Nitrocellulose-coated microarrays and manufacturing processes

Abstract

The invention relates to microarrays for the immobilization of biomolecules and methods for their production. Furthermore, the invention relates to the use of a microarray for the immobilization of biomolecules and for the analysis of biomolecules contained in a sample.

Description

[0001] The invention relates to microarrays for the immobilization of biomolecules and methods for their production. Furthermore, the invention relates to the use of a microarray for the immobilization of biomolecules and for the analysis of biomolecules contained in a sample.

[0002] Various microarrays and biochips are known in practice that enable the immobilization of biomolecules contained in a sample in a very small space and their presentation for further analysis. In this regard, it should be noted that the term "sample" in the context of this disclosure is to be understood in the broadest sense and thus includes, among other things, both body fluids to be analyzed, such as blood, blood plasma, urine, saliva, etc., as well as tissue, tissue sections, or whole cells isolated therefrom, but also laboratory-produced samples, such as cell layers, cell extracts, cell culture media and supernatants, nucleic acid or protein solutions, etc., whereby such a sample can be immobilized and ultimately analyzed by immobilizing the biomolecules it contains on the surface of the array or chip.

[0003] Reactive coatings for the immobilization of biomolecules, for example, on the surface of a microarray or biochip, allow a large number of different samples to be applied in defined and distinct areas ("spots") in a very small space (usually with the help of robots). The biomolecules to be immobilized act as capture probes in a spot and come into direct contact with the reactive coating of a microarray or biochip. This contact specifically immobilizes them in the respective spot for further steps of an analysis procedure. For example, in a subsequent step, biomolecules corresponding to the capture probes can be immobilized in the respective spot through their interaction with the respective capture probes. There, they can be detected qualitatively but also quantitatively using excitable dyes that, upon excitation, emit detectable radiation (so-called fluorophores).In the context of the present disclosure, the term “microarray” expressly includes (a) solid substrates with a coating that allows the immobilization of biomolecules, including the application / spotting of primary capture probes, but does not yet have immobilized biomolecules, as well as (b) solid substrates with a coating on which biomolecules, such as primary capture probes, are already applied.

[0004] DNA microarrays (or DNA chips), for example, usually consist of solid substrates, usually glass plates, comprising a layer of chemically fixed, different DNA molecules as capture probes, each in discrete spots. Through nucleic acid hybridization, the capture probes in a spot interact only with complementary DNA molecules from a complex sample to be analyzed, so that the complementary DNA molecules are specifically immobilized in the respective spot. The binding of two complementary DNA molecules can then be optically detected and quantified in the subsequent analysis process, e.g., using fluorescent markers that are coupled to the sample DNA during analysis. Microarrays are used, for example, to investigate the expression of a very large number of genes simultaneously or to diagnose specific gene mutations.

[0005] In addition, various protein microarrays (or protein chips) are known, which can be used in various fields of biological research and diagnostics to investigate complex protein interactions and functions. For example, some antibody microarrays use antibodies immobilized in spots to detect the presence and concentration of certain proteins specifically recognized by the antibodies in a sample. Peptide microarrays comprise peptides immobilized in spots, usually on a solid substrate, which can be used to study the interactions between proteins and specific peptide sequences. Similarly, in protein chip microarrays, purified proteins are applied directly to the chip to analyze their interactions with other proteins or biomolecules. Such protein chip microarrays are particularly used in functional genome analysis and pharmacology, for example.Another type of protein microarray is reverse-phase protein arrays (RPPAs), which enable the quantitative analysis of protein expression in cell lysates. Protein function microarrays, in turn, can be used to test protein functions on a large scale. For example, they can be used to identify enzyme activities or evaluate protein-ligand interactions.

[0006] To ensure the immobilization of biomolecules, especially nucleic acids and / or proteins contained in a sample, on a solid substrate suitable for the array, the substrate surface must exhibit suitable binding properties for the respective biomolecules to be bound. Various reactive surfaces and coatings are known in this regard. For example, protein and nucleic acid molecules can react the amino groups of an aminosilanized surface with the carboxyl groups of proteins or nucleic acids, thus immobilizing them on the surface. Chemical activation or functionalization of glass surfaces can be used to provide reactive amino groups and / or epoxy groups for binding nucleic acids and / or proteins to the surface. Polystyrene and polypropylene surfaces can also be chemically activated or functionalized for the binding of biomolecules.

[0007] Furthermore, microarrays with a nitrocellulose coating for the immobilization of biomolecules are known. The binding of biomolecules to nitrocellulose occurs through a combination of weak intermolecular forces, likely dominated by hydrophobic interactions and van der Waals forces. This property of nitrocellulose is used in the form of stand-alone nitrocellulose membranes in a wide range of biomedical applications. Further functionalizations can also give nitrocellulose coatings a high density of carbonyl groups, which form bonds by reacting with amino groups in nucleic acids and proteins. These properties of nitrocellulose have been utilized in biotechnology for many years, for example, in the production of Western blots (protein), Southern blots (DNA), or Northern blots (RNA).

[0008] While nitrocellulose membranes have already proven themselves in many diagnostic and analytical applications and procedures due to their robustness and practicality, nitrocellulose coatings for microarrays are not without their problems. One disadvantage of known nitrocellulose coatings, for example, is an unfavorable "signal-to-noise ratio," i.e., the ratio of the background signal strength to the signal strength of an immobilized biomolecule to be detected. The background signal strength (also called "background noise") of a ready-to-use microarray or biochip consists of the intrinsic fluorescence signal of the coated substrate and the background signal of the coating generated by nonspecific binding or immobilization of dye. This can thus overwhelm the specific detection signal of the immobilized biomolecule to be detected and impair the sensitivity of the diagnosis.This disadvantage can be further compounded by the fact that known microarray nitrocellulose coatings often require relatively large spot diameters to immobilize a certain amount of sample material. Furthermore, known nitrocellulose coatings are often opaque and thus – depending on the layer thickness – only partially transmit light, which further limits their use in optical analysis methods.

[0009] Reactive solid-state substrates used as microarrays are typically equipped with functional aldehyde, carboxyl, epoxy, or amino groups that can form a covalent bond with the biomolecules to be analyzed, permanently attaching them to the substrate surface. Although these methods work quite well for small biomolecules, they tend to be less effective for larger probe molecules such as antibodies, as their detection can often depend on the orientation and flexibility of the biomolecule to be analyzed. Such conventional covalent attachment methods typically bind only a few biomolecules as capture probes in a 2-dimensional layer (2D layer; "monolayer") to the surface of the solid-state substrate, resulting in low binding capacity and difficult signal detection.Since there is relatively little capture probe present on the surface of such a 2D array, such systems also exhibit a low signal-to-noise ratio for a positive binding reaction between the biomolecule and the staining agent. Furthermore, specific antibody binding often cannot be maintained without sufficient hydration and support in or on the coated or functionalized surface of the solid substrate. In 2D microarray formats, in which antibodies serve as "capture probes" for the biomolecules to be analyzed, the lifetime is therefore limited, and diagnostic or analytical results may fluctuate depending on the age of the array.

