DE102011006278B4 - Coating layers with biofunctionalized surface and method for biofunctionalization of lacquer layers - Google Patents

Abstract

Method for biofunctionalization of a lacquer layer, comprising the following steps: (I) providing a lacquer layer (S) which has free functional groups (B) on its surface, and (II) immobilizing a multiplicity of one or more different biomolecules on the surface of the lacquer layer ( S), so that the biomolecules are covalently bound to the surface of the lacquer layer (S).

Description

• (I) Bereitstellen einer Lackschicht (S), die auf ihrer Oberfläche freie funktionelle Gruppen (b) besitzt, und
• (II) Immobilisieren einer Vielzahl eines oder mehrerer unterschiedlicher Biomoleküle an der Oberfläche der Lackschicht (S), so dass die Biomoleküle kovalent an die Oberfläche der Lackschicht (S) gebunden werden.

The present invention relates to novel methods for biofunctionalization of a lacquer layer, comprising the following steps:

• (I) providing a lacquer layer (S) having on its surface free functional groups (b), and
• (II) immobilizing a plurality of one or more different biomolecules on the surface of the lacquer layer (S), so that the biomolecules are covalently bound to the surface of the lacquer layer (S).

The present invention also relates to lacquer layers with a biofunctionalized surface, which can be produced by a method according to the invention.

Furthermore, the present invention relates to articles comprising or consisting of an article and a lacquer layer according to the invention associated with the article.

Furthermore, the present invention also relates to various uses of a lacquer layer according to the invention.

• – Aufbringen einer Lackschicht auf den Gegenstand und
• – Biofunktionalisieren der (aufgebrachten) Lackschicht mittels eines erfindungsgemäßen Verfahrens.

The present invention also relates to a method of providing an article having a biofunctional surface, the method comprising the steps of:

• Applying a lacquer layer on the object and
• - Biofunktionalisieren the (applied) lacquer layer by means of a method according to the invention.

Further aspects of the present invention will become apparent from the following description, in particular the examples described and the appended claims.

A "lacquer layer" is a layer of lacquer that is applied to or applied to an article. According to DIN EN 971-1, a varnish is a liquid, pasty or powdered, pigmented coating material which gives a covering coating with protective, decorative or specific technical properties. These include clearcoats that form a transparent coating with protective, decorative or specific technical properties (ISO 4618: 2006).

A "biomolecule" is a natural product, d. H. a chemical substance that is formed by an organism to fulfill biological functions. In the context of the present text, biomolecules are in particular proteins, peptides, carbohydrates, lipids, nucleic acids, hormones, amino acids and nucleotides.

In the context of the present text, "immobilization" is to be understood as a process in which (bio) molecules are bound to a lacquer layer surface via specific chemical interactions. In the context of the present invention, the biomolecules are covalently bonded to the surface of the lacquer layer (S) during immobilization. A covalent bond (also called atomic bond, electron pair bond or homo-polar bond) is a form of chemical bond in which the atoms form at least one (binding) pair of electrons between them. As described below, one or more linkers are preferably used here.

For the purposes of the present invention, the term "biofunctionalization" is understood to mean the immobilization of biomolecules onto a lacquer layer surface while retaining the (at least) partial or complete activity or function of the biomolecule. Accordingly, a "biofunctionalized surface" is a surface to which biomolecules are covalently and functionally bound so that they can perform a particular function.

Biofunctionalized surfaces have great future and application potential. Conventionally, in particular, plastics are combined with biomolecules (eg proteins and nucleic acids). As a result, the material to be biofunctionalized should retain the functionality of the respective biomolecule in order to utilize its properties in combination with the material. Of particular importance here is the immobilization of biomolecules without loss of functionality. However, the discrepancy between the stability of the material to be functionalized and the susceptibility of the biomolecule poses a particular problem.

The methods described in the prior art for applying biomolecules on paint layer surfaces use only non-specific surface couplings, z. B. by immersion in or Spray with a biomolecule-containing solution. For example, in the field of medical diagnosis, antibodies are non-specifically immobilized on plastic surfaces to be used for ELISA tests. In this case, however, often the problem arises that the coupling is poorly reproducible and thus from test to test a low reproducibility at relatively high variance occurs. Unspecific couplings also result in the further disadvantage that a large amount of biomolecules is necessary for sufficient immobilization of biomolecules.

Furthermore, unspecific coupling strategies have the disadvantage that biomolecules (eg antibodies or enzymes) can bind to the surface because of the non-directional coupling with their reactive center (antibody: paratope, enzyme: reactive domain). This is accompanied by a reduction or a total loss of function (of the biomolecule).

The object of the present invention was therefore to provide a method by means of which coating layers can be reliably biofunctionalized, in particular while maintaining the function of the biomolecules used, wherein preferably the above-mentioned problems are avoided.

Another object of the present invention is to provide corresponding biofunctionalized lacquer layers.

Other objects of the present invention are to provide articles having such a lacquer layer and to provide advantageous uses of such lacquer layers.

Another object of the present invention is to provide a method of providing an article having a biofunctional surface.

Other objects of the present invention will become apparent from the following description and in particular the appended claims.

• (I) Bereitstellen einer Lackschicht (S), die auf ihrer Oberfläche freie funktionelle Gruppen (b) besitzt, und
• (II) Immobilisieren einer Vielzahl eines oder mehrerer unterschiedlicher Biomoleküle an der Oberfläche der Lackschicht (S), so dass die Biomoleküle kovalent an die Oberfläche der Lackschicht (S) gebunden werden.

The primary object is achieved by a method for biofunctionalization of a lacquer layer, comprising the following steps:

• (I) providing a lacquer layer (S) having on its surface free functional groups (b), and
• (II) immobilizing a plurality of one or more different biomolecules on the surface of the lacquer layer (S), so that the biomolecules are covalently bound to the surface of the lacquer layer (S).

For the terms "lacquer layer", "Immobilize", "Biomolecules" and "covalent" the above applies in each case.

Based on the prior art, it was not obvious to provide such a method. In particular, since a lacquer should conventionally not have any reactive groups on the surface in terms of its actual, intended function, so that it does not react with the environment.

