RU2780656C2 - Three-dimensional polymer meshes and their use - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a method for the production of a three-dimensional hydrogel mesh having one or more transport channels. The method includes: (a) treatment of a mixture including: (i) at least two types of monovalent alkali metal ions having a total concentration of at least 500 mM, (ii) water-soluble polymer chains, (iii) fragments of a crosslinking reagent, and (iv) optionally probe molecules, under conditions providing the formation of salt crystals, optionally in which the mixture is on the surface of substrate, and optionally in which conditions providing the formation of salt crystals contain dehydration of the mixture or cooling of the mixture, as long as the relative salt content in the mixture does not increase to exceed the solubility limit, thereby forming a mixture containing one or more salt crystals; (b) crosslinking of polymer chains in the mixture containing one or more salt crystals, thereby forming hydrogel containing one or more salt crystals; and (c) interaction of hydrogel containing one or more salt crystals with a solvent, in which one or more salt crystals are soluble, thereby dissolving salt crystals; and thereby obtaining a three-dimensional hydrogel mesh, which contains one or more transport channels. A three-dimensional mesh, an array, a method for its production, and a method for determination of whether the analyzed substance is contained in a sample are also proposed.

EFFECT: proposed three-dimensional meshes can link larger amount of the analyzed substance coming through transport channels, which provides higher sensitivity in the detection of the analyzed substance and quantitative determination in a wider range of concentrations of the analyzed substance.

17 cl, 17 dwg, 2 ex

Description

1. STATE OF THE ART

In a U.S. publication No. 2008/0293592 describes a method for the covalent immobilization of probe biomolecules on organic surfaces using photochemically reactive crosslinking reagents. In practice, the method has been shown to be useful because it allows the immobilization of probe biomolecules on non-reactive surfaces such as silanized glass substrates and substrates made from standard industrial plastics. The polymer is used in the method described in US 2008/0293592 to provide a type of three-dimensional network with which probe biomolecules can be associated on the surface of the network or within the network. Compared to an organic surface, on which biomolecules-probes are immobilized only in two-dimensional form, three-dimensional immobilization of biomolecules in polymer and/or copolymer networks provides a higher density of biomolecules-probes on the organic surface. This increases the amount of analyte that can bind to a unit area of the organic surface. Thus, the use of the surface as a biological sensor provides higher measurement accuracy and high measurement speed.

However, the disadvantage of the methods and polymer networks described in U.S. 2008/0293592 is that analyte molecules or analyte components that bind to probe biomolecules located on or near the surface of the polymer network can block the network. Subsequent analyte molecules or analyte components, as well as probe biomolecules that have not yet bound, located at a greater distance from the surface of the grid in its inner part, can no longer participate in binding.

Thus, improved polymer networks are needed.

2. SUMMARY OF THE INVENTION

The present invention relates to three-dimensional polymer networks, including cross-linked polymer chains, for example, water-soluble polymer chains and one or more transport channels. Transport channels provide molecules in solution, such as analyte molecules, with access to the polymer chains in the network. In some sites, polymer chains are crosslinked to probe molecules and transport channels provide more surface area for analytes to bind to probe molecules.

The networks are preferably covalently bonded to the surface. As indicated in the previous paragraph, one or more probes, such as a biomolecule, can be immobilized on the surface of the mesh and throughout the interior of the mesh and provide a sensor to detect the presence and/or measure the amount of an analyte in the sample. For example, probe nucleic acids can be used to detect complementary nucleic acids contained in a sample, and probe antibodies can be used to detect antigens contained in a sample. The meshes of the present invention provide faster hybridization of a given amount of analyte than meshes without transport channels because the transport channels can effectively increase the surface area of the mesh, exposing more sample probes at a given time. In addition, the meshes of the present invention can bind more analyte than the same volume of a mesh without transport channels, because the transport channels reduce or eliminate the hindrance that the analyte or other sample components bind to probes at the surface. or close to the mesh block access to probes located in the interior of the mesh. than possible when using a grid that does not contain transport channels, i.e. it is possible to increase the signal-to-noise ratio compared to using grids that do not contain transport channels, since a given amount of analyte can be concentrated in a smaller volume of the grid. Another advantage of the meshes of the present invention is that the large amount of analyte delivered by the transport channels allows for quantitation over a wider range of analyte concentrations than meshes without transport channels.

The present invention also relates to arrays comprising a plurality of three-dimensional grids of the present invention and a substrate. The arrays of the present invention can be used to detect and/or measure one or more analytes simultaneously in one or more samples. The arrays of the present invention can be washed and reused, and provide significant cost savings compared to a single use of the arrays. Another advantage of the arrays proposed in the present invention is that they can be obtained in a simple manner, since all the components necessary to obtain a separate mesh can be applied as a single mixture to the surface of the substrate during the preparation procedure.

The present invention also relates to methods for obtaining three-dimensional meshes and arrays proposed in the present invention. The three-dimensional networks of the present invention can be obtained by crosslinking a polymer in the presence of at least two types of salt crystals, preferably acicular salt crystals and compact salt crystals, and then dissolving the salt crystals to form transport channels in the crosslinked polymer network. Without being limited by theory, the inventors of the present invention believe that the presence of compact salt crystals during crosslinking results in a spongy polymer with short channels through which pass long channels formed due to the presence of needle-like salt crystals during crosslinking.

The present invention also relates to methods for using the three-dimensional grids and arrays of the present invention to detect and/or measure the content of an analyte in a sample, preferably a liquid sample.

3. BRIEF DESCRIPTION OF THE DRAWINGS

In FIG. 1 is a schematic representation of a mixture that includes a biomolecule probe (1) and a polymer (3) containing two photochemically reactive groups (4) in one molecule dissolved in an aqueous salt solution (as described in section 4.3.1).

In FIG. 2 is a cross-sectional view of a mixture droplet (5), such as that shown in FIG. 1, having a surface (10) located on the spot (7) of the surface (2) which is located on the holder (6). The surface is preferably the surface of an organic substrate or a substrate containing organic matter. The surface is preferably rigid. The holder may be a heating holder or a cooling holder providing controlled crystallization of salts in an aqueous salt solution.

In FIG. 3 is a sectional view of the system shown in FIG. 2, after heating the mixture and when (a) needle-shaped salt crystals (8) extending from the nuclei of crystallization (9), and (b) compact crystals (14) formed in the salt solution.

In FIG. 4 is a sectional view of the system shown in FIG. 3 after drying and irradiating the mixture with radiation in the visible region of the spectrum (11) to form a polymer network (15) having a surface (16).

In FIG. 5 is a schematic representation of the mixture shown in FIG. 1 after exposure to radiation in the visible region of the spectrum.

In FIG. 6 is a sectional view of the system shown in FIG. 4, after dissolving the salt crystals in the solvent (12) with the formation of transport channels in the form of long channels (13) and short channels (19).

In FIG. 7 shows the reaction path for the formation of a copolymer (sodium dimethylacrylamide-methacryloyloxybenzophenone-4-vinylbenzenesulfonate).

In FIG. 8 is a perspective view of a biochip (17) showing polymer meshes (15) in spots (7) arranged in a matrix of rows and columns. The chip preferably has an organic surface.

In FIG. 9 is a top view of the biochip (17) shown in FIG. 8, in which each polymer mesh (15) has a diameter (D) and in which the rows and columns are separated by a distance Y and a distance X, respectively, measured from the center points of the polymer meshes (15).

In FIG. 10 shows a biosensor (17') comprising a flexible substrate belt (18) on which polymer networks (15) having a diameter (D) are located in spots (7) separated by a distance X measured from the centers of the polymer networks.

In FIG. 11A-11C show a biochip with 6 rows (A-F) and 6 columns (1-6) in polymer meshes before (FIG. 11A) and after (FIG. 11B) drying, which shows the salt concentrations used to obtain the polymer networks of rows D and E shown in Fig. 11V. Polymer grid lines D and E was obtained using an aqueous solution of salt containing sodium phosphate and potassium phosphate. The remaining rows were prepared using an aqueous salt solution containing only sodium phosphate at a concentration of 350 mm. The polymer grids of rows D and E look more rounded and uniform.

In FIG. 12A-12B show the results of hybridization of the PCR reaction product using S. aureus and E. coli specific primer pairs with arrays corresponding to FIG. 12A - fig. 12V. In FIG. 12A shows hybridization with an array of PCR product amplified from 100 copies of S. aureus DNA. In FIG. 12B shows hybridization with an array of PCR product amplified from 100 copies of E. coli DNA. In FIG. 12B is a probe map for the arrays shown in FIG. 12A and in FIG. 12B. E. coli = E. coli probe; Salmonella = Salmonella probe (with which the E. coli PCR product shows some cross-reactivity); Allstaph = Staphylococcus probe cuvette; S. aureus=S. aureus probe; PCR control = probe for detecting an internal control for the PCR amplification process; LL = beacons, which are fluorophore-labeled oligonucleotides linked to polymer chains in networks used as an array of controls.

In FIG. 13 is a quantitative description of fluorescence signals from hybridization of a PCR product amplified using S. aureus and E. coli specific primer pairs in the absence of background noise template. #1 represents spot D1, E1; #2 represents spot D1, E2; #3 represents spot D1, E3; #4 represents spot D1, E4; #5 represents spot D1, E5; and #6 represents spot D1, E6.

4. DETAILED DESCRIPTION OF THE INVENTION

4.1. 3D polymer meshes

The three-dimensional meshes of the present invention include a cross-linked polymer, such as the polymer according to Rendl et al., 2011, Langmuir 27:6116-6123 or US 2008/0293592, the contents of which are incorporated herein by reference in their entirety. . The three-dimensional grids of the present invention further include one or more transport channels and optionally may further include one or more probes immobilized on the grid, for example by cross-linking with polymer chains.

The meshes of the present invention may have apertures of size (measured in the hydrated state of the mesh) 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). "Net hydrated state" means that the net is in an equilibrium state of water absorption, i.e. in an aqueous solution, it absorbs as much water as it desorbs from it.

Resins that can be used to form meshes are described in section 4.1.1. Crosslinkers that can be used to create meshes are described in section 4.1.2. The characteristics of one or more transport channels are described in section 4.1.3. Probes that can be immobilized on grids are described in Section 4.1.4.

4.1.1. Polymers

The three-dimensional networks of the present invention may include a crosslinked homopolymer, a copolymer, mixtures of homopolymers, mixtures of copolymers, or mixtures of one or more homopolymers and one or more copolymers. The term "polymer" as used in the present invention includes homopolymers and/or copolymers. The term "copolymer" as used in the present invention includes polymers polymerized from two or more types of monomers (eg, biopolymers, terpolymers, quaternary copolymers, etc.). The copolymers include alternating copolymers, periodic copolymers, random copolymers, random copolymers, block copolymers, linear copolymers, and branched copolymers. The three-dimensional meshes of the present invention may include any combination of the above types of polymers. Reagents and techniques for preparing such polymers are known in the art (see, for example, Ravve, A., Principles of Polymer Chemistry, Springer Science + Business Media, 1995; Cowie, JMG, Polymers: Chemistry & Physics of Modern Materials, 2 ^nd Edition, Chapman & Hall, 1991; Chanda, M., Introduction to Polymer Science and Chemistry: A Problem-Solving Approach, 2nd ^Edition , CRC Press, 2013; Nicholson, JW, The Chemistry of Polymers, 4th ^Edition , RSC Publishing , 2012; the contents of which are incorporated herein by reference in their entirety).

