the X-axis and level shown on the Y-axis. The fluorescence intensity profiles represented by these micrographs demonstrate that binding of TET-labeled oligonucleotides to a hvdrogel surface depletes the oligonucleotides in the interstitial areas between the micropillars.

[0099] With reference to FIGS. 12A-12C, to demonstrate the ability to capture and release target library molecules using the above-described example implementation, a PhiX library was incubated with biotinylated P5 and P7 primers (1:10). FIG. 12A depicts hybridizing P7° and P5° regions of sequencing library molecules (502 and504 respectively) to biotinylated P5 and P7 oligonucleotides (506 and 508 respectively). FIG. 12B depicts capturing sequencing library molecules 502 and 504 with streptavidin- functionalized hydrogel pillars 452, which are attached to the surface of flow cell 400. FIG. 12C depicts seeding bound sequencing library molecules 502 and 504 by incubation at 85°C to denature hybridized biotinylated primers 506 and 508 and then ramping the temperature to 20°C to allow hybridization of library molecules 502 and 504 to surface primers 510 and 512 respectively. The hybridized PhiX library (1pM) was then incubated in either an untreated flow cell (control) or streptavidin-pillar patterned flow cell. After incubation with library molecules hybridized to biotin-primers, flow cells were washed with HT-1, followed by seeding (80°C 53min, 60°C Smin, 40°C 2min and 20°C 2min), first extension (AMS-1, 50°C 5min) and 24 cycles of bridge amplification. While the control flow cell showed no clusters (see FIGS. 13A-13B), the streptavidin-pillar flow cells showed high cluster density (see FIGS. 13C-13F), demonstrating successful capture of a library hybridized to biotin-P3/biotin-P7.

[00100] FIG. 13A is a bright field micrograph of an untreated flow cell (control) and FIG. 13B is a fluorescence micrograph (488 nm) of a SYTOX (ThermoFisher Scientific) stained untreated flow cell (control) showing no clusters. FIG. 13C is a bright field micrograph of a flow cell having streptavidin micropillars and FIG. 13D is a fluorescence micrograph (488 nm) of a SYTOX-stamed flow cell having streptavidin micropillars. FIG. 13E is a bright field micrograph of the hydrogel micropillar of FIG. 13C. FIG. 13F is a fluorescence micrograph (488 nm) of the SYTOX-stained micropillar of FIG. 13D. The fluorescence micrographs of SYTOX-stained flow cells demonstrate that while untreated flow cells show no clusters, the flow cell having streptavidin-hydrogel pillars demonstrates high cluster density and pillar “footprints” where hydrogel micropillars had been patterned. Furthermore, when patterning only one channel (narrow channel) of a flow cell with streptavidin pillars, clusters show a gradient in density within the same flow cell, where cluster density close to pillars is higher. FIG. 14A depicts flow cell 400 in cartridge 460, wherein streptavidin micropillars have been formed in narrow channel 422, but not in wide channel 422. When streptavidin pillars are pattemed only in the narrow channel of a MiSeq™ flow cell, cluster density forms a gradient from #igh close to the micropillars (narrow channel) to low far away from the micropillars (wide channel). FIG. 14B is a micrograph of wide channel 424 stained with SYTOX dye after 24 cycles of bridge amplification and FIG. 14C is a micrograph of narrow channel 422 stained with SYTOX dye after 24 cycles of bridge amplification. The descriptions below provide some additional examples related to the methods provided herein. They are not necessarily part of the non-limiting working examples provided above.

[0101] FIG. 15 is a flowchart depicting a first method for making functionalized three- dimensional polymer structures on a flow cell. Method 1500 comprises loading a polymer precursor solution into a flow cell at block 1502. wherein the polymer precursor solution includes a monomer, a crosslinker, a photoinitiator, and a functionalized polymer such as, for example, PAZAM containing azide moieties, and wherein the flow cell includes at least one channel for receiving the polymer precursor solution, and wherein the at least one channel has an upper interior surface and a lower interior surface; placing a photomask over the at least one channel at block 1504, wherein the photomask includes a series of apertures formed therein; and illuminating the polymer precursor solution through the photomask with a light source at block 1506, wherein the light source emits light at a wavelength that activates the photoinitiator, and wherein activation of the photoinitiator polymerizes the polymer precursor solution underneath the apertures in the photomask and forms three-dimensional polymer structures extending from the upper interior surface to the lower interior surface of the at least one channel.

[0102] FIG. 16 is a flowchart depicting a second method for making functionalized three- dimensional polymer structures on a flow cell. Method 1600 comprises loading a polymer precursor solution into a flow cell at block 1602, wherein the polymer precursor solution includes a monomer, a crosslinker, a photoinitiator, and PAZAM containing azide moieties, and wherein the flow cell includes at least one channel for receiving the polymer precursor solution, and wherein the at least one channel has an upper interior surface and a lower interior surface: placing a photomask over the at least one channel at block 1604, wherein the photomask includes a series of apertures formed therein; illuminating the polymer precursor solution through the photomask with a light source at block 1606, wherein the light source emits light at a wavelength that activates the photoinitiator, and wherein activation of the photoinitiator polvmerizes the polymer precursor solution undemeath the apertures in the photomask and forms three-dimensional polymer structures extending from the upper interior surface to the lower interior surface of the at least one channel; reacting a biotin-PEG-alkyne complex with the azide moieties in the PAZAM in the three-dimensional polymer structures using an azide-alkyne click reaction at block 1608; binding streptavidin to the biotin in the biotin-PEG-alkyne complex at block 1610; and binding biotinylated capture oligonucleotides to the streptavidin at block 1612, wherein the biotinylated capture oligonucleotides are specific for target molecules of interest in a sequencing library.

[0103] FIG. 17 is a flowchart depicting a third method for making functionalized three- dimensional polymer structures on a flow cell. Method 1700 comprises loading a polymer precursor solution into a flow cell at block 1702, wherein the polymer precursor solution includes a monomer, a crosslinker, a photoinitiator, and a streptavidin-labeled acrylamide monomer, and wherein the flow cell includes at least one channel for receiving the polymer precursor solution, and wherein the at least one channel has an upper interior surface and a lower interior surface; placing a photomask over the at least one channel at block 1704, wherein the photomask includes a series of apertures formed therein; illuminating the polymer precursor solution through the photomask with a light source at block 1706, wherein the light source emits light at a wavelength that activates the photoinitiator, and wherein activation of the photoinitiator polymerizes the polymer precursor solution underneath the apertures in the photomask and forms three-dimensional polymer structures extending from the upper interior surface to the lower interior surface of the at least one channel; binding biotinylated capture oligonucleotides to the streptavidin in the three-dimensional polymer structures at block 1708, wherein the biotinylated capture oligonucleotides are specific for target molecules of interest in a sequencing library and bind thereto; and eluting the bound target molecules and seeding the eluted target molecules on the surfaces of the flow cell having oligonucleotides bound thereto at block 1710.