The prior art has hitherto described no suitable (coupling) systems which are suitable for immobilizing biomolecules in a targeted manner on the surface of a lacquer layer. This certainly plays a role that it was in the context of the present invention on the way to the immobilization of biomolecules according to the invention to overcome a number of hurdles, z. B. in connection with the generation of functional groups on the paint layer surface (as described below).

Coating systems are complex compositions of different substances which have to meet certain requirements, which makes the generation of (reactive) surface groups a challenge. Because when creating reactive surface groups there is always the danger that the actual paint is no longer suitable for conventional purposes. This is due to the fact that by adding and / or modifying individual constituents, if appropriate, lacquers can no longer harden and / or the mechanical coating properties can be changed (for example, too soft or too brittle).

Although different methods are described in the prior art, which deal with chemical-covalent coupling of biomolecules on certain material surfaces. These include the coupling of biomolecules via silanization of glass or oxide surfaces, the attachment of biomolecules via SH groups to gold vapor-deposited surfaces and the generation of reactive groups on z. B. polymer surfaces by plasma methods. However, the above-mentioned methods are not described in connection with the biofunctionalization of enamel layers. In addition, these methods would not or only partially suitable for biofunctionalization of paint layers. Thus, for example, wet-chemical processes can lead to the destruction of the lacquer layer due to the solvents used (eg when using acetone or NMP as solvent).

DE 100 25 174 A1 describes, for example, a process for producing an electrode coated with biomolecules, which consists of a plastic bound electrically conductive material. D. Dankbar's PhD thesis (2005, University of Tübingen, "Photochemical surface modification for bioanalytics on plastic substrates") deals with surface modifications on plastic substrates by means of photoactivatable crosslinkers. In an Internet excerpt to the doctoral student award of the Leibnitz Institute for Polymer Research Dresden e. V., dated Nov. 12, 2010, refers to a dissertation which deals with the conductivity of plastics and describes the preparation of materials having functional properties, namely the antistatic conductivity of intrinsically insulating isomers. However, a method for the biofunctionalization of a lacquer layer in the context of the present invention is not described in the prior art.

• (I.1) Bereitstellen einer ausgehärteten Lackschicht (s1), die auf ihrer Oberfläche freie funktionelle Gruppen (a) besitzt, und Binden einer oder mehrerer eine oder mehrere funktionelle Gruppen (b) besitzender Verbindung(en) (B) an die Oberfläche der ausgehärteten Lackschicht (s1), wobei die Verbindung(en) (B) vorzugsweise kovalent an die Oberfläche der Lackschicht (s1) gebunden wird bzw. werden, so dass eine Lackschicht (S) entsteht, die auf ihrer Oberfläche freie funktionelle Gruppen (b) besitzt, oder
• (I.2) Bereitstellen einer noch nicht ausgehärteten Lackschicht (s2) und Aufbringen einer oder mehrerer eine oder mehrere funktionelle Gruppen (b) besitzender Verbindung(en) (B) auf die Oberfläche der noch nicht ausgehärteten Lackschicht (s2), so dass nach dem Aushärten der Lackschicht (s2) eine Lackschicht (S) entsteht, die auf ihrer Oberfläche freie funktionelle Gruppen (b) besitzt.

The lacquer layer (S) mentioned in step (I) is preferably produced in the context of a method according to the invention or is preferably preparable by

• (I.1) providing a cured lacquer layer (s1) having on its surface free functional groups (a), and binding one or more of one or more functional groups (b) possessing compound (s) (B) to the surface of hardened lacquer layer (s1), wherein the compound (s) (B) is preferably covalently bound to the surface of the lacquer layer (s1) or, so that a lacquer layer (S) is formed, the free functional groups on its surface (b) owns, or
• (I.2) providing a not yet cured lacquer layer (s2) and applying one or more one or more functional groups (b) possessing compound (s) (B) on the surface of the not yet cured lacquer layer (s2), so that after the hardening of the lacquer layer (s2) produces a lacquer layer (S) which has free functional groups (b) on its surface.

When providing a lacquer layer (s1) according to alternative (I.1) described above, care must preferably be taken that the properties of the lacquer are not adversely affected as described above. The skilled person can test this in the context of routine and comparative experiments. Within the scope of the present invention, the free functional groups (a) of the lacquer layer (s1) can be provided, for example, by the hardener used and / or the binder (crosslinker) of the lacquer system used. In this case, for example (as preferred according to the invention), an undercrosslinked coating system can be used in which the amount of binder (eg polyols) in the cured state is greater than the amount of hardener (eg hardener with (free) polyisocyanate groups). This advantageously offers the advantage that not every (free) functional group of the binder is bound / crosslinked, resulting in an undercrosslinked system with free functional groups (a), which are located (inter alia) on the surface of the cured lacquer layer (s1), so that they can bind to one or more functional groups (b) there. Further details on preferred constituents of the lacquers preferably to be used in connection with the present invention are explained below in the context of various (preferred according to the invention) embodiments and examples.

A particularly preferred aspect of the present invention relates to a process according to the invention as described above in the context of alternative (1.1), where one, several or all of the free functional groups (a) is or are selected from the group consisting of hydroxyl, isocyanate and , (primary) amino, carboxyl, epoxy and thiol group.

Preferably, the free functional groups are (a) functional groups of compounds selected from the group consisting of:
secondary amines, thiols, N-hydroxysuccinimides, acrylates, methacrylates, azides, alkynes, carboxylic acids, acid chlorides, acid anhydrides, epoxides and carbamate esters.

The free functional groups (a) are particularly preferably hydroxy and / or isocyanate groups.

Another, preferred aspect of the present invention relates to a method as described above in the context of alternative (I.2), wherein the or one, several or all compound (s) (B) by means of ultrasonic atomization on the surface of the not yet cured lacquer layer (s2) is sprayed or become.

Further preferred methods according to the invention for applying compound (s) (B) to the surface of the not yet cured lacquer layer (s2) are: dipcoats, spincoats or printing methods (ink or bubble jet, aerosol printing, screen printing, dispenser-based printing and the like). ).

