Method and coating system for the immobilization of biomolecules

Abstract

The invention relates to methods for producing a coated solid substrate for immobilizing biomolecules, as well as coating systems for immobilizing biomolecules. Furthermore, the invention relates to a solid substrate for immobilizing biomolecules produced by a method according to the invention and comprising the coating system, as well as a functional solution to be used in the method for producing a component of the coating system. Finally, the invention relates to the use of the coating system for immobilizing biomolecules, as well as to a microarray or biochip comprising the coating system for analyzing biomolecules contained in a sample.

Description

Method for the production of a coated solid substrate for immobilising biomolecules and coating systems for immobilising biomolecules. Furthermore, the invention relates to a solid-state substrate for immobilizing biomolecules produced by a method according to the invention and comprising the coating system and a functional solution for producing a component of the coating system. Finally, the invention relates to the use of the coating system for immobilizing biomolecules, and also to a microarray or biochip comprising the coating system for analyzing biomolecules contained in a sample.Various microarrays and biochips are known from practice, which make it possible to immobilize biomolecules contained in a sample in the closest space and to present them for further analysis. In this regard, it is to be noted that the term "sample" in the context of this disclosure is to be understood in the broadest sense and thus includes, inter alia, both body fluids to be analyzed, such as, for example, blood, blood plasma, urine, saliva, etc., and also tissues, tissue sections or whole cells isolated therefrom, but also comprises samples produced by laboratory techniques, such as, for example, cell layers, cell extracts, cell culture media and supernatants, nucleic acid or protein solutions, etc., wherein such a sample can be immobilized on the surface of the array or chip by the immobilization of the biomolecules contained therein and can ultimately be analyzed.Reactive coatings for immobilizing biomolecules, for example on microarrays or biochips, make it possible to apply a large number of different samples in very confined spaces (regularly with the aid of robots) in defined and mutually delimited regions ("spots"), wherein the biomolecules to be analyzed in the respective samples come into contact with the coating and are specifically immobilized in the respective spot for further steps of the analysis method by this contact.DNA microarrays (or else DNA chips) for example regularly consist of solid-state substrates, usually glass plates, which comprise a layer with chemically fixed DNA molecules, which in turn interacts with complementary DNA molecules of a sample by nucleic acid hybridization, such that the complementary DNA molecules are specifically immobilized in the region of the respective spot. The binding of two complementary DNA molecules can then be optically detected and quantified in the further analysis method, for example by means of fluorescence markers which are coupled to the sample DNA during the analysis. Microarrays are thus used, for example, to examine the expression of a very high number of genes simultaneously or to diagnose particular gene mutations.Biochips can represent biologically / medically relevant properties and reactions as biosensors by (a) comprising a coating on which certain biomolecules of a sample, e.g. nucleic acids, peptides, proteins such as enzymes or antibodies, are immobilized and (b) containing an integrated microprocessor which evaluates the signals of the immobilized bio-molecules.Various coatings for immobilizing biomolecules are likewise known from practice.Conventional methods of coating solid state substrates for the fabrication of microarrays and biochips for biological sample analysis include any known methods for applying tie layers of complex coating systems, including via spray, spray and / or spin technology.In this connection, the use of UV-curable coatings for immobilizing biomolecules is likewise known.A method for producing a reactive, UV-curable coating for immobilizing biomolecules in the context of an enzyme-linked immunosorbent assay (ELISA) is described, for example, in EP 2 532 639 A1. The described coating for immobilizing peptides is produced using UV-reactive cycloalkanes, in particular bicyclononynes, by means of CLICK chemistry.A further method for producing a reactive, UV-curable coating for immobilizing biomolecules, in particular nucleic acids and proteins, is described in US 2005 / 0074478 A1. For producing the described coating, acrylamide polymers as well as acrylate polymers are used which are curable by means of UV radiation, so that the coating ultimately comprises acrylamide-acrylate copolymers, wherein the acrylate polymers contain functional groups suitable for immobilizing biomolecules.UV-curable coatings known from the prior art for immobilizing biomolecules on surfaces provided with the coating, such as the surfaces of microarrays or biochips, for example, are regularly limited in their binding capacity for biomolecules, so that detection against non-specific background fluorescence is often a challenge in practice.Furthermore, conventional UV lamps, typically medium pressure mercury lamps, are generally used for curing such coating systems. Since a large portion of the applied energy of the lamp is not converted into UV radiation, but into visible light and infrared radiation, i.e. heat, conventional lamps must be cooled. Here, too, contact with the lamp is at risk for burning. In addition, when conventional UV lamps are used for curing coating systems, ozone usually forms amounts of the irritant gas relevant to health. Ozone which is produced must not escape into the working space in hazardous concentration and must accordingly be sucked off, carried away or absorbed at the point of formation.It is therefore an object of the present invention to configure and further develop a method for producing a solid substrate coated with a coating of the type mentioned at the beginning in such a way that a coating system having a high binding capacity for biomolecules with low background fluorescence is producible and provided, and also to provide a correspondingly coated solid substrate and also a functional solution for producing a component of the coating system. A further object of the present invention is to specify both a use of the coating system for immobilizing biomolecules on a surface of a microarray or of a biochip provided with the coating system and a microarray or biochip comprising the coating system, in particular for analyzing biomolecules contained in a sample. In this regard, it is to be noted that the term "sample" in the context of this disclosure is to be understood in the broadest sense and thus includes, inter alia, both body fluids to be analyzed, such as, for example, blood, blood plasma, urine, saliva, etc., and also tissues, tissue sections or whole cells isolated therefrom, but also comprises samples produced by laboratory techniques, such as, for example, cell layers, cell extracts, cell culture media and supernatants, nucleic acid or protein solutions, etc., wherein such a sample can be immobilized on the surface of the array or chip by the immobilization of the biomolecules contained therein and can ultimately be analyzed.In one embodiment, the present invention achieves the above-mentioned objects with a method for producing a coated solid substrate for immobilizing biomolecules, comprising the following steps: (a) applying an adhesion promoter layer to at least one surface of the solid substrate, wherein the contact angle of water on the adhesion promoter layer is preferably between 50° and 60°; (b) providing a functional solution comprising:a crosslinking degree modulator comprising: acrylate monomers and / or acrylate oligomers having one acrylate ester group per molecule, preferably tetrahydrofurfuryl methacrylate monomers and / or oligomers; and / or 2-phenoxyethyl acrylate monomers and / or oligomers; and / or acrylate monomers and / or acrylate oligomers having more than one, preferably more than two or more than three acrylate ester groups per molecule, in particular comprising: trimethylolpropane ethoxy triacrylate monomers and / or oligomers; dipentaerythritol penta / hexacrylate monomers and / or oligomers; and / or functional dendrimers and / or dendrons, preferably highly branched bis(hydroxymethyl)propionic acid, acetylene, highly branched bis(hydroxymethyl)propionic acid, azide and / or highly branched bis(hydroxymethyl)propionic acid, hydroxyl and / or highly branched poly(amidoamine) dendrimers and / or dendrons;an immobilization modulator which provides reactive groups for immobilizing biomolecules, preferably an immobilization modulator comprising: ester groups, preferably N-hydroxysuccinimide ester groups, glycidyl ester groups or isocyanate ester groups; ether groups, preferably glycidyl ether groups; epoxy groups; aldehyde groups; free carboxyl and / or free amino groups, and combinations thereof; anda photoinitiator having an absorption maximum in the wavelength range between 315 and 400 nm, preferably a photoinitiator comprising: methyl phenylglyoxylate and diphenyl-(2,4,6-trimethylbenzoyl)phosphine oxide; ethylphenyl(2,4,6-trimethylbenzoyl)phosphinates; or phenylbis(2,4,6-trimethylbenzoyl)phosphine oxides;(c) applying the functional solution to the adhesion promoter layer; and (d) curing the functional solution by means of LED UV-A radiation in the wavelength range between 315 and 400 nm to form an immobilizing agent covalently connected to the adhesion promoter layer, wherein the LED UV-A radiation brings about a photochemically induced free-radical chain polymerization of the acrylate monomers and / or acrylate oligomers present in the functional solution and a 3-dimensional polymer structure of the immobilizing agent forms by covalent crosslinking of the acrylate monomers and / or acrylate oligomers, in and on which reactive groups for immobilizing biomolecules are arranged.Preferably, the adhesion promoter layer in step (a) is applied to at least one surface of the solid substrate by means of wet-chemical coating and / or chemical vapor deposition. Advantageously, the adhesion promoter layer predominantly comprises epoxysilane, (3-mercaptopropyl)trimethoxysilane, 3-(trimethoxysilyl)propyl methacrylate, polycyanurates, methacrylate-phosphate esters or isocyanate-based surface additives.In particular (3-mercaptopropyl)trimethoxysilane, 3-(trimethoxysilyl)propyl methacrylate, and isocyanate-based