Process for creating three-dimensional polymer networks

Abstract

The present disclosure provides processes for making three-dimensional cross-linked polymer networks having transport channels, processes for making arrays comprising the three-dimensional networks, arrays comprising the three-dimensional networks, and uses of the three-dimensional networks and arrays.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 356,171, filed June 28, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] 2. Background technology WO 2017 / 103128 and WO 2018 / 234253, the entire contents of which are incorporated herein by reference, describe three-dimensional polymer networks comprising cross-linked polymer chains and one or more transport channels. The transport channels allow molecules in solution, such as analyte molecules, to access the polymer molecules within the network. The polymer chains can be cross-linked to probe molecules, and the transport channels provide a larger surface area for binding the analyte to the probe molecules.

[0003] The networks described in WO 2017 / 103128 and WO 2018 / 234253 allow for faster hybridization of a given amount of analyte than networks lacking transport channels, because the transport channels effectively increase the surface area of the network, allowing more probes to be exposed to the sample in a given amount of time. Furthermore, the transport channels reduce or eliminate the problem of sample analytes or other components bound to probes at or near the surface of the network blocking access to probes located inside the network, allowing the networks to bind more analytes than networks without transport channels of the same volume. Another advantage of the networks of WO 2017 / 103128 and WO 2018 / 234253 is that the large analyte loading enabled by the transport channels allows for more sensitive analyte detection than would be possible in networks without transport channels, i.e., a given amount of analyte can be concentrated in a smaller network volume, thereby improving the signal-to-noise ratio compared to networks without transport channels. Yet another advantage of the networks of WO 2017 / 103128 and WO 2018 / 234253 is that the high analyte loading enabled by the transport channels allows for the quantification of a wider range of analyte concentrations compared to networks without transport channels.

[0004] WO 2017 / 103128 and WO 2018 / 234253 describe various processes for creating three-dimensional polymer networks with transport channels. Exemplary processes can include the following steps: (a) exposing a mixture containing an aqueous salt solution, a polymer, and a cross-linking agent to salt crystal-forming conditions, (b) exposing the mixture to cross-linking conditions to cross-link the polymer and form a cross-linked polymer network, and (c) contacting the cross-linked polymer network with a solvent to dissolve the salt crystals and form one or more channels.

[0005] 3. Summary of the Invention The present disclosure provides a new process for producing a three-dimensional network having one or more transport channels.The present disclosure is based in part on the surprising discovery that the uniformity of three-dimensional networks and the arrays comprising them can be improved by using a relatively low concentration of polymer chains and / or salts when producing the network.The present disclosure is also based in part on the surprising discovery that the uniformity of networks and arrays can be improved by using a relatively low cross-linking energy to cross-link polymer chains during the production of the network.

[0006] When relatively high concentrations of polymer chains and / or salts are used in the mixtures used to create three-dimensional networks of the type described in WO 2017 / 103128 and WO 2018 / 234253, it is believed that components may prematurely precipitate from the mixture, reducing the uniformity of arrays containing the three-dimensional networks and adversely affecting their functionality. Furthermore, it is believed that reducing the concentration of polymer chains and / or salts can reduce or avoid premature precipitation, resulting in improved array uniformity and functionality. Furthermore, it is believed that reducing the crosslinking energy can facilitate extraction of non-crosslinked material, resulting in more uniform arrays.

[0007] Thus, in some aspects, the present disclosure provides processes for making three-dimensional networks in which the polymer and / or salt concentrations are lower than those described in WO 2017 / 103128 and WO 2018 / 234253, and / or the cross-linking energy is lower than those described in WO 2017 / 103128 and WO 2018 / 234253. Exemplary processes for making three-dimensional networks are described in Section 5.1 below and in specific embodiments 1-221.

[0008] The present disclosure also provides a process for fabricating an array comprising a plurality of three-dimensional hydrogel networks. Exemplary processes for fabricating the array are described in Section 5.1 below and in specific embodiments 222-230.

[0009] The present disclosure also provides three-dimensional networks, a plurality of three-dimensional networks, and arrays comprising the three-dimensional networks and substrates of the present disclosure, produced by the processes of the present disclosure. Exemplary three-dimensional networks, a plurality of three-dimensional networks, and arrays are described in Sections 5.2 and 5.3 below, and in specific embodiments 231-296.

[0010] The present disclosure also provides processes for detecting and / or measuring analytes in a sample, preferably a liquid sample, using the three-dimensional networks and arrays of the present disclosure. Exemplary processes for using the three-dimensional networks and arrays are described in Sections 5.4 and 5.5 below, and in specific embodiments 297-348.

[0011] The present disclosure also provides kits useful for producing the three-dimensional networks and / or arrays of the present disclosure. Exemplary kits are described in Section 5.6 below and in specific embodiments 349-351. [Brief explanation of the drawings]

[0012] [Figure 1A] Figure 1 shows the hybridization signals and coefficients of variation (%CV) of three-dimensional networks made using different concentrations of phosphate, polymer, and probe, and different cross-linking energies (Example 1). Figure 1A shows the results for probe AllCan1. [Figure 1B] Figure 1B shows the hybridization signals and coefficients of variation (%CV) for three-dimensional networks created using different concentrations of phosphate, polymer, and probe, and different cross-linking energies (Example 1). Figure 1B shows the results for probe Sau-71p. [Figure 2] Figure 2 shows the printing scheme for the three-dimensional networks of Example 2 made using different concentrations of phosphate, polymer, and probe, and different cross-linking energies. Polymer concentrations are shown along the left side of the figure, phosphate concentrations are shown along the top of the figure, and probe concentrations are shown along the bottom of the figure. Rows A and J contained spatial control spots (cc) for orienting the plate. [Figure 3A] FIG. 10 shows a Cy3 fluorescent image of the three-dimensional network of Example 2. [Figure 3B] FIG. 1 shows a Cy5 fluorescent image of the three-dimensional network of Example 2. [Figure 4A] FIG. 1 shows the Cy3 signal intensity of the probe E. coli-1637p measured for the three-dimensional network of Example 2. [Figure 4B] FIG. 1 shows the Cy5 signal intensity of the probe E. coli-1637p measured for the three-dimensional network of Example 2. [Figure 5A] Figure 1 shows the coefficient of variation (%CV) using Cy3 fluorescence for the three-dimensional networks of Example 2. Data shown is for the probe E. coli-1637p. [Figure 5B] Figure 1 shows the coefficient of variation (%CV) using Cy5 fluorescence for the three-dimensional networks of Example 2. Data shown is for the probe E. coli-1637p. [Figure 6A] FIG. 1 shows the Cy3 signal intensity of the probe Entb-132p measured for the three-dimensional network of Example 2. [Figure 6B] FIG. 1 shows the Cy5 signal intensity of the probe Entb-132p measured for the three-dimensional network of Example 2. [Figure 7A] Figure 1 shows the coefficient of variation (%CV) using Cy3 fluorescence for the three-dimensional networks of Example 2. Data shown is for the probe Ent-132p. [Figure 7B]Figure 1 shows the coefficient of variation (%CV) using Cy5 fluorescence for the three-dimensional networks of Example 2. Data shown is for the probe Ent-132p.

[0013] 5. MODE FOR CARRYING OUT THE INVENTION 5.1. Processes for Fabricating Three-Dimensional Polymer Networks and Arrays In one aspect, the disclosed process for making a three-dimensional polymer network includes: (a) exposing a mixture comprising an aqueous salt solution, a polymer, a cross-linking agent, and, optionally, one or more probes to salt crystal-forming conditions; (b) exposing the mixture to cross-linking conditions to cross-link the polymer and form a cross-linked polymer network; and (c) contacting the cross-linked polymer network with a solvent to dissolve the salt crystals and form one or more transport channels.

[0014] The concentration of polymer chains in the mixture can be selected so that precipitation of the polymer chains from the mixture does not occur before the formation of salt crystals (e.g., as observed by visual inspection, e.g., via a microscope or digital image). For example, if precipitation of the polymer is observed before the formation of salt crystals during step (a), the concentration of the polymer can be reduced until precipitation is no longer observed before the formation of salt crystals. Alternatively or additionally, the salt concentration can be adjusted to reduce or avoid premature precipitation of the polymer. In some embodiments, the concentrations of the polymer and salt are selected so that the polymer and salt co-precipitate during step (a).

[0015] The process can further include forming a mixture by combining the aqueous salt solution, polymer, cross-linking agent, and optionally one or more probes, and / or can further include applying the mixture to a substrate (e.g., a substrate described in Section 5.3) before exposing the mixture to salt crystal-forming conditions. If the polymer being used has a pre-attached cross-linking agent (e.g., when using a copolymer polymerized from monomers that include a cross-linking agent), forming the mixture can include combining the aqueous salt solution with the polymer and optionally one or more probes.

[0016] The mixture can be applied to the substrate, for example, by spraying the mixture onto the surface of the substrate (e.g., at 1024 sites on the surface) before exposing the mixture to salt crystal-forming conditions. The mixture can be applied to the surface, for example, using a DNA chip spotter or an inkjet printer. In a preferred embodiment, the mixture is sprayed using an inkjet printer. This allows the mixture to be easily and quickly applied to multiple spots on the substrate. The spots can be arranged, for example, in the form of a matrix of several rows and / or columns. Preferably, the salt content in the mixture during printing is below its solubility limit so that the mixture does not crystallize in the print head of the printer. The volume of mixture applied to each spot can be, for example, 100 pl, 200 pl, 300 pl, 400 pl, 500 pl, 750 pl, 1 nl, 2 nl, 3 nl, 4 nl, or 5 nl, or can be selected from a range bounded by any two of the aforementioned values (e.g., 100 pl to 5 nl, 100 pl to 1 nl, 300 pl to 1 nl, 200 pl to 750 nl, 100 pl to 500 pl, 200 pl to 2 nl, 500 pl to 2 nl, 1 nl to 2 nl, etc.). In a preferred embodiment, the spot volume is 200 pl to 4 nl.

[0017] The diameter of each spot depends on the composition of the mixture, the volume of the mixture applied, and the surface chemistry of the substrate. Spot diameters typically range from 80 μm to 1000 μm and can be obtained by varying the parameters described above. In various embodiments, the spot diameter is 80 μm, 100 μm, 120 μm, 140 μm, 160 μm, 180 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1000 μm, or a range bounded by any two of the preceding embodiments. For example, the diameter is selected from 80 μm to 200 μm, 100 μm to 120 μm, 120 μm to 140 μm, 120 μm to 180 μm, 140 μm to 160 μm, 160 μm to 180 μm, 180 μm to 200 μm, 120 μm to 200 μm, 100 μm to 400 μm, 160 μm to 600 μm, or 120 μm to 700 μm, etc. In a preferred embodiment, the diameter is in the range of 100 μm to 200 μm or a subrange thereof.

[0018] Polymers, crosslinkers, salts, and probes that can be used to create the networks are described in Sections 5.1.1, 5.1.2, 5.1.3, and 5.1.4, respectively. Suitable salt crystal formation conditions are described in Section 5.1.5. Suitable crosslinking conditions are described in Section 5.1.6. Suitable solvents for dissolving salt crystals are described in Section 5.1.7.

[0019] In some embodiments, the polymer used in the process has at least one crosslinker group per polymer molecule. In preferred embodiments, the polymer has at least two crosslinker groups per molecule. In particularly preferred embodiments, the polymer has at least two photoreactive crosslinker groups per molecule. In these embodiments, separate polymer and crosslinker molecules are not required.

[0020] Polymers The three-dimensional networks of the present disclosure can comprise crosslinked homopolymers, copolymers, mixtures of homopolymers, mixtures of copolymers, or mixtures of one or more homopolymers with one or more copolymers. As used herein, the term "polymer" includes both homopolymers and / or copolymers. As used herein, the term "copolymer" includes polymers polymerized from two or more types of monomers (e.g., bipolymers, terpolymers, quaterpolymers, etc.). Copolymers include alternating copolymers, periodic copolymers, statistical copolymers, random copolymers, block copolymers, linear copolymers, and branched copolymers. The three-dimensional networks of the present disclosure can comprise any combination of the aforementioned types of polymers. Reagents and methods for making such polymers are known in the art (e.g., Ravve, A., Principles of Polymer Chemistry, Springer Science+Business Media, 1995; Cowie, J.M.G., Polymers: Chemistry & Physics of Modern Materials, 2000). nd Edition,Chapman&Hall,1991;Chanda,M.,Introduction to Polymer Science and Chemistry:A Problem-Solving Approach,2 nd Edition,CRC Press,2013;Nicholson,JW,The Chemistry of Polymers,4 th Edition, RSC Publishing, 2012; the contents of each of which are incorporated herein by reference in their entirety).

[0021] Preferred polymers are hydrophilic and / or contain hydrophilic groups. The polymers used in the process of the present disclosure are preferably water-soluble. Thus, in some embodiments, the polymer comprises a water-soluble polymer chain. In one embodiment, the polymer is a copolymer polymerized from two or more monomers selected to provide a desired level of water solubility. For example, the water solubility of the copolymer can be controlled by changing the amount of input monomer, such as sodium 4-vinyl sulfonate, used to make the copolymer.

[0022] When crosslinked, water-soluble polymers form water-swellable gels or hydrogels. Hydrogels absorb aqueous solutions through hydrogen bonding with water molecules. The total absorbency and swelling capacity of hydrogels can be controlled by the type and degree of crosslinking agent used to create the gel. Low-crosslink density polymers generally have a higher absorption capacity and swell to a greater extent than high-crosslink density polymers, but the gel strength of high-crosslink density polymers is stronger and they can maintain their network shape even under moderate pressure.

[0023] The ability of a hydrogel to absorb water is a factor of the ionic concentration of the aqueous solution. In certain embodiments, the hydrogel of the present disclosure can absorb up to 50 times its weight in deionized distilled water (5-50 times its own volume) and up to 30 times its weight in saline (4-30 times its own volume). The reduced absorbency in saline is due to the presence of valent cations, which interfere with the polymer's ability to bind water molecules.

[0024] The three-dimensional network of the present disclosure can include copolymers polymerized from one, two, three, or more than three types of monomers, where one, two, three, or more types of monomers have polymerizable groups independently selected from acrylate groups (e.g., acrylate, methacrylate, methyl methacrylate, hydroxyethyl methacrylate, ethyl acrylate, 2-phenyl acrylate), acrylamide groups (e.g., acrylamide, methacrylamide, dimethyl acrylamide, ethyl acrylamide), itaconate groups (e.g., itaconate, 4-methyl itaconate, dimethyl itaconate), and styrene groups (e.g., styrene, 4-methyl styrene, 4-ethoxy styrene). Preferred polymerizable groups are acrylate, methacrylate, ethacrylate, 2-phenyl acrylate, acrylamide, methacrylamide, itaconate, and styrene. In some embodiments, one of the monomers used to prepare the copolymer, such as sodium 4-vinylbenzenesulfonate, is incorporated.

[0025] The polymers used to create the networks of the present disclosure can contain at least one, at least two, or more than two crosslinker groups per molecule. Crosslinker groups are groups that covalently bond the polymer molecules of the network to each other and, optionally, to the probe and / or substrate. Copolymers polymerized from two or more monomers (e.g., monomers having polymerizable groups independently selected from those described in the preceding paragraph), at least one of which contains a crosslinker, are suitable for creating the three-dimensional networks of the present disclosure. Exemplary crosslinkers are described in Section 5.1.2. A preferred monomer containing a crosslinker is methacryloyloxybenzophenone (MABP) (see Figure 7 of WO 2018 / 234253).

