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EP1646672A1 - Reseaux de biomolecules immobilisees sur des surfaces formant des hydrogels, leur production et leur utilisation - Google Patents

Reseaux de biomolecules immobilisees sur des surfaces formant des hydrogels, leur production et leur utilisation

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Publication number
EP1646672A1
EP1646672A1 EP04740781A EP04740781A EP1646672A1 EP 1646672 A1 EP1646672 A1 EP 1646672A1 EP 04740781 A EP04740781 A EP 04740781A EP 04740781 A EP04740781 A EP 04740781A EP 1646672 A1 EP1646672 A1 EP 1646672A1
Authority
EP
European Patent Office
Prior art keywords
groups
biomolecules
star
surface layer
prepolymer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04740781A
Other languages
German (de)
English (en)
Inventor
Haitao Rong
Stefano Levi
Jürgen GROLL
Thomas Ameringer
Claudia Mourran
Martin Möller
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Henkel AG and Co KGaA
Original Assignee
SusTech GmbH and Co KG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SusTech GmbH and Co KG filed Critical SusTech GmbH and Co KG
Publication of EP1646672A1 publication Critical patent/EP1646672A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G18/00—Polymeric products of isocyanates or isothiocyanates
    • C08G18/06—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen
    • C08G18/08—Processes
    • C08G18/10—Prepolymer processes involving reaction of isocyanates or isothiocyanates with compounds having active hydrogen in a first reaction step
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G18/00—Polymeric products of isocyanates or isothiocyanates
    • C08G18/06—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen
    • C08G18/28—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen characterised by the compounds used containing active hydrogen
    • C08G18/40—High-molecular-weight compounds
    • C08G18/48—Polyethers
    • C08G18/4833—Polyethers containing oxyethylene units
    • C08G18/4837—Polyethers containing oxyethylene units and other oxyalkylene units
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L2203/00—Applications
    • C08L2203/02—Applications for biomedical use

Definitions

  • the present invention relates to arrays of immobilized biomolecules on hydrogel-forming surfaces, their production and use.
  • the present invention also relates to devices based on the arrays, in particular chips, microfluid devices and dipsticks.
  • a typical example of a so-called "high density array” is the GeneChip from Affymetrix, an oligonucleotide array with typically over 400 different capture samples that are built up base by base on the glass wafer.
  • the chips are characterized by a very high information density of up to 40,000 oligonucleotides / cm 2.
  • the individual "spots" are rectangular.
  • Low density arrays are available, for example, from Genometrix.
  • a maximum of 250 spots with a diameter of 50 ⁇ m are printed with a capillary pin bundle of up to 1000 individual capillaries into the cavities of a 96-well microtiter plate.
  • Conventional techniques are used for coupling to the surface, which are based in particular on amino silanizations and NHS-activated haptens, epoxy-activated surfaces, carbodiimide couplings on carboxyl groups and biotin / streptavidin bonds (cf. WO 97/18226; EP 0910570 A1; WO 98/29736).
  • the quality of these biochips is not sufficient for satisfactory use, especially in medical diagnostics. There are several reasons for this.
  • a central problem is the unspecific adsorption of biomolecules, especially proteins and cells on surfaces. This leads to the fact that contact with the analyte can cause non-specific coverage of the surface of the array or biochip with proteins and cell material, which leads to a reduction in the selectivity towards the target and ultimately to destruction of the biochip / array.
  • proteins and antibodies there is also the problem that they can lose their activity due to non-specific interaction with the substrate surface and, in the worst case, denature.
  • surfaces are required which, on the one hand, allow a targeted connection of the biomolecules and, on the other hand, have an extremely low tendency towards non-specific binding of such molecules.
  • Arrays are known from WO 02/059372 which have a large number of hydrogel cells on a support surface which have an immobilized binding site for proteins.
  • No. 6,174,683 describes the production of biochips which have covalently bound biomolecules, in which a biomolecule is derivatized with a hydrogel-forming prepolymer based on polyurethane in solution, the polymerization of the derivatized prepolymer is triggered in the solution and the solution of the polymerizing prepolymer is microspotted. Techniques on the surface of a suitable support. The biochips produced in this way have a better lifespan, but are comparatively complex to manufacture and unsatisfactory in terms of non-specific interactions with proteins.
  • the present invention is therefore based on the object of providing a suitable surface for the production of an array which is distinguished by a low tendency towards the non-specific interaction of biomolecules and which on the other hand allows selective coupling of biomolecules.
  • these surfaces are characterized by a particularly low affinity for non-specific binding of biomolecules.
  • these surfaces have a large number of functional groups R, which can react either directly or after activation with the functional groups R 'usually present in biomolecules, such as NH 2 , OH or SH, with the formation of bonds, so that there is a covalent bond between the Biomolecules come to the surface, whereby an immobilization of the biomolecules on the surface is achieved.
  • the activation of the biomolecules in a manner known per se can be used, ie the introduction of functional groups R 'into the biomolecules which react with the functional groups R on the surface to form bonds.
  • Another alternative is the covalent attachment of substances that a biomolecule can bind via a coordinative or affinitive bond, ie for which the biomolecule has one or more binding sites.
  • substances are also referred to below as adhesion promoters or coupling agents.
  • the present invention thus relates to arrays of immobilized biomolecules which are coupled to an organic surface layer which is essentially composed of crosslinked star-shaped prepolymers P, the prepolymers P having on average at least 4 polymer arms A which are water-soluble in themselves.
  • the present invention also relates to a method for producing such an array, comprising:
  • an organic surface layer on the surface of a carrier the surface layer being essentially composed of cross-linked star-shaped prepolymers which on average have at least 4 polymer arms A, which are water-soluble in and of themselves, and have a multiplicity of functional groups R on their surface, and ii. Immobilize the biomolecules in several spatially separated areas on the surface of the surface layer.
  • the immobilization of the biomolecules is usually carried out by applying the biomolecules which either have a functional group R 'which react with the functional groups R of the surface to form bonds, or by applying biomolecules which have a binding site for a covalently bonded to the surface Have binding partners (adhesion promoters).
  • the biomolecules are applied to the already crosslinked surface layer.
  • the biomolecules are applied during or before the crosslinking of the prepolymers forming the surface layer. If an adhesion promoter is used to attach the biomolecules, this can also be applied to the already cross-linked surface layer or before or during the cross-linking of the surface layer.
  • the arrays produced are distinguished by an extraordinarily low tendency to absorb non-specific proteins. This is probably due to the fact that the surface layer swells with water, ie forms a hydrogel.
  • the surface layer has a large number of functional groups in high surface density on its surface, which can be used for the immobilization of the biomolecules. Due to the structure of the layer, this surface layer is also extremely stable against aging and mechanical stress.
  • the biomolecules are also covalently bonded to the surface or by means of a covalently bonded adhesion promoter and are therefore not washed off.
  • the arrays are therefore very robust and have high long-term stability.
  • the arrays can be encapsulated and / or automatically Integrate analyzers.
  • biomolecules immobilized as a spot have a uniform coating, ie they are homogeneous. Good discrimination rates are achieved for oligonucleotide arrays and a high dynamic range is made available for hybridization experiments. Intra and interarray variations are small. This enables the use of the arrays in the diagnostic / analytical area. Proteins, including antibodies and enzymes, do not lose their activity when coupled to the surface layer. A preferred embodiment of the invention therefore relates to arrays of immobilized proteins, including antibodies and enzymes.
  • array denotes an arrangement of defined places, in particular a local assignment of certain substances to defined places, different places being able to be assigned the same or different substances.
  • a defined place corresponds to a certain area, the value of which is advantageously less than an intra-assay variance 15%, preferably less than 7% or an interassay variance of advantageously less than 20%, preferably less than 15% and in particular less than 10%, is subject to when comparing two areas with one another assigned a substance type, although it is also conceivable to assign mixtures of two or more types of substance to a location.
  • Two-dimensional arrays are preferred.
  • the locations advantageously form a regular two-dimensional pattern of fields. Within an array, each type of substance or each type of substance mixture e in field or multiple fields.
  • biochip here designates an array of biomolecules that are immobilized on a solid support, ie bound in a localized manner.
  • the binding can be based on covalent, ionic, coordinative or affinitive interactions, for example protein-ligand or protein-protein interactions, or on Mixed forms of the aforementioned interactions are based.
  • biomolecules denotes any biochemical and biological substances, both as individual molecules and as several interacting molecules. Examples include: • nucleic acids, in particular oligonucleic acids, for example single and / or double-stranded, linear, branched or circular DNA, cDNA, RNA, PNA (peptide nucleic acid), LNA (locked nucleic acid), PSNA (phosphothioate nucleic acid); • Antibodies, in particular human, animal, polyclonal, monoclonal, recombinant, antibody fragments, for example Fab, Fab ', F (ab) 2 , synthetic;
  • Proteins e.g. Allergens, inhibitors, receptors;
  • Enzymes e.g. Peroxidases, alkaline phosphatases, glucose oxidase, nucleases;
  • haptens e.g. Pesticides, hormones, amino acids, antibiotics, pharmaceuticals, dyes, synthetic receptors, receptor ligands.
  • biomolecule defines the ability of a substance to be able to interact with a biological sample or a part thereof, in particular the analyte.