Preferred polymers are hydrophilic and/or containing hydrophilic groups. The polymer is preferably soluble in water. In one embodiment, the polymer is a copolymer that is polymerized from two or more types of monomers selected to provide the desired degree of solubility in water. For example, the solubility of the copolymer in water can be controlled by changing the amount of charged monomer, such as sodium 4-vinylsulfonate, used to form the copolymer.

After cross-linking, water-soluble polymers form water-swellable gels or hydrogels. Hydrogels absorb aqueous solutions through hydrogen bonds with water molecules. The overall absorption and swelling capacity of the hydrogel can be controlled by changing the type and amount of crosslinkers used to form the gel. Low cross-link density polymers typically have higher absorbent capacity and swell more than high cross-link density polymers, but the gel strength of high cross-link density polymers is generally higher and can maintain the shape of the network even under moderate pressure.

The ability of a hydrogel to absorb water depends on the concentration of ions in an aqueous solution. In some embodiments, the hydrogel of the present invention can absorb up to 50 times its weight in deionized distilled water (volume 5 to 50 times its volume) and up to 30 times its weight in saline (volume , 4-30 times its volume). The reduced ability to absorb saline is due to the presence of monovalent cations, which prevent the polymer from binding to water molecules.

The three-dimensional network of the present invention may include a copolymer that is polymerized from one, two, three, or more than three types of monomers, where one, two, three, or more than three types of monomers include a polymerizable group independently selected from the group consisting of an acrylate group ( e.g., acrylate, methacrylate, methyl methacrylate, hydroxyethyl methacrylate, ethyl acrylate, 2-phenyl acrylate), acrylamide group (e.g. styrene, 4-methylstyrene, 4-ethoxystyrene). Preferred polymerizable groups are acrylate, methacrylate, ethacrylate, 2-phenylacrylate, acrylamide, methacrylamide, taconate and styrene. In some embodiments, one of the monomers used to form the copolymer is, for example, sodium 4-vinylbenzenesulfonate.

The polymer used to obtain the network proposed in the present invention may include at least one, at least two or more than two crosslinking groups in one molecule. A linking group is a group that covalently links the polymer molecules of the network to each other and optionally to the probes and/or substrate. Copolymers that are polymerized from two or more monomers (for example, monomers containing a polymerizable group independently selected from those described in the previous paragraph), at least one of which includes a crosslinking agent, are suitable for obtaining a three-dimensional network proposed in the present invention . Typical crosslinkers are described in section 4.1.2. A preferred monomer comprising a crosslinker is methacryloyloxybenzophenone (MABP) (see FIG. 7).

In a preferred embodiment, the copolymer is a biopolymer or a terpolymer including a crosslinker. In a particularly preferred embodiment, the copolymer is a (sodium dimethylacrylamide-methacryloyloxybenzophenone-4-vinylbenzenesulfonate) copolymer (see FIG. 7).

4.1.2. Crosslinking reagents

Crosslinkers (or crosslinkers) suitable for producing crosslinks in three-dimensional networks include those activated by ultraviolet radiation (eg, long wavelength UV radiation), visible light, and heat. Typical UV activated crosslinkers include benzophenone, thioxanthones (eg thioxanthen-9-one, 10-methylphenothiazine) and benzoin esters (eg benzoin methyl ester, benzoin ethyl ester). Exemplary visible-activated crosslinkers include ethyleosin, eosin Y, rose bengal, camphorquinone, and erythrosine. Typical heat-activated crosslinkers include 4,4'-azobis(4-cyclopentanoic) acid and 2,2-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride and benzoyl peroxide. Other crosslinking agents known in the art may also be used, for example, which may form radicals or other reactive groups upon irradiation.

4.1.3. Transport channels

Three-dimensional grids proposed in the present invention contain one or more transport channels.

Transport channels can provide access to the interior of the grid. Although the transport channels may have a relatively large cross section, the mesh may remain mechanically stable since the size of the mesh openings may be significantly smaller than the cross section of the transport channel.

The transport channels may form some type of highway through which analytes can rapidly enter and exit the interior of the grid. The transport of analytes in the transport channels can proceed by diffusion and/or convection.

Transport channels are formed when a network is created by crosslinking polymer chains in the presence of salt crystals, as described in section 4.3. After the salt crystals are washed out, transport channels remain.

Without being limited by theory, the authors of the present invention believe that the methods of obtaining networks proposed in the present invention, lead to the formation of at least two types of salt crystals, formed from different metal ion-salt pairs. When the salt crystals are washed out, at least two types of transport channels remain, in accordance with the principle of loss of shape. Transport channels allow analytes to enter the interior of the mesh and specifically bind to the probe located inside the mesh. In addition, the transport channels allow unbound analytes to exist within the mesh after washing, which reduces the intensity of the non-specific signal of the analytes introduced into the mesh.

It is assumed that one type of transport channel is a long channel formed from needle-shaped salt crystals. As used in the present invention, a "long channel" is an elongated passage in a mesh that (1) is substantially straight and (2) in the hydrated state of the mesh has a minimum cross section that is at least 300 nm and a length that is at least 3 times, preferably 5 times and more preferably at least 10 times the minimum passage area. For example, the length of the long channel may be 5-15 times, 5-10 times, or 10-15 times the minimum section of the long channel. A long channel that is "generally straight" is one that extends from the nucleation point in one direction without changing direction by more than 45 degrees in either direction, i.e. in the X, Y, or Z direction. Since the long channels are formed from acicular crystals that extend from a common nucleation point, the grids of the present invention may include groups of (for example, 5, 10, or more) long channels that converge into a point located in the grid corresponding to the starting point of crystallization nucleation. Long channels are usually located so that they are directed from the surface of the grid inward, the lateral distance between the long channels decreases.

It is assumed that in other objects, one type of transport channel is a short channel, for example, formed from cubic or rod-shaped crystals. As used in the present invention, a "short channel" is an elongated passage in the mesh that (1) is substantially straight and (2) in the hydrated state of the mesh has a minimum cross section that is preferably at least 10 times the size of the mesh openings, and a length , which is at least 3 times (e.g., may range from 1 to 2.75 times, 1 to 2.5 times, 1 to 2 times, or 1 to 1.5 times) greater minimum cross section. A short channel that is "generally straight" is one that extends from the nucleation point in one direction without changing direction by more than 45 degrees in either direction, ie. in the X, Y, or Z direction. To maintain the strength of the mesh, the short channel preferably has a section equal to no more than 1/20 of the width or diameter of the mesh, for example, for a mesh that is in the form of a "spot" in the array and has a diameter of 200 microns , the cross section of the short channel is preferably at most 10 μm, and for a spot in the array that has a diameter of 100 μm, the cross section of the short channel is preferably at most 5 μm. In some aspects, the short channel cross section is 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 micron or more. The short channels in the grid may have approximately (eg, within +/- 10% or +/- 25%) the same diameter or different diameters. In preferred embodiments, the short channels in the grid have a diameter between any two of the above values, for example, it can be in the range from 100 nm to 10 µm, from 50 nm to 1 µm, from 500 nm to 5 µm, from 250 nm to 10 µm, etc. etc.

Without being limited by theory, the authors of the present invention believe that the short channels form a sponge polymer through which the long channels pass.

4.1.4. Probes

The probe immobilized on the mesh of the present invention may be a biomolecule or a molecule that binds to a biomolecule, eg a component of a specifically interacting complementary binding component (receptor/ligand) system. For example, probes may include nucleic acids and their derivatives (such as RNA, DNA, looped nucleic acids (LNA) and peptide nucleic acids (PNA)), proteins, peptides, polypeptides and their derivatives (such as glucosamine, antibodies, antibody fragments, and enzymes), lipids (eg phospholipids, fatty acids such as arachidonic acid, monoglycerides, diglycerides and triglycerides), carbohydrates, enzyme inhibitors, enzyme substrates, antigens and epitopes. The probes can also include larger and more complex structures such as liposomes, membranes and membrane fragments, cells, cell lysates, cell fragments, spores and microorganisms.

A specifically interacting system of complementary binding components can be based, for example, on the interaction of a nucleic acid with a complementary nucleic acid, the interaction of PNA with a nucleic acid, or the interaction of an enzyme/substrate, receptor/ligand, lectin/sugar, antibody/antigen, avidin/biotin or streptavidin/ biotin.

Nucleic acid probes can be DNA or RNA, eg an oligonucleotide or aptamer, ZNA, PNA, or DNA containing a methacrylate group at the 5' end (5' Acrydite™). Oligonucleotide probes may include, 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 up to 25, from 16 to 20, from 15 to 40, from 15 to 45, from 15 to 50, from 15 to 60, from 20 to 55, from 18 to 60, from 20 to 50, from 30 to 90, from 20 up 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 up to 150 nucleotides. In preferred embodiments, the probe oligonucleotide comprises 15 to 60 nucleotides.

When a probe nucleic acid is used, all or only a portion of the probe may be complementary to the target sequence. The portion of the probe complementary to the target sequence preferably comprises at least 12 nucleotides, and more preferably at least 15, at least 18, or at least 20 nucleotides. In probe nucleic acids greater than 40 or 50 nucleotides, the portion of the probe complementary to the target sequence may be at least 25, at least 30, or at least 35 nucleotides.

An antibody may be, for example, a polyclonal, monoclonal, or chimeric antibody, or an antigen-binding fragment (i.e., "antigen-binding portion") or one chain thereof, fusion proteins comprising the antibody, and any other modified configuration of the immunoglobulin molecule. , which includes an antigen recognition site, including, for example, without limitation, single-chain (scFv) and domain antibodies (for example, human, camel or shark domain antibodies), maxibodies, minibodies, intraantibodies, diabodies, tribodies, tetrabodies, vNAR and bis- scFv (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 a subclass thereof) and antibodies should not be assigned to any particular class. Depending on the amino acid sequence of the heavy chain C-domain, immunoglobulin antibodies can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), for example, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . "Antibodies" also includes any of the above types of antibody/immunoglobulin.

The 3D grids of the present invention may include one probe type or more than one probe type (eg 2, 3, 4 or 5 or more types). Three-dimensional networks may include more than one type of probe for the same target (eg, antibodies that bind different epitopes on the same target) and/or include probes that bind multiple targets.

The grids may include a labeled (eg, fluorescently labeled) control probe molecule, which can be used, for example, to determine the amount of probe contained in the grid.

Probes may be distributed over the grid (eg, on the surface and in the interior of the grid). Preferably, at least one probe is not located on the surface of the grid and is in contact with at least one channel. The probe thus positioned is then directly accessible to the analyte molecules or analyte components via the channel. In some embodiments, most of the probes are located in the interior of the grid.

One or more probes can be immobilized on the grid covalently or non-covalently. For example, the probe may be cross-linked with a cross-linked polymer, or the probe may be non-covalently attached to the network (eg, by binding to a molecule covalently attached to the network). In a preferred embodiment, one or more probes are crosslinked with a crosslinked polymer. In some embodiments, most of the probes are covalently bonded within the mesh (eg, such that at least a portion of the probes are in contact with the transport channel).