surface additives can serve as adhesion promoters for the immobilizing agent via chemical interaction with the substrate surface and thus connect it to the substrate surface, for example via hydrogen bonds or alternatively via covalent addition of the isocyante group to a Lewis-acidic hydrogen on the substrate surface.Furthermore, triazine-containing polycyanurates can form adhesion promoter layers in the form of a thin film which adsorbs to the substrate surface by chemical adhesion interactions. In particular, the polycyanurates adsorb glass substrates (SiO 2 on Si (100)) by electron interactions between the trioxy-triazine rings of the cyanurates and the naturally oxidized Si single crystals. On oxidic aluminum oxide surfaces (Al 2 O 3) the Lewis acidic OH groups serve as electron acceptors, the common electron pairs at O and N acting as electron donors.In step (c), the functional solution is preferably applied to the adhesion promoter layer by means of spin coating, airless spray application, ultrasonic spray application or ink jet methods.The functional solution in step (d) is cured preferably by means of LED UV-A radiation in the wavelength range from 340 to 390 nm or 350 to 390 nm or 360 to 390 nm or 370 to 390 nm or 380 to 390 nm or 385 nm or 360 to 380 nm or 360 to 370 nm or 365 nm.By using LED UV-A emitters, in particular in the high-frequency UV-A wavelength range from 340 to 390 nm, many disadvantages of conventional UV emitters are avoided. In particular, the formation of harmful ozone is practically avoided, so that smaller working safety measures are necessary and persons producing the coating system are exposed to lower risks. In addition, conventional UV emitters are regularly emitters containing mercury, which require greater work protection and disposal costs per se. LED-UV-A emitters often contain specially developed micro-optics for focusing the UV-A radiation, so that more UV-A-mediated energy strikes the functional solution, so that many photoinitiators (here radical formers) are activated.In embodiments in which the functional solution is applied by means of spin coating methods, curing is typically carried out by means of an LED planar emitter at a wavelength of 365 nm. In this case, the photoinitiator of the functional solution is advantageously a photoinitiator having an absorption maximum of about 365 nm and a comparatively high radical efficiency factor (f). The high free radical yield is required for optimum through-curing of the immobilizing agent.In embodiments in which the functional solution is applied by means of ink-jet methods, curing is typically effected by means of an LED UV lamp integrated in the ink-jet printer used at a wavelength of approximately 385 nm. In this case, the photoinitiator of the functional solution is advantageously a photoinitiator having an absorption maximum of about 385 nm and again a comparatively high radical efficiency factor (f). A particular advantage of the ink-jet method is that both the method step of applying the functional solution and the step of curing take place in a device, namely an ink-jet printer.In a further embodiment, the present invention achieves the aforementioned objects with a coating system for immobilizing biomolecules on a surface provided with the coating system, comprising at least (a) an adhesion promoter layer and (b) an immobilizing agent covalently bonded to the adhesion promoter layer, characterized in that the immobilizing agent has a 3-dimensional polymer structure formed by covalent crosslinking of acrylate monomers and / or acrylate oligomers of a crosslinking degree modulator, wherein the 3-dimensional polymer structure is cured by a radical chain polymerization which is photochemically induced by means of UV-A radiation and contains reactive groups for immobilizing biomolecules.In this regard, it should be mentioned that the immobilizing agent of the coating system according to the invention substantially comprises monomers and / or oligomers, such as dipentaerythritol penta / hexaacrylate, 2-hydroxyethyl 2-methacrylate, glycidyl methacrylate and (methyl phenylglyoxylate and diphenyl-(2,4,6-trimethylbenzoyl)phosphine oxide) monomers and / or oligomers. Advantageously, the coating system can be produced in a particularly cost-effective manner, since-in contrast to the use of copolymer-based raw materials-by the use of monomer- or oligomer-based raw materials-additional, partly cost-intensive synthesis, processing and analysis steps are dispensed with. In addition, the use of monomer- or oligomer-based raw materials is overall more environmentally compatible or friendly, in particular also because monomer- or oligomer-based raw materials can typically be processed directly on account of their lower viscosity and chemical structure, whereas copolymer-based raw materials are dissolved in normally toxic, organic solvents such as tetrahydrofuran before use.The curing of the coating system, and in particular of the immobilizing agent based on functional acrylate and methacrylate monomers or oligomers, is based on the principle of photochemically induced free-radical chain polymerization. This is usually carried out at room temperature under oxygen conditions or under inert conditions with a residual oxygen of <50 ppm by absorption of photons in the wavelength range from 200 nm to 400 nm. The basic requirement of radical chain polymerization is the presence of free radicals. Although it is technically possible to break up the π-component of the C=C double formation directly, it would be necessary for this purpose at a dissociation energy of about 6.3 eV at a wavelength of 197 nm. Short wavelength radiation of this order of magnitude would severely limit the application by ozone formation in the presence of oxygen and a small penetration depth of a few micrometers. In order to be able to operate in a longer-wave spectral range, it is thus necessary to use so-called photoinitiators.It is therefore also particularly advantageous that the curing of the coating system according to the invention can be carried out by means of LED UV lamps on the basis of the use of acrylate monomers or oligomers having free acrylate functional groups. In contrast, the curing of coating systems which are produced on the basis of copolymer-based acrylate raw materials frequently requires the use of mercury-containing UV lamps, since steric hindrances have to be overcome. Through the use of LED UV-radiationLamps have other advantages, such as:• Extremely Rapid and Complete Curing of the Coating• Very small use of volatile solvents is necessary• no ozone formation during UV irradiation (high working safety, no negative influence on the further coating process)• Narrow spectral distribution: efficiency better than in a mercury-containing UV lamp• Life of up to 50,000 hours - mercury-containing UV lamps usually have a life of only 10,000 to 15,000 hours• Environmentally Friendly: No Mercury• Low energy consumption• Low heat development: curing in the case of heat-sensitive substrates such as plastics, for example PMMA (polymethyl methacrylate), is possible.Biomolecules to be immobilized are usually contained in a sample, often aqueous.The term "biomolecule" in the context of this disclosure comprises both high molecular macromolecules (such as proteins, nucleic acids, polysaccharides or lipids) and low molecular weight compounds, which are for example the building blocks of the high molecular weight macromolecules (such as amino acids, nucleotides, sugars or fatty acids). Typical biomolecules to be immobilized include nucleic acids, in particular deoxyribonucleic acids (DNA) or ribonucleic acids (RNA); peptides; or proteins, in particular enzymes or antibodies. Nucleic acids to be analyzed can be, in particular, DNA or RNA oligonucleotides.In a manner according to the invention, it has first been recognized that the specific binding capacity of a coating system for immobilizing biomolecules can be realized in a surprisingly simple manner by virtue of the immobilizing agent having a 3-dimensional polymer structure. A 3-dimensional polymer structure is understood to mean a structure which is formed by 3-dimensional crosslinking of the acrylate monomers and / or oligomers of the crosslinking degree modulator, such that the immobilizing agent provides a greater number of reactive groups per unit area, compared with a monolayer of reactive polymers, and the specific binding capacity of the coating is thus greater. In particular, the immobilizing agent can be configured as a 3-dimensional hydrogel such that biomolecules contained in a sample are immobilized in the 3-dimensional polymer structure by reaction with the reactive groups. The 3-dimensionality of the polymer structure can be demonstrated by the swelling behavior of a coating via the layer thickness determination by means of quartz crystal microbalance and dissipation (QCM-D) measurement method.According to an advantageous embodiment, the coating system according to the invention for immobilizing biomolecules can be characterized in that:at least some of the acrylate monomers and / or acrylate oligomers of the crosslinking degree modulator are: tetrahydrofurfuryl methacrylate monomers and / or oligomers; and / or 2-phenoxyethyl acrylate monomers and / or oligomers; orat least some of the acrylate monomers and / or acrylate oligomers of the crosslinking degree modulator comprise more than one, preferably more than two or more than three acrylate ester groups per molecule, in particular comprising: trimethylolpropane ethoxy triacrylate monomers and / or oligomers; dipentaerythritol penta / hexacrylate monomers and / or oligomers; and / orthe immobilizing agent comprises highly branched, functional dendrimers and / or dendrons, preferably highly branched bis(hydroxymethyl)propionic acid acetylene, highly branched bis(hydroxymethyl4propionic acid azide, highly branched bis(hydroxymethyl)propionic acid hydroxyl and / or highly branched poly(amidoamine) dendrimers and / or dendrons.Preferably, the immobilizing agent of the coating system according to the invention comprises:an additional raw material, preferably comprising hexanediol diacrylate, poly(ethylene glycol) or polythiol; and / ora hydrophilicity modulator, preferably 2-hydroxyethyl-2-methacrylate; and / orradicals of a starting radical of a photoinitiator, preferably of methyl phenylglyoxylate, of diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, of ethyl phenyl(2,4,6-trimethylbenzoyl)phosphinates or of phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxides; and / oran immobilization modulator which provides the reactive groups for immobilizing biomolecules, preferably an immobilization modulator comprising: ester groups, preferably N-hydroxysuccinimide ester groups, glycidyl