[0010] Conventional methods for coating solid-state substrates for the production of microarrays and biochips for the analysis of biological samples include all known methods for applying functional layers using dip, spray, and / or spin coating technology. Various coating methods are also known for coating solid-state substrates with a nitrocellulose coating for use as microarrays. However, the reliable application of consistently reproducible layers regularly requires the use of various solvents and detergents, as well as a sequence of complex process steps.

[0011] For example, DE 602 18 695 T2 describes a biosensor and corresponding measurement method. Furthermore, JP 000003788519 B2 describes a device for determining the efficacy of a test compound against a biochemical system. JP 002004004071 A describes multilayer reagent test samples and a method for quantifying glycated protein in physiological samples using the same. Furthermore, JP 000003863559 B2 describes recombinant expression vectors encoding papillomavirus L1 and L2 proteins, methods for producing such recombinant proteins, and methods for using such recombinant proteins.

[0012] An object of the present invention is therefore to design and further develop a microarray for immobilizing biomolecules of the type mentioned above in such a way that the microarray has a high specific binding capacity with a small spot diameter, a favorable signal-to-noise ratio, and high sensitivity at low concentrations of the biomolecules to be immobilized in a sample to be analyzed. In this regard, it should be noted that the term "sample" in the context of this disclosure is to be understood in the broadest sense and thus includes, among other things, both body fluids to be analyzed, such as blood, blood plasma, urine, saliva, etc., as well as tissue, tissue sections, or whole cells isolated therefrom, but also laboratory-produced samples, such as cell layers, cell extracts, cell culture media and supernatants, nucleic acid or protein solutions, etc.wherein biomolecules contained in such a sample can be immobilized and ultimately analyzed by direct or indirect attachment to the immobilization layer of the microarray.

[0013] In one embodiment, the present invention achieves the above-mentioned object with a microarray for immobilizing biomolecules, comprising (a) a glass or glass-ceramic substrate, and (b) a nitrocellulose coating, wherein the nitrocellulose coating is arranged at least in regions on a first planar surface of the substrate, and wherein the nitrocellulose coating serves as an immobilization region for biomolecules;characterized in that the layer thickness of the nitrocellulose coating is between 30 and 150 nm, and the nitrocellulose coating is optically clear and has a root mean square (RMS) roughness of at least 0.5 nm.

[0014] A particularly advantageous property of the nitrocellulose coating of the aforementioned microarray is its persistently rough surface texture despite its thin layer thickness of between 10 and 150 nm. As mentioned above, nitrocellulose coatings are well known. Nitrocellulose coatings and membranes with layer thicknesses of several micrometers (µm) are often three-dimensional and porous, and thus exhibit a high degree of roughness (see Sauer U. Analytical Protein Microarrays: Advancements Towards Clinical Applications. Sensors (Basel). 2017 Jan 29;17(2):256. doi: 10.3390 / s17020256. PMID: 28146048; PMCID: PMC5335935.).While the three-dimensional and porous design of such known nitrocellulose coatings increases the binding capacity, it also regularly leads to disadvantageously high background fluorescence on such coated glass or glass-ceramic substrates, resulting in only a low signal-to-noise ratio. Among other things, the dynamic range of a microarray is limited by a low signal-to-noise ratio. However, a wide dynamic range is important to ensure accurate and comprehensive detection and analysis of biomolecules present in a sample at different concentrations or expression levels. If the dynamic range is too narrow, important information can be lost, especially when analyzing biomolecules with large differences in concentration or expression levels.

[0015] In addition, the surface properties of a nitrocellulose coating influence the morphology of the samples applied in individual spots on a microarray. For known nitrocellulose coatings and membranes with layer thicknesses of several micrometers (µm), spot diameters of more than 200 µm, often up to 400 µm, are regularly required to apply a sufficient sample volume per spot.

[0016] In accordance with the invention, it was first recognized that the signal-to-noise ratio of a nitrocellulose coating can be increased in an astonishingly simple manner by reducing the layer thickness while simultaneously forming a microstructure on the surface of the nitrocellulose coating. Since the smaller layer thickness reduces the nonspecific binding capacity compared to a thicker 3D nitrocellulose layer, the aforementioned surface microstructure of the thin nitrocellulose coating can provide a larger number of reactive groups per unit area compared to a monolayer of reactive groups, thus achieving an advantageous specific binding capacity. Furthermore, such a configuration expands the dynamic range of a microarray according to the invention compared to a microarray with a 2D monolayer of reactive epoxy groups.

[0017] Preferably, the layer thickness of the nitrocellulose coating of the microarray according to the invention is between 10 and 100 nm, in particular between 20 and 100 nm, in particular between 30 and 100 nm, in particular between 30 and 90 nm, in particular between 35 and 85 nm, in particular between 40 and 80 nm, in particular between 45 and 75 nm, or is approximately 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm or 85 nm.

[0018] Microarrays with nitrocellulose coatings with a layer thickness of less than 200 nm are also known. However, the thin nitrocellulose coatings of such known microarrays are essentially smooth.

[0019] Despite the small layer thickness, the nitrocellulose coating of the microarray according to the invention has, as already mentioned, a root mean square (RMS) roughness of at least 0.5 nm, preferably between 0.5 and 2 nm, in particular between 0.75 and 2 nm, in particular between 1 and 2 nm, in particular between 1.25 and 2 nm, in particular between 1.25 and 1.75 nm, in particular between 1.45 and 1.65 nm or of approximately 1.3 nm, 1.4 nm, 1.5 nm, 1.6 nm or 1.7 nm.

[0020] The root mean square value (RMS or Sq) is often used in relation to surface properties and in metrology to describe the roughness of a surface. The RMS value indicates how much a surface deviates from an ideally smooth surface.

[0021] The formula for calculating the RMS value for the roughness of a surface is: Sq=1a∫∫a(Z(x,y))2dxdy

[0022] The value Z(x,y) denotes the height Z of the surface above a reference surface at the coordinates (x,y). A low RMS value indicates that the surface is relatively smooth and homogeneous, while a higher RMS value indicates a textured or rougher surface with larger deviations.

