Patent Description:
Testing samples for the presence of analytes of interest can be important in a variety of applications, including disease diagnostics, food and water safety and environmental surveillance. A variety of analytical methods can be performed on samples to determine if a sample contains a particular analyte.

One area of application of such analytical methods is in pharmaceutical companies nowadays applying high throughput drug discovery systems which enable automated, highly miniaturized screening of hundreds of thousands of samples to discover pharmacologically active compounds interacting with therapeutic targets on the molecular level. There is a strong need for a reliable early-on evaluation of the pharmacological potential of these compounds in order to determine the most promising candidates for the costly drug discovery. As a lot of compounds are available in only small amounts, it is of particular importance that the techniques utilized provide highly specific and sensitive detection of the analyte of interest. There is also a continued need for sensitive diagnostic and prognostic methods of detecting analytes. For example, an important parameter in tuberculosis diagnostics (interferon gamma) is detected and quantified millions of times in diagnostic laboratories worldwide. There is also high demand for diagnostic applications in cancer samples that may or may not contain cancer cells and to assess the level of cancer in patients before, during and/or after treatment. Similarly, autoimmune diseases is another area in which there exists a continued need for diagnostic, prognostic, and therapeutic analytical methods.

Such analytical methods are carried out on assay substrates. An assay substrate is a surface upon which various chemical and/or biological analyses can be performed. Examples of assay substrates include microarray plates, glass plates, and microtiter plates. A microtiter plate is a flat plate that has multiple containers or "wells" formed in its surface. Each well can be used as a small test tube into which various materials can be placed to perform biochemical analyses. One illustrative use of microtiter plates includes enzyme-linked immunosorbent assay (ELISA).

<NPL>) relates to a process for immobilization of biomolecules on polymer substrates based on surface-attached polymer networks. <CIT> relates to three-dimensional crosslinked polymer networks, arrays comprising the networks, processes for making the networks, and uses of the networks and arrays. <NPL>) relates to surface-attached dual-functional hydrogel for controlled cell adhesion based on poly(N,N-dimethylacrylamide). <CIT> relates to photo-crosslinked hydrogel surface coatings using a polymeric or copolymeric precursor that includes, respectively, monomer subunits that have a photo-crosslinkable functionality and monomer subunits that have a chemically selective functionality for binding a biomolecular analyte. <CIT> is in the field of immunochemistry and organic chemistry and relates to means and methods for immobilizing a molecule, such as an antigen to a solid support. <CIT> relates to polymers having orthogonal reactive groups and solid supports comprising polymers immobilized thereto. <CIT> relates to a cell culture system or assembly, flask, plate and dish for growing, viewing and evaluating cells; and methods of use. In recent years, the target of cell analysis has become subdivided from cell colony level into one cell level. Attempts are being made to capture cells one by one, identify, sort, collect, and culture the cells, and perform analytical techniques. As the method for identifying and capturing the cells, a method is adopted in which secretions from the cells are separated, target secretions are detected, and then the cell secreting the target secretions is screened out. For the single-cell analysis described above, a technique of comprehensively capturing and analyzing cells is required. The ELISpot (Enzyme-linked-immuno-Spot) test is a sensitive method to measure cell activation at the single cell level. This cell activation releases cytokines, which are then made visible as colored dots (spots) in the ELISpot using monoclonal antibodies. A well-known problem with analytical techniques such as ELISpot technology is the creation of clearly defined spots after the detection reaction has been completed. Spot generation is influenced, among other things, by high non-specific background signals, a "washing out" of the edges and the mixing of the individual spots into clusters. Here, however, cells that are close together can also cause interference of the spots that form.

Therefore, there is a need in the art for high immobilized concentrations of the capture antibodies leading to a rapid local binding of the antigens secreted by the cells in the immediate vicinity of the cell body, which minimizes the problems with sensitivity such as the washing out of the spots in the ELISpot technology and reducing so-called "specific background signals" on the detection surface. Furthermore, there is a need to combine such sensitive analytical techniques with their ability to specifically capture, separate and identify the secretions by individual cells.

The present invention relates to functionalized hydrogel coatings of assay plates. Disclosed herein is a novel method of manufacturing a surface-bonded continuous layer of a copolymer that can be applied to assay plates used in analytical or diagnostic applications. The present invention provides a container comprising such a layer and uses thereof.

The present invention involves the techniques of hydrogels, click chemistry, and photomasks for photolithographic structuring using a photosensitive radical reaction. In the present invention, it has been proven that such structures on surface-bonded continuous copolymer layers of the invention enhance the capturing and separation of agents and/or objects, in particular cells. It has surprisingly been found that the present invention reduces non-specific background reactions and allows for an increase in speed, specificity and sensitivity of the detection of analytes. Accordingly, the means and methods provided by the present invention are particularly useful in analytical and/or diagnostic applications, including immunoaffinity assays, ELISA or ELISpot assay.

In certain aspects of the invention, the copolymer is characterized by a monolithic, topographical modification of the surface through a unique, temporally and spatially selective exposure to UV light. After this exposure, either a UV-reactive side chain or the click-reactive side chain is retained to bind a biomolecule. The surface can be made up of polystyrenes, cyclic olefin polymers, poly(methyl methacrylates) or polycarbonates. The copolymer layer (or hydrogel layer) can be used topographically for the separation of objects, in particular cells. For example, circular column structures (diameter and/or width <NUM>-<NUM>, height <NUM>-<NUM>) with circular distances (diameter <NUM>-<NUM>) in a hexagonal arrangement can be used. For the production of the copolymer (or hydrogel) layer, the polymer can be dissolved in a solvent, in particular ethanol, and dispensed, e.g., into a container. After the ethanol has evaporated, the polymer layer can be cross-linked to form a hydrogel by means of UV light (e.g., <NUM>-<NUM>) and attached to the surface of the bottom end of the container. If the polymer offers a bond-reactive group (e.g., azide), a previously modified biomolecule (e.g., an antibody which has been labeled with an alkaline group, e.g., dibenzocyclooctyne (DBCO)) can be coupled to it. This biomolecule can then bind the analyte that is to be detected in, e.g., an immunoaffinity assay. The analyte can then be bound to another epitope in these assays with a second enzyme-labeled antibody. In a further step, the enzyme can then initiate a color reaction on the substrate.

Disclosed herein is a method of manufacturing a surface-bonded continuous layer of a copolymer with a synthetic one-component system comprising at least one photo-reactive side group for a C, H-insertion cross-linking ["CHic"] reaction; and at least one click binding-reactive side group ["Click"] for (bio-)functionalization of the copolymer layer. Further disclosed herein is a method of manufacturing a container having a surface-bonded continuous layer of such a copolymer with a synthetic one-component system comprising at least one photo-reactive side group for a C, H-insertion cross-linking ["CHic"] reaction; and at least one click binding-reactive side group ["Click"] for (bio-) functionalization of the copolymer layer. Also disclosed herein are methods of 3D functionalization of a container using the surface-bonded continuous layer of a copolymer of the invention. Disclosed herein is a surface-bonded continuous layer of a copolymer manufactured by the methods disclosed herein. ln particular, the present invention provides a container, as defined by the appended claims, manufactured by the methods disclosed herein. The present invention further provides uses of the manufactured container or a container assembly or an analytical device comprising the container of the invention in analytical and/or diagnostic applications, as defined by the appended claims.

The present invention provides in particular:.

Further disclosed herein is a method of manufacturing a surface-bonded continuous layer of a copolymer, comprising the steps:.

Disclosed herein, but not forming part of the invention as defined by the appended claims, is a method of manufacturing a container having a continuous layer of a copolymer bonded to the surface of the bottom end of the container, comprising the steps:.

The _container is formed of polymeric material and has an open top end, a closed bottom end, and at least one wall extending between said ends.

The amount of copolymer applied to the surface is at least <NUM>µg/mm<NUM>, preferably at least <NUM>-<NUM>µg/mm<NUM>.

The method may further comprise a step of photostructuring the surface-bonded copolymer layer by a spatially selective exposure of the copolymer layer to light, preferably UV light.

The photostructuring generates hill-like structures on the continuous copolymer layer while maintaining a monolithic topography, preferably wherein the hill-like structures exhibit a hexagonal arrangement.

The hill-like structures have a diameter of <NUM>-<NUM> and/or a height of <NUM>-<NUM>.

Disclosed herein is a surface-bonded continuous layer of a copolymer obtainable by the method of as described herein.

The copolymer layer may be photostructured while maintaining a monolithic topography, wherein the copolymer layer may have hill-like structures, wherein the hill-like structures may exhibit a hexagonal arrangement.

The at least three different monomer units comprise a hydrophilic monomer (with or without partial charge) as another of the at least three different monomer units, preferably a hydrophilic acryl monomer (with or without partial charge), and/or wherein at least one monomer is a photoreactive crosslinker, and/or wherein the at least one click binding-reactive side group is an azide or a derivative thereof.

