EP1230213A1 - Article a surface activee pour l'immobilisation de macromolecules, et procede de fabrication d'un tel article - Google Patents

Article a surface activee pour l'immobilisation de macromolecules, et procede de fabrication d'un tel article

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Publication number
EP1230213A1
EP1230213A1 EP01925501A EP01925501A EP1230213A1 EP 1230213 A1 EP1230213 A1 EP 1230213A1 EP 01925501 A EP01925501 A EP 01925501A EP 01925501 A EP01925501 A EP 01925501A EP 1230213 A1 EP1230213 A1 EP 1230213A1
Authority
EP
European Patent Office
Prior art keywords
dendrimeric
dendrimers
article
macromolecules
basic units
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Withdrawn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
EP01925501A
Other languages
German (de)
English (en)
Inventor
Rüdiger BENTERS
Christof Niemeyer
Dieter WÖHRLE
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.)
Chimera Biotec GmbH
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Chimera Biotec GmbH
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Filing date
Publication date
Application filed by Chimera Biotec GmbH filed Critical Chimera Biotec GmbH
Publication of EP1230213A1 publication Critical patent/EP1230213A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C335/00—Thioureas, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C335/04—Derivatives of thiourea
    • C07C335/16—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C335/20—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B01J2219/00529—DNA chips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/0061—The surface being organic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00614—Delimitation of the attachment areas
    • B01J2219/00617—Delimitation of the attachment areas by chemical means
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00623—Immobilisation or binding
    • B01J2219/00626—Covalent
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00632—Introduction of reactive groups to the surface
    • B01J2219/00637—Introduction of reactive groups to the surface by coating it with another layer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00639—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium
    • B01J2219/00641—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium the porous medium being continuous, e.g. porous oxide substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00639—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium
    • B01J2219/00644—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium the porous medium being present in discrete locations, e.g. gel pads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
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    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00702—Processes involving means for analysing and characterising the products
    • B01J2219/00707—Processes involving means for analysing and characterising the products separated from the reactor apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11—Compounds covalently bound to a solid support
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof

Definitions

  • the present invention relates to articles, in particular sensors, with an activated surface for immobilizing, in particular, bioorganic macromolecules, and to processes for their production.
  • the methods according to the invention include, in particular, methods for generating activated sensor surfaces for the highly efficient covalent immobilization, in particular of bioorganic macromolecules.
  • silicon dioxide surfaces can be activated by coating with an N-alkylamino-silane for the coupling of bioorganic macromolecules (Chrisey, L; O ' Ferrall, CE; Spargo, BJ; Dulcey, CS; Calvert, JM Nucleic Acids Research 24/15 3040-3047 (1996)).
  • the macromolecule to be immobilized contains thiol groups, direct coupling through chemisorption to the gold surface is possible, cf. DE 19807339 A1.
  • Glass surfaces can be functionalized by using suitable silanes. Glass surfaces activated with epoxyalkylsilane are frequently used to immobilize hydroxyl- or amino-functionalized biomolecules (see US Pat. No. 5,919,626), while thiolated macromolecules can be coupled to thiolsilane-activated surfaces via disulfide bridges (see US Pat. No. 5,837,860). Another possibility is to use surfaces coated with avidin / streptavidin, which have a high affinity for biotinylated substances (cf. DE 3640412 A1; DE 19724787 A1).
  • Beier et al. describes a method with which it is possible to increase the number of potential binding sites for macromolecules on the sensor surface through the in-situ construction of branched linker systems (see Beier, M .; Hoheisel, JD Vol27 / No9, 1970-1977 (1999 )).
  • silation, RFPE radio frequency plasma discharge in ammonia, etc.
  • a first (partial) object of the present invention was to provide an article, in particular a sensor, with an activated surface that has a high density reactive coupling groups and also has a high physical-chemical stability to thermal and chemical regeneration steps.
  • a third (partial) object of the present invention was to provide a corresponding method for producing an article (in particular a sensor) with a bioorganic macromolecule immobilized on the surface.
  • the first (partial) object is achieved by an article with an activated surface for immobilizing, in particular, bioorganic macromolecules, comprising an substrate with a surface, a dendrimeric framework linked to the substrate surface and a number of first linked to the dendrimeric framework
  • Coupling groups e.g. NHS or isothiocyanate groups
  • the article can in particular be a (bio) sensor or sensor element, such as a DNA microarray, a protein array of antibodies, receptors and / or enzymes, a test array of peptides, peptoids, or low-molecular compounds such as Act pharmacophores or other active ingredients.
  • a (bio) sensor or sensor element such as a DNA microarray, a protein array of antibodies, receptors and / or enzymes, a test array of peptides, peptoids, or low-molecular compounds such as Act pharmacophores or other active ingredients.
  • the dendrimeric scaffold can comprise a large number of identical or different first coupling groups (e.g. NHS esters, isothiocyanate groups, nucleic acid strands or the like).
  • first coupling groups e.g. NHS esters, isothiocyanate groups, nucleic acid strands or the like.