[0023] The average roughness of a surface over its entire three-dimensional structure can be determined by the mean of the absolute deviations (S a ) must be specified. Sa=1a∫∫a|Z(x,y)|(dx)dy

[0024] The mean value of the absolute deviations of the roughness (S a) of the rough nitrocellulose coating of the microarray according to the invention is preferably between 0.3 and 1 nm, in particular between 0.3 and 0.9 nm, in particular between 0.3 and 0.7 nm, in particular between 0.4 and 0.7 nm, in particular between 0.5 and 0.7 nm, in particular between 0.5 and 0.6 nm, or is approximately 0.4 nm, 0.45 nm, 0.5 nm, 0.55 nm, 0.6 nm, 0.65 nm or 0.7 nm.

[0025] Another parameter for characterizing the roughness of a surface is the measurable peak-to-valley height (S z ), i.e. the maximum difference in altitude between the highest summit height (S p ) and the greatest valley depth (S v ) a rough surface. Sz=Sp−Sv

[0026] The peak-to-valley height of the roughness (S z) of the rough nitrocellulose coating of the microarray according to the invention is preferably between 150 and 300 nm, in particular between 150 and 275 nm, in particular between 150 and 250 nm, in particular between 175 and 250 nm, in particular between 190 and 230 nm, in particular between 210 and 230 nm or is approximately 190 nm, 200 nm, 215 nm, 220 nm, 225 nm, 230 nm or 240 nm.

[0027] As described in Example 1, the surface quality of the nitrocellulose coating of the microarray according to the invention differs significantly in terms of its microstructure from the surface quality of known microarrays with nitrocellulose coatings of comparably thin layer thicknesses. The aforementioned roughness of the surface of the nitrocellulose coating of a microarray according to the invention is the objectively measurable expression of this advantageous microstructure.

[0028] The nitrocellulose coating of the microarray according to the invention is optically clear. "Optically clear" with respect to the nitrocellulose coating here means that the transmittance of the microarray essentially corresponds to that of the glass or glass-ceramic substrate without the nitrocellulose coating, and the coating itself has a transmittance of more than 90% in the wavelength range between 400 and 700 nm. As described in Example 2, the microarray according to the invention has a transmittance in the wavelength range between 300 and 800 nm of at least 85%, preferably between 85 and 99%, in particular between 90 and 99%, in particular between 90 and 95%, or the transmittance is approximately 85%, 87.5%, 90%, 92.5%, 95%, or 97.5%.

[0029] A glass or glass-ceramic substrate of the microarray according to the invention itself (i.e. without the nitrocellulose coating) also has a transmittance of at least 85% in the wavelength range between 300 and 800 nm, preferably between 85 and 99%, in particular between 90 and 99%, in particular between 90 and 95%, or the transmittance is approximately 85%, 87.5%, 90%, 92.5%, 95% or 97.5%.

[0030] In one embodiment, the microarray according to the invention is preferably designed such that - the layer thickness of the rough nitrocellulose coating is between 45 and 75 nm, - the nitrocellulose coating has a Root Mean Square (RMS) roughness of between 1.45 and 1.65 nm, - the mean value of the absolute deviations of the roughness (Sa) of the rough nitrocellulose coating is between 0.5 and 0.6 nm, - the peak-to-valley height of the roughness (Sz) of the rough nitrocellulose coating is between 210 and 230 nm, and - the microarray has a transmittance in the wavelength range between 300 and 800 nm of between 90 and 95%.

[0031] With regard to a glass substrate to be used in the aforementioned microarrays, it is conceivable that this is selected from: soda-lime glasses, borosilicate glasses, quartz glasses and / or alkali-free alumino-borosilicate glasses.

[0032] Preferably, the glass substrate to be used in the microarray according to the invention can have the following composition / components corresponding to a lithium aluminum silicate glass (in weight %): SiO2 55-69 Al2O3 18-25 Li2O 3-5 Na2O+K2O 0-30 MgO+CaO+SrO+BaO 0-5 ZnO 0-4 TiO2 0-5 ZrO2 0-5 TiO2+ZrO2+SnO2 2-6 P2O5 0-8 F 0-1 B2O3 0-2

[0033] Again, the glass substrate to be used in the microarray according to the invention may preferably comprise the following components (in weight %): SiO2 57-66 Al2O3 18-23 Li2O 3-5 Na2O+K2O 3-25 MgO+CaO+SrO+BaO 1-4 ZnO 0-4 TiO2 0-4 ZrO2 0-5 TiO2+ZrO2+SnO2 2-6 P2O5 0-7 F 0-1 B2O3 0-2

[0034] Again, the glass substrate to be used in the microarray according to the invention may preferably comprise the following components (in weight %): SiO2 57-63 Al2O3 18-22 Li2O 3.5-5 Na2O+K2O 5-20 MgO+CaO+SrO+BaO 0-5 ZnO 0-3 TiO2 0-3 ZrO2 0-5 TiO2+ZrO2+SnO2 2-5 P2O5 0-5 F 0-1 B2O3 0-2

[0035] Again, the glass substrate to be used in the microarray according to the invention may preferably comprise the following components corresponding to a soda-lime silicate glass (in weight %): SiO2 40-81 Al2O3 0-6 B2O3 0-5 Li2O+Na2O+K2O 5-30 MgO+CaO+SrO+BaO+ZnO 5-30 TiO2+ZrO2 0-7 P2O5 0-2

[0036] Again, the glass substrate to be used in the microarray according to the invention may preferably comprise the following components (in weight %): SiO2 50-81 Al2O3 0-5 B2O3 0-5 Li2O+Na2O+K2O 5-28 MgO+CaO+SrO+BaO+ZnO 5-25 TiO2+ZrO2 0-6 P2O5 0-2

[0037] Again, the glass substrate to be used in the microarray according to the invention may preferably comprise the following components (in weight %): SiO2 50-76 Al2O3 0-5 B2O3 0-5 Li2O+Na2O+K2O 5-25 MgO+CaO+SrO+BaO+ZnO 5-20 TiO2+ZrO2 0-5 P2O5 0-2

[0038] Again, the glass substrate to be used in the microarray according to the invention can preferably comprise the following components corresponding to a borosilicate glass (in % by weight): SiO2 60-85 Al2O3 0-10 B2O3 5-20 Li2O+Na2O+K2O 2-16 MgO+CaO+SrO+BaO+ZnO 0-15 TiO2+ZrO2 0-5 P2O5 0-2

[0039] Again, the glass substrate to be used in the microarray according to the invention may preferably comprise the following components (in weight %): SiO2 63-84 Al2O3 0-8 B2O3 5-18 Li2O+Na2O+K2O 3-14 MgO+CaO+SrO+BaO+ZnO 0-12 TiO2+ZrO2 0-4 P2O5 0-2