Further disclosed herein is a method of separating dispersed particles and/or objects, comprising a surface-bonded continuous layer of a copolymer according to the invention. The dispersed particles and/or objects may be cells.

Disclosed herein is the method as described above, wherein step (a) comprises:.

In accordance with the present invention, particularly provided is a container formed of polymeric material and having an open top end, a closed bottom end, and at least one wall extending between said ends, characterized in that the surface of the bottom end has a surface-bonded continuous copolymer (hydrogel) layer having a monolithic topography, wherein the copolymer comprises at least three different monomer units, wherein.

The surface-bonded continuous copolymer (hydrogel) layer may have hill-like structures, preferably wherein the hill-like structures exhibit a hexagonal arrangement. More specifically, the hill-like structures may have a diameter of <NUM>-<NUM> and/or a height of <NUM>-<NUM>. The container may comprise an optically transparent part, and/or the container may have an essentially tubular shape and/or has a conical shape towards the bottom end of the container, preferably the container is a laboratory test tube, e.g. a well of a microplate. Still further, the hydrophilic monomer without partial charge may be a hydrophilic acryl monomer, and/or the at least one click binding-reactive side group may be an azide or a derivative thereof. The container may be used in analytical and/or diagnostic applications, preferably in an immuno-affinity assay, ELISA or ELISpot assay. More specifically, the container may be used for (a) separation of dispersed particles and/or objects, preferably the dispersed particles and/or objects are cells; or (b) the selective covalent attachment and/or capturing of biochemical reagents to the copolymer layer.

As further described herein, in various preferred embodiments of the present invention, the container formed of polymeric material and having an open top end, a closed bottom end, and at least one wall extending between said ends, is characterized in that the surface of the bottom end has a surface-bonded continuous copolymer layer extending to said at least one wall and is further characterized in that the surface-bonded continuous layer is generated by crosslinking a copolymer to a monolithic topography (or structure), wherein the copolymer comprises at least three different monomer units as describe elsewhere herein. Accordingly, in various embodiments, the container (assembly) as well as the uses thereof disclosed herein may be characterized in that the surface-bonded continuous copolymer layer is generated (or formed) on the (solid) surface by (a step of) crosslinking the copolymer to (generate or form a) a monolithic topography (or structure). As described elsewhere herein and as demonstrated in the present invention, the monolithic topography (or structure) of the continuous copolymer layer bonded to the surface of a container is maintained despite photostructuring. Accordingly, as further described herein, in various preferred aspects and embodiments of the present invention, the surface-bonded continuous copolymer layer is photostructured while crosslinking and is thereby maintaining a monolithic topography (or structure), preferably the copolymer layer has hill-like structures, preferably the hill-like structures exhibit a hexagonal arrangement and are generated on top but within the monolithic topography.

Disclosed herein, but not part of the claimed invention, is a method of manufacturing a surface-bonded continuous layer of a copolymer with a synthetic one-component system comprising at least one photo-reactive side group for a C, H-insertion cross-linking ["CHic"] reaction; and at least one click binding-reactive side group ["Click"] for (bio-)functionalizing of the copolymer layer. In particular, disclosed herein is a method of manufacturing a surface-bonded continuous layer of a copolymer, comprising the steps:.

The method disclosed herein involves obtaining a surface-bonded continuous layer of a copolymer subsequent to steps (a), (b), and (c).

In various preferred embodiments of the present invention, the monomer unit having a photo-reactive side group for a "CHic" reaction (or being a photo-reactive cross-linker with a side group for a CHic reaction) can react independently of the other (residual) two of the at least three monomer units, for a "CHic" reaction. Accordingly, in various preferred embodiments of the present invention, the monomer unit having at least one click binding-reactive side group ["Click"] for (bio-)functionalizing of the copolymer layer, is different from the monomer unit having a photo-reactive side group for a "CHic" reaction (or being a photo-reactive cross-linker with a side group for a CHic reaction), and can react independently of the monomer unit having a photo-reactive side group for a "CHic" reaction (or being a photo-reactive cross-linker with a side group for a CHic reaction).

Accordingly, in various preferred embodiments of the present invention, the monomer unit having a photo-reactive side group for a "CHic" reaction (or being a photo-reactive cross-linker with a side group for a CHic reaction) is a monomer unit distinct (and/or different) from the monomer unit having at least one click binding-reactive side group ["Click"] for (bio-)functionalizing of the copolymer layer, and vice versa. In various preferred embodiments, the monomer unit having a photo-reactive side group for a "CHic" reaction (or being a photo-reactive cross-linker with a side group for a CHic reaction) may not exhibit the function or reaction of the monomer unit having at least one click binding-reactive side group ["Click"] for (bio-)functionalizing of the copolymer layer, and vice versa.

As used herein, the term "copolymer" preferably refers to alternating polymers formed by the polymerization reaction of at least two different monomers, i.e., two, three or more different monomers. Thus, the term "copolymer" as used herein does not exclude, e.g., so-called terpolymers obtained from co-polymerization of three different monomers. The term "alternating copolymer" as used herein refers to a polymer having two or more different types of monomers joined together in the same polymer chain wherein the different monomers are arranged in a random (statistical) order.

In various preferred embodiments, the copolymer is a one-component compound, preferably wherein the copolymer is an alternating copolymer. A component as used herein may be one of a collection of chemically-independent constituents of a system. The number of components represents the minimum number of independent species necessary to define the composition of all phases of the system. Accordingly, one-component compound as used herein means that the copolymer represents a compound that requires only one of the independent monomers of the copolymer in order to define all phases of the compound. Accordingly, the term "one-component system" means that all of the components of the copolymer which are required for the subsequent steps are already contained in the one-component copolymer. The term "monomer" as used herein can refer to a chemical (or material thereof) characterized as having a molecule (or unit) that can bind to other molecules. Large numbers of monomer units can bind to form polymers.

In various embodiments, the copolymer comprises at least one monomer unit that is a photo-reactive side group for a C, H-insertion cross-linking reaction. In preferred embodiments, the photo-reactive side group for a C, H-insertion cross-linking reaction is a photoreactive crosslinker. C, H-insertion reactions concern the insertion reaction of e.g. carbenes, nitrenes or ketyl biradicals into a carbon-hydrogen bond. This organic reaction is of some importance in the synthesis of new organic compounds in which only one reactive group is contained, which after activation reacts with the neighboring polymer chains through C, H-insertion reactions. In such reactions the cross-linking units can be incorporated into the polymer matrix and induce the linking of the involved polymer chains after thermal or UV-activation by (formal) C, H-insertion. Upon imparting heat or light into the film, the cross-linker units can be activated and generate a reactive intermediate, which depending on the chemical nature of cross-linker reacts with almost any groups in the neighboring chains through a C, H-insertion reaction (CHic = C, H-insertion based crosslinking). This crosslinking reaction pathway offers the advantage that there is no need that two reaction partners meet each other before connecting as one reaction partner - the C, H group - is present in abundance. The term "photoreactive crosslinker" refers to photo-activable reactive groups for binding proteins, nucleic acids and other biomolecules in an irreversible manner after activated by ultraviolet or visible light. As described herein, the terms "photoreactive crosslinker" and "photochemical crosslinker" may be used interchangeably. Examples of photoreactive crosslinkers include, but are not limited to, arylazides (aryl azides), azido-methylcoumarins, benzophenones, anthraquinones, certain diazo compounds, diazirines, and psoralen derivatives. In preferred embodiments, the photoreactive crosslinker used in the present invention is methacryloyloxybenzophenone (MABP). In other preferred embodiments, the copolymer is an alternating copolymer. In still other preferred embodiments, the copolymer is a one-component compound. In further preferred embodiments, the copolymer is a one-component compound, preferably wherein the copolymer is an alternating copolymer.

The copolymer comprises at least one monomer unit that is a click binding-reactive side group ["Click"]. Binding-reactive side group may include for example, chemically reactive groups, biologically reactive groups or bioaffinity agents. The term "Click" as used herein generally refers to 'click chemistry', i.e., any bioorthogonal reaction that enables the efficient formation of a specific product within a highly complex chemical environment. In particular, it refers to a chemical reaction which allows for the incorporation of one or more reactive tag(s) onto a biomolecular target and the subsequent high selectivity in tag derivatization within a complex biological sample. Click chemistry results in the formation of such substances quickly and reliably. That is, the reactions are efficient, high yielding, and tolerant of various solvents and functional groups.