[0026] In preferred embodiments, the copolymer is a bipolymer or terpolymer containing a crosslinker. In a particularly preferred embodiment, the copolymer comprises p(dimethylacrylamide-co-methacryloyl-benzophenone-co-sodium 4-vinylbenzenesulfonate) (see Figure 7 of WO 2018 / 234253). In some embodiments, the copolymer comprises dimethylacrylamide (DMAA), methacryloyloxybenzophenone (MABP), and sodium 4-vinylbenzenesulfonate (SSNa), with the polymer comprising 2.5-7.5 mol% MABP (e.g., 2.5-5 mol%, 3-6 mol%, or 4-7.5 mol%), 2-5 mol% SSNa (e.g., 2-3 mol%, 2-4 mol%, or 3-5 mol%), and the remainder DMAA. In some embodiments, the polymer comprises DMAA, MABP, and SSNa in a 92.5:5:2.5 molar ratio.

[0027] Polymers of various molecular weights can be used in the processes for making three-dimensional networks described herein. For example, in some embodiments, the average molecular weight of the polymer can be in the range of 100 kDa to 600 kDa, e.g., 100 kDa to 500 kDa, 100 kDa to 400 kDa, 100 kDa to 300 kDa, 100 kDa to 200 kDa, 200 kDa to 600 kDa, 200 kDa to 500 kDa, 200 kDa to 400 kDa, 200 kDa to 300 kDa, 300 kDa to 600 kDa, 300 kDa to 500 kDa, 300 kDa to 400 kDa, 400 kDa to 600 kDa, 400 kDa to 500 kDa, or 500 kDa to 600 kDa. In some embodiments, the average molecular weight of the polymer is 300 kDa. Unless the context requires otherwise, the term "average molecular weight" as used herein refers to the weight average molecular weight.

[0028] In some embodiments, the concentration of the polymer (e.g., a copolymer of DMAA, MABP, and SSNa) in the mixture is less than 1 mg / ml. For example, the concentration of the polymer in some embodiments is between 0.01 mg / ml and less than 1 mg / ml, e.g., between 0.01 mg / ml and 0.5 mg / ml, between 0.01 mg / ml and 0.4 mg / ml, between 0.01 mg / ml and 0.3 mg / ml, between 0.01 mg / ml and 0.2 mg / ml, between 0.01 mg / ml and 0.1 mg / ml, between 0.05 mg / ml and 1 mg / ml, or between 0.05 mg / ml and 0.5 mg / ml. The concentration of the polymer can range from 0.05 mg / ml to 0.4 mg / ml, 0.05 mg / ml to 0.3 mg / ml, 0.05 mg / ml to 0.2 mg / ml, 0.05 mg / ml to 0.1 mg / ml, 0.1 mg / ml to 1 mg / ml, 0.1 mg / ml to 0.5 mg / ml, 0.1 mg / ml to 0.4 mg / ml, 0.1 mg / ml to 0.3 mg / ml, or 0.1 mg / ml to 0.2 mg / ml. In some embodiments, the concentration of the polymer is 0.1 mg / ml. A relatively low concentration of polymer can reduce or eliminate premature precipitation of the polymer during production of the three-dimensional polymer network.

[0029] 5.1.2. Crosslinking Agents Cross-linking reagents (or cross-linking agents) suitable for creating cross-links in the three-dimensional network include those activated by ultraviolet light (e.g., short-wave or long-wave UV light), visible light, and heat. Exemplary cross-linking agents activated by UV light include benzophenone, thioxanthone (e.g., thioxanthen-9-one, 10-methylphenothiazine), and benzoin ethers (e.g., benzoin methyl ether, benzoin ethyl ether). Exemplary cross-linking agents activated by visible light include ethyl eosin, eosin Y, rose bengal, camphorquinone, and erythrodin. Exemplary cross-linking agents activated by heat include 4,4'-azobis(4-cyanopentanoic) acid, 2,2-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, and benzoyl peroxide. Other cross-linking agents known in the art, such as those capable of forming radicals or other reactive groups upon irradiation, may also be used.

[0030] In a preferred embodiment, the crosslinker moiety comprises a benzophenone moiety.

[0031] Salt The polymer networks of the present disclosure are characterized by transport channels that arise when the polymer is crosslinked in a mixture containing salt crystals formed from an aqueous solution containing one or more types of salt, such as one, two, or at least two types of salt. In some embodiments, the three-dimensional networks of the present disclosure are fabricated using salt-forming needle-shaped salt crystals (e.g., as described in WO 2017 / 103128). In other embodiments, the three-dimensional networks of the present disclosure are fabricated using salt-forming needle-shaped salt crystals and salt-forming cubic or rod-shaped crystals (e.g., as described in WO 2018 / 234253).

[0032] The one or more salts are preferably selected for their compatibility with the one or more probes. Ideally, each salt has one or more of the following characteristics: (i) the salt is not toxic to the probe (e.g., the salt does not denature the probe), (ii) the salt does not chemically react with the probe, (iii) the salt does not attack fluorophores, such as cyanine dyes, suitable for optically marking the probe, and / or (iv) the salt does not react with the analyte, detection molecule, and / or binding partner bound thereto. Preferably, at least one of the salts forms needle-like crystals.

[0033] In some embodiments, the salt solution comprises a monovalent cation. + Cations and phosphate ions PO4 3- It may contain disodium hydrogen phosphate and / or sodium dihydrogen phosphate, which releases sodium phosphate. Sodium phosphate dissolves readily in water and forms colorless crystals.

[0034] In some embodiments, the mixture includes dipotassium hydrogen phosphate (KHPO) and / or potassium dihydrogen phosphate (KHPO). These salts are highly soluble in water and therefore can form a correspondingly large number of needle-shaped salt crystals in the mixture.

[0035] In preferred embodiments, the aqueous salt solution contains phosphate ions (e.g., a sodium phosphate solution) at a concentration of less than 125 mM to 350 mM, e.g., 125 mM to 340 mM, 125 mM to 250 mM, 150 mM to 340 mM, 150 mM to 300 mM, 150 mM to 250 mM, 200 mM to 340 mM, 200 mM to 300 mM, 200 mM to 250 mM, 225 mM to 340 mM, 225 mM to 300 mM, or 225 mM to 250 mM. In some embodiments, the concentration of phosphate in the aqueous salt solution is 250 mM.

[0036] In some embodiments, the aqueous salt solution contains a single type of monovalent cation, for example, sodium or potassium cations.

[0037] In other embodiments, the aqueous salt solution contains at least two types of monovalent cations, such as two types of alkali metal cations. Alkali metal cations that can be used include sodium and potassium cations, although other alkali metal cations, such as lithium cations, can also be used.

[0038] In some embodiments, the aqueous salt solution contains sodium and potassium cations and / or has a total monovalent cation concentration such that, when combined with the polymer solution and optional probe solution (before crosslinking), the resulting mixture has a total monovalent cation concentration of at least 500 mM. In certain embodiments, the sodium ion concentration in the mixture is at least 250 mM, can range from 250 mM to 500 mM, and more preferably is in the range of 300 mM to 400 mM. In certain embodiments, the sodium ion concentration in the mixture is 350 mM. In some embodiments, the potassium ion concentration in the mixture is preferably at least 150 mM, preferably is in the range of 150 mM to 500 mM, more preferably is in the range of 200 mM to 400 mM, and even more preferably is in the range of 250 mM to 350 mM.

[0039] In some embodiments, the aqueous salt solution can be a sodium phosphate buffer containing both disodium hydrogen phosphate and sodium dihydrogen phosphate, optionally supplemented with dipotassium hydrogen phosphate (KHPO) and / or potassium dihydrogen phosphate (KHPO). In one embodiment, a sodium phosphate buffer containing both disodium hydrogen phosphate and sodium dihydrogen phosphate and a potassium phosphate buffer containing both dipotassium hydrogen phosphate and potassium dihydrogen phosphate are made separately and combined into a single aqueous solution before or after mixing with the polymer and / or probe solution.

[0040] In general, the aqueous salt solution preferably has a pH in the range of 6 to 9, more preferably in the range of 7 to 8.5. In certain exemplary embodiments, the pH is 7.5, 8, or 8.5, most preferably 8.

[0041] For networks containing protein-based probe biomolecules, the aqueous salt solution can include phosphate buffered saline ("PBS") and / or monovalent sulfate cations.

[0042] Probe Probes that can be immobilized on the networks of the present disclosure include biomolecules and molecules that bind to biomolecules, such as complementary binding partners (receptors / ligands) that interact specifically with the system. For example, probes can include nucleic acids and their derivatives (e.g., RNA, DNA, locked nucleic acids (LNA), and peptide nucleic acids (PNA)), proteins, peptides, polypeptides and their derivatives (e.g., glucosamine, antibodies, antibody fragments, and enzymes), lipids (e.g., phospholipids, fatty acids such as arachidonic acid, monoglycerides, diglycerides, and triglycerides), carbohydrates, enzyme inhibitors, enzyme substrates, antigens, and epitopes. Probes can also include larger complex structures such as liposomes, membranes and membrane fragments, cells, cell lysates, cell fragments, spores, and microorganisms.

[0043] Specific interacting systems of complementary binding partners can be based, for example, on nucleic acid-complementary nucleic acid interactions, PNA-nucleic acid interactions, or enzyme / substrate, receptor / ligand, lectin / sugar, antibody / antigen, avidin / biotin or streptavidin / biotin interactions.

[0044] The nucleic acid probe can be DNA or RNA, such as an oligonucleotide or aptamer, LNA, PNA, or DNA containing a methacryl group at the 5' end (5' Acrydite™). The oligonucleotide probe can be, for example, 12 to 30, 14 to 30, 14 to 25, 14 to 20, 15 to 30, 15 to 25, 15 to 20, 16 to 30, 16 to 25, 16 to 20, 15 to 40, 15 to 45, 15 to 50, 15 to 60, 20 to 55, 18 to 60, 20 to 50, 30 to 90, 20 to 100, 20 to 60, 40 to 80, 40 to 100, 20 to 120, 20 to 40, 40 to 60, 60 to 80, 80 to 100, 100 to 120, or 12 to 150 nucleotides in length. In a preferred embodiment, the oligonucleotide probe is 15 to 60 nucleotides in length.

[0045] When using a nucleic acid probe, all or only a part of the probe can be complementary to the target sequence.The part of the probe that is complementary to the target sequence is preferably at least 12 nucleotides long, more preferably at least 15, at least 18, or at least 20 nucleotides long.For nucleic acid probes longer than 40 or 50 nucleotides, the part of the probe that is complementary to the target sequence can be at least 25, at least 30, or at least 35 nucleotides long.When using modified nucleic acid probes such as LNA or PNA, the part of the probe that is complementary to the target sequence can in some embodiments be shorter than 12 nucleotides, because these modified molecules have increased binding energy to their complementary nucleic acids.

[0046] The antibody can be, for example, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, or an antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof, a fusion protein comprising the antibody, and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site, including, but not limited to, single-chain (scFv) and domain antibodies (e.g., human, camelid, and shark domain antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, vNARs, and bis-scFvs (see, for example, Hollinger and Hudson, 2005, Nature Biotech 23:1126-1136). Antibodies include antibodies of any class, such as IgG, IgA, or IgM (or subclass thereof), and antibodies do not have to be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. "Antibody" also encompasses any of each of the foregoing antibody / immunoglobulin types.

[0047] The three-dimensional networks of the present disclosure can include a single species of probe or two or more species of probes (e.g., two, three, four, or five or more species). The three-dimensional networks can include more than one probe to the same target (e.g., antibodies that bind to different epitopes of the same target) and / or can include probes that bind to multiple targets.

[0048] The network can include, for example, labeled (eg, fluorescently labeled) control probe molecules that can be used to measure the amount of probe present in the network.

[0049] The probes can be distributed throughout the network (e.g., on the surface and inside the network). Preferably, at least one probe is spaced from the surface of the network and adjacent to at least one transport channel. The probes thus located can directly access the analyte molecule or analyte component through the transport channel. In some embodiments, the majority of the probes are located inside the network.

[0050] One or more probes can be immobilized on the network covalently or non-covalently.For example, the probe can be cross-linked to a cross-linking polymer, or the probe can be non-covalently bound to the network (for example, by binding to a molecule that is covalently bound to the network).In preferred embodiments, one or more probes are cross-linked to a cross-linking polymer.In some embodiments, most of the probes are covalently bound to the interior of the network (for example, so that at least a portion of the probes are adjacent to the transport channel).

[0051] Without being bound by theory, the inventors believe that the processes described herein for producing three-dimensional networks in the presence of salt crystals (particularly phosphate crystals) may result in a higher concentration of probe molecules at or near the interface between the polymer and the transport channel due to electrostatic interactions between the probe molecules (particularly nucleic acid probe molecules) and the salt crystals. Thus, in some embodiments of the present invention, the present disclosure provides networks according to the present disclosure in which the probe density is greater at the interface between the polymer and the transport channel than in regions of the polymer not adjacent to the transport channel. In various embodiments, the probe density is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher at the interface between the polymer and the transport channel than in regions of the polymer not adjacent to the transport channel.

[0052] The density of the probe molecules in the network can be verified using the following procedure.

[0053] The network is contacted with an aqueous liquid at room temperature, for example, in a bowl. The liquid contains multiple nanoparticles attached to sites that interact with the probe molecules in the network, such as streptavidin if the probe molecules are biotinylated. The size of the nanoparticles is smaller than the mesh size of the network and smaller than the smallest cross section of at least one type of transport channel in the network to allow the nanoparticles to be distributed throughout the polymer. Suitable nanoparticles are quantum dots with a diameter of 2 to 5 nanometers.

[0054] The incubation period is selected so that the network in the liquid is fully hydrated, i.e., so that the network absorbs, on average, the same amount of water as it releases. The incubation period can be, for example, 1 hour. The penetration of the nanoparticles into the network can be accelerated by moving the network and / or the liquid during incubation, for example, by vibrating the network and / or the liquid, preferably by ultrasound.

[0055] After the incubation is complete, the liquid is separated from the network, for example, by draining the liquid from the bowl or removing the network from the bowl.

[0056] The hydrated network is then frozen, for example, with liquid nitrogen. The frozen network can then be cut into thin slices along parallel cutting planes using a cryomicrotome. The cutting planes are arranged transverse to the longitudinal extension of the transport channel and penetrate the transport channel. Cutting is preferably performed using a liquid nitrogen-cooled diamond blade. The slice thickness can be, for example, about 100 nm or 200 nm.

[0057] Using a microscope, the nanoparticles located within the disks obtained by cutting the frozen network are localized. If necessary, the nanoparticles can be fluorescently and optically enhanced to better distinguish them from the network. Nanoparticle localization can be performed using appropriate software with image processing methods. To examine the disks, a confocal microscope, laser scanning microscope, or electron microscope equipped with fluorescence optics is preferably used.

[0058] The nanoparticle geometry and / or positional information thus obtained can be used to create, with the aid of a computer, a three-dimensional geometric model of the nanoparticle distribution within the network. This model can then be used to determine whether the nanoparticle distribution reflects a higher density of probe molecules near the site of the transport channel.