  • a certain type of biomolecule can be called a ligand.
  • Ligands interact with and in particular bind - preferably specifically - to certain targets.
  • Reactive groups R in the sense of this invention are those which react with nucleophiles in an addition reaction including a Michael reaction or in a substitution reaction, e.g. B. isocyanate groups, (meth) acrylic groups, vinylsulfone groups, oxirane groups, oxazoline groups, aldehyde groups, carboxylic acid groups, carboxylic acid ester and carboxylic anhydride groups, carboxylic acid and sulfonic acid halide groups, but also the complementary, nucleophile-reacting groups such as alcoholic OH groups, primary and secondary amino groups, thiol groups and the like.
  • active ester groups of the formula -C (O) OX in which X is pentafluorophenyl, pyrrolidin-2,5-dione-1-yl, benzo-1,2,3-triazol-1-yl or one, are particularly preferred as examples of carboxylic ester groups
  • Reactive groups in the sense of the invention are also nucleophilic groups which react with the aforementioned electophilic groups to form bonds, eg. B. NH 2 , SH, or OH, but especially NH 2 . It goes without saying that a surface according to the invention either has predominantly electrophilic groups or nicleophilic groups, and in the case of NCO / NH 2 both groups can also be present side by side.
  • vinyl sulfone Complementary group R vinyl sulfone Complementary group
  • C C double bonds
  • R reactive groups
  • C C double bonds
  • acrylic groups also vinyl ether and vinyl ester groups
  • activated C C double bonds
  • N N double bonds
  • allyl groups in the sense of an en reaction or with conjugated diolefin Groups react in the sense of a Diels-Alder reaction.
  • Examples of groups which can react with allyl groups in the sense of an en reaction or with dienes in the sense of a Diels-Alder reaction are maleic acid and fumaric acid groups, maleic acid ester and fumaric acid ester groups, cinnamic acid ester groups, propiolic acid (ester) groups, Maleic acid amide and fumaric acid amide groups, maleimide groups, azodicarboxylic acid ester groups and 1, 3,4-triazoline-2,5-dione groups.
  • the surface of the organic surface layer has functional groups which are accessible to an addition or substitution reaction by nucleophiles.
  • Preferred groups R are isocyanate, isothiocyanate, active esters, NHS esters, aldehyde groups, acrylic and methacrylic groups, so that the biomolecules are bound to the surface via one of the following functional groups, selected from amide groups, urethane groups, imino groups, urea groups, thio urethane groups, ester groups, thiourea groups or sulfoethyl groups and optionally via a spacer.
  • a particularly preferred embodiment relates to arrays in which the surface layers have reactive isocyanate groups as R groups. These are deactivated prior to the use according to the invention after the coupling of the biomolecules by treatment with water to form amino groups.
  • the surface has a large number of nucleophilic groups, in particular NH 2 groups.
  • Star-shaped polymers are understood to mean those polymers which have a plurality of polymer chains bonded to a low-molecular central unit, the low-molecular central unit generally having 4 to 100 skeletal atoms, such as C atoms, N atoms or O atoms. Accordingly, the star-shaped polymers used according to the invention can be described by the following general formula I:
  • n is an integer with a value of at least 4, e.g. B. 4 to 12, in particular 5 to 12 and especially 6 to 8;
  • Z stands for a low molecular weight n-valent organic radical as central unit, which generally has 4 to 100, preferably 5 to 50 framework atoms, in particular 6 to 30 framework atoms.
  • the central unit can have both aliphatic and aromatic groups. It stands for example for one of at least 4-valent alcohol, e.g. B. a 4- to 12-valent, preferably an at least 5-valent, especially a 6- to 8-valent alcohol, e.g. B.
  • pentaerythritol dipentaerythritol, a sugar alcohol such as erythritol, xylitol, mannitol, sorbitol, maltitol, isomaltulose, isomaltitol, trehalulose, or the like derived residue;
  • A is a hydrophilic polymer chain which as such is water soluble
  • B is a chemical bond or a divalent, low molecular weight organic radical with preferably 1 to 20, in particular 2 to 10, carbon atoms, for example one C 2 -C 10 alkylene group, a phenylene or a naphthylene group or a C 5 - C 10 cycloalkylene group, where the phenylene, naphthylene and the cycloalkylene group additionally one or more, for. B. 1, 2, 3, 4, 5, or 6 substituents, e.g. B. alkyl groups with 1 to 4 carbon atoms, alkoxy groups with 1 to 4 carbon atoms or halogen have granules; and
  • FR is a reactive group which can react with a complementary reactive functional group FR "or with itself to form bonds.
  • Possible reactive groups FR and complementary reactive groups FR 1 are the functional groups mentioned in connection with the groups R or R 'in Table 1.
  • the groups FR are isocyanate groups.
  • the star-shaped prepolymers have on average at least 4, preferably at least 5 and in particular at least 6 polymer arms, but often not more than 16, in particular not more than 12 and especially not more than 10 polymer arms.
  • the star-shaped prepolymers particularly preferably have 6 to 8 polymer arms A.
  • the number average molecular weight of the polymer arms is generally in the range from 200 to 20,000 daltons, preferably in the range from 300 to 10,000 daltons, in particular in the range from 400 to 8000 daltons and especially in the range from 500 to 5000 daltons.
  • the star-shaped prepolymer has a number average molecular weight in the range of at least 1500 daltons, preferably 2000 to 100000 daltons, in particular 2500 to 50,000 daltons and especially 3000 to 30,000 daltons.
  • the molecular weight of the star-shaped polymers can be determined in a manner known per se by means of gel permeation chromatography, in which case commercially available columns, detectors and evaluation software can be used. If the number of end groups per polymer molecule is known, the molecular weight can also be determined by titration of the end groups FR, in the case of isocyanate groups, for. B. by reaction with a defined amount of a secondary amine such as dibutylamine and subsequent titration of the excess amine with acid.
  • a secondary amine such as dibutylamine
  • the hydrogel-forming properties of the surface layer are guaranteed by the water solubility of the polymer arms A. They are usually guaranteed when the molecular structure, ie at least the type of repeat units, preferably also the molecular weight of the polymer arm, corresponds to a polymer whose solubility in water is at least 1% by weight and preferably at least 5% by weight (at 25 ° C. and 1 bar) ,
  • polymers with sufficient water solubility examples include poly-C 2 -C - alkylene oxides, polyoxazolines, polyvinylalcohols, homo- and copolymers containing at least 50 wt .-% N-vinylpyrrolidone in copolymerized form, homo- and copolymers containing at least 30 weight % Copolymerized hydroxyethyl (meth) acrylate, hydroxypropyl (meth) acrylate, acrylamide, methacrylamide, acrylic acid and / or methacrylic acid, hydroxylated polydienes and the like.
  • the polymer arms A are preferably derived from poly-C 2 -C 4 -alkylene oxides and are selected in particular from polyethylene oxide, polypropylene oxide and polyethylene oxide / polypropylene oxide copolymers, which may have a block or a statistical arrangement of the repeating units.
  • Star-shaped prepolymers whose polymer arms A are derived from polyethylene oxides or from polyethylene oxide / polypropylene oxide copolymers with a propylene oxide content of not more than 50% are particularly preferred.
  • the prepolymers used according to the invention are e.g. T. known, e.g. B. from WO 98/20060, US 6,162,862, Götz et al., Macromol. Mater. Closely. 2002, 287, p. 223, Bartelink et al. J. Polymer Science 2000, 38, p. 2555, DE 10216639.0 and DE 10203937 (polyether star polymers), Chujo Y. et al., Polym. J.
  • the star-shaped prepolymers used in accordance with the invention are generally produced by functionalizing suitable star-shaped prepolymer precursors which already have the prepolymer structure described above, ie at least 4 water-soluble polymer arms, and which each have a functional group FR "at the ends of the polymer arms, which can be converted into one of the aforementioned reactive groups FR.
  • prepolymer precursors are known from the prior art, for. B. from US 3,865,806, US 5,872,086, US 6,162,862, Polym. J. 1992, 24 (11), 1301-1306, WO 01/55360 and commercially available, e.g. B. in the case of star-shaped poly-C 2 -C 4 -alkylene oxides under the trade names VORANOL®, TERRALOX®, SYNALOX® and DOWFAX® from Dow-Chemical Corporation, SORBETH® from Glyco-Chemicals Inc. and GLUCAM® from Amerchol Corp. or can be prepared by known methods in polymer chemistry by polymerizing suitable monomers in the presence of polyfunctional initiators, e.g. B.
  • the star-shaped prepolymer precursors can in principle be functionalized in analogy to known functionalization processes of the prior art.
  • those prepolymer precursors which have A OH groups at the ends of the polymer arms A are particularly suitable as starting materials.
  • the conversion of OH groups into amino groups can e.g. B. in analogy to that of Skarzewski, J. et al. Monatsh. Chem. 1983, 114, 1071-1077 method described.
  • the OH groups in a conventional manner, (z. B. by Organikum, 15th ed., VEB, Berlin 1981 S. 241ff .; J. March, Advanced Organic Synthesis 3rd ed. S. 382 ff.
