WO2017042262A1

WO2017042262A1 - Biosensor chip

Info

Publication number: WO2017042262A1
Application number: PCT/EP2016/071164
Authority: WO
Inventors: Stefan Arnold; Christian Meyer
Original assignee: Bpc Arnold Gmbh Biopharma Consulting
Priority date: 2015-09-11
Filing date: 2016-09-08
Publication date: 2017-03-16
Also published as: DE102015115359A1; DE102015115359B4

Abstract

The present invention relates to a biosensor chip comprising a metal-coated substrate, a cross-linked polymer layer and at least one group attached to the surface by means of the binding sites, said chip comprising individual, defined, spatially separate regions with differing degrees of derivatization as measuring regions (spots). The present invention further relates to a method for producing the biosensor chip, to the use of said biosensor chip for interaction analysis, in screening methods, or as a microarray in surface plasmon resonance (SPR) imaging, and also relates to an SPR imager which contains the biosensor chip according to the invention.

Description

biosensor chip

The present invention relates to a biosensor chip, a method for producing the biosensor chip, the use of the biosensor chip for interaction analysis, in screening methods or as a microarray in the imaging

Surface Plasmon Resonance (SPR) and a Surface Plasmon Resonance Imager, which contains the biosensor chip according to the invention. The invention also relates to a chromatographic phase which is obtainable through the use of the biosensor chip.

The detection of, for example, bioorganic macromolecules via affinity reactions to biosensors is widespread. This requires substrate surfaces that are coated with an interaction partner of the interacting biomolecules (analyte / solid phase method). In order to be able to immobilize this reaction partner permanently on a substrate surface, it must first be chemically modified in such a way that reactive coupling groups are provided for immobilization. These coupling groups are typically reactive hydroxyl, amino, carboxyl, acyl halide, aldehyde, isothiocyanate or epoxy groups covalently linked to the surface via spacers.

The provision of coupling groups for immobilization can be carried out, for example, via so-called anchor molecules. WO 98/40739 A1 describes a step-by-step construction of a dilute bonding layer in which, in a first step, cystamine is applied to a gold surface and the provision of maleimide as a coupling group is achieved by a second chemical reaction. A commonly chosen strategy for immobilizing an interacting partner is based on films of organic monolayers (Bain and Whitesides, Angew Chem, 101, (1989), 522-528; Zhong and Porter, Anal. Chem. (1995), 709A-715A ). For producing such films, there are known processes in which functionalized alkylthiols are chemisorbed on gold (Bardea, A., Dagan, A. Willner, I. Anal. Chim. Acta 385, (1998), 33-43). The long-chain molecules are packed as a highly ordered monolayer (self-assembled monolayer, SAM) on the solid phase.

The prior art also often uses hydrophilic polymers as a substrate coating to increase the number of potential binding sites for macromolecules. These include polyethers, e.g. Polyethylene glycol, polysaccharides, e.g. Dextran, polyalcohols, e.g. Polyvinyl alcohol, or polyamides, such. Polyacrylamide. For biosensing purposes, US Pat. No. 5,436,161 discloses the use of thin hydrogel layers of carboxylated polysaccharides, such as carboxymethyldextran.

DE 100 36 907 A1 describes a biospecific coating in which a hydrophilic polymer layer is applied to a gold-coated glass substrate. The functionalized polymer chains of the polymer layer are brush-like, ie oriented perpendicular to the substrate surface, and a dextran layer is further laid over this polymer layer.

Preferred fields of use for such analyte

On the one hand, solid phase conjugates are purification processes in which ligands to be isolated from complex mixtures can be bound to the immobilized analyte; On the other hand, such conjugates are in the analytic / diagnostic field, in particular in the context of screening methods used, for example for the detection of certain ligands in biological fluids or for diagnostic methods in the field of DNA technology. In the latter case, solid phase conjugates are increasingly being used as biosensor chips.

The interaction analysis is also used for example in the pharmaceutical industry to search for active ingredients. It is important to analyze the largest possible number of different samples in as short a time as possible in order to be able to simulate any process conditions as quickly as possible. There is a growing interest in developing test systems for preliminary studies or "trouble shooting" studies, which are capable of simultaneously subjecting a large number of identical and different molecules to a specific test method for the quantification of biospecific binding properties, and a direct one To allow comparison with other possible variants to identify specific molecules, for example by means of high-throughput screening (HTS), a commonly used method for studying binding properties is surface plasmon resonance (SPR) highly sensitive measurement of the mass change that occurs during the interaction of the mobile with the immobilized binding partner on the surface of the biosensor chip.

Closely related to such a parallelization is the simultaneous automation of the test procedures as well as the direct transferability of the results to suitably structured chromatography phases. Furthermore, find such

Interaction analysis also for the genome study (polymorphism (SNP) or expression pattern analysis) or in the agrochemical industry as well as in food analysis use.

Neumann, et al. ("SPR-based Fragment Screening: Advantages and Application", Current Topics in Edical Chemistry, 2007, 7, 1630-1642) describe a biosensor chip for screening small molecule fragments (100 to 300 daltons) using SPR technology, thereby becoming labeled fragments covalently bound via spacer molecules to a highly ordered monolayer (SAM) deposited on a gold surface by means of pintool spotting.The biosensor chips prepared in this way are then used for high-throughput SPR (HT-SPR) to bind specific target molecules, eg proteins, enzymes, receptors , etc. However, this system has the disadvantage that specific binding environments, such as in / on chromatography phases, are not present and thus no representative binding parameters can be obtained.