Particularly preferred is a method of the invention (as described above), wherein one, several or all of the free functional groups (b) on the surface of the lacquer layer (S) is selected from the group consisting of amino, carbonyl, carboxyl , Epoxy, aldehyde, hydroxy and thiol group. Particularly preferably, the or one of the free functional groups (b) is an amino group.

Particularly preferred is a method according to the invention described above, wherein the or one, several or all compound (s) (B) is selected from the group consisting of silane derivatives, preferably aminosilanes, more preferably beta, alpha and gamma-alkoxysilanes, particularly preferably 3-aminopropyltriethoxysilane (APTMS) and 3-aminopropylsilanetriol (APTS), in particular from the group consisting of silane derivatives, preferably aminosilanes, more preferably beta-, alpha- and gamma-alkoxysilanes, in particular (3-acryloxypropyl ) trimethoxysilane (in particular because of the functional vinyl group), (3-mercaptopropyl) trimethoxysilane (in particular because of the functional thiol group, especially for coupling of biopolymers via polycondensation to form a thioether bond), (3-triethoxysilyl) undecanal (in particular because of the functional aldehyde group, which advantageously - depending on the embodiment of the method - the (add (3-aminopropyl) diisopropylethoxysilane or (3-aminopropyl) dimethylethoxysilane (which advantageously has a particularly favorable orientation of the free functional groups for the purposes of the present invention (b) on the surface of the lacquer layer (S)), N-phenylaminopropyltrimethoxysilane (especially because of the benzene ring which can function as a functional group), 2- (2-pyridylethyl) thiopropyltrimethoxysilane, (6-azicosulfonylhexy) triethoxysilane (especially because of the suitability to further Used in the context of an azide-alkyne cyclo-step reaction), 5,6-epoxyhexyltriethoxysilane (in particular because of the functional epoxy group), (3-isocyananopropyl) triethoxysilane (in particular because of the isocyanate group, which is advantageously used for the coupling of biopolymers to form a Isourethanbindung suitable), 3-aminopropyltriethoxysilane (APTMS), 3-aminopropyl ilanetriol (APTS) and (3-aminopropyl) trimethoxysilane.

As described in the introduction, a "biomolecule" is a chemical substance as defined in the introduction. However, it is particularly preferred according to the invention if the or one, several or all of the (different) biomolecule (s) is / are selected from the group consisting of proteins (in particular antibodies and enzymes), peptides, lipids, carbohydrates, nucleic acids, Hormones, amino acids and nucleotides, more preferably from the group consisting of biopolymers. Depending on the desired application, the person skilled in the above-mentioned group can also use / immobilize a mixture of two or more of these biomolecules.

The biomolecules to be immobilized according to the invention preferably have one or more free hydroxy, carboxy, thiol and / or amino group (s) which can be used for the purposes of the binding or reaction mechanisms preferred in the context of the present invention; can.

It is understood that within the scope of the present invention, if necessary, the person skilled in the art, the functional groups (b) or the compounds (B) depending on the biomolecules to be immobilized or the (free) functional groups thereof or in dependence of any linkers to be used (as described below) and, optionally, depending on the free functional groups (a). The same applies in each case to the further compounds to be used according to the invention.

Within the scope of a method according to the invention (as described above), the biomolecules are preferably bound either covalently (directly) to one or more free functional groups (b) on the surface of the lacquer layer (S) (alternative (II.1)) or in each case via one or more linkers (L) covalently bound to one or more free functional groups (b) on the surface of the lacquer layer (S) (alternative (II.2)).

• (II.1) (die) Biomoleküle jeweils kovalent an eine oder mehrere freie funktionelle Gruppen (b) auf der Oberfläche der Lackschicht (S) gebunden werden und/oder
• (11.2) (die) Biomoleküle jeweils über einen oder mehrere Linker (L) kovalent an eine oder mehrere freie funktionelle Gruppen (b) auf der Oberfläche der Lackschicht (S) gebunden werden.

Accordingly, according to the invention, a method as described above is preferred, wherein

• (II.1) the biomolecules are each covalently bound to one or more free functional groups (b) on the surface of the lacquer layer (S) and / or
• (11.2) the biomolecules are each covalently bound via one or more linkers (L) to one or more free functional groups (b) on the surface of the lacquer layer (S).

In the context of the present text, "linkers" are short to long-chain organic molecules (as described below in the context of various examples) via which the biomolecules to be immobilized-depending on the process design-can be bound to the surface of the lacquer layer. In the context of the present invention, linkers represent a connection between reactive groups on the lacquer surface and reactive groups of the biomolecules. In the context of the present invention, linkers can be used not only to couple the biomolecules, but also advantageously to biomolecules from the surface of the lacquer layer immobilized or immobilized, so as not to interfere with or maximize the activity of the biomolecules.

In the case of the alternative (II.2) described above, it is particularly preferred if one or more, or all, of the linkers (L) is or are selected from the group consisting of homobifunctional linkers and heterobifunctional linkers.

• – N-Hydroxysuccinimid (NHS)-Ester, z. B. Sulfo-NHS, Dithiobis(succinimidylpropionat) (DSP), Disuccinimidylsuberat (DSS) (insbesondere für die Ankopplung mit einer Amino-Gruppe) oder N,N-Disuccinimidylcarbonat (insbesondere für die Ankopplung mit einer Hydroxyl-Gruppe),
• – Imidoester, z. B. Dimethyladipimidat (DMA) oder Dimethylpimelimidat (DMP) (insbesondere für die Ankopplung mit einer Amino-Gruppe),
• – Sulfhydryl reaktive Linker, z. B. 1,4-di-[3-(2-pyridyldithio)propionamido]butat, Bis-maleimidohexane (insbesodner für die Ankopplung mit einer Thiol-Gruppe),
• – Hydrazide, z. B. Adipicsäure-Dihydrazide oder Carbohydrozide (insbesondere für die Ankopplung mit einer Carbonyl-Gruppe),
• – Aldehyde, vorzugsweise Polyaldehyde, insbesondere Dialdehyde, besonders bevorzugt 1,5-Pentandial,
• – Polyisocyanate, vorzugsweise Diisocyanate, besonders bevorzugt Hexamethylendiisocyanat,
• – Isophorondiisocyanat, 1,4-Cyclohexyldiisocyanat, Toluylendiisocyanat, Diphenylmethandiisocyanat und Polymeres Diphenylmethandiisocyanat.