[0102] The foregoing description is provided to enable a person skilled in the art to practice the various configurations described herein. While the subject technology has been particularly described with reference to the various figures and configurations, it should be understood that these are for illustration purposes only and should not be taken as limiting the scope of the subject technology.

[0104] All literature and similar material cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety. In the event that one or more of the incorporated references and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.

[0105] As used herein, the singular forms "a," "an," and "the," refer to both the singular as well as plural, unless the context clearly indicates otherwise. The term "comprising" as used herein is synonymous with "including," "containing," or “characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. Unless context indicates otherwise, the recitations of numerical ranges by endpoints mclude all numbers subsumed within that range. Furthermore, references to “one implementation” are not intended to be interpreted as excluding the existence of additional implementations that also incorporate the recited features. Moreover, unless explicitly stated to the contrary, implementations “comprising” or “having” an element or a plurality of elements having a particular property may include additional elements whether or not they have that property.

[0106] The terms “substantially” and “about” used throughout this specification are used to describe and account for small fluctuations, such as due to variations in processing. For example, they can refer to less than or equal to £5%, such as less than or equal to £2%, such as less than or equal to +1%, such as less than or equal to £0.5%, such as less than or equal to £0.2%, such as less than or equal to +0. 1%, such as less than or equal to 0.05%, and/or 0%.

[0107] There may be many other ways to implement the subject technology. Various functions and elements described herein may be partitioned differently from those shown without departing from the scope of the subject technology. Various modifications to these implementations may be readily apparent to those skilled in the art, and generic principles defined herein may be applied to other implementations. Thus. many changes and modifications may be made to the subject technology, by one having ordinary skill in the art, without departing from the scope of the subject technology. For instance, different numbers of a given module or unit may be employed, a different type or types of a given module or unit may be emploved, a given module or unit may be added, or a given module or unit may be omitted.

[0108] Underlined and/or italicized headings and subheadings are used for convenience only, do not limit the subject technology, and are not referred to in connection with the interpretation of the description of the subject technology. All structural and functional equivalents to the elements of the various implementations described throughout this disclosure that are known or later come to be known to those of ordinary skill in the art are expressly incorporated herein by reference and intended to be encompassed by the subject technology. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the above description.

[0109] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein.

[0110] The disclosure also includes the following clauses:

1. A method for making on-flow cell three-dimensional polymer structures having functionalized surfaces. comprising: loading a polymer precursor solution into a flow cell, wherein the polymer precursor solution includes a monomer, a crosslinker, a photoinitiator, and a functionalized polymer, and wherein the flow cell includes at least one channel for receiving the polymer precursor solution, and wherein the at least one channel has an upper interior surface and a lower interior surface; and illuminating the polymer precursor solution through a photomask with light at a wavelength that activates the photoinitiator, wherein the photomask includes a series of apertures formed therein, wherein the photomask has been placed over an exterior surface of the channel, and wherein activation of the photoinitiator polymerizes at least some of the polymer precursor solution underneath the apertures in the photomask and forms three- dimensional polymer structures extending from the upper interior surface to the lower interior surface of the at least one channel.

2. The method of clause 1, further comprising reacting a bi-functional linker, having a first end and a second end, with the functionalized polymer, wherein the first end of the bi- functional linker 1s chemically or enzymatically attached to the functionalized polymer, and wherein the second end of the bi-functional linker selectively binds predetermined types of molecules.

3. The method of clause 2, wherein the functionalized polymer is poly(N-(5- azidoacetamidylpentyl) acrylamide-co-acrylamide (PAZAM) containing azide moieties and wherein the bi-functional linker is a biotin-PEG-alkyne complex, and the method further comprising reacting the biotin-PEG-alkyne complex with the azide moieties in the PAZAM using an azide-alkyne click reaction a click reaction.

4. The method of clause 3, further comprising binding streptavidin to the biotin in the biotin- PEG-alkyne complex.

5. The method of clause 4, further comprising binding biotinylated capture oligonucleotides to the streptavidin, wherein the biotinylated capture oligonucleotides are specific for targets of interest in a sequencing library.

6. The method of any of clauses 1-5, further comprising washing unpolymerized polymer precursor solution out of the flow cell.

7. The method of any of clauses 1-6, further comprising cleaving at least some the three- dimensional polymer structures from the flow cell using heat, cleaving chemicals, or a combination of heat and cleaving chemicals.

8. The method of any of clauses 1-7, wherein the flow cell has oligonucleotides of predetermined lengths and sequences bound to both the upper and lower interior surfaces of the at least one channel, and wherein the oligonucleotides include primers adapted for nucleic acid amplification.

9. The method of any of clauses 1-8, wherein the polymer is a hydrogel.

10. The method of any one of clauses 1-9, wherein the monomer is the compound of formula T: ie

I wherein each R? is independently hydrogen or (C,.) alkyl.

I1. The method of any one of clauses 1-10, wherein the crosslinker is a compound of formula II: & =

II wherein: each n is independently an integer from 1-6; and each R' is independently a hydrogen or (C,.) alkyl.

12. The method of any one of clauses 1-11. wherein the photoinitiator is a diazosulfonate initiator; a monoacylphosphineoxide (MAPO) salt; a bisacylphosphineoxide (BAPO) salt; or combinations thereof.

13. The method of any of clauses 1-12 wherein the monomer is acrylamide, the crosslinker is N‚N'Bis(acryloyl)cystamine (BACy), and the photoinitiator is lithium phenyl-2.4.6- trimethylbenzoylphosphinate (LAP).