The object of the present invention was to provide a biosensor chip, in particular a biosensor chip for use in SPR technology, a significant increase in throughput compared with the prior art for the identification of suitable polymer coatings, various residual variations and combinations or a suitable degree of derivatization of the chip surface allows. As a result, preliminary studies on process development with determination of optimal binding, washing and elution conditions can be carried out very quickly. A further object was the provision of a biosensor chip, with which any process conditions can be simulated quickly and the binding behavior can be investigated in order to ensure a direct transfer of the results obtained to appropriately constructed chromatography phases. The object is achieved by a biosensor chip comprising a metal-coated substrate;

a polymer layer applied to the metal-coated substrate and having on its surface functional groups as binding sites;

at least one radical which is applied to the surface of the polymer layer via the binding sites,

wherein the polymers of the polymer layer are crosslinked with each other and wherein the biosensor chip further comprises individual defined, spatially limited areas on which the at least one residue is applied so that on each of the areas a predetermined different degree of derivatization by a different saturation of the binding sites with the at least one Rest is set, wherein at least two individual areas are present with the same derivatization and the same degree of derivatization.

A biosensor chip in the sense of the invention is a solid-phase support which consists of a substrate and which according to the invention comprises a polymer layer to which residues are attached which are capable of binding to specific target molecules, e.g. Biomolecules, bind. The target molecules can be detected and / or influenced in this way, so that biochemical detections or label-free optical biomolecular interaction analyzes, in particular

Surface plasmon resonance (SPR).

By combining the biosensor chip according to the invention with SPR imaging, binding kinetics and binding affinities of the target molecules bound to the residual or modified polymer coating on the surface of the biosensor chip of the invention can be quantitatively determined and compared with each other in one measurement step, and the results obtained can be directly established Chromatography phases are transferred. The system is thus an ideal model system for a chromatography phase and can be used for trouble-shooting studies, as well as for screening a significantly increased number of phase or residue combinations, which is a prerequisite for solving complex binding problems.

The spatial configuration of the biosensor chip for use in the invention is not limited, although in particular for sensor applications preferably planar, two-dimensionally extended structures are used. Depending on the field of application, however, three-dimensionally shaped shapes, such as spheres or networks, may also be considered.

Preferred substrates according to the invention are those materials which are suitable for biotechnological

Problems or analyzes on sensory functional surfaces come into question. Reflection optical methods, such as surface plasmon resonance, the substrate is preferably made of a laser beam transparent material selected from the group consisting of plastics, for example polymethyl methacrylate, polyethylene terephthalate, polycarbonate and cycloolefin polymer, metals, metal oxides and silicates, e.g. Glass, quartz or ceramics.

According to one embodiment of the invention, it is preferred that the substrate is coated with a metal. Preferably, the metal is selected from the group consisting of gold, silver, copper, platinum, palladium and aluminum and combinations thereof. Gold is especially preferred.

According to the invention, a polymer layer is applied to the metal-coated substrate in which the polymers networked with each other. The crosslinked polymer layer may be covalently bonded to the metal-coated substrate surface, or via non-covalent interactions. Preferably, the polymer layer is bound via non-covalent interactions to the metal-coated substrate surface.

A covalent bond of the polymer layer to the substrate can take place via anchor molecules. Suitable anchor molecules comprise at least two functional units which are present at opposite ends of the armature and which, on the one hand, allow the connection to the surface of the metal-coated substrate and, on the other hand, the bonding of the polymer layer. The functional group for attachment to the metal-coated substrate is selected from the group consisting of -SH, -SS, -SeH, -SeSe, and -COSH. The functional group for binding to the polymer layer is selected from the group of typical coupling reagents, for example consisting of an epoxide, a hydroxyl, a carboxyl, a succinimidyl or an alkoxy group. Preferred anchor molecules are succinimidyl groups.

Possible non-covalent interactions of the polymer layer with the metal-coated substrate include: hydrogen bonds, dipole-dipole

Interactions, van der Waals interactions, charge-transfer interactions, e.g. π-π interactions, ionic interactions, coordinate bonding, e.g. at

Transition metals, and combinations of these interactions.

The polymer layer preferably comprises at least one polymer containing amino groups. Most preferred is polyvinylamine. Other suitable polyamines are, for example, polyethyleneimine, polyallylamine and the like. But also Polymers having other functional groups than amino groups, such as polyvinyl alcohol, polyvinyl acetate, polyacrylic acid, polymethacrylic acid or their

Precursor polymers such as polymaleic anhydride, polyamides or polysaccharides (cellulose, dextran, pullolan, etc.) and copolymers thereof are suitable.

The polymer to be crosslinked preferably has an average molecular weight in the range of 5,000 to 1,000,000 g / mol, more preferably 7500 to 250,000 g / mol, and most preferably 10,000 to 100,000 g / mol.

The polymer can be applied to the metal-coated substrate surface by any coating method known to a person skilled in the art of polymer chemistry, such as, for example, vapor deposition, adsorption, polymerization from the liquid, gas or plasma phase, spin coating, surface condensation, wetting, Dipping, spraying, soaking, steaming, applying electric fields or pressure, as well as methods based on molecular self-assembly, such as liquid crystals or Langmuir-Blodgett film formation. In this case, the polymer can be applied directly to the substrate as a single layer or multiple layer, or by stepwise repeated application of individual layers.

According to the invention, the polymers of the polymer layer are crosslinked with one another. According to a preferred embodiment of the invention, the degree of crosslinking of the crosslinked polymer layer is at least 5%, based on the number of crosslinkable groups in the polymer layer. Further preferably, the degree of crosslinking is from 5% to 30%, more preferably from 5 to 20%, and most preferably from 10 to 15%, based on the number of crosslinkable groups in the polymer layer. In a preferred embodiment of the invention, the polymers of the polymer layer are covalently crosslinked by at least one chemical crosslinking reagent. The degree of crosslinking can be set very easily by the stoichiometric amount of the at least one crosslinking reagent used for crosslinking the polymer, it being assumed that almost 100 mol% of the crosslinking agent form crosslinks. This can be demonstrated by analytical methods. For example, the degree of crosslinking can be determined by MAS NMR spectroscopy and quantitative determination of the amount of crosslinking reagent relative to the amount of polymer. This is the preferred method. The degree of crosslinking can also be determined by IR spectroscopy, for example by the COC or OH oscillation using a calibration curve. Both methods represent standard analytical methods for a person skilled in the art.