Particular preference is given to (homobifunctional) linkers selected from the group consisting of

• N-hydroxysuccinimide (NHS) ester, e.g. Sulfo-NHS, dithiobis (succinimidylpropionate) (DSP), disuccinimidyl suberate (DSS) (especially for coupling with an amino group) or N, N-disuccinimidyl carbonate (especially for coupling with a hydroxyl group),
• - Imidoester, z. Dimethyl adipimidate (DMA) or dimethylpimelimidate (DMP) (especially for coupling with an amino group),
• Sulfhydryl reactive linkers, e.g. For example, 1,4-di- [3- (2-pyridyldithio) propionamido] butoxide, bis-maleimidohexanes (especially for coupling with a thiol group),
• - Hydrazides, z. B. adipic acid dihydrazides or carbohydrocides (especially for coupling with a carbonyl group),
• Aldehydes, preferably polyaldehydes, in particular dialdehydes, more preferably 1,5-pentanedial,
• Polyisocyanates, preferably diisocyanates, more preferably hexamethylene diisocyanate,
• - Isophorone diisocyanate, 1,4-cyclohexyl diisocyanate, tolylene diisocyanate, diphenylmethane diisocyanate and polymer diphenylmethane diisocyanate.

Also suitable as (heterobifunctional) linkers are, for example, N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) or succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC).

• – eine, mehrere oder sämtliche der freien funktionellen Gruppen (a) Hydroxy-Gruppen sind,
• – die bzw. eine, mehrere oder sämtliche Verbindung(en) (B) ein Aminosilan ist bzw. Aminosilane sind, besonders bevorzugt, wobei die bzw. eine Verbindung (B) 3-Aminopropyltriethoxysilan (APTMS) ist,
• – eine, mehrere oder sämtliche der freien funktionellen Gruppen (b) auf der Oberfläche der Lackschicht (S) Amino-Gruppen sind, und
• – die Biomoleküle jeweils über einen Linker (L) kovalent an eine oder mehrere freie funktionelle Gruppen (b) auf der Oberfläche der Lackschicht (S) gebunden werden, wobei der Linker (L) vorzugsweise ein Polyaldehyd ist, vorzugsweise ein Dialdehyd, besonders bevorzugt 1,5-Pentandial.

A particularly preferred aspect in the context of the present invention relates in summary to a method according to the invention according to alternative (I.1) (as described above), wherein

• One, several or all of the free functional groups (a) are hydroxy groups,
• The one or more or all the compound (s) (B) is an aminosilane or aminosilanes are particularly preferred, the compound or (B) being 3-aminopropyltriethoxysilane (APTMS),
• - One, several or all of the free functional groups (b) on the surface of the lacquer layer (S) are amino groups, and
• - The biomolecules are each bound via a linker (L) covalently to one or more free functional groups (b) on the surface of the lacquer layer (S), wherein the linker (L) is preferably a polyaldehyde, preferably a dialdehyde, particularly preferably 1 , 5-pentanedial.

In the following, such a process is explained in more detail by means of selected, particularly preferred examples.

First, in a first step, a cured lacquer layer (s1) is provided which has free functional groups (a) on its surface. The paint system used is preferably a two-component polyurethane system. By way of the mixing ratio of the coating system constituents, free functional groups (a) (here: preferably hydroxy groups) can be produced on the surface of the lacquer layer.

The paint system is preferably provided by using binders and hardeners preferably in the ratio of 4: 1 to 8: 1 by weight, more preferably in the ratio of about 6: 1, be mixed with each other and optionally with other ingredients. For example, at a mixing ratio of binder (here: Bayhydrol A 242) to hardener (in this case: Bayhydur XP 2570) of 6: 1 according to stoichiometric calculation, that in 100 g of a corresponding mixture of binder and hardener 0.5 g of hydroxyl Groups are included. Thus, the OH content is 0.5 wt .-%, based on the total weight of the amounts of binder and hardener. At this mixing ratio, it is advantageously possible to produce an excess of hydroxy groups (as functional groups (a) in the context of the present invention) on the paint layer surface, which serve as a starting point for the functionalization of the paint surface and finally for the coupling reactions of biomolecules, as in US Pat Framework of the present text described in more detail.

The lacquer layer may be applied to any surface (article) suitable for the purposes of the present invention. For the purposes of the following explanations, a coil-coating sheet is considered, which is coated with a corresponding layer of lacquer (has been) and therefore on its surface free functional groups (a) has (here: hydroxy groups).

The free functional groups (a) are preferably allowed to react with a compound (B), preferably one amino silane. The compound (B) has one or more functional groups (b) as described above. As compound (B), 3-aminopropyltrimethoxysilane (APTMS) or 3-aminopropylsilsantriol is preferably used. It is particularly preferable to use APTMS (preferably in anhydrous toluene).

Reaktionsschema 1: Schematische Darstellung einer Silanisierungsreaktion an einer OH-funktionellen Oberfläche mit APTMS in wasserfreiem Toluol In this case, advantageously, a functionalization of the free. functional groups (a) (here: hydroxy groups) on the lacquer layer surface by means of silanization, as shown in the following reaction scheme:

Scheme 1: Schematic representation of a silanization reaction on an OH-functional surface with APTMS in anhydrous toluene

In the silanization reaction, hydroxy groups on the paint layer surface react with APTMS to form an aminosilanized surface. To test the silanization reaction that has taken place, it is preferable to use surface-sensitive analysis methods such as X-ray photoelectron spectroscopy (XPS) and FT-ATR spectroscopy. On the basis of the characteristic bands of C-N and N-H vibrations in the IR spectra (in FT-ATR spectroscopy), the amino groups of the silanes, which serve as confirmation of a modification made, can be detected at the lacquer surface. The XPS technique the detection of changes in the elemental composition at the paint layer surface. Thus, in the present case (after the silanization reaction) a higher carbon content and a lower oxygen atom content as well as silicon atoms are detected.

After the aminosilization of the lacquer layer surface, the lacquer layer surface has free functional groups (b) (as described above), namely (here :) amino groups.