As further described herein, in certain embodiments, "click chemistry" refers to reactions that may have one or more of the following characteristics: (<NUM>) exhibit functional group orthogonality (i.e., the functional portion reacts only with a reactive site that is complementary to the functional portion, without reacting with other reactive sites); (<NUM>) the resulting bond is irreversible (i.e., once the reactants have been reacted to form products) or in some instances the resulting bond may be reversible (i.e., revert back to reactants under appropriate conditions); (<NUM>) stereospecificity; (<NUM>) physiological stability; (<NUM>) a single reaction product; (<NUM>) substantially no byproducts.

As further described herein, in certain embodiments, the monomer unit that is a click binding-reactive side group ["Click"] may be considered as monomer unit being, or comprising, an orthogonal click binding-reactive (side) group (i.e., "orthogonal ["Click"] monomer unit").

In various embodiments, click binding reaction includes, without being limited thereto, nucleophilic substitutions, particularly to a small strained ring (e.g., epoxides, aziridines, aziridinium ions, and episulfonium ions), addition to carbon-carbon double or triple bonds (e.g., thiol-ene, tiol-yne reactions, dihydroxilation, epoxidation, and aziridination), cycloadditions (e.g., [<NUM>+<NUM>] cycloadditions, [<NUM>+<NUM>] cycloadditions, and Diels-Alder), "non-aldol"-type carbonyl chemistry (e.g., formation of ureas, thioureas, aromatic heterocycles, oxime ethers, hydrazones, and amides). Accordingly, in various embodiments, the at least one click binding-reactive side group ["Click"] for (bio-) functionalizing of the copolymer layer may provide for, or effect, a click binding reaction of any one of nucleophilic substitutions, particularly to a small strained ring (e.g., epoxides, aziridines, aziridinium ions, and episulfonium ions), addition to carbon-carbon double or triple bonds (e.g., thiol-ene, tiol-yne reactions, dihydroxilation, epoxidation, and aziridination), cycloadditions (e.g., [<NUM>+<NUM>] cycloadditions, [<NUM>+<NUM>] cycloadditions, and Diels-Alder), and "non-aldol"-type carbonyl chemistry (e.g., formation of ureas, thioureas, aromatic heterocycles, oxime ethers, hydrazones, and amides). In various embodiments, cycloadditions are preferred, more preferably [<NUM>+<NUM>] cycloadditions, even more preferably <NUM>,<NUM> dipolar cycloadditions. In various embodiments, cycloaddition with an azide (group), or a derivative thereof, is preferred, including but not limited to (Cu(l) catalyzed) azide-alkyne cycloadditions. Examples of addition reactions include addition reactions to carbon-carbon double bonds such as epoxidation and dihydroxylation. Nucleophilic substitution examples can include nucleophilic substitution to strained rings such as epoxy and aziridine compounds. Other examples can include formation of ureas and amides.

The term 'biofunctionalization' of the copolymer as used herein refers to the formation of click binding-reactive groups on the copolymer for modulating the interaction and/or immobilization of biomolecules. In preferred embodiments, the click binding-reactive side group ["Click"] used in the invention is an azide or its derivative thereof. The term "azide" as used herein refers to any compound having the N moiety therein. The azide may be a metal azide wherein the metal is an alkali metal such as potassium, sodium, lithium, rubidium or cesium. The metal may be a transition metal such as, but not limited to, iron, cobalt, nickel, copper or zinc. It is understood that certain metallic azides may be formed in solution by mixing sodium azide or the like with a metallic salt such as, for example, copper sulfate. The azide of the present invention may also be an organic azide or ammonium azide. In various preferred embodiments, the azide is N-(<NUM>-azidopropyl)methacrylamide (AzMA). In various preferred embodiments, the copolymer is an alternating copolymer. In other preferred embodiments, the copolymer is a one-component compound. In further preferred embodiments, the copolymer is a one-component compound, preferably wherein the copolymer is an alternating copolymer.

In other embodiments, the copolymer comprises a hydrophilic monomer unit with or without partial charge. The hydrophilic monomer unit with or without partial charge includes hydrophilic acryl monomers, preferably acrylamides and its derivatives. The term acrylamide as used herein includes unsubstituted acrylamide and derivatives thereof, such as methacrylamide, and the like, as well as N-substituted derivatives thereof. In preferred embodiments, the acrylamide as used herein is dimethylacrylamide (DMAA). In other preferred embodiments, the copolymer is an alternating copolymer. In other preferred embodiments, the copolymer is a one-component compound. In further preferred embodiments, the copolymer is a one-component compound, preferably wherein the copolymer is an alternating copolymer.

The copolymer comprises at least one monomer unit that is a photo-reactive side group for a C, H-insertion cross-linking reaction, preferably a photoreactive cross linker, and more preferably a methacryloyloxybenzophenone (MABP), and at least one monomer unit is a click binding-reactive side group ["Click"], preferably an azide or its derivatives, more preferably N-(<NUM>-azidopropyl)methacrylamide (AzMA). In various preferred embodiments, the copolymer is an alternating copolymer. In other preferred embodiments, the copolymer is a one-component compound. In further preferred embodiments, the copolymer is a one-component compound, preferably wherein the copolymer is an alternating copolymer.

The copolymer comprises three monomer units, as exemplified in <FIG> and explained in Example <NUM>. In various embodiments, the copolymer comprises at least one monomer unit that is a photo-reactive side group for a C, H-insertion cross-linking reaction, preferably a photoreactive cross linker and more preferably a methacryloyloxybenzophenone (MABP), and at least one monomer unit that is a click binding-reactive side group ["Click"], preferably an azide or its derivatives, more preferably N-(<NUM>-azidopropyl)methacrylamide (AzMA), and a hydrophilic monomer unit with or without partial charge, including hydrophilic acryl monomers, preferably, e.g., acrylamides and its derivatives, more preferably dimethylacrylamide (DMAA). In preferred embodiments, the copolymer is an alternating copolymer. In other preferred embodiments, the copolymer is a one-component compound. In further preferred embodiments, the copolymer is a one-component compound, preferably wherein the copolymer is an alternating copolymer.

<FIG> exemplifies the proportion of individual monomer units in percentages used in the method of manufacturing the copolymer of the invention. DMAA is represented by x%, MABP as y% and AzMA as z%. In various embodiments, the proportion of the hydrophilic monomer unit with or without partial charge, including hydrophilic acryl monomers, preferably acrylamides and its derivatives, preferably dimethylacrylamide (DMAA), is between <NUM>% to <NUM>%, preferably between <NUM>% and <NUM>%, between <NUM>% and <NUM>%, between <NUM>% and <NUM>%, between <NUM>% and <NUM>%, between <NUM>% and <NUM>%, preferably <NUM>%, or <NUM>%, or <NUM>%, or <NUM>%, and more preferably <NUM>%. Each alternative represents a separate embodiment of the invention. In other embodiments, the proportion of the at least one monomer unit that is a photo-reactive side group for a C, H-insertion cross-linking reaction, including a photoreactive cross linker, preferably a methacryloyloxybenzophenone (MABP), is between <NUM>% and <NUM>%, between <NUM>% and <NUM>%, between <NUM>% and <NUM>%, preferably between <NUM>% and <NUM>%, and most preferably <NUM>%. Each alternative represents a separate embodiment of the invention. In other embodiments, the proportion of the at least one monomer unit that is a click binding-reactive side group ["Click"], including an azide or its derivatives, preferably N-(<NUM>-azidopropyl)methacrylamide (AzMA), is between <NUM>% and <NUM>%, preferably <NUM>%, or <NUM>% or <NUM>% or <NUM>%, and more preferably <NUM>%.

As further described herein, in certain embodiments, the polymers described herein are "random co-terpolymers", i.e., that the polymers comprise three different (monomer) (sub)units connected in random order. In some embodiments, the at least three monomer units of a copolymer of the invention, which may be considered as a random co-terpolymer, may be represented by the following general structure (in accordance with <FIG>):
<CHM>
wherein X, Y and Z independently represent the three monomer units, and a, b and c are integers representing the number, or proportion, of each monomer unit within the polymer. Specifically, X, Y and Z independently represent the hydrophilic monomer unit with or without partial charge as described elsewhere herein (X), the monomer unit that is a photo-reactive side group for a C, H-insertion cross-linking reaction, including a photoreactive cross linker, as described elsewhere herein (Y), and the monomer unit that is a click binding-reactive side group ["Click"], including an azide or its derivatives, as described elsewhere herein (Z), respectively. The number or proportion of the hydrophilic monomer unit with or without partial charge may be between <NUM>% to <NUM>%, preferably between <NUM>% and <NUM>%, between <NUM>% and <NUM>%, between <NUM>% and <NUM>%, between <NUM>% and <NUM>%, between <NUM>% and <NUM>%, preferably <NUM>%, or <NUM>%, or <NUM>%, or <NUM>%, and more preferably <NUM>%, while the proportion of the at least one monomer unit that is a photo-reactive side group for a C, H-insertion cross-linking reaction may be between <NUM>% and <NUM>%, between <NUM>% and <NUM>%, between <NUM>% and <NUM>%, preferably between <NUM>% and <NUM>%, and most preferably <NUM>%, and the proportion of the at least one monomer unit that is a click binding-reactive side group ["Click"] may be between <NUM>% and <NUM>%, preferably <NUM>%, or <NUM>% or <NUM>% or <NUM>%, and more preferably <NUM>%.