As a result of the crosslinking according to the invention, a macromolecular surface is built up from the surface-fixed polymer layer, which has two decisive advantages in comparison to conventional linear linker systems with brush-like polymer chains. Due to the (covalent) crosslinking of the polymer layer, the surfaces on the one hand have increased physical-chemical stability and thus enable the loss-free regeneration of the surfaces loaded with (bioorganic) macromolecules (for example biosensor surfaces). On the other hand, however, the polymer layer still has a high flexibility of the polymer chains, and the associated properties of a high solvent absorption capacity and a high diffusivity. The polymer layer is therefore in a homogeneous, biocompatible, soft and gel-like state, which allows the target molecules (biomolecules) completely or partially in immerse the polymer layer. In combination with the used functionalization of the polymer layer, as described below, quasi-three-dimensional are on the entire surface of the biosensor chip according to the invention

Created interaction areas that allow an optimal and multiple contact with the binding sites of the target molecules, resulting in a high sensitivity in heterogeneous affinity reactions.

The at least one crosslinking agent for crosslinking the polymer layer is preferably selected from the group consisting of dicarboxylic acids, diamines, diols and bis-epoxides, for example 1, 10-decanedicarboxylic acid,

Ethylene glycol diglycidyl ether (EGDGE) or 4,4'-

Biphenyl bicarboxylic acid. In a preferred embodiment, the at least one crosslinking reagent is a linear, conformationally flexible molecule having a length of between 1 and 20 atoms.

The polymer film of the present invention still has the ability to swell or shrink despite being crosslinked, with the actual thickness of the film being highly dependent on the solvent used.

According to the invention, the crosslinked polymer has functional groups. By the term "functional groups" as used in the present invention are meant atomic groups belonging to the crosslinked polymer on the surface of the substrate or to the crosslinkable polymer during the preparation of the polymer layer on the surface of the substrate. The functional groups preferably contain at least one weak bond and / or one heteroatom, more preferably one group exhibiting nucleophilic or electrophilic properties Accordingly, the term "functional group" encompasses all chemical structures that contribute to the formation of covalent bonds but also non-covalent interactions may be involved. According to the invention, the functional groups thus also represent chemical binding sites on the surface of the polymer layer.

Preferred functional groups are primary and secondary amino groups as well as hydroxyl, carboxy or ester groups. Depending on the acidity / basicity of the surrounding medium, amino groups may be present as protonated ammonium ions, and carboxy groups as deprotonated carboxylate ions.

According to the invention, the functional groups on the surface of the crosslinked polymer layer serving as binding sites are at least partially substituted / derivatized with at least one radical. The residues have the function of targeting certain target molecules, e.g. Biomolecules bind by a chemical interaction. The chemical interaction is here selected from the group consisting of hydrophobic interactions, hydrophilic interactions, cation exchange, anion exchange,

Size exclusion and / or metal ion chelation.

The term "derivatization" encompasses any chemical reaction capable of imparting a specific residue on the surface of the polymer layer, in particular by reaction of the functional groups serving as binding sites on the surface of the polymer layer with a derivatizing reagent which has the desired residue or a precursor compound thereof. The conversion of one functional group to another, reactive functional group should also be covered by the term "derivatization". For the purposes of the present invention, the term "radical" is understood to mean any uniquely definable chemical entity or a uniquely identifiable, usually recurring arrangement of chemical units of the same or different nature, which are on a nano-scale to a complex or a region with high and / or selective affinity to at least one complementary structure or surface region of at least one target molecule, provided that the affinity is stronger than a van der Waals interaction with CH or CH2 units of the polymer chains of the polymer layer on the surface of the biosensor chip. The remainder may be wholly synthetic or natural, or a combination thereof. In addition, the remainder may comprise more than one uniquely definable chemical moiety, including those chemical moieties that are relatively unreactive, such as alkyl or alkenyl moieties, but which are still useful in forming hydrophobic or dispersive interactions.

According to the invention only a certain proportion of the functional groups is derivatized in each case, whereby the surface of the biosensor chip has individual defined, spatially limited areas on which the at least one residue is applied so that on the areas in each case a predetermined different degree of derivatization by a different saturation of the binding sites with at least one remainder set.

In the context of the present invention, the term "degree of derivatization" means that a certain percentage of the functional groups present on the surface of the polymer layer is deliberately reacted with the appropriately selected at least one radical. A complete derivatization (degree of derivatization = 100%) would mean 100% of the functional groups on the surface of the polymer layer are substituted / derivatized by a radical.

In a preferred embodiment of the present invention, the saturation of the binding sites with a residue of> 0% to 100, the saturation of the binding sites with the at least one residue, and thus the degree of derivatization, in individually defined areas on the surface of the biosensor chip different be so that spatially limited measuring areas (spots) are formed with desired different degrees of derivatization on the surface of the biosensor chip.

In a further embodiment according to the invention, a first residue and a second residue, which is different from the first residue, are applied to the surface of the polymer layer, wherein the two different residues in the individually defined, spatially separated areas of the biosensor chip in each case in different ratios or Concentrations may be applied to each other. In this way, several residues with desired degrees of derivatization can be present on the biosensor chip.

According to the invention, at least two individual regions with the same derivatization and the same degree of derivatization are present on the surface of the biosensor chip in order to enable at least a twofold determination to increase the measurement accuracy.

The two individual regions with the same derivatization and the same degree of derivatization are preferably not adjacent to the surface of the biosensor chip. In a preferred embodiment, on the surface of the biosensor chip at least three individual regions with the same derivatization and the same degree of derivatization are present in order to allow at least a triple determination, whereby the measurement accuracy can be further increased.