[0059] When the mixture used to produce the three-dimensional network includes oligonucleotide probe molecules, the concentration of the probe molecules in some embodiments ranges from 5 μM to 35 μM, e.g., 15 μM to 25 μM, 5 μM, 20 μM, or 35 μM, or any range bounded by any two of the foregoing values.

[0060] 5.1.5. Salt crystal formation conditions The salt crystal-forming conditions can include dehydrating or cooling the mixture until the relative salt content in the mixture increases beyond the solubility limit, meaning the mixture becomes supersaturated with salt. This promotes the formation of salt crystals from crystal buds located within the mixture volume toward the surface of the mixture. Without being bound by theory, it is believed that the use of an aqueous solution containing at least two different monovalent metal ions results in the formation of at least two different types of salt crystals.

[0061] The mixture can be dehydrated by heating the mixture, exposing the mixture to a vacuum, and / or reducing the humidity of the atmosphere surrounding the mixture.

[0062] The mixture can be heated by placing the mixture on a heated substrate or surface (e.g., about 50°C to about 70°C), by heating the substrate or surface on which the mixture is placed (e.g., about 50°C to about 70°C), and / or by contacting the mixture with hot gas (e.g., air, nitrogen, or carbon dioxide having a temperature higher than that of the mixture) so that water evaporates from the mixture. Contact with hot gas can be achieved, for example, by placing the mixture in a heated oven. During transport to the heated oven, the mixture can be maintained at a relative humidity of 40% or more, e.g., approximately 60%, although higher relative humidities, even as high as 75% or higher, are feasible. Mixtures with higher potassium ion concentrations can tolerate lower relative humidities, and mixtures with lower potassium salt concentrations are preferably maintained at higher relative humidities during transport.

[0063] The mixture can also be heated to activate the heat-activatable crosslinking agent, if present, thereby crosslinking the polymer.

[0064] In some embodiments, the temperature of the heated substrate and / or the air used to dehydrate the mixture is at least 20°C higher than the temperature of the mixture before heating the mixture, but less than 100°C.

[0065] The mixture can be cooled by placing the mixture on a cooled substrate or surface (e.g., about 5°C to about 15°C), by cooling the substrate or surface on which the mixture is placed (e.g., about 5°C to about 15°C), and / or by contacting it with a cryogenic gas (e.g., air, nitrogen, or carbon dioxide having a temperature lower than that of the mixture). Upon cooling, the temperature-dependent solubility limit of the salt in the mixture decreases until the mixture is eventually supersaturated with the salt. In some embodiments, the mixture is cooled by incubating it in a low-humidity (e.g., temperature between 0°C and 10°C, relative humidity <40%) cryogenic chamber.

[0066] The temperature in the mixture is preferably maintained above the dew point of the ambient air surrounding the mixture during the formation of one or more salt crystals, thereby preventing the mixture from being diluted with water condensed from the ambient air, which may result in a decrease in the relative salt content in the mixture.

[0067] 5.1.6.Crosslinking conditions Crosslinking conditions can be selected based on the type of crosslinker used. For example, when using a crosslinker activated by ultraviolet light (e.g., benzophenone, thioxanthone, or benzoin ether), the crosslinking conditions can include exposing the mixture to ultraviolet (UV) light. In some embodiments, UV light having a wavelength of about 250 nm to about 360 nm is used (e.g., 260±20 nm or 355±20 nm). The use of lower energy / longer wavelength UV light (e.g., 360 nm UV light vs. 254 nm UV light) may require longer exposure times. When using a crosslinker activated by visible light (e.g., ethyl eosin, eosin Y, rose bengal, camphorquinone, or erythridine), the crosslinking conditions can include exposing the mixture to visible light. When using a heat-activated crosslinker (e.g., 4,4'-azobis(4-cyanopentanoic) acid and 2,2-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, or benzoyl peroxide), the crosslinking conditions can include exposing the mixture to heat.

[0068] The length and strength of the crosslinking conditions can be selected to result in crosslinking of polymer molecules to other polymer molecules, crosslinking of polymer molecules to probe molecules (if present), and crosslinking of polymer molecules to substrate molecules or organic molecules present on the substrate (if present). The length and strength of the crosslinking conditions for the probe-containing mixture can be determined experimentally, for example, to balance the robustness and inherent nature of the immobilization of the probe molecules.

[0069] In some embodiments, when UV crosslinking is used (e.g., crosslinking with 254 nm UV light), the dose of crosslinking energy is 1 J / cm 2For example, in some embodiments, the crosslinking energy is less than 0.4 J / cm 2 ~0.9J / cm 2 , 0.5J / cm 2 ~0.9J / cm 2 , 0.6J / cm 2 ~0.9J / cm 2 , 0.7J / cm 2 ~0.9J / cm 2 , 0.4J / cm 2 ~0.8J / cm 2 , 0.5J / cm 2 ~0.8J / cm 2 , 0.6J / cm 2 ~0.8J / cm 2 , 0.7J / cm 2 ~0.8J / cm 2 , 0.4J / cm 2 ~0.7J / cm 2 , 0.5J / cm 2 ~0.7J / cm 2 , 0.6J / cm 2 ~0.7J / cm 2 , 0.4J / cm 2 ~0.6J / cm 2 , 0.5J / cm 2 ~0.6J / cm 2 , or 0.4 J / cm 2 ~0.5J / cm 2 In some embodiments, 0.7 J / cm 2 of UV energy dose is used.

[0070] 5.1.7. Dissolution of salt crystals After cross-linking the polymer, salt crystals can be dissolved in a solvent such that at least one transport channel is formed within the network.

[0071] When the crystals contain needle-shaped salt crystals, they can form long, narrow channels extending from the surface and / or near the surface of the network to the interior of the network. Without being bound by theory, it is believed that the use of two types of monovalent salt cations during crystal formation results in at least two types of crystals: small crystals and needle-shaped crystals. It is believed that dissolution of the small crystals results in short channels perforated by long channels resulting from dissolution of the needle-shaped crystals, creating a sponge-like effect in the network.

[0072] When arrays produced by the disclosed methods are used as biological sensors, high measurement accuracy and high measurement dynamics are permitted.

[0073] The solvent for dissolving one or more salt crystals can be selected to be compatible with the polymer and probe (if present) (e.g., the solvent can be selected so as not to dissolve the polymer and probe). Preferably, the solvent used is an aqueous buffer solution such as dilute phosphate buffer. Methanol, ethanol, propanol, or a mixture of these liquids can be added to the buffer solution to facilitate the removal of unbound polymer from the network.

[0074] After removal of the salt crystals, the network can be collapsed by drying and rehydrated, which is beneficial for transport and stabilization of the probe biomolecules.

[0075] 5.2. Three-dimensional polymer networks and multiple three-dimensional polymer networks In one aspect, the present disclosure provides a three-dimensional polymer network produced by the process described herein. The three-dimensional network comprises a cross-linked polymer, one or more transport channels, and can optionally further comprise one or more probes immobilized on the network, for example, by cross-linking to the polymer chains. Probes that can be immobilized on the network are described in Section 5.1.4.

[0076] The networks of the present disclosure can have a mesh size (measured in the hydration state of the network) of, for example, 5 to 75 nm (e.g., 10 to 20 nm, 10 to 30 nm, 10 to 40 nm, 10 to 50 nm, 20 to 30 nm, 20 to 40 nm, 20 to 50 nm, 30 to 40 nm, 30 to 50 nm, or 40 to 50 nm). By "hydration state of the network" is meant that the network is in equilibrium with respect to water absorption, i.e., it absorbs the same amount of water in aqueous solution as it releases.

[0077] The transport channels can provide access to the interior of the network, and although the transport channels can have a relatively large cross section, the mesh size of the network can be significantly smaller than the transport channel cross section, so that the network remains mechanically stable.

[0078] The transport channels can form a kind of highway through which analytes can rapidly move in and out of the interior of the network. Transport of analytes can occur within the transport channels by diffusion and / or convection.

[0079] Transport channels are formed when a network is formed by cross-linking polymer chains in the presence of salt crystals, as described in Section 5.1. After washing away the salt crystals, the transport channels remain.

[0080] The three-dimensional network of the present disclosure can include one or more types of transport channels. When the salt crystals formed in the process for creating the three-dimensional hydrogel network described herein are washed away, transport channels are left behind, according to the principle of "lost" morphology. The transport channels allow analytes to penetrate the interior of the network and specifically bind to probes located within the network. Furthermore, the transport channels allow unbound analytes to remain within the network after washing, reducing the amount of nonspecific signal from "clogged" analytes within the network.

[0081] One type of transport channel that can be present in the three-dimensional networks of the present disclosure is considered to be a long channel created from needle-shaped salt crystals. As used herein, a "long channel" is an elongated passage within the network that (1) is substantially straight and (2) has, in the hydrated state of the network, a minimum cross-section of at least 300 nm and a length at least three times, preferably five times, and more preferably at least ten times the minimum cross-section of the passage. For example, the length of a long channel can be 3 to 15 times, 5 to 10 times, or 10 to 15 times the minimum cross-section of the long channel. A "substantially straight" long channel is a channel that extends from a nucleation point in one direction without changing direction by more than 45 degrees in any direction (i.e., X, Y, or Z). Because long channels arise from needle-shaped crystals formed from a common nucleation point, a network of the present disclosure can include a group of long channels (e.g., 5, 10, or more) that converge at a point located within the network corresponding to the original nucleation point of crystallization. The long channels are typically arranged such that the lateral distance between the long channels decreases from the surface toward the interior of the network.

[0082] Another type of transport channel that can be present in the three-dimensional networks of the present disclosure is considered to be a short channel formed, for example, from cubic or rod-shaped crystals. As used herein, a "short channel" is a passage in a network that (1) is substantially straight and (2) has a minimum cross-section, in the hydrated state of the network, that is preferably at least 10 times the mesh size of the network and a length that is less than 3 times the minimum cross-section of the passage (e.g., 1x to 2.75x, 1x to 2.5x, 1x to 2x, or 1x to 1.5x). A "substantially straight" short channel is a channel that extends from the nucleation point in one direction without changing direction by more than 45 degrees in any direction (i.e., X, Y, or Z). To maintain network strength, the short channel preferably has a cross-section that is no more than one-twentieth of the network width or diameter. For example, for a network in the form of "spots" on a 200 μm diameter array, the cross-section of the short channel is preferably no more than 10 μm, and for spots on a 100 μm diameter array, the cross-section of the short channel is preferably no more than 5 μm. In certain aspects, the cross-sections of the short channels are about 20 nm or more, about 50 nm or more, about 100 nm or more, about 250 nm or more, at least 500 nm or more, or about 1 μm or more. The short channels within the network can have approximately the same diameter (e.g., + / - 10% or + / - 25%) or different diameters. In certain embodiments, the short channels within the network have diameters ranging between any two of the aforementioned dimensions, such as 100 nm to 10 μm, 50 nm to 1 μm, 500 nm to 5 μm, 250 nm to 10 μm, etc.

[0083] Without wishing to be bound by theory, the inventors believe that if the mixture used to create the network includes an aqueous salt solution with components capable of forming different metal ion-salt ion pairings, a sponge polymer can be made with short channels interpenetrated by long channels.

[0084] In some embodiments, the three-dimensional networks of the present disclosure comprise long and short channels, while in other embodiments, the three-dimensional networks of the present disclosure comprise only long channels (e.g., when a single salt is included in the aqueous salt solution).

[0085] In another aspect, the present disclosure provides a plurality (e.g., a plurality of 2 or more, 5 or more, 10 or more, or 20 or more, and / or up to 50, up to 100, or up to 1000) of three-dimensional polymer networks as described herein. In some embodiments, the individual members of the plurality of three-dimensional polymer networks are located on a single array. In other embodiments, the individual members are located on two or more separate arrays. For example, the array can be an array as described in Section 5.3.

[0086] The members of the multiple three-dimensional networks can have a high degree of uniformity with each other. For example, when contacted with an analyte (e.g., a fluorescently labeled oligonucleotide) that can bind to probe molecules present in multiple three-dimensional networks, the measurement signals of the three-dimensional networks can be relatively similar, for example, having a coefficient of variation of less than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, or 5%. In some embodiments, the coefficient of variation is less than 25%, but at least 1%, at least 2%, at least 5%, or at least 10%; or less than 20%, but at least 1%, at least 2%, at least 5%, or at least 10%; or less than 15%, but at least 1%, at least 2%, at least 5%, or at least 10%; or less than 10%, but at least 1%, at least 2%, or at least 5%; or less than 9%, but at least 1%, at least 2%, or at least 5%; or less than 8%, but at least 1%, at least 2%, or at least 5%; or less than 7%, but at least 1%, at least 2%, or at least 5%; or less than 6%, but at least 1%, at least 2%, or at least 5%; or less than 5%, but at least 1% or at least 2%.

[0087] In the case of multiple networks with fluorescently labeled probes, the probes can be used to assess uniformity without (or in addition to) binding labeled analytes. In some embodiments, when exciting fluorescently labeled probes present in multiple three-dimensional networks, the coefficient of variation of the measured signal is less than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, or 5%. In some embodiments, the coefficient of variation is less than 25%, but at least 1%, at least 2%, at least 5%, or at least 10%; or less than 20%, but at least 1%, at least 2%, at least 5%, or at least 10%; or less than 15%, but at least 1%, at least 2%, at least 5%, or at least 10%; or less than 10%, but at least 1%, at least 2%, or at least 5%; or less than 9%, but at least 1%, at least 2%, or at least 5%; or less than 8%, but at least 1%, at least 2%, or at least 5%; or less than 7%, but at least 1%, at least 2%, or at least 5%; or less than 6%, but at least 1%, at least 2%, or at least 5%; or less than 5%, but at least 1% or at least 2%.

[0088] Arrays The three-dimensional networks of the present disclosure can be located (e.g., deposited) on a substrate and are preferably immobilized thereon (e.g., by covalent crosslinks between the network and the substrate). Multiple networks can be immobilized on a substrate to form an array useful, for example, as a biochip.

[0089] Suitable substrates include organic polymers, such as cycloolefin copolymers (COCs), polystyrene, polyethylene, polypropylene, polycarbonate, and polymethyl methacrylate (PMMA, Plexiglas®). Ticona sells one suitable COC under the trade name Topas®. Inorganic materials (e.g., metals, glass) can also be used as substrates. Such substrates can be coated with organic molecules to enable crosslinking between the network and the surface of the substrate. For example, inorganic surfaces can be coated with self-assembled monolayers (SAMs). SAMs can themselves be completely non-reactive and therefore can comprise or consist of, for example, pure alkylsilanes. Other substrates may also be suitable for crosslinking into three-dimensional networks, as long as they can enter into stable bonds with organic molecules during free radical processes (e.g., organoboron compounds).

[0090] The substrate can be rigid or flexible. In some embodiments, the substrate is in the shape of a plate (e.g., a rectangular plate, a square plate, a circular disk, etc.). For example, the substrate can include a microwell plate, and the three-dimensional network can be located within the wells of the plate.