14. The method of any of clauses 1-12, wherein the polymer precursor solution includes polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N‚N'- Bis(acryloyl)cystamine (BACYy), PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate} (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co- glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L- aspartic acid), poly(L- glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrvlate, diallylamine, triallvlamine, divinyl sulfone, diethyleneglycol diallyl ether, ethyleneglycol diacrylate, polymethyleneglycol diacrylate, polyethyleneglvcol diacrylate, trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetracrylate, or combinations or mixtures thereof.

15. The method of any of clauses 1-12 wherein the polymer precursor solution includes polyethylene glycol (PEG)-thiol/PEG-acrylate; acrylamide/N.N'-Bis(acryloylDcystamime (BACy). PEG/polypropylene oxide (PPO); or combinations thereof.

16. The method of any of clauses 1-15, wherein the photomask is polyethylene terephthalate .

17. The method of any of clauses 1-16, wherein the photomask is laminated to the upper surface of the flow cell.

18. The method of any of clauses 1-17, further comprising a light source, wherein the light source 1s an ultraviolet light source.

19. The method of any of clauses 1-18. wherein the three-dimensional polymer structures are cylindrical. 19 20. A method for making on-flow cell three-dimensional polymer structures having functionalized surfaces, comprising: loading a hydrogel precursor solution into a flow cell, wherein the hydrogel precursor solution includes a monomer, a crosslinker, a photoinitiator, and PAZAM containing azide moieties, and wherein the flow cell includes at least one channel for receiving the hydrogel precursor solution, and wherein the at least one channel has an upper interior surface and a lower interior surface: placing a photomask over the at least one channel, wherein the photomask includes a series of apertures formed therein; and illuminating the hydrogel precursor solution through the photomask with light at a wavelength that activates the photoinitiator, and wherein activation of the photoinitiator polymerizes at least some of the hydrogel precursor solution underneath the apertures in the photomask and forms three-dimensional hydrogel structures extending from the upper interior surface to the lower interior surface of the at least one channel; reacting a biotin-PEG-alkyne complex with the azide moieties in the PAZAM in the three- dimensional polymer structures using an azide-alkyne click reaction: binding streptavidin to the biotin in the biotin-PEG-alkyne complex: and binding biotinylated capture oligonucleotides to the streptavidin, wherein the biotinylated capture oligonucleotides are specific for target molecules of interest in a sequencing library.

21. The method of clause 20, wherein the monomer is the compound of formula I:

NS

I wherein each R? is independently hydrogen or (C,.) alkyl.

22. The method of any one of clauses 20-21, wherein the crosslinker is a compound of formula II: 2 2

II wherein: each n is independently an integer from 1-6; and each R! is independently hydrogen or (C,.) alkyl.

23. The method of any one of clauses 20-22, wherein the photoinitiator is a diazosulfonate initiator; a monoacylphosphineoxide (MAPO) salt: a bisacylphosphineoxide (BAPO) salt; or combinations or mixtures thereof.

24. The method of any of clauses 20-23, wherein the monomer is acrylamide, the crosslinker is N.N'- Bis(acryloyl)cystamme ~~ (BACy), and the photoinitiator is lithium phenyl-2.4.6- trimethylbenzoylphosphinate (LAP).

25. The method of any of clauses 20-24, wherein the polymer precursor solution includes polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N.N'- Bis(acryloyl)cystamine (BACy), PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly{methyl methacrylate) (PMMA). poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co- glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L- aspartic acid), poly(L- glutamic acid), polvlysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethvleneglycol diallyl ether, ethyleneglyeol diacrylate, polymethyleneglycol diacrylate, polvethyleneglvcol diacrylate, trimethylopropoane trimethacrylate, cthoxylated trimethylol triacrvlate, or ethoxvlated pentaerythritol tetracrylate, or combinations or mixtures thereof.

26. The method of any of clauses 20-24, wherein the polymer precursor solution includes polyethylene glycol (PEG)-thiol/PEG-acrylate; acrylamide / N,N'- Bis(acryloyvl)cystamine (BACy); PEG/polypropylene oxide (PPO); or combinations thereof.

27. The method of any of clauses 20-26, wherein the photomask comprises a polyester film.

28 The method of any of clauses 20-27, wherein the photomask is laminated to the upper surface of the flow cell.

29 The method of any of clauses 20-28, wherein the light source is an ultraviolet light source.

30. The method of any of clauses 20-29, wherein the three-dimensional polymer structures are cylindrical.

31. A method for making on-flow cell three-dimensional polymer structures having functionalized surfaces, comprising: loading a polymer precursor solution into a flow cell, wherein the polymer precursor solution includes a monomer, a crosslinker, a photoinitiator, and a streptavidin-labeled acrylamide monomer, and wherein the flow cell includes at least one channel for receiving the polymer precursor solution, and wherein the at least one channel has an upper interior surface and a lower interior surface, and wherein oligonucleotides of predetermined lengths are bound to both the upper and lower surfaces of the at least one channel; placing a photomask over the at least one channel, wherein the photomask includes a series of apertures formed therein; illuminating the polymer precursor solution through the photomask with light at a wavelength that activates the photoinitiator, and wherein activation of the photoinitiator polymerizes at least some of the polymer precursor solution underneath the apertures in the photomask and forms three-dimensional polymer structures extending from the upper interior surface to the lower interior surface of the at least one channel; selectively binding biotinylated capture oligonucleotides to the streptavidin in the three- dimensional polymer structures, wherein the biotinylated capture oligonucleotides are specific for target molecules of interest in a library and bind thereto; and eluting the bound target molecules and seeding the eluted target molecules on the surfaces of the flow cell having oligonucleotides bound thereto,

32. The method of clause 31, wherein the monomer is the compound of formula I: 2

I wherein each R? is independently hydrogen or (C.6) alkyl.

33. The method of any one of clauses 31-32, wherein the crosslinker is a compound of formula II:

RY NS

II wherein: cach n is independently an integer from 1-6: and each R' is independently hydrogen or (C.) alkyl.

34. The method of any one of clauses 31-33, wherein the photoinitiator is a diazosulfonate initiator, a monoacylphosphineoxide (MAPO) salt; a bisacylphosphineoxide (BAPO) salt; or combinations or mixtures thereof.

35. The method of any of clauses 31-34, wherein the monomer is acrylamide, the crosslinker 1s N.N'-Bis(acryloyl)cystamine (BACy), and the photoinitiator is lithium phenyl-2,4,6- trimethylbenzovlphosphinate (LAP).