In certain embodiments, the number or proportion of the individual monomer units described above may represent the molar ratio (molar % or mole %) of the individual monomer units in the (random) copolymer, relative to molar ratio (molar % or mole %) of the other monomer (sub)units in the polymer.

For ease of illustration, the above structure depicts a linear connectivity of X, Y and Z; however, it is to be emphasized that (random) copolymers (e.g., random co-terpolymers) of the present invention are not limited to copolymers having the depicted connectivity of monomer units, and the monomer units in a (random) copolymer may be connected in any random sequence, and the copolymer and co-terpolymer may be branched.

According to the method of manufacturing the copolymer layer described herein above, the copolymer according to the embodiments described herein above is dissolved in a suitable solvent for the production of the copolymer (or hydrogel) layer. The term "solvent" as used herein refers to an organic solvent, a mixture of organic solvents, an organic solvent or mixture of organic solvents and water containing salts or no salts buffered or non-buffered. "Organic solvent" refers to, e.g., hydrocarbons such as, e.g., hexane, cyclo-hexane, heptane, toluene, xylenes, ketones such as, e.g., tert-butyl methyl ketone, methyl isopropyl ketone, <NUM>-butanone, acetone, <NUM>-methyl-<NUM>-pentanone, ethers such as, e.g., diethylether, tert-butyl methyl ether (MTBE), isopropyl methyl ether, dioxane, dibutylether, dioxolane, anisole, and tetrahydrofuran (THF), nitriles such as, e.g., acetonitrile and <NUM>-hydroxypropionitrile, polar solvents such as, e.g., dimethyl sulfoxide (DMSO), sulfolane, N,N'-dimethylpropyleneurea (DMPU), glyoxal, acids such as, e.g., acetic acid and formic acid, aldehydes such as, e.g., acetaldehyde, halogenated hydrocarbons such as, e.g., dichloromethane (DCM), trichloroethane, chloroform, chloroben-zene, dichlorobenzene, and dichloroethane, esters such as, e.g., ethyl acetate (EtOAc), isopropyl acetate, or tert-butyl acetate, straight or branched alcohols such as, e.g., <NUM>-methyl-<NUM>-butanol, tert-butanol, methanol, ethanol, n-propanol, n-butanol, and <NUM>-propanol. The term "solvent" as used herein refers to buffered (e.g., phosphate, acetate), non-buffered water, or buffered or non-buffered water containing a water miscible organic solvent such as acetone, tetrahydrofuran (THF), <NUM>-propanol, ethanol, t-butanol, dimethylformamide, dimethyl sulfoxide, or <NUM>-methyl-<NUM>-pentanone or ethers, such as tert-butylmethyl ether (MTBE), saturated or not saturated with water. The solvent for the production of the hydrogel is may be ethanol, or <NUM>-propanol. The dissolved copolymer in the solvent may be applied to a surface at an amount of at least <NUM>µg/mm<NUM> or <NUM>µg/well to at least <NUM>-<NUM>µg/mm<NUM> or <NUM>µg/well. In the next step, the surface is heated to a temperature range between <NUM> to <NUM>, preferably <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, and <NUM> to <NUM>, even more preferably, <NUM>, or <NUM> or <NUM>, or <NUM> or <NUM>.

According to the method of manufacturing a surface-bonded continuous layer of a copolymer of the invention, the cross-linking of the copolymer and surface bond may be achieved by a radical reaction using UV-light. As used herein, the term "surface bonded" refers to a substituent that is substantially bonded, either covalently or ionically, primarily or only to the outer surface of a material. The term "surface" refers to a solid surface, i.e., a material having a rigid or semi-rigid surface. As used herein, the method of manufacturing a surface-bonded continuous layer of a copolymer includes providing a surface on which the copolymer layer is bonded. Surface as used herein is preferably a polymeric material. The polymeric material preferably consists of polystyrenes, cyclic olefin polymers, poly(methyl methacrylates) or polycarbonates. The (solid) surface may be of plastic material, or may be of glass material.

The surface-bond may be a covalent bond. This means that there is no reaction equilibrium but an irreversible binding of the synthetic copolymer of the invention to a surface. The radical reaction may take place between the monomer unit of the synthetic copolymer that is a photo-reactive side group and the surface. The copolymer layer may be surface-bonded by a selective UV exposure. As further described herein, the copolymer layer is surface-bonded by photoactivation, which refers to the activation or control of a chemical reaction by light, in particular exposure to UV light. The selective UV exposure may be spatial and temporal. The selective UV exposure allows the copolymer to form a cross-linked copolymer and allows covalent bonding to the surface in order to produce a monolithic coating via the photo-reactive side group of a photoreactive crosslinker described herein for a C, H-insertion cross-linking reaction. The UV exposure may be at a wavelength of between <NUM> to <NUM>, preferably <NUM> and the energy of <NUM> J/cm<NUM>. The copolymer layer can be surface-bonded and crosslinked by a single exposure, the exposure duration or the energy input plays an important role and also influences the properties of the coating in particular the number of crosslinking points and degree of swelling of the layer.

The degree of swelling may be between <NUM> and <NUM>, preferably between <NUM> and <NUM>. The term "degree of swelling" is defined as follows: ((volume of crosslinked copolymer) + (volume of a compound used in swelling)) / (volume of crosslinked copolymer). The term "volume of a compound (solvent) used in swelling" refers to the volume of a compound (solvent) accumulated in the crosslinked copolymer.

As described herein, "photoreactive crosslinking" or "photochemical crosslinking" refer to the light-induced formation of a covalent bond between two moieties, molecules or other physical entities. Two physical entities that are "photoreactive crosslinked" or "photochemical crosslinked" to each other represent the product of a photo-crosslinking reaction.

The method of manufacturing a surface-bonded copolymer layer of the invention provides for a continuous layer. This is demonstrated in <FIG> that confirms that the cross-linked copolymer is also present between the hills. The term "continuous" as used herein means a coating of the surface-bonded layer of copolymer having an uninterrupted extension in along a surface. Other ways to describe continuously include but are not limited to "constantly" or "reoccurring in rapid succession". Accordingly, as used herein, the term "continuous" refers to a layer that covers an entire surface without gaps or bare spots that reveal material underlying the layer. The term "continuous" as used herein is meant to encompass the surface-bonded layer of copolymer having unintentional, intermittent, isolated interruptions or gaps (e.g., from a manufacturing defect) from a discontinuous deposit. These gaps or bare spots on the surface may be less than about <NUM> % of the total surface area. Accordingly, the method of manufacturing a surface-bonded, continuous copolymer layer may comprise creating a solution from a single copolymer using a solvent, wherein the solvents preferably consisting of alcoholic solutions, in particular ethanol and <NUM>-propanol, but also acetonitrile or water, or aqueous mixtures with other solvents; and the dissolved copolymer is applied to the surface preferably by using spraying and dispensing techniques; wherein the applied mass of the copolymer is at least <NUM>µg/mm<NUM> or <NUM>µg/well to at least <NUM>-<NUM>µg/mm<NUM> or <NUM>µg/well; and the wetting of the surface is controlled by the temperature and/or concentration of the polymer solution; and after evaporation of the solvent, the copolymer layer is cross-linked by means of UV light, preferably UV-A (<NUM>-<NUM>), and bound to the surface.

Also disclosed herein is a method of manufacturing a container having a surface-bonded continuous layer of a copolymer with a synthetic one-component system comprising at least one photo-reactive side group for a C, H-insertion cross-linking ["CHic"] reaction; and at least one click binding-reactive side group ["Click"] for (bio-) functionalizing of the copolymer layer. In particular, disclosed herein is a method of manufacturing a container having a continuous layer of a copolymer bonded to the surface of the bottom end of the container, comprising the steps:.

The method may involve obtaining a container having a continuous layer of a copolymer bonded to the surface of the bottom end of the container subsequent to steps (a), (b), and (c). The container may be formed of polymeric material and has an open top end, a closed bottom end, and at least one wall extending between said ends. In various embodiments, the amount of copolymer applied to the surface is at least <NUM>µg/mm<NUM>, preferably at least <NUM>-<NUM>µg/mm<NUM>.