In a next step, the biomolecules to be immobilized-preferably in each case via a linker (L) -are covalently bonded to one or more free functional groups (b) on the surface of the lacquer layer. Here, the binding of the biomolecules to the free functional groups (b) (here: amino groups) is preferably carried out by using a dialdehyde as a linker (L). Particularly preferred is the use of glutaraldehyde (1,5-pentanedial) as a linker (L). Alternatively preferred is the use of homobifunctional coupling reagent HDI (1,6 hexamethylene diisocyanate) as linker (L) (see also below).

As mentioned above, any biomolecule suitable for the purposes of the present invention can be immobilized. For the purposes of the following discussion, however, a model protein (here: BSA-FITC), which is covalently bound via glutaraldehyde as a linker (L) to the surface of the lacquer layer, as explained in more detail below. The model protein is a fluorescently labeled native bovine serum albumin. This advantageously enables detection by means of fluorescence microscopy.

To attach the biomolecule to the (amino-functionalized) lacquer layer surface, the lacquer layer surface is preferably reacted with glutaraldehyde.

Reaktionsschema 2: Darstellung der Anbindung eines Modellproteins (BSA-FITC mittels Glutaraldehyd als Linker. In this case, a condensation reaction takes place by nucleophilic addition between a carbonyl group of the glutaraldehyde and the primary amino group (of the silane) on the lacquer layer surface. The resulting compound stabilizes with elimination of water to form a Schiff base. Upon addition of the biomolecule, it is covalently bound (via the linker) to the lacquer layer surface. For this purpose, the object provided with the lacquer layer (here by way of example coil coating tin) is preferably brought into a solution of the model protein to be bound (here: 0.1 mg BSA-FITC / 5 ml PBS buffer pH 7.4), preferably for a period of about 4 hours. The general reaction mechanism is shown in Reaction Scheme 2:

Scheme 2: Representation of the binding of a model protein (BSA-FITC using glutaraldehyde as a linker.

• – eine, mehrere oder sämtliche der freien funktionellen Gruppen (a) Isocyanat-Gruppen sind,
• – die bzw. eine, mehrere oder sämtliche Verbindung(en) (B) ein Aminosilan ist bzw. Aminosilane sind, besonders bevorzugt, wobei die bzw. eine Verbindung (B) 3-Aminopropylsilantriol ist,
• – eine, mehrere oder sämtliche der freien funktionellen Gruppen (b) auf der Oberfläche der Lackschicht (S) Amino-Gruppen sind, und
• – die Biomoleküle jeweils über einen Linker (L) kovalent an eine oder mehrere freie funktionelle Gruppen (b) auf der Oberfläche der Lackschicht (S) gebunden werden, wobei der Linker (L) vorzugsweise ein Polyisocyanat ist, besonders bevorzugt Hexamethylendiisocyanat.

A further, particularly preferred aspect of the present invention relates in summary to an inventive method according to alternative (I.1) (as described above), wherein

• One, several or all of the free functional groups (a) are isocyanate groups,
• The one or more or all the compound (s) (B) is an aminosilane or aminosilanes are particularly preferred, the compound or (B) being 3-aminopropylsilanetriol,
• - One, several or all of the free functional groups (b) on the surface of the lacquer layer (S) are amino groups, and
• - The biomolecules are each bound via a linker (L) covalently to one or more free functional groups (b) on the surface of the lacquer layer (S), wherein the linker (L) is preferably a polyisocyanate, more preferably hexamethylene diisocyanate.

Such a method will be explained below with reference to selected, particularly preferred examples.

First of all, a hardened lacquer layer (s1) which has free functional groups (a) on its surface is also provided in a first step. The paint system used is preferably a two-component polyurethane system. By way of the mixing ratio of the coating system constituents, free functional groups (a) (in this case preferably isocyanate groups) can be produced on the (undercrosslinked) lacquer layer surface. The preferred mixing ratio of the total amount of binder to the total amount of hardener is 4: 1.

Again, the lacquer layer is basically applied to any surface (article) suitable for the purposes of the present invention.

The free functional groups (a) are preferably allowed to react with a compound (B), preferably with an aminosilane. The compound (B) has one or more functional groups (b) as described above. Preference is given to using (3-aminopropyltrimethoxysilane (APTMS) or 3-aminopropylsilanetriol as compound (B).) 3-aminopropylsilane triol is particularly preferred in the context of this exemplary embodiment.

Reaktionsschema 3: Schematische Darstellung einer Silanisierungsreaktion an einer NCO-funktionellen Lackschichtoberfläche mit Aminopropylsilantriol. In this case, advantageously, a functionalization of the free functional groups (a) (here: isocyanate groups) on the lacquer layer surface by means of silanization, wherein the reaction is preferably carried out with a strong base, for. DBU, as summarized in the following reaction scheme:

Scheme 3: Schematic representation of a silanization reaction on an NCO-functional lacquer layer surface with aminopropylsilane triol.

In a next step, the biomolecules to be immobilized - again preferably in each case via a linker (L) - are covalently bound to one or more free functional groups (b) (here: amino groups) on the surface of the lacquer layer. In this case, the binding of the biomolecules to the free functional groups (b) preferably takes place by using a polyisocyanate as linker (L). Particularly preferred is the use of homobifunctional coupling reagent HDI (1,6 hexamethylene diisocyanate) as a linker (L), as described in more detail below.

Reaktionsschema 4: Verwendung von Hexamethylendiisocyanat als Linker. The lacquer layer surface is preferably mixed with a 3% 1,6-hexamethylene diisocyanate solution. Since the reaction rate of hexamethylene diisocyanate is relatively low at room temperature, dibutyltin dilaurate (DBTL) is preferably used as the catalyst, as shown in summary in the following reaction scheme:

Scheme 4: Use of hexamethylene diisocyanate as a linker.

When the lacquer layer surface is mixed with HDI, the amino group of the silane reacts with the diisocyanate to form a urea bond.

Preferably, after a reaction time of about one hour, it is washed with methyl tertiary butyl ether (MTBE) to remove unbound HDI.