ester groups or isocyanate ester groups; ether groups, preferably glycidyl ether groups; epoxy groups; aldehyde groups; free carboxyl and / or free amino groups, and combinations thereof.Further preferably, the 3-dimensional polymer structure of the immobilizing agent by curing a functional solution is comprising:between 12 and 32 percent by weight of the crosslinking degree modulator;between 11 and 16 percent by weight of the hydrophilicity modulator;between 7 and 18 percent by weight of the additional raw material;between 20 and 50 weight percent of the immobilization modulator; andbetween 3.5 and 6.5 percent by weight of the photoinitiator,This can be produced.In a particularly advantageous manner, the UV-A radiation is an LED-UV-A radiation in the wavelength range from 315 to 400 nm, preferably in the wavelength range from 340 to 390 nm or 350 to 390 nm or 360 to 390 nm or 370 to 390 nm or 380 to 390 nm or 385 nm or 360 to 380 nm or 360 to 370 nm or 365 nm. By using LED UV-A emitters, in particular in the high-frequency UV-A wavelength range from 340 to 390 nm, many disadvantages of conventional UV emitters are avoided. Since LED UV emitters emit the UV-A energy, do not contain mercury, do not produce heat virtually no ozone, overall less work protection measures are necessary and persons producing the coating system are exposed to significantly lower risks.Preferably, the adhesion promoter layer predominantly comprises derivatives of epoxysilane, (3-mercaptopropyl)trimethoxysilane, 3-(trimethoxysilyl)propyl methacrylate, polycyanurates, methacrylate-phosphate esters or isocyanate-based surface additives, which are covalently bonded to acrylate monomers and / or acrylate oligomers of the immobilizing agent. In a particularly advantageous manner, during the binding reaction covalently with the acrylate monomers and / or oligomers and, for example, an amino or thiol group of a biomolecule, no condensation product is generated which could remain in the coating system.The adhesion promoter layer can advantageously be applied to a surface by chemical vapor deposition. Alternatively, the adhesion promoter layer can be applied by means of a wet chemical coating method, in particular a dipping, casting, injection or pressure coating method, to a correspondingly prepared substrate surface (for example to a cleaned and activated glass substrate surface). Both the application by chemical vapor deposition and by a wet chemical coating method enables the rapid and cost-effective production of a substrate provided with the adhesion promoter layer. Suitable and already known methods for applying the immobilizing agent to the adhesion promoter layer are spin coating, slot die coating, airless spray application, ultrasonic spray application or ink jet methods. In particular, the use of an immobilizing agent that can be cured by means of UV-A radiation-photochemically induced radical polymerization enables the immobilizing agent to be applied particularly advantageously by the inkjet method. The ink-jet method enables the production of microarrays and biochips provided with the coating system according to the invention in a particularly efficient and simple manner.The combination according to the invention of an adhesion promoter layer with the immobilizing agent provides a coating system, wherein the contact angle of water on the immobilizing agent is optionally greater than 60°, preferably between 60° and 70°. By means of the preferably adjusted contact angle, relatively large volumes of aqueous sample solutions in spots with a small diameter, for example a diameter of between 140 and 170 μm, can be applied to the surface provided with the coating system, which in turn ensures a high concentration of biomolecules to be analyzed per spot.The combination of such a large number of biomolecules per spot and the advantageously high binding capacity of the immobilizing agent leads to a high signal intensity of the biomolecules to be analyzed in the subsequent analysis methods, which significantly facilitates their evaluation.The coating system preferably has a solvent resistance to organic solvents, in particular to acetone, toluene or methanol, so that it has no detectable damage after treatment with organic solvents, in particular after treatment with toluene or methanol for 30 minutes at 60° C. in each case. This robustness of the coating system enables its use in a wide variety of different analytical methods.In a further embodiment, the present invention achieves the aforementioned objects by a solid-state substrate for immobilizing biomolecules, which is produced by the aforementioned method according to the invention and comprises at least one surface provided with the aforementioned coating system according to the invention, wherein the substrate is preferably a glass substrate or plastic substrate or a silicon substrate or an oxidic substrate and is suitable for producing a microarray or a biochip.The solid-state substrate is preferably a microarray or biochip for immobilizing biomolecules. As already mentioned, the biomolecules to be immobilized for analysis are usually contained in a sample, often aqueous. Typical biomolecules to be analyzed include nucleic acids, in particular deoxyribonucleic acids (DNA) or ribonucleic acids (RNA); peptides; or proteins, in particular enzymes. Nucleic acids to be analyzed can be, in particular, DNA or RNA oligonucleotides.For labeling and detection of immobilized biomolecules, stimulable colorants are usually used which emit detectable radiation again after excitation, so-called fluorophores. Suitable fluorophores include, for example:Hydroxycoumarin325386Bluemethoxycoumarin360410BlueAlexa fluoro345442Blueaminocoumarin aminocoumarin aminocoumarin aminocoumarin aminocoumarin aminocoumarin aminocoumarin aminocoumarin aminocoumarin aminocoumarin aminocoumarin aminocoumarin amino350445BlueCy2490510Dark Green GreenFAM495516Dark Green GreenAlexa fluoro 488494517Light green greenFluorescein FITC495518Light green greenAlexa fluoro 430430545Light green greenAlexa fluoro 532530555Light green greenHEX535556Light green greenCy3550575YellowTRITC547572YellowAlexa fluoro 546556573YellowAlexa fluoro 555556573YellowR-phycoerythrin (PE)480;565578YellowRhodamine Red-X560580OrangeTamara565580Red RedCy3.5 581581596Red RedRox575602Red RedAlexa fluoro 568578603Red RedRed 613480;565613Red RedTexas Red615615Red RedAlexa fluoro 594590617Red RedAlexa fluoro 633621639Red RedAllophycocyanin Allophycocyanin650660Red RedAlexa fluoro 633650668Red RedCy5650670Red RedAlexa fluoro 660663690Red RedName NameAbsorbing wavelengthEmitting WavelengthVisible colorCy5.5675694Red RedTrRed490;675695Red RedAlexa fluoro 680679702Red RedCy7743770Red RedCyanine fluorophores are particularly suitable for labeling DNA or RNA molecules, including the known fluorophores Cy2, Cy3, Cy3B, Cy 3.5, Cy5, Cy5.5 or Cy7. Preferably, a microarray or biochip comprising a surface provided with the aforementioned coating system according to the invention has only a low background intensity. This low background intensity ensures that signals emitted by the fluorophores in the wavelength range of approximately 500 nm to 575 nm can be detected substantially without disruptive background signals of the substrate itself. The sensitivity of a microarray or biochip produced on the basis of the solid-state substrate according to the invention is thus advantageously high.The person skilled in the art knows how the background intensity is determined in a fluorescence-based measurement system compared to the signal intensity to be determined. For example, a microplate reader from TECAN can be used for this purpose, in which both the background intensity and the signal intensity to be determined of a microarray or biochip produced on the basis of the solid-state substrate according to the invention are effected via an optical system. In this system, the substrate surface is first irradiated by a light source having a wavelength of 532 nm in the excitation mode. This results in fluorescence emitted from the glass surface. The relevant emission wavelength is selected by an optical filter, for example with a wavelength of 575 nm. The intensity of the emitted light can then be determined by means of a detector. In such fluorescence measurements, depending on the requirement, a signal amplification "gain" can be set, which can naturally greatly influence fluorescence measurements. In this respect, the skilled person knows that it is precisely the determination of this parameter in each measurement system that is important in order to be able to determine a low background intensity compared to the signal intensity to be determined.A solid-state substrate which can be provided with the coating system according to the invention in order to be used as a microarray or biochip emits only a very low fluorescence signal before the coating system is applied. If, after application of the adhesion promoter layer, an intrinsic fluorescence with a signal strength of about 40±10 relative fluorescence units (rfu) is determined upon excitation with a wavelength of 532 nm, the substrate is also suitable for application of the immobilizing agent, since the background signal strength of the ready-to-use microarray or biochip, which results from the intrinsic fluorescence signal of the coated substrate and the background signal generated by non-specific binding or immobilization of coloring agent, then after excitation with a wavelength of 532 nm is usually still significantly less than 150 rfu. Example 1 demonstrates that (a) the intrinsic fluorescence of a suitable glass substrate is 22 ± 0.5 rfu, (b) the intrinsic fluorescence of the glass substrate with the adhesion promoter layer applied by chemical vapor deposition is 26.2 ± 1.0 rfu, and (c) the intrinsic fluorescence of the glass substrate with the coating system of the invention is 68.4 ± 1.4 rfu. In contrast, the intrinsic fluorescence of a known microarray provided with a conventional coating system is 172.8±20.1 rfu in a direct comparison and is thus more than 2.5 times higher.With regard to a plastic substrate to be used in the aforementioned solid-state substrates, microarrays or biochips, this can be a polymethyl methacrylate (PMMA) substrate.With regard to a glass substrate to be used in the aforementioned solid-state substrates, microarrays or biochips, it is conceivable that this is selected from: soda-lime glasses, borosilicate glasses, quartz glasses and / or alkali-free aluminoborosilicate glasses.Preferably, the glass of a glass substrate to be used in the solid-state substrate according to the invention can have the following composition of components corresponding to a lithium aluminosilicate glass (in % by weight):SiO 255-69Al 2 O 318-25Li 2 