Without being limited by theory, the authors believe that the methods described in section 4.3 for obtaining three-dimensional networks in the presence of salt crystals (especially phosphate crystals) can lead to a higher concentration of probe molecules at or near the interface between the polymer and the channel. due to electrostatic interactions between probe molecules (especially nucleic acid probe molecules) and salt crystals. Therefore, in some embodiments, the implementation of the present invention relates to grids proposed in the present invention, in which the density of the probes at the interface between the polymer and channels is greater than in areas of the polymer that are not in contact with the channel. In various embodiments, the density of the probes at the interface between the polymer and the transport channels is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% more, than in the areas of the polymer that are not in contact with the transport channel.

The density of probe molecules in the grid can be checked using the following method:

The grid is introduced into interaction with an aqueous liquid at room temperature, for example, in a Cup. The fluid contains a plurality of nanoparticles attached to a moiety that interacts with probe molecules in the network, such as streptavidin if the probe molecules are biotinylated. The size of the nanoparticles is smaller than the size of the mesh openings and less than the minimum cross section of at least one type of transport channel in the mesh, which ensures the distribution of the nanoparticles over the polymer. Suitable nanoparticles are quantum dots with a diameter of 2-5 nm.

The incubation period is chosen so that the mesh in the liquid is fully hydrated, i.e. so that the mesh absorbs, on average, the same amount of water as it emits. The incubation period may be, for example, 1 hour. Penetration of the nanoparticles into the mesh can be accelerated by moving the mesh and/or liquid during incubation, for example by vibrating the mesh and/or liquid, preferably by means of ultrasonic waves.

After incubation is complete, the liquid is separated from the mesh, for example by draining the liquid from the dish or removing the mesh from the dish.

The hydrated grid is then frozen, for example with liquid nitrogen. The frozen mesh can then be cut into parallel planes using a cryomicrotome to obtain thin sections. The cut planes are located perpendicular to the longitudinal length of the transport channel and pass into the transport channel. The cutting is preferably carried out with a diamond blade cooled with liquid nitrogen. The slice thickness may be, for example, about 100 nm or 200 nm.

Using a microscope, the positions of nanoparticles located on disks obtained by cutting a frozen grid are determined. The nanoparticles can be fluorescent and optionally illuminated to make them easier to distinguish from the grid. The positions of the nanoparticles can be determined using suitable software, including an image processing technique. It is preferable to use a confocal laser scanning microscope with fluorescent optics or an electron microscope to examine the discs.

Information about the geometry and/or positions of the nanoparticles obtained in this way can be used by a computer to build a three-dimensional geometric model of the distribution of nanoparticles in a grid. The model can then be used to determine if the nanoparticle distributions reflect the presence of a higher density of probe molecules near the walls of the transport channels.

4.2. Arrays

The three-dimensional networks of the present invention can be positioned (eg deposited) on a substrate and are preferably immobilized on the substrate (eg by covalently linking the network and the substrate). A plurality of grids can be immobilized on a substrate to form an array useful, for example, as a biochip.

Suitable substrates include organic polymers such as cycloolefin copolymers (CCO), polystyrene, polyethylene, polypropylene and polymethyl methacrylate (PMMA, Plexiglas®). Ticona sells a suitable central heating system under the trade name Topas®. Inorganic materials (eg metal, glass) can also be used as a substrate. Such substrates can be coated with organic molecules to provide crosslinking between the network and the surface of the substrate. For example, self-aggregated monolayers (SAMs) can be deposited on inorganic surfaces. The CAMs themselves may be completely non-reactive and, for example, include or consist of pure alkylsilanes. Other substrates may also be suitable for cross-linking with a three-dimensional network if they can participate in stable bonds with organic molecules in free radical reactions (for example, organoboron compounds).

The substrate may be rigid or elastic. In some embodiments, the substrate is in the form of a tablet (eg, a rectangular tablet, a square tablet, a round disk, etc.). For example, the substrate may include a microplate and three-dimensional grids may be placed in the wells of the plate.

The individual grids may be located at different spots on the surface of the substrate, for example in a matrix including a plurality of columns and rows. In the embodiment shown in FIG. 11, the grids are arranged on 36 spots grouped in 6 columns and 6 rows. Arrays are provided with varying numbers of rows and columns, the number of each of which can be independently selected (for example, 2 to 64 columns and 2 to 64 rows). The columns may be separated by a distance X and the rows may be separated by a distance Y (eg as shown in FIG. 12) so that a grid of spots is formed on which individual grids can be placed. X and Y can be chosen such that the grids located on the lattice spots do not touch each other in the dehydrated state and do not touch each other in the hydrated state. The X and Y dimensions 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 distances 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 from 500 µm to 1 mm). In some embodiments, X and Y are both about 500 microns. In other embodiments, X and Y are both 500 µm.

In some embodiments, the implementation of the substrate is ribbon-like (for example, as shown in Fig. 10). The grids may be arranged in a single row extending in the longitudinal direction of the ribbon-like organic surface, or may be arranged in multiple rows extending in the longitudinal direction of the ribbon-like surface. The rows and columns in such band arrays may have X and Y lattice sizes as described above.

Each individual mesh may occupy an area of the array surface that is circular or mostly circular. Typically, the diameter of the area on the surface of the array covered with individual grids (ie, spot diameter) is from 80 µm to 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 selected from a range between any two of the above values, for example, 80 µm to 200 µm, 100 µm to 120 µm, 120 µm to 140 µm, 120 µm to 180 µm, 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., etc. In a preferred embodiment, the diameter is in the range of 100 µm to 200 µm or a subrange thereof.

The arrays of the present invention typically contain at least 8 separate 3D meshes. In some objects, arrays include 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 3D meshes. In some embodiments, the arrays of the present invention comprise 24, 48, 96, 128, 256, 512, 1024, 2048, 4096, or 8192 individual meshes, or contain a number of 3D meshes selected from a range between any two of the above. values, for example, 8 to 128, 8 to 512, 24 to 8192, 24 to 4096, 48 to 2048, 96 to 512, 128 to 1024, 24 to 1024, 48 to 512, 96 to 1024 or 128 to 512 3D meshes, etc., etc. In a preferred embodiment, the number of 3D meshes in the array is in the range of 8 to 1024. In a particularly preferred embodiment, the number of 3D meshes in the array is in the range of 25 to 400.

The individual grids that comprise the arrays of the present invention may contain the same or different probes (for example, each grid may contain a different set of probes, multiple grids may contain the same set of probes, and other grids may contain a different set or different sets of probes, or all grids can contain the same set of probes). For example, grids located in the same row of the matrix may contain the same probes, and grids located in different rows of the matrix may contain different probes.

Typically, for individual array meshes, spot diameters and/or individual mesh volumes differ from each other by no more than 20%, no more than 15%, no more than 10%, or no more than 5%.

In some embodiments, the arrays include one or more individual grids (eg, spots in the array) with one or more control oligonucleotides or probe molecules. Control oligonucleotides can be labeled, e.g., fluorescently labeled, for use as a spatial control (for spatial orientation of the array) and/or to determine the number of probe molecules associated with the grids, for example, when washing and reusing the array of the present invention ( i.e. for "reusability control"). The spatial and reusability control probes may be the same or different.

The same spot in the array or different spots in the array may further include an unlabeled probe that is complementary to the known target. When used for hybridization studies, by determining the intensity of the hybridization signal of an unlabeled probe with a labeled target, the efficiency of the hybridization reaction can be determined. If a separate grid (i.e. a spot in the array) is used for reusability control and/or spatial control and hybridization control, a fluorescent moiety, different from the fluorescent moiety for reusability control probes, can be used to label the target molecule. or spatial control.

In some embodiments, the arrays of the present invention 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 (for example, from 5 to 20 times, 5 to 30 times, 10 to 50 times, 10 to 20 times, 10 to 30 times, 20 to 40 times, or 40 to 50 times, preferably reusing the array 10 to 50 times). The array can be washed with a salt solution under conditions that ensure denaturation (for example, at low salt concentration and high temperature). For example, between uses, the array can be washed with 1-10 mm phosphate buffer at a temperature equal to 80-90°C. The wash temperature can be selected based on the extent (Tm) of the target:probe hybrid.

The integrity of an array can be determined using a "reusability control" probe. The reusability probe may be fluorescently labeled or may be formed by hybridization with a fluorescently labeled complementary nucleic acid. The fluorescent label of a fluorescently labeled reusability control probe can discolor on repeated excitation before the integrity of the nucleic acid is compromised; in such cases, any additional reuses may include the detection of hybridization with a fluorescently labeled complementary nucleic acid as a control. Typically, the array of the present invention is stable for at least 6 months.

In various embodiments, the fluorescence signal intensity of the fluorescently labeled reusability probe is at least 99%, 95% 90%, 80%, 70%, 60%, or 50% of its initial fluorescence signal intensity after 5, 10, 20, 30, 40 or 50 uses. Preferably, the reusability probe's fluorescence signal intensity is at least 75% of its fluorescence signal intensity after 5 or 10 uses. The array can continue to be reused as long as the fluorescence signal intensity of the reusability control probe is at least 50% of its fluorescence signal intensity, for example, after 20, 30, 40, or 50 reuses. The intensity of the control probe fluorescence signal can be determined after every reuse, after every second reuse, after every third reuse, after every fourth reuse, after every fifth reuse, after every sixth reuse, after every seventh reuse, after every eighth reuse, after every ninth reuse, after every tenth reuse, or a combination of the above. For example, the signal intensity can be determined periodically after 5 or 10 repetitions at the beginning and the frequency of determination is increased with increasing number of repetitions so that it is determined after each repetition after a certain number (for example, 5, 10, 20, 30, 40 or 50) uses. In some embodiments, the detection rate averages once after 1, 1.5, 2, 2.5, 3, 4, 5, or 10 uses, or averages between any two of the above values, such as once after 1-2 uses, once after 1-1.5 uses, once after 1-3 uses, or once after 1.5-3 uses.

It should be noted that the terms "spatial control", "reusability control", and "hybridization control" are included for convenience and reference, and are not intended to indicate that probes for "spatial control", "reusability control" and "hybrid control" can be used by themselves.

4.3. Methods for obtaining three-dimensional polymer networks

In one aspect, the methods of the present invention for producing three-dimensional polymer networks comprise (a) treating a mixture comprising an aqueous salt solution, a polymer, a cross-linking agent, and optionally one or more probes under conditions to form salt crystals, (b) treating the mixture under crosslinking conditions to crosslink the polymer to form a crosslinked polymer network; and (c) reacting the crosslinked polymer network with a solvent to dissolve the salt crystals and form one or more transport channels.

The methods may further include the step of forming a mixture by combining an aqueous salt solution, a polymer, a crosslinker, and optionally one or more probes, and/or further include the step of applying the mixture to a substrate (e.g., the substrate described in Section 4.2) prior to exposing the mixture to conditions providing salt formation. If the polymer used contains a pre-attached crosslinker (for example, when using a copolymer polymerized from a monomer comprising a crosslinker), then the step of forming a mixture may include combining an aqueous salt solution with the polymer and optionally with one or more probes.

The mixture may be applied to the substrate prior to the mixture being treated under conditions to form a salt, for example by spraying the mixture onto the surface of the substrate (eg, 1024 centers on the surface). 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 provides a simple and quick application of the mixture to a large number of spots on the substrate. The spots may be arranged, for example, in the form of a matrix in several rows and/or columns. It is preferable that the salt content of the mixture during printing is less than the solubility limit value, so that the mixture does not crystallize in the printer's print head. The volume of the mixture applied to individual spots can be, for example, 100pl, 200pl, 300pl, 400pl, 500pl, 750pl, 1nl, 2nl, 3nl, 4nl or 5nl, or may be selected from a range between any two of the above values (for example, from 100 pl to 5 nl, from 100 pl to 1 nl, from 300 pl to 1 nl, from 200 pl to 750 nl, from 100 pl to 500 pl, from 200 pl to 2 nl, 500 pl to 2 nl, 1 nl to 2 nl, etc., etc.). In preferred embodiments, the spot volume is between 200 pl and 4 nL.