[0091] Individual networks can be located in distinct spots on the surface of the substrate, for example, in a matrix including multiple columns and rows. Arrays with different numbers of rows and columns, each of which can be independently selected, are contemplated (e.g., 2 to 64 columns and 2 to 64 rows). Columns can be separated by a distance X, and rows can be separated by a distance Y, to form a grid of spots from which individual networks can be located (e.g., as shown in Figure 9 of WO 2018 / 234253). X and Y can be selected so that networks located in the grid spots do not contact each other in the dehydrated state, and do not contact each other in the hydrated state. The dimensions X and Y can be the same or different. In some embodiments, X and Y are the same. In some embodiments, X and Y are different. In some embodiments, X and Y are independently selected from a distance of at least about 500 μm (e.g., 500 μm to 5 mm, 500 μm to 4 mm, 500 μm to 3 mm, 500 μm to 2 mm, or 500 μm to 1 mm). In some embodiments, X and Y are both about 500 μm. In other embodiments, X and Y are both 500 μm.

[0092] In some embodiments, the substrate is strip-shaped (e.g., as shown in Figure 10 of WO 2018 / 234253). The network can be arranged as a single column extending along the length of the strip-shaped organic surface, or as multiple columns extending along the length of the strip-shaped surface. The rows and columns of such a strip-shaped array can have grid dimensions X and Y as described above.

[0093] Each individual network can cover an area on the surface of a circular or substantially circular array. Typically, the diameter of the area on the surface of the array covered by an individual network (i.e., spot diameter) is between 80 μm and 1000 μm. In various embodiments, the spot diameter is 80 μm, 100 μm, 120 μm, 140 μm, 160 μm, 180 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1000 μm, or a range bounded by any two of the foregoing embodiments. For example, the diameter is selected from 80 μm to 200 μm, 100 μm to 120 μm, 120 μm to 140 μm, 120 μm to 180 μm, 140 μm to 160 μm, 160 μm to 180 μm, 180 μm to 200 μm, 120 μm to 200 μm, 100 μm to 400 μm, 160 μm to 600 μm, or 120 μm to 700 μm, etc. In a preferred embodiment, the diameter is in the range of 100 μm to 200 μm or a subrange thereof.

[0094] Arrays of the present disclosure typically have at least 8 individual three-dimensional networks. In certain aspects, arrays have at least 16, at least 24, at least 48, at least 96, at least 128, at least 256, at least 512, or at least 1024 individual three-dimensional networks. In some embodiments, arrays of the present disclosure have 24, 48, 96, 128, 256, 512, 1024, 2048, 4096, or 8192 individual networks, or have a number of three-dimensional networks selected from a range bounded by any two of the foregoing embodiments, such as 8-128, 8-512, 24-8192, 24-4096, 48-2048, 96-512, 128-1024, 24-1024, 48-512, 96-1024, or 128-512 three-dimensional networks. In preferred embodiments, the number of three-dimensional networks on the array ranges from 8 to 1024. In particularly preferred embodiments, the number of three-dimensional networks on the array ranges from 25 to 400.

[0095] The individual networks comprising the arrays of the present disclosure can have the same or different probes (e.g., each network can have a unique probe set, multiple networks can have the same probe set, other networks can have different probe set(s), or all of the networks can have the same probe set). For example, networks located in the same row of the matrix can include the same probes, while networks located in different rows of the matrix can have different probes.

[0096] Typically, individual networks on the array differ from each other by no more than 20%, no more than 15%, no more than 10%, or no more than 5% in spot diameter and / or network volume.

[0097] In some embodiments, the array comprises one or more individual networks (e.g., spots on the array) with one or more control oligonucleotides or probe molecules. The control oligonucleotides can be labeled, e.g., fluorescently labeled, for use as a spatial control (to spatially orient the array) and / or to quantify the amount of probe molecules bound to the network, for example, when the arrays of the present disclosure are washed and reused (i.e., as "reusable controls"). The spatial and reusable control probes can be the same or different probes.

[0098] The same spot on the array or a different spot on the array can further contain an unlabeled probe complementary to a known target.When used in a hybridization assay, the efficiency of the hybridization reaction can be determined by determining the signal intensity of the hybridization of the unlabeled probe to the labeled target.When individual networks (i.e., spots on the array) are used as both reusable and / or spatial controls and hybridization controls, the target molecules can be labeled with a fluorescent moiety that is different from the fluorescent moiety of the reusable control or spatial control probe.

[0099] In some embodiments, arrays of the present disclosure can be reused at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, or at least 50 times (e.g., 5-20 times, 5-30 times, 10-50 times, 10-20 times, 10-30 times, 20-40 times, or 40-50 times, preferably including reusing the array 10-50 times). The array can be washed with a salt solution under denaturing conditions (e.g., low salt concentration and high temperature). For example, the array can be washed with 1-10 mM phosphate buffer at 80-90°C between uses. The temperature of the wash can be selected based on the length (Tm) of the target:probe hybrid.

[0100] The integrity of the array can be determined by a "reusable control" probe. The reusable control probe can be fluorescently labeled or detected by hybridization with a fluorescently labeled complementary nucleic acid. The fluorescent label of the fluorescently labeled reusable control probe can be bleached by repeated excitation before the integrity of the nucleic acid is compromised. In such cases, any further reuse can include detection of hybridization to a fluorescently labeled complementary nucleic acid as a control. Typically, the arrays of the present invention are stable for at least six months.

[0101] In various embodiments, the fluorescently labeled reusable control probe retains at least 99%, 95%, 90%, 80%, 70%, 60%, or 50% of its initial fluorescent signal intensity after 5, 10, 20, 30, 40, or 50 uses. Preferably, the reusable control probe retains at least 75% of its fluorescent signal intensity after 5 or 10 uses. The array can continue to be reused until the reusable control probe retains at least 50% of its fluorescent signal intensity, for example, after 20, 30, 40, or 50 reuses. The fluorescent signal intensity of the control probe can be tested after every reuse, every other reuse, every third reuse, every fourth reuse, every fifth reuse, every sixth reuse, every seventh reuse, every eighth reuse, every ninth reuse, every tenth reuse, or any combination thereof. For example, signal strength can initially be tested periodically for 5 or 10 reuses, with the frequency of testing increasing with the number of reuses, such that testing is performed after each reuse after a certain number of uses (e.g., 5, 10, 20, 30, 40, or 50). In some embodiments, the frequency of testing is an average of once per 1, 1.5, 2, 2.5, 3, 4, 5, or 10 uses, or an average within a range bounded by any two of the foregoing values, e.g., once per 1-2 uses, once per 1-1.5 uses, once per 1-3 uses, or once per 1.5-3 uses.

[0102] It should be noted that the nomenclature "spatial control," "reusable control," and "hybridization control" is included for convenience and reference purposes and does not imply a requirement that the probes referred to as "spatial control," "reusable control," and "hybridization control" be used as is.

[0103] 5.4. How to use the 3D network The network and array of the present disclosure can be used to determine the presence or absence of an analyte in a sample, preferably a liquid sample. Thus, the present disclosure provides a method for determining whether an analyte is present in one or more samples, comprising contacting one or more samples with a network or array of the present disclosure, which comprises probe molecules capable of binding to the analyte, and detecting the binding of the analyte to the probe molecules, thereby determining whether the analyte is present in one or more samples. When an array comprising different types of probes capable of binding to different types of analytes is used in this method, the presence of different types of analytes can be determined by detecting the binding of the different types of analytes to the probes. In some embodiments, the method further comprises quantifying the amount of analyte(s) bound to the array.

[0104] The analyte can be, for example, a nucleic acid, such as a polymerase chain reaction (PCR) amplicon. In some embodiments, the PCR amplicon is amplified from a biological or environmental sample (e.g., blood, serum, plasma, tissue, cells, saliva, sputum, urine, cerebrospinal fluid, pleural effusion, milk, tears, stool, sweat, semen, whole cells, cellular components, cell smears, or extracts or derivatives thereof). In some embodiments, the nucleic acid is labeled (e.g., fluorescently labeled).

[0105] Analytes placed on the surface of the network can penetrate into the interior of the network through the transport channels to specifically bind to the probes (e.g., biomolecules) covalently bound to the polymer. When the array of the present disclosure with fixed networks is used as a biological sensor, high measurement accuracy and high measurement dynamics are permitted.

[0106] The networks and arrays of the present disclosure can be regenerated after use as biosensors and can be used several times (e.g., 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, or at least 50 times). If the probe molecule is DNA, this can be achieved, for example, by heating the network(s) in 1x phosphate-buffered saline to a temperature of 80°C to 90°C for about 10 minutes. The phosphate-buffered saline can then be replaced with fresh phosphate-buffered saline to wash the denatured DNA from the network(s). If the probe molecule of the network(s) or array is an antigen, the network(s) or array can be regenerated by contacting the network(s) with 0.1N NaOH for about 10 minutes. The 0.1N NaOH can then be replaced with phosphate-buffered saline to wash the antigen from the network. Thus, some embodiments of methods for using the networks and arrays of the present disclosure include using a network or array that has been washed before contacting with one or more samples.

[0107] 5.5. Applications of the Arrays of the Present Disclosure The arrays of the present invention have immediate application to problems related to health and disease in humans and non-human animals because they achieve economical determination of the qualitative and quantitative presence of nucleic acids in a sample.

[0108] In these applications, preparations containing target molecules are derived or extracted from biological or environmental sources according to protocols known in the art.Target molecules can be derived or extracted from cells and tissues of all taxonomic classes (including all phyla and classes of viruses, bacteria and eukaryotes, prokaryotes, protists, plants, fungi, and animals).Animal can be vertebrates, mammals, primates, and especially humans.Blood, serum, plasma, tissue, cells, saliva, sputum, urine, cerebrospinal fluid, pleural effusion, milk, tears, feces, sweat, semen, whole cells, cell components, and cell smears are suitable sources of target molecules.

[0109] The target molecule is preferably a nucleic acid that has been amplified (eg, by PCR) from any of the aforementioned sources.

[0110] The arrays of the invention can include probes useful for detecting human or non-human animal pathogens, such probes comprising oligonucleotides at least partially complementary to bacterial, viral or fungal targets, or any combination of bacterial, viral and fungal targets.

[0111] The arrays of the invention may comprise probes useful for detecting gene expression in human or non-human animal cells, e.g., gene expression associated with a disease or disorder such as cancer, cardiovascular disease, or metabolic disease, for the purpose of diagnosing a subject, monitoring a subject's treatment, or prognosing a subject's outcome. Gene expression information can then track the progression or regression of the disease, and such information can be useful for monitoring the success of initial treatment or changes in the course of treatment.

[0112] 5.6.Kit In another aspect, the present disclosure provides a kit comprising a mixture comprising a salt solution described herein, a polymer described herein, a crosslinker moiety described herein (which may be covalently attached to the polymer), a substrate described herein, and, optionally, a probe molecule described herein. The kit of the present disclosure can be used, for example, to make the three-dimensional networks and / or arrays described herein.

[0113] 6. Working Example 6.1. Example 1: Effect of Process Parameters on Three-Dimensional Networks and Arrays: Study 1 Arrays with three-dimensional networks containing transport channels were fabricated according to the process described in WO 2017 / 103128. Variability was observed between the resulting three-dimensional networks when a mixture containing sodium phosphate with a phosphate concentration of 341 mM, 1 mg / mL of the cross-linked polymer poly(dimethylacrylamide) copolymer, 5% methacryloyl-benzophenone copolymer, 2.5% sodium 4-vinylbenzenesulfonate, and oligonucleotide probes was used. When the source of variability was investigated, it was surprisingly discovered that the polymer sometimes precipitated prematurely from the mixture before salt crystals formed, forming a polymer cap on top of the mixture. It was further discovered that the polymer cap was sometimes, but not always, removed during subsequent washing, contributing to the variability between three-dimensional networks on a given array.

[0114] Following the discovery that polymers can prematurely separate from mixtures, we investigated the effects of polymer concentration, buffer (salt) concentration, cross-linking energy, and oligonucleotide probe concentration on 3D network and array variability. Specifically, the following parameters were evaluated: [Table 1]

[0115] The signal intensity and coefficient of variation (CV) were measured for three-dimensional networks fabricated with different polymer concentrations, phosphate concentrations, probe concentrations, and crosslinking energies. The results are shown in Figure 1A-1B.

[0116] The most important factors contributing to the variability observed using the original mixture were found to be polymer concentration and crosslinking energy. High polymer concentrations were found to result in a relatively large amount of non-uniform three-dimensional network. Without being bound by theory, this was attributed to the formation of polymer precipitates in the mixture and incomplete extraction of the precipitates after crosslinking. Again, without being bound by theory, it is believed that using lower crosslinking energy reduces the degree of crosslinking, facilitating the extraction of unbound material and thus resulting in a more uniform three-dimensional network on the array.

[0117] It was further discovered that reducing the phosphate concentration improved the variability between three-dimensional networks (reduced CV) and reduced the risk of premature precipitation of components from the mixture.

[0118] 6.2. Example 2: Effect of Process Parameters on Three-Dimensional Networks and Arrays: Study 2 A similar study to that in Example 1 was carried out using the probes E. coli-1637p and Entb-132p. [Table 2]

[0119] The printing scheme for fabricating the three-dimensional network of the study is shown in Figure 2A.

[0120] Figure 3A shows a Cy3 fluorescence image of a three-dimensional network without bound analyte, while Figure 3B shows a Cy5 fluorescence image of a three-dimensional network containing Cy5-labeled analyte molecules. Visual inspection revealed that the three-dimensional networks fabricated using 341 mM buffer with probe concentrations of 20 μM and 35 μM had more uniform spot morphology compared to the other three-dimensional networks. For example, the three-dimensional networks fabricated using 341 mM buffer with probe concentrations of 20 μM and 35 μM generally exhibited less of a halo effect, which, without being bound by theory, is believed to be an indication that the three-dimensional networks were not completely uniform.

[0121] The Cy3 signal intensity of the probe E. coli-1637p is plotted in Figure 4A. The 3D network fabricated at a polymer concentration of 0.01 mg / ml exhibited relatively low signal intensity, whereas polymer concentrations of 0.1 mg / ml, 0.26 mg / ml, and 0.5 mg / ml exhibited similar, higher signals. The 3D network fabricated at a 5 μM oligonucleotide concentration generally exhibited the lowest signal, whereas 3D networks fabricated at 20 μM and 35 μM exhibited similar signals. The 3D networks fabricated at buffer concentrations of 244 mM and 341 mM exhibited similar signal intensities at each oligonucleotide concentration. Increasing the crosslinking energy was found to increase signal intensity.

[0122] The Cy5 signal intensity of the probe E. coli-1637p is shown in Figure 4B. The 3D network prepared at 5 μM oligonucleotide concentration showed the lowest signal, while 35 μM showed a signal slightly higher than 20 μM. The 3D network prepared at each oligonucleotide concentration and with a buffer concentration of 341 mM showed the highest signal intensity. It was found that increasing the crosslinking energy decreased the signal intensity.

[0123] The coefficient of variation, which provides a measure of the 3D network uniformity, for the 3D networks using the E. coli-1637p probe is shown in Figure 5A (Cy3) and Figure 5B (Cy5). As shown in Figure 5A, the 3D network made with 0.01 mg / ml of polymer showed a high %CV, but at 0.7 J / cm 2 The cross-linking energy is 1 J / cm 2 As shown in Figure 5B, the %CV was better than that of 1 J / cm. 2 The three-dimensional networks made with 0.4 J / cm2 showed relatively high %CV, whereas the three-dimensional networks made with 5 μM oligonucleotide had relatively high %CV for all parameter combinations. 2 The three-dimensional network fabricated with a crosslinking energy of 0.7 J / cm showed good signal intensity, but 2 Relatively good %CV was observed for the three-dimensional network prepared with crosslinking energy of .