The remainder can be chosen freely depending on the target molecule. The radical may be a straight, branched or cyclic aliphatic group, or an aromatic or heteroaromatic group which may be substituted or unsubstituted. Preferably, the residue contains heteroatoms, for example N, O, P, S atoms and the like, which are suitable for interacting with the target molecule to be bound.

In particular, the radical may be a monovalent linear aliphatic hydrocarbon group having 1 to 40 carbon atoms, or a branched or cyclic aliphatic hydrocarbon group having 3 to 40 carbon atoms, wherein one or more CH 2 units in this group are represented by O, S, -S (O) 2 ~, -C (O) NH- or -C (S) NH- may be substituted, one or more hydrogen atoms by F, Cl, Br, -CN or -NC may be substituted and said groups one or more double bonds between two carbon atoms can contain.

Particularly preferred monovalent linear aliphatic hydrocarbon groups having 1 to 40 carbon atoms, or branched or cyclic aliphatic hydrocarbon groups having 3 to 40 carbon atoms are selected from the group: methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec Butyl (1-methylpropyl), tert-butyl, isopentyl, n-pentyl, tert-pentyl (1,1-dimethylpropyl), 1, 2-dimethylpropyl, 2,2-dimethylpropyl (neopentyl), 1-ethylpropyl, 2-methylbutyl, n-hexyl, iso-hexyl, 1, 2-dimethylbutyl, 1-ethyl-1-methylpropyl, 1 Ethyl 2-methylpropyl, 1, 1, 2-trimethylpropyl, 1,2,2-

Trimethylpropyl, 1-ethylbutyl, 1-methylbutyl, 1,1-

Dimethylbutyl, 2, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, 1-hexylnonyl, n-hexadecyl, 1-hexyl-decyl, n-heptadecyl, n-octadecyl, n-nonadecyl, - (CH 2) 2oCH 3, (CH 2) 2iCH 3, - (CH 2) 22 CH 3, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-ethylhexyl, trifluoromethyl, pentafluoroethyl, 2, 2, 2-trifluoroethyl, ethenyl , Propenyl, butenyl, pentenyl, cyclopentenyl, hexenyl, cyclohexenyl, heptenyl, cycloheptenyl, octenyl or cyclooctenyl, where one or more, preferably one, CH 2 unit in said group is represented by 0, S, -S (O) 2 -, C ( 0) NH- or -C (S) NH-, and wherein one or more hydrogen atoms may be substituted by F, Cl, Br, -CN or -NC, with F and -CN being preferred.

Further suitable as radicals aliphatic hydrocarbon groups having 1 to 40 carbon atoms or branched or cyclic aliphatic hydrocarbon groups having 3 to 40 carbon atoms are described in European Patent Application EP 2570183 AI, the relevant disclosure of which is hereby incorporated by reference.

In another embodiment, the moiety may be a monovalent mono- or polycyclic aromatic ring system having from 6 to 28 aromatic ring atoms wherein one or more hydrogen atoms is represented by D, F, Cl, OH, C 1 to C 6 alkyl, ci to C 6 alkoxy , NH 2, -NO 2, -B (OH) 2, -CN or -NC.

Particularly preferred aromatic ring systems are: biphenyl, triphenyl, naphthyl, anthryl, binaphthyl, phenanthryl, Dihydrophenanthryl, pyrene, dihydropyrene, chrysene, perylene, tetracene, pentacene, benzpyrene, fluorine, indene and ferrocenyl.

In a further preferred embodiment, the remainder is a monovalent mono- or polycyclic heteroaromatic ring system having 5 to 28 aromatic ring atoms, wherein the heteroaromatic ring system contains at least one heteroatom selected from N, O, S and Se (the remaining atoms are carbon atoms).

Particularly preferred heteroaromatic ring systems are, for example, 5-membered rings, e.g. Pyrrole, pyrazole, imidazole, 1, 2, 3-triazole, 1, 2, 4-triazole, tetrazole, furan, thiophene, selenophene, oxazole, isoxazole, 1,2-diazole, 1,3-diazole, 1, 2, 3-oxadiazole, 1, 2, 4-oxadiazole, 1, 2, 5-oxadiazole, 1,3,4-oxadiazole, 1, 2, 3-thiadiazole, 1, 2, 4-thiadiazole, 1, 2, 5 Thiadiazole and 1, 3, -thiadiazole, 6-membered rings, such as Pyridine, pyridazine, pyrimidine, pyrazine, 1, 3, 5-triazine, 1, 2, 4-triazine, 1, 2, 3-triazine, 1, 2, 4, 5-tetrazine, 1, 2, 3, 4 Tetrazine, 1,2,3,5-tetrazine, or fused groups such as indole, isoindole, indolizine, indazole, benzimidazole, benzotriazole, purines, naphthimidazole, phenanthrimidazole, pyridimidazole, pyrazine imidazole, quinoxaline imidazole, benzoxazole, naphthoxazole, anthroxazole, phenanthroxazole, Isoxazole, benzothiazole,

Benzofuran, isobenzofuran, dibenzofuran, quinoline, isoquinoline, pteridine, benzo-5,6-quinoline, benzo-6,7-quinoline, benzo-7,8-quinoline, benzoisoquinoline, acridine, phenothiazine, phenoxazine, benzopyridazine, benzopyrimidine, quinoxaline, phenazine,

Naphthyridine, azacarbazole, benzocarboline, phenanthridine, phenanthroline, thieno [2, 3b] thiophene, thieno [3, 2b] thiophene, dithienothiophene, isobenzothiophene, dibenzothiophene,

Benzothiadiazothiophene or combinations of these groups. Most preferred are imidazole, benzimidazole and pyridine. Further suitable as radicals monovalent mono- or polycyclic aromatic ring systems having 6 to 28 aromatic ring atoms and other suitable monovalent mono- or polycyclic heteroaromatic ring systems having 5 to 28 aromatic ring atoms are described in the European patent application EP 2570185 AI, to the relevant disclosure hereby fully reference is taken.