The diameter of individual spots depends on the composition of the mixture, the volume of the applied mixture and the chemical characteristics of the surface of the substrate. Spot diameters are typically in the range of 80 µm to 1000 µm and can be achieved by varying the above parameters. In various embodiments, spot diameters are 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 may be selected from a range between any two of the above values, for example, 80 µm to 200 µm, 100 µm to 120 µm, 120 µm to 140 µm, 120 µm to 180 µm, 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. ., etc. In a preferred embodiment, the diameter is in the range of 100 µm to 200 µm, or a subrange thereof.

Suitable polymers, crosslinkers and probes that can be used in the methods of the present invention are described in sections 4.1.1, 4.1.2 and 4.1.4, respectively. In some embodiments, the implementation of the polymer used in the methods contains at least one crosslinking group in one molecule of the polymer. In a preferred embodiment, the polymer contains at least two crosslinking groups in one molecule. In a particularly preferred embodiment, the polymer contains at least two photochemically reactive groups per molecule. In these embodiments, separate polymer and crosslinker molecules are not required.

Suitable salts that may be included in the mixture are described in section 4.3.1. Suitable conditions for salt formation are described in section 4.3.2. Suitable conditions for crosslinking are described in section 4.3.3. Suitable solvents for dissolving salt crystals are described in section 4.3.4.

4.3.1. Salt

The polymer networks of the present invention are characterized by transport channels that are formed when the polymers are crosslinked in a mixture containing salt crystals formed from an aqueous solution containing at least two types of salts.

The salts are preferably chosen for their compatibility with one or more probes. Ideally, the salt has one or more of the following characteristics: (i) the salt is non-toxic to the probes (e.g., the salt does not denature the probes), (ii) the salt does not chemically react with the probes, (iii) the salt does not react with fluorophores, such as cyanine dyes, which are suitable for optically labeling probes, (iv) the salt does not chemically react with analytes, detection molecules, and/or components that bind to them. Preferably, at least one of the salts forms needles.

In a preferred embodiment, the salt solution includes at least two types of monovalent cations, for example two types of alkali metal cations. Alkali metal cations that can be used include sodium cations and potassium cations, although other alkali metal cations such as lithium cations can also be used.

For an optimal signal-to-noise ratio for detecting nucleic acids of interest, the aqueous salt solution preferably contains sodium and potassium cations and/or has such a total concentration of monovalent cations that when combined with the polymer solution and optional probe solution (before crosslinking), the resulting mixture has a complete a monovalent cation concentration of at least 500 mM. In preferred embodiments, the concentration of sodium ions in the mixture is at least 250 mM and may be in the range of 250 mM to 500 mM, more preferably in the range of 300 mM to 400 mM. In a preferred embodiment, the concentration of sodium ions in the mixture is 350 mm. The concentration of potassium ions in the mixture is preferably at least 150 mM and preferably in the range of 150 mM to 500 mM, more preferably in the range of 200 mM to 400 mM, and even more preferably in the range of 250 mM to 350 mM.

An aqueous salt solution can be prepared using disodium hydrogen phosphate (Na ₂ HPO ₄ ) and/or sodium dihydrogen phosphate (NaH ₂ PO ₄ ), which, in aqueous solution, releases Na ⁺ cations and PO ₄ ^3- phosphate ions. An aqueous salt solution can also be prepared using potassium hydrogen phosphate (K ₂ HPO ₄ ) and/or potassium dihydrogen phosphate (KH ₂ PO ₄ ).

Preferably, the aqueous salt solution may be a sodium phosphate buffer containing disodium hydrogen phosphate and sodium dihydrogen phosphate with the addition of potassium hydrogen phosphate (K ₂ HPO ₄ ) and/or potassium dihydrogen phosphate (KH ₂ PO ₄ ). In one embodiment, the sodium phosphate buffer containing disodium hydrogen phosphate and sodium dihydrogen phosphate and the potassium phosphate buffer containing potassium hydrogen phosphate and potassium dihydrogen phosphate are prepared separately and combined in the same aqueous solution before or after mixing with the polymer and/or probe solutions.

Generally, the aqueous salt solution preferably has a pH value in the range of 6 to 9, and more preferably in the range of 7-8.5. In some exemplary embodiments, the pH is 7.5, 8, or 8.5, most preferably 8.

For grids containing protein-based bioprobes, the aqueous salt solution may include phosphate buffered saline ("PBS") and/or monovalent cation sulfate.

4.3.2. Salt crystal formation conditions Salt crystal formation conditions may include the formation of at least one salt crystal, preferably an acicular salt crystal, in the mixture by dehydrating the mixture or cooling the mixture until the relative salt content of the mixture increases beyond the solubility limit, and this means that the mixture is supersaturated with salt. This stimulates the growth of salt crystals towards the surface of the mixture from a crystallization nucleus located in the bulk of the mixture. Without being limited by theory, it can be assumed that the use of aqueous solutions containing at least two different monovalent metal ions leads to the formation of at least two different types of salt crystals.

The mixture can be dehydrated by heating the mixture, evacuating the mixture, and/or reducing the humidity of the atmosphere around the mixture.

The mixture can be heated (for example, from about 50°C to about 70°C) by placing the mixture on a heating substrate or surface, heating the substrate or surface on which the mixture is placed (for example, from about 50°C to about 70°C), and/or reacting the mixture with a hot gas (eg, air, nitrogen, or carbon dioxide at a temperature above the temperature of the mixture) so that water is evaporated from the mixture. Hot gas contact, for example, can be achieved by placing the mixture in a heating furnace. During transfer to the heating oven, the mixture can be maintained at a humidity of 40% or more, for example at a relative humidity of about 60%, although higher relative humidity, even as high as 75% or more, are also suitable. Mixtures with higher concentrations of potassium ion can withstand lower relative humidity and mixtures with lower concentrations of potassium salt during transportation are preferably kept at higher relative humidity.

In some embodiments, the temperature of the heating substrate and/or the air used to dehydrate the mixture is 20°C or more above the temperature of the mixture prior to heating the mixture, but below 100°C.

The mixture can be cooled (for example, from about 5°C to about 15°C) by placing the mixture on a cooling pad or surface, by cooling the substrate or surface on which the mixture is placed (for example, from about 5°C to about 15°C) and/or reacting the mixture with a cold gas (eg, air, nitrogen, or carbon dioxide at a temperature below the temperature of the mixture). Upon cooling, the temperature-dependent limit of solubility of the salt in the mixture decreases until the mixture eventually becomes supersaturated with salt. This stimulates the formation of one or more salt crystals, preferably acicular. In some embodiments, the implementation of the mixture is cooled by keeping in a cold chamber with low humidity (for example, at a temperature of from 0°C to 10°C, relative humidity < 40%).

During the formation of one or more salt crystals, the temperature in the mixture is preferably maintained above the dew point temperature of the atmosphere around the mixture. This prevents the mixture from diluting with water condensing from the atmosphere, which can lead to a decrease in the relative salt content of the mixture.

4.3.3. Conditions for crosslinking

Crosslinking conditions can be selected based on the type of crosslinker used. For example, when using a cross-linking agent activated by ultraviolet radiation (eg, benzophenone, thioxanthone, or benzoin simple ether), the conditions for cross-linking may include exposure of the mixture to ultraviolet (UV) radiation. In some embodiments, UV radiation having a wavelength of about 250 nm to about 360 nm (eg, 260 ± 20 nm or 355 ± 20 nm) is used. The use of lower energy/longer wavelength UV (eg 360 nm UV versus 254 nm UV) may require longer exposure times. When using a crosslinking agent activated by visible light (eg, ethyleosin, eosin Y, rose bengal, camphorquinone, or erythrosine), crosslinking conditions may include exposure of the mixture to visible light. When using a thermally activated crosslinking agent (for example, 4,4'-azobis(4-cyclopentanoic)acid and 2,2-azobis[2-(2-imidazolin-2-yl)propan]dihydrochloride or benzoyl peroxide), the conditions for crosslinking are may include heating the mixture.

The duration and intensity of exposure to the crosslinking conditions can be selected to ensure crosslinking of polymer molecules with molecules of another polymer, crosslinking of polymer molecules with probe molecules (if present), and crosslinking of polymer molecules with substrate molecules or organic molecules located on the substrate (if present). The duration and intensity of exposure to crosslinking conditions for a mixture containing probes can be determined experimentally, for example, to balance the strength of immobilization and the nativeness of the probe molecules.

4.3.4. Dissolution of salt crystals

After crosslinking the polymer, the salt crystals can be dissolved in the solvent such that at least one transport channel is formed in the network. Without being limited 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, compact crystals and acicular crystals. The dissolution of the compact crystals presumably results in short channels, which result in a sponge-like effect in the network through which the long channels formed by the dissolution of the acicular crystals pass.

By using the array obtained by the method of the present invention as a biological sensor, high measurement accuracy and high measurement speed become possible.

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

Once the salt crystals are removed, the network may collapse due to drying and may be rehydrated. Drying the mesh is useful for transporting and stabilizing probe biomolecules.

4.3.5. Ways to use 3D meshes

The grids and arrays of the present invention can be used to determine the presence or absence of an analyte in a sample, preferably in a liquid sample. Therefore, the present invention relates to methods for determining whether an analyte is contained in a sample, or a plurality of samples, comprising interacting with a grid or array of the present invention, including probe molecules that are capable of binding to the analyte, the sample, or plurality of samples, and detecting binding the analyte to the probe molecules, thereby determining whether the analyte is contained in the sample or a plurality of samples. If the methods use arrays containing different types of probes capable of binding to different types of analyte, then the presence of different types of analytes can be determined by detecting the binding of different types of analytes to the probes. In some embodiments, the methods further include the step of determining the amount of analyte or analytes associated with the array.

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 fluid, milk, tears, feces, sweat, semen, whole cells, cell component, cell swab, or extract or derivative thereof). In some embodiments, the nucleic acid is labeled (eg, fluorescently labeled).

The analyte, placed on the surface of the mesh, can penetrate into the interior of the mesh through a channel for specific binding to a probe (eg, a biomolecule) covalently bound there to a polymer. By using the arrays of the present invention with grids immobilized thereon as a biological sensor, high measurement accuracy and high measurement speed become possible.

The grids and arrays of the present invention can be regenerated after use as a biosensor and can be used multiple 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). once). If the probe molecules are DNA, this can be achieved, for example, by heating the grid(s) in 1x phosphate buffered saline at 80°C to 90°C for about 10 minutes. The phosphate buffered saline can then be replaced with new phosphate buffered saline to wash out the denatured DNA from the mesh(es). If the probe molecules of the grid(s) or array are antigens, the grid(s) or array can be regenerated by treating the grid(s) with 0.1N. NaOH solution for about 10 minutes. Then 0.1 n. the NaOH solution can be replaced with phosphate buffered saline to wash antigens out of the mesh. Thus, some embodiments of the mesh and array methods of the present invention involve the use of a mesh or array that has been washed prior to interaction with a sample or multiple samples.