[0124] The Cy3 signal intensity of the probe Entb-132p is plotted in Figure 6A, and a similar profile to that in Figure 4A was observed.

[0125] The Cy5 signal intensity of the probe Entb-132p is shown in Figure 6B. The three-dimensional network made with a polymer concentration of 0.01 mg / ml exhibits a relatively low signal intensity, while polymer concentrations of 0.1 mg / ml, 0.26 mg / ml, and 0.5 mg / ml exhibit similar, higher signals. The three-dimensional network made with a 5 μM oligonucleotide concentration exhibited the lowest signal, with 35 μM exhibiting a signal slightly higher than 20 μM. The three-dimensional network made with a buffer concentration of 341 mM at each oligonucleotide concentration exhibited the highest signal intensity. Increasing the crosslinking energy decreased the signal intensity, reaching 0.4 J / cm. 2 showed relatively high signal intensity.

[0126] The coefficients of variation of the three-dimensional network using the Entb-132p probe are shown in Figure 7A (Cy3) and Figure 7B (Cy5). The profile shown in Figure 7A is similar to the profile shown in Figure 5A. 1 J / cm 2 The three-dimensional network fabricated with a crosslinking energy of 0.4 J / cm showed good signal intensity, but 2 and 0.7 J / cm 2 Relatively good %CV was observed for the three-dimensional network prepared with crosslinking energy of .

[0127] 6.3. Example 3: Comparison of 3D networks created with different parameters Following the studies of Examples 1 and 2, arrays made using the following parameters were compared for array functionality and production run yield. [Table 3]

[0128] The functionality of arrays produced using the original and new parameters was comparable, but the yield of production runs was better with the new process parameters (data not shown).