  • sensors should often be able to detect a wide variety of analytes, and it then makes sense to offer these different analytes different first coupling groups.
  • the substrate surface is linked to the dendrimeric scaffold via a linking unit, which (in the manufacture of the article, see below) by reaction of an initiator group assigned to the substrate surface with a complementary functional group assigned to the dendrimeric scaffold.
  • a linking unit which (in the manufacture of the article, see below) by reaction of an initiator group assigned to the substrate surface with a complementary functional group assigned to the dendrimeric scaffold.
  • the linking unit is an amide.
  • the dendrimeric skeleton can in particular comprise the following dendrimeric basic units, which can be crosslinked in the dendrimeric skeleton: starburst dendrimers and their in particular chemically or biochemically modified derivatives, metallodendrimers, carbosilane dendrimers, polysilane dendrimers, glycosyl-containing saccharide chargers and dendrimers - Dendrimers and their derivatives, peptide and oligopeptide dendrimers and their derivatives as well as nucleotide and oligonucleotide dendrimers and their derivatives.
  • the dendrimeric framework advantageously carries first coupling groups for immobilizing, in particular, bioorganic macromolecules or other substances which (during the manufacture of the article, see below) can be formed by reacting a functional group assigned to the dendrimeric framework with a homo- or heterobifunctional linker substance (linker molecule) , If the functional group assigned to the dendrimeric skeleton is, for example, an amino function and the linker substance is the homobifunctional substance phenylene-1,4-diisothiocyanate, the dendrimeric skeleton carries an isothiocyanate group as the first coupling group. Further examples of linker substances are given below in connection with the explanation of the production process according to the invention.
  • the dendrimeric framework advantageously comprises dendrimeric basic units linked to the substrate surface, which are cross-linked to one another, for example with the aid of bifunctional linker substances (linker molecules).
  • the second (partial) object is achieved by a method for producing an article with an activated surface for immobilizing, in particular, bioorganic macromolecules, with the following steps:
  • dendrimeric basic units in particular the basic units of a dendrimeric framework
  • the article produced is then usually an article as described in more detail above.
  • the substrate surface is usually first modified with a reactive initiator group, ie. the initiator group is connected to an (unmodified) substrate surface, before the (modified) substrate surface is then linked (and thus further modified) to the dendrimeric framework in a subsequent step.
  • the substrate surface can be modified using standard methods, cf. Fig. 6.
  • the reactive, surface-bound initiator group can be selected from one of the following chemically reactive groups: hydroxyl, amino, carboxyl, acyl halide, ester, aldehyde, epoxy or thiol group. It can also be selected from one of the following biologically or chemically reactive groups: disulfides, metal chelates, nucleotide and oligonucleotides, peptides or haptens, such as, for example, biotin, digoxigenin, dinitrophenyl groups or similar groups.
  • the (unmodified) substrate surface to which the reactive initiator group is coupled will preferably be selected from the following group: consists of: carrier materials based on metals, semimetals, semiconductor materials, metal and non-metal oxides; glasses; plastics; organic and inorganic polymers; organic and inorganic films and gels, in particular as a coating for one of the aforementioned materials.
  • the dendrimeric framework which is usually linked to it via the reactive initiator group of a modified substrate surface, can comprise several identical or different functional groups per basic dendrimer unit. These are preferably aminoalkyl, hydroxyalkyl or carboxyalkyl groups which are bonded in the periphery of the dendrimeric base unit and can be converted into the first coupling group with the aid of the linker substance.
  • the dendrimeric basic units can preferably be selected from the following group, which consists of: starburst dendrimers and their chemically and biochemically modified derivatives, metallodendrimers, glycosyl-containing dendrimers, saccharide and oligosaccharide dendrimers and their derivatives, peptide and oligopeptide dendrimers and their derivatives and nucleotide and oligonucleotide dendrimers and their derivatives.
  • the dendrimers to which the basic dendrimeric units correspond in the finished article according to the invention are generally synthesized in a conventional manner before carrying out the process according to the invention (Zeng, F .; Zimmerman, SC; Chem. Rev. 97, 1681-1712 (1997)) and provided for performing the method according to the invention.
  • the dendrimeric basic units ie the educt dendrimers or the dendrimeric skeleton formed therefrom
  • the dendrimeric basic units are equipped with a number of coupling groups for immobilization, in particular bioorganic macromolecules, by reacting functional groups assigned to the dendrimeric basic unit with a homo- or heterobifunctional linker substance (linker molecule).
  • the dendrimeric base units can be equipped with the first coupling groups before or after the substrate surface is linked to the dendrimeric base units.
  • homobifunctional linker substances that can be used are: photochemically, chemically, biochemically or biologically active compounds such as, for example, dicarboxylic acids and their anhydrides, disuccinimidyl glutarate,
  • heterobifunctional linker substances can be used in particular: photochemically, chemically, biochemically or biologically active compounds such as, for example, 3 - [(2-aminoethyl) dithio] propionic acid, ⁇ / - ⁇ -
  • a process design (and accordingly the resulting articles) is particularly preferred in which the surface-bound dendrimeric base units are crosslinked with the aid of homobifunctional linkers, with the exception of carboxylic acid anhydrides, so that a polymeric thin film is formed Basic dendrimeric units are created.