[0040] Again, the glass substrate to be used in the microarray according to the invention may preferably comprise the following components (in weight %): SiO2 63-83 Al2O3 0-7 B2O3 5-18 Li2O+Na2O+K2O 4-14 MgO+CaO+SrO+BaO+ZnO 0-10 TiO2+ZrO2 0-3 P2O5 0-2

[0041] Again, the glass substrate to be used in the microarray according to the invention may preferably comprise the following components (in weight %): SiO2 60-70 Al2O3 1-10 B2O3 1-10 K2O 1-10 Na2O 1-10 ZnO 1-10 TiO2 1-10

[0042] Again, the glass substrate to be used in the microarray according to the invention may preferably comprise the following components corresponding to an alkali aluminum silicate glass (in weight %): SiO2 40-75 Al2O3 10-30 B2O3 0-20 Li2O+Na2O+K2O 4-30 MgO+CaO+SrO+BaO+ZnO 0-15 TiO2+ZrO2 0-15 P2O5 0-10

[0043] Again, the glass substrate to be used in the microarray according to the invention may preferably comprise the following components corresponding to a low-alkali aluminum silicate glass (in weight %): SiO2 50-70 Al2O3 10-27 B2O3 0-18 Li2O+Na2O+K2O 5-28 MgO+CaO+SrO+BaO+ZnO 0-13 TiO2+ZrO2 0-13 P2O5 0-9

[0044] Again, the glass substrate to be used in the microarray according to the invention may preferably comprise the following components (in weight %): SiO2 55-68 Al2O3 10-27 B2O3 0-15 Li2O+Na2O+K2O 4-27 MgO+CaO+SrO+BaO+ZnO 0-12 TiO2+ZrO2 0-10 P2O5 0-8

[0045] Again, the glass substrate to be used in the microarray according to the invention may preferably comprise the following components (in weight %): SiO2 50-75 Al2O3 7-25 B2O3 0-20 Li2O+Na2O+K2O 0-4 MgO+CaO+SrO+BaO+ZnO 5-25 TiO2+ZrO2 0-10 P2O5 0-5

[0046] Again, the glass substrate to be used in the microarray according to the invention may preferably comprise the following components (in weight %): SiO2 52-73 Al2O3 7-23 B2O3 0-18 Li2O+Na2O+K2O 0-4 MgO+CaO+SrO+BaO+ZnO 5-23 TiO2+ZrO2 0-10 P2O5 0-5

[0047] Again, the glass substrate to be used in the microarray according to the invention may preferably comprise the following components (in weight %): SiO2 53-71 Al2O3 7-22 B2O3 0-18 Li2O+Na2O+K2O 0-4 MgO+CaO+SrO+BaO+ZnO 5-22 TiO2+ZrO2 0-8 P2O5 0-5

[0048] It goes without saying that the respective glass components of the listed glass compositions must total 100% by weight. Nevertheless, the glasses used in the invention, in particular the glasses described above, can be modified. For example, the color of the respective glass can be altered by adding color oxides.

[0049] In advantageous embodiments, the glass substrates according to the invention are produced using particularly pure raw materials in order to minimize fluorescence under UV and / or visible light illumination. In particular, the use of raw materials with a very low iron content has proven advantageous for this purpose. The glasses produced in this way therefore advantageously contain particularly few impurities, in particular very little iron.

[0050] Those skilled in the art know how to determine the background intensity relative to the signal intensity to be determined in a fluorescence-based measurement system. 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 the microarray according to the invention are determined via an optical system. In this system, the immobilization area is first irradiated by a light source with a wavelength of 532 nm in excitation mode. This results in fluorescence emitted from the surface. The relevant emission wavelength is selected using an optical filter, for example, with a wavelength of 575 nm. The intensity of the emitted light can then be determined using a detector.In such fluorescence measurements, a signal amplification (gain) can be adjusted as required, which can, of course, significantly influence the fluorescence measurements. Therefore, experts know that setting this parameter is particularly important in any measurement system in order to be able to determine a low background intensity compared to the signal intensity to be determined.

[0051] Preferably, the glass or glass-ceramic substrate of the microarray itself emits only a very low fluorescence signal after excitation with a wavelength of 532 nm, so that the background signal strength of the microarray, which results from the intrinsic fluorescence signal of the coated substrate and the background signal generated by non-specific binding or immobilization of dye on the nitrocellulose coating, after excitation with a wavelength of 532 nm and a gain of 140 usually remains at significantly less than 100 relative fluorescence units (rfu), in particular between 10 and 50 rfu.

[0052] In particular, the intrinsic fluorescence of the microarray is less than 50, preferably between 15 and 40, relative fluorescence units at an excitation wavelength of 532 nm and a gain of 140.

[0053] Example 3 shows that the intrinsic fluorescence of a microarray increases with increasing nitrocellulose layer thickness, that the intrinsic fluorescence of a microarray according to the invention with a nitrocellulose coating having a layer thickness of less than 150 nm is advantageously low and, in particular, differs advantageously from the intrinsic fluorescence of a microarray with a nitrocellulose coating having a layer thickness of several micrometers.

[0054] Furthermore, the nitrocellulose coating of the microarray according to the invention is characterized by its stable bond to the glass or glass-ceramic substrate, so that the microarray is stable when stored between 4 and 40°C, in particular between 10 and 30°C, in particular between 18 and 25°C, and long storage stability of the microarray can be ensured even at room temperature. Preferably, the microarray remains stable for more than 2 months, in particular for more than 4 months, more than 6 months, more than 12 months, more than 18 months, or more than 24 months when stored between 4 and 40°C, in particular between 10 and 30°C, in particular between 18 and 25°C.

[0055] In certain embodiments of the microarray according to the invention, the first planar surface of the substrate, on which the nitrocellulose coating is arranged at least in part, is not functionalized. In the context of this application, this means that the surface has not been chemically modified to specifically improve the binding properties compared to the nitrocellulose coating. Nevertheless, the microarrays exhibit the characteristic stability.

[0056] In alternative embodiments of the microarray according to the invention, the first planar surface of the substrate, on which the nitrocellulose coating is arranged at least in regions, is functionalized. Optionally, the functionalized first planar surface of the substrate comprises: - functional ester groups, preferably N-hydroxysuccinimide ester groups, glycidyl ester groups or isocyanate ester groups; - functional ether groups, preferably glycidyl ether groups; - functional epoxy groups; - aldehyde functional groups; - functional free carboxyl groups; and / or - functional free amino groups; and combinations thereof.

[0057] Preferably, the first planar surface of the substrate is functionalized by means of an adhesion promoter layer, which - functional ester groups, preferably N-hydroxysuccinimide ester groups, glycidyl ester groups or isocyanate ester groups; - functional ether groups, preferably glycidyl ether groups; - functional epoxy groups; - functional aldehyde groups; - functional free carboxyl groups; and / or - functional free amino groups; and combinations thereof.