4.4. The use of arrays proposed in the present invention

Since the arrays of the present invention provide an economical means of detecting and quantifying the presence of nucleic acids in a sample, it is of direct relevance to the health and disease concerns of humans and non-human animals.

In these applications, the formulation containing the target molecule is prepared or extracted from biological or environmental sources according to protocols known in the art. Target molecules can be obtained or extracted from cells and tissues of organisms of all taxonomic classes, including viruses, bacteria and eukaryotes, prokaryotes, unicellular organisms, plants, fungi and animals of all types and classes. The animals may be vertebrates, mammals, primates, and preferably humans. Blood, serum, plasma, tissue, cells, saliva, sputum, urine, cerebrospinal fluid, pleural fluid, milk, tears, feces, sweat, semen, whole cells, cell component, and cell swabs are suitable sources of target molecules.

Target molecules are preferably nucleic acids amplified (eg, by PCR) from any of the above sources.

The arrays of the present invention may include probes that are useful for detecting human and non-human pathogens. Such probes include oligonucleotides that are at least partially complementary to bacterial, viral, or fungal targets, or any combination of bacterial, viral, and fungal targets.

The arrays of the present invention may include probes useful for detecting gene expression in human and non-human cells, for example gene expression associated with a disease or disorder such as cancer, cardiovascular disease, or metabolic disease, to diagnosing the subject, monitoring the subject's treatment, or predicting the outcome of the subject's treatment. Gene expression information can then provide tracking of disease progression or remission, and such information can help monitor completion of treatment or modify initial treatment delivery.

5. TYPICAL PROTOCOLS

The following exemplary protocols, using the numbers found in the accompanying drawings, are within the scope of the present invention and can be used for polymers, crosslinkers, and probes of sections 4.1.1, 4.1.2, and 4.1.4, respectively. Other polymers (including copolymers) and crosslinking groups suitable for use in the procedures described below are described in Rendl et al., 2011, Langmuir 27:6116-6123 and US 2008/0293592, the contents of which are incorporated herein by reference. In one embodiment, a polymer blend of section 6.2 is used.

5.1. Typical protocol 1

After the formation of crystal nuclei (8), (14), spots (7) in the UV crosslinker are immediately irradiated with UV radiation (11) (see Fig. 4), so that the probe biomolecules (1) covalently bind to the polymer (3) and the polymer (3) binds to the organic surface (2) and crosslinks (see Fig. 5). Care must be taken that the dried, cross-linked mixture (5) does not absorb moisture and re-liquid.

Then the dried, cross-linked mixture (5) is introduced into interaction with the solvent (12) for the crystals (8), so that on the places where the crystals (8), (14) were located, in the grid (15), including the polymer (3) and biomolecule probes (1), channels (13) are formed (see Fig. 6), long (13) and short (19) channels are formed. The solvent (12) is then removed. The long channels (13) may extend from the surface (16) of the mesh (15) into the interior of the mesh (15). The solvent (12) in which the salt crystals (8), (14) are dissolved is chosen so that it is compatible with the probe biomolecule (1) and also with the polymer (3). Preferably an aqueous solvent (12) is used.

5.2. Typical Protocol 2

A mixture (5) containing a polymer (3), biomolecule probes (1) and an aqueous salt solution is spotted on an organic surface (2) located on a plate using a standard device for applying DNA spots on a chip (for example, Science, Germany). The volumes applied by printing on each spot (7) are from 0.5 to 4 nl (see Fig. 2). The tablet with spots (7) on the surface (2), preferably organic, is placed on a cooling holder (6) (see Fig. 3). Temperatures from 5°C to 15°C are suitable. The liquid of these spots is cooled to supersaturation of the buffer, which almost immediately leads to the formation of crystal nuclei. After the formation of nuclei, needle-shaped salt crystals (8) can extend from at least one crystallization nucleus (9) located in the volume of mixture (5) to the surface (10) of mixture (5). In addition, the formation of shorter cubic or rod-shaped crystals (14) is presumably occurring (see Fig. 3). After printing, these targets are placed in an oven (eg at 70° C.) to dry completely. After the formation of crystal nuclei, the spots in the UV crosslinker are immediately irradiated with UV radiation (11) (see Fig. 4), so that the probe biomolecules (1) covalently bind to the polymer (3) and the polymer (3) covalently binds to the organic surface ( 2) and stitched together. Care must be taken that the dried, cross-linked mixture does not absorb moisture and re-liquid.

Then the dried, cross-linked mixture (5) is introduced into interaction with the solvent (12) to dissolve the crystals (8), (14), so that at the places where the crystals (8), (14) were located, in the grid (15), including polymer (3) and probe biomolecules (1), transport channels are formed, for example, long channels (13) and short channels (19). The solvent (12) is then removed. The long channels (13) may extend from the surface (16) of the mesh (15) into the interior of the mesh (15). The solvent (12), in which the salt crystals (8), (14) are dissolved, is chosen so that it is compatible with the probe biomolecule (1) and with the polymer (3). Preferably an aqueous solvent (12) is used.

As can be seen in FIG. 6, a plurality of long channels (13) and short channels (19) can be formed in the grid (15). The long channels (13) may extend from the surface (16) of the mesh (15) to at least one point located in the mesh (15). The long channels (13) can be arranged in such a way that, starting from the surface (16) towards the inside, the lateral distance between the channels (13) decreases.

5.3. Typical Protocol 3

A mixture (5) containing polymer (3), biomolecule probes (1) and an aqueous salt solution is printed on the surface (2), preferably organic, of the tablet under normal conditions, at a humidity in the range of 40-80%, preferably 50 -70%. The mixture may contain, for example, 350 mM sodium phosphate, pH 8 and 250-300 mM potassium phosphate, pH 8. The volumes printed on each spot (7) are between 0.5 and 4 nL. Humidity in the printing chamber causes the spots (7) to remain liquid without crystal formation (i.e. no nucleation occurs). The tablet is then placed in a container, such as a cardboard box. Covers are put on for transfer to the tablet. The spotted tablet (7) is then placed in a drying oven or hot plate to rapidly induce nucleation so that salt needles (8) extend from at least one nucleation (9) located in the volume of the mixture towards surfaces (10) of mixture (5). In addition, the formation of shorter cubic or rod-shaped crystals is expected (14).

The temperature of the oven/hotplate should be 20°C or more above the printing temperature. Temperatures above 100°C are optional.

After drying, the mixture is irradiated to cross-link the polymer (3), probe biomolecules (1), and the organic surface (2).

Then the dried, cross-linked mixture (5) is introduced into interaction with the solvent (12) to dissolve the crystals (8), (14), so that at the places where the crystals (8), (14) were located, in the grid (15), including polymer (3) and probe biomolecules (1), long channels (13) and short channels (19) are formed. The solvent (12) is then removed. The long channels (13) may extend from the surface (16) of the mesh (15) into the interior of the mesh (15). The solvent (12), in which the salt crystals (8), (14) are dissolved, is chosen so that it is compatible with the probe biomolecules (1) and with the polymer (3). Preferably an aqueous solvent (12) is used.

5.4. Typical Protocol 4

Alternatively, a plate with spots (7) on a preferably organic surface (2) prepared as described in a typical protocol 3 can be cooled to allow nucleation by placing it in a cold chamber with low humidity (e.g. < 10°C). C, relative humidity < 40%). Drying can be carried out by reducing the humidity or by vacuuming after nucleation has begun. After the formation of nuclei, needle-shaped salt crystals (8) can extend from at least one crystallization nucleus (9) located in the volume of mixture (5) towards the surface (10) of mixture (5). The tablet with spots (7) on the organic surface (2) is placed in an oven at 60°-70°C for 1 hour to completely dry the spot. The spots (7) are irradiated with 1.00 J UV at 254 nm in a UV crosslinker, i. Stratalinker 2400. To do this, the patch plate (7) can be placed in the center of the chamber, with its short side parallel to the chamber door. The lid is then removed and the crosslinking agent begins to act. After the end of the operation of the device, the array is removed and closed with a lid.

Alternatively, other UV crosslinkers at the same wavelength (eg 240-270 nm) or greater wavelengths, eg 360 nm, can be used.

Then the mixture (5) is introduced into interaction with the solvent (12) to dissolve the crystals (8), (14), so that at the places where the crystals (8), (14) were located, in the grid (15), including the polymer ( 3) and probe biomolecules (1), long channels (13) and short channels (19) are formed. The solvent (12) is then removed. The long channels (13) may extend from the surface (16) of the mesh (15) into the interior of the mesh (15). The solvent (12), in which the salt crystals (8), (14) are dissolved, is chosen so that it is compatible with the probe biomolecules (1) and with the polymer (3). Preferably an aqueous solvent (12) is used.

6. EXAMPLES

6.1. Background

Polymer networks prepared essentially as described in the present invention, but containing sodium phosphate buffer ("NaPi") at a concentration of 350 mM, without a second salt, can be dried and undergo a phase transformation if, during drying on a hot plate humidity is not maintained at 60% or more. This is due to the fact that at lower humidity crystallization can proceed in an uncontrolled manner.

It would be desirable to increase the concentration of sodium phosphate to avoid uncontrolled crystallization. However, the concentration of NaPi cannot be increased significantly, as NaPi crystals can then deposit in the printer and block the print head.

Various salts (sodium chloride, sodium bromide) were used in test experiments in combination with NaPi. But none of them resulted in more significant signals than NaPi alone. Typically, hybridization signals from these spots were weak (data not shown).

Other attempts to minimize drying at normal levels of humidity have included testing phosphate buffered saline, sodium citrate buffer, or potassium phosphate buffer instead of NaPi, resulting in weaker hybridization signals in polymer networks.

However, when the NaPi buffer was backed up with potassium phosphate buffer (as described in section 6.2), stabilization of the liquid spots was observed without signal reduction, especially when the potassium phosphate concentration exceeded 150 mM.

During these experiments, the following was unexpectedly discovered. At higher concentrations of potassium phosphate (150 mM or more), the background signal ("noise") decreased and at concentrations of 200 mM, there was almost no noise from the hybridization reaction (as described in section 6.3). The reason for this effect is most likely that the short channels lead to a spongy polymer matrix through which the long sodium phosphate channels pass. Then the combination of these two structures improves not only the kinetics of inclusion (hybridization), but also the kinetics of deactivation (washout of unbound or weakly bound material). A less intense background signal reduces the background signal and therefore improves the LOF (limit of detection) in any test performed using polymer meshes prepared with the simultaneous use of sodium phosphate and potassium phosphate.

6.2. Example 1: Formation of 3D Polymer Networks

A 10 mg/mL polymer stock solution was prepared by dissolving 10 mg of the crosslinked copolymer dimethylacrylamide-5% methacryloyloxybenzophenone-2.5% sodium 4-vinylbenzenesulfonate in 1.0 ml of DNase-free water. This is done by vigorous shaking and agitation for about 5 minutes until all visible polymer is dissolved. The stock solution is then wrapped in foil to protect from light and placed in a refrigerator overnight to ensure complete dissolution of the polymer and reduce the amount of foam. The polymer contains at least two photochemically reactive groups in one molecule.