In a further embodiment of the present invention, the radical is either an organic cationic group or an organic protonatable group, that is, a group which may be present in solution as a cationic group. Preferably, this group is in an aqueous solution having a pH in the range of 6 to 8 in cationic form, that is, in protonated form. The term "organic group" is to be understood not only those groups containing hydrogen and carbon, but also those containing nitrogen and hydrogen, such as amines.

Other organic protonatable or organic cationic groups which are suitable as radicals are described in European Patent Application EP 2570182 A1, the disclosure of which is hereby fully incorporated by reference.

In another embodiment of the present invention, the residue is a deprotonatable group or an anionic group, that is, a group which may be in solution as an anionic group. Preferably, this group is in an aqueous solution with a pH range between 6 and 8 completely or partially in anionic form, that is in deprotonated form before. Also included are polar groups which have a hydrogen atom which is replaced by stronger Bases can be cleaved, these hydrogen atoms are preferably bonded to a heteroatom.

Further deprotonatable or anionic groups which are suitable as radicals are described in European Patent Application EP 2570181 A1, the disclosure of which is hereby fully incorporated by reference.

In a further embodiment of the present invention, the remainder is a monovalent mono- or polycyclic aromatic ring system having 6 to 28 aromatic ring atoms or a monovalent mono- or polycyclic aromatic ring system having 5 to 28 aromatic ring atoms, as described above, having an anionic group or a deprotonatable group as described above is substituted. Preferably, these groups are present in an aqueous solution having a pH range between 6 and 8 completely or partially as ionic groups. These groups also include polar groups having a hydrogen atom which can be cleaved off by means of stronger bases, these hydrogen atoms preferably being bonded to a heteroatom.

Such radicals are especially in the European

Patent application EP 2570184 AI described, the relevant disclosure of which is hereby fully incorporated by reference.

In contrast to the residues, the functional groups are primarily not intended to interact with analytes, although it can not be completely ruled out that they nevertheless interact with the analytes. The present invention is further directed to a method of making the biosensor described above comprising the steps of:

a) activating the surface of the metal-coated substrate;

b) applying a polymer layer to the metal-coated substrate;

c) crosslinking the polymers of the polymer layer;

d) applying the at least one remainder to the surface of the polymer layer, wherein the at least one remainder is applied such that in each case a predetermined different degree of derivatization is set on individually defined, spatially delimited regions of the polymer layer; and

e) optionally sequential application of further radicals to the polymer layer, wherein the further radicals are each applied in different concentrations and different ratios to the at least one radical according to step d) on the individual defined, spatially limited regions of the polymer layer.

The material used for the substrate depends on the measuring method used. Be reflection-optical

Methods, e.g. the surface plasmon resonance used, the substrate is preferably made of a material transparent to a laser beam material from the group comprising plastics, for example polymethyl methacrylate,

Polyethylene terephthalate, polycarbonate and cycloolefin polymer, metals, metal oxides and silicates such as glass, quartz or ceramics. In this connection, it is desirable that the substrate is formed of a material which does not exhibit anisotropy with respect to the polarization. The metal layer may be applied to the substrate both with the aid of an intermediate layer serving for adhesion promotion, such as a chromium layer, as well as in direct contact with the substrate. The metal used for the metal film is not particularly limited as long as it is used to produce

Surface plasmon resonance is suitable. Preferably, the metal is selected from the group consisting of gold, silver, copper, platinum, palladium and aluminum and combinations thereof. Gold is especially preferred.

The thickness of the metal layer can be chosen freely. Preferably, however, the thickness of the metal layer is in the range of 0.1 nm to 500 nm, and more preferably in the range of 1 to 200 nm. If the thickness of the metal layer exceeds 500 nm, the effect of surface plasmon resonance can not be sufficiently detected. In the case where a chromium layer or the like is interposed between the substrate and the metal film, the thickness of the inserted layer is preferably in the range of 0.1 nm to 10 nm.

The metal layer may be formed on the substrate by known methods such as vapor deposition, sputtering, ion plating, electroplating and non-electrolytic plating.

In order to activate the metal-coated substrate surface, it is first modified according to the invention with a reactive initiator group, ie the initiator group is combined with an (unmodified) substrate surface before the (modified) substrate surface is then combined with the polymer in a subsequent step (and thus further modified). The modification of the substrate surface can be done using standard methods. The reactive, surface-bound initiator group may be selected from one of the following chemically reactive groups: hydroxyl, amino, carboxyl, acyl halide, ester, aldehyde, epoxy or thiol group. It may also be selected from any one of the following biologically or chemically reactive groups: disulfides, metal chelates, nucleotides and

Oligonucleotides, peptides or haptens such as biotin, digoxigenin, dinitrophenyl or similar groups.

If the polymer layer is to be covalently bonded to the activated metal-coated substrate surface, the attachment takes place via anchor molecules from the group of typical coupling reagents, for example consisting of an epoxide, a hydroxyl, a carboxyl, a succinimidyl or an alkoxy group. Preferred anchor molecules are succinimidyl groups. The anchor molecules are applied by reaction of the metal surface with functional groups of the anchor molecules, e.g. -SH, -SS, -SeH, -SeSe, and -COSH groups.

The polymer can be applied to the metal-coated substrate surface by any coating method known to a person skilled in the art of polymer chemistry, such as, for example, vapor deposition, adsorption, polymerization from the liquid, gas or plasma phase, spin coating, surface condensation, wetting, Dipping, spraying, soaking, steaming, applying electric fields or pressure, as well as methods based on molecular self-assembly, such as liquid crystals or Langmuir-Blodgett film formation. The polymer may in this case be applied directly to the substrate as a single layer or multiple layer, or by stepwise repeated application of individual layers. After application of the crosslinkable polymer on the surface of the metal-coated substrate, a crosslinking step follows according to the invention. To generate the polymeric network (derivatized or underivatized), any method known to a person skilled in the art can be used.