As used herein, the term "container" refers to any hollow rigid object suitable for supporting and containing a product. The container is optionally provided with a sealing area around the perimeter of the opening of the container, a rim or at least around the perimeter of the opening of the container, such as a sealing flange, suitable for being sealed to the heat sealable layer of a film forming the lid of the container. The containers of the present invention are suitable for use as a container for any kind of matter, but preferably for liquids or other compositions which are fluid. The term container does not imply a particular intended use for the article. For example, the term "container" as used herein encompasses articles destined to comprise a surface-bonded continuous layer of a copolymer of the invention. As used herein the term "container" may also be interpreted as "well". The term "well" as used herein is a sample holder such as a small test tube where a biological or other sample is placed in microtiter plate. Accordingly, the term 'container assembly' or 'analytical device' according to the invention may refer to microtiter plates. "Microtiter plates" (also referred to as microplates and microwell plates) generally are a substantially flat plate with multiple wells arranged in an array. Microtiter plates can be configured with from about <NUM> to about <NUM> wells. Microtiter plates have multiple uses including but not limited to holding and transporting liquids, performing biological or chemical reactions, combinations thereof and the like. Microtiter plates frequently are used in research or diagnostic procedures including high throughput protocols.

The container having a surface-bonded continuous layer of a copolymer forming part of the invention is characterized in that the copolymer comprises at least one monomer unit that is a photo-reactive side group for a C, H-insertion cross-linking reaction. In preferred embodiments, the photo-reactive side group for a C, H-insertion cross-linking reaction is a photoreactive crosslinker. C, H-insertion reactions concern the insertion reaction of, e.g., carbenes, nitrenes or ketyl biradicals into a carbon-hydrogen bond. This organic reaction is of some importance in the synthesis of new organic compounds in which only one reactive group is contained, which after activation reacts with the neighboring polymer chains through C, H-insertion reactions. In such reactions the cross-linking units can be incorporated into the polymer matrix and induce the linking of the involved polymer chains after thermal or UV-activation by (formal) C, H-insertion. Upon imparting heat or light into the film, the cross-linker units can be activated and generate a reactive intermediate, which depending on the chemical nature of cross-linker reacts with almost any groups in the neighboring chains through a C, H-insertion reaction (CHic = C, H-insertion based crosslinking). This crosslinking reaction pathway offers the advantage that there is no need that two reaction partners meet each other before connecting as one reaction partner - the C, H group - is present in abundance. The term "photoreactive crosslinker" refers to photo-activable reactive groups for binding proteins, nucleic acids and other biomolecules in an irreversible manner after activated by ultraviolet or visible light. Examples of photoreactive crosslinkers include, but are not limited to, arylazides (aryl azides), azido-methyl-coumarins, benzophenones, anthraquinones, certain diazo compounds, diazirines, and psoralen derivatives. In various embodiments, the photoreactive crosslinker is an aryl azide having an R-N<NUM> structure, wherein R is an aromatic ring (e.g., an aromatic hydrocarbon, such as phenyl, naphthyl, thienyl, etc.). Photoactivation results in the azide functional group forming a reactive intermediate that can form covalent bonds, for example by insertion into carbon-hydrogen (C-H) bonds as described elsewhere herein.

In a preferred embodiment, the photoreactive crosslinker used in the present invention is methacryloyloxybenzophenone (MABP). In preferred embodiments, the copolymer is an alternating copolymer. In other preferred embodiments, the copolymer is a one-component compound. In further preferred embodiments, the copolymer is a one-component compound, preferably wherein the copolymer is an alternating copolymer.

The container having a surface-bonded continuous layer of a copolymer forming part of the invention is further characterized in that the copolymer comprises at least one monomer unit that is a click binding-reactive side group ["Click"]. Binding-reactive side group may include for example, chemically reactive groups, biologically reactive groups or bioaffinity agents. The term "click" as used herein generally refers to 'click chemistry' i.e. any bioorthogonal reaction that enables the efficient formation of a specific product within a highly complex chemical environment. In particular, it refers to a chemical reaction which allows for the incorporation of one or more reactive tag(s) onto a biomolecular target and the subsequent high selectivity in tag derivatization within a complex biological sample. Click chemistry results in the formation of such substances quickly and reliably. That is, the reactions are efficient, high yielding, and tolerant of various solvents and functional groups. The term 'biofunctionalization' of the copolymer as used herein refers to the formation of click binding-reactive groups on the copolymer for modulating the interaction and/or immobilization of biomolecules. In preferred embodiments, the click binding-reactive side group ["Click"] used in the invention is an azide or its derivative thereof. The term "azide" as used herein refers to any compound having the N moiety therein. The azide may be a metal azide wherein the metal is an alkali metal such as potassium, sodium, lithium, rubidium or cesium. The metal may be a transition metal such as, but not limited to, iron, cobalt, nickel, copper or zinc. It is understood that certain metallic azides may be formed in solution by mixing sodium azide or the like with a metallic salt such as, for example, copper sulfate. The azide of the present invention may also be an organic azide or ammonium azide. In preferred embodiments, the azide is N-(<NUM>-azidopropyl)methacrylamide (AzMA). In other preferred embodiments, the copolymer is an alternating copolymer. In further preferred embodiments, the copolymer is a one-component compound. In still further preferred embodiments, the copolymer is a one-component compound, preferably wherein the copolymer is an alternating copolymer.

In various embodiments, the container having a surface-bonded continuous layer of a copolymer is characterized in that the copolymer comprises a hydrophilic monomer unit with or without partial charge. The hydrophilic monomer unit with or without partial charge includes hydrophilic acryl monomers, preferably acrylamides and its derivatives. The term acrylamide as used herein includes unsubstituted acrylamide and derivatives thereof, such as methacrylamide, and the like, as well as N-substituted derivatives thereof. In preferred embodiments, the acrylamide as used herein is dimethylacrylamide (DMAA). In preferred embodiments, the copolymer is an alternating copolymer. In other preferred embodiments, the copolymer is a one-component compound. In further preferred embodiments, the copolymer is a one-component compound, preferably wherein the copolymer is an alternating copolymer. In preferred embodiments, the container having a surface-bonded continuous layer of a copolymer is characterized in that the copolymer comprises at least one monomer unit that is a photoreactive cross linker, preferably a methacryloyloxybenzophenone (MABP), and an azide or its derivatives, more preferably N-(<NUM>-azidopropyl)methacrylamide (AzMA) as at least one monomer unit providing for the click binding-reactive side group ["Click"]. In preferred embodiments, the copolymer is an alternating copolymer. In other preferred embodiments, the copolymer is a one-component compound. In further preferred embodiments, the copolymer is a one-component compound, preferably wherein the copolymer is an alternating copolymer.

In preferred embodiments, the container having a surface-bonded continuous layer of a copolymer is characterized in that the copolymer comprises three monomer units as shown in <FIG> and explained in Example <NUM>. In particular, the copolymer comprises at least one monomer unit that is a photo-reactive side group for a C, H-insertion cross-linking reaction, preferably a photoreactive cross linker and more preferably a methacryloyloxybenzophenone (MABP), and at least one monomer unit that is a click binding-reactive side group ["Click"], preferably an azide or its derivatives, more preferably N-(<NUM>-azidopropyl)methacrylamide (AzMA), and a hydrophilic monomer unit with or without partial charge, including hydrophilic acryl monomers, preferably acrylamides and its derivatives, preferably dimethylacrylamide (DMAA). In preferred embodiments, the copolymer is an alternating copolymer. In other preferred embodiments, the copolymer is a one-component compound. In further preferred embodiments, the copolymer is a one-component compound, preferably wherein the copolymer is an alternating copolymer.

<FIG> exemplifies the proportion of individual monomer units in percentages that can be used in the method of manufacturing the container having a surface-bonded continuous layer of a copolymer of the invention according to any of the embodiments disclosed herein. DMAA is represented by x%, MABP as y% and AzMA as z%. In various embodiments, the composition of the hydrophilic monomer unit with or without partial charge, including hydrophilic acryl monomers, preferably acrylamides and its derivatives, preferably dimethylacrylamide (DMAA), is between <NUM>% to <NUM>%, preferably between <NUM>% and <NUM>%, between <NUM>% and <NUM>%, between <NUM>% and <NUM>%, between <NUM>% and <NUM>%, between <NUM>% and <NUM>%, preferably <NUM>%, or <NUM>%, or <NUM>%, or <NUM>%, and more preferably <NUM>%. Each alternative represents a separate embodiment of the invention. In other embodiments, the composition of the at least one monomer unit that is a photo-reactive side group for a C, H-insertion cross-linking reaction, preferably a photoreactive cross linker, and more preferably a methacryloyloxybenzophenone (MABP) is between <NUM>% and <NUM>%, between <NUM>% and <NUM>%, between <NUM>% and <NUM>%, preferably between <NUM>% and <NUM>%, and more preferably <NUM>%. Each alternative represents a separate embodiment of the invention. In further embodiments, the composition of at least one monomer unit that is a click binding-reactive side group ["Click"], preferably an azide or its derivatives, more preferably N-(<NUM>-azidopropyl)methacrylamide (AzMA), is between <NUM>% and <NUM>%, preferably <NUM>%, or <NUM>% or <NUM>% or <NUM>%, and more preferably <NUM>%.