  • a halogenating agent such as thionyl chloride, sulfuryl chloride, thionyl bromide, phosphorus tribromide, phosphorus oxychloride, oxalyl chloride and the like, optionally in the presence of an auxiliary base such as pyridine or triethylamine, in the corresponding halide, or with methanesulfonyl chloride in the corresponding mesylate transferred.
  • the halogen compound or the mesylate thus obtained is then converted into the corresponding azide using an alkali metal azide, preferably in an aprotic polar solvent such as dimethyl sulfoxide, dimethylformamide, dimethylacetamide or N-methylpyrrolidone.
  • an alkali metal azide preferably in an aprotic polar solvent such as dimethyl sulfoxide, dimethylformamide, dimethylacetamide or N-methylpyrrolidone.
  • the azide is then with Hydrogen is converted into the amino compound in the presence of a transition metal catalyst or with a complex hydride such as lithium aluminum hydride.
  • prepolymers which carry oxirane groups at the ends of the polymer arms A can be achieved, for example, by reacting prepolymer precursors which have A OH groups at the ends of the polymer arms with glycidyl chloride.
  • Prepolymers which carry A (meth) acrylic groups at the ends of the polymer arms can be produced, for example, by esterifying prepolymer precursors which carry A OH groups at the ends of the polymer arms with acrylic acid or methacrylic acid or by reacting the OH- Groups with acrylic chloride or with methacrylic chloride, or with the anhydrides of acrylic acid or methacrylic acid in analogy to known processes.
  • prepolymer precursors which carry A NH 2 groups at the ends of the polymer arms the NH 2 groups can be reacted with acrylic acid or methacrylic acid, with their anhydrides or with their acid chlorides.
  • the preparation of (meth) acrylate-terminated prepolymers can e.g. B. in analogy to that of Cruise et al. Biomaterials 1998, 19, 1287-1294 and Han et al. Macromolecules 1997, 30, 6077-6083 for the modification of polyether diols and triols described methods take place.
  • Prepolymers which carry thiol groups at the ends of polymer arms A can be prepared, for example, by reacting prepolymer precursors which carry halogen atoms at the ends of polymer arms A with thioacetic acid and subsequent hydrolysis in analogy to that in Houben-Weyl, Methods of Organic Chemistry, Ed. E. Müller, 4th ed., Vol. 9, p. 749, G. Thieme, Stuttgart 1955.
  • aromatic diisocyanates such as toluene-2,4-diisocyanate, toluene-2,6-diisocyanate, commercially available mixture of toluene-2,4- and -2,6-diisocyanate (TDI), m-phenylene diisocyanate, 3, 3 'diphenyl-4,4' -biphenylendiisocyanat, 4,4'- biphenylene diisocyanate, biphenylene diisocyanate 4,4'-diphenylmethane diisocyanate, 3,3 'dichloro-4,4'-, cumene-2,4-diisocyanate, 1, 5-naphthalene diisocyanate, p-xylylene diisocyanate, p-phenylene diisocyanate, 4-methoxy-1 , 3-phenylene diisocyanate, 4-chloro-1,3-phenylene diisocyanate, 4-bromo-1,
  • diisocyanates whose isocyanate groups differ in their reactivity, such as toluene-2,4-diisocyanate, toluene-2,6-diisocyanate, and a mixture of toluene-2,4- and -2,6-diisocyanate and ice and trans-isophorone.
  • Star-shaped prepolymers with aliphatic diisocyanate groups are particularly preferred, in particular those obtained by adding IPDI to the chain ends of OH-group-terminated star-shaped prepolymer precursors.
  • the star-shaped prepolymers are naturally reacted with the diisocyanate in such a way that a diisocyanate unit is added to each chain end of the star molecules, the second isocyanate group of the diisocyanate remaining free.
  • each end group of the star molecules is provided with a free isocyanate group via a urethane linkage.
  • Methods for this are e.g. from US 5,808,131, WO 98/20060 and US 6,162,862, Bartelink CF et al., J. Polymer Science 2000, 38, 2555-2565 and Götz et al., Macromolecular Materials and Engineering, 2002, 287, 223-230 ,
  • the prepolymer precursor for avoiding the formation of multimeric adducts, ie adducts in which two or more prepolymers are linked to one another via diisocyanate units is added to an excess of the diisocyanate for this purpose.
  • the excess is at least 10 mol%, based on the Stoichiometry of the reaction, ie at least 1, 1 mol, preferably a little. at least 2 moles and in particular at least 5 moles of diisocyanate and especially at least 10 moles of diisocyanate per mole of functional group in the prepolymer precursor.
  • the reaction is preferably carried out under controlled reaction conditions, ie the addition of the prepolymer precursor takes place under the reaction conditions so slowly that heating of the reactor contents by more than 20 K is avoided.
  • the prepolymer precursor is preferably reacted with the diisocyanate in the absence of a solvent or diluent.
  • the reaction can take place in the absence or in the presence of small amounts of conventional catalysts which promote the formation of urethanes.
  • Suitable catalysts are, for example, tertiary amines such as diazabicyclooctane (DABCO) and organotin compounds, e.g. B. dialkyltin (IV) salts of aliphatic carboxylic acids such as dibutyltin dilaurate and dibutyltin dioctoate.
  • the amount of catalyst is usually not more than 0.5 wt .-%, based on the prepolymer precursor, for. B. 0.01 to 0.5 wt .-%, in particular 0.02 to 0.3 wt .-%. In a preferred procedure, no catalyst is used.
  • the required reaction temperatures naturally depend on the reactivity of the prepolymer precursor used, the diisocyanate, and if used on the type and amount of the catalyst used. It is generally in the range from 20 to 100 ° C. and in particular in the range from 35 to 80 ° C. It goes without saying that the reaction of the prepolymer precursor with the diisocyanate takes place in the absence of moisture ( ⁇ 2000 ppm, preferably ⁇ 500 ppm).
  • the reaction mixture thus obtained is generally worked up by distilling off the excess diisocyanate, preferably under reduced pressure.
  • the reaction products obtained predominantly contain the star-shaped prepolymer which has isocyanate groups at the ends of the polymer arms.
  • the proportion of the star-shaped prepolymer is generally at least 70% by weight, preferably at least 80% by weight, of the reaction product.
  • the remaining constituents of the reaction product are essentially dimers and, to a small extent, trimers, which in these quantities are also suitable for producing the coatings according to the invention. 1 b
  • the organic surface layer is usually arranged on the surface of a carrier.
  • the provision of the organic surface essentially composed of crosslinked star-shaped prepolymers P therefore usually comprises the deposition of the star-shaped prepolymers P on the surface of the support and subsequent crosslinking of the reactive groups FR of the prepolymers P.
  • the steps of deposition and crosslinking can, if desired, be carried out repeatedly become. This leads to thicker layers.
  • the method of depositing the star-shaped prepolymers P comprises the following steps: i.a. Deposition of the star-shaped prepolymer P on the surface of a support by applying a solution of at least one star-shaped prepolymer, which on average has at least four polymer arms A, which are in themselves soluble in water and carry a reactive functional group FR at their free ends, on which Surface of the support; i.b. subsequently carrying out a linking reaction of the reactive groups FR with one another and removing the solvent; and i.e. optionally activating the functional groups R on the surface of the surface layer.
  • step i.a. a surface layer is obtained which is composed of uncrosslinked prepolymers and which has a large number of functional groups FR on its surface.
  • a linking reaction of the reactive groups FR to one another is carried out in this surface layer, the linking reaction being able to take place after the application and removal of the solvent by subsequently treating the surface layer with a crosslinking agent or when removing the solvent.
  • biomolecules can be applied before, during or after the linking-crosslinking reaction of the prepolymers. This results in 4 variants of the method according to the invention, which are explained in more detail below.
  • the biomolecules will be applied to an already completely cross-linked surface layer.
  • the functional groups R of the surface layer are activated before the application of the biomolecules by treatment with a suitable compound in order to have sufficient activity towards the functional groups FR 'of the biomolecules or an affinity for them to reach the binding sites of the biomolecules.
  • any excess FR, ie groups not occupied by biomolecules will then be deactivated again, in the case of NCO groups or isothiocyanate groups by treatment with water or steam.
  • the method according to the invention comprises:
  • an organic surface layer on a support which is essentially composed of non-crosslinked star-shaped prepolymers which on average have at least 4 polymer arms A, which are in themselves water-soluble and which have reactive groups FR at their ends, one surface layer being obtained Has a large number of functional groups FR; ii. Applying biomolecules which have functional groups FR ', which react with the functional groups FR of the surface to form bonds, in several spatially separated areas on the surface, and iii. Performing a crosslinking reaction;
  • an organic surface layer on a support which is essentially composed of non-crosslinked star-shaped prepolymers which on average have at least 4 polymer arms A, which are in themselves water-soluble and which have reactive groups FR at their ends, one surface layer being obtained Has a large number of functional groups FR;
  • an activator substance or an adhesion promoter
  • which has functional groups FR ' which react with the functional groups FR of the surface to form bonds on the surface, iii.
  • steps ii and iii or ii and iv can be carried out simultaneously or in a narrow temporal connection, ie step ii can be carried out while the networking in step iii or. iv. takes place.