36. The method of any of clauses 31-35, wherein the polymer precursor solution includes polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N N'- Bis(acryloyl)cystamine (BACy), PEG, polypropylene oxide (PPO). polyacrylic acid, polv(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), polv(N-isopropvlacrvlamide) (PNIPAAm), polv(lactic acid) (PLA), poly(lactic-co- glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L- aspartic acid), poly(L- glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamme. divinyl sulfone, diethyleneglycol diallyl ether, ethyleneglycol diacrylate, polvmethyleneglycol diacrylate, polyethyleneglyeol diacrylate, trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate. or ethoxylated pentaerythritol tetracrylate, or combinations or mixtures thereof.

37. The method of any of clauses 31-35, wherein the polymer precursor solution includes polyethylene glycol (PEG)-thiol/PEG-acrylate; acrylamide /N.N'- Bis(acrvloyl)cystamine (BACy); PEG/polypropylene oxide (PPO): or combinations thereof.

38. The method of any of clauses 31-37, wherein the photomask is polyethylene terephthalate.

39. The method of any of clauses 31-38, wherein the photomask is laminated to the upper surface of the flow cell.

40. The method of any of clauses 31-39, wherein the light source 1s an ultraviolet light source.

41. The method of any of clauses 31-40, wherein the three-dimensional polymer structures are cylindrical.

42. A flow cell, comprising: a channel, wherein the channel includes an upper interior surface having primers coated thereon and a lower interior surface having primers coated thereon; and reversible, permeable, three-dimensional polymer structures in the channel from a polymer precursor solution, wherein the three-dimensional polymer structures extend from the upper interior surface of the channel to the lower interior surface of the channel.

43. The flow cell of clause 42, further comprising a photomask placed over an outer exterior surface of the channel.

44. The flow cell of any of clauses 42-43, wherein the three-dimensional polymer structures include hydrogels.

45. The flow cell of any of clauses 42-44, wherein the flow cell, polymer precursor solutions, and photomask are provided in a kit.

20200210 Sequence listing.txt

SEQUENCE LISTING <110> Illumina, Inc, San Diego, California, US <120> ON-FLOW CELL THREE-DIMENSIONAL POLYMER STRUCTURES HAVING FUNCTIONALIZED SURFACES <130> P168530NL00/38/JED <140> NL2024528 <141> 2019-12-20 <160> 2 <170> BiSSAP 1.3.6 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide P5 <400> 1 aatgatacgg cgaccaccga 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide P7 <400> 2 caagcagaag acggcatacg a 21 Page 1

Claims (45)