[0058] The term "biomolecule" in the context of this disclosure encompasses both high-molecular-weight macromolecules (such as proteins, nucleic acids, polysaccharides, or lipids) and low-molecular-weight compounds that are, for example, the building blocks of high-molecular-weight macromolecules (such as amino acids, nucleotides, sugars, or fatty acids). Typically, the biomolecules to be immobilized are: 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, in particular, be DNA or RNA oligonucleotides.

[0059] In the context of this disclosure, biomolecules to be immobilized are typically contained in an aqueous sample.

[0060] As already described, the microstructure caused by the roughness of the surface of the nitrocellulose coating surprisingly leads to an advantageously increased binding capacity of the membrane for biomolecules to be immobilized and improved spot morphology compared to other nitrocellulose coatings with a comparably low layer thickness.

[0061] In one embodiment, the immobilization region defined by the nitrocellulose coating can encompass the entire planar surface of the substrate. In such embodiments, the entire surface is thus capable of immobilizing biomolecules. However, as is known, one advantage of microarrays is the ability to apply a large number of different samples in defined and distinct areas ("spots") in a very small space (usually with the aid of robots). In certain embodiments, a large number of individual, distinct spots can be arranged in the immobilization region. In this respect, the surface properties of the nitrocellulose coating of the microarray according to the invention and the resulting spot morphology are also advantageous.

[0062] Due to the advantageous surface properties of the nitrocellulose coating, relatively large volumes of aqueous sample solutions can be applied in spots with a small diameter, for example a diameter of between 130 and 170 µm, to the surface provided with the nitrocellulose coating, which in turn ensures an advantageously high concentration of biomolecules per spot.

[0063] In particular, a spotting volume of 500 picoliters (pl) applied in the immobilization area has a spot diameter of between 130 and 170 µm.

[0064] The combination of such a large number of biomolecules per spot and the advantageously high binding capacity of the nitrocellulose coating leads to a high signal intensity of biomolecules to be analyzed in the subsequent analysis procedures, which significantly facilitates their evaluation.

[0065] Excitable dyes, called fluorophores, are commonly used to label and detect immobilized biomolecules. These dyes emit detectable radiation upon excitation. Suitable fluorophores include, for example: Name AbsorbierendeWellenlänge Emittierende Wellenlänge Sichtbare Farbe Hydroxycoumarin 325 386 Blue methoxycoumarin 360 410 Blue Alexa fluor 345 442 Blue aminocoumarin 350 445 Blue Cy2 490 510 dark green FAM 495 516 dark green Alexa fluor 488 494 517 Light green Fluorescein FITC 495 518 Light green Alexa fluor 430 430 545 Light green Alexa fluor 532 530 555 Light green HEX 535 556 Light green Cy3 550 575 Yellow TRITC 547 572 Yellow Alexa fluor 546 556 573 Yellow Alexa fluor 555 556 573 Yellow R-phycoerythrin (PE) 480;565 578 Yellow Rhodamine Red-X 560 580 Orange Tamara 565 580 Red Cy3.5 581 581 596 Red Rox 575 602 Red Alexa fluor 568 578 603 Red Red 613 480;565 613 Red Texas Red 615 615 Red Alexa fluor 594 590 617 Red Alexa fluor 633 621 639 Red Allophycocyanin 650 660 Red Alexa fluor 633 650 668 Red Cy5 650 670 Red Alexa fluor 660 663 690 Red Cy5.5 675 694 Red TruRed 490;675 695 Red Alexa fluor 680 679 702 Red Cy7 743 770 Red

[0066] Cyanine fluorophores, including the well-known fluorophores Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, and Cy7, are particularly suitable for labeling DNA or RNA molecules. A microarray according to the invention preferably 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 essentially without interfering background signals from the substrate itself. Advantageously, the sensitivity of a microarray or biochip produced based on the solid-state substrate according to the invention is thus high.

[0067] Due to the low intrinsic fluorescence of the microarray according to the invention, the advantageous binding capacity with low non-specific binding and simultaneously improved spot morphology on the nitrocellulose coating in the immobilization region of the microarray according to the invention, a particularly advantageous high signal-to-noise ratio is achieved for samples with a low concentration of biomolecules to be immobilized.

[0068] For example, the microarray according to the invention allows the reliable detection and analysis of biomolecules even at antibody concentrations of 0.01 to 0.1 mg / ml.

[0069] A further object of the present invention is to provide a method for producing the aforementioned microarray and to provide a microarray produced in this way.

[0070] In various embodiments of the invention, the microarrays already described, which are provided with a nitrocellulose coating, can be produced in an astonishingly simple and cost-effective manner by a method according to the invention.

[0071] In one embodiment, the present invention achieves the above-mentioned object with a method for producing a microarray according to the invention, the method comprising at least: (a) providing a coating solution; (b) applying the coating solution to a glass or glass-ceramic substrate by immersing the substrate in the coating solution and then withdrawing it from the coating solution; (c) depositing a nitrocellulose coating from the coating solution onto a first planar surface of the substrate by drying, wherein the drying advantageously comprises evaporating an organic solvent of the coating solution and the nitrocellulose coating has a layer thickness of between 10 and 150 nm, characterized in that the coating solution: ▪ between 0.2 and 5.0 wt.% of high-purity nitrocellulose in an organic solvent; ▪ between 0.5 and 5.0 wt.% (3-glycidyloxypropyl)trimethoxysilane (GPTS); and ▪ between 0.3 and 20.0 wt.% water, and the deposited nitrocellulose coating is optically clear and has a root mean square (RMS) roughness of at least 0.5 nm.

[0072] The coating solution preferably comprises: ▪ between 0.5 and 1.5 wt%, preferably between 0.6 and 1.4 wt% or between 0.7 and 1.3 wt% or between 0.8 and 1.2 wt% or between 0.9 and 1.1 wt% or 1 wt% of high purity nitrocellulose in an organic solvent; ▪ between 2.0 and 4.0 wt%, preferably between 2.1 and 3.9 wt% or between 2.2 and 3.8 wt% or between 2.3 and 3.7 wt% or between 2.4 and 3.6 wt% or between 2.5 and 3.5 wt% or between 2.6 and 3.4 wt% or between 2.7 and 3.3 wt% or between 2.8 and 3.2 wt% or between 2.9 and 3.1 wt% or 3 wt% GPTS; and ▪ between 0.3 and 15.0 wt.%, preferably between 0.5 and 10.0 wt.% or between 1.0 and 9.0 wt.% or between 2.0 and 8.0 wt.% or between 3.0 and 7.0 wt.% or between 4.0 and 6.0 wt.% or between 4.1 and 5.9 wt.% or between 4.2 and 5.8 wt.% or between 4.3 and 5.7 wt.% or between 4.4 and 5.6 wt.% or between 4.5 and 5.5 wt.% or between 4.6 and 5.4 wt.% or between 4.7 and 5.3 wt.% or between 4.8 and 5.2 wt.% or between 4.9 and 5.1 wt.% or 5 wt.% water.