As mentioned above, any biomolecule suitable for the purposes of the present invention can be immobilized. For the purposes of the following explanations, however, a model protein (BSA-FITC) is also used in the context of this exemplary embodiment, which is bound covalently to the surface of the lacquer layer via HDI as linker (L) (see above).

Reaktionsschema 5: Darstellung der Anbindung eines Modellproteins (BSA-FITC) mittels HDI als Linker.For this purpose, the objects provided with the lacquer layer are preferably brought into a solution of the model protein to be bound (here: 0.1 mg BSA-FITC / 5 ml PBS buffer pH 7.4) directly after the attachment of HDI or after washing. preferably for a period of about 4 hours. The model proteins are covalently bound by formation of a urea bond to the free isocyanate groups (of the linker HDI), as shown in the following reaction scheme:

Scheme 5: Representation of the connection of a model protein (BSA-FITC) by means of HDI as a linker.

• – die bzw. eine, mehrere oder sämtliche Verbindung(en) (B) ein Aminosilan ist bzw. Aminosilane sind, besonders bevorzugt, wobei die bzw. eine Verbindung (B) 3-Aminopropyltriethoxysilan (APTMS) ist,
• – eine, mehrere oder sämtliche der freien funktionellen Gruppen (b) auf der Oberfläche der Lackschicht (S) Amino-Gruppen sind, und
• – die Biomoleküle jeweils kovalent an eine oder mehrere freie funktionelle Gruppen (b) auf der Oberfläche der Lackschicht (S) gebunden werden, wobei die Biomoleküle vor dem Binden an die freien funktionellen Gruppen (b) optional aktiviert werden.

Another preferred aspect of the present invention relates to an inventive method according to alternative (1.2) (as described above), wherein

• The one or more or all the compound (s) (B) is an aminosilane or aminosilanes are particularly preferred, the compound or (B) being 3-aminopropyltriethoxysilane (APTMS),
• - One, several or all of the free functional groups (b) on the surface of the lacquer layer (S) are amino groups, and
• - The biomolecules are each covalently bound to one or more free functional groups (b) on the surface of the lacquer layer (S), wherein the biomolecules are optionally activated before binding to the free functional groups (b).

By "activated" it is meant herein that the biomolecules are altered in such a way that they are converted into an intermediate suitable for the reaction (for binding to the free functional groups (b)). For example, in the case of substitution reactions, it depends on the quality of the leaving group whether the reaction proceeds or not. When a hydroxy group, which is a poor leaving group, is first converted to an ester, a good leaving group, substitutions with, for example, halides can be easily realized.

In the following, such a process is explained in more detail by means of selected, particularly preferred examples.

To provide a (not yet cured) lacquer layer (s2), an aqueous 2-K acrylate / melamine resin system is preferably used as the lacquer system.

Preferably, one or more or all of compound (s) (B) (here exemplified by 3-aminopropyltrimethoxysilane) are sprayed by ultrasonic atomization onto the surface of the not yet cured lacquer layer (s2). As a result, silanes are advantageously bound to the matrix of the paint system during the polymerization of the paint system. The result can be checked, for example, by means of XPS measurements (see above).

The immobilization of biomolecules is carried out in the context of this embodiment, preferably by means of carbodiimide method, wherein the biomolecules are activated before binding to the free functional groups (b). In the context of this exemplary embodiment, the biomolecules to be immobilized are preferably (one or more different) proteins. For the purposes of the following explanations, a model protein (here: horseradish peroxidase) is used in the context of this exemplary embodiment, which is covalently bonded to the surface of the lacquer layer. The success of the immobilization can be advantageously detected in conjunction with the chromogen 3,3 ', 5,5'-tetramethylbenzidine (TMB) by means of a (blue) color reaction. By means of this test it was also confirmed in investigations of the present invention (by positive blue staining) that the protein immobilized on the surface of the lacquer layer (here: horseradish peroxidase) has advantageously retained its original function (compare also application example III).

In the case of the carbodiimide method (see above), an activation reaction of the carboxyl group of the protein (1) (see reaction scheme 7) with N-ethyl-N- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) (2) is initially formed of the intermediate "O-acylisourea derivative" (3). The reaction step of attaching the proteins to the functional groups (b) (here: amino groups) is preferably carried out in a slightly acidic medium, since this advantageously the protonation of the amino groups is ensured and so an electrophilic attack of amino-groups is ensured.

The O-acylisourea groups of the resulting intermediate are highly reactive and readily hydrolyzable. If the coupling reaction (as preferred according to the invention) is carried out in an aqueous medium, there is a risk that two side reactions take place in the coupling of proteins that on the 1,3 rearrangement of O-Acylisoharnstoffs in N-acyl urea derivative (4) and the hydrolysis Recovery of the free acid groups (5) are due.

Reaktionsschema 7: Schematische Darstellung der Aktivierung einer Carboxyl-Gruppe eines (zu immobilisierenden) Proteins.

Scheme 7: Schematic representation of the activation of a carboxyl group of a protein (to be immobilized).

The rearrangement is more stable and prevents the reaction to form the desired attachment of the proteins. In order to avoid this side reaction, the O-acylisourea groups are preferably reacted with N-hydroxysuccinimide (NHS) (6) as active esters. In addition, the addition of NHS advantageously increases the reaction rate.

Subsequently, a condensation reaction by the nucleophilic attack of a free primary amino group of the silane (8), d. H. a compound (B) to form an amide group (9) and the elimination of the urea (7) (see reaction scheme 7). The proteins to be immobilized can then be covalently bound to the lacquer layer surface via this reactive amide compound.

The present invention also relates to a biofunctionalized lacquer layer. Such a lacquer layer has on its surface a multiplicity of covalently bound biomolecules. The biomolecules can be identical or different. For the biomolecules and the type of connection to the lacquer layer surface, the above applies accordingly. Accordingly, the biomolecules are preferably covalently bound to one or more functional groups (b) (as described above). Preferably, one or more, or all of the groups (b) are derived from a compound (B) (as described above), which in turn are preferably covalently bound to the paint layer surface via functional groups (a) (as described above). For preferred lacquer layers, compounds, the use of linkers or linker systems and preferred groups, the above statements also apply accordingly.