O3-5Na 2 O+K 2 O0-30MgO+CaO+SrO+BaO0-5ZnO0-4TiO 20-5ZrO 20-5TiO2+SnO 2+ SnO 22-6P 2 O 50-8F. F0-1B 2 O 30-2Again preferably, the glass of a glass substrate to be used in the solid-state substrate according to the invention can have the following components (in % by weight):SiO 257-66Al 2 O 318-23Li 2 O3-5Na 2 O+K 2 O3-25MgO+CaO+SrO+BaO1-4ZnO0-4TiO 20-4ZrO 20-5TiO2+SnO 2+ SnO 22-6P 2 O 50-7F. F0-1B 2 O 30-2Again preferably, the glass of a glass substrate to be used in the solid-state substrate according to the invention can have the following components (in % by weight):SiO 257-63Al 2 O 318-22Li 2 O3.5-5Na 2 O+K 2 O5-20MgO+CaO+SrO+BaO0-5ZnO0-3TiO 20-3ZrO 20-5TiO2+SnO 2+ SnO 22-5P 2 O 50-5F. F0-1B 2 O 30-2Again preferably, the glass of a glass substrate to be used in the solid substrate according to the invention can have the following components corresponding to a soda-lime silicate glass (in % by weight):SiO 240-81Al 2 O 30-6B 2 O 30-5Li 2 O+Na 2 O+K 2 O5-30MgO+CaO+SrO+BaO+ZnO5-30TiO 2+ ZrO 20-7P 2 O 50-2Again preferably, the glass of a glass substrate to be used in the solid-state substrate according to the invention can have the following components (in % by weight):SiO 250-81Al 2 O 30-5B 2 O 30-5Li 2 O+Na 2 O+K 2 O5-28MgO+CaO+SrO+BaO+ZnO5-25TiO 2+ ZrO 20-6P 2 O 50-2Again preferably, the glass of a glass substrate to be used in the solid-state substrate according to the invention can have the following components (in % by weight):SiO 250-76Al 2 O 30-5B 2 O 30-5Li 2 O+Na 2 O+K 2 O5-25MgO+CaO+SrO+BaO+ZnO5-20TiO 2+ ZrO 20-5P 2 O 50-2Again preferably, the glass of a glass substrate to be used in the solid-state substrate according to the invention can have the following components corresponding to a borosilicate glass (in % by weight):SiO 260-85Al 2 O 30-10B 2 O 35-20Li 2 O+Na 2 O+K 2 O2-16MgO+CaO+SrO+BaO+ZnO0-15TiO 2+ ZrO 20-5P 2 O 50-2Again preferably, the glass of a glass substrate to be used in the solid-state substrate according to the invention can have the following components (in % by weight):SiO 263-84Al 2 O 30-8B 2 O 35-18Li 2 O+Na 2 O+K 2 O3-14MgO+CaO+SrO+BaO+ZnO0-12TiO 2+ ZrO 20-4P 2 O 50-2Again preferably, the glass of a glass substrate to be used in the solid-state substrate according to the invention can have the following components (in % by weight):SiO 263-83Al 2 O 30-7B 2 O 35-18Li 2 O+Na 2 O+K 2 O4-14MgO+CaO+SrO+BaO+ZnO0-10TiO 2+ ZrO 20-3P 2 O 50-2Again preferably, the glass of a glass substrate to be used in the solid-state substrate according to the invention can have the following components (in % by weight):SiO 260-70Al 2 O 31-10B 2 O 31-10K 2 O1-10Na 2 O1-10ZnO1-10TiO 21-10Again preferably, the glass of a glass substrate to be used in the solid-state substrate according to the invention can have the following components corresponding to an alkali aluminosilicate glass (in % by weight):SiO 240-75Al 2 O 310-30B 2 O 30-20Li 2 O+Na 2 O+K 2 O4-30MgO+CaO+SrO+BaO+ZnO0-15TiO 2+ ZrO 20-15P 2 O 50-10Again preferably, the glass of a glass substrate to be used in the solid-state substrate according to the invention can have the following components corresponding to a low-alkali aluminosilicate glass (in % by weight):SiO 250-70Al 2 O 310-27B 2 O 30-18Li 2 O+Na 2 O+K 2 O5-28MgO+CaO+SrO+BaO+ZnO0-13TiO 2+ ZrO 20-13P 2 O 50-9Again preferably, the glass of a glass substrate to be used in the solid-state substrate according to the invention can have the following components (in % by weight):SiO 255-68Al 2 O 310-27B 2 O 30-15Li 2 O+Na 2 O+K 2 O4-27MgO+CaO+SrO+BaO+ZnO0-12TiO 2+ ZrO 20-10P 2 O 50-8Again preferably, the glass of a glass substrate to be used in the solid-state substrate according to the invention can have the following components (in % by weight):SiO 250-75Al 2 O 37-25B 2 O 30-20Li 2 O+Na 2 O+K 2 O0-4MgO+CaO+SrO+BaO+ZnO5-25TiO 2+ ZrO 20-10P 2 O 50-5Again preferably, the glass of a glass substrate to be used in the solid-state substrate according to the invention can have the following components (in % by weight):SiO 252-73Al 2 O 37-23B 2 O 30-18Li 2 O+Na 2 O+K 2 O0-4MgO+CaO+SrO+BaO+ZnO5-23TiO 2+ ZrO 20-10P 2 O 50-5Again preferably, the glass of a glass substrate to be used in the solid-state substrate according to the invention can have the following components (in % by weight):SiO 253-71Al 2 O 37-22B 2 O 30-18Li 2 O+Na 2 O+K 2 O0-4MgO+CaO+SrO+BaO+ZnO5-22TiO 2+ ZrO 20-8P 2 O 50-5It goes without saying that the total of the respective glass constituents of the glass compositions listed must be 100% by weight. Nevertheless, the glasses to be used in the invention, in particular the glasses described above, can again be modified. Thus, for example, the color of the respective glass can be changed by addition of color oxides.In advantageous embodiments, the glass substrates according to the invention are produced using particularly pure raw materials in order to minimize the fluorescence under illumination with UV radiation and / or radiation in visible light. In particular, the use of raw materials with a very low iron content has proven to be advantageous for this purpose. The glasses produced in this way thus advantageously contain particularly few impurities, in particular little iron.In a further embodiment, the present invention achieves the aforementioned objects with a microarray or biochip for immobilizing biomolecules, preferably for analyzing biomolecules contained in a sample, comprising the coating system according to the invention and / or the solid-state substrate according to the invention, wherein the biomolecules are preferably: nucleic acids, in particular deoxyribonucleic acids (DNA) or ribonucleic acids (RNA); peptides; or proteins, in particular enzymes.In a further embodiment, the present invention achieves the above-mentioned objects with a functional solution for use in a method according to the invention and / or for use in the production of an immobilizing agent of the coating system according to the invention, at least comprising:a crosslinking degree modulator comprising: acrylate monomers and / or acrylate oligomers having one acrylate ester group per molecule, preferably tetrahydrofurfuryl methacrylate monomers and / or oligomers; and / or 2-phenoxyethyl acrylate monomers and / or oligomers; and / or acrylate monomers and / or acrylate oligomers having more than one, preferably more than two or more than three acrylate ester groups per molecule, in particular comprising: trimethylolpropane ethoxy triacrylate monomers and / or oligomers; dipentaerythritol penta / hexacrylate monomers and / or oligomers; and / or highly branched functional dendrimers and / or dendrons, preferably highly branched bis(hydroxymethyl)propionic acid acetylene, highly branched bis(hydroxymethyl)propionic acid azide and / or highly branched bis(hydroxymethyl)propionic acid hydroxyl and / or highly branched poly(amidoamine) dendrimers and / or dendrons;an immobilization modulator which provides reactive groups for immobilizing biomolecules, preferably an immobilization modulator comprising: ester groups, preferably N-hydroxysuccinimide ester groups, glycidyl ester groups or isocyanate ester groups; ether groups, preferably glycidyl ether groups; epoxy groups; aldehyde groups; free carboxyl and / or free amino groups, and combinations thereof; anda photoinitiator having an absorption maximum in the wavelength range between 315 and 400 nm, preferably a photoinitiator comprising: methyl phenylglyoxylate and diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide; ethyl phenyl(2,4,6-trimethylbenzoyl)phosphinates; phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxides; or 2-hydroxy-2-methylpropiophenones.Highly branched, functional dendrimers and / or dendrons preferably comprise highly branched bis(hydroxymethyl)propionic acid acetylene, highly branched bis(hydroxymethyl)propionic acid azide and / or highly branched bis(hydroxymethyl4propionic acid hydroxyl and / or highly branched poly(amidoamine) dendrimers and / or dendrons, such as, for examplebis(hydroxymethyl)propionic acid acetylene dendrimers (bis-MPA acetylene dendrimers; polymer factory; product code: PFD-G1-TMP-ACETYLENES) are monodisperse multifunctional frameworks having a trimethylolpropane (TMP) core and an exact number of surface groups. These alkyne functional dendrimers can be used for various click chemistry reactions such as CuAAC and thiol-Yne coupling;bis(hydroxymethyl4propionic acid azide dendrimers (bis-MPA azide dendrimers; polymer factory; product code: PFD-G1-TMP-AZIDE) are monodisperse multifunctional frameworks having a trimethylolpropane (TMP) core and an exact number of azide surface groups. The azide functional dendrimers can also be used for click chemistry reactions such as CuAAC or SPAAC;bis(hydroxymethyl4propionic acid hydroxyl-dendrones (bis-MPA hydroxyl-dendrones; polymer factory; product code: PFd-G2-acetylene-OH) are monodisperse, multifunctional frameworks having a central acetylene group and an exact number of hydroxyl surface groups. The central acetylene group can be used for click chemistry conjugations such as CuAAC and thiol-in coupling to produce high functionality materials;a highly branched poly(amidoamine) which is considered an analog of PAMAM dendrimers. It has primary amine end groups and carboxylic acids, which allows a variety of reactions for easy modification or network formation.Bis(hydroxymethyl)propionic acid polyesters - and poly(amidoamine) frameworks, are biodegradable and have low cytotoxicity.The abovementioned functional solution preferably further comprises:containing an additional raw material, preferably comprising hexanediol diacrylate, poly(ethylene glycol) or polythiol; and / ora hydrophilicity modulator, preferably 2-hydroxyethyl-2-methacrylate.The crosslinking degree modulators specified here allow the crosslinking degree of the acrylate monomers and / or oligomers within the functional solution to be adjusted by means of radical chain polymerization which is photochemically induced by means of UV-A radiation. In combination with the immobilization modulators which provide the reactive groups for interaction with the biomolecules to be immobilized, the solvent and temperature resistance, the scratch resistance, the polymer shrinkage and the direct adjustment of the background intensity, but also of the immobilizing agent which is configured as the 3-dimensional binder capacity, can therefore be adjusted as a function of the functionality, structure and concentration of the respectively selected crosslinking degree and immobilization modulators. In particular, when crosslinking degree modulators having more than one, preferably more than two or more than three acrylate ester groups per molecule are used, the 3-dimensional polymer structure of the immobilizing agent can be adjusted advantageously. Note that a balance