[0073] In further embodiments of the process according to the invention, the coating solution comprises in particular: ▪ between 0.8 and 1.2 wt.% of high-purity nitrocellulose in an organic solvent; ▪ between 2.8 and 3.2 wt% GPTS; and ▪ between 4.8 and 5.2 wt% water.

[0074] In yet further embodiments of the process according to the invention, the coating solution comprises in particular: ▪ 1 wt.% high-purity nitrocellulose in an organic solvent; ▪ 3 wt% GPTS; and ▪ 5 wt% water.

[0075] In addition, the pulling speed during immersion is between 5 and 15 cm per minute, in particular between 8 and 12 cm per minute, in particular 10 cm per minute, at a coating solution temperature of between 18 and 25°C. Surprisingly, the above-mentioned relative proportions are in the ranges: ▪ of between 0.2 and 3.0 wt.% of high-purity nitrocellulose in an organic solvent; ▪ of between 0.5 and 5.0 wt.% (3-glycidyloxypropyl)trimethoxysilane (GPTS); and ▪ of between 0.3 and 20.0 wt.% water, necessary to apply the nitrocellulose coating with the properties described for the microarray according to the invention to at least a first planar surface of the glass or glass-ceramic substrate.

[0076] Preferably, drying to deposit the nitrocellulose from the coating solution onto the surface is carried out over a period of at least 5 minutes at a temperature between 18 and 70°C, preferably over a period of 20 minutes at a temperature of 60°C.

[0077] In various embodiments, the organic solvent can be amyl acetate, isopropanol, 1-propanol, tetrahydrofuran, or dimethyl sulfoxide. The aprotic non-polar organic solvent amyl acetate has proven particularly suitable for dissolving high-purity nitrocellulose.

[0078] The method according to the invention enables the production of large quantities of the microarrays according to the invention with particularly high stability in an astonishingly simple manner.

[0079] Furthermore, the present invention solves the above-mentioned problem with a microarray for the immobilization of biomolecules produced by the above-mentioned inventive method.

[0080] A further object of the present invention is to provide a use of the microarray both for the immobilization of biomolecules and for the analysis of biomolecules contained in a sample.

[0081] In one embodiment, the present invention achieves the above-mentioned objects by using a microarray according to the invention for immobilizing biomolecules in an immobilization region, wherein the biomolecules are preferably: nucleic acids, in particular deoxyribonucleic acids or ribonucleic acids; peptides; or proteins, in particular enzymes or antibodies.

[0082] In a further embodiment, the present invention achieves the above-mentioned objects by using a microarray according to the invention for analyzing biomolecules contained in a sample, wherein the biomolecules are preferably: nucleic acids, in particular deoxyribonucleic acids or ribonucleic acids; peptides; or proteins, in particular enzymes or antibodies.

[0083] 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 embodiments of the invention with reference to the drawings. In conjunction with the explanation of the preferred embodiments of the invention with reference to the drawings, generally preferred embodiments and developments of the teaching are also explained. The drawings show: Fig.Figure 1 shows, according to Example 1, atomic force microscopy images of the surface properties of different nitrocellulose coatings, which define the immobilization area of ​​various microarrays. (a) Surface properties of the nitrocellulose coating of the microarray according to the invention (SCHOTT NC-Array); (b) Surface properties of a comparably thin nitrocellulose coating of a known microarray (PATH® Protein Microarray Slide); (c) Surface properties of a nitrocellulose coating with a layer thickness in the micrometer range of a known microarray (ONCYTE® SuperNOVA). Fig. Figure 2 shows - according to Example 1 - the relevant roughness parameters of the nitrocellulose coating of the microarray according to the invention of the Fig. 1(a) with those of the nitrocellulose coating of the known microarray of Fig.1(b) in comparison. (a) Root Mean Square (RMS) roughness; (b) mean of the absolute deviations of the roughness (S a ); (c) peak-to-valley height of roughness (S z ). Fig. Figure 3 shows - according to Example 2 - the transmission spectrum of a cleaned glass substrate (reference); the corresponding glass substrate provided with the nitrocellulose coating of the microarray according to the invention (SCHOTT NC-Array), a known microarray with a comparably thin nitrocellulose coating (PATH® Protein Microarray Slide), and a known microarray with a nitrocellulose coating with a layer thickness in the micrometer range (ONCYTE® SuperNOVA). Fig. Figure 4 shows - according to Example 3 - the results of the background intensity measurements of microarrays coated with nitrocellulose as a function of the nitrocellulose layer thickness. Fig.Figure 5 shows - according to Example 4 - the results of background intensity measurements of the microarray according to the invention (SCHOTT NC-Array) in comparison to three known microarray formats (SCHOTT Slide E and ONCYTE® and PATH® from Grace Biolabs). Fig. Figure 6 shows - according to Example 5 - the spot morphology of a sample applied to the nitrocellulose coating of the microarray according to the invention compared to the spot morphology of a corresponding sample on a thick nitrocellulose coating. Fig. Figure 7 shows - according to Example 6 - the extension of the dynamic range of the microarray according to the invention (SCHOTT NC array) compared to a 2D epoxy microarray (SCHOTT Slide E).

[0084] With regard to further advantageous embodiments of the microarray according to the invention for immobilizing biomolecules, the method according to the invention for producing a microarray for immobilizing biomolecules, the microarray produced by means of the method for immobilizing biomolecules and the uses of the microarrays according to the invention, reference is made to the general part of the description and to the appended examples and claims in order to avoid repetition. Example 1

[0085] Comparative analysis of the nitrocellulose coating. Atomic force microscopy images were acquired in intermittent mode in air at room temperature at a speed of 1 Hz. Roughness was determined for the entire image at a resolution of 256x256 pixels.

[0086] Fig.Figure 1(a) illustrates the advantageous structure of the surface of the coating of a microarray according to the invention at a layer thickness of 72 nm compared to that in Fig. 1(b) shows the smooth surface of a comparably thin nitrocellulose coating of the known microarray “PATH® Protein Microarray Slide” with a layer thickness of 30 nm and the Fig. 1(c) shows the surface texture of a 3-dimensionally structured, porous nitrocellulose coating in the micrometer range (layer thickness 12.5µm) of the known microarray “ONCYTE® SuperNOVA”.