[0129] 7. Specific Embodiments The present disclosure is illustrated by the following specific embodiments. 1. A process for producing a three-dimensional hydrogel network, comprising: (a) a mixture (optionally located on a surface of a substrate), (i) aqueous salt solutions; (ii) a water-soluble polymer chain; (iii) a crosslinker moiety, and (iv) exposing the mixture, optionally including the probe molecule, to salt crystal forming conditions; thereby forming a mixture containing one or more salt crystals; (b) crosslinking water-soluble polymer chains in the mixture containing one or more salt crystals, thereby forming a hydrogel containing one or more salt crystals; and (c) contacting the hydrogel containing the one or more salt crystals with a solvent in which the one or more salt crystals are soluble, thereby dissolving the salt crystals; thereby forming a three-dimensional hydrogel network, Optionally, A) the concentration of the water-soluble polymer chains in the mixture of step (a) is such that precipitation of the water-soluble polymer chains from the mixture does not occur prior to the formation of one or more salt crystals in step (a); and / or B) the concentration of the water-soluble polymer chains in the mixture of step (a) is such that the water-soluble polymer chains and salt crystals co-precipitate in step (a); and / or C) The process wherein the concentration of the water-soluble polymer chains in the mixture of step (a) is less than 1 mg / ml. 2. The process of embodiment 1, wherein the concentration of the water-soluble polymer chains in the mixture of step (a) is such that precipitation of the water-soluble polymer chains from the mixture does not occur prior to the formation of one or more salt crystals in step (a). 3. The process of embodiment 1 or embodiment 2, wherein the concentration of the water-soluble polymer chains in the mixture of step (a) is such that the water-soluble polymer chains and salt crystals co-precipitate during step (a). 4. The process of any one of embodiments 1 to 3, wherein the concentration of the water-soluble polymer chains in the mixture of step (a) is less than 1 mg / ml. 5. The process of any one of embodiments 1 to 4, wherein the concentration of the water-soluble polymer chains in the mixture of step (a) ranges from a lower limit ("polymer lower limit") that is at least 0.01 mg / ml to an upper limit ("polymer upper limit") that is less than 1 mg / ml. 6. The process of embodiment 5, wherein the polymer lower limit is 0.01 mg / ml. 7. The process of embodiment 5, wherein the polymer lower limit is 0.05 mg / ml. 8. The process of embodiment 5, wherein the polymer lower limit is 0.1 mg / ml. 9. The process of any one of embodiments 5 to 8, wherein the polymer upper limit is 0.5 mg / ml. 10. The process of any one of embodiments 5 to 8, wherein the polymer upper limit is 0.4 mg / ml. 11. The process of any one of embodiments 5 to 8, wherein the polymer upper limit is 0.3 mg / ml. 12. The process of any one of embodiments 5 to 8, wherein the polymer upper limit is 0.2 mg / ml. 13. The process of any one of embodiments 5 to 8, wherein the polymer upper limit is 0.1 mg / ml. 14. The process of embodiment 5, wherein the concentration of the water-soluble polymer chains in the mixture of step (a) is 0.1 mg / ml. 15. The process of any one of embodiments 1 to 14, wherein the crosslinker moiety is covalently attached to the water-soluble polymer chain. 16. The process of any one of embodiments 1 to 15, wherein the crosslinker moiety is photoreactive. 17. The process of embodiment 16, wherein the crosslinking comprises exposing the mixture containing one or more salt crystals to UV light. 18. The process of embodiment 17, wherein the crosslinking comprises exposing the mixture containing the one or more salt crystals to UV light having a wavelength of 254 nm. 19. The crosslinker moiety is photoreactive and step (b) is 1 J / cm 2 19. The process of any one of the preceding embodiments, comprising crosslinking the water-soluble polymer chains with a UV light energy dose of less than 1000 W / m. 20. Step (b) is at least 0.4 J / cm 2 (the "lower crosslinking energy limit") to 1 J / cm 2 20. The process of embodiment 19, comprising crosslinking the water-soluble polymer chains with a UV light energy dose ranging up to an upper limit ("upper crosslinking energy limit") that is less than 21. The minimum crosslinking energy is 0.4 J / cm 2 21. The process of embodiment 20, wherein 22. The minimum crosslinking energy is 0.5 J / cm 2 21. The process of embodiment 20, wherein 23. The minimum crosslinking energy is 0.6 J / cm 221. The process of embodiment 20, wherein 24. The minimum crosslinking energy is 0.7 J / cm 2 21. The process of embodiment 20, wherein 25. Upper limit of crosslinking energy is 0.9 J / cm 2 25. The process of any one of embodiments 20 to 24, wherein 26. Upper limit of crosslinking energy is 0.8 J / cm 2 25. The process of any one of embodiments 20 to 24, wherein 27. Upper limit of crosslinking energy is 0.7 J / cm 2 25. The process of any one of embodiments 20 to 24, wherein 28. Step (b) is 0.7 J / cm 2 21. The process of embodiment 20, comprising crosslinking the water-soluble polymer chains with a UV energy dose of 29. The process of any one of embodiments 1 to 28, wherein the aqueous salt solution comprises phosphate ions. 30. The process of embodiment 29, wherein the concentration of phosphate ions in the mixture of step (a) ranges from a lower limit ("phosphate lower limit") that is at least 125 mM to an upper limit ("phosphate upper limit") that is less than 350 mM. 31. The process of embodiment 30, wherein the phosphate lower limit is 125 mM. 32. The process of embodiment 30, wherein the phosphate lower limit is 150 mM. 33. The process of embodiment 30, wherein the phosphate lower limit is 200 mM. 34. The process of embodiment 30, wherein the phosphate lower limit is 225 mM. 35. The process of any one of embodiments 30 to 34, wherein the phosphate upper limit is 340 mM. 36. The process of any one of embodiments 30 to 34, wherein the phosphate upper limit is 300 mM. 37. The process of any one of embodiments 30 to 34, wherein the phosphate upper limit is 250 mM. 38. The process of embodiment 30, wherein the concentration of phosphate ions in the mixture of step (a) is 250 mM. 39. The process of any one of embodiments 1 to 38, wherein the aqueous salt solution comprises a sodium phosphate solution. 40. The process of embodiment 39, wherein the aqueous salt solution comprises a solution produced by a process comprising dissolving disodium hydrogen phosphate, sodium dihydrogen phosphate, or a combination thereof in water or an aqueous solution. 41. The process of any one of embodiments 1 to 40, wherein the salt crystal-forming conditions result in the formation of one or more needle-shaped crystals such that one or more long channels are produced after dissolution of the salt crystals. 42. The process of any one of embodiments 1 to 28, wherein the mixture of step (a) comprises at least two types of monovalent metal ions having a total concentration of at least 500 mM. 43. The process of embodiment 42, wherein the mixture of step (a) comprises at least two types of monovalent metal ions having a total concentration of 500 mM to 1000 mM. 44. The process of embodiment 43, wherein the total concentration of monovalent metal ions in the mixture of step (a) is 550 mM to 800 mM. 45. The process of embodiment 44, wherein the total concentration of monovalent metal ions in the mixture of step (a) is 600 mM to 750 mM. 46. The process of any one of embodiments 42 to 45, wherein the mixture of step (a) comprises two types of monovalent metal ions. 47. The process of embodiment 46, wherein the concentration of each of the two monovalent ions is at least 150 mM or at least 200 mM. 48. The process of embodiment 46 or embodiment 47, wherein the monovalent metal ions are selected from sodium ions, potassium ions, and lithium ions. 49. The process of embodiment 46 or embodiment 47, wherein the monovalent metal ions are sodium ions and potassium ions. 50. The process of embodiment 49, wherein the concentration of sodium ions in the mixture of step (a) is at least 300 mM. 51. The process of embodiment 50, wherein the concentration of sodium ions in the mixture of step (a) is 300 mM to 500 mM. 52. The process of embodiment 51, wherein the concentration of sodium ions in the mixture of step (a) is 300 mM to 400 mM. 53. The process of embodiment 52, wherein the concentration of sodium ions in the mixture of step (a) is 350 mM. 54. The process of any one of embodiments 49 to 53, wherein the potassium ion concentration in the mixture of step (a) is 150 mM to 500 mM. 55. The process of embodiment 54, wherein the concentration of potassium ions in the mixture of step (a) is 175 mM to 400 mM. 56. The process of embodiment 55, wherein the concentration of potassium ions in the mixture of step (a) is 200 mM to 350 mM. 57. The process of embodiment 56, wherein the concentration of potassium ions in the mixture of step (a) is 250 mM to 350 mM. 58. The process of any one of embodiments 42 to 45, wherein the mixture of step (a) comprises three types of monovalent metal ions. 59. The process of embodiment 58, wherein the concentrations of at least two of the monovalent ions are at least 150 mM each or at least 200 mM each. 60. The process of embodiment 58 or embodiment 59, wherein the monovalent metal ions are sodium ions, potassium ions, and lithium ions. 61. The process of embodiment 60, wherein the concentration of sodium ions in the mixture of step (a) is at least 250 mM. 62. The process of embodiment 61, wherein the concentration of sodium ions in the mixture of step (a) is 250 mM to 500 mM. 63. The process of embodiment 62, wherein the concentration of sodium ions in the mixture of step (a) is 300 mM to 400 mM. 64. The process of embodiment 63, wherein the concentration of sodium ions in the mixture of step (a) is 350 mM. 65. The process of any one of embodiments 60 to 64, wherein the potassium ion concentration in the mixture of step (a) is 150 mM to 500 mM. 66. The process of embodiment 65, wherein the concentration of potassium ions in the mixture of step (a) is 200 mM to 400 mM. 67. The process of embodiment 66, wherein the concentration of potassium ions in the mixture of step (a) is 250 mM to 350 mM. 68. The process according to any one of embodiments 42 to 67, wherein the concentration of phosphate ions in the mixture is at least 250 mM. 69. The process of embodiment 68, wherein the concentration of phosphate ions in the mixture is 250 mM to 1000 mM. 70. The process of embodiment 69, wherein the concentration of phosphate ions in the mixture is 550 mM to 800 mM. 71. The process of embodiment 70, wherein the concentration of phosphate ions in the mixture is 600 mM to 750 mM. 72. The process of any one of embodiments 42 to 71, wherein the salt crystal-forming conditions result in the formation of one or more needle-shaped crystals such that one or more long channels are produced after dissolution of the salt crystals. 73. The process according to any one of embodiments 42 to 72, wherein the salt crystal-forming conditions result in the formation of one or more small crystals, such that after dissolution of the salt crystals, one or more short channels are produced. 74. The process of any one of embodiments 1 to 73, wherein the water-soluble polymer chain comprises a homopolymer chain. 75. The process of any one of embodiments 1 to 73, wherein the water-soluble polymer chain comprises a copolymer chain. 76. The process of any one of embodiments 1 to 73, wherein the water-soluble polymer chains comprise a mixture of homopolymer chains and copolymer chains. 77. The process of any one of embodiments 74 to 76, wherein the water-soluble polymer chains comprise polymer chains polymerized from one or more species of monomer. 78. The process of embodiment 77, wherein each of the monomers comprises a polymerizable group independently selected from an acrylate group, a methacrylate group, an ethacrylate group, a 2-phenylacrylate group, an acrylamide group, a methacrylamide group, an itaconate group, and a styrene group. 79. The process of embodiment 78, wherein at least one monomer species in the water-soluble polymer contains a methacrylate group. 80. The process of embodiment 79, wherein at least one monomer species containing a methacrylate group is methacryloyloxybenzophenone (MABP). 81. The process of any one of embodiments 77, wherein the water-soluble polymer comprises a polymer polymerized from dimethylacrylamide (DMAA), methacryloyloxybenzophenone (MABP), and sodium 4-vinylbenzenesulfonate (SSNa). 82. The process of embodiment 81, wherein the water-soluble polymer comprises a polymer polymerized from dimethylacrylamide (DMAA), methacryloyloxybenzophenone (MABP), and sodium 4-vinylbenzenesulfonate (SSNa), and comprises 2.5 to 7.5 mol % MABP, 2 to 5 mol % SSNa, and residual DMAA. 83. The process of embodiment 81, wherein the water-soluble polymer comprises a polymer polymerized from dimethylacrylamide (DMAA), methacryloyloxybenzophenone (MABP), and sodium 4-vinylbenzenesulfonate (SSNa) in a DMAA:MABP:SSNa molar ratio of 92.5:5:2.5. 84. The process of any one of embodiments 1 to 83, wherein the water-soluble polymer chain is a copolymer chain comprising a crosslinker moiety. 85. The process of embodiment 84, wherein the water-soluble polymer chain comprises at least two crosslinker moieties per polymer molecule. 86. The process of any one of embodiments 1 to 85, wherein the crosslinker moiety is selected from benzophenone, thioxanthone, benzoin ether, ethyl eosin, eosin Y, rose bengal, camphorquinone, erythrosine, 4,4' azobis(4-cyanopentanoic) acid, 2,2-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, and benzoyl peroxide. 87. The process of embodiment 86, wherein the crosslinker moiety is a benzophenone moiety. 88. The process of any one of embodiments 1 to 87, wherein the average molecular weight of the water-soluble polymer chains is at least 100 kDa. 89. The process of embodiment 88, wherein the average molecular weight of the water-soluble polymer chains is at least 200 kDa. 90. The process of embodiment 88, wherein the average molecular weight of the water-soluble polymer chains is at least 300 kDa. 91. The process of embodiment 88, wherein the average molecular weight of the water-soluble polymer chains is at least 400 kDa. 92. The process according to any one of embodiments 88 to 91, wherein the average molecular weight of the water-soluble polymer chains is 600 kDa or less. 93. The process according to any one of embodiments 88 to 91, wherein the average molecular weight of the water-soluble polymer chains is 500 kDa or less. 94. The process according to any one of embodiments 88 to 90, wherein the average molecular weight of the water-soluble polymer chains is 400 kDa or less. 95. The process according to any one of embodiments 88 to 90, wherein the average molecular weight of the water-soluble polymer chains is 300 kDa or less. 96. The process of any one of embodiments 1 to 87, wherein the average molecular weight of the water-soluble polymer chain is between 200 kDa and 400 kDa. 97. A process for producing a three-dimensional hydrogel network, comprising: (a) a mixture (optionally located on a surface of a substrate), (i) an aqueous salt solution containing phosphate ions at a concentration ranging from a lower limit of at least 125 mM (the "phosphate lower limit") to an upper limit of less than 350 mM (the "phosphate upper limit"); (ii) a water-soluble polymer chain at a concentration of less than 1 mg / ml, the polymer comprising a polymer polymerized from dimethylacrylamide (DMAA), methacryloyloxybenzophenone (MABP), and sodium 4-vinylbenzenesulfonate (SSNa); and (iii) exposing the mixture, optionally including the probe molecule, to salt crystal forming conditions; thereby forming a mixture containing one or more salt crystals; (b) crosslinking water-soluble polymer chains in the mixture containing one or more salt crystals, thereby forming a hydrogel containing one or more salt crystals; and (c) contacting the hydrogel containing the one or more salt crystals with a solvent in which the one or more salt crystals are soluble, thereby dissolving the salt crystals; thereby forming a three-dimensional hydrogel network. 98. The process of embodiment 97, wherein the concentration of the water-soluble polymer chains in the mixture of step (a) ranges from a lower limit ("polymer lower limit") that is at least 0.01 mg / ml to an upper limit ("polymer upper limit") that is less than 1 mg / ml. 99. The process of embodiment 98, wherein the polymer lower limit is 0.01 mg / ml. 100. The process of embodiment 98, wherein the polymer lower limit is 0.05 mg / ml. 101. The process of embodiment 98, wherein the polymer lower limit is 0.1 mg / ml. 102. The process of any one of embodiments 98 to 101, wherein the polymer upper limit is 0.5 mg / ml. 103. The process according to any one of embodiments 98 to 101, wherein the upper polymer limit is 0.4 mg / ml. 104. The process according to any one of embodiments 98 to 101, wherein the upper polymer limit is 0.3 mg / ml. 105. The process according to any one of embodiments 98 to 101, wherein the upper polymer limit is 0.2 mg / ml. 106. The process according to any one of embodiments 98 to 101, wherein the upper polymer limit is 0.1 mg / ml. 107. The process of embodiment 97, wherein the concentration of the water-soluble polymer chains in the mixture of step (a) is 0.1 mg / ml. 108. The process according to any one of embodiments 97 to 107, wherein the phosphate lower limit is 125 mM. 109. The process according to any one of embodiments 97 to 107, wherein the phosphate lower limit is 150 mM. 110. The process according to any one of embodiments 97 to 107, wherein the phosphate lower limit is 200 mM. 111. The process according to any one of embodiments 97 to 107, wherein the phosphate lower limit is 225 mM. 112. The process according to any one of embodiments 97 to 111, wherein the phosphate upper limit is 340 mM. 113. The process according to any one of embodiments 97 to 111, wherein the phosphate upper limit is 300 mM. 114. The process according to any one of embodiments 97 to 111, wherein the phosphate upper limit is 250 mM. 115. The process according to any one of embodiments 97 to 107, wherein the concentration of phosphate ions in the mixture of step (a) is 250 mM. 116. The process of any one of embodiments 97 to 115, wherein the aqueous salt solution comprises a sodium phosphate solution. 117. The process of embodiment 116, wherein the aqueous salt solution comprises a solution produced by a process comprising dissolving disodium hydrogen phosphate, sodium dihydrogen phosphate, or a combination thereof in water or an aqueous solution. 118. The process according to any one of embodiments 97 to 117, wherein the crosslinking comprises exposing the mixture containing one or more salt crystals to UV light. 119. The process of embodiment 118, wherein the crosslinking comprises exposing the mixture containing one or more salt crystals to UV light having a wavelength of 254 nm. 120. Step (b) is 1 J / cm 2120. The process of any one of embodiments 97 to 119, comprising crosslinking the water-soluble polymer chains with a UV light energy dose of less than 1000 W / m. 121. Step (b) is at least 0.4 J / cm 2 (the "lower crosslinking energy limit") to 1 J / cm 2 121. The process of embodiment 120, comprising crosslinking the water-soluble polymer chains with a UV light energy dose ranging up to an upper limit ("upper crosslinking energy limit") that is less than 122. Lower limit of crosslinking energy is 0.4 J / cm 2 122. The process of embodiment 121, wherein 123. Lower limit of crosslinking energy is 0.5 J / cm 2 122. The process of embodiment 121, wherein 124. The lower limit of crosslinking energy is 0.6 J / cm 2 122. The process of embodiment 121, wherein 125. The lower limit of crosslinking energy is 0.7 J / cm 2 122. The process of embodiment 121, wherein 126. Upper limit of crosslinking energy is 0.9 J / cm 2 126. The process of any one of embodiments 121 to 125, wherein 127. Upper limit of crosslinking energy is 0.8 J / cm 2 126. The process of any one of embodiments 121 to 125, wherein 128. Upper limit of crosslinking energy is 0.7 J / cm 2 126. The process of any one of embodiments 121 to 125, wherein 129. Step (b) is 0.7 J / cm 2 121. The process of embodiment 120, comprising crosslinking the water-soluble polymer chains with a UV energy dose of 130. The process of any one of embodiments 97 to 129, wherein the water-soluble polymer comprises a polymer polymerized from dimethylacrylamide (DMAA), methacryloyloxybenzophenone (MABP), and sodium 4-vinylbenzenesulfonate (SSNa), and comprises 2.5 to 7.5 mol % MABP, 2 to 5 mol % SSNa, and residual DMAA. 131. The process of any one of embodiments 97 to 129, wherein the water-soluble polymer comprises a polymer polymerized from dimethylacrylamide (DMAA), methacryloyloxybenzophenone (MABP), and sodium 4-vinylbenzenesulfonate (SSNa) in a DMAA:MABP:SSNa molar ratio of 92.5:5:2.5. 132. The process according to any one of embodiments 97 to 131, wherein the average molecular weight of the water-soluble polymer chains is at least 100 kDa. 133. The process according to embodiment 132, wherein the average molecular weight of the water-soluble polymer chains is at least 200 kDa. 134. The process according to embodiment 132, wherein the average molecular weight of the water-soluble polymer chains is at least 300 kDa. 135. The process according to embodiment 132, wherein the average molecular weight of the water-soluble polymer chains is at least 400 kDa. 136. The process according to any one of embodiments 132 to 135, wherein the average molecular weight of the water-soluble polymer chains is 600 kDa or less. 137. The process according to any one of embodiments 132 to 135, wherein the average molecular weight of the water-soluble polymer chains is 500 kDa or less. 138. The process according to any one of embodiments 132 to 135, wherein the average molecular weight of the water-soluble polymer chains is 400 kDa or less. 139. The process according to any one of embodiments 132 to 135, wherein the average molecular weight of the water-soluble polymer chains is 300 kDa or less. 140. The process according to any one of embodiments 97 to 118, wherein the average molecular weight of the water-soluble polymer chain is between 200 kDa and 400 kDa. 141. The process of any one of embodiments 1 to 140, wherein the salt crystal-forming conditions comprise dehydrating the mixture. 142. The process of embodiment 141, comprising dehydrating the mixture by heating the mixture, exposing the mixture to a vacuum, reducing the humidity of the atmosphere surrounding the mixture, or a combination thereof. 143. The process of embodiment 142, comprising dehydrating the mixture by exposing the mixture to a vacuum. 144. The process of embodiment 142, comprising dehydrating the mixture by heating the mixture. 145. The process of embodiment 144, wherein heating the mixture comprises contacting the mixture with a gas having a temperature higher than the temperature of the mixture. 146. The process of any one of embodiments 1 to 141, wherein the salt crystal-forming conditions comprise cooling the mixture until the mixture is supersaturated with the salt. 147. The process of embodiment 146, comprising cooling the mixture by contacting the mixture with a gas having a temperature lower than that of the mixture. 148. The process of any one of embodiments 1 to 147, wherein the temperature of the mixture in step (a) is maintained above the dew point of the atmosphere surrounding the mixture. 149. The process of any one of embodiments 1 to 148, wherein the pH of the aqueous salt solution is in the range of 6 to 9. 150. The process of embodiment 149, wherein the pH of the aqueous salt solution is in the range of 7 to 8.5. 151. The process of embodiment 150, wherein the pH of the aqueous salt solution is 8. 152. The process of any one of the preceding embodiments, further comprising, prior to step (a), forming a mixture. 153. The process according to any one of embodiments 1 to 152, wherein the solvent is water or an aqueous buffer. 154. The process of embodiment 153, wherein the solvent is water. 155. The process of embodiment 153, wherein the solvent is an aqueous buffer. 156. The process of embodiment 155, wherein the aqueous buffer comprises phosphate, methanol, ethanol, propanol, or a mixture thereof. 157. The process of any one of embodiments 1 to 156, wherein the mixture of step (a) further comprises a probe molecule. 158. The process of embodiment 157, wherein at least some, most or all of the probe molecules comprise nucleic acids, nucleic acid derivatives, peptides, polypeptides, proteins, carbohydrates, lipids, cells, ligands or combinations thereof. 159. The process according to embodiment 158, wherein at least some of the probe molecules comprise a nucleic acid or a nucleic acid derivative. 160. The process according to embodiment 158, wherein at least a majority of the probe molecules comprise nucleic acids or nucleic acid derivatives. 161. The process according to embodiment 158, wherein all of the probe molecules comprise nucleic acids or nucleic acid derivatives. 162. The process of embodiment 157, wherein at least some, most, or all of the probe molecules comprise an antibody, an antibody fragment, an antigen, an epitope, an enzyme, an enzyme substrate, an enzyme inhibitor, a nucleic acid, or a combination thereof. 163. The process of embodiment 162, wherein at least some of the probe molecules comprise nucleic acids. 164. The process of embodiment 162, wherein at least a majority of the probe molecules comprise nucleic acids. 165. The process of embodiment 162, wherein all of the probe molecules comprise nucleic acids. 166. The process according to any one of embodiments 163 to 165, wherein the nucleic acid is an oligonucleotide. 167. The process according to embodiment 166, wherein the oligonucleotide is 12 to 30 nucleotides in length. 168. The process according to embodiment 166, wherein the oligonucleotide is 14 to 30 nucleotides in length. 169. The process of embodiment 166, wherein the oligonucleotide is 14 to 25 nucleotides in length. 170. The process of embodiment 166, wherein the oligonucleotide is 14 to 20 nucleotides in length. 