  • star-shaped prepolymer P is usually applied as a solution to the surface of the carrier. Accordingly, these process variants naturally also include the removal of the solvent. The removal can take place before or during the crosslinking reaction, but always before applying the activator substance and before applying the biomolecules.
  • the crosslinking is generally carried out by reacting the reactive groups FR of the prepolymers, which are arranged at the free ends of the water-soluble polymer arms A, with one another or with complementary-reactive groups FR 'with the formation of bonds.
  • the complementary-reactive groups FR ' can be located either at the ends of the water-soluble arms A of star-shaped prepolymers or in different crosslinking substances (crosslinkers).
  • Examples of deposition processes are the immersion of the surface to be coated in the solution of the prepolymer as well as the spin coating - the solution of the prepolymer is applied to the surface to be coated rotating at high speed. It goes without saying that for the production of ultra-thin coatings, the coating measures are usually carried out under dust-free conditions.
  • the substrates are immersed in a solution of the star polymer in a suitable solvent and then the solution is run off, so that a thin liquid film with a thickness that is as uniform as possible remains on the substrate. This is then dried. The resulting film thickness depends on the concentration of the star polymer solution.
  • the networking is then triggered.
  • the initially non-rotating substrate is generally completely wetted with the solution of the star-shaped prepolymer.
  • the substrate to be coated is rotated at high speeds, usually at least 50 rpm, often at least 500 rpm, for example 500 to 30,000 rpm, preferably above 1000 rpm, e.g. B. 1000 to 10000 U / min, and particularly preferably 3000 to 6000 U / min, set in rotation, wherein the solution is largely spun off and a thin coating film remains on the surface of the substrate.
  • networking is also triggered here.
  • the concentration is usually at least 0.001 mg / ml, preferably at least 0.01 mg / ml, in particular at least 0.1 mg / ml, particularly preferably at least 1 mg / ml.
  • the concentration of the prepolymer in the solution will generally not exceed 500 mg / ml, preferably 250 mg / ml and in particular 100 mg / ml.
  • the thickness of the coating can of course be controlled via the concentration.
  • this will generally have a layer thickness in the dry state ( ⁇ 10% water) of at least 2 nm, preferably at least 5 nm and in particular at least 10 nm (measured by means of ellipsometry according to that in Guide to using WVASE 32 TM, JA Woollam Co. Ind., Lincoln NE USA 1998).
  • a layer thickness in the dry state ( ⁇ 10% water) of at least 2 nm, preferably at least 5 nm and in particular at least 10 nm (measured by means of ellipsometry according to that in Guide to using WVASE 32 TM, JA Woollam Co. Ind., Lincoln NE USA 1998).
  • coating thicknesses in the dry state ( ⁇ 10% water) of in particular 500 ⁇ m and especially 100 ⁇ m will not be exceeded in order not to impair the function of the biochips.
  • Advantageous layer thicknesses are in particular in the range from 10 nm to 50 ⁇ m (dry).
  • solvents which have no or only a low reactivity towards the functional groups FR of the prepolymer to be dissolved are suitable for the preparation of the solutions of the prepolymers P.
  • those are preferred which have a high vapor pressure and are therefore easy to remove.
  • Such solvents are therefore preferably have a boiling point below 150 ° C and preferably below 120 C C at atmospheric pressure.
  • suitable solvents are aprotic solvents, e.g. B.
  • ethers such as tetrahydrofuran (THF), dioxane, diethyl ether, tert-butyl methyl ether, aromatic hydrocarbons such as xylenes and toluene, acetonitrile, propionitrile and mixtures of these solvents.
  • protic solvents such as water or alcohols, e.g. B. methanol, ethanol, n-propanol, isopropanol, n-butanol and tert-butanol, and mixtures thereof with aprotic solvents.
  • the prepolymers can be crosslinked in different ways.
  • the prepolymers can be reacted with a crosslinking agent.
  • all compounds with 2 or more functional groups FR ' are suitable as crosslinking agents. net, which react with the functional groups FR of the prepolymer to form bonds.
  • the organic surface is provided by a method in which the linking of the reactive groups FR is triggered by adding a compound V1 which has at least two reactive groups FR 'per molecule which are in contact with the reactive groups FR of the star-shaped prepolymer reacts to form bonds.
  • the polyfunctional compounds V1 can be low molecular weight compounds, e.g. B. aliphatic or cycloaliphatic diols, triols and tetraols, e.g. B. ethylene glycol, butanediol, diethylene glycol, triethylene glycol, trimethylolpropane, pentaerythritol and the like, aliphatic or cycloaliphatic diamines, triamines or tetramines, eg. B.
  • the low molecular weight polyfunctional compounds generally have a molecular weight of ⁇ 500 g / mol.
  • the polyfunctional compound V1 can already be contained in the solution of the prepolymer which is used in the process according to the invention. Then react in the initially created surface layer of largely uncrosslinked prepolymers, e.g. B. when drying or heating the layer, the reactive groups FR 'of the crosslinking agent with the reactive groups FR of the prepolymer and in this way form a layer of crosslinked prepolymers, which as a rule still have a large number of functional groups FR and / or FR' has on their surface.
  • the compound V1 will accordingly have at least two dienophilic groups and vice versa. If the prepolymers have reactive groups which undergo an en reaction, the compound V1 will have at least two allylic double bonds. As a rule, in such systems for the production of the coatings, solutions are used which contain both the prepolymer and the compounds. gen V1 included. The crosslinking then takes place when the coating obtained primarily is dried, if appropriate after heating.
  • prepolymers are also suitable as polyfunctional compounds V1 which have at least four polymer arms A, which are in themselves soluble in water and have a reactive functional group FR 'at their free ends, which react with the reactive groups FR of the prepolymer to form bonds.
  • solutions of at least two different prepolymers can also be used according to the invention, in which one prepolymer has reactive groups FR and the other reactive groups FR 'complementary thereto. In this way too, a layer of crosslinked prepolymers is obtained, which generally still has a large number of functional groups FR and / or FR 'on its surface.
  • the linking of the reactive groups R is triggered by adding a sufficient amount of a compound V2 which reacts with a part of the reactive groups FR to form reactive groups FR ', which then reacts with the remaining reactive groups FR below React bond formation.
  • crosslinking can be triggered, for example, by treating the coated article with water, e.g. B. by storage in a damp atmosphere or under water.
  • some of the isocyanate groups react to form amino groups, which in turn react with the remaining isocyanate groups to form bonds, forming a layer of crosslinked prepolymers.
  • the crosslinking agent V2 is therefore water here. Coatings produced in this way, when freshly produced, still have free isocyanate groups. In the event of prolonged exposure to moisture, the isocyanate groups arranged on the surface are converted into amino groups.
  • solutions of the prepolymer in water or in a mixture of water with one or more water-miscible solvents are used.
  • suitable water-miscible solvents are, in particular, those which do not react with the isocyanate groups or react very slowly than water.
  • Examples include cyclic ethers such as tetrahydrofuran and dioxane, and also N-alkylamides such as N-methylpyrrolidone, dimethylformamide and dimethylacetamide.
  • the mixing ratio of water and solvent is generally in the range from 1: 100 to 100: 1, preferably in the range from 50: 1 to 1:10 and especially in the range 20: 1 to 1: 1.
  • This variant is particularly suitable for the production of thicker layers.
  • the concentration of prepolymer is then preferably in the range from 0.1 to 500 mg / ml, in particular in the range from 0.5 to 200 mg / ml and especially in the range from 1 to 100 mg / ml.
  • a solution of a star-shaped prepolymer for example as a monolayer, is first applied to the surface to be coated in the manner described above, if necessary a partial crosslinking of the reactive groups FR is carried out and then at least one further star-shaped prepolymer 2 is applied to the so applied surface, which has at least four polymer arms A, which are in themselves soluble in water and have at their free ends a reactive functional group FR ', which have a complementary to the reactive groups R of the first prepolymer reactivity. If necessary, the remaining reactive groups FR 'are then crosslinked again. This process can be repeated one or more times. In this way it is possible to produce multilayer coatings in a targeted manner.
  • the layer-by-layer method can be implemented in a particularly elegant manner with those prepolymers which have reactive groups FR which react with compounds V2 to form reactive groups R 'which have a reactivity that is complementary to the groups FR.
  • groups FR are isocyanate groups.
  • the connection V2 is water.
  • NH 2 groups then form as complementarily reactive groups FR '. If the crosslinking of the first layer with the compound V2 is triggered, the coating obtained has free groups FR '(for example amino groups) on its surface. These then react with the groups FR (for example isocyanate groups) of the prepolymers applied in a second coating process to form bonds
  • the surface layers can also contain water-soluble polysaccharides, such as hyaluronates, heparins, alginates or z. B. contain dextrans.
  • Such coatings are produced by depositing the prepolymer and water-soluble polysaccharide on the surface to be finished.
  • prepolymers with functional groups R reactive toward OH functions, e.g. B. isocyanate groups then the polysaccharide acts as a crosslinker. Polysaccharide and prepolymer then together form the hydrogel-forming organic surface layer.
  • the weight ratio of polysaccharide to star shaped prepolymer in the coating will generally not exceed a value of 1: 1 and is, for example, in the range from 1: 1000 to 1: 1 and especially in the range from 1: 100 to 1: 2.