CONCLUSIES l. Werkwijze voor het vervaardigen van doorstrommgscel driedimensionale polymeerstructuren welke functionele vlakken heeft, omvattende: - het laden van een polymeeruitgangsstofoplossing in een stromingscel, waarbij de polymeeruitgangsstofoplossmg een monomeer, een vernetter, en een foto-initiator, en een gefunctionaliseerd polymeer omvat, en waarbij de stromingscel ten minste één kanaal voor het ontvangen van de polvmeeruitgangsstofoplossing omvat, en waarbij het ten minste één kanaal een bovenste binnenvlak en een onderste binnenvlak heeft: en - het belichten van de polymeeruitgangsstofoplossing door een gepatroneerd fotomasker met licht bij een golflengte welke de foto-initiator activeert, waarbij het fotomasker een reeks openingen daarin gevormd omvat, en waarin activatie van de foto-mitiator ten minste deels de polymeeruitgangsstofoplossing beneden de openingen in het fotomasker polymeriseert en driedimensionale polymeerstructuren vormt welke uitstrekken van het bovenste binnenvak tot het onderste binnenvlak van het ten minste één kanaal.CONCLUSIONS l. Method for making flow cell three-dimensional polymer structures having functional planes, comprising: loading a polymer precursor solution into a flow cell, wherein the polymer precursor solution comprises a monomer, a crosslinker, and a photoinitiator, and a functionalized polymer, and wherein the flow cell at least one channel for receiving the polymer precursor solution, and wherein the at least one channel has an upper inner surface and a lower inner surface: and - exposing the polymer precursor solution through a patterned photomask with light at a wavelength which activates the photoinitiator wherein the photomask comprises a series of apertures formed therein, and wherein activation of the photo-mitiator at least partially polymerizes the polymer precursor solution below the apertures in the photomask and forms three-dimensional polymer structures extending from the upper inner pocket to the lower inner surface of the at least one channel. 2. Werkwijze volgens conclusie 1, aanvullend omvattend het reageren van een bifunctionele verbinder, hebbende een eerste uiteinde en een tweede uitemde. met het gefunctionaliseerd polymeer, waarbij het eerste uiteinde van de bifunctionele verbinder chemisch of enzymatisch gebonden wordt met het gefunctionaliseerd polymeer, en waarbij het tweede uiteinde van de bifunctionele verbinder selectief vooraf bepaalde type moleculen bindt.The method of claim 1, additionally comprising reacting a bifunctional linker having a first end and a second end. with the functionalized polymer, wherein the first end of the bifunctional linker is chemically or enzymatically bonded to the functionalized polymer, and wherein the second end of the bifunctional linker selectively binds predetermined type of molecules. 3. Werkwijze volgens conclusie 2, waarbij het gefunctionaliseerd polymeer poly (N-(5- azidoacetamidylpentyl)acrylamide-co-acrylamide (PAZAM) is bevattend azide delen en waarbij de bifunctionele verbinder een biotine-PEG-alkyn complex is. en de werkwijze aanvullend het reageren van het biotine-PEG-alkyn complex met de azide delen in het PAZAM gebruik makend van een azide-alkyn klikreactie (een klikreactie).The method of claim 2, wherein the functionalized polymer is poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide (PAZAM) containing azide moieties and wherein the bifunctional linker is a biotin-PEG-alkyne complex. reacting the biotin-PEG-alkyne complex with the azide moieties in the PAZAM using an azide-alkyne click reaction (a click reaction). 4. Werkwijze volgens conclusie 3, aanvullend het binden van streptavidine aan het biotine in het biotine-PEG-alkyn complex omvattend.The method of claim 3, additionally comprising binding streptavidin to the biotin in the biotin-PEG-alkyne complex. 5. Werkwijze volgens conclusie 4, aanvullend het binden van gebiotinyleerde grijp-oligonucleotiden aan het streptavidine, waarbij de gebiotinyleerde grijp-oligonucleotiden specifiek zijn voor doelen van belang in een sequentiebibliotheek.The method of claim 4, additionally binding biotinylated grab oligonucleotides to the streptavidin, wherein the biotinylated grab oligonucleotides are specific for targets of interest in a sequence library. 6. Werkwijze volgens één van de conclusies 1 - 5, aanvullend het wassen van ongepolymeriseerde polymeeruitgangsstofoplossing uit de stromingscel omvattend.A method according to any one of claims 1 to 5, additionally comprising washing unpolymerized polymer precursor solution from the flow cell. 7. Werkwijze volgens één van de conclusies 1 - 6, aanvullend het splitsen van ten minste enkele van de driedimensionale polymeerstructuren uit de stromingscel gebruik makend van warmte, splitschemicaliën, of een combinatie van warmte en splitschemicalién omvattend.The method of any one of claims 1-6, additionally comprising cleaving at least some of the three-dimensional polymer structures from the flow cell using heat, cleavage chemicals, or a combination of heat and cleavage chemicals. 8. Werkwijze volgens één van de conclusies 1 - 7, waarbij de stromingscel oligonucleotiden heeft van vooraf bepaalde lengtes en sequenties gebonden aan zowel de bovenste en onderste binnenvlakken van het ten minste één kanaal, en waarbij de oligonucleotiden primers hebben aangepast voor nucleïne zuur vermeerdering.The method of any one of claims 1 to 7, wherein the flow cell has oligonucleotides of predetermined lengths and sequences bound to both the upper and lower inner surfaces of the at least one channel, and wherein the oligonucleotides have primers adapted for nucleic acid amplification. 9. Werkwijze volgens één van de conclusies 1 - 8, waarbij het polymeer een hydrogel is.A method according to any one of claims 1-8, wherein the polymer is a hydrogel. 19, Werkwijze volgens één van de conclusies 1 - 9, waarbij het monomeer de stof is met de formule I: Ns: We ye N° 8 RpProcess according to any one of claims 1 to 9, wherein the monomer is the substance of the formula I: Ns : We ye N° 8 Rp I waarbij elke R° onafhankelijk waterstof of (C,.¢) alkyl is. 11, Werkwijze volgens één van de conclusies 1 - 10, waarbij de vernetter een stof is met de formule II: ord ml Ee ay ml waarbij: elke n onafhankelijk een geheel getal van 1 — 6 1s; en elke R' onafhankelijk waterstof of (C'1.) alkyl is.I wherein each R 0 is independently hydrogen or (C 1-6 ) alkyl. A method according to any one of claims 1 to 10, wherein the crosslinker is a substance of the formula II: ord ml Ee ay ml wherein: each n is independently an integer from 1 to 6 1s; and each R' is independently hydrogen or (C'1) alkyl. 12. Werkwijze volgens één van de conclusies 1 - 11, waarbij de foto-initiator een diazosulfonaat-initiator; een monoacylfosfineoxide (MAPO) zout: een bisacylfosfineoxide (BAPO) zout; of combinaties daarvan is.A method according to any one of claims 1 to 11, wherein the photoinitiator is a diazosulfonate initiator; a monoacylphosphine oxide (MAPO) salt: a bisacylphosphine oxide (BAPO) salt; or combinations thereof. 