Also disclosed herein is a method of preparing a modified (solid) surface, wherein the surface is a surface as defined elsewhere herein, the method comprising:.

The modified (solid) surface may have a surface-bonded continuous layer of a copolymer as described elsewhere herein. Embodiments described elsewhere herein may be applied or implemented into the method of preparing a modified (solid) surface accordingly.

The methods disclosed herein may further comprise a step of photostructuring the surface-bonded copolymer layer by a spatially selective exposure of the copolymer layer to light, preferably UV light. The photostructuring generates hill-like structures, more specifically 3D hill-like structures, on the continuous copolymer layer while maintaining a monolithic topography, wherein the (3D) hill-like structures may exhibit a hexagonal arrangement. The (3D) hill-like structures may have a diameter and/or width of <NUM>-<NUM> and/or a height of <NUM>-<NUM>.

The selective UV exposure may involve the use of a photomask in order to photostructure the surface-bonded copolymer layer. As used herein, the term photostructure means that the surface bonded, continuous copolymer layer, which when spatially and temporally exposed to ultraviolet (UV) radiation/light of a sufficient intensity compared to the regions that are not exposed, leads to comparatively deep, well defined etched features called microstructures. The term "microstructure" as used herein is defined to have its conventional meaning, for example, the microscopic structure of a material including, phase boundaries, orientations, size scale and surface morphology including structures that have small features of dimensions measured in the micrometer range or patterned structures comprising "microscale" features. The terms "microscale", "micro-sized" or "micro-range" are to be interpreted broadly to include any dimensions that are in the range of about <NUM> to about <NUM>.

The photostructuring of the surface-bonded copolymer layer may be carried out by a selective UV exposure involving the use of a photomask. The term "photomask" is intended to mean any material having varying or non-uniform barrier properties to ultra-violet light or other electromagnetic radiation used to photopolymerize the copolymer layer of the invention. A preferred photomask material comprises an electromagnetic barrier material having a design which perforates (or is cut out of) the material. Upon application of electromagnetic radiation on one side of the photomask, a pattern of electromagnetic radiation is emitted from the opposite side of the photomask. The emitted pattern preferably comprises "shadow portions" (having virtually no electromagnetic radiation) and electromagnetic radiation portions: together the two portions can form an intricate pattern of electromagnetic radiation. The photomask can comprise openings of any size and shape known to a skilled person in the art.

The photomask used in the UV exposure as described above can be used to obtain a monolithic topography of the (surface-bonded) continuous copolymer (hydrogel) layer. The term "topography" as described herein refers to the relief features or surface configuration of an area or adjacent areas and encompasses depth which includes, but is not limited to, a geometrical depth and/or a depth of structure, or physical characteristic (physical attribute). Topography as used herein may include ridges, cones, pillars, hills, valleys, grooves, pits, dimples, depressions, bumps, convexity, concavity, ribs, protrusions, curves, raised surfaces or other surface topography. The terms "pillar" or "pillar-shaped" is to be interpreted broadly to include any substantially upright longitudinal body where the length is much greater than the width and where the width dimension is relatively constant throughout the length dimension. The terms "cone" or "cone-shaped" is to be interpreted broadly to include any substantially upright longitudinal body where the length is much greater than the width and where the width dimension decreases as the structure protrudes out of the surface where it is fixed. The terms "hills" or "hill-like structure" is to be interpreted to include a protrusion out of a surface where the width is much greater than the height and where the width dimension decreases as the structure protrudes out of the surface where it is fixed. The UV exposure as described above provides for a monolithic topography of the (surface-bonded) continuous copolymer (hydrogel) layer, wherein the monolithic topography is preferably characterized by (3D) hill-like structures in a hexagonal arrangement. Such (3D) hill-like structures may comprise a height between <NUM> to <NUM>, preferably between <NUM> to <NUM>, preferably between <NUM> to <NUM>, preferably between <NUM> to <NUM>, preferably between <NUM> to <NUM>, preferably between <NUM> to <NUM>, preferably between <NUM> to <NUM>, preferably between <NUM> to <NUM>, preferably at least <NUM> and preferably <NUM>, even more preferably <NUM>.

The photomask used in the UV exposure creates may create topography comprising circular openings separated by circular distances in a hexagonal arrangement. The photomask used in the UV exposure may comprise circular openings with a diameter and/or width between <NUM> to <NUM>, preferably <NUM> to <NUM>, even more preferably <NUM> to <NUM>, and most preferably <NUM>. Furthermore, the photomask used in the UV exposure may comprise circular distances with a diameter between <NUM> to <NUM>, preferably between <NUM> to <NUM>, even more preferably between <NUM> to <NUM>, and most preferably <NUM>. Accordingly, the photomask used in the UV exposure may comprise circular openings separated by circular distances in a hexagonal arrangement, wherein the circular openings may comprise a diameter of <NUM>, and wherein the circular openings may be separated by circular distances with a diameter of <NUM>.

Disclosed herein is a continuous, surface-bonded, photostructured, copolymer layer according to the methods described herein, wherein the copolymer may be an alternating copolymer, or. _a one-component compound. The copolymer may be a one-component compound, wherein the copolymer is an alternating copolymer.

Further disclosed herein is a method of 3D functionalization of a container using the surface-bonded continuous layer of a copolymer of the invention. 3D functionalization of a container as used herein refers to selective spatial and temporal exposure of the copolymer layer using a photomask to form a 3D functionalized copolymer layer. Accordingly, disclosed herein is a method of producing a copolymer layer with tailored 3D surface properties by functionalization. As described elsewhere herein, the method of manufacturing a container comprising a surface-bonded continuous copolymer layer may include selective UV exposure that allows the copolymer to form a cross-linked copolymer and further may allow covalent bonding to the surface in order to produce a monolithic coating via the photo-reactive side group of a photo reactive crosslinker described herein for a C, H-insertion cross-linking reaction.

Disclosed herein is a surface-bonded continuous layer of a copolymer manufactured or obtainable or obtained by any of the methods disclosed herein. The surface-bonded continuous layer of a copolymer manufactured by the methods disclosed herein shows a very high, reproducible binding capacity due to the click-binding reactive molecules disclosed herein. In various embodiments, the surface-bonded continuous layer of the invention provides a binding capacity of any of between <NUM> pmol to <NUM> pmol, between <NUM> pmol to <NUM> pmol, <NUM> pmol to <NUM> pmol, <NUM> pmol to <NUM> pmol, <NUM> pmol to <NUM> pmol, and even more than <NUM> pmol. These values provide for an increase in binding specificity by a factor of <NUM> to <NUM> times as compared to other copolymer layers known in the art. In a related embodiment, the binding capacity of the copolymer layer is directly dependent on the proportion of azide used. In various embodiments, azide at a concentration of <NUM> mol% gives the maximum binding capacity. The binding capacity is <NUM> pmol without any azide group.

The surface-bonded continuous layer of a copolymer manufactured by the embodiments of the methods disclosed herein shows a significantly improved mechanical stability as shown in Example <NUM> and <FIG>.

The surface-bonded continuous layer of a copolymer manufactured by the embodiments of the methods disclosed herein specifically with the 3D functionalization of photostructures explained herein are surprisingly sufficient to separate objects, particles and/ or cells. In various embodiments, these structures can separate particles with a diameter of <NUM> µm as shown in <FIG> and <FIG>. In such embodiments, the cells can be separated over an interval of time wherein the separation is achieved between <NUM> to <NUM> minutes, <NUM> to <NUM> minutes, <NUM> to <NUM> minutes, preferably between <NUM> to <NUM> minutes. In such embodiments, the particles fall may largely into the micro-pockets created by the photostructured copolymer layer of the invention.

The surface-bonded continuous layer of a copolymer manufactured by the methods disclosed herein results in an improved sensitivity in detecting the analytes of interest. In various embodiments, the sensitivity is improved by a factor of <NUM> as compared to other copolymer layers known in the art. In such embodiments, the detection sensitivity of the copolymer layer of the invention is at least <NUM>µg / ml.

The invention provides a container having a surface-bonded continuous layer of a copolymer, as defined by the appended claims, manufactured or obtainable or obtained by the methods disclosed herein. Preferably, the container has the continuous layer of a copolymer bonded to the surface of the bottom end of the container. In various embodiments, the container is formed of polymeric material and/or may have an open top end, a closed bottom end, and at least one wall extending between said ends. The surface of the bottom end may have a surface-bonded continuous layer of a copolymer comprising at least three different monomer units and including.