  • the surfaces of inert materials will often be chemically activated before the actual finishing.
  • This can be done, for example, by treating the surface to be coated with acid or alkali, by oxidation (flaming), by electron radiation or by plasma treatment with an oxygen-containing plasma, as described by P. Chevallier et al. J. Phys. Chem. B 2001, 105 (50), 12490-12497; in JP 09302118 A2; in DE 10011275; or by D. Klee, et al. Adv. Polym. Be. 1999, 149, 1-57.
  • the surface can also be activated by means of plasma treatment with a plasma containing NH 3 , as described in US Pat. Nos. 6,017,577, 6,040,058 and 6,080,488.
  • the surface of the support can also be treated with compounds which, on the one hand, have a known good adhesion to the surface and which, moreover, have functional groups FR 'which are complementary to the functional groups FR of the star-shaped prepolymer.
  • Such connections are also referred to below as adhesion promoters.
  • Suitable groups FR ' depending on the reactive group FR of the prepolymer: isocyanate, aldehyde, amino, hydroxyl, mercapto and epoxy groups, furthermore groups which react in the sense of a Michael addition, dienophilic groups, the Diels-Alder Enter addition reactions, electron-deficient double bonds, which react with allylic double bonds in a Diels-Alder addition or ene reaction; activated ester groups; oxazoline; as well as vinyl groups, specifically enter into a free radical addition.
  • trialkoxyaminoal- kylsilanes such as triethoxyaminopropylsilane and N [(3-triethoxysilyl) propyl] ethylenediamine
  • trialkoxyalkyl-3-glycidyl ether silanes such as triethoxypropyl-3-glycidyl ether silane
  • trialkoxyalkyl mercaptane such as triethoxypropyl mercaptane methanekylsiloxane triloxysiloxane triloxysilane triloxysiloxane
  • - (meth) acrylamidoalkanes such as 1-triethoxysilyl-3-acryloxypropane.
  • Polyoxidic polyammonium groups are also suitable as adhesion-promoting groups for oxidic materials and plastic materials.
  • Examples of such compounds are polyammonium compounds with free primary amine groups, such as those used for. B. by J. Scheerder, JFJ Engbersen, and DN Reinhoudt, Recl. Trav. Chim. Pays-Bas 1996, 115 (6), 307-320, and von Decher, Science 1997, 277, 1232-1237 for this purpose, furthermore polylysine and polyethyleneimine.
  • the adhesion promoters are preferably applied as a monolayer to the surface to be equipped. Such monolayers can be achieved in a manner known per se by treating the surfaces to be coated with dilute solutions of the compounds, for. B. according to the immersion process described above or by means of spin coating. Solvents and concentrations correspond to the information given for the application of the prepolymers. It is often advisable to carry out the surface treatment with the abovementioned adhesion promoters following activation by flame, by electron radiation or by plasma treatment.
  • Carriers in the sense of the invention are generally materials with a rigid or semi-rigid surface and in this sense can be described as solid. Planar surfaces are common. In certain cases, however, it can be advantageous to use profiled beams. Elevations and depressions, such as steps, grooves, channels, notches, for example V-notches or mesa structures, are possible. These structures can be arranged on the surface in such a way that they appropriately influence the immobilization of the biomolecules. Channels can be used to carry liquid. At least parts of the surface of such carriers correspond to the specifications according to the invention.
  • the carrier materials are generally not subject to any restrictions.
  • suitable surface materials are oxidic surfaces, e.g. B. silicates such as glass, quartz, silicon dioxide such as in silica gels, or ceramics, further semimetals such as silicon, semiconductor materials, metals and metal alloys such as steel, polymers such as polyvinyl chloride, polyethylene, polymethylpentene, polypropylene, polyester, fluoropolymers (e.g. Teflon®), polyamides, polyurethanes, poly (meth) acrylates, blends and composites of the aforementioned materials.
  • the surface of the carrier on which the organic surface layer is to be provided has a low roughness.
  • the R a value which is characteristic of the roughness is advantageously not more than 100 nm and in particular not more than 50 nm.
  • a carrier can be varied and depends primarily on the type of use of the carrier to be described. For example, it can be slides, microtiter plates, tubes, chips, dipsticks, stamps, caps, particles, strands, in particular fiber bundles, spherical bodies, such as spheres or spheres, precipitation products, membranes, gels, sheets, containers, capillaries, disks, foils or Trade records. Wafer formats can also serve as carriers, which can be separated if desired. The carriers can also be provided with further useful components, for example chips can be encapsulated. According to a particular embodiment, a body, in particular in the form of a dip stick, is closed with a closure means, e.g.
  • the body is preferably in the form of a stick, at one end of which the closure means and at the other end of which the biomolecules are expediently immobilized. At least part of the surface of the body thus complies with the requirements of the invention.
  • the body can be formed from the polymer based on polycycloolefin or a suitable composite material with corresponding surface parts.
  • a plurality of dipsticks can also be connected to one another - also in accordance with the embodiment explained above.
  • the surface of the carrier available for the coupling can have customary dimensions, e.g. B. 0.5 to 50 cm 2 , in particular 1 cm 2 to 25 cm 2 .
  • a typical dimension is 20 mm x 70 mm or 26 mm x 76 mm or intermediate formats, half formats etc.
  • the biomolecules are immobilized on the organic surface layer in a manner known per se by binding, ie coupling the biomolecules to that on the The surface of this layer contains reactive functional groups R.
  • the connection can be made both to a surface layer which has not yet been crosslinked and to an already crosslinked surface layer.
  • the surface has a large number of the groups FR composed of the star-shaped prepolymer.
  • these are isocyanate groups.
  • the groups R can be converted into functional groups R2 which react with the functional groups of the biomolecule to form bonds, for example in epoxy groups, in isocyanate groups, in active esters, in NHS esters, in Michael systems such as acrylic amide -Groups, vinyl sulfone groups, acrylic ester groups or maleimide groups or in aldehydes.
  • This is achieved, for example, by applying activator compounds which, in addition to a suitable functional group R 'which reacts with the functional groups R of the surface to form bonds, have a further functional group R2 which has the reactivity required for binding the biomolecule.
  • the activator compound can be applied in a manner which leads to a more or less uniform activation of the entire surface.
  • an organic surface is obtained which, in a uniformly distributed manner, has a large number of reactive groups R2 which are suitable for forming bonds with the reactive groups R 'of the biomolecules.
  • the activator compound can also be applied only in those areas (places) in which the biomolecules are to be coupled to the array. In this way a surface is obtained which has a multiplicity of regions in which the surface carries reactive groups R2, whereas the surface outside these regions carries no or essentially no groups R2. ,__O
  • activator compounds is particularly useful when the surface provided has a large number of nucleophilic groups of comparatively low reactivity, for example SH, OH and NH 2 groups.
  • Such surfaces are obtained in particular when the star-shaped prepolymers have such a group or when the reactive groups R of the star-shaped prepolymers are converted into such a group during crosslinking, for example in the case of isocyanate-terminated prepolymers when crosslinked with water (see above).
  • deactivating compounds for example low-molecular nucleophilic compounds such as water, alcohols, ammonia, primary amines or dilute alkali.
  • the deactivation is usually carried out by washing the surface with the deactivating compound or a solution of the deactivating compound in an inert solvent. Excess biomolecules that are not coupled to the surface are also removed.
  • the biomolecules are functionalized with suitable reactive groups R3, which react with the reactive groups R of the surface to form bonds.
  • the groups R3 are then usually electrophilic functional groups which form a bond with the nucleophilic groups R on the surface.
  • Suitable groups R3 are selected, for example, from active ester groups, N-hydroxysuccinimide ester groups, isocyanate groups, isothiocyanate groups, (meth) acrylic groups and vinylsulfone groups. This procedure is particularly useful when the surface provided has a large number of nucleophilic groups R, for example SH, OH and NH 2 groups.
  • Such surfaces are obtained in particular when the star-shaped prepolymers have such a group or when the reactive groups R of the star-shaped prepolymers are converted into such a group during crosslinking, for example in the case of iso (thio) cyanate-terminated prepolymers when crosslinked with water ,
  • the biomolecules can be functionalized in a manner known per se.
  • the biomolecule binds to an activator compound covalently bound on the surface.
  • This type of connection can be a covalent, ionic, coordinative or affinitive bond, for example in the sense of a protein ligand or a protein-protein bond.
  • the activator compound in addition to the ⁇ ) group (s) R 'necessary for binding to the surface, has at least one functional group or at least one binding group Center that has a connection of the functional group or the binding center of the biomolecule to be immobilized.
  • it is a binding center that can form a coordinative bond to the biomolecule, which is mediated via a transition metal atom.
  • these are transition metal atoms of the third period and especially those that can form divalent ions, such as in copper (II), nickel (II), zinc (II), cobalt (II) and iron (II).
  • Such activator compounds preferably bind the metal ion in a chelating manner, ie the activator compounds have at least 2, preferably at least 3, e.g. B. 3, 4 or 5 functional groups that can form a coordinative bond to the above metal ions.
  • the spatial arrangement of the functional groups is preferably selected so that the connection in the form of a chelate is carried out over several and in particular all functional groups of the activator compound.