is found between the resistance of the immobilizing agent and the binding capacity of the immobilizing agent for biomolecules. For example, when using crosslinking degree modulators, the binding capacity of the immobilizing agent decreases with increasing branching, while the crosslinking degree increases. As the degree of crosslinking increases, the immobilizing agent becomes more compact and the accessibility of biomolecules to the reactive groups in the coating decreases.In order to be able to induce the photochemical free-radical chain polymerization of the acrylate monomers or oligomers by means of UV-A radiation, the functional solution also comprises a photoinitiator. In particular, the photoinitiator is distinguished by the high yield in the generation of free radicals on irradiation in UV-A radiation in the wavelength range between 315 and 400 nm. Photoinitiators are known and a preferred, commercially available photoinitiator comprises methyl phenylglyoxylate and diphenyl-(2,4,6-trimethylbenzoyl)phosphine oxide (5.8% by weight) and has an absorption maximum at a wavelength of about 365 nm. An alternative but likewise suitable photoinitiator comprises ethyl phenyl(2,4,6-trimethylbenzoyl)phosphinates and has an absorption maximum at a wavelength of about 385 nm. Another likewise suitable photoinitiator comprises phenylbis(2,4,6-trimethylbenzoyl)phosphine oxides and has an absorption maximum at a wavelength of about 370 nmIn particular embodiments in which the functional solution comprises an additional raw material, the additional raw material preferably serves to define the flexibility of the immobilizing agent after curing, in particular the additional raw material leads to an advantageous shrinkage of the immobilizing agent during curing, wherein the additional raw material supports the 3-dimensional polymer structure of the immobilizing agent via its swelling behavior and at the same time can prevent non-specific binding of other constituents of the samples. In addition, the reduction of non-specific bonds is achieved by increasing the polar proportion of the free surface energy. Increasing the polarity of the coating prevents hydrophobic interactions between the biomolecules and the coating.In order to adjust the contact angle of water on the immobilizing agent so that it is optionally greater than 60°, preferably between 60° and 70°, the functional solution preferably additionally comprises a hydrophilicity modulator. The hydrophilicity modulator additionally improves the adhesion of the coating system to the surface and reduces non-specific attachment of biomolecules. In some embodiments, the substrate adhesion of the coating system is formed due to the formation of hydrogen bonds between free SiOHxgroups and hydroxyl groups of the hydrophilicity modulator 2-hydroxyethyl-2-methacrylate.The functional solution preferably comprises:between 12 and 32 percent by weight of the crosslinking degree modulator;between 11 and 16 percent by weight of the hydrophilicity modulator;between 7 and 18 percent by weight of the additional raw material;between 20 and 50 weight percent of the immobilization modulator; andbetween 3.5 and 6.5 percent by weight of the photoinitiator.As already described, the aforementioned solid-state substrate is preferably a microarray or biochip for immobilizing biomolecules. As likewise already described, the biomolecules to be immobilized on the solid-state substrates according to the invention are preferably nucleic acids, in particular deoxyribonucleic acids (DNA) or ribonucleic acids (RNA); peptides; or proteins, in particular enzymes.The method according to the invention makes it possible in a surprisingly simple manner to produce large numbers of the solid-state substrates, microarrays and biochips according to the invention with particularly high resistance to aqueous solutions and solvents such as methanol, toluene and / or acetone, even at elevated temperature.In a further embodiment, the present invention solves the aforementioned problems by using a coating system according to the invention for immobilizing biomolecules on a surface of a microarray or a biochip provided with the coating system, wherein the biomolecules are preferably: nucleic acids, in particular deoxyribonucleic acids or ribonucleic acids; peptides; or proteins, in particular enzymes.There are now various possibilities for advantageously embodying and developing the teaching of the present invention. For this purpose, reference is made on the one hand to the claims subordinate to claim 1 and on the other hand to the following explanation of preferred exemplary embodiments of the invention on the basis of the drawing. In conjunction with the explanation of the preferred exemplary embodiments of the invention on the basis of the drawing, preferred embodiments and refinements of the teaching are also generally explained. The drawings show FIG. 1 schematically illustrates the steps of the inventive method of manufacturing a coating system on the surface of a solid substrate. Figure 2 shows a bar graph showing, according to Example 2, the polar portion of the free surface energy and the fluorescence intensity of an immobilized fluorescently labeled protein as a function of the concentration of 2-hydroxyethyl-2-methacrylate (HEMA) in the immobilizing agent. FIG. 3 shows, according to Example 3, the transmission spectrum of a cleaned solid substrate (here a glass slide; sample 1), of a solid substrate having the adhesion promoter layer of the coating system according to the invention (here a corresponding glass slide having a 3-(trimethoxysilyl)propyl methacrylate-based adhesion promoter layer; sample 2), of a solid substrate having the coating system according to the invention (sample 3) and of a corresponding solid substrate having a known copolymer-based coating system (sample 4). FIG. 4 shows, according to Example 4, representative light micrographs (magnification 20x) of the coating regions of a surface of a glass substrate provided with the coating system according to the invention after aging in solvent: (a) after aging for 48 h at room temperature RT in toluene; (b) after aging for 48 h at room temperature RT in methanol; and (c) after aging for 48 h at room temperature RT in acetone. Fig. 5 shows, according to Example 5, the results of cross-cut tests concerning a known coating system (comparative sample) applied to a glass substrate and two samples of the coating system according to the invention applied to respective glass substrates; i.o.= okay; n.i.o.= not okay FIG. 6 shows, according to Example 6, the results of the contact angle measurement of water on adhesion promoter layers based on 3-methacryloxypropyltrimethoxysilane and as a function of the silane concentration. FIG. 7 shows, according to Example 6, the results of the background intensity measurements of adhesion promoter layers based on 3-methacryloxypropyltrimethoxysilane and as a function of the silane concentration and the temperature. Fig. 8 shows, according to Example 7, a schematic representation of the DNA molecules bound upon hybridization of a DNA sample to be analyzed in the 3-dimensional polymer structure of the immobilizing agent. Figure 9 shows, according to Example 7, the results of a comparative investigation of the oligonucleotide binding capacity of a coating system according to the invention (with 3D polymer structure) and of a 2D epoxy coating as a function of concentration. Figure 10 shows, according to Example 8, results of the measurement of non-specific binding of proteins on a coating system according to the invention (with 3D polymer structure) in comparison with a 2D epoxy coating. Figure 11 shows, according to Example 8, results of the measurement of non-specific binding of proteins on a coating system according to the invention (with 3D polymer structure) in comparison with a 2D epoxy coating. FIG. 12 shows, according to Example 9, the relationship between the increased contact angle of water on a coating system according to the invention and the spot diameter of an applied sample. FIG. 13 shows, according to Example 9, the increased binding capacity of the coating system according to the invention per spot.With regard to further advantageous configurations of the coating system according to the invention for immobilizing biomolecules, of the solid-state substrate according to the invention for immobilizing biomolecules, of the microarray according to the invention or biochips for immobilizing biomolecules, of the functional solution according to the invention for producing an immobilizing agent and of the method according to the invention for producing a coated solid-state substrate, reference is made to the general part of the description and to the appended examples and claims in order to avoid repetitions.FIG. 1 schematically illustrates the steps of the inventive method of producing a coating system ( 1) on a surface ( 2) of a solid substrate ( 3). First, a tie layer (4) comprising methacrylate organo silane or thiol organo silane monomers is applied to a surface (2) of a glass substrate (3), preferably by chemical vapor deposition (CVD). Catalyzed hydrolysis and condensation of the silyl ether compound of the methacrylate or thiolsilane adsorbed on the surface ( 2) results in free (i.e. reactive) acrylate groups ( 5) or free (i.e. reactive) thiol groups ( 6) remaining when methacrylate organosilane monomers or thiol organosilane monomers are used (FIG. 1( a)). Through trimethoxy or triethoxysilyl groups, the methacrylate organo silane or thiol organo silane monomers bond to the surface of the glass substrate. These reactive groups (5, 6) of the adhesion promoter layer can form covalent bonds with constituents of the immobilizing agent (7) to be applied, and thus impart the optimum adhesion strength of the immobilizing agent (7) on the adhesion promoter layer (4) on the one hand and indirectly on the surface (2) of the glass substrate (3) on the other hand. In the next step, the functional solution provided is applied to the adhesion promoter layer and cured by means of free-radical chain polymerization which is photo-chemically induced by means of LED UV-A radiation in the wavelength range between 315 and 400 nm. In particular, intermolecular crosslinking takes place between the reactive groups (5, 6) of the adhesion promoter layer (4) with constituents of the functional solution, such that the 3-dimensional polymer structure of the immobilizing agent forms, in and on which reactive epoxy groups (8) are arranged. The reactive epoxy groups (9) can react with amino groups of the biomolecules to be immobilized through epoxy opening and thus immobilize the biomolecules.ExamplesThe composition of immobilizing agents according to the invention can be