The copolymer (hydrogel) layer of the container may be photostructured while maintaining a monolithic topography, preferably the copolymer (hydrogel) layer has hill-like structures, more specifically 3D hill-like structures. Preferably, the hill-like structures exhibit a hexagonal arrangement. As described elsewhere herein and as demonstrated in the present invention, in various embodiments, the surface-bonded continuous copolymer layer is photostructured while crosslinking and is thereby maintaining a monolithic topography, preferably the copolymer (hydrogel) layer has hill-like structures, preferably the hill-like structures exhibit a hexagonal arrangement and are generated on top but within the monolithic topography.

As further described herein, in certain embodiments, the (3D) hill-like structures of the copolymer (hydrogel) layer having a monolithic topography are periodically separated from each other, or are arranged in a periodically separated order on the copolymer (hydrogel) layer. More specifically, in certain embodiments, (3D) the hill-like structures of the copolymer (hydrogel) layer having a monolithic topography are periodically spatially-separated from each other, or are arranged in a periodically spatially-separated order on the copolymer (hydrogel) layer.

As further described herein, in certain embodiments, the copolymer (hydrogel) layer of the invention (and as comprised by the container) may be described as a (3D) patterned, or (3D) hexagonal-shaped, or (3D) hexagonal-patterned, copolymer (hydrogel) layer due to the (3D) photostructuring of the surface-bonded copolymer (hydrogel) layer by selective UV exposure described elsewhere herein.

As further described herein, in certain embodiments, the (3D) hill-like structures of the copolymer (hydrogel) layer having a monolithic topography are circular-shaped hills or circular-shaped hill-like structures. In various embodiments, the (3D) hill-like structures of the copolymer (hydrogel) layer having a monolithic topography are circular-shaped column-like hills or circular-shaped column-like hill structures.

As further described herein, in certain embodiments, the (3D) hill-like structures of the copolymer (hydrogel) layer having a monolithic topography are hexagonal-shaped hills or hexagonal-shaped hill-like structures. In various embodiments, the (3D) hill-like structures of the copolymer (hydrogel) layer having a monolithic topography are hexagonal-shaped column-like hills or hexagonal-shaped column-like hill structures.

The container having a surface-bonded continuous layer of a copolymer manufactured by the embodiments of the methods disclosed herein shows a very high, reproducible binding capacity due to the click-binding reactive molecules disclosed herein. In various embodiments, the surface-bonded continuous layer of the invention provides a binding capacity between <NUM> pmol to <NUM> pmol, between <NUM> pmol to <NUM> pmol, <NUM> pmol to <NUM> pmol, <NUM> pmol to <NUM> pmol, <NUM> pmol to <NUM> pmol, or even more than <NUM> pmol. These values provide for an increase in binding specificity by a factor of <NUM> to <NUM> times as compared to other copolymer layers known in the art. In further embodiments, the binding capacity of the copolymer layer is directly dependent on the proportion of azide used. In various embodiments, azide at a concentration of <NUM> mol% gives the maximum binding capacity. The binding capacity is <NUM> pmol without any azide group.

The container having a surface-bonded continuous layer of a copolymer manufactured by the embodiments of the methods disclosed herein shows a significantly improved mechanical stability as shown in Example <NUM> and <FIG>.

The container having a surface-bonded continuous layer of a copolymer manufactured by the embodiments of the methods disclosed herein specifically with the 3D functionalization of photostructures explained herein are surprisingly sufficient to separate objects, particles and/ or cells. In various embodiments, these structures can separate particles with a diameter of <NUM> as shown in <FIG> and <FIG>. In such embodiments, the cells can be separated over an interval of time wherein the separation is achieved between <NUM> to <NUM> minutes, <NUM> to <NUM> minutes, <NUM> to <NUM> minutes, preferably between <NUM> to <NUM> minutes. In such embodiments, the particles fall largely into the micro-pockets created by the photostructured copolymer layer of the invention.

The container having a surface-bonded continuous layer of a copolymer manufactured by the embodiments of the methods disclosed herein results in reduced non-specific background reactions and an improved sensitivity in detecting the analytes of interest. In various embodiments, the sensitivity is improved by a factor of <NUM> as compared to other copolymer layers known in the art. In such embodiments, the detection sensitivity of the copolymer layer of the invention is at least <NUM>µg / ml.

In the present invention, the container may comprise an optically transparent part. In various embodiments, the container has an essentially tubular shape and/or has a conical shape towards the bottom end of the container, preferably the container is a laboratory test tube, e.g. a well of a microplate.

As further described herein, the container may be a vessel, or reaction vessel, separating or isolating one reagent or reaction (e.g., a binding reaction) from another, or providing a space in which a reaction can take place. Non-limiting examples of (reaction) vessels useful in connection with the present invention include: laboratory test tubes, flow cells, wells of a multiwell plate; microscope slides; tubes (e.g., capillary tubes) etc..

The present invention provides a container assembly, or an analytical device, comprising one or more of a surface-bonded continuous layer of a copolymer as disclosed herein, or one or more of a container as disclosed herein.

The present invention provides the use of the surface-bonded continuous layer of a copolymer according to the invention, or the container of the invention, or the container assembly or analytical device of the invention, in analytical and/or diagnostic applications, preferably for use in an immunoaffinity assay, ELISA or ELISpot assay. In various embodiments, the said use is for separation of dispersed particles and/or objects, preferably wherein the dispersed particles and/or objects are cells.

The present invention provides a method of using the surface-bonded continuous layer of a copolymer according to the invention, or the container of the invention, or the container assembly or analytical device of the invention, in analytical and/or diagnostic applications, preferably for use in an immunoaffinity assay, ELISA or ELISpot assay.

The term "immunoaffinity" means techniques by which materials are isolated by virtue of their content of immunogenic epitopes. In a well-known immunoaffinity assay, ELISA, a capture antibody is printed on the bottom of a well in a microtiter plate. A microtiter plate is a flat plate that has multiple containers or "wells" formed in its surface. Each well can be used as a small test tube into which various materials can be placed to perform biochemical analyses. In a preferred embodiment, the bottom of the container, wells and/or test tube is optically transparent. By the term "optically transparent" as used herein is meant an object having a transmittance of at least <NUM>% at a wavelength in the visible spectrum (<NUM>-<NUM>). The capture antibody used in ELISA has specificity for a particular antigen for which the assay is being performed. A sample to be analyzed is added to the well containing the capture antibody, and the capture antibody "captures" or immobilizes the antigen contained in the sample. A detect antibody is then added to the well, which also binds and/or forms a complex with the antigen. Further materials are then added to the well which cause a detectable signal to be produced by the detect antibody. For example, when light of a specific wavelength is shone upon the well, the antigen/antibody complexes will fluoresce. The amount of antigen in the sample can be inferred based on the magnitude of the fluorescence. In another example, a compound can be added to the well that causes the detect antibody to emit light within a predetermined wavelength (e.g., <NUM>-<NUM>). This light can be read by a charge-coupled device (CCD) camera to measure the optical brightness of the emitted light. Example <NUM> demonstrates the use of the surface-bonded continuous layer of a copolymer forming part of the method disclosed herein in an ELISA plate by measuring IL-<NUM> as an analyte of interest.

The ELISpot (Enzyme-linked-immuno-Spot) test is a sensitive method to measure cell activation at the single cell level. This cell activation releases cytokines, which are then made visible as colored dots (spots) in the ELISpot using monoclonal antibodies. With regard to the detection of cytokines that were secreted by cells, the ELISPOT method appears to be more sensitive by up to a factor of <NUM> than a comparable ELISA method. A well-known problem with analytical techniques such as ELISPOT technology is the creation of clearly defined spots after the detection reaction has been completed. Spot generation is influenced, among other things, by high non-specific background signals, a "washing out" of the edges and the mixing of the individual spots into clusters. Here, however, cells that are close together can also cause interference of the spots that form. The present invention has surprisingly overcome this problem by providing a container having a surface-bonded, continuous layer of copolymer that is capable of separation of dispersed particles and/or objects, preferably wherein the dispersed particles and/or objects are cells. The term "cell" as used herein may be any cell type. In certain embodiments, the cells are mammalian cells.

The use of the surface-bonded continuous layer of a copolymer manufactured by the method of the invention surprisingly provides for reduced non-specific background reactions as illustrated in Examples <NUM> and <NUM>. As illustrated in Example <NUM>, the surface-bonded continuous layer of a copolymer manufactured by the method of the invention provides for about twice as many spots as carried out without the copolymer of the invention in a reference plate. In addition, the detection signals, i.e., the color spots, are spatially clearly separated, sharper and smaller than on the reference plate (<FIG>).