  • suitable activator compounds which are able to coordinate bind metal atoms and which simultaneously have a reactive group R 'include, for example, aromatic orthohydroxy aldehydes such as salicylaldehyde, functionalized pyridine and quinoline compounds such as 8-hydroxyquinoline, dipicolylamine, N-2-pyridylmethylaminoacetate , furthermore polycarboxylic acids with preferably at least 2, for example 2, 3, 4, 5 or 6, carboxyl groups, in particular compounds which contain at least 2, for. B.
  • the activator compounds which can form complexes with transition metal ions, can be applied to the surface in a complex form or, preferably, in an uncomplicated form. If uncomplexed compounds V are used, a complexation is then carried out by rinsing with a solution containing metal ions. If appropriate, after the application of the compound V or after the complexation of the compound V bound to the surface, a washing step will be introduced in order to to remove excess compound V and / or excess metal ions.
  • carboxylic acids as activator compounds, they can also be used in carboxyl-protected form, ie. H. use as ester.
  • Suitable protective groups for carboxyl groups are known to the person skilled in the art from the relevant literature, preference being given to those groups which can be split off under conditions which do not impair or even destroy the hydrogel layer.
  • An example of this is the tert-butyl group, which is thermally, for. B. can be removed by heating to 150-220 ° C.
  • the solutions used for complexation are usually aqueous solutions of water-soluble salts of the metal ions, e.g. Solutions of chlorides, nitrates, sulfates, acetates etc.
  • the concentration of the metal ions in the solution is generally in the range from 1 mM to 1 M.
  • the pH of the solution is in the Rule 5 to 12, in particular 6 to 11.
  • the solutions may optionally contain weak complexing agents, for example Ammonia to ensure the solubility of the metal ions at higher pH values.
  • the activator compounds have, in addition to the reactive group R ′ required for binding to the surface, a group which can bind to the biomolecule via at least 2 and preferably via at least 3 hydrogen bonds.
  • groups are, for example, oligonucleotide groups with at least 2, preferably at least 3, e.g. B. 3 to 100 and in particular 3 to 20 nucleotide bases. Biomolecules which have an oligonucleotide sequence complementary thereto can bind to these groups, the proportion of complementary bases being at least 50%.
  • the activator compounds used are substances which are known to have an affinitive interaction with biomolecules.
  • suitable activator compounds of this type are ligands, for example haptens and antibodies, which are functionalized, that is to say have a group R 'such that they can react with the surface to form a covalent bond.
  • This also includes chemical compounds which, in addition to at least one reactive group R ', have a further molecular group or amino acid sequence which can bind to the biomolecule in the sense of an affinitive interaction, for example a protein ligand or protein-protein interaction.
  • biotin derivatives which are modified with a group R 'and thus an anchor for Form biomolecules that have a streptavidin or avidin sequence, as well as streptavidin and avidin derivatives that are modified with a group R ', so that they are an anchor for biotinylated biomolecules.
  • the surfaces provided in step i have a large number of reactive, typically electrophilic groups R, which are qualified for binding biomolecules, without activation being necessary.
  • reactive groups R typically electrophilic groups
  • examples of this are, in particular, isocyanate groups which can be crosslinked in a simple manner by the action of water, and on the other hand have a high reactivity towards nucleophilic groups, for example SH, OH and NH 2 groups.
  • the surface layer obtained in this way then still has sufficient amounts of isocyanate groups as groups R which permit coupling of those biomolecules which have one or more nucleophilic groups R ', for example SH, OH and NH 2 groups.
  • Partial crosslinking is achieved in a particularly simple manner by applying the star-shaped prepolymer containing isocyanate groups to the support in an aqueous solvent as described above, at least partially removing the solvent while maintaining a coating, and immediately afterwards, but at the latest after 2 h, preferably not later than 1 h, the biomolecules are applied to the surface thus obtained in spatially separated areas on the surface thus generated.
  • excess isocyanate groups are then deactivated by treatment with water or aqueous solutions.
  • the biomolecules have at least one reactive group R 'which is qualified for coupling to the reactive groups R of the surface.
  • This group can be present in the actual biomolecule or can be connected to the biomolecule via a spacer, ie the biomolecule has been functionalized in a manner known per se, so that it has one or more reactive groups R 'which are separated from the actual biomolecule via a molecular group ( Spacer) is spatially separated.
  • Suitable spacers are, for example, homobifunctional compounds, such as diamines, diacids, e.g. Dicarboxylic acids, or ethylene glycol oligomers, or heterobifunctional compounds, such as amino acids, e.g. ⁇ -alanine, glycine, 6-aminocapric acid or v-aminobutyric acid, arylacetylenes, biotins, avidines, strepavidines, oligosaccharides, e.g. from riboses or deoxyriboses, nucleic acids, especially oligo- (d) A or - (d) T, fatty acids, phosphoric acid esters and derivatives and / or combinations thereof.
  • homobifunctional compounds such as diamines, diacids, e.g. Dicarboxylic acids, or ethylene glycol oligomers
  • heterobifunctional compounds such as amino acids, e.g. ⁇ -alanine, gly
  • Spacers can also be constructed from several parts (linkers) which are linked to one another by covalent and / or non-covalent bonds.
  • covalently linkable parts of a spacer are heterobifunctional elements, such as amino acids, for example 6-aminocaproic acid or ⁇ -aminobutyric acid, of which several can be connected to one another to extend a spacer.
  • non-covalently linkable parts of a spacer are molecules between which affinity bonds can form, such as between biotin and avidin or streptavidin or their analogs.
  • Spacers can have one or more binding sites for biomolecules (e.g. dendrimers).
  • biomolecules e.g. dendrimers
  • the above-mentioned heterobifunctional elements for example, generally have one, two or three such binding sites, while molecules that form affinity bonds, such as avidin or streptavidin, have multiple binding sites. The latter can lead to an advantageous increase in immobilized biomolecules per unit area.
  • Spacers can also have variable binding parts which offer different binding possibilities depending on their respective configuration. This applies in particular to affinity bonds, the binding affinity of which can vary depending on the configuration of the binding partners involved.
  • Preferred spacers are based on diamines of the formula III
  • n1 corresponds to a value from 1 to 50 and in particular from 5 to 20 and n2 corresponds to a value from 2 to 100 and preferably 5 to 50
  • R 1 is the amino acid naturally occurring for ⁇ -C substituents, in particular represents hydrogen and CrC 4 alkyl, where several R 1 radicals can be the same or different (in the case of n1 ⁇ 1) and the sugar is in particular ribose and deoxyribose, preferably in the D form.
  • n1 corresponds in particular to a value from 2 to 6 and preferably from 3 while n2 corresponds in particular to a value from 2 to 20, preferably 4 to 10, for example 6.
  • the functional group R ' is then arranged at the end of the spacer distal to the biomolecule.
  • spacers to biomolecules
  • Covalently attached spacers in particular via -O-, -NH- or -S- attached spacers, are preferred.
  • Biomolecules can specify certain functionalities for this purpose, but they can also be modified appropriately.
  • proteins can be Bind sulfide or carboxy groups, and nucleic acids via 5'- or 3'-OH groups.
  • proteins can be reductively modified in a manner known per se in order to convert disulfide bridges into free sulfide groups, and nucleic acids can be reacted for example with known and also commercially available reagents in order to convert terminal OH groups into groups having amino groups.
  • nucleic acids are bound to the spacer via their respective 5 'ends. This can advantageously be done using polyethylene glycol spacers, in particular the spacers of formula VII.
  • the biomolecules are applied to the organic surface layer by means of a stamping technique, the so-called ⁇ CP method.
  • ⁇ CP method An overview of such processes can be found in Xia et al., Chem. Ref. 1999, 99, pp. 1823-1848, Appl. Chem. Int. ed. 1998, 37, pp. 550-575.
  • a An overview of the application of the ⁇ CP technique for the application of biomolecules can be found in Kane et al., Biomaterials 1999, 20, pp. 2363-2376.
  • a stamp which has a large number of elevations, is wetted with a solution of the biomolecule that is to be immobilized.
  • the stamp is then pressed onto the organic surface layer.
  • a pattern (array) of immobilized biomolecules is obtained on the organic surface layer.
  • the arrangement and number of places of immobilized biomolecules on the organic surface layer corresponds to the arrangement and number of elevations on the stamp surface.
  • the stamp surface is formed from an elastic material in order to ensure the best possible transfer of the biomolecules to the organic surface layer.
  • Suitable stamps are e.g. B. from the publications of Xia et al. (see above), Bernard et al., Adv. Mater. 2000, 12, 1067-1070, and Kane et al. (see above).
  • the number and arrangement of the elevations depends on the geometry of the array.
  • the elevations are columnar with a circular, ellipsoidal, rectangular or strip-shaped end face. Circular or ellipsoidal geometries are preferred.
  • the individual elevations have an average diameter in the range from 0.1 ⁇ m to 100 ⁇ m.
  • the diameter here refers to twice the average distance between the boundary line of the end face and its center point. In the case of a rectangular arrangement, the diameter specified here corresponds approximately to the mean value of the diagonals and heights of the end face.