In a preferred embodiment of the invention, the crosslinking of the polymer layer takes place by means of at least one crosslinking reagent and the polymers are covalently crosslinked with one another. The at least one crosslinking reagent is preferably selected from the group consisting of dicarboxylic acids, diamines, diols and bis-epoxides, for example 1, 10-decanedicarboxylic acid, ethylene glycol diglycidyl ether (EGDGE) or, 'biphenylbicarboxylic acid. In a preferred embodiment, the at least one crosslinking reagent is a linear, conformationally flexible molecule having a length of between 1 and 20 atoms.

According to the invention, the crosslinkable polymer forms a polymer layer on the substrate surface. The term "polymeric layer" or "polymeric film" refers to the presence of a two- or preferably three-dimensional synthetic or biosynthetic polymeric network of at least one layer, but usually between a few and a few tens of molecular polymer layers. The polymer layer may consist of a chemically homogeneous composition, or of at least two different types of polymer whose chains penetrate each other (for example, polyacrylic acid and a polyamine).

In one possible embodiment of the invention, the polymer layer, the polymers of which are crosslinked to one another by covalent bonds, is bound to the underlying metal-coated substrate by non-covalent interactions, that is, the polymer layer is bound to the metal-coated substrate only by physical and / or chemical adsorption.

In a further embodiment of the invention, the crosslinkable polymer is first adsorbed on the surface of the metal-coated substrate and then crosslinked. However, the crosslinking of the polymer can also be carried out only after the derivatization of the functional groups with the at least one radical.

The degree of crosslinking of the polymer layer is preferably in the range of 5% to 30%, based on the number of functional groups available for crosslinking. Crosslinking is preferably by condensation of functional groups, but any other method known in polymer chemistry can be used, including radical and photochemical methods. The crosslinking can be carried out by reaction between functional groups of a polymer (intramolecular) and / or adjacent polymers (intermolecular). In addition, the crosslinking can also take place directly between functional groups of the polymers involved without the addition of a crosslinking reagent. This is particularly the case when copolymers or polymer blends are used which have at least two different functional groups which can react with each other, such as amino groups and carboxyl groups, which can react with each other after activation to form an amide bond. The crosslinking is preferably carried out by forming a covalent CN bond, for example an amide, urethane, urea or secondary or tertiary amine bond, which are formed by reaction of activated carboxylic acids or epoxides with amines. The intramolecular and intermolecular crosslinking of the polymer layer forms a stable two-, preferably three-dimensional polymer network.

Although crosslinking can be produced by any method known in the art, including non-selective methods based on free radical formation, such as electrochemical, photoluminescent or by-pass

(Ionizing) radiation-induced methods, the crosslinking is preferably carried out only between functional groups of the polymer using at least one cross-linking agent which is capable of entering into a condensation reaction with said functional groups.

Due to the degree of crosslinking, which is not too highly selected, the polymer film crosslinked according to the invention has on the one hand the ability to swell or shrink, on the other hand a stable two- or preferably three-dimensional network is formed by the intra- or intermolecular crosslinking of the polymer layers, whereby a desorption from the surface of the metal coated substrate is prevented. A further increase in stability can be achieved by covalently bonding the chains of the polymer layer to the substrate material.

In the context of the present invention, the term "degree of crosslinking" is defined as the maximum number of crosslinks formed by the crosslinking reaction, based on the total number of functional groups available for crosslinking. If, as preferred, bifunctional reagents are used in the crosslinking, the degree of crosslinking thus reflects the molar ratio between the amount of crosslinking reagent involved in the crosslinking reaction and the number of crosslinking reagents functional groups available for crosslinking in the polymer (in this case, this means that two functional groups are necessary to form a crosslink), provided that the crosslinking reaction proceeds almost quantitatively. In principle, however, both the formation of crosslinks within a polymer chain or between two different polymer chains is possible as well as the formation of terminal, non-crosslinked side chains by an incomplete cross-linking.

As previously described, the crosslinkable polymer contains functional groups that can be substituted or derivatized before or after application of the polymer to the metal-coated substrate, or before or after crosslinking of the polymer by the at least one moiety.

According to the invention, respectively defined subregions of the functional groups which are available on the polymer surface of the biosensor as binding sites of the at least one residue are derivatized by the at least one or the optionally more derivatization steps. This means that, taking into account the reactivities of different functional groups and reagents, in each case a specifically selected percentage of the functional groups present in the underivatized polymer, which may be the same or different, are converted into functional groups which correspond to the at least one selected radical are derivatized. In a preferred embodiment of the present invention, the saturation of the binding sites with the at least one residue, which corresponds to the degree of derivatization, is between 0% and 100% corresponding to the best solution of the separation problem. As has already been described above, the at least one residue according to the procedure described above is applied according to the invention to individual defined, spatially limited regions of the polymer layer in each case in different concentrations, and further radicals can be sequentially applied to the individually defined, spatially delimited regions of the polymer layer in each case be applied in different concentrations and ratios to the at least one remainder, so that the biosensor chip comprises individual defined, spatially limited areas in which the at least one residue is present with the desired degrees of derivatization, whereby spatially defined measurement areas (spots) on the surface of the biosensor chip forms become.

The application of the at least one remainder to the surface of the biosensor chip while maintaining the desired molar ratios which are necessary for setting the desired degree of derivatization in the respective spatially separate measuring range is preferably carried out using a pipetting device, a spotting device, a micropipetting device, a micro-printer head with a Microchannel liquid processing unit or an inkjet process. By means of these devices or methods, a precise localization of the individual defined, spatially limited areas on the surface of the biosensor chip is possible.