As illustrated in Example <NUM>, the surface-bonded continuous layer of a copolymer manufactured by the method of the invention provides for covalent attachment and/or capturing of biochemical reagents according to the analytical test used. The term biochemical reagent composition as used herein refers to a composition as a reagent for the detection, analysis or use as a substrate or intermediate in synthetic, biochemical, or immunological reactions e.g. emission reagents include luminescent, fluorescent, or phosphorescent materials. Other emission reagents include colloidal gold, colloidal silver, other colloidal metals and non-metals, quantum dots, other fluorescent nanoparticles, plasmon resonant particles, grating particles, photonic crystals reagents and the like. Furthermore, absorption reagent refers to any material that causes absorbance of the excitation light. Example <NUM> illustrates the covalent attachment of streptavidin-dibenzocyclooctin. In this way, the methods as disclosed herein provide for testing the binding specificity of any biochemical reagent on the copolymer manufactured by the method of the invention as disclosed herein.

The means and methods of the as disclosed herein provide numerous advantages including, but not limited to:
The binding capacity clearly exceeds that of other 3D coatings, which are usually only produced by individual branched polymer chains. The invention provides for a high, reproducible, specific binding capacity for any, but specifically selected molecules.

Due to the increased specific binding capacity, small, spatially clearly recognizable spots could be achieved in the ELISpot. This also increased sensitivity.

The modified copolymer surface by the compositions of the invention is characterized by increased chemical and mechanical stability compared to the otherwise usual filter base plates.

The copolymer forming part of the invention significantly reduces non-specific background reactions. Fewer washing and blocking steps are necessary or can be carried out with milder reagents.

Using photomasks and a unique UV exposure, the polymer can be specifically shaped into a microstructured hydrogel. This also made it possible to separate and selectively distribute objects or cells.

The invention is illustrated in the following examples. These examples are provided for illustrative purposes only and are not to be considered to limit the scope of the invention to any extent.

<FIG> shows an example of a structural formula of the synthetic one-component compound (copolymer) with which the plastic surface can be modified accordingly. The letters x, y and z indicate the variable proportions of the monomer units. An advantageous composition would be, for example, x = <NUM> mol%, y = <NUM> mol% and z = <NUM> mol%. The number average molar mass is, for example, <NUM>,<NUM> / mol. The one-component compound is dissolved in pure ethanol, whereby a concentration of <NUM> / ml is obtained. Then <NUM>µl of this solution are pipetted onto the bottom of a container, here the bottom of a well of a microtiter plate made of polystyrene, which was heated to <NUM> ° C. The volume of the solution spreads out on the surface of the bottom of the well, with the solvent evaporating rapidly at the same time. When the edge of the well is reached, the solvent has evaporated, a uniform layer having formed on the surface. <FIG> shows a schematic representation of a coating of microtiter plates using a manual dispensing process. Further application examples for the production of the modified surface with ethanol as solvent, a concentration of <NUM> / ml and a volume of <NUM>µl are summarized in Table <NUM>.

The coatings obtained from the method of Example <NUM> are exposed by UV exposure at a wavelength of, for example, <NUM> with an energy of, for example, <NUM> J / cm <NUM>, as a result of which the one-component compound is crosslinked and covalently bonded to the plastic surface, so that a monolithic coating is produced.

If a photomask is used in the UV exposure, which has hexagonal, circular openings with a diameter of, for example, <NUM>, the openings being separated by circular distances with a diameter of <NUM>, a monolithic topography with a columnar structure on the plastic surface can be obtained which is shown in <FIG>.

In order to demonstrate the formation of a monolithic coating by this process, the topography was examined using an atomic force microscope [atomic force microscopy (AFM)]. <FIG> shows an example of the result of such a measurement. A total layer thickness of <NUM> µm can be seen from the height profile shown. Furthermore, a continuous coating is achieved, since there is also cross-linked copolymer between the columns. This was an unexpected but desirable result because it created a complete, coherent layer. This could be confirmed by measurements on the mechanical profilometer (<FIG>).

Binding capacity measurements of these surfaces showed a very high, reproducible binding of click-binding-reactive molecules compared to other 3D coatings, which are usually generated by single branched polymer chains (<FIG>). Binding capacities of more than <NUM> pmol / well were achieved, with azide contents of approximately <NUM> mol% in the copolymer. A comparable coating of a product on the market has a binding capacity of about <NUM> pmol / well. An increase by a factor of <NUM> could thus be achieved. A straight line of origin results for a copolymer without clickable groups, i.e. the binding capacity is, as expected, <NUM> pmol / well.

The modified plastic surface shows a significantly improved mechanical stability compared to the Filter floor panels for ELISpot tests. This was tested by specifically scratching with a pipette (tip diameter about <NUM>) on the plate bottoms (<FIG>). The modified plastic surface withstands high pressure (about <NUM>/mm<NUM>, <FIG>, whereby the plate bottom of the filter base plate breaks through (<FIG>. With light pressure (about <NUM>/mm<NUM>) with the pipette on the plate bottom, the monolithic coating only leads to a reduction in the signals (<FIG>), whereas unwanted flat signals are generated with the reference plate (<FIG>.

The structures obtained in this way can be used very well to separate, for example, polystyrene particles with a diameter of <NUM> on the modified plastic surface (<FIG>). The sedimentation process of Peripheral Blood Mononuclear Cells (PBMC) is shown in <FIG>. It can be seen that PMBCs that hit a column migrate into a micro-pocket in a relatively short time and can thus be separated. The unique and unexpected thing about it is that the particles/cells could be separated purely by the topography. The entire surface consists only of the one-component compound and both the columns and the micro-pockets have the same chemical structure.

Cell separation during the ELISpot test is illustrated in <FIG>. Cell clusters can be seen on the unstructured one-component copolymer. In the case of the microtiter plate base coated and structured with the one-component copolymer, the cells fall largely into the micro-pockets intended for this purpose.

Coating with the one-component compound results in a (diagnostic) sensitivity of the ELISpot test that is improved by a factor of about <NUM>. This was shown by a titration series of PHA-M (stimulates cell proliferation), whereby, compared to the reference plate (<NUM>µg / ml), <NUM>µg / ml was still detectable with the coating (Table <NUM>). <FIG> shows, for example, a test series with <NUM>µg / ml PHA-M, in which it can be seen that with the ELISpot assay carried out in the same way, about twice as many spots are obtained. The comparatively lower non-specific background coloration in the coated well is also clear. In addition, the detection signals, i.e. the color spots, are spatially clearly separated, sharper and smaller than on the reference plate. The usual spot diameter on the reference plate is <NUM> - <NUM>, which corresponds to other standard ELISpot plates. Spots with a diameter of around <NUM> can be clearly assigned to exactly one cell on the structured one-component copolymer (<FIG>).

The surface bonded continuous copolymer layer used in this example is similar to Examples <NUM> and <NUM> with minor modifications. The copolymer is also dissolved in ethanol and with a dispenser (e.g., Multidrop from Thermo Fisher Scientific) in a <NUM> / mL solution that has been heated to <NUM>, with <NUM>µL dispensed evenly per well on microtiter plates (MTP) from Greiner Bio One (<NUM>). After the ethanol has evaporated, the MTP are irradiated with UV light (UV table TFX-<NUM> from Vilber-Lourmat) until after one minute the polymer has cross-linked. With repeated washing with ethanol, unbound polymer strands are removed. The azide hydrogel MTP thus obtained is coated in a second step with streptavidin-dibenzocyclooctyne (SA-DBCO) from Microcoat Biotechnologie GmbH. A biotinylated antibody against IL-<NUM> with <NUM>µg / well is bound to the corresponding SA plate (also Microcoat Biotechnologie GmbH). Solutions with different concentrations of human IL-<NUM> are placed on this plate as samples. This IL-<NUM> is in turn bound with an antibody against IL-<NUM> which has been labeled with peroxidase. A color reaction is initiated by means of TMB, by means of which the wells are stained in accordance with the amount of bound IL-<NUM>. This color reaction is stopped with dilute sulfuric acid and measured in an MTP reader (e.g., from Biorad) at <NUM> to <NUM>. The IL-<NUM> ELISA works very well (see <FIG>) with an LOD of <NUM> pg / mL and a LOQ of <NUM> pg / mL. This also shows the very low background of the hydrogel plates, which is comparable to a pure buffer measurement in an MTP.

Claim 1:
A container formed of polymeric material and having an open top end, a closed bottom end, and at least one wall extending between said ends, characterized in that the surface of the bottom end has a surface-bonded continuous copolymer layer having a monolithic topography, wherein the copolymer comprises at least three different monomer units, wherein
(i) one of the at least three different monomer units is a photo-reactive cross-linker with a side group for a C, H-insertion cross-linking ["CHic"] reaction;
(ii) one of the at least three different monomer units has a click binding-reactive side group ["Click"] for (bio-)functionalizing of the copolymer layer; and
(iii) one of the at least three different monomer units is a hydrophilic monomer without partial charge.