[0087] Fig. 2 shows the advantageously adjusted roughness parameters RMS, S a and S zof a microarray according to the invention in comparison to the known "PATH® Protein Microarray Slide". The roughness values ​​were determined from atomic force microscopy images with an edge length of 30 µm x 30 µm. The roughness of the surface of the microarray "ONCYTE® SuperNOVA" is not shown. However, as already shown when considering Fig. As can be seen in Figure 1(c), the surface roughness of the "ONCYTE® SuperNOVA" microarray is very high. In fact, the measurable RMS roughness value is over 80 nm. Example 2

[0088] Comparative transmission properties. The transmission was measured using spectrophotometry. The sample was placed perpendicular to the optical axis in the beam path. Since the sample is coated on both sides, the beam passes through both layers. The transmission values ​​therefore apply to the entire sample; for one layer, they are likely to be even higher.

[0089] The transmission profile of the microarray according to the invention is shown in Fig. 3. Advantageously, the transmission of the SCHOTT_NC microarray coated with nitrocellulose according to the invention is almost identical to that of the uncoated reference substrate. PATH® and ONCYTE® exhibit significantly lower transmission. Example 3

[0090] Measurement of background intensity as a function of nitrocellulose layer thickness. To determine the layer thickness, the layer was partially removed mechanically and the layer thickness was measured at three different points using a white light interferometer. The stated value is the mean and standard deviation of the measurement points across at least five samples produced from different batches. The corresponding background intensity was determined according to Example 4.

[0091] The background fluorescence of nitrocellulose-coated microarrays increases - as in Fig.4 – increases with layer thickness. For comparison, the background fluorescence of ONCYTE® is shown, which exhibits significantly higher background fluorescence. Example 4

[0092] Comparative measurement of background intensity. The background intensity was measured at an excitation wavelength of 532 nm and a pixel size of 10 µm using a fluorescence scanner across the entire sample, excluding the 5 mm edge area. The measured intensity can be adjusted by varying the gain and is kept constant for comparison across different samples.

[0093] Fig.Figure 5 shows the background fluorescence of the SCHOTT_NC coating compared to SCHOTT Slide-E and the nitrocellulose-coated ONCYTE® and PATH® microarrays from Grace Biolabs, before and after a hybridization reaction. The microarrays were scanned with a gain of 150 and a wavelength of 532 nm. The background fluorescence scale is in logarithmic units. Example 5

[0094] Comparative spot morphology. To assess the spot morphology, the spatially resolved emission of the surface was plotted in 2D and 3D surface plots. A homogeneous intensity distribution across the entire spot is advantageous here.

[0095] Fig. Figure 6 illustrates the advantageous spot morphology of 500 picoliter spots in an array on the thin nitrocellulose coating of a SCHOTT_NC microarray according to the invention with a spot diameter of approximately 150 µm ( Fig.6 a)), compared to the spot diameter of approximately 200 µm on a 3 µm thick nitrocellulose coating ( Fig. 6 b)). Example 6

[0096] Comparative determination of the dynamic range. Within the dynamic range, the intensities of the spots depend on the protein solution used. Outside the dynamic range, a saturation of the intensity is evident.

[0097] Fig. Figure 7 shows 3D surface plots of the inventive microarray with a thin nitrocellulose coating at a Root Mean Square (RMS) roughness of at least 0.5 nm (SCHOTT_NC, Fig. 7 a)) applied spots as a function of the protein concentration (Rabbit anti-Goat IgG) to determine the dynamic range. As can be seen, the dynamic range of the microarray according to the invention is significantly higher compared to a functionalized 2-dimensional epoxy array ( Fig. 7 b) significantly expanded.

[0098] Finally, it should be expressly pointed out that the above-described embodiments of the microarray according to the invention for immobilizing biomolecules, the method according to the invention for producing a microarray for immobilizing biomolecules, the microarray produced by means of the method for immobilizing biomolecules and the uses of the microarrays according to the invention serve only to discuss the claimed teaching, but do not limit it to the embodiments.