found in the following table:IngredientsPercentage (% by weight)Dipentaerythritol penta / hexaacrylate21,22-Hydroxyethyl-2-methacrylate13,5Poly(ethylene glycol)15,4Glycidyl methacrylate44,2Methyl phenylglyoxylate and diphenyl-(2,4,6-trimethylbenzoyl)phosphine oxide (5.8% by weight) or ethyl phenyl(2,4,6-trimethylbenzoyl)phosphinates5,8SUM:100(Hexanediol diacrylate) HDDA (flexibility)10,4Trimethylolpropane ethoxy triacrylates29,8Glycidyl methacrylate41,82-Hydroxyethyl-2-methacrylate14,02,4,6-Trimethylbenzoyldiphenylphosphine oxide4,0SUM:100Tetrahydrofurfuryl acrylate11-12Trimethylolpropane ethoxy triacrylates41 - 54Glycidyl methacrylate14 - 272-Hydroxyethyl-2-methacrylate14Ethylphenyl(2,4,6-trimethylbenzoyl)phosphinates6-8SUM:100Example 1Comparative Intrinsic Fluorescence Determination: (a) a suitable glass substrate (BOROFLOAT 33, thickness 1.0±0.05 mm; SCHOTT AG, Mainz, Germany, (b) the glass substrate provided with a methacryloxypropyltrimethoxysilane-based adhesion promoter layer by means of chemical vapor deposition (CVD), (c) the glass substrate provided with a coating system according to the invention, wherein the coating system comprises a methacryloxypropyltrimethoxysilane-based adhesion promoter layer and an immobilizing agent according to formulation 2.5 by means of CVD, and (d) a microarray available on the market comprising a conventional coating system (TRIDIA HD, Surmodics IVD, Inc, Eden Prairie, USA).Determination method is performed as follows:Device settings:Excitation wavelength532 nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nm nmEmission wavelength575 nm (Cy3 filter)Amplification of the Signal_PM (Photomultiplier tube) Gain200Scan Resolution10 μmMeasurement results: Measurement results(a) BOROFLOAT 33220,50,02(b) BOROFLOAT 33 with adhesion promoter layer26,21,00,04(c) BOROFLOAT 33 with coating system68,41,40,02(d) TRIDIA HD172,820,10,12Example 2In general, the optimum functionality of a microarray and its evaluation largely depends on the intrinsic fluorescence of the solid-state substrate provided with a coating or a coating system and the specific binding behavior of the coating system of the microarray. In order to reduce non-specific binding and thus an increased background signal after incubation with a sample, the immobilizing agent was modified via the incorporation of the raw material 2-hydroxyethyl-2-methacrylate (HEMA) in order to increase the polar fraction of the free surface energy in steps and was investigated by means of NSB tests (NSB=non-specific binding).The specific and non-specific binding behavior of a coating system is tested with an NSB test in which a glass substrate provided with the coating system is incubated with a fluorescence-labeled protein mix (DY-547P1). By increasing the polar portion of the free surface energy by the incorporation of HEMA, less non-specific binding on the coating system can be achieved.The intensities shown in Figure 2 were normalized to the intensities of the cleaned glass substrate without the coating system (negative control).Material:- Goat serum DY-547P11xPBS / 0.1% Tween 20Deionized waterResults: As can be seen from an experiment, a coating system without a hydrophilicity modulator (here without HEMA; negative control) has very strong non-specific binding behavior. Advantageously, the signal intensity of the immobilized fluorescently labeled protein decreases continuously with the increase in the HEMA concentration in the immobilizing agent. The decrease in signal intensity of the immobilized fluorescently labeled protein is presumably due to the decrease in hydrophobic protein-solid interactions. The coating surface can interact with the adsorbed proteins in a microscopic as well as on a macroscopic scale and can also influence their conformation. First, consideration should be given to precisely those factors which greatly influence the properties of the solid surface. An important driving force for the adsorption of the proteins is the hydrophobic interaction with the solid surface. Generally, the hydrophobic properties make the highest contribution to protein adsorption compared to electrostatics and depend on surface polarity. In this context, investigations regarding the resistance to protein adsorption in oligoether-based self-assembled monolayers (SAM, engl. Self-assembled monolayer) with factors such as internal hydrophilicity, terminal hydrophilicity and lateral density of the SAM layer shows that increasing hydrophobicity of the oligo(ethylene glycol) layer due to the introduced hydrophobic end groups enhances the adsorption of the protein fibrinogen.In summary, it can be stated that the immobilization is dependent on the following parameters:the physical nature of the support (e.g. porosity, shape etc.)the chemical nature of the support (chemical composition, in particular crosslinking, branching, number of functional groups, reactivity of the functional groups, etc.)the nature of the linkage or the binding chemistrythe conformation of the protein during immobilization and after immobilization; the type and length of the spacerthe properties of the medium during the attachment of the selected proteinthe individual properties of the selected protein;the number of bonds between the protein and the carrierdistributing the protein on or in the carrier.Example 3In order to make possible the measurements of signal intensities of immobilized fluorescently labeled biomolecules through a glass substrate provided with the coating system, the coating system "LED UV coating" should have a high transparency. Since UV coating systems are cured with the aid of short-wave radiation, it can be assumed that these are at least partially transparent to ultraviolet radiation, since otherwise the radiation cannot propagate in the bulk layer. In this context, the question of transparency and of the UV activity of the coating is to be clarified by measuring the extinction or transmission at the respective wavelength ranges in the UV / Vis spectrum.Materials:A SPECORF200 spectrometer from Fa. Analytics - Jena was used and the transmission spectra measured are summarized in FIG. 2.Results:As can be seen from the results shown in FIG. 3, the application of the coating system according to the invention to a glass substrate does not lead to a disadvantageous restriction of the transmission (see transmission sample 3 in FIG. 2 ). In particular, the coating system according to the invention (sample 3 in FIG. 2 ) has transmission values of comparable magnitude to a coating system already available on the market (sample 4 in FIG. 2 ).Example 4Since in cytodiagnostics organic solvents such as toluene, methanol or acetone are used as fixing agents, an advantageous coating system must have good solvent resistance.The following steps were carried out to test the solvent resistance of the coating system according to the invention:Step No. 1: Before the removal of storage - Scanning by means of Tecan Scanner (Control)Step No. 2: KW measurement on a contact angle measuring instrument from KrüssStep No. 3: Deposition of substrates in the following organic solvents, such as toluene, methanol and acetone, at room temperature RT for 48 hoursStep No. 4: After aging, dry in a stream of nitrogenStep No. 5: After drying in a substrate holder or rack in an oven at a temperature of 30°C for 30 minStep No. 6: Cooling to RT for 20 min Step No. 7: Measurement of background fluorescence by means of Tecan scanner (result)Step No. 8: Light microscopic (LM) Evaluation of durability: LM photographs at 2.5x and 20x magnification taken of treated and untreated sites. In this case, the coating with the glass pencil should be intentionally violated so that the coating side can also be visually recognized on the image.Step No. 9: Contact angle (HC) measurements on the treated and untreated areas.Step No. 10: Documentation of the test procedure including. EvaluationResults:As shown in FIGS. 4a) to 4c), the coating system according to the invention has very good solvent resistance to toluene, methanol and acetone at room temperature.Example 5In order to test the adhesion of the coating system to the solid surface, the cross-cut test was carried out in the formulated coating on the basis of DIN EN ISO 2409. The requirement for the coating with regard to the adhesive strength was the achievement of a cross-cut characteristic value of 0-1. It can be carried out easily and does not require great expenditure on apparatus and time. The coating surface is scratched with a cutter until the substrate is reached. The sample is then rotated through approximately 90° and the process is repeated. This results in a scribe pattern on the sample. In the case of hard substrates, an adhesive strip is then placed parallel to a cut edge over the grid pattern and pressed on. After a time of at most 5 minutes, the adhesive tape is uniformly peeled off at an angle of 60° within 0.5 seconds to 1.0 second. The resulting crosscut is then observed with a magnifying glass and documented by a light micrograph. There are six grid intersection characteristics.Crosscut characteristic value 0 (GT0) means that the pattern has remained completely unchanged and detachment or raggedness of the cut edges cannot be observed at any point. With a grid cut characteristic value of 5, more than 65% of the grid cut surface is detachedIn principle, a poor adhesive strength of the coating system can result from the fact that the adhesion promoter layer has not undergone sufficient bonding to the substrate (adhesive bonding), or that the immobilizing agent in the bulk of the coating system has not undergone sufficient bonding to the adhesion promoter layer (cohesive bonding). This cross-cut test does not allow any statement on which of the two processes is responsible for this in the case of poor adhesion.Results:As shown in FIG. 5, the samples (Sample 1_Sl No. 1 and Sample 1_Sl No. 2) of the coating system according to the invention have an advantageous GT value of 1.Example 6To ensure the adhesive strength and the wetting behavior of the coating system according to the invention on a cleaned glass surface, the adhesion promoter layer (organosilane layer based on 3-(trimethoxysilyl)propyl methacrylate) was tested for its suitability in this respect. In order to achieve an optimum contact surface between the coating and the glass surface, at which the adhesion interactions can be transferred, the adhesion promoter layer (primer layer) must primarily have very good wetting behavior with respect to the immobilizing agent (LED-UV coating). The optimum wetting behavior of the adhesion promoter layer is achieved in the test system carried out by the following:a contact angle of water of 55°±5° on the adhesion promoter layer; anda background intensity of 40±10 rfu,Set.A