13. Werkwijze volgens één van de conclusies 1 - 12, waarbij het monomeer acrylamide is. de vernetter N, N'-bis(acryloyl)cystamine (BACy) is, en de foto-initiator lithium fenyl-2.4.6- trimethylbenzoylfosfinaat (LAP) is.The method of any one of claims 1 to 12, wherein the monomer is acrylamide. the crosslinker is N,N'-bis(acryloyl)cystamine (BACy), and the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). 14. Werkwijze volgens één van de conclusies 1 - 12, waarbij de polymeeruitgangsstofoplossing polyethyleenglycol (PEG)-thiol, PEG-acrylaat, acrylamide, N, N°-bis(acryloyl)cystamime (BACy), PEG, propyleenglycol (PPO), polvacrylzuur, poly(hydroxyethylmethacrylaat) (PHEMA), poly(methylmethacrylaat) (PMMA), poly(N-isopropvlacrvlamide) (PNIPA Am), polymelkzuur (PLA), poly(melkzuur-co-glycolzuur), polycaprolacton (PCL), poly(vinylsulfonzuur) (PVSA), poly(L-asparaginezuur), poly(L-glutaminezuur), polylysine, agar, agarose, alginaat, heparine, alginaatsulfaat, dextransulfaat, hyaluronan, pectine, carrageen, gelatine, chitosan, cellulose, collageen, bisacrylamide, diacrylaat, diallylamine, triallylamine, divinylsulfon, diethyleenglycoldiallylether. ethyleenglycoldiacrylaat, polymethyleenglycoldiacrylaat, polyethyleenglycoldiacrylaat, trimethylolpropaantrimethacrylaat, geethoxyleerd trimethyloltriacrylaat, geethoxyleerd pentaerythritoltetraacrylaat, of combinaties daarvan omvat.A process according to any one of claims 1 to 12, wherein the polymer precursor solution is polyethylene glycol (PEG) thiol, PEG-acrylate, acrylamide, N, N°-bis(acryloyl)cystamime (BACy), PEG, propylene glycol (PPO), polyvacrylic acid , poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropyl acrylide) (PNIPA Am), polylactic acid (PLA), poly(lactic-co-glycolic acid), polycaprolactone (PCL), poly(vinyl sulfonic acid ) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate , diallylamine, triallylamine, divinyl sulfone, diethylene glycol diallyl ether. ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylolpropane trimethacrylate, ethoxylated trimethylol triacrylate, ethoxylated pentaerythritol tetraacrylate, or combinations thereof. 15. Werkwijze volgens één van de conclusies 1 - 12, waarbij de polymeeruitgangsstofoplossing polyethyleenglycol (PEG)-thiol / PEG-acrylaat, acrylamide / N, N’-bis(acryloyl)cystamine (BACy), PEG / propyleenglycol (PPO), of combinaties daarvan omvat.The method of any one of claims 1 to 12, wherein the polymer precursor solution is polyethylene glycol (PEG)-thiol/PEG-acrylate, acrylamide/N, N'-bis(acryloyl)cystamine (BACy), PEG/propylene glycol (PPO), or combinations thereof. 16. Werkwijze volgens één van de conclusies 1 - 15, waarbij het fotomasker polyethyleentereftalaat is.A method according to any one of claims 1 to 15, wherein the photomask is polyethylene terephthalate. 17. Werkwijze volgens één van de conclusies 1 - 16, waarbij het fotomasker is gelamineerd tot aan het bovenste buitenvlak van de strommgscel.The method of any one of claims 1 to 16, wherein the photomask is laminated to the upper outer surface of the flow cell. 18. Werkwijze volgens één van de conclusies 1 - 17, aanvullend een lichtbron omvattend. waarbij de lichtbron een ultraviolet lichtbron is.A method according to any one of claims 1 to 17, additionally comprising a light source. wherein the light source is an ultraviolet light source. 19. Werkwijze volgens één van de conclusies 1 - 18, waarbij de driedimensionale polymeerstructuren cilindrisch zijn.A method according to any one of claims 1 to 18, wherein the three-dimensional polymer structures are cylindrical. 20. Werkwijze voor het vervaardigen van doorstromingscel driedimensionale polymeerstructuren, omvattende:A method of manufacturing flow cell three-dimensional polymer structures, comprising: het laden van een hydrogel-uitgangsstofoplossing in een stromingscel, waarbij de hydrogel-uitgangsstofoplossing een monomeer, een vernetter, en een foto-initiator, en PAZAM bevattend azide delen omvat, en waarbij de stromingscel ten minste één kanaal voor het ontvangen van de hvdrogeluitgangsstofoplossing omvat, en waarbij het ten minste één kanaal een bovenste binnenvlak en een onderste binnenvlak heeft; het plaatsen van cen fotomasker over het ten minste één kanaal, waarbij het ten minste één kanaal een reeks openingen daarin gevorm heeft; en het belichten van de hydrogel-uitgangsstofoplossing door een gepatroneerd fotomasker met licht bij cen golflengte welke de foto-initiator activeert, en waarbij activatie van de foto-initiator ten minste deels de hydrogel-uitgangsstofoplossmg beneden de openingen in het gepatroneerd fotomasker polymeriseert en driedimensionale hydrogelstructuren vormt welke uitstrekken van het bovenste binnenvlak tot het onderste binnenvlak van het ten minste één kanaal: het reageren van een biotine-PEG-alkyn complex met de azide delen in het PAZAM in de driedimensionale polymeerstructuren gebruik makend van een azide-alkyn klikreactie: het binden van streptavidine aan het biotine in het biotine-PEG-alkyn complex; en het binden van gebiotinyleerde grijp-oligonucleotiden aan het streptavidine, waarbij de gebiotinyleerde grijp-oligonucleotiden specifiek zijn voor doelen van belang in een sequentiebibliotheek..loading a hydrogel precursor solution into a flow cell, wherein the hydrogel precursor solution comprises a monomer, a crosslinker, and a photoinitiator, and PAZAM-containing azide moieties, and wherein the flow cell comprises at least one channel for receiving the hydrogel precursor solution and wherein the at least one channel has an upper inner surface and a lower inner surface; placing a photomask over the at least one channel, the at least one channel having a series of apertures formed therein; and exposing the hydrogel precursor solution through a patterned photomask with light at one wavelength which activates the photoinitiator, and wherein activation of the photoinitiator at least partially polymerizes the precursor hydrogel solution below the openings in the patterned photomask and three-dimensional hydrogel structures forms which extend from the upper inner surface to the lower inner surface of the at least one channel: reacting a biotin-PEG-alkyne complex with the azide moieties in the PAZAM in the three-dimensional polymer structures using an azide-alkyne click reaction: binding from streptavidin to the biotin in the biotin-PEG-alkyne complex; and binding biotinylated grab oligonucleotides to the streptavidin, wherein the biotinylated grab oligonucleotides are specific for targets of interest in a sequence library. 21. Werkwijze volgens conclusie 20, waarbij het monomeer de stof is met de formule I:The method of claim 20, wherein the monomer is the substance of the formula I: I waarbij elke R° onafhankelijk waterstof of (C,.