The present invention therefore also relates, as a result, in particular also to a (inventive) lacquer layer having a biofunctionalized surface, which can be produced by a method as described above. For preferred embodiments of a lacquer layer or the method according to the invention, what has been said above applies accordingly.

Accordingly, a lacquer layer according to the invention is particularly preferred, the biomolecules being selected from the group consisting of proteins, peptides, lipids, carbohydrates, nucleic acids, hormones, amino acids and nucleotides. Incidentally, what has been said above for the biomolecules applies correspondingly.

The present invention also relates to an article comprising or consisting of an article and a lacquer layer (as described above) associated with the article.

For preferred embodiments of the lacquer layer, the above applies accordingly.

Preference is given to an article according to the invention, wherein the lacquer layer partially or completely surrounds the article.

Further preferred is an inventive article as described above, wherein the article (at least in one section) preferably has a planar, curved, curved or three-dimensional surface course, which is partially or completely coated with the biofunctionalized paint system according to the invention.

The article is preferably selected from the group consisting of sensors, biochips, bioreactors or components of a bioreactor and implants.

A further aspect of the present invention relates to the use of a lacquer layer or an article according to the invention, each described above, for diagnostic purposes for immunological detection methods, for on-chip laboratory applications, for purification of mixtures containing biomolecules for testing of substances for impurities, for cell culture purposes, for the production of an implant or a biofunctionalized medical device, for the production of one or as a biocatalytic and / or bioactive surface.

• – Aufbringen einer Lackschicht auf den Gegenstand und
• – Biofunktionalisieren der Lackschicht mittels eines (erfindungsgemäßen) Verfahrens wie vorstehend beschrieben.

Another aspect of the present invention relates to a method of providing an article having a biofunctional surface comprising the steps of:

• Applying a lacquer layer on the object and
• - Biofunktionalisieren the lacquer layer by means of a (inventive) method as described above.

For the method or the paint layer as well as preferred embodiments thereof, in each case the above statement applies correspondingly.

Examples:

Application Example I: Biofunctionalization of a Lacquer Layer with an Antifreeze Protein (AFP = Anti Frost Protein)

The immobilization of an AFP (type I protein or peptide sequence of this protein) to the lacquer layer surface was carried out in the context of this example with the coupling reagent glutaraldehyde, which is preferably used in particular in the 2K polyurethane system for the coupling of proteins.

First, the functionalization of a lacquer layer surface (based on an undercrosslinked lacquer system (as described above)) with free hydroxy groups by means of silanization with (3-aminopropyl) trimethoxysilane.

The amino groups generated on the lacquer layer surface after silanization were then reacted with a dialdehyde (glutaraldehyde) to give imines. For this purpose, the silanized lacquer layer was placed in a 3% glutaraldehyde solution (GDA) in 100 mM pH 7.0 sodium phosphate buffer for four hours. Subsequently, the samples were washed extensively with deionized water.

The lacquer layer was then placed directly into a solution of the AFP to be bound (here Type I) (1.16 mg of AFP-type 1/10 ml PBS buffer) for 4 hours, the coupling reaction of the second carbonyl group of glutaraldehyde and a primary Amino group of the protein expired to form the second imine group.

After four hours of incubation, samples of the varnish layer (to evaluate the result) were each washed five times with 15 ml of PBS buffer on the shaker (shaking frequency 250 min-1).

The successful biofunctionalization could be demonstrated in a tire chamber. In comparison with a lacquer layer not modified according to the invention, the thickness of the frost layer on the lacquer layer surface (according to the invention) was reduced by more than 30 percent.

Example of Use II: Biofunctionalization of a Lacquer Layer with a Fluorescence-Labeled Bovine Serum Albumin Protein (BSAFITC)

Immobilization of a BSA-FITC to the lacquer layer surface was also carried out with glutaraldehyde in the context of this example.

First, the functionalization of a lacquer layer surface (based on an undercrosslinked lacquer system (as described above)) with free hydroxy groups by means of silanization with (3-aminopropyl) trimethoxysilane.

The amino groups generated on the lacquer layer surface after silanization were then reacted with a dialdehyde (glutaraldehyde) to give imines. For this purpose, the silanized lacquer layer was placed in a 3% glutaraldehyde solution (GDA) in 100 mM pH 7.0 sodium phosphate buffer for four hours. Subsequently, the samples were washed extensively with deionized water.

The lacquer layer was then extensively washed with deionized water and immediately after 4 hours in a solution of BSA-FITC to be immobilized (5 mg / ml in PBS buffer (PBS = phosphate buffered saline)), wherein the coupling reaction of the second carbonyl group of Glutaraldehyds and a primary amino group of the protein to form the second imine group expires.

After four hours of incubation, samples of the varnish layer (to evaluate the result) were each washed five times with 15 ml of PBS buffer on the shaker (shaking frequency 250 min-1).

The successful biofunctionalization could be detected with the help of a fluorescence microscope. After several washings (over several hours) to remove non-covalently bound components, a fluorescence signal was observed on the sample. This demonstrates that (functional) biomolecules are covalently bound to the surface. A control in which all reactions except the reaction with glutaraldehyde were carried out showed no bright green fluorescence compared to the sample according to the invention.

Example of Use III: Biofunctionalization of a Lacquer Layer with the Horseradish Peroxidase Enzyme (HRP)

The immobilization of the enzyme horseradish perosidase (HRP) to the lacquer layer surface was carried out analogously to the conditions explained in the application examples I and II.

HRP is an enzyme commonly used in biotechnology / biochemistry as a marker enzyme. It belongs to the family of peroxidases and is able to catalyze reactions with peroxides. For coupling, a concentration of 2.8 mg per mL PBS buffer was used.

To demonstrate successful coupling / immobilization, the HRPbiofunctionalized lacquer layer surface was first washed several times with PBS buffer to remove non-covalently bound components. Subsequently, the chromogenic substance 3,3'5,5'-tetramethylbenzidine (TMB) was added. HRP converts TMB (as a substrate) from a colorless to a blue substance. This reaction can be quenched with 1 M sulfuric acid, changing the color from blue to yellow. The color development can be measured quantitatively at a wavelength of 450 nm.