In particular, in the case that a sequential application of multiple residues is desired, the micro-print head with a micro-channel liquid processing unit for each measuring range over a Reaktionskämmer on each associated micro-channels automatically feed an individual reaction mixture with the corresponding residues, whereby a sequential application is achieved for the respective measuring range.

In a further embodiment, the biosensor chip may be formed as an array having a multiplicity of fields or measuring ranges on a planar solid-phase carrier. Each of these fields can be used in accordance with the previously described, individually defined, spatially separated areas on the surface of the biosensor chip as a separate measuring surface and. According to the invention, these measuring surfaces differ in each case in the type or concentration of the at least one residue applied thereto, the individual measuring surfaces each having the desired degree of derivatization.

A further aspect of the invention therefore relates to the use of the biosensor chip for producing a chromatographic phase, for interaction analysis, in screening methods or as a microarray in surface plasmon resonance imaging.

The spatial structure of the resulting array can be predetermined by a mechanical structuring of the carrier. If structured carrier plates are used as biosensor surfaces in the present invention, they preferably have a multiplicity of regularly arranged, position-adaptable fields for providing binding sites, these fields being located in cavities of only shallow depth. Preferably, the cavities have a depth of 20 to 100 μπι. This provides a liquid barrier while keeping the surface area as low as possible to minimize any unspecific adsorption phenomena. Furthermore, these fields include the inventive Polymer layer which provides the functional groups as binding sites for the application of the at least one residue, wherein the polymer used according to the invention can be chosen freely in each field. The polymer layer is preferably bonded to the surface of the carrier plate via non-covalent interactions or covalent bonds.

With the aid of fields / measuring ranges designed in this way, it is possible to avoid or minimize disadvantages with regard to non-specific binding. According to the invention, at least two of these fields have the same derivatization in order to allow at least a twofold determination to increase the measurement accuracy. In addition, the production of such a carrier plate can be made inexpensive by using the methods and materials of photolithography and etching techniques used in semiconductor technology.

By using the biosensor chip of the invention in combination with SPR technology, binding of target molecules, e.g. Biomolecules to be measured directly to certain residues or residual combinations. The SPR technology makes it possible, with suitable chemical compatibility of the applied on the surface of the biosensor chip at least a residue with the liquid-contacting surfaces of the SPR imager online monitoring and quantification of the molecules bound to the biosensor chip.

SPR spectroscopy is a well-known and important method for the detection of molecules, in particular biomolecules, and for the determination of chemical reactions on surfaces. Surface plasmon resonance (SPR) is the result of the interaction between electromagnetic waves and the free electron gas of a conducting surface. The Surface plasmon resonance is based on total internal reflection at the interface between a dielectric and a metal layer, that is, between two media whose dielectric constants have different signs. A strongly attenuated surface electromagnetic wave propagates along the metal layer. Within the volume of the damped electromagnetic wave, changes in concentration, for example of biomolecules, are detected in the form of surface refractive index changes. At the same time, this leads to a change in the resonance condition of the electromagnetic light wave reflected at the metal layer. Any refractive index change in the medium adjacent to the irradiated surface results in a shift in resonance and, consequently, a change in the intensity of the reflected beam, which can be measured simultaneously and spatially resolved, for example, by means of a CCD camera or array of photodetectors.

By combining the biosensor according to the invention with imaging SPR, the binding of target molecules to different residues or residual combinations, which has been carried out correspondingly simultaneously on the individual spot, can be measured simultaneously on a biosensor chip.

By means of online monitoring, that is, the measurement of the binding by means of imaging SPR in real time while an analyte containing the target molecules flows through a flow cell in which the biosensor chip according to the invention is attached with the bindable residues, is in a measurement process, a rapid quantification the target molecules bound to the biosensor chip according to the invention possible, which can be quickly obtained information about the decoupled binding behavior of a residue or a residue combination with the target molecule, which in turn for the individual possible variants a direct Comparison of the binding constants depending on the selected degree of derivatization in the individual defined regions of the biosensor allows.

The direct transferability of the measurement results obtained to suitably structured chromatography phases is ensured due to the high degree of conformity of the three-dimensional binding sites on the biosensor chip according to the invention and the chromatography phases. The invention thus also relates to a chromatography phase which can be prepared by the use according to the invention of the biosensor chip.

Another aspect of the present invention is therefore also an SPR imager comprising the biosensor chip according to the invention.

The invention allows a screening approaches (96 phases per day) applied today to identify suitable polymer coatings (type of polymer or copolymer, degree of crosslinking, layer thickness), ligands and in particular ligand combinations and the

Degree of derivatization a significant multiplication of the throughput (up to 96 phases in 10 minutes). Due to the rapid quantitative determination of the binding kinetics and the binding affinities of the residues used or

Restekombinationen and the simple production of phases on the biosensor chip according to the invention corresponding phase libraries can be created. The screening of the identified residues or residual combinations can then be carried out on existing or correspondingly newly synthesized phases of the phase library.

This allows preliminary studies to be carried out on the process development and determination of optimal binding, washing and elution conditions without great expenditure of time on chromatography phases equipped with appropriate residues are transferable. Thereby, an ideal model system for the chromatography phase can be obtained, which can be used for "trouble shooting" studies, as it can simulate any process conditions and the binding behavior can be investigated without loss of time. By increasing the screening rate, the present invention is also suitable to solve complex binding tasks, since in these cases a significantly increased number of phases or residues or residual combinations must be screened.

In the following, the invention will be explained with reference to figures and examples, which are not to be understood as limiting the scope of protection.