contact angle measuring instrument from Krüss was used for the water contact angle measurements. As the test liquid, double-distilled water was used. To measure the static contact angle, a drop volume of 1-3 μl was deposited continuously on the sample surface to be examined at the dossing speed of 2.0-4.0 μl / min. The contact angle analysis was automatically performed by the Krüss software "ADVANCE".Without these parameters being reached, a coating system regularly exhibits effects of wetting disturbances, for example in the form of crater formation in the immobilizing agent. For this reason, the relationship between the silane concentration of the adhesion promoter layer and the contact angle of the water was systematically investigated in order to define an optimum process condition for applying the adhesion promoter layer to a cleaned substrate surface (silanization of a cleaned substrate surface). Furthermore, the formulation of the adhesion promoter layer is adapted in such a way as to keep the intrinsic fluorescence of the adhesion promoter layer low. A low intrinsic fluorescence of the adhesion promoter layer contributes to a low background intensity of the entire coating system and ensures that signals emitted by the immobilized, fluorescence-labeled biomolecules can be detected without disturbing background signals of the coating system itself. Since the low intrinsic fluorescence of the adhesion promoter layer adds to the likewise low intrinsic fluorescence of the immobilizing agent, the coating system according to the invention has a very low background intensity.Results:As shown in Figure 6, the desired contact angle of water of 55±5° is achieved with a silane concentration in a range of 0.005 to 0.01 mol / L and at a temperature of between 60 and 90°C. As is also shown in FIG. 7, adhesion promoter layers according to the invention achieve the required low background intensity of 40±10 rfu.Example 7Free-radical chain polymerization, for example of poly(ethylene glycol) diacrylates (PEG), poly(ethylene glycol) methacrylates (PEG), polythiols, produces PEG diacrylates and PEG methacrylates which give water-soluble, neutral polymers and highly water-swellable hydrogels. It can thus be ensured that an aqueous DNA solution can also penetrate into the 3-dimensional polymer structure of the coating system and can bind a greater number of biomolecules than would be possible with a polymer monolayer.FIG. 8 schematically shows a hydrogel with immobilized DNA probe molecules, it being intended to illustrate that such a coating system comprising a 3-dimensional (3-D) polymer structure has a greater binding capacity for DNA probe molecules than a polymer monolayer (2-D). As a result, amplification of the emitted fluorescence signal can be achieved using the coating system according to the invention after successful hybridization.Results: As shown in FIG. 9, the coating system according to the invention with reactive epoxy groups for immobilizing biomolecules (left bars in each case) has a higher oligonucleotide binding capacity than a known 2D epoxy coating (reference; right bars in each case). Furthermore, it can be seen that the adsorption of the known 2D epoxy coating at a probe concentration of 5 μmol / L is close to saturation, whereas this is not yet the case with the 3-D LED UV coating according to the invention, but instead allows a further increase in the adsorption under the same conditions.Example 8In general, diagnostic coating systems are modified physically or chemically in such a way that specific immobilization of biomolecules and thus the precision in diagnostic results can be ensured. As already shown in Example 2, certain embodiments of the coating system according to the invention contain HEMA in order to keep non-specific immobilization of biomolecules low.Material and Method:PBS buffer with pH 7.2 - pH 7,6PBS 0.05% Tween 20- Skimmed milk powder solutionGoat serum labeled with the dye Dy547 (goat serum-NH2)For a comparative test of the non-specific binding of biomolecules to a coating system according to the invention, the following steps were carried out:Glass substrates (slides) provided with the coating system were prescanned prior to incubation using Tecan ScannerLS 400 to analytically determine the background intensity of the coating prior to testing.After prescanning, the glass substrates provided with the coating system were blocked on a shaker at RT for 3 h incubation using a lean milk powder solution.After blocking, the incubation solution (and thus the coated slides) was added with Goat serum Dy547, and further incubated on a shaker at 4°C in the refrigerator.After incubation, the washing process was carried out on the shakerFor drying, the slides were dry-centrifuged for 5 min at 1,300 rpm and RT.After drying, the incubated slides were measured by means of Tecan scanner LS 400 to determine signal intensity of the nonspecifically bound Goat serum Dy547.As can be seen from FIGS. 10 and 11, the signal intensity of the immobilized Goat serum Dy547 on the 3D LED UV epoxy coating according to the invention is about 68 to 71% lower at 451 or 425 rfu compared with the 2D epoxy coating at 1487 rfu (FIG. 10 ) or at 455 or 484 rfu compared with the 2D epoxy coating at 1490 rfu (FIG. 11 ).Example 9To adjust the contact angle of an aqueous sample on the coating system according to the invention, 2-hydroxyethyl-2-methacrylate was used as hydrophilicity modulator in the immobilizing agent.To compare the 3D epoxy coating system according to the invention with the known 2D epoxy coating (reference), the following experimental steps were carried out in parallel on both coatings: (1) measurement of the intrinsic fluorescence; (2) spotting of samples containing synthetically produced, single-stranded DNA fragments as biomolecules to be immobilized and an already fluorescence-labeled DNA fragment as positive control (3) immobilization of the biomolecules on the immobilizing agent (30 minutes); (4) measurement of the background fluorescence; (5) hybridization with complementary CY3-labeled oligonucleotides in an automated microarray hybridization stations (HS 4800 Tecan; about 2.5 hours) using the following washing solutions: 1st washing solution: Pre Hyb 2nd washing solution: 2xSS, 0.2% SDS 3. washing solution: 2xSS 4. washing solution: 0.2xSS 5. washing solution: deionized water 6th washing solution: 0.1% SDSFIG. 12 shows that, as a result of a higher contact angle for water on a coated surface of a solid substrate, as expected, an aqueous sample has a smaller spot diameter for the same volume.Figure 13 shows that the coating system, in comparison with the known 2D epoxy coating (reference), can, however, not only record the higher sample density made possible by the increased contact angle on the smaller surface, but can also bind permanently, so that a significantly higher fluorescence intensity is measured. The increased binding capacity of the coating system can be attributed to the 3-dimensional polymer structure of the immobilizing agent.Finally, it should be expressly pointed out that the above-described exemplary embodiments of the coating system according to the invention for immobilizing biomolecules, of the solid-state substrate according to the invention for immobilizing biomolecules, of the microarray according to the invention or biochips for immobilizing biomolecules, of the functional solution according to the invention for producing an immobilizing agent and of the method according to the invention for producing a coated solid-state substrate serve merely to explain the teaching claimed, but do not restrict it to the exemplary embodiments.References included in the specificationThis list of documents cited by the applicant has been produced in an automated manner and is only included for the better information of the reader. The list is not part of the German patent application or utility model application. The DPMA does not take any adhesion for any faults or omissions.Patent Literature citedEP 2 532 639 A1

[0009] US 2005 / 0074478 A1

[0010]

Claims

A method for producing a coated solid substrate for immobilizing biomolecules, comprising the following steps: (a) applying a bonding layer to at least one surface of the solid substrate, wherein the contact angle of water on the bonding layer is preferably between 50° and 60°; (b) providing a functional solution comprising: - a crosslinking degree modulator comprising: acrylate monomers and / or acrylate oligomers having one acrylate ester group per molecule, preferably tetrahydrofurfuryl methacrylate monomers and / or oligomers; and / or 2-phenoxyethyl acrylate monomers and / or oligomers; and / or acrylate monomers and / or acrylate oligomers having more than one, preferably more than two or more than three acrylate ester groups per molecule, in particular comprising: trimethylolpropane ethoxy triacrylate monomers and / or oligomers; Dipentaerythritol penta / hexacrylate monomers and / or oligomers; and / or functional dendrimers and / or dendrons, preferably highly branched bis(hydroxymethyl)propionic acid acetylene, highly branched bis(hydroxymethyl)propionic acid azide and / or highly branched bis(hydroxymethyl)propionic acid hydroxyl and / or highly branched poly(amidoamine) dendrimers and / or dendrons; - an immobilization modulator which provides reactive groups for immobilizing biomolecules, preferably an immobilization modulator comprising: ester groups, preferably N-hydroxysuccinimide ester groups, glycidyl ester groups or isocyanate ester groups; ether groups, preferably glycidyl ether groups; epoxy groups; aldehyde groups; free carboxyl and / or free amino groups, and combinations thereof; and - a photoinitiator having an absorption maximum in the wavelength range between 315 and 400 nm, preferably a photoinitiator comprising: methyl phenylglyoxylate and diphenyl-(2,4,6-trimethylbenzoyl)phosphine oxide; ethylphenyl(2,4,6-trimethylbenzoyl)phosphinates; or phenylbis(2,4,6-trimethylbenzoyl)phosphine oxides; (c) applying the functional solution to the adhesion promoter layer; (d) curing the functional solution by means of LED-UV-A radiation in the wavelength range between 315 and 400 nm to form an immobilizing agent covalently bonded to the adhesion promoter layer, wherein the LED-UV-A radiation causes a photochemically induced free-radical chain polymerization of the acrylate monomers and / or acrylate oligomers present in the functional solution and a 3-dimensional polymer structure of the immobilizing agent is formed by covalent crosslinking of the acrylate monomers and / or acrylate oligomers, in and on which reactive groups for immobilizing biomolecules are arranged.The method according to claim 1, wherein the functional solution further comprises: - an additional