The biofunctionalized lacquer layer surface showed an optically visible blue color and a measurable with the aid of a UV-VIS spectrometer. Implementation of the substance TMB. This demonstrates that the immobilized enzyme was not impaired in its activity or retained the functionality of HRP.

Claims (19)

Method for biofunctionalization of a lacquer layer, comprising the following steps: (I) providing a lacquer layer (S) having on its surface free functional groups (b), and (II) immobilizing a plurality of one or more different biomolecules on the surface of the lacquer layer (S), so that the biomolecules are covalently bound to the surface of the lacquer layer (S). The method of claim 1, wherein the lacquer layer (S) according to step (I) is prepared or can be prepared by (I.1) providing a cured lacquer layer (s1) having on its surface free functional groups (a), and Binding one or more one or more functional groups (b) possessing compound (s) (B) to the surface of the cured lacquer layer (s1), wherein the compound (s) (B) preferably covalently bonded to the surface of the lacquer layer (s1) to become a paint layer (S) having free functional groups (b) on its surface, or (I.2) to provide a not yet cured paint layer (s2) and to apply one or more functional ones Groups (b) possessing compound (s) (B) on the surface of the not yet cured lacquer layer (s2), so that after curing of the lacquer layer (s2) a lacquer layer (S) is formed, on their surface free functional groups (b ) owns. A method according to claim 2, alternative (I.1), wherein one, several or all of the free functional groups (a) is selected from the group consisting of hydroxy, isocyanate, (primary) amino, carboxyl, Epoxy and thiol group. A method according to claim 2, alternative (1.2), wherein the or one, several or all compound (s) (B) by means of ultrasound atomization is sprayed onto the surface of the not yet cured lacquer layer (s2). Method according to one of the preceding claims, wherein one, several or all of the free functional groups (b) on the surface of the lacquer layer (S) is selected from the group consisting of amino, carbonyl, carboxyl, epoxy, aldehyde , Hydroxy and thiol group. Method according to one of claims 2 to 5, wherein the or one, several or all compound (s) (B) is selected from the group consisting of silane derivatives, preferably aminosilanes, more preferably beta, alpha and gamma-alkoxysilanes, more preferably 3-aminopropyltriethoxysilane (APTMS) and 3-aminopropylsilane triol (APTS). Method according to one of the preceding claims, wherein the or one, several or all biomolecules is selected from the group consisting of proteins, peptides, lipids, carbohydrates, nucleic acids, hormones, amino acids, nucleotides. Method according to one of the preceding claims, wherein (II.1) the biomolecules are each covalently bonded to one or more free functional groups (b) on the surface of the lacquer layer (S) and or (II.2) the biomolecules in each case via one or more linkers (L) are covalently bonded to one or more free functional groups (b) on the surface of the lacquer layer (S). The method of claim 8, alternative (112), wherein the one or more or all of the linkers (L) are selected from the group consisting of N-hydroxysuccinimide (NHS) esters, imidoesters, sulfhydryl reactive linkers, hydrazides, Aldehydes, preferably polyaldehydes, especially dialdehydes, more preferably 1,5-pentanedial, polyisocyanates, preferably diisocyanates, more preferably hexamethylene diisocyanate, isophorone diisocyanate, 1,4-cyclohexyl diisocyanate, tolylene diisocyanate, diphenylmethane diisocyanate and polymer diphenylmethane diisocyanate. The method of claim 2, alternative (I.1), wherein One, several or all of the free functional groups (a) are hydroxy groups, or one, several or all compound (s) (B) is an aminosilane or aminosilanes are particularly preferred, the or a Compound (B) is 3-aminopropyltriethoxysilane (APTMS), - one, several or all of the free functional groups (b) on the surface of the lacquer layer (S) are amino groups, and - the biomolecules are each covalent via a linker (L) be bound to one or more free functional groups (b) on the surface of the lacquer layer (S), wherein the linker (L) is preferably a polyaldehyde, preferably a dialdehyde, more preferably 1,5-pentanedial. The method of claim 2, alternative (I.1), wherein One, several or all of the free functional groups (a) are isocyanate groups, The one or more or all the compound (s) (B) is an aminosilane or aminosilanes are particularly preferred, the compound or (B) being 3-aminopropylsilanetriol, - One, several or all of the free functional groups (b) on the surface of the lacquer layer (S) are amino groups, and - The biomolecules are each bound via a linker (L) covalently to one or more free functional groups (b) on the surface of the lacquer layer (S), wherein the linker (L) is preferably a polyisocyanate, more preferably hexamethylene diisocyanate. The method of claim 2, alternative (1.2), wherein The one or more or all the compound (s) (B) is an aminosilane or aminosilanes are particularly preferred, the compound or (B) being 3-aminopropyltriethoxysilane (APTMS), - One, several or all of the free functional groups (b) on the surface of the lacquer layer (S) are amino groups, and - The biomolecules are each covalently bound to one or more free functional groups (b) on the surface of the lacquer layer (S), wherein the biomolecules are optionally activated before binding to the free functional groups (b). Lacquer layer with a biofunctionalized surface, producible by a method according to one of the preceding claims. Lacquer layer according to claim 13, wherein the biomolecules are selected from the group consisting of proteins, peptides, lipids, carbohydrates, nucleic acids, hormones, amino acids, nucleotides. Article comprising or consisting of - an object and A lacquer layer connected to the article according to one of claims 13 or 14. The article of claim 15, wherein the lacquer layer partially or completely encloses the article. An article according to any one of claims 15 or 16, wherein the article is selected from the group consisting of sensors, biochips, bioreactors or components of a bioreactor and implants. Use of a lacquer coating according to any one of claims 13 or 14 or an article according to any one of claims 15 to 17 for diagnostic purposes for immunological detection methods, for on-chip laboratory applications, for purification of mixtures containing biomolecules for testing substances on impurities, for cell culture purposes, for the production of an implant or a biofunctionalized medical device, for the production of one or as a biocatalytic and / or bioactive surface. A method of providing an article having a biofunctional surface comprising the steps of: Applying a lacquer layer on the object and Biofunctionalizing the lacquer layer according to one of claims 1 to 12.