Various mixtures containing 10mg/mL polymer (PDMAA-5%MABP-2.5%SSNa), probe biomolecules (including DNA oligonucleotides with fluorescent Cy3 fragment), and aqueous saline with 350mM sodium phosphate buffer and in some cases with a variable amount of potassium phosphate by printing applied to the organic surface of the tablet at equal to 65% humidity. Volumes of 1.6 nL were printed onto each spot using a Scienion Sciflex printer. Then the tablet was placed in a container, a cardboard box. Lids were then placed on the shipping plate having an organic surface. Then the plate with spots on the organic surface was placed in a drying oven or hot plate (70°C) to initiate the formation of salt crystals. After drying for 1 hour, the plate was irradiated to crosslink the polymer, probe biomolecules, and the organic surface.

After printing, the plate was washed with 10 mM NaPi buffer to remove unbound material and then dried and stored. The plate was scanned with a Sensovation Fluorescence scanner to visually assess the morphology of the spots. The resulting images are shown in Fig. 11A (after washing) and in FIG. 11B (after drying).

The spots in rows D and E include variable amounts of potassium phosphate (as shown in FIG. 11B) and the spots in the remaining rows were generated with a saline solution containing only sodium phosphate buffer. The incorporation of potassium phosphate resulted in more uniform and rounded polymer networks than for stains made using sodium phosphate alone (Fig. 11A - Fig. 11B). The inclusion of potassium phosphate also always controlled the crystallization when the relative humidity was not increased, eg about normal atmospheric humidity of about 40%.

6.3. Example 2: Quality of hybridization of polymer networks

The arrays described in Example 1 were obtained using S. aureus or E. coli detection probes.

S. aureus and E. coli amplification primer pairs were used in PCR reactions with 100 copies of S. aureus and E. coli genomic DNA, respectively, as templates, and the PCR products were hybridized to arrays containing S. aureus and E. coli probes, respectively. The results are shown in FIG. 12A-12B. In FIG. 12A shows hybridization with an array of PCR product amplified from 100 copies of S. aureus DNA. In FIG. 12B shows hybridization with an array of PCR product amplified from 100 copies of E. coli DNA. The probe map for the arrays shown in FIG. 12A and FIG. 12B is shown in FIG. 12V. This study shows that the signals from polymer networks obtained using an aqueous salt solution containing a potassium phosphate buffer as well as a buffer based on phosphate are comparable to the signal from polymer networks obtained using an aqueous salt solution containing only sodium phosphate.

Surprisingly, it has been found that polymer networks prepared using an aqueous salt solution containing potassium phosphate have reduced background "noise" compared to polymer networks prepared using an aqueous salt solution containing only sodium phosphate, as shown in FIG. 13. See FIG. 13 which shows quantitative data for fluorescence signals from hybridization of a PCR product amplified using the same S. aureus or E. coli specific primer pairs in the absence of a template. Thus, any hybridization signal is background "noise". Background "noise" is absent for polymer networks obtained in the presence of potassium phosphate at higher concentrations.

7. PREFERRED EMBODIMENTS

The following preferred embodiments are examples of the present invention.

1. A method for obtaining a three-dimensional hydrogel network, including: (a) processing a mixture (optionally located on the surface of a substrate, under conditions that ensure the formation of salt crystals, including:

(i) at least two types of monovalent metal ions having a total concentration of at least 500 mM,

(ii) water-soluble polymer chains,

(iii) crosslinker fragments and

(iv) optionally probe molecules and

thereby forming a mixture containing one or more salt crystals;

(b) treating the mixture containing the one or more salt crystals under crosslinking conditions, thereby forming a hydrogel containing the one or more salt crystals; and

(c) reacting a hydrogel containing one or more salt crystals with a solvent in which one or more salt crystals are soluble, thereby dissolving the salt crystals;

thereby forming a three-dimensional hydrogel network.

2. The method according to embodiment 1 wherein the mixture contains at least two types of monovalent metal ions having a total concentration of 500 mM to 1000 mM.

3. The method according to embodiment 2 wherein the total concentration of monovalent metal ions in the mixture is between 550 mM and 800 mM.

4. The method according to embodiment 3 wherein the total concentration of monovalent metal ions in the mixture is between 600 mM and 750 mM.

5. The method according to any one of embodiments 1-4, wherein the mixture contains two types of monovalent metal ions.

6. The method according to embodiment 5, wherein the concentration of each monovalent ion is at least 150 mM or at least 200 mM.

7. The method according to Embodiment 5 or Embodiment 6, wherein the monovalent metal ions are selected from the group consisting of sodium ions, potassium ions and lithium ions.

8. The method according to Embodiment 5 or Embodiment 6, wherein the monovalent metal ions are sodium ions and potassium ions.

9. The method according to embodiment 8 wherein the sodium ion concentration is at least 300 mM.

10. The method of Embodiment 9 wherein the sodium ion concentration is 300 mM to 500 mM.

11. The method according to Embodiment 10 wherein the sodium ion concentration is 300 mM to 400 mM.

12. The method according to embodiment 11 wherein the sodium ion concentration is from 350 mM.

13. The method according to any one of embodiments 8-12, wherein the concentration of potassium ions is 150 mM to 500 mM.

14. The method of Embodiment 13 wherein the potassium ion concentration is 175 mM to 400 mM.

15. The method according to Embodiment 14 wherein the potassium ion concentration is 200 mM to 350 mM.

16. The method of Embodiment 15 wherein the potassium ion concentration is 250 mM to 350 mM.

17. The method according to any one of embodiments 1-4, wherein the mixture contains three types of monovalent metal ions.

18. The method of Embodiment 17 wherein the concentration of at least two monovalent ions is at least 150 mM each or at least 200 mM each.

19. The method according to Embodiment 17 or Embodiment 18, wherein the monovalent metal ions are sodium ions, potassium ions, and lithium ions.

20. The method of Embodiment 19 wherein the sodium ion concentration is at least 250 mM.

21. The method of Embodiment 20 wherein the sodium ion concentration is 250 mM to 500 mM.

22. The method of Embodiment 21 wherein the sodium ion concentration is 300 mM to 400 mM.

23. The method of Embodiment 22 wherein the sodium ion concentration is 350 mM.

24. The method according to any one of embodiments 19-23, wherein the concentration of potassium ions is 150 mM to 500 mM.

25. The method of Embodiment 24 wherein the potassium ion concentration is 200 mM to 400 mM.

26. The method of Embodiment 25 wherein the potassium ion concentration is 250 mM to 350 mM.

27. A method according to any one of embodiments 1-26 which further comprises, up to step (a), forming a mixture.

28. The method of Embodiment 27 wherein the formation of the mixture comprises combining an aqueous salt solution containing monovalent metal cations and one or more solutions containing water-soluble polymer chains, crosslinker moieties and, if present, optional probe molecules. .

29. The method of Embodiment 28 wherein the water-soluble polymer chains, crosslinker moieties are in the same solution.

30. The method of Embodiment 29 wherein the crosslinked moieties are covalently linked to polymer chains.

31. A method according to any one of embodiments 29-30, wherein the aqueous salt solution has a pH value in the range of 6 to 9.

32. The method of Embodiment 31 wherein the aqueous salt solution has a pH in the range of 7 to 8.5.

33. The method of Embodiment 32 wherein the aqueous salt solution has a pH value of 8.

34. The method according to any one of embodiments 29-33, wherein the aqueous salt solution comprises a solution prepared by a procedure comprising dissolving disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, sodium sulfate, potassium sulfate, or a combination thereof in water or aqueous solution.

35. The method according to embodiment 34 wherein the aqueous salt solution is prepared by a procedure comprising dissolving disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, or a combination thereof in water or an aqueous solution.

36. The method of Embodiment 35 wherein the aqueous salt solution is prepared by a procedure comprising dissolving disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate and potassium dihydrogen phosphate, or combinations thereof, in water.

37. A method according to any one of embodiments 1-36 wherein the concentration of phosphate ions in the mixture is at least 250 mM.

38. The method according to embodiment 37 wherein the concentration of phosphate ions in the mixture is 250 mM to 1000 mM.

39. The method according to embodiment 38 wherein the concentration of phosphate ions in the mixture is 550 mM to 800 mM.

40. The method of Embodiment 39 wherein the concentration of phosphate ions in the mixture is 600 mM to 750 mM.

41. A method according to any one of embodiments 1-40, wherein the conditions to form the salt crystal result in the formation of one or more acicular crystals such that one or more long channels are formed after the salt crystals are dissolved.

42. A method according to any one of embodiments 1-41, wherein the conditions to form the salt crystal result in the formation of one or more compact crystals such that one or more short channels are formed after the salt crystals are dissolved.

43. The method according to any one of embodiments 1-42, wherein the conditions to form the salt crystal include dehydration of the mixture.

44. A method according to embodiment 43 which includes dehydrating the mixture by heating the mixture, evacuating the mixture, reducing the humidity of the atmosphere around the mixture, or a combination thereof.

45. The method according to embodiment 44 which comprises dehydrating the mixture by evacuating the mixture.

46. The method according to embodiment 44 which comprises dehydrating the mixture by heating the mixture.

47. A method according to embodiment 46 wherein heating the mixture comprises contacting the mixture with a gas whose temperature is higher than the temperature of the mixture.

48. A method according to any one of embodiments 1-42, wherein the conditions to form the salt crystal include cooling the mixture until the mixture is supersaturated with salt.

49. The method according to embodiment 48 which comprises cooling the mixture by reacting the mixture with a gas whose temperature is lower than the temperature of the mixture.

50. A method according to any one of embodiments 1-49, wherein the temperature of the mixture in step (a) is maintained above the dew point temperature of the atmosphere around the mixture.

51. A method according to any one of embodiments 1-50, wherein the crosslinker moieties are activated with ultraviolet (UV) radiation and the crosslinking conditions include treating the mixture with ultraviolet radiation.

52. A method according to any one of embodiments 1-50, wherein the crosslinker moieties are activated with visible light and the crosslinking conditions include exposing the mixture to visible light.

53. A method according to any one of embodiments 1-50, wherein the crosslinker moieties are heat activated and the crosslinking conditions include heating the mixture.

54. The method according to any one of embodiments 1-53, wherein the water-soluble polymer chains comprise homopolymer chains.

55. The method according to any one of embodiments 1-54, wherein the water-soluble polymer chains comprise copolymer chains.

56. The method according to any one of embodiments 1-54, wherein the water-soluble polymer chains comprise a mixture of homopolymer and copolymer chains.

57. The method according to any one of embodiments 54-56, wherein the water-soluble polymer chains comprise polymer chains polymerized from one or more types of monomers.

58. The method according to Embodiment 57, wherein each type of monomer contains a polymerizable group independently selected from the group consisting of 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.

59. The method of Embodiment 58 wherein at least one type of monomer in the water-soluble polymer contains a methacrylate group.

60. The method of Embodiment 59 wherein at least one type of monomer containing a methacrylate group is methacryloyloxybenzophenone (MABP).

61. The method of any one of embodiments 57, wherein the water-soluble polymer comprises a polymer obtained by copolymerization of dimethylacrylamide (DMAA), methacryloyloxybenzophenone (MABP) and sodium 4-vinylbenzenesulfonate (SSNa).

62. The method according to any one of embodiments 1-61, wherein the water-soluble polymer chains are copolymer chains containing crosslinker moieties.

63. The method of Embodiment 62 wherein the water-soluble polymer chains contain at least two crosslinker moieties per polymer molecule.

64. The method according to any one of embodiments 1-63, wherein the crosslinker moieties are selected from the group consisting of benzophenone, thioxanthone, benzoin ester, ethyl eosin, eosin Y, rose bengal, camphorquinone, erythrosin, 4,4'-azobis(4 -cyclopentanoic) acid, 2,2-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride and benzoyl peroxide.