raw material, preferably comprising hexanediol diacrylate, poly(ethylene glycol) or polythiol; and / or - a hydrophilicity modulator, preferably 2-hydroxyethyl-2-methacrylate.The method according to claim 2, wherein the functional solution comprises: - between 12 and 32 weight percent of the crosslinking degree modulator; - between 11 and 16 weight percent of the hydrophilicity modulator; - between 7 and 18 weight percent of the additional raw material; - between 20 and 50 weight percent of the immobilization modulator; and - between 3.5 and 6.5 weight percent of the photoinitiator.The method according to any of the preceding claims, wherein: - the application of the adhesion promoter layer to at least one surface of the solid substrate in step (a) is effected by means of wet chemical coating and / or chemical vapor deposition; and / or - the adhesion promoter layer predominantly comprises epoxysilane, (3-mercaptopropyl)trimethoxysilane, 3-(trimethoxysilyl)propyl methacrylate, polycyanurates, methacrylate-phosphate esters or isocyanate-based surface additives; and / or - the application of the functional solution to the adhesion promoter layer in step (c) is effected by means of spin coating, slot die coating, airless spray application or ink jet methods; and / or - curing the functional solution in step (d) by means of LED UV-A radiation in the wavelength range from 340 to 390 nm or 350 to 390 nm or 350 to 390 nm or 360 to 390 nm or 370 to 390 nm or 380 to 390 nm or 385 nm or 360 to 380 nm or 360 to 370 nm or 365 nm.The method according to any of the preceding claims, wherein the solid-state substrate is a microarray or biochip for immobilizing biomolecules, preferably: nucleic acids, in particular deoxyribonucleic acids or ribonucleic acids; peptides; or proteins, in particular enzymes.Coating system for immobilizing biomolecules on a surface provided with the coating system, comprising at least (a) an adhesion promoter layer and (b) an immobilizing agent covalently bonded to the adhesion promoter layer, characterized in that the immobilizing agent has a 3-dimensional polymer structure formed by covalent crosslinking of acrylate monomers and / or acrylate oligomers of a crosslinking degree modulator, the 3-dimensional polymer structure being cured by a radical chain polymerization which is photochemically induced by means of UV-A radiation and comprising reactive groups for immobilizing biomolecules.The coating system according to claim 6, characterized in that: - at least some of the acrylate monomers and / or acrylate oligomers of the crosslinking degree modulator are: tetrahydrofurfuryl methacrylate monomers and / or oligomers; and / or 2-phenoxyethyl acrylate monomers and / or oligomers; or - at least some of the acrylate monomers and / or acrylate oligomers of the crosslinking degree modulator contain more than one, preferably more than two or more than three acrylate ester groups per molecule, in particular comprising: trimethylolpropane ethoxy triacrylate monomers and / or oligomers; dipentaerythritol penta / hexacrylate monomers and / or oligomers; and / or - the immobilizing agent comprises highly branched functional dendrimers and / or dendrons, preferably highly branched bis(hydroxymethyl)propionic acid, acetylene, highly branched bis(hydroxymethyl)propionic acid, azide and / or highly branched bis(hydroxymethyl)propionic acid, hydroxyl and / or highly branched poly(amidoamine) dendrimers and / or dendrons.The coating system according to claim 6 or claim 7, characterized in that: - the immobilizing agent comprises an additional raw material, preferably comprising hexanediol diacrylate, poly(ethylene glycol) or polythiol; and / or - the immobilizing agent comprises a hydrophilicity modulator, preferably 2-hydroxyethyl-2-methacrylate; and / or - the immobilizing agent comprises residues of a starting radical of a photoinitiator, preferably methyl phenylglyoxylate, diphenyl-(2,4,6-trimethylbenzoyl)phosphine oxide, ethyl (2,4,6-trimethylbenzoyl)phenylphosphinate, or phenyl-bis(2,4,6-trimethylbenzoyl)phosphine oxides; and / or - the immobilizing agent comprises an immobilizing modulator which provides the reactive groups for immobilizing biomolecules, preferably an immobilizing modulator comprising: ester groups, preferably N-hydroxysuccinimide ester groups, glycidyl ester groups or isocyanate ester groups; ether groups, preferably glycidyl ether groups; epoxy groups; aldehyde groups; free carboxyl and / or free amino groups, and combinations thereof.The coating system according to any of the preceding claims 6 to 8, wherein the 3-dimensional polymer structure of the immobilizing agent is producible by curing a functional solution comprising: - between 12 and 32 weight percent of the crosslinking degree modulator; - between 11 and 16 weight percent of the hydrophilicity modulator; - between 7 and 18 weight percent of the additional raw material; - between 20 and 50 weight percent of the immobilizing modulator; and - between 3.5 and 6.5 weight percent of the photoinitiator.The coating system according to any one of the preceding claims 6 to 9, wherein the UV-A radiation is an LED-UV-A radiation in the wavelength range of 315 to 400 nm, preferably in the wavelength range of 340 to 390 nm or 350 to 390 nm or 350 to 390 nm or 360 to 390 nm or 370 to 390 nm or 380 to 390 nm or 385 nm or 360 to 380 nm or 360 to 370 nm or 365 nm.The coating system according to any of the preceding claims 6 to 10, wherein the adhesion promoter layer predominantly comprises derivatives of epoxysilane, (3-mercaptopropyl)trimethoxysilane, 3-(trimethoxysilyl)propyl methacrylate, polycyanurates, methacrylate phosphate esters or isocyanate-based surface additives covalently linked to acrylate monomers and / or acrylate oligomers of the immobilizing agent.The coating system according to any of the preceding claims 6 to 11, wherein the adhesion promoter layer is able to be applied to a surface by chemical vapor deposition, and / or wherein the immobilizing agent is able to be applied to the adhesion promoter layer by spin coating, slot-coating, airless spray application or ink jet methods, and wherein the contact angle of water on the immobilizing agent is optionally greater than 60°, preferably between 60° and 70°.The coating system according to any of the preceding claims 6 to 12, wherein the coating system has a solvent resistance to organic solvents, in particular to acetone, toluene or methanol, so that it does not have any detectable damage after a treatment with organic solvents, in particular after a treatment with toluene or methanol for 30 minutes at 60°C in each case.Solid-state substrate for immobilising biomolecules produced by a method according to one of Claims 1 to 5 and comprising at least one surface provided with a coating system according to one of Claims 6 to 13, the substrate preferably being a glass substrate or plastic substrate or silicon substrate or an oxidic substrate, and being suitable for producing a microarray or a biochip.The solid-state substrate according to claim 14, wherein the solid-state substrate is a microarray or biochip for immobilizing biomolecules, preferably for analyzing biomolecules contained in a sample, and wherein the biomolecules preferably comprise: nucleic acids, in particular deoxyribonucleic acids or ribonucleic acids; peptides; or proteins, in particular enzymes, and the microarray or biochip has a background intensity of less than 150 relative fluorescence units at an excitation wavelength of 532 nm.Functional solution for use in a method according to any of claims 1 to 5 and / or for use in the preparation of an immobilizing agent of the coating system according to any of claims 6 to 13, at least comprising: - a crosslinking degree modulator comprising: acrylate monomers and / or acrylate oligomers having one acrylate ester group per molecule, preferably tetrahydrofurfuryl methacrylate monomers and / or oligomers; and / or 2-phenoxyethyl acrylate monomers and / or oligomers; and / or acrylate monomers and / or acrylate oligomers having more than one, preferably more than two or more than three acrylate ester groups per molecule, in particular comprising: trimethylolpropane ethoxy triacrylate monomers and / or oligomers; dipentaerythritol penta / hexacrylate monomers and / or oligomers; and / or functional dendrimers and / or dendrons, preferably highly branched bis(hydroxymethyl4propionic acid acetylene, highly branched bis(hydroxymethyl4propionic acid azide and / or highly branched bis(hydroxymethyl4propionic acid hydroxyl and / or highly branched poly(amidoamine) dendrimers and / or dendrons; - an immobilization modulator which provides reactive groups for immobilizing biomolecules, preferably an immobilization modulator comprising: ester groups, preferably N-hydroxysuccinimide ester groups, glycidyl ester groups or isocyanate ester groups; ether groups, preferably glycidyl ether groups; epoxy groups; aldehyde groups; free carboxyl and / or free amino groups, and combinations thereof; and - a photoinitiator having an absorption maximum in the wavelength range between 315 and 400 nm, preferably a photoinitiator comprising: methyl phenylglyoxylate and diphenyl-(2,4,6-trimethylbenzoyl)phosphine oxide; ethylphenyl(2,4,6-trimethylbenzoyl)phosphinates; or phenylbis(2,4,6-trimethylbenzoyl)phosphine oxides.The functional solution according to claim 16, further comprising: - an additional raw material, preferably comprising hexanediol diacrylate, poly(ethylene glycol) or polythiol; and / or - a hydrophilicity modulator, preferably 2-hydroxyethyl-2-methacrylate.The functional solution according to claim 17 comprising: - between 12 and 32 weight percent of the crosslinking degree modulator; - between 11 and 16 weight percent of the hydrophilicity modulator; - between 7 and 18 weight percent of the additional raw material; - between 20 and 50 weight percent of the immobilization modulator; and - between 3.5 and 6.5 weight percent of the photoinitiator.Use of a coating system according to one of claims 6 to 13 for immobilizing biomolecules on a surface of a microarray or a biochip provided with the coating system, wherein the biomolecules are preferably: nucleic acids, in particular deoxyribonucleic acids or ribonucleic acids; peptides; or proteins, in particular enzymesMicroarray or biochip for immobilizing biomolecules, preferably for analyzing biomolecules contained in a sample, comprising the coating system according to any of claims 6 to 13 and / or the solid-state substrate according to claim 14 or 15, wherein the biomolecules are preferably: nucleic acids, in particular deoxyribonucleic acids (DNA) or ribonucleic acids (RNA); peptides; or proteins, in particular enzymes.

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