171. The process of embodiment 166, wherein the oligonucleotide is 15 to 30 nucleotides in length. 172. The process of embodiment 166, wherein the oligonucleotide is 15 to 25 nucleotides in length. 173. The process according to embodiment 166, wherein the oligonucleotide is 15 to 20 nucleotides in length. 174. The process of embodiment 166, wherein the oligonucleotide is 16 to 30 nucleotides in length. 175. The process of embodiment 166, wherein the oligonucleotide is 16 to 25 nucleotides in length. 176. The process of embodiment 166, wherein the oligonucleotide is 16 to 20 nucleotides in length. 177. The process of embodiment 166, wherein the oligonucleotide is 15 to 40 nucleotides in length. 178. The process according to embodiment 166, wherein the oligonucleotide is 15 to 45 nucleotides in length. 179. The process of embodiment 166, wherein the oligonucleotide is 15 to 50 nucleotides in length. 180. The process of embodiment 166, wherein the oligonucleotide is 15 to 60 nucleotides in length. 181. The process of embodiment 166, wherein the oligonucleotide is 20 to 55 nucleotides in length. 182. The process of embodiment 166, wherein the oligonucleotide is 18 to 60 nucleotides in length. 183. The process of embodiment 166, wherein the oligonucleotide is 20 to 50 nucleotides in length. 184. The process of embodiment 166, wherein the oligonucleotide is 30 to 90 nucleotides in length. 185. The process of embodiment 166, wherein the oligonucleotide is 20 to 100 nucleotides in length. 186. The process according to embodiment 166, wherein the oligonucleotide is 20 to 120 nucleotides in length. 187. The process according to embodiment 166, wherein the oligonucleotide is 20 to 40 nucleotides in length. 188. The process of embodiment 166, wherein the oligonucleotide is 20 to 60 nucleotides in length. 189. The process of embodiment 166, wherein the oligonucleotide is 40 to 80 nucleotides in length. 190. The process of embodiment 166, wherein the oligonucleotide is 40 to 100 nucleotides in length. 191. The process of embodiment 166, wherein the oligonucleotide is 40 to 60 nucleotides in length. 192. The process of embodiment 166, wherein the oligonucleotide is 60 to 80 nucleotides in length. 193. The process of embodiment 166, wherein the oligonucleotide is 80 to 100 nucleotides in length. 194. The process of embodiment 166, wherein the oligonucleotide is 100 to 120 nucleotides in length. 195. The process of embodiment 166, wherein the oligonucleotide is 12 to 150 nucleotides in length. 196. The process according to any one of embodiments 166 to 195, wherein the concentration of the oligonucleotide in the mixture of step (a) ranges from 5 μM to 35 μM. 197. The process of embodiment 196, wherein the concentration of the oligonucleotide in the mixture of step (a) is 15 μM to 25 μM. 198. The process of embodiment 196, wherein the concentration of the oligonucleotide in the mixture of step (a) is 5 μM. 199. The process of embodiment 196, wherein the concentration of the oligonucleotide in the mixture of step (a) is 20 μM. 200. The process of embodiment 196, wherein the concentration of the oligonucleotide in the mixture of step (a) is 35 μM. 201. The process of any one of embodiments 1 to 200, further comprising, prior to step (a), applying the mixture to a surface of a substrate. 202. The process of embodiment 201, wherein the mixture is applied in a volume of 100 pl to 5 nl. 203. The process of embodiment 201, wherein the mixture is applied in a volume of 100 pl to 1 nl. 204. The process of embodiment 201, wherein the mixture is applied in a volume of 200 pl to 1 nl. 205. The process according to embodiment 201, wherein the mixture is applied in a volume of 1 nl to 2 nl. 206. The process according to embodiment 201, wherein the mixture is applied in a volume of 1.5 nl to 2 nl. 207. The process of any one of embodiments 201 to 206, wherein applying the mixture to the substrate comprises spraying the mixture onto the surface of the substrate. 208. The process of embodiment 207, wherein the mixture is sprayed by an inkjet printer. 209. The process of any one of embodiments 201 to 208, wherein the substrate comprises an organic polymer or an inorganic material having a self-assembled monolayer of organic molecules on its surface. 210. The process of embodiment 209, wherein the substrate comprises an organic polymer. 211. The process of embodiment 210, wherein the organic polymer is selected from cycloolefin copolymers, polystyrene, polyethylene, polypropylene, polycarbonate, and polymethyl methacrylate. 212. The process of embodiment 211, wherein the substrate comprises polymethyl methacrylate, polystyrene, or a cycloolefin copolymer. 213. The process of embodiment 211, wherein the substrate comprises polystyrene. 214. The process of embodiment 209, wherein the substrate comprises an inorganic material having an alkylsilane self-assembled monolayer on the surface thereof. 215. The process of any one of embodiments 201 to 214, wherein the substrate comprises a microwell plate. 216. The process of any one of embodiments 201 to 215, wherein the polymer is crosslinked to the surface in step (b). 217. The process of embodiment 216, wherein a surface-crosslinked water-swellable polymer is produced. 218. The process of embodiment 217, wherein the water-swellable polymer is capable of absorbing up to 50 times its weight in deionized distilled water. 219. The process of embodiment 217 or embodiment 218, wherein the water-swellable polymer is capable of absorbing 5 to 50 times its own volume of deionized distilled water. 220. The process according to any one of embodiments 217 to 219, wherein the water-swellable polymer is capable of absorbing up to 30 times its weight in saline. 221. The process according to any one of embodiments 217 to 220, wherein the water-swellable polymer is capable of absorbing 4 to 30 times its own volume of saline. 222. A process for making an array, comprising generating a plurality of three-dimensional hydrogel networks in discrete spots on the surface of the same substrate by the process according to any one of embodiments 1 to 221. 223. The process according to embodiment 222, wherein a three-dimensional hydrogel network is simultaneously generated. 224. The process according to embodiment 222, wherein the three-dimensional hydrogel network is produced continuously. 225. The process according to any one of embodiments 222 to 224, further comprising crosslinking a plurality of three-dimensional hydrogel networks to the surface of the substrate. 226. A process for producing an array, comprising positioning a plurality of three-dimensional hydrogel networks produced or obtainable according to the process of any one of embodiments 1 to 221 at discrete spots on the surface of the same substrate. 227. The process according to any one of embodiments 222 to 226, further comprising crosslinking a plurality of three-dimensional hydrogel networks to the surface. 228. A process for producing an array, comprising positioning a plurality of three-dimensional hydrogel networks produced or obtainable according to the process described in any one of embodiments 196 to 221 at discrete spots on the surface of the same substrate. 229. The process of embodiment 228, wherein the positioning comprises applying a mixture that causes a three-dimensional hydrogel network to form in discrete spots. 230. The process according to any one of embodiments 222 to 229, wherein the spots are arranged in columns and / or rows. 231. A three-dimensional hydrogel network produced or obtainable by a process according to any one of embodiments 1 to 221. 232. A plurality of three-dimensional hydrogel networks, each having a surface and an interior, comprising (a) a cross-linked polymer, (b) one or more channels, and (c) a probe molecule immobilized within the network, wherein when contacted with an analyte capable of binding to the probe molecule and producing a signal, the measured signal of the plurality of three-dimensional hydrogel networks has a coefficient of variation of less than 25%. 233. A plurality of three-dimensional networks according to embodiment 232, having a coefficient of variation of less than 20%. 234. A plurality of three-dimensional networks according to embodiment 232, having a coefficient of variation of less than 15%. 235. A plurality of three-dimensional networks according to embodiment 232, having a coefficient of variation of less than 10%. 236. A plurality of three-dimensional networks according to embodiment 232, having a coefficient of variation of less than 9%. 237. A plurality of three-dimensional networks according to embodiment 232, having a coefficient of variation of less than 8%. 238. A plurality of three-dimensional networks according to embodiment 232, having a coefficient of variation of less than 7%. 239. A plurality of three-dimensional networks according to embodiment 232, having a coefficient of variation of less than 6%. 240. A plurality of three-dimensional networks according to embodiment 232, having a coefficient of variation of less than 5%. 241. A plurality of three-dimensional networks according to any one of embodiments 232 to 240, wherein the coefficient of variation is at least 1%. 242. A plurality of three-dimensional networks according to any one of embodiments 232 to 240, wherein the coefficient of variation is at least 2%. 243. A plurality of three-dimensional networks according to any one of embodiments 232 to 239, wherein the coefficient of variation is at least 5%. 244. A plurality of three-dimensional networks according to any one of embodiments 232 to 234, wherein the coefficient of variation is at least 10%. 245. A plurality of three-dimensional hydrogel networks, each having a surface and an interior, comprising (a) a cross-linked polymer, (b) one or more channels, and (c) a fluorescent probe molecule immobilized within the network, wherein when the fluorescent probe is excited to produce a signal, the measured signal of the plurality of three-dimensional hydrogel networks has a coefficient of variation of less than 25%. 246. A plurality of three-dimensional networks according to embodiment 245, having a coefficient of variation of less than 20%. 247. A plurality of three-dimensional networks according to embodiment 245, having a coefficient of variation of less than 15%. 248. A plurality of three-dimensional networks according to embodiment 245, having a coefficient of variation of less than 10%. 249. A plurality of three-dimensional networks according to embodiment 245, having a coefficient of variation of less than 9%. 250. A plurality of three-dimensional networks according to embodiment 245, having a coefficient of variation of less than 8%. 251. A plurality of three-dimensional networks according to embodiment 245, having a coefficient of variation of less than 7%. 252. A plurality of three-dimensional networks according to embodiment 245, having a coefficient of variation of less than 6%. 253. A plurality of three-dimensional networks according to embodiment 245, having a coefficient of variation of less than 5%. 254. A plurality of three-dimensional networks according to any one of embodiments 245 to 253, wherein the coefficient of variation is at least 1%. 255. A plurality of three-dimensional networks according to any one of embodiments 245 to 253, wherein the coefficient of variation is at least 2%. 256. A plurality of three-dimensional networks according to any one of embodiments 245 to 252, wherein the coefficient of variation is at least 5%. 257. A plurality of three-dimensional networks according to any one of embodiments 245 to 247, wherein the coefficient of variation is at least 10%. 258. A plurality of three-dimensional networks according to any one of embodiments 232 to 257, which is a plurality of three-dimensional networks according to any one of embodiments 245 to 257. 259. A plurality of three-dimensional networks according to any one of embodiments 232 to 258, wherein the probe molecules are oligonucleotide probes. 260. The plurality of three-dimensional networks of any one of embodiments 232 to 259, wherein each three-dimensional network comprises at least five channels. 261. A plurality of three-dimensional networks according to any one of embodiments 232 to 259, wherein each three-dimensional network comprises at least 10 channels. 262. A plurality of three-dimensional networks according to any one of embodiments 232 to 261, wherein each three-dimensional network comprises a plurality of channels that converge at an interior point of the network, such that the lateral distance between the channels decreases from the surface towards the interior point. 263. A plurality of three-dimensional networks according to any one of embodiments 232 to 262, comprising long channels. 264. A plurality of three-dimensional networks according to any one of embodiments 232 to 262, comprising long channels and short channels. 265. The plurality of three-dimensional networks of any one of embodiments 232 to 264, wherein the three-dimensional networks are located on an array. 266. A plurality of three-dimensional networks according to any one of embodiments 232 to 264, wherein the three-dimensional networks are located on separate arrays. 267. The plurality of three-dimensional networks of any one of embodiments 232 to 265, comprising at least 2, at least 5, at least 10, at least 20, at least 100, and / or up to 1000 individual three-dimensional networks. 268. An array comprising a plurality of three-dimensional hydrogel networks according to embodiment 231 on a substrate. 269. An array comprising a plurality of three-dimensional hydrogel networks according to any one of embodiments 232 to 267 on a substrate. 270. An array produced or obtainable by a process according to any one of embodiments 222 to 230. 271. The array according to any one of embodiments 268 to 270, comprising at least eight three-dimensional hydrogel networks. 272. The array according to any one of embodiments 268 to 270, comprising at least 16 three-dimensional hydrogel networks. 273. The array according to any one of embodiments 268 to 270, comprising at least 24 three-dimensional hydrogel networks. 274. The array according to any one of embodiments 268 to 270, comprising at least 48 three-dimensional hydrogel networks. 275. The array according to any one of embodiments 268 to 270, comprising at least 96 three-dimensional hydrogel networks. 276. The array according to any one of embodiments 268 to 270, comprising at least 128 three-dimensional hydrogel networks. 277. The array according to any one of embodiments 268 to 270, comprising at least 256 three-dimensional hydrogel networks. 278. The array according to any one of embodiments 268 to 270, comprising at least 512 three-dimensional hydrogel networks. 279. The array according to any one of embodiments 268 to 270, comprising at least 1024 three-dimensional hydrogel networks. 280. The array of any one of embodiments 268 to 270, comprising 24 to 8192 three-dimensional hydrogel networks. 281. The array of any one of embodiments 268 to 270, comprising 24 to 4096 three-dimensional hydrogel networks. 282. The array of any one of embodiments 268 to 270, comprising 24 to 2048 three-dimensional hydrogel networks. 283. The array of any one of embodiments 268 to 270, comprising 24 to 1024 three-dimensional hydrogel networks. 284. The array of any one of embodiments 268 to 270, comprising 24 three-dimensional hydrogel networks. 285. The array of any one of embodiments 268 to 270, comprising 48 three-dimensional hydrogel networks. 286. The array of any one of embodiments 268 to 270, comprising 96 three-dimensional hydrogel networks. 287. The array of any one of embodiments 268 to 270, comprising 128 three-dimensional hydrogel networks. 288. The array of any one of embodiments 268 to 270, comprising 256 three-dimensional hydrogel networks. 289. The array of any one of embodiments 268 to 270, comprising 512 three-dimensional hydrogel networks. 290. The array of any one of embodiments 268 to 270, comprising 1024 three-dimensional hydrogel networks. 291. The array according to any one of embodiments 268 to 290, wherein the three-dimensional hydrogel network comprises probe molecules, and two or more three-dimensional hydrogel networks comprise different species of probe molecules. 292. The array according to any one of embodiments 268 to 291, wherein the three-dimensional hydrogel network comprises a probe molecule, and two or more three-dimensional hydrogel networks comprise the same type of probe molecule. 293. The array according to any one of embodiments 268 to 290, wherein the three-dimensional hydrogel networks comprise probe molecules, and each of the three-dimensional hydrogel networks comprises the same type of probe molecule. 294. The array of any one of embodiments 268 to 293, wherein the plurality of three-dimensional hydrogel networks comprises one or more three-dimensional hydrogel networks comprising labeled control probe molecules. 295. The array of embodiment 294, wherein the labeled control probe molecules are fluorescently labeled. 296. The array of any one of embodiments 268 to 295, wherein the substrate comprises a microwell plate, and each well of the microwell plate contains only a single three-dimensional hydrogel network. 297. A method for determining whether an analyte is present in a sample, comprising: (a) contacting the three-dimensional hydrogel network according to embodiment 231 or the array according to any one of embodiments 268 to 296, which comprises probe molecules capable of binding to an analyte, with a sample; and (b) detecting binding of the analyte to the probe molecules in the three-dimensional hydrogel network or array, thereby determining whether the analyte is present in the sample. 298. The method of embodiment 297, further comprising washing the network or array containing the probe molecules between steps (a) and (b). 299. The method of embodiment 297 or embodiment 298, further comprising, prior to step (a), contacting the network or array comprising the probe molecules with a blocking reagent. 300. The method of any one of embodiments 297 to 299, further comprising quantifying the amount of analyte bound to the three-dimensional hydrogel network or array comprising the probe molecule. 301. A method for determining whether an analyte is present in each sample in a plurality of samples, comprising: (a) contacting the array according to any one of embodiments 268 to 296, comprising probe molecules capable of binding to an analyte, with a sample; and (b) detecting binding of the analyte to the probe molecules in the array, thereby determining whether the analyte is present in each of the plurality of samples. 302. A method for determining whether an analyte is present in each sample in a plurality of samples, comprising: (a) contacting the array according to any one of embodiments 268 to 296, comprising probe molecules capable of binding to the analyte and comprising control probe molecules, the array having been used and washed before step (a), with the sample; and (b) detecting binding of the analyte to the probe molecules in the array, thereby determining whether the analyte is present in each of the plurality of samples. 303. A method for determining whether two or more analytes are present in a sample, comprising: (a) contacting the array according to any one of embodiments 268 to 296, comprising different species of probe molecules capable of binding to different species of analytes, with a sample; and (b) detecting binding of the analyte to the probe molecules in the array, thereby determining whether two or more species of analyte are present in the sample. 304. A method for determining whether two or more analytes are present in a sample, comprising: (a) contacting the array according to any one of embodiments 268 to 296, comprising probe molecules of different species capable of binding to analytes of different species and comprising control probe molecules, the array having been used and washed before step (a), with a sample; and (b) detecting binding of the analyte to the probe molecules in the array, thereby determining whether two or more species of analyte are present in the sample. 305. (a) the array substrate comprises a microwell plate; (b) each well of the microwell plate contains only a single three-dimensional hydrogel network; and (c) The method of any one of embodiments 301 to 304, wherein contacting the array with the sample comprises contacting each well with only a single sample. 306. The method of any one of embodiments 301 to 305, further comprising washing the array containing the probe molecules between steps (a) and (b). 307. The method of any one of embodiments 301 to 306, further comprising, prior to step (a), contacting the array containing the probe molecules with a blocking reagent. 308. The method of any one of embodiments 301 to 307, further comprising quantifying the amount of analyte(s) bound to the array. 309. The method of any one of embodiments 297 to 308, further comprising reusing the array. 310. The method of embodiment 309, wherein the array is reused at least five times. 311. The method of embodiment 309, wherein the array is reused at least 10 times. 312. The method of embodiment 309, wherein the array is reused at least 20 times. 313. The method of embodiment 309, wherein the array is reused at least 30 times. 314. The method of embodiment 309, wherein the array is reused at least 40 times. 315. The method of embodiment 309, wherein the array is reused at least 50 times. 316. The method of embodiment 310, comprising reusing the array 5 to 20 times. 317. The method of embodiment 310, comprising reusing the array 5 to 30 times. 318. The method of embodiment 310, comprising reusing the array 10 to 50 times. 319. The method of embodiment 310, comprising reusing the array 10 to 20 times. 320. The method of embodiment 310, comprising reusing the array 10 to 30 times. 321. The method of embodiment 310, comprising reusing the array 20 to 40 times. 322. The method of embodiment 310, comprising reusing the array 40 to 50 times. 323. The method of any one of embodiments 309 to 322, comprising washing the array between reuses. 324. The method of embodiment 323, wherein the array is washed under denaturing conditions. 325. The method of embodiment 324, wherein the denaturing conditions comprise exposing the array to heat. 326. The method of embodiment 324, wherein the denaturing conditions comprise exposing the array to a low salt concentration. 327. The method of embodiment 324, wherein the denaturing conditions comprise exposing the array to both heat and low salt concentration. 328. The method of embodiment 324, wherein the denaturing conditions are removed before reuse. 329. The method of embodiment 328, wherein the denaturing conditions comprise exposing the array to heat, and reducing the temperature before reuse. 330. The method of embodiment 328, wherein the denaturing conditions comprise exposing the array to a low salt concentration, and increasing the salt concentration before reuse. 331. The method of embodiment 328, wherein the denaturing conditions include exposing the array to both heat and low salt concentration, and the temperature is reduced and the salt concentration is increased before reuse. 332. The method of any one of embodiments 309 to 331, wherein the array comprises at least one three-dimensional hydrogel network comprising a fluorescently labeled oligonucleotide as a reusability control. 333. The method of embodiment 332, comprising testing the fluorescent signal intensity. 334. The method of embodiment 333, wherein the reusable control retains at least 70% of its initial fluorescent signal intensity after 10 uses. 335. The method of embodiment 334, wherein the reusable control retains at least 50% of its signal intensity after 20 uses. 336. The method according to any one of embodiments 332 to 335, wherein the array is no longer reused after the reusable control has lost more than 50% of its signal intensity. 337. The method of any one of embodiments 297 to 336, wherein the analyte is a nucleic acid. 338. The method of embodiment 337, wherein the nucleic acid is a polymerase chain reaction (PCR) amplicon. 339. The method of embodiment 337, wherein the PCR amplicon is amplified from a biological or environmental sample. 340. The method of embodiment 339, wherein the PCR amplicon is amplified from a biological sample. 341. The method of embodiment 339, wherein the PCR amplicon is amplified from an environmental sample. 342. The method of embodiment 340, wherein the biological sample is blood, serum, plasma, tissue, cells, saliva, sputum, urine, cerebrospinal fluid, pleural effusion, milk, tears, stool, sweat, semen, whole cells, cellular components, cell smears, or extracts or derivatives thereof. 343. The method of embodiment 342, wherein the biological sample is mammalian blood, serum or plasma or an extract thereof. 344. The method of embodiment 343, wherein the biological sample is human or bovine blood, serum or plasma or an extract thereof. 345. The method of embodiment 342, wherein the biological sample is milk or an extract thereof. 346. The method of embodiment 345, wherein the biological sample is milk or an extract thereof. 347. The method of any one of embodiments 337 to 346, wherein the nucleic acid is labeled. 348. The method of embodiment 347, wherein the nucleic acid is fluorescently labeled. 349. A kit, (a) A mixture containing: (i) Salt solution; (ii) water-soluble polymers; (iii) a crosslinker moiety, optionally covalently attached to a water-soluble polymer; and (iv) optionally, a probe molecule; and (b) a substrate; The kit, wherein the concentration of the water-soluble polymer chains in the mixture is less than a saturation concentration of the water-soluble polymer chains, optionally less than 1 mg / ml, and the mixture comprises phosphate ions at a concentration ranging from 125 mM to less than 350 mM. 350. The kit of embodiment 349, wherein the concentration of the water-soluble polymer in the mixture is 0.1 mg / ml and the concentration of the phosphate in the mixture is 250 mM. 351. The kit of any one of embodiments 349 to 350, wherein the water-soluble polymer chain comprises a polymer polymerized from dimethylacrylamide (DMAA), methacryloyloxybenzophenone (MABP), and sodium 4-vinylbenzenesulfonate (SSNa), optionally in a DMAA:MABP:SSNa molar ratio of 92.5:5:2.5.