¢) alkyl is.I wherein each R 0 is independently hydrogen or (C 1-6 ) alkyl. 22. Werkwijze volgens één van de conclusies 20 - 21, waarbij de vernetter een stof is met de formule II:A method according to any one of claims 20 to 21, wherein the crosslinker is a substance of the formula II: II waarbij: elke n onafhankelijk een geheel getal van 1 -G is; en elke R! onafhankelijk waterstof of (C,) alkyl is.II wherein: each n is independently an integer from 1 -G; and every R! independently hydrogen or (C 1 ) alkyl. 23. Werkwijze volgens één van de conclusies 20 - 22, waarbij de foto-initiator een diazosulfonaat- initiator; een monoacylfosfineoxide (MAPO) zout; een bisacylfosfineoxide (BAPO) zout: of combinaties daarvan is.The method of any one of claims 20 to 22, wherein the photoinitiator is a diazosulfonate initiator; a monoacylphosphine oxide (MAPO) salt; a bisacylphosphine oxide (BAPO) salt: or combinations thereof. 24. Werkwijze volgens één van de conclusies 20 - 23, waarbij het monomeer acrylamide is, de vernetter N,N'-bis(acryloyl)eystamine (BACy) is, en de foto-initiator lithium feny1-2,4,6- trimethylbenzovlfosfinaat (LAP) is.A method according to any one of claims 20 to 23, wherein the monomer is acrylamide, the crosslinker is N,N'-bis(acryloyl)eystamine (BACy), and the photoinitiator is lithium phenyl1-2,4,6-trimethylbenzoylphosphinate. (LAP). 25. Werkwijze volgens één van de conclusies 20 - 24, waarbij de polymeeruitgangsstofoplossing polyethyleenglycol (PEG)-thiol, PEG-acrylaat, acrylamide, N, N'°-bis(acryloyl)cystamine (BACy), PEG, propyleenglycol (PPO). polyacrylzuur, poly (hvdroxvethylmethacrylaat) (PHEMA), poly(methylmethacrylaat) (PMMA), poly(N-isopropylacrylamide) (PNIPA Am), polymelkzuur (PLA), poly(melkzuur-co-glycolzuur), polycaprolacton (PCL), poly(vinylsulfonzuur) (PV SA), poly(L-asparagine zuur), poly(L-glutaminezuur), polylysine, agar, agarose, alginaat, heparine, alginaatsulfaat, dextransulfaat, hyaluronan, pectine, carrageen, gelatine, chitosan, cellulose, collageen, bisacrylamide, diacrylaat, diallylamine, triallylamine, divinylsulfon, diethyleenglycoldiallylether, ethyleenglycoldiacrylaat, polymethyleenglvcoldiacrylaat, polyethyleenglycoldiacrylaat, trimethvlolpropaantrimethaerylaat, geethoxyleerd trimethyloltriacrylaat, geethoxvleerd pentaerythritoltetraacrylaat, of combinaties daarvan omvat.The method of any one of claims 20 to 24, wherein the polymer precursor solution is polyethylene glycol (PEG) thiol, PEG-acrylate, acrylamide, N,N'°-bis(acryloyl)cystamine (BACy), PEG, propylene glycol (PPO). polyacrylic acid, poly(hydroxethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPA Am), polylactic acid (PLA), poly(lactic-co-glycolic acid), polycaprolactone (PCL), poly( vinylsulfonic acid) (PV SA), poly(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylpropane trimethaerylate, ethoxylated trimethylol triacrylate, ethoxylated combination pentaerythritol, or combination thereof. 26. Werkwijze volgens één van de conclusies 20 - 24, waarbij de polymeeruitgangsstofoplossing polyethyleenglycol (PEG)-thiol / PEG-acrylaat, acrylamide / N, N’-bis(acryloyl)cystamine (BACy), PEG / propyleenglycol (PPO), of combinaties daarvan omvat.The method of any one of claims 20 to 24, wherein the polymer precursor solution is polyethylene glycol (PEG)-thiol/PEG-acrylate, acrylamide/N, N'-bis(acryloyl)cystamine (BACy), PEG/propylene glycol (PPO), or combinations thereof. 27. Werkwijze volgens één van de conclusies 20 - 26, waarbij het fotomasker een polyester film omvat.A method according to any one of claims 20 to 26, wherein the photomask comprises a polyester film. 28. Werkwijze volgens één van de conclusies 20 - 27, waarbij het fotomasker is gelamineerd op het bovenste buitenvlak van de stromingscel.A method according to any one of claims 20 to 27, wherein the photomask is laminated to the upper outer surface of the flow cell. 29. Werkwijze volgens één van de conclusies 20 - 28, waarbij de lichtbron een ultraviolet lichtbron is.A method according to any one of claims 20 to 28, wherein the light source is an ultraviolet light source. 30. Werkwijze volgens één van de conclusies 20 - 29, waarbij de driedimensionale polymeerstructuren cilindrisch zijn.A method according to any one of claims 20 to 29, wherein the three-dimensional polymer structures are cylindrical. 31. Werkwijze voor het vervaardigen van doorstromingscel driedimensionale polymeerstructuren welke functionele vlakken heeft, omvattende: — het laden van een polymeeruitgangsstofoplossing in een stromingscel.31. A method of manufacturing flow cell three-dimensional polymer structures having functional planes, comprising: - loading a polymer precursor solution into a flow cell. — waarbij de polymeeruitgangsstofoplossing een monomeer, een vernetter, een foto-initiator, en een streptavidine-bevattend acrylamide monomeer omvat, en — waarbij de stromingscel ten minste één kanaal voor het ontvangen van de polymeeruitgangsstofoplossing omvat, en waarbij het ten minste één kanaal cen bovenste binnenvlak en een onderste binnenvlak heeft, en waarbij oligonucleotiden van vooraf bepaalde lengten zijn gebonden aan zowel het bovenste en onderste vlak van het ten minste één kanaal; — het plaatsen van een fotomasker over het ten minste één kanaal, waarbij het ten minste één kanaal een reeks openingen daarin gevorm heeft: — het belichten van de polymeeruitgangsstofoplossing door het fotomasker met licht bij een golflengte welke de foto-initiator activeert, en waarbij activatie van de foto-initiator ten minste deels de polvmeeruitgangsstofoplossing beneden de openingen in het fotomasker polymeriseert en driedimensionale polymeerstructuren vormt welke uitstrekken van het bovenste binnenvlak tot het onderste binnenvlak van het ten minste één kanaal: — het selectief binden van gebiotinyleerde grijp -oligonucleotiden aan het streptavidine in de driedimensionale polymeerstructuren, waarbij de gebiotinyleerde grijp-oligonucleotiden specifiek zijn voor doelmoleculen van belang in een bibliotheek en daaraan binden: en — het elueren van de gebonden doelmoleculen en het nederzetten van de gebonden doelmoleculen op de vlakken van de stromingscel hebbende oligonucleotiden gebonden daarop.