Example 1: Production of a biosensor chip

For coating, gold-coated biosensor chips functionalized with N-succinimide spacers were used (e.g., GWC open-platform SPRchip ™, HORIBA Chip CS SPRi-BioChip ™). This modification of the chip surface allows a direct coupling of polymers via amino groups. a) Immobilization of Polymer to the NHS-Activated Sensor Chip:

The biosensor chip is incubated in an 8-10% solution of polyvinylamine (molecular weight 20-50 kDa) in 100 mM sodium phosphate buffer pH7 at 25 ° C for 120 min. The chip is then washed once in water and once in isopropanol. b) Cross-linking of the polymer in defined surface areas.

Nanovolume applicators (eg Wasatch Microfluidics CFM, Arrayit NanoPrint ™ LM60 or HORIBA SPRi Arrayer ™) were selected to select 48 spots (3 spots each) 5 times a 0.56 mM Ethylenglycoldiglycidylether solution in isopropanol and incubated for 15 min at room temperature. After that, the chip is 10s each with

1. isopropanol 2. 0.5M trifluoroacetic acid in water 3. water 4. dimethylformamide 5. 0.5M trifluoroacetic acid

Dimethylformamide 6. Rinsed dimethylformamide. c) derivatization of the polymer surface with 2 ligands

The ligands 3-indolylpropanoic acid and urocanic acid were mixed in different ratios in DMF with activator reagent HBTU and buffer triethyamine according to the mixing scheme below and the 48 ranges (4 times 11 different mixing ratios, 4 spots with DMF / HBTU mixture without ligands) 3 times as under b) described with each 5nl mixture applied. The application was repeated 2 times after every 15 minutes.

Table 1

Scheme Apply the reactions to the biosensor chip Example 2: IgG binding on the biosensor chip at pH 7.4 in running buffer 1

The biosensor chip prepared as described in Example 1 is introduced into a suitable Surface Plasmon Resonance Imager device (eg GWC SPRimagerOII, Horiba SPRI-PLEX II 115 or comparable systems), equilibrated with running buffer 1 and the time-dependent binding (Figure 2) of IgG in running buffer 1 (10 M / ml IgG in 100 mM NaKHP0 ₄ pH7.) At the different regions coated with different ligand ratios (spots, Figure 1). Detachment of the bound IgG was carried out by injection of regeneration solutions RP1 (150 mM sodium acetate pH .3 + 100 mM NaCl) and RP2 (400 mM acetic acid).

The negative control reaction 12 (dashed line) shows almost no binding, while different plasmon resonance intensities and therefore IgG bonds can be observed in the various different derivatization reactions with variable ratios of the ligands (Table 1). Regeneration with Regeneration Solution 1 (RPI) was not sufficient to completely detach the bound IgG. Only regeneration solution 2 (RP2) completely dissolves the bound IgG molecules. The traces then return to the baseline (0% reflectivity).

The experimental design thus allows a comparative determination of the suitability of different ligand combinations or ratios for the reversible binding of the target molecule IgG.

Averaged representation (n = 3) of the adsorption kinetics of IgG (10 g / ml in 100 mM NaKHPO ₄ pH 7.4) to the 12 different derivatized areas on the biosensor chip (3 spots each). After IgG binding, regeneration solution 1 (RPl: 150 mM Sodium acetate pH .3 + 100 mM NaCl) and then regenerated with regeneration solution 2 (RP2: 400 mM acetic acid).

Claims

claims

1. biosensor chip comprising

a metal-coated substrate;

at least one radical which is applied to the surface of the polymer layer via the binding sites, characterized in that the polymers of the polymer layer are crosslinked with one another and wherein the biosensor chip further comprises individual defined, spatially limited regions on which the at least one radical is applied in each case a predetermined different degree of derivatization is set by a different saturation of the binding sites with the at least one residue on the individual defined regions, at least two individual regions having the same derivatization and the same degree of derivatization being present.

2. Biosensorchip according to claim 1, characterized in that the metal coating of the substrate is selected from the group consisting of gold, silver, copper, platinum, palladium and aluminum, and combinations thereof.

3. Biosensorchip according to claim 2, characterized in that the metal coating of the substrate is a gold coating.

4. Biosensorchip according to claim 1 or 2, characterized in that the polymers of the polymer layer by at least one chemical crosslinking agent is covalently crosslinked with each other.

5. Biosensorchip according to claim 4, characterized in that the chemical cross-linking reagent is selected from the group consisting of dicarboxylic acids, diamines, diols and bis-epoxides.

6. Biosensorchip according to one of claims 1 to 5, characterized in that the polymer layer consists of a polyamine or a polyvinylamine or a polyamine-polyvinylamine copolymer.

7. Biosensorchip according to one of claims 1 to 6, characterized in that the crosslinked polymer layer is covalently bound or via non-covalent interactions to the metal-coated substrate surface.

8. Biosensorchip according to one of claims 1 to 7, characterized in that the saturation of the binding sites with the at least one residue is between 0% and 100%.

9. biosensor chip according to one of claims 1 to 8, characterized in that a first remainder and a second remainder, which is different from the first remainder, are applied to the surface of the polymer layer.

10. Biosensorchip according to one of claims 1 to 9, characterized in that the biosensor chip comprises at least three individual areas with a same derivatization and a same degree of derivatization.

11. A method for producing a biosensor chip according to any one of claims 1 to 10, comprising the steps of a) activating the surface of the metal-coated substrate;

b) applying a polymer layer to the metal-coated substrate;

c) crosslinking the polymers of the polymer layer;

12. A method for producing a biosensor chip according to claim 11, characterized in that the at least one remainder is applied by means of a pipetting device, a spotting device, an icropipettiervorrichtung, a micro-printhead with Microkanal- liquid processing unit or an ink-jet process while maintaining the desired molar ratios ,

13. Use of a biosensor chip according to one of claims 1 to 10 for the preparation of a chromatography phase, for interaction analysis, in screening methods or as a microarray in the imaging surface plasmon resonance.

14. A surface plasmon resonance imager comprising a biosensor chip according to one of claims 1 to 10.