[0130] While various particular embodiments have been illustrated and described, it will be understood that various modifications can be made without departing from the spirit and scope of the present disclosure(s).

[0131] 8. Citation of References All publications, patents, patent applications, and other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes. In the event of a conflict between the teachings of one or more references incorporated herein and the present disclosure, the teachings of the present disclosure are intended.

Claims

1. A process for fabricating a three-dimensional hydrogel network, (a) A mixture (optionally located on the surface of the substrate), (v) Salt solution, (vi) Water-soluble polymer chain, (vii) Crosslinking agent portion, and (viiii) Optionally, a mixture containing a probe molecule is exposed to salt crystal formation conditions. This results in the formation of a mixture containing one or more salt crystals; (b) Crosslinking the water-soluble polymer chain in the mixture containing one or more salt crystals to form a hydrogel containing one or more salt crystals; and (c) Contacting the hydrogel containing one or more salt crystals with a solvent in which the one or more salt crystals are soluble, thereby dissolving the salt crystals; It includes, thereby forming the three-dimensional hydrogel network, Here: A) The concentration of the water-soluble polymer chain in the mixture in step (a) is such that precipitation of the water-soluble polymer chain from the mixture does not occur before the formation of one or more salt crystals in step (a); and / or B) The concentration of the water-soluble polymer chain in the mixture in step (a) is such that the water-soluble polymer chain and salt crystals coprecipitate in step (a); and / or C) A process in which the concentration of the water-soluble polymer chain in the mixture of step (a) is less than 1 mg / ml.

2. The process according to claim 1, wherein the concentration of the water-soluble polymer chain in the mixture in step (a) is such that precipitation of the water-soluble polymer chain from the mixture does not occur before the formation of one or more salt crystals in step (a).

3. The process according to claim 1, wherein the concentration of the water-soluble polymer chain in the mixture in step (a) is such that the water-soluble polymer chain and salt crystals coprecipitate during step (a).

4. The process according to claim 1, wherein the concentration of the water-soluble polymer chain in the mixture of step (a) is less than 1 mg / ml.

5. The process according to claim 1, wherein the concentration of the water-soluble polymer chain in the mixture of step (a) is in the range from a lower limit of at least 0.01 mg / ml ("polymer lower limit") to an upper limit of less than 1 mg / ml ("polymer upper limit").

6. The process according to claim 5, wherein the lower limit of the polymer is 0.01 mg / ml, 0.05 mg / ml, or 0.1 mg / ml, and / or the upper limit of the polymer is 0.5 mg / ml, 0.4 mg / ml, 0.3 mg / ml, 0.2 mg / ml, or 0.1 mg / ml, and optionally the concentration of the water-soluble polymer chain in the mixture of step (a) is 0.1 mg / ml.

7. The crosslinking agent portion is photoreactive, and step (b) is 1 J / cm ２ The process according to claim 1, comprising crosslinking the water-soluble polymer chain with a UV light energy dose of less than 1.

8. Step (b) is at least 0.4 J / cm ２ From the lower limit ("bridge energy lower limit") of 1 J / cm² ２ The process according to claim 7, comprising crosslinking the water-soluble polymer chain with UV light energy doses up to an upper limit ("crosslinking energy upper limit") which is less than 1.

9. The lower limit of the crosslinking energy is 0.4 J / cm ２ , 0.5 J / cm ２ , 0.6 J / cm ２ , or 0.7 J / cm ２ , and / or the upper limit of the crosslinking energy is 0.9 J / cm ２ , 0.8 J / cm ２ , or 0.7 J / cm ２ , optionally, step (b) comprises crosslinking the water-soluble polymer chain with a UV energy dose of 0.7 J / cm ２ , The process according to claim 8.

10. The process according to claim 1, wherein the salt aqueous solution contains phosphate ions.

11. The process according to claim 10, wherein the concentration of phosphate ions in the mixture of step (a) is in the range from a lower limit of at least 125 mM ("phosphate lower limit") to an upper limit of less than 350 mM ("phosphate upper limit").

12. The process according to claim 11, wherein the lower limit of the phosphate is 125 mM, 150 mM, 200 mM, or 225 mM, and / or the upper limit of the phosphate is 340 mM, 300 mM, or 250 mM, and optionally, the concentration of phosphate ions in the mixture of step (a) is 250 mM.

13. The process according to claim 1, wherein the salt aqueous solution contains a sodium phosphate solution.

14. The process according to claim 1, wherein the salt crystal formation conditions result in the formation of one or more needle-shaped crystals such that one or more long channels are produced after the dissolution of the salt crystals.

15. The process according to claim 1, wherein the mixture in step (a) comprises at least two types of monovalent metal ions having a total concentration of at least 500 mM, and optionally, the mixture in step (a) comprises at least two types of monovalent metal ions having a total concentration of 500 mM to 1000 mM.

16. The process according to claim 15, wherein the salt crystal formation conditions result in the formation of one or more needle-shaped crystals such that one or more long channels are produced after the dissolution of the salt crystals.

17. The process according to claim 15, wherein the salt crystal formation conditions result in the formation of one or more small crystals such that one or more short channels are produced after the dissolution of the salt crystal.

18. The process according to claim 1, wherein the water-soluble polymer comprises a polymer polymerized from dimethylacrylamide (DMAA), methacryloyloxybenzophenone (MABP), and sodium 4-vinylbenzenesulfonate (SSNa), and optionally comprises 2.5 to 7.5 mol% MABP, 2 to 5 mol% SSNa, and the remainder DMAA.

19. A process for fabricating a three-dimensional hydrogel network, (a) A mixture (optionally located on the surface of the substrate), (iv) A salt solution containing phosphate ions in a concentration ranging from a lower limit of at least 125 mM ("lower phosphate limit") to an upper limit of less than 350 mM ("upper phosphate limit") (v) A water-soluble polymer chain at a concentration of less than 1 mg / ml, comprising a polymer polymerized from dimethylacrylamide (DMAA), methacryloyloxybenzophenone (MABP), and sodium 4-vinylbenzenesulfonate (SSNa), and (vi) Optionally, expose a mixture containing a probe molecule to salt crystal formation conditions. This results in the formation of a mixture containing one or more salt crystals; (b) Crosslinking the water-soluble polymer chain in the mixture containing one or more salt crystals to form a hydrogel containing one or more salt crystals; and (c) Contacting the hydrogel containing one or more salt crystals with a solvent in which the one or more salt crystals are soluble, thereby dissolving the salt crystals; A process comprising, thereby forming the three-dimensional hydrogel network.

20. The process according to claim 19, wherein the concentration of the water-soluble polymer chain in the mixture of step (a) is in the range from a lower limit of at least 0.01 mg / ml ("polymer lower limit") to an upper limit of less than 1 mg / ml ("polymer upper limit").

21. The process according to claim 20, wherein the lower limit of the polymer is 0.01 mg / ml, 0.05 mg / ml, or 0.1 mg / ml, and / or the upper limit of the polymer is 0.5 mg / ml, 0.4 mg / ml, 0.3 mg / ml, 0.2 mg / ml, or 0.1 mg / ml.

22. The process according to claim 19, wherein the concentration of the water-soluble polymer chain in the mixture of step (a) is 0.1 mg / ml.

23. The process according to claim 19, wherein the lower limit of the phosphate is 125 mM, 150 mM, 200 mM, or 225 mM, and / or the upper limit of the phosphate is 340 mM, 300 mM, or 250 mM.

24. The process according to claim 19, wherein the concentration of phosphate ions in the mixture in step (a) is 250 mM.

25. The process according to claim 19, wherein the salt aqueous solution contains a sodium phosphate solution.

26. Process (b) is 1 J / cm ２ The process according to claim 19, comprising crosslinking the water-soluble polymer chain with a UV light energy dose of less than 100%.

27. Step (b) is at least 0.4 J / cm ２ From the lower limit ("bridge energy lower limit") of 1 J / cm² ２ The process according to claim 26, comprising crosslinking the water-soluble polymer chain with UV light energy doses up to an upper limit ("crosslinking energy upper limit") which is less than 100.

28. The lower limit of the crosslinking energy is 0.4 J / cm ２ , 0.5 J / cm ２ , 0.6 J / cm ２ , or 0.7 J / cm ２ And / or the crosslinking energy limit is 0.9 J / cm². ２ , 0.8 J / cm ２ , or 0.7 J / cm ２ The process according to claim 27.

29. Process (b) is 0.7 J / cm ２ The process according to claim 26, comprising crosslinking the water-soluble polymer chain with a UV energy dose.

30. The process according to claim 19, wherein the water-soluble polymer comprises a polymer polymerized from dimethylacrylamide (DMAA), methacryloyloxybenzophenone (MABP), and sodium 4-vinylbenzenesulfonate (SSNa), and further comprises 2.5 to 7.5 mol% of MABP, 2 to 5 mol% of SSNa, and the remainder of DMAA.

31. The process according to claim 19, wherein the water-soluble polymer comprises a polymer polymerized from dimethylacrylamide (DMAA), methacryloyloxybenzophenone (MABP), and sodium 4-vinylbenzenesulfonate (SSNa) in a DMAA:MABP:SSNa molar ratio of 92.5:5:2.

5.

32. The process according to claim 1, wherein the salt crystal formation conditions optionally include heating the mixture, exposing the mixture to a vacuum, reducing the humidity of the atmosphere surrounding the mixture, or a combination thereof, thereby dehydrating the mixture.

33. The process according to claim 1, wherein the salt crystal formation conditions optionally include cooling the mixture until the mixture is supersaturated with salt by contacting the mixture with a gas having a temperature lower than the temperature of the mixture.

34. The process according to claim 1, wherein the mixture of step (a) further comprises probe molecules.

35. The process according to claim 34, wherein at least a portion, most, or all of the probe molecules comprises nucleic acids.

36. The process according to claim 35, wherein the nucleic acid is an oligonucleotide.

37. The process according to claim 36, wherein the concentration of the oligonucleotide in the mixture of step (a) is in the range of 5 μM to 35 μM.

38. The process according to claim 1, further comprising the step of applying the mixture to the surface of a substrate before step (a).

39. The process according to claim 38, wherein the mixture is applied in a volume of 100 pl to 5 nl.

40. A process for fabricating an array, comprising generating a plurality of three-dimensional hydrogel networks at discrete spots on the surface of the same substrate by the process described in claim 1.

41. A process for fabricating an array, comprising positioning a plurality of three-dimensional hydrogel networks, which can be produced or obtained according to any one of claims 1 to 39, at discrete spots on the surface of the same substrate.

42. A three-dimensional hydrogel network produced or obtainable by the process described in claim 1.

43. A plurality of three-dimensional hydrogel networks having a surface and an interior, each comprising (a) a crosslinked polymer, (b) one or more channels, and (c) probe molecules immobilized within the network, wherein when in contact with an analyte capable of binding to the probe molecules and producing a signal, the measured signal of the plurality of three-dimensional hydrogel networks has a coefficient of variation of less than 25%.

44. The plurality of three-dimensional networks according to claim 43, wherein the coefficient of variation is less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, or less than 5%, and / or at least 1% or at least 2%.

45. Multiple three-dimensional networks according to claim 43, wherein each three-dimensional network includes a plurality of channels that converge at a point inside the network, thereby decreasing the lateral distance between the channels from the surface toward the point inside.

46. An array comprising a plurality of three-dimensional hydrogel networks as described in claim 42 on a substrate.

47. An array comprising a plurality of three-dimensional hydrogel networks as described in claim 43 on a substrate.

48. An array produced or obtainable by the process described in claim 40.

49. A method for determining whether an analyte is present in a sample, (a) Contacting the sample with a three-dimensional hydrogel network according to claim 42 or an array according to any one of claims 46 to 48, which includes probe molecules capable of binding to the analyte; and (b) A method comprising detecting the binding of the analyte to the probe molecules in the three-dimensional hydrogel network or array, thereby determining whether the analyte is present in the sample.

50. It's a kit, (a) Mixtures containing the following: (i) Salt solution; (ii) Water-soluble polymers; (iii) A crosslinking agent portion optionally covalently bonded to the water-soluble polymer; and (iv) Optionally, a probe molecule; and (b) base material; Includes, A kit wherein the concentration of the water-soluble polymer chain in the mixture is less than 1 mg / ml, and the mixture contains phosphate ions in a concentration ranging from 125 mM to less than 350 mM.

51. The kit according to claim 50, wherein the concentration of the water-soluble polymer in the mixture is 0.1 mg / ml, and the concentration of the phosphate in the mixture is 250 mM.

52. The kit according to claim 50, wherein the water-soluble polymer chain comprises a polymer polymerized from dimethylacrylamide (DMAA), methacryloyloxybenzophenone (MABP), and sodium 4-vinylbenzenesulfonate (SSNa) in an optional DMAA:MABP:SSNa molar ratio of 92.5:5:2.5.

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