65. The method of Embodiment 64 wherein the crosslinker moieties are benzophenone moieties.

66. The method according to any one of embodiments 1-65, wherein the solvent is water or an aqueous buffer.

67. The method of Embodiment 66 wherein the solvent is water.

68. The method of Embodiment 66 wherein the solvent is an aqueous buffer.

69. The method of Embodiment 68 wherein the aqueous buffer comprises phosphate, methanol, ethanol, propanol, or a mixture thereof.

70. A method according to any one of embodiments 1-69, wherein the mixture in step (a) further comprises probe molecules.

71. The method of embodiment 70, wherein at least some, most, or all of the probe molecules comprise a nucleic acid, nucleic acid derivative, peptide, polypeptide, protein, carbohydrate, lipid, cell, ligand, or combination thereof.

72. The method of Embodiment 71, wherein at least some of the probe molecules comprise a nucleic acid or nucleic acid derivative.

73. The method of Embodiment 71 wherein at least a majority of the probe molecules comprise a nucleic acid or nucleic acid derivative.

74. The method of Embodiment 71 wherein all probe molecules comprise a nucleic acid or nucleic acid derivative.

75. The method of embodiment 70, 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.

76. The method of embodiment 75 wherein at least some of the probe molecules comprise a nucleic acid.

77. A method according to embodiment 75 wherein at least a majority of the probe molecules comprise a nucleic acid.

78. The method of embodiment 75 wherein all probe molecules comprise a nucleic acid.

79. A method according to any one of embodiments 76-78, wherein the nucleic acid is an oligonucleotide.

80. The method of embodiment 79 wherein the oligonucleotide comprises 12 to 30 nucleotides.

81. The method of embodiment 79 wherein the oligonucleotide comprises 14 to 30 nucleotides.

82. The method of embodiment 79 wherein the oligonucleotide comprises 14 to 25 nucleotides.

83. The method of embodiment 79 wherein the oligonucleotide comprises 14 to 20 nucleotides.

84. The method of embodiment 79 wherein the oligonucleotide comprises 15 to 30 nucleotides.

85. The method of embodiment 79 wherein the oligonucleotide comprises 15 to 25 nucleotides.

86. The method of embodiment 79 wherein the oligonucleotide comprises 15 to 20 nucleotides.

87. The method of embodiment 79 wherein the oligonucleotide comprises 16 to 30 nucleotides.

88. The method of embodiment 79 wherein the oligonucleotide comprises 16 to 25 nucleotides.

89. The method of embodiment 79 wherein the oligonucleotide comprises 16 to 20 nucleotides.

90. The method of embodiment 79 wherein the oligonucleotide comprises 15 to 40 nucleotides.

91. The method of embodiment 79 wherein the oligonucleotide comprises 15 to 45 nucleotides.

92. The method of embodiment 79 wherein the oligonucleotide comprises 15 to 50 nucleotides.

93. The method of embodiment 79 wherein the oligonucleotide comprises 15 to 60 nucleotides.

94. The method of embodiment 79 wherein the oligonucleotide comprises 20 to 55 nucleotides.

95. The method of embodiment 79 wherein the oligonucleotide comprises 18 to 60 nucleotides.

96. The method of embodiment 79 wherein the oligonucleotide comprises 20 to 50 nucleotides.

97. The method of embodiment 79 wherein the oligonucleotide comprises 30 to 90 nucleotides.

98. The method of embodiment 79 wherein the oligonucleotide comprises 20 to 100 nucleotides.

99. The method of embodiment 79 wherein the oligonucleotide comprises 20 to 120 nucleotides.

100. The method of embodiment 79 wherein the oligonucleotide comprises 20 to 40 nucleotides.

101. The method of embodiment 79 wherein the oligonucleotide comprises 20 to 60 nucleotides.

102. The method of embodiment 79 wherein the oligonucleotide comprises 40 to 80 nucleotides.

103. The method of embodiment 79 wherein the oligonucleotide comprises 40 to 100 nucleotides.

104. The method of embodiment 79 wherein the oligonucleotide comprises 40 to 60 nucleotides.

105. The method of embodiment 79 wherein the oligonucleotide comprises 60 to 80 nucleotides.

106. The method of embodiment 79 wherein the oligonucleotide comprises 80 to 100 nucleotides.

107. The method of embodiment 79 wherein the oligonucleotide comprises 100 to 120 nucleotides.

108. The method of embodiment 79 wherein the oligonucleotide comprises 12 to 150 nucleotides.

109. The method according to any one of embodiments 1-108, further comprising up to step (a) the step of applying the mixture to the surface of the substrate.

110. The method of embodiment 109 wherein the mixture is applied in a volume of 100 pl to 1 nl.

111. The method of embodiment 109 wherein the mixture is applied in a volume of 100 pl to 1 nl.

112. The method of embodiment 109 wherein the mixture is applied in a volume of 200 pl to 1 nl.

113. A method according to any one of embodiments 109-112 wherein the step of applying the mixture to the substrate comprises spraying the mixture onto the surface of the substrate.

114. The method of Embodiment 113 wherein the mixture is sprayed with an inkjet printer.

115. A method according to any one of embodiments 109-114 wherein the substrate comprises an organic polymer or an inorganic material containing a self-aggregated monolayer of organic molecules on the surface.

116. The method of embodiment 115 wherein the substrate comprises an organic polymer.

117. The method of Embodiment 116 wherein the organic polymer is selected from the group consisting of cycloolefin copolymers, polystyrene, polyethylene, polypropylene, polycarbonate, and polymethyl methacrylate.

118. The method of embodiment 117 wherein the substrate comprises polymethyl methacrylate, polystyrene, or cycloolefin copolymers.

119. The method of embodiment 115 wherein the substrate comprises an inorganic material containing an alkylsilane self-aggregated monolayer on the surface.

120. A method according to any one of embodiments 109-119 wherein the substrate comprises a microplate.

121. A method according to any one of embodiments 109-120 wherein the polymer is crosslinked to the surface in step (b).

122. The method of Embodiment 121 produces a water-swellable polymer that is cross-linked to a surface.

123. The method of Embodiment 122 wherein the water-swellable polymer can absorb up to 50 times its weight of deionized distilled water.

124. The method of Embodiment 122 or Embodiment 123 wherein the water-swellable polymer can absorb 5 to 50 times its volume of deionized distilled water.

125. The method of any one of embodiments 122-124 wherein the water-swellable polymer can absorb up to 30 times its weight of saline.

126. The method of any one of embodiments 122-125, wherein the water-swellable polymer can absorb 4-30 times its volume of saline.

127. A method of obtaining an array, including the formation of a plurality of three-dimensional hydrogel networks in a manner corresponding to any of embodiments 1-126, on separate spots on the surface of the same substrate.

128. The method according to embodiment 127 wherein the three-dimensional hydrogel networks are formed simultaneously.

129. The method according to embodiment 127, wherein three-dimensional hydrogel networks are formed sequentially.

130. A method according to any one of embodiments 127-129, further comprising stitching a plurality of 3D hydrogel networks to the surface of a substrate.

131. A method of obtaining an array, including placing a plurality of three-dimensional hydrogel networks obtained or obtained by a method corresponding to any of embodiments 1-126, on separate spots on the surface of the same substrate.

132. A method according to any one of embodiments 127-131, further comprising stitching three-dimensional hydrogel networks to a surface.

133. A method of obtaining an array, including placing a plurality of three-dimensional hydrogel networks obtained or obtained by the method corresponding to any of embodiments 109-126, on separate spots on the surface of the same substrate.

134. The method of Embodiment 133 wherein the placement comprises applying mixtures that form three-dimensional hydrogel networks to individual spots.

135. A method according to any one of embodiments 127-134 wherein the spots are arranged in columns and/or rows.

136. Three-dimensional hydrogel network obtained or obtained by the method corresponding to any of embodiments 1-126.

137. An array including a plurality of 3D hydrogel networks according to any of the embodiments 136 on a substrate.

138. An array obtained or obtained by a method corresponding to any of embodiments 127-135.

139. An array corresponding to Embodiment 137 or Embodiment 138, which includes at least 8 3D hydrogel meshes.

140. An array corresponding to embodiment 137 or embodiment 138, which includes at least 16 3D hydrogel meshes.

141. An array corresponding to embodiment 137 or embodiment 138, which includes at least 24 3D hydrogel meshes.

142. An array corresponding to Embodiment 137 or Embodiment 138, which includes at least 48 3D hydrogel meshes.

143. An array corresponding to Embodiment 137 or Embodiment 138, which includes at least 96 3D hydrogel meshes.

144. An array corresponding to Embodiment 137 or Embodiment 138, which includes at least 128 3D hydrogel meshes.

145. An array corresponding to Embodiment 137 or Embodiment 138, which includes at least 256 3D hydrogel meshes.

146. An array corresponding to Embodiment 137 or Embodiment 138, which includes at least 512 3D hydrogel meshes.

147. An array corresponding to embodiment 137 or embodiment 138, which includes at least 1024 3D hydrogel meshes.

148. An array corresponding to Embodiment 137 or Embodiment 138, which includes 24 to 8192 3D hydrogel meshes.

149. An array corresponding to Embodiment 137 or Embodiment 138, which includes 24 to 4096 3D hydrogel meshes.

150. An array corresponding to Embodiment 137 or Embodiment 138, which includes 24 to 2048 3D hydrogel meshes.

151. An array corresponding to Embodiment 137 or Embodiment 138, which includes 24 to 1024 3D hydrogel meshes.

152. An array corresponding to Embodiment 137 or Embodiment 138, which includes from 24 3D hydrogel meshes.

153. An array corresponding to Embodiment 137 or Embodiment 138, which includes 48 3D hydrogel meshes.

154. An array corresponding to Embodiment 137 or Embodiment 138, which includes 96 3D hydrogel meshes.

155. An array corresponding to Embodiment 137 or Embodiment 138, which includes 128 3D hydrogel meshes.

156. An array corresponding to Embodiment 137 or Embodiment 138, which includes 256 3D hydrogel meshes.

157. An array corresponding to Embodiment 137 or Embodiment 138, which includes 512 3D hydrogel meshes.

158. An array corresponding to embodiment 137 or embodiment 138, which includes 1024 3D hydrogel meshes.

159. An array according to any one of embodiments 137-158, wherein the 3D hydrogel networks include probe molecules and two or more 3D hydrogel networks include the same types of probe molecules.

160. An array according to any one of embodiments 137-159, wherein the 3D hydrogel networks include probe molecules and two or more 3D hydrogel networks include the same types of probe molecules.

161. An array according to any one of embodiments 137-158, wherein the 3D hydrogel networks include probe molecules and each of the 3D hydrogel networks includes the same types of probe molecules.

162. An array according to any one of embodiments 137-161, wherein the plurality of 3D hydrogel networks includes one or more 3D hydrogel networks comprising labeled control probe molecules.

163. Array corresponding to option 162, in which the labeled control probe molecules are fluorescently labeled.

164. An array according to any one of embodiments 137-163, wherein the substrate comprises a microplate and each well of the microplate contains no more than one 3D hydrogel mesh.