  • the size of the surveys is of minor importance. As a rule, however, it will not fall below a value of 0.5 ⁇ m and in particular 1 ⁇ m. As a rule, it is in the range from 1 ⁇ m to 500 ⁇ m, with a ratio of the height of the elevation h to the diameter d of h / d ⁇ 5, in particular ⁇ 2, being guaranteed for reasons of stability.
  • the distance between the elevations is generally in the range from 0.1 ⁇ m to 100 ⁇ m, in particular 1 to 100 ⁇ m and especially 5 to 50 ⁇ m, depending on the desired surface pattern of the array. Corresponding dimensions also apply to stamp surfaces with strip or ribbon-shaped elevations.
  • Elastic materials are generally suitable as stamp materials.
  • the stamp materials are materials containing polydimethylsiloxane, as are already used for this purpose in the prior art. Occasionally it has proven advantageous to hydrophilize the stamp surface, for example by treatment with oxygen plasma as described by Donzel et al., in Adv. Mater. 2001, 13, pp. 1164-1167.
  • the concentration of the biomolecules in the solution is in the range from 1 mmol / ml to 1 ⁇ mol / ml.
  • Non-contact methods such as piezo dispensers and pressure pulse dispensers can also be used.
  • the control of temperature and relative humidity to avoid satellite spots has proven to be useful.
  • the grounding of the carrier material is useful for dissipating electrostatic charge.
  • capillary dispensers which apply the liquid with a thin capillary, for example with an inner diameter of 1.5 to 5 ⁇ m
  • pin and ring technology can also be used.
  • the polarity of the medium to be dispensed can be used to ensure an optimal tear-off, e.g. by adding salt or solvents such as ethanol or 1-propanol.
  • the amount of liquid dispensed is important because it determines the area on which the biomolecules are immobilized, in particular the spot size.
  • amounts of liquid in the femto to nanoliter range are applied. For example, spots with a diameter of approximately 150 to 200 ⁇ m are available with a contact-free amount of approximately 10 nl to 2 ⁇ l. Spots with diameters in the range of 10 ⁇ m can be created by applying amounts of approximately 0.01 nl to 0.1 ⁇ l using capillary dispensers.
  • the solvent is generally first evaporated. This can normally take place at room temperature because of the relatively small amounts of liquid, but can also be influenced by the selection of suitable higher or lower temperatures. Accordingly, the coupling reaction essentially does not take place in solution, although a residual solvent content can be advantageous.
  • a washing process is generally carried out. The purpose of this washing process is in particular to remove uncoupled biomolecules. In line with the solution previously used to apply the biomolecules, aqueous solvents or solvent mixtures are also used for washing. In principle, the above statements apply accordingly.
  • washing is carried out with a solvent or solvent mixture which contains a surfactant, preferably a nonionic surfactant.
  • a surfactant preferably a nonionic surfactant.
  • Polyalkoxylated, in particular polyethoxylated, fatty acid esters of polyols, in particular of glycerol or sorbitol, for example the sorbitan fatty acid esters sold under the trade name Tween® are particularly preferred.
  • the use of ethoxylated sorbitan hexylaurate has proven to be expedient.
  • the concentration of surfactant in the washing solution is expediently chosen so that on the one hand an effective removal of uncoupled biomolecules is ensured, and on the other hand the washing solution is compatible with the immobilized biomolecules. Concentrations in the range from 0.01% by weight to 10% by weight and preferably 0.05% by weight to 2% by weight have proven to be expedient.
  • the concentration of the biomolecules in the solution to be applied is variable and depends in particular on the desired density and molecular weight of immobilized biomolecules. Concentrations in the range of 1 nmol / ml to 1 ⁇ mol / ml can be used, in certain cases in the lower concentration range the surface is not saturated with biomolecules, while in the upper concentration range this is the case and excess biomolecules due to saturation not paired, can then be removed. Concentrations above at least 10 nmol / ml and in particular at least 20 nmol / ml have proven to be expedient for the purpose of saturating the surface with a satisfactory homogeneity of the immobilized biomolecules.
  • washing steps can follow.
  • organic solvents in particular lower alcohols, for example isopropanol, which can then subsequently be removed again by rinsing with water.
  • the surface is dried under customary conditions, conveniently at ambient temperature and, if desired, by blowing dry with air or inert gas.
  • arrays are obtained which have a large number of sites with immobilized biomolecules.
  • more than 10, 60, 100, 600, 1000, 5000, 10000, 40000, 100000, 400000 or 1000000 defined places (areas) can be arranged on an array per cm 2 carrier surface.
  • arrays according to the invention have 5-10000 spaces / mm 2 .
  • a special embodiment is arrays with 5 to 100 places / mm 2 and in particular 10 to 50 places / mm 2 (low density).
  • Another special embodiment is arrays with 100-10000 spaces / mm 2 and in particular 500-2500 spaces / mm 2 (high density).
  • the same or different biomolecules can be immobilized in different places.
  • the locations at which biomolecules are immobilized, in particular fields of immobilized biomolecules, can have different geometries.
  • the shape can be arbitrary, for example circular, rectangular, square or elliptical.
  • the area is preferably less than 1 mm 2 , in particular less than 10000 ⁇ m 2 , and very particularly preferably less than 1000 ⁇ m 2 .
  • biomolecules are immobilized as spots, i.e. as essentially circular fields.
  • spots preferably have diameters in a range from 1 ⁇ m to 1000 ⁇ m, in particular 2 ⁇ m to 50 ⁇ m or 50 ⁇ m to 500 ⁇ m.
  • spots with diameters below 200 ⁇ m and especially below 50 ⁇ m or 20 ⁇ m can advantageously be realized with the ⁇ CP technology described above.
  • the biomolecules are nucleic acids, in particular oligonucleotides with a length of 2 to 500 bases.
  • the immobilized oligonucleotides are able to bind to a target nucleic acid. In this sense, the immobilized oligonucleotides are also referred to as probes.
  • the probes are usually single-stranded oligomers. DNA, RNA and nucleic acid analogs and derivatives, such as, optionally modified, PNA, LNA and PSNA as well as modified DNA or RNA can be used.
  • the minimum length of the probes depends on the complexity of the sample, in particular the number of bases of nucleic acids to be detected, but also on the type of nucleic acid used as a probe and the thermodynamic stability that can be achieved between sample and probe.
  • probes usually have a length of 8 to 60, preferably 13 to 25 and in particular 13 bases for DNA, 8 to 60, preferably 13 to 25 and in particular 13 bases for DNA-LNA hybrids, 8 to 60, preferably from 13 to 25 and in particular from 13 bases for PSNA, from 6 to 30, preferably from 8 to 18 and in particular from 9 bases for LNA, and from 6 to 18, preferably from 8 to 18 and in particular from 9 bases for PNA on.
  • nucleic acids to be detected e.g. Amplificates, in particular PCR amplificates, viruses, plasmids and microorganisms, in particular for applications such as the detection of mutations, SNPs or methylations (epigenetics)
  • Amplificates in particular PCR amplificates, viruses, plasmids and microorganisms, in particular for applications such as the detection of mutations, SNPs or methylations (epigenetics)
  • PCR amplificates in particular PCR amplificates
  • viruses e.g. PCR amplificates
  • viruses e.g. PCR amplificates
  • viruses e.g. PCR amplificates
  • viruses e.g. PCR amplificates
  • viruses e.g. PCR amplificates
  • viruses e.g. PCR amplificates
  • viruses e.g. PCR amplificates
  • viruses e.g., viruses, plasmids and microorgan
  • RNA unamplified RNA
  • Longer probes for example in the range of 16-60 mer and in particular 25-50 mer for synthetic probes, are expedient for cDNA of total amplified RNA, unamplified total genomic DNA and amplified total genomic DNA, in particular for applications such as gene expression determinations or the detection of genomic amplifications or deletions or the probes consist of PCR products, cDNA, plasmids or DNA from lysed bacteria, which can also have a number of bases beyond this.
  • a test probe generally has a base sequence which is complementary to a specific target sequence of a nucleic acid to be detected or, according to another aspect, can hybridize specifically with the target sequence.
  • a control probe can in particular have the function of a normalization, mismatch, housekeeping, sample preparation, hybridization or amplification control and accordingly have a base sequence which is complementary to a base sequence of a reference nucleic acid, a nucleic acid to be detected with a similar base sequence, a constitutively expressed nucleic acid, for example a nucleic acid derived from the ß 2 microglobulin, ß-actin, GAPDH, PBGD, ubiquitin, tubulin or transferrin receptor gene, a species-specific nucleic acid or an amplificate.
  • the immobilized biomolecules are provided with a marking in accordance with the above particular embodiment, a quality control of the array can now follow.
  • the array can be measured with a detection system corresponding to the marking.
  • the coupling efficiency and thus the immobilization of the biomolecules can be assessed in terms of area, homogeneity and density via the measured amount of immobilized marking.
  • the present invention also relates to the use of arrays according to the invention for analytical and in particular diagnostic purposes.
  • arrays according to the invention are suitable both for non-competitive and for competitive analytical methods (assays).
  • the substance to be analyzed interacts with the biomolecules immobilized on the surface.
  • the analyte is marked beforehand, but marking is also possible after the analyte has already interacted with the immobilized biomolecules, for example by means of primer extension or rolling-cycle PCR. In these cases, a measurement signal is obtained which is greater the more analyte is present.