Claims

[1] Microarray for the immobilization of biomolecules, comprising (a) a glass or glass-ceramic substrate, and (b) a nitrocellulose coating, wherein the nitrocellulose coating is arranged at least in regions on a first planar surface of the substrate, and wherein the nitrocellulose coating serves as an immobilization region for biomolecules; characterized by , that the layer thickness of the nitrocellulose coating is between 10 and 150 nm, the nitrocellulose coating is optically clear and the nitrocellulose coating has a root mean square (RMS) roughness of at least 0.5 nm. [2] The microarray according to claim 1, wherein the layer thickness of the nitrocellulose coating is between 10 and 100 nm, in particular between 20 and 100 nm, in particular between 30 and 100 nm, in particular between 30 and 90 nm, in particular between 35 and 85 nm, in particular between 40 and 80 nm, in particular between 45 and 75 nm or is approximately 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm or 85 nm. [3] The microarray according to claim 1 or claim 2, characterized by that the nitrocellulose coating has a Root Mean Square (RMS) roughness of between 0.5 and 2 nm, in particular between 0.75 and 2 nm, in particular between 1 and 2 nm, in particular between 1.25 and 2 nm, in particular between 1.25 and 1.75 nm, in particular between 1.45 and 1.65 nm or of approximately 1.3 nm, 1.4 nm, 1.5 nm, 1.6 nm or 1.7 nm. [4] The microarray according to any one of the preceding claims, characterized bythat the mean value of the absolute deviations of the roughness (S a ) of the nitrocellulose coating is between 0.3 and 1 nm, in particular between 0.3 and 0.9 nm, in particular between 0.3 and 0.7 nm, in particular between 0.4 and 0.7 nm, in particular between 0.5 and 0.7 nm, in particular between 0.5 and 0.6 nm or is approximately 0.4 nm, 0.45 nm, 0.5 nm, 0.55 nm, 0.6 nm, 0.65 nm or 0.7 nm. [5] The microarray according to any one of the preceding claims, characterized by that the peak-to-valley height of the roughness (S z ) of the nitrocellulose coating is between 150 and 300 nm, in particular between 150 and 275 nm, in particular between 150 and 250 nm, in particular between 175 and 250 nm, in particular between 190 and 230 nm, in particular between 210 and 230 nm or is approximately 190 nm, 200 nm, 215 nm, 220 nm, 225 nm, 230 nm or 240 nm. [6] The microarray according to any one of the preceding claims, characterized bythat the microarray has a transmittance in the wavelength range between 300 and 800 nm of at least 85%, preferably between 85 and 99%, in particular between 90 and 99%, in particular between 90 and 95%, or the transmittance is approximately 85%, 87.5%, 90%, 92.5%, 95% or 97.5%. [7] The microarray according to any one of the preceding claims, characterized by , that - the layer thickness of the nitrocellulose coating is between 45 and 75 nm, - the nitrocellulose coating has a Root Mean Square (RMS) roughness of between 1.45 and 1.65 nm, - the mean value of the absolute deviations of the roughness (S a ) of the nitrocellulose coating is between 0.5 and 0.6 nm, - the peak-to-valley height of the roughness (S z ) of the nitrocellulose coating is between 210 and 230 nm, and - the microarray has a transmittance in the wavelength range between 300 and 800 nm of between 90 and 95%. [8] The microarray according to any one of the preceding claims, characterized by that the first planar surface of the substrate is functionalized, and optionally - functional ester groups, preferably N-hydroxysuccinimide ester groups, glycidyl ester groups or isocyanate ester groups; - functional ether groups, preferably glycidyl ether groups; - functional epoxy groups; - aldehyde functional groups; - functional free carboxyl groups; and / or - functional free amino groups; and combinations thereof. [9] The microarray according to claim 8, characterized by that the first planar surface of the substrate is functionalized by means of an adhesion promoter layer, which - functional ester groups, preferably N-hydroxysuccinimide ester groups, glycidyl ester groups or isocyanate ester groups; - functional ether groups, preferably glycidyl ether groups; - functional epoxy groups; - aldehyde functional groups; - functional free carboxyl groups; and / or - functional free amino groups; and combinations thereof. [10] The microarray according to any one of the preceding claims, characterized by that the intrinsic fluorescence of the microarray is less than 100 rfu, preferably between 10 and 50 rfu, relative fluorescence units at an excitation wavelength of 532 nm and a gain of 150. [11] The microarray according to any one of the preceding claims, wherein the biomolecules are: nucleic acids, in particular deoxyribonucleic acids or ribonucleic acids; peptides; or proteins, in particular enzymes or antibodies. [12] The microarray according to any one of the preceding claims, characterized by that a spotting volume of 500 pl applied in the immobilization area has a spot diameter of between 130 and 170 µm. [13] The microarray according to any one of the preceding claims, characterized by that the microarray is stable when stored between 4 and 40°C, in particular between 10 and 30°C, in particular between 18 and 25°C, preferably for more than 2 months, more than 4 months, more than 6 months, more than 12 months, more than 18 months or more than 24 months. [14] A method for producing a microarray according to any one of the preceding claims, comprising at least (a) providing a coating solution; (b) applying the coating solution to a glass or glass-ceramic substrate by immersing the substrate in the coating solution and then withdrawing it from the coating solution; (c) depositing a nitrocellulose coating from the coating solution onto a first planar surface of the substrate by drying, wherein the drying advantageously comprises evaporating an organic solvent of the coating solution and the nitrocellulose coating has a layer thickness of between 10 and 150 nm, characterized in that the coating solution: ▪ between 0.2 and 3.0 wt.% of high-purity nitrocellulose in an organic solvent; ▪ between 0.5 and 10.0 wt% (3-glycidyloxypropyl)trimethoxysilane (GPTS); and ▪ comprises between 0.3 and 20.0 wt.% water, and the deposited nitrocellulose coating is optically clear and has a root mean square (RMS) roughness of at least 0.5 nm. [15] The method according to claim 14, characterized by that the coating solution: ▪ between 0.5 and 1.5 wt%, preferably between 0.6 and 1.4 wt% or between 0.7 and 1.3 wt% or between 0.8 and 1.2 wt% or between 0.9 and 1.1 wt% or 1 wt% of high purity nitrocellulose in an organic solvent; ▪ between 2.0 and 4.0 wt%, preferably between 2.1 and 3.9 wt% or between 2.2 and 3.8 wt% or between 2.3 and 3.7 wt% or between 2.4 and 3.6 wt% or between 2.5 and 3.5 wt% or between 2.6 and 3.4 wt% or between 2.7 and 3.3 wt% or between 2.8 and 3.2 wt% or between 2.9 and 3.1 wt% or 3 wt% GPTS; and ▪ between 0.3 and 15.0 wt.%, preferably between 0.5 and 10.0 wt.% or between 1.0 and 9.0 wt.% or between 2.0 and 8.0 wt.% or between 3.0 and 7.0 wt.% or between 4.0 and 6.0 wt.% or between 4.1 and 5.9 wt.% or between 4.2 and 5.8 wt.% or between 4.3 and 5.7 wt.% or between 4.4 and 5.6 wt.% or between 4.5 and 5.5 wt.% or between 4.6 and 5.4 wt.% or between 4.7 and 5.3 wt.% or between 4.8 and 5.2 wt.% or between 4.9 and 5.1 wt.% or 5 wt.% of water. [16] The method according to claim 15, characterized by that the coating solution: ▪ between 0.8 and 1.2 wt.% of high purity nitrocellulose in an organic solvent; ▪ between 2.8 and 3.2 wt% GPTS; and ▪ contains between 4.8 and 5.2 wt% water. [17] The method according to claim 16, characterized by that the coating solution: ▪ 1 wt.% of high purity nitrocellulose in an organic solvent; ▪ 3 wt% GPTS; and ▪ Contains 5% water by weight. [18] The method according to any one of the preceding claims 14 to 17, characterized by that the pulling speed during immersion and subsequent withdrawal is between 5 and 15 cm per minute, in particular between 8 and 12 cm per minute, in particular 10 cm per minute at a temperature of the coating solution of between 18 and 25°C. [19] The method according to any one of the preceding claims 14 to 18, characterized by that the drying takes place over a period of at least 5 minutes at a temperature between 18 and 90°C, preferably over a period of 20 minutes at a temperature of 60°C. [20] The method according to any one of the preceding claims 14 to 19, characterized bythat the method comprises the functionalization of at least a first planar surface of the substrate and wherein the functionalization of the surface takes place (i) during the application of the coating solution or (ii) before the application of the coating solution in a separate step by applying an adhesion promoter layer. [21] The method according to any one of the preceding claims 14 to 20, characterized by that the organic solvent is amyl acetate, isopropanol, 1-propanol, tetrahydrofuran, or dimethyl sulfoxide. [22] Microarray for immobilizing biomolecules prepared according to a method of the preceding claims 14 to 21. [23] Use of a microarray according to any one of claims 1 to 13 or 22 for immobilizing biomolecules in an immobilization region, wherein the biomolecules are preferably: nucleic acids, in particular deoxyribonucleic acids or ribonucleic acids; peptides; or proteins, in particular enzymes or antibodies. [24] Use of a microarray according to any one of claims 1 to 13 or 22 for the analysis of biomolecules contained in a sample, wherein the biomolecules are preferably: nucleic acids, in particular deoxyribonucleic acids or ribonucleic acids; peptides; or proteins, in particular enzymes or antibodies.

Images