— wherein the polymer precursor solution comprises a monomer, a crosslinker, a photoinitiator, and a streptavidin-containing acrylamide monomer, and — wherein the flow cell comprises at least one channel for receiving the polymer precursor solution, and wherein the at least one channel in an upper inner surface and a lower inner surface, and wherein oligonucleotides of predetermined lengths are bound to both the upper and lower surfaces of the at least one channel; — placing a photomask over the at least one channel, the at least one channel having a series of apertures formed therein: — exposing the polymer precursor solution through the photomask with light at a wavelength which activates the photoinitiator, and wherein activation of the photoinitiator at least partially polymerizes the polymer precursor solution below the apertures in the photomask and forms three-dimensional polymer structures extending from the upper inner surface to the lower inner surface of the at least one channel: — selectively binding biotinylated grab oligonucleotides to the streptavidin in the three-dimensional polymer structures, wherein the biotinylated grab oligonucleotides are specific for target molecules of interest in a library and bind to them: and — eluting the bound target molecules and depositing the bound target molecules on the faces of the flow cell having bound oligonucleotides den thereon. 32. Werkwijze volgens conclusie 31, waarbij het monomeer de stof is met de formule I:The method of claim 31, wherein the monomer is the substance of the formula I: MN “Re NSMN “Re NS I waarbij elke R° onafhankelijk waterstof of (Cs) alkyl is.I wherein each R 0 is independently hydrogen or (C 5 ) alkyl. 33, Werkwijze volgens één van de conclusies 31 - 32, waarbij de vernetter een stof is met de formule II: So Ri33. A method according to any one of claims 31 to 32, wherein the crosslinker is a substance of the formula II: So Ri II waarbij: elke n onafhankelijk een geheel getal van 1 —61s;en elke R' onafhankelijk waterstof of (C,) alkyl is.II wherein: each n is independently an integer from 1 to 61s; and each R' is independently hydrogen or (C 1 ) alkyl. 34. Werkwijze volgens één van de conclusies 31 - 33, waarbij de foto-initiator een diazosulfonaat- initiator; een monoacylfosfineoxide (MAPO) zout; een bisacylfosfineoxide (BAPO) zout: of combinaties daarvan is.The method of any one of claims 31 to 33, wherein the photoinitiator is a diazosulfonate initiator; a monoacylphosphine oxide (MAPO) salt; a bisacylphosphine oxide (BAPO) salt: or combinations thereof. 35, Werkwijze volgens één van de conclusies 31 - 34, waarbij het monomeer acrylamide is, de vernetter N,N'-bis(acryloyl)cystamine (BACy) is, en de foto-initiator lithium feny1-2,4.6- trimethylbenzoylfosfinaat (LAP) is.The method of any one of claims 31 to 34, wherein the monomer is acrylamide, the crosslinker is N,N'-bis(acryloyl)cystamine (BACy), and the photoinitiator is lithium phenyl1-2,4,6-trimethylbenzoylphosphinate (LAP ). 36. Werkwijze volgens één van de conclusies 31 - 35, waarbij de polymeeruitgangsstofoplossing polyethyleenglycol (PEG)-thiol, PEG-acrylaat, acrylamide, N, N’°-bis(acryloyl)cystamine (BACy), PEG, propyleenglycol (PPO). polyacrylzuur, poly(hydroxyethylmethacrylaat) (PHEMA), poly(methylmethacrylaat) (PMMA), poly(N-isopropylacrylamide) (PNIPA Am), polymelkzuur (PLA), poly(melkzuur-co-glycolzuur), polvcaprolacton (PCL), poly (vinylsulfonzuur) (PVSA), poly(L-asparaginezuur), poly(L-glutaminezuur), polylysine, agar, agarose, alginaat, heparine, alginaatsulfaat, dextransulfaat, hyaluronan, pectine, carrageen, gelatine, chitosan, cellulose, collageen, bisacrylamide, diacrylaat, diallylamine. triallvlamine, divinylsulfon, diethyleenglvcoldiallylether, ethyleenglycoldiacrylaat, polymethyleenglycoldiacrylaat, polyethyleenglycoldiacrylaat, trimethylolpropaantrimethacrylaat, geethoxyleerd trimethyloltriacrylaat, geethoxyleerd pentaerythritoltetraacrylaat, of combinaties daarvan omvat.The method of any one of claims 31 to 35, wherein the polymer precursor solution is polyethylene glycol (PEG) thiol, PEG acrylate, acrylamide, N,N'°-bis(acryloyl)cystamine (BACy), PEG, propylene glycol (PPO). polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPA Am), polylactic acid (PLA), poly(lactic-co-glycolic acid), polycaprolactone (PCL), poly( vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine. triallvlamine, divinyl sulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylolpropane trimethacrylate, ethoxylated trimethylol triacrylate, ethoxylated pentaerythritol tetraacrylate, or combinations thereof. 37, Werkwijze volgens één van de conclusies 31 - 35, waarbij de polymeeruitgangsstofoplossing polyethyleenglycol (PEG)-thiol / PEG-acrvlaat, acrylamide / N, N’°-bis(aeryloyl}cystamine (BA Cy), PEG / propyleenglycol (PPO). of combinaties daarvan omvat.The method of any one of claims 31 to 35, wherein the polymer precursor solution is polyethylene glycol (PEG)-thiol/PEG-acrylate, acrylamide/N, N'°-bis(aeryloyl}cystamine (BA Cy), PEG/propylene glycol (PPO) or combinations thereof. 38. Werkwijze volgens één van de conclusies 31 - 37, waarbij het fotomasker polyethyleentereftalaat is.A method according to any one of claims 31 to 37, wherein the photomask is polyethylene terephthalate. 39. Werkwijze volgens één van de conclusies 31 - 38, waarbij het fotomasker is gelamineerd op het bovenste vlak van de stromingscel.The method of any one of claims 31 to 38, wherein the photomask is laminated to the top face of the flow cell. 40. Werkwijze volgens één van de conclusies 31 - 39, waarbij de lichtbron een ultraviolet lichtbron is.A method according to any one of claims 31 to 39, wherein the light source is an ultraviolet light source. 41. Werkwijze volgens één van de conclusies 31 - 40, waarbij de driedimensionale polymeerstructuren cilindrisch zijn.A method according to any one of claims 31 to 40, wherein the three-dimensional polymer structures are cylindrical. 42. Strommgscel, omvattend: een kanaal, waarbij het kanaal een bovenste binnenvlak hebbende primers bedekt daarop en een onderste binnenvlak hebbende primers bedekt daarop omvattend; en - reversibele, permeabele, driedimensionale polymeerstructuren in het kanaal uit een polymeeruitgangsstofoplossing, waarbij de driedimensionale polymeerstructuren uitstrekken van het bovenste binnenvlak van het kanaal tot het onderste binnenvlak van het kanaal.A flow cell, comprising: a channel, the channel having an upper inner surface covering primers thereon and having a lower inner surface covering primers covered thereon; and - reversible, permeable, three-dimensional polymer structures in the channel from a polymer precursor solution, wherein the three-dimensional polymer structures extend from the upper inner surface of the channel to the lower inner surface of the channel. 43. Stromingscel volgens conclusie 42, aanvullend een fotomasker geplaatst over een buitenste buitenoppervlak van het kanaal omvattend.The flow cell of claim 42, additionally comprising a photomask disposed over an outer outer surface of the channel. 44. Stromingscel volgens één van de conclusies 42 - 43, waarbij de driedimensionale polymeerstructuren hydrogels omvatten.The flow cell of any one of claims 42 to 43, wherein the three-dimensional polymer structures comprise hydrogels. 45. Stromingscel volgens één van de conclusies 42 - 44, waarbij de stromingscel, polymeeruitgangsstofoplossingen en fotomasker verschaft zijn in een uitrusting.The flow cell of any one of claims 42 to 44, wherein the flow cell, polymer precursor solutions and photomask are provided in a kit.