  • the interaction of the sample with the substances immobilized on the surface changes the fluorescence of the immobilized substances (weakening, amplification, for example molecular beacons) or changes the activity of an enzyme and this change is registered as a measurement signal .
  • non-competitive assays are hybridization reactions of PCR products or labeled DNA / RNA on immobilized on the surface.
  • a labeled substance (marker) is added to the sample which has similar binding properties to the biomolecules immobilized on the surface as the analyte itself. There is a competitive reaction between analyte and marker for the limited number of binding sites on the surface. A signal is obtained which is lower the more analyte is present.
  • competitive assays are immunoassays (ELISA) and receptor assays).
  • Figure 1 Fluorescence microscope image of a fluorescence-labeled nucleotide array with spots at a distance of 20 ⁇ m and a spot diameter of 5 ⁇ m.
  • Figure 2a Fluorescence microscope image of an array with stripe-shaped areas of an immobilized biotin-streptavidin system with fluorescent marker, the stripes being 5 ⁇ m wide and 10 ⁇ m apart between the stripes.
  • Figure 2b Intensity distribution of the fluorescence of the array from Figure 2a along a line perpendicular to the strips.
  • Figure 30 Fluorescence microscope image of an array with punctiform areas of an immobilized green fluorescent protein
  • IPDI isophorone diisocyanate
  • the prepolymer precursors used are commercially available 6-armed polyalkylene ethers (hereinafter referred to as polyols) which have been prepared by anionic ring-opening polymerization from ethylene oxide and / or propylene oxide using sorbitol as initiator.
  • the polyol used was dried to a residual water content of less than 350 ppm before use. Remains of the for Production of the polyols used alkali metal hydroxide was bound by neutralization with phosphoric acid.
  • the polyol was slowly added via a pump (approx. 80 ml / h), so that the reaction temperature did not deviate by more than 10 K from the specified temperature.
  • the elution diagrams were adapted for evaluation on two Gaussian curves, the proportion of bi- / trimer being determined via the area ratios.
  • the unreacted OH groups were determined by reacting the prepolymers with acetic anhydride in pyridine and titrating excess acid (hydrolysis of the unreacted acetic anhydride) with NaOH.
  • the star-shaped polyether polyol used is a 6-arm statistical poly (ethylene / propylene oxide) with an EO / PO ratio of 80/20 with a molecular weight of 3100 g / mol.
  • phosphoric acid 0.05% by weight was added to the polyol and heated to 80 ° C. in vacuo with stirring.
  • the isophorone diisocyanate (IPDI) used was distilled in vacuo before use (95 ° C / 0.01 mbar) 125 ml (0.59 mol) were placed in a reactor and heated to 50 ° C. in an inert gas atmosphere. The dried and degassed polyol (20 g, 6.45 mmol) was then added slowly (approx.
  • the substrate surfaces were first pretreated.
  • the substrates pre-cleaned by water and acetone were treated in a plasma system of the type TePla 100-E from the company Plasma Systems in oxygen plasma for 2 minutes (pressure: 1 mbar, 50W) and then washed with water and acetone.
  • the pre-cleaned substrates can also be treated with oxygen under a 40 w UV lamp with a wavelength of 185 nm for 10 min (distance between substrate surface and Light source 2 mm), and then washed with water and acetone.
  • the substrates can also be stored for 1 hour at 60 ° C. in a mixture of concentrated aqueous ammonia, hydrogen peroxide (25% by weight) and water in a volume ratio of 1: 1: 5 without pre-cleaning. Then they were rinsed several times with water. The substrates treated in this way were then stored in deionized water.
  • the pretreated substrates were coated with an aminosilian monolayer.
  • the sample taken from the water and blown dry with nitrogen was transferred to a glove box.
  • the substrates were stored in a 0.5% (v / v) solution of N- (3- (trimethoxysilyl) propyl) ethylenediamine or (3-aminopropyl) trimethoxylsilane in dry toluene for 16 h, then thoroughly with toluene washed and dried in a glove box using a filtered nitrogen stream before use in a nitrogen atmosphere.
  • G1 Solution of 3'-amino-terminated-5'-fluorescence-labeled oligonucleotide (from Molecular Probes) in PBS buffer with a concentration of 100 pmol / ⁇ l.
  • G2 Solution of 3'-amino-terminated oligonucleotide (from Molecular Probes) in PBS buffer with a concentration of 100 pmol / ⁇ l.
  • G3 Solution of 5'-fluorescence-labeled oligonucleotide (opposite strand to the oligonucleotide used in G2; from Molecular Probes) in PBS buffer with a concentration of 100 pmol / ⁇ l.
  • Example 2 a) The prepolymer from Preparation Example 1 was dissolved in a concentration of 10 mg / ml in dry tetrahydrofuran. Then an amino-functionalized silicon carrier, produced according to II, was coated by means of spin coating. For this purpose, the non-rotating, dried substrate was first completely wetted with the prepolymer solution before the solution was spun off at a final speed of 4000 rpm for 40 seconds. The coating was then dry. b) 2 spots of 1 ⁇ l of solution G1 and 2 spots of 1 ⁇ l of solution G2 were applied to the coating produced in this way using a micropipette. The mean diameter of the Spots was about 200 ⁇ m.
  • the array thus obtained was then kept overnight in an atmosphere saturated with water vapor.
  • the coating was then washed with PBS (pH 7.5) and stored in a mixture of 10 ⁇ l G3, 10 ⁇ l Tween20 with 10 ml double PBS at 4 ° C. overnight, and then washed with double PBS.
  • the substrates were then measured with a fluorescence scanner. The intensity of the fluorescence of the respective spots G1 and G2 was determined. The G2 spots before hybridization served as a reference. The results are shown in Table 2.
  • Example 3 Prepolymer from Preparation Example 1 with a concentration of 10 mg / ml in tetrahydrofuran / water 1: 1 (v / v).
  • Example 4 Prepolymer from Preparation Example 1 with a concentration of 1 mg / ml in dry tetrahydrofuran.
  • Example 5 Prepolymer from Preparation Example 1 with a concentration of 1 mg / ml in tetrahydrofuran / water 1: 1 (v / v).
  • Example 6 a) The prepolymer from Preparation Example 2 was dissolved in a concentration of 10 mg / ml in dry tetrahydrofuran. Then an amino-functionalized silicon carrier 4b
  • the non-rotating, dried substrate was first completely wetted with the prepolymer solution before the solution was spun off at a final speed of 4000 rpm for 40 seconds.
  • Example 7 Array with stripe-shaped regions of a biotin-streptavidin system
  • Example 8 Production of a protein array via complex formation
  • the support thus obtained was then kept overnight in an atmosphere saturated with water vapor and washed with toluene.
  • the carrier obtained was 10 min. stored at 200 C C under vacuum (0.02 mbar) and after cooling for 5 min. immersed in a solution of NiCI 2 in water (5 mg / ml) and washed with water.
  • the carrier treated in this way was then dissolved in a solution of a His-tagged green fluorescent protein in PBS buffer (0.03 mg / ml) for 10 min. immersed and then washed with PBS buffer.
  • the array obtained was examined by fluorescence microscopy. The pattern shown in Figure 3 was obtained.

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Abstract

L'invention concerne des réseaux de biomolécules immobilisées qui sont couplées à une couche superficielle organique constituée essentiellement de prépolymères en forme d'étoiles réticulés les uns avec les autres, présentant en moyenne au moins quatre bras de polymères A qui, en soi, sont hydrosolubles. L'invention concerne également des dispositifs qui présentent de tels réseaux. Elle concerne en outre un procédé de production de tels réseaux et l'utilisation de tels réseaux et dispositifs à des fins de diagnostic et/ou d'analyse.
EP04740781A 2003-07-18 2004-07-08 Reseaux de biomolecules immobilisees sur des surfaces formant des hydrogels, leur production et leur utilisation Withdrawn EP1646672A1 (fr)

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DE2003132849 DE10332849A1 (de) 2003-07-18 2003-07-18 Arrays immobilisierter Biomoleküle auf Hydrogel-bildenden Oberflächen, deren Herstellung und deren Verwendung
PCT/EP2004/007474 WO2005014695A1 (fr) 2003-07-18 2004-07-08 Reseaux de biomolecules immobilisees sur des surfaces formant des hydrogels, leur production et leur utilisation

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US5275838A (en) * 1990-02-28 1994-01-04 Massachusetts Institute Of Technology Immobilized polyethylene oxide star molecules for bioapplications
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US6509098B1 (en) * 1995-11-17 2003-01-21 Massachusetts Institute Of Technology Poly(ethylene oxide) coated surfaces
US6174683B1 (en) * 1999-04-26 2001-01-16 Biocept, Inc. Method of making biochips and the biochips resulting therefrom
US6664061B2 (en) * 1999-06-25 2003-12-16 Amersham Biosciences Ab Use and evaluation of a [2+2] photoaddition in immobilization of oligonucleotides on a three-dimensional hydrogel matrix
DE60137523D1 (de) * 2000-10-26 2009-03-12 Biocept Inc Dreidimensionales format aufweisender biochip
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