165. A method for determining whether an analyte is contained in a sample, including:

(a) interaction of a three-dimensional hydrogel network corresponding to the embodiment 136 or an array corresponding to any of the embodiments - 164, including probe molecules that are capable of binding to the analyte in the sample; and

(b) detecting the binding of the analyte to the probe molecules in the three-dimensional hydrogel network or array, thereby determining whether the analyte is contained in the sample.

166. The method of embodiment 165, which further comprises washing the grid or array comprising probe molecules between steps (a) and (b).

167. The method of Embodiment 165 or Embodiment 166, which further comprises reacting a mesh or array including probe molecules with a blocking reagent prior to step (a).

168. The method according to any of embodiments 165-167, further comprising determining the amount of analyte associated with a three-dimensional hydrogel network or array comprising probe molecules.

169. A method for determining whether an analyte is contained in each sample of a plurality of samples, including:

(a) interacting with an array corresponding to any of embodiments 137-167, including probe molecules that are capable of binding to the analyte in the samples; and

(b) detecting the binding of the analyte to the probe molecules in the array, thereby determining whether the analyte is contained in each sample of the plurality of samples.

170. A method for determining whether an analyte is contained in each sample of a plurality of samples, including:

(a) reacting an array according to any one of embodiments 137-164 comprising probe molecules that are capable of binding to the analyte in the samples and comprising control probe molecules, in which the array was used and washed to step (a); and

(b) detecting the binding of the analyte to the probe molecules in the array, thereby determining whether the analyte is contained in each sample of the plurality of samples.

171. A method for determining whether a sample contains more than one type of analyte, including:

(a) interacting with an array corresponding to any of embodiments 137-164 containing different types of probe molecules that are capable of binding to different types of analytes in the sample; and

(b) detecting binding of analytes to probe molecules in the array, thereby determining if the sample contains more than one type of analyte.

172. A method for determining whether a sample contains more than one type of analyte, including:

(a) interaction of an array according to any one of embodiments 137-164, comprising different types of probe molecules that are capable of binding to different types of analytes in the sample and including control probe molecules, in which the array was used and washed up to step (a) ; and

(b) detecting binding of analytes to probe molecules in the array, thereby determining if the sample contains more than one type of analyte.

173. A method according to any one of embodiments 169-172, wherein:

(a) the array substrate includes a microplate;

(b) each well of the microplate contains no more than one three-dimensional hydrogel mesh; and

(c) the interaction of the array with the samples includes the interaction of each well with no more than one sample.

174. The method of any one of embodiments 169-173, which further comprises washing the array comprising probe molecules between steps (a) and (b).

175. The method of any one of embodiments 169-174, which further comprises reacting an array comprising probe molecules with a blocking reagent prior to step (a).

176. A method according to any one of embodiments 169-175, further comprising determining the amount of analyte or analytes associated with the array.

177. A method according to any of embodiments 165-176, further comprising reusing the array.

178. The method of embodiment 177 wherein the array is reused at least 5 times.

179. The method of embodiment 177 wherein the array is reused at least 10 times.

180. The method of embodiment 177 wherein the array is reused at least 20 times.

181. The method of embodiment 177 wherein the array is reused at least 30 times.

182. The method of embodiment 177 wherein the array is reused at least 40 times.

183. The method of embodiment 177 wherein the array is reused at least 50 times.

184. The method of embodiment 178 which includes reusing the array 5 to 20 times.

185. The method of embodiment 178 which includes reusing the array 5 to 30 times.

186. The method of embodiment 178 which includes reusing the array 10 to 50 times.

187. The method of embodiment 178 which includes reusing the array 10 to 20 times.

188. The method of embodiment 178 which includes reusing the array 10 to 30 times.

189. The method of embodiment 178 which includes reusing the array 20 to 40 times.

190. The method of embodiment 178 which includes reusing the array 40 to 50 times.

191. A method according to any one of embodiments 177-190 which includes flushing the array between reuses.

192. The method of Embodiment 191 wherein the array is washed under denaturing conditions.

193. The method of Embodiment 192, wherein the denaturing conditions include heating the array.

194. The method of embodiment 192 wherein the conditions for denaturation include treating the array with low concentrations of salt.

195. A method according to embodiment 192 wherein the conditions for denaturing include heating the array and treating the array with low salt concentrations.

196. The method of embodiment 192 wherein the conditions for denaturation are eliminated before reuse.

197. The method of Embodiment 196, wherein the denaturing conditions include heating the array, and wherein the temperature is reduced prior to reuse.

198. The method of Embodiment 196, wherein the denaturing conditions include treating the array with low salt concentrations, and wherein the salt concentration is increased prior to reuse.

199. The method of Embodiment 196, wherein the denaturing conditions include heating the array and treating the array with low salt concentrations, and wherein the temperature is reduced and the salt concentration is increased prior to reuse.

200. The method of any one of embodiments 177-199 wherein the array includes at least one 3D hydrogel mesh comprising a fluorescently labeled oligonucleotide to control reusability.

201. The method of embodiment 200 which includes determining the intensity of a fluorescence signal.

202. The method of Embodiment 201 wherein the intensity of the fluorescence signal for reuse control after 10 uses is at least 70% of the original value.

203. The method of Embodiment 202 wherein the intensity of the fluorescence signal for reuse control after 20 uses is at least 50% of the original value.

204. The method of any one of embodiments 200-203, wherein the array is no longer reused after the fluorescence signal intensity of the reusability control probe is less than 50% of the original value.

205. A method according to any of embodiments 165-204, wherein the analyte is a nucleic acid.

206. The method of embodiment 205 wherein the nucleic acid is a polymerase chain reaction (PCR) amplicon.

207. The method of embodiment 205 wherein the PCR amplicon is amplified from a biological or environmental sample.

208. The method of embodiment 207 wherein the PCR amplicon is amplified from a biological sample

209. The method of embodiment 207 wherein the PCR amplicon is amplified from an environmental sample.

210. The method of embodiment 208 wherein the biological sample is blood, serum, plasma, tissue, cells, saliva, sputum, urine, cerebrospinal fluid, pleural fluid, milk, tears, feces, sweat, semen, whole cells, component cells, cell smear, or an extract or derivative thereof.

211. The method of embodiment 210 wherein the biological sample is mammalian blood, serum, or plasma, or an extract thereof.

212. The method of embodiment 211 wherein the biological sample is human or bovine blood, serum, or plasma, or an extract thereof.

213. The method of embodiment 210 wherein the biological sample is milk or an extract thereof.

214. The method of embodiment 213 wherein the biological sample is cow's milk or an extract thereof.

215. A method according to any of embodiments 205-214 wherein the nucleic acid is labeled.

216. The method of embodiment 215 wherein the nucleic acid is fluorescently labeled.

While various specific embodiments have been illustrated and described, it should be understood that various changes may be made to the present invention without departing from the spirit and scope of the present invention.

8. NOTICE OF LINKS

All publications, patents, patent applications, and other documents referred to in the specification are hereby incorporated by reference in their entirety by reference to the entire subject matter, to the same extent as if each individual publication, patent, patent application, and other document were individually listed as included in the present invention as a reference for all objects. In the event that there is a discrepancy between the provisions of one or more of the references included in the present invention and the present invention, the provisions of the present invention shall apply.

Claims (32)

1. A method for obtaining a three-dimensional hydrogel network having one or more transport channels, including: (a) processing a mixture comprising: (i) at least two types of monovalent alkali metal ions having a total concentration of at least 500 mM, (ii) water-soluble polymer chains, (iii) crosslinker fragments, and (iv) optionally probe molecules, under conditions allowing the formation of salt crystals, optionally in which the mixture is on the surface of the substrate, and optionally in which the conditions providing formation of salt crystals include dehydration of the mixture or cooling of the mixture until the relative salt content in the mixture increases to exceed the solubility limit, thus thereby forming a mixture containing one or more salt crystals; (b) crosslinking polymer chains in a mixture containing one or more salt crystals, thereby forming a hydrogel containing one or more salt crystals; and (c) reacting a hydrogel containing one or more salt crystals with a solvent in which one or more salt crystals are soluble, thereby dissolving the salt crystals; thereby forming a three-dimensional hydrogel network that contains one or more transport channels. 2. The method of claim 1, wherein the mixture contains at least two types of monovalent alkali metal ions having a total concentration of 500 mM to 1000 mM. 3. The method of claim 2, wherein the mixture comprises sodium ions at a concentration of 200 mM or more and potassium ions at a concentration of 150 mM or more, optionally wherein: (a) the concentration of sodium ions is in the range from 300 mm to 400 mm; and (b) the concentration of potassium ions is in the range from 200 mm to 350 mm. 4. The method according to any one of paragraphs. 1-3 which further comprises, up to step (a), forming a mixture, optionally by combining an aqueous salt solution containing monovalent alkali metal cations and one or more solutions containing water-soluble polymer chains, crosslinker moieties and, if they are contained, optional probe molecules. 5. The method of claim 4, wherein the aqueous salt solution has a pH value in the range of 6 to 9. 6. The method according to claim 4 or 5, wherein the aqueous salt solution is prepared by a procedure comprising dissolving disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate and potassium dihydrogen phosphate, or combinations thereof, in water. 7. The method according to any one of paragraphs. 1-6, wherein the water-soluble polymer chains include methacrylate groups and at least two crosslinker moieties per molecule, optionally wherein the crosslinker moieties are benzophenone moieties. 8. The method of claim 7 wherein the water-soluble polymer chains are polymerized from dimethylacrylamide (DMAA), methacryloyloxybenzophenone (MABP) and sodium 4-vinylbenzenesulfonate (SSNa). 9. The method according to any one of paragraphs. 1-8, wherein the mixture in step (a) comprises probe molecules, optionally wherein the probe molecules are nucleic acid molecules. 10. Three-dimensional mesh obtained or obtained by the method according to any one of paragraphs. 1-9. 11. A three-dimensional network comprising cross-linked water-soluble polymer chains and one or more probe molecules, and having one or more long transport channels and one or more short transport channels, optionally, in which the probe molecules are nucleic acid molecules , wherein (a) each long transport channel is substantially straight and in the hydrated state of the mesh has a minimum cross section which is at least 300 nm and a length which is at least 3 times the minimum passage cross section; and (b) each short transport channel is substantially straight and, when hydrated, the mesh has a length that is less than 3 times its minimum passage area. 12. The 3D mesh of claim 11, wherein at least one short transport channel is traversed by one or more long transport channels. 13. An array including a plurality of three-dimensional grids according to any one of paragraphs. 10-12, wherein (a) the 3D meshes are immobilized on the substrate and (b) each of the 3D meshes is in a separate spot on the substrate, optionally in which the array can be reused at least 10 times. 14. The method of obtaining an array, including obtaining a plurality of three-dimensional hydrogel networks by the method according to any one of paragraphs. 1-9 on individual spots on the surface of the same substrate, and cross-linking the meshes to the substrate in step (b). 15. A method for determining whether an analyte is contained in a sample, comprising: (a) the interaction of a three-dimensional hydrogel network according to any one of paragraphs. 10-12 or the array of claim 13, said network or array includes probe molecules that are capable of binding to an analyte in the sample; and (b) detecting the binding of the analyte to the probe molecules in the three-dimensional hydrogel network or array, thereby determining whether the analyte is contained in the sample. 16. The method of claim 15 wherein the mesh or array has been used and washed at least 10 times before step (a) or which further comprises reusing the mesh or array at least 10 times after step (b). 17. The method of claim 16, which further comprises quantifying analyte binding to probe molecules in a three-dimensional grid.

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