  • the linker substances are preferably but not necessarily the same ones that are used to generate the coupling groups; reference is made to the above explanations regarding preferred linker substances.
  • a process design (and accordingly the resulting articles) is particularly preferred in which the first coupling group is generated with the aid of heterobifunctional linkers or with carboxylic acid anhydrides.
  • each functional group of the dendrimeric base unit is converted into an active coupling group; reference is made to the above explanations regarding preferred linker substances.
  • dendrimers dendrimeric basic units
  • linker substances in particular with a view to the substances to be immobilized and with a view to good crosslinking of the dendrimeric basic units.
  • a macromolecular surface is built up from the surface-fixed dendrimers by covalent crosslinking.
  • the surfaces generated in this way have two decisive advantages compared to common linear linker systems. Due to the (covalent) cross-linking of the immobilized dendrimers, the surfaces have an increased physicochemical stability and thus enable the loss-free regenerability of the surfaces loaded with bioorganic macromolecules (for example sensor surfaces). Furthermore, the immobilization efficiency is drastically increased by using polyfunctionalized dendrimers.
  • a process design is particularly preferred in which the construction of the dendrimeric framework and thus an essential step in the production of articles according to the invention, for example the construction of aminodendrimer Sensor surfaces, via a sandwich preparation.
  • two substrates activated in each case for example by aminosilylation and subsequent activation using, for example, disuccinimidyl glutarate, for example two glass slides with an area of approximately 3 ⁇ 8 cm (any other sizes can also be used), are put together in pairs that the immobilization of the dendrimer can take place in the space between the two substrates (for example glass substrates).
  • the sandwich preparation can also be used to produce the first coupling group by applying a saturated solution of the linker substance, for example glutaric anhydride or 1,4-phenylenediisothiocyanate, to the substrates coated with dendrimeric units
  • a particularly advantageous embodiment of the method according to the invention consists in initially modifying large planar substrates, the area of which corresponds, for example, to a multiple of a desired chip surface, using the steps described above with dendrimer layers. Typically, this is done in such a way that a grid of the chip formats to be produced is first applied to known glass, for example 100 cm ⁇ 100 cm large glass panes (or corresponding panes of other substrates; significantly larger panes can also be used) by breaking edges are generated on which the glass pane (or the other substrate) can later be divided into the final chip format.
  • the chemical modification is then usually carried out by carrying out the steps described above (and in the examples below), for example cleaning, silylation and activation steps, for example in immersion baths.
  • the dendrimer is advantageously applied using the sandwich technique mentioned above, and the final activation of the dendrimers by means of homo- or heterobifunctional linkers is preferably carried out in sandwich or immersion treatment.
  • the large-area substrate wafers are then separated into the final format.
  • the large-area dendrimer-coated disks can first be cut up and only then activated as required using bifunctional linkers.
  • a cross-linked dendrimeric framework with free coupling groups as a macromolecular (intermediate) layer on a substrate surface (see FIG. 1), whereby (a) the number of potential binding sites for the immobilization of bioorganic macromolecules or other substances, (b) the physicochemical stability of the surface is improved and (c) the regenerability of the substrate surfaces (carrier) modified with the bioorganic macromolecules is improved.
  • the present invention also relates to articles which are obtainable by the process according to the invention (including the special process designs specified above).
  • the articles according to the invention comprise functionalized solid phases and can be used, for example, as sensor elements, reactor elements and elements with electrical, electronic or optical functions.
  • the articles functionalized with bioorganic molecules can also be used advantageously as solid phases in enzyme reactors, in which a high level of physical and chemical resistance and high occupancy densities are required.
  • resistant surfaces can also be used advantageously to build up electronic and optically active elements, for example for high-resolution displays.
  • the third (sub) task is finally solved by a method for producing an article with a (macro or other) molecule immobilized on the surface, with the following step:
  • bioorganic macromolecules In addition to bioorganic macromolecules, other substances such as macromolecular colloids and nanoparticles or low-molecular compounds such as pharmacological substances, hormones, antigens or other active substances can also be immobilized on appropriately prepared articles (a or b) according to the invention.
  • the invention also relates to articles comprising a (macro or other) molecule linked to the dendrimeric backbone.
  • the (macro) molecule can be, for example, one selected from the group consisting of: antibodies, in particular the classes IgG, IgM, IgA, enzymes; receptors; Membrane proteins, glycol proteins; carbohydrates; nucleic acids; such as. DNA, RNA, peptide nucleic acid (PNA), pyranosyl ribonucleic acid (pRNA).
  • other substances includes in particular organic-chemical or inorganic-chemical groups with specific or also potentially active pharmacological, catalytic (for example photocatalytically active substances such as porphyrins and their derivatives or catalysts for the stereoselective transformation of organic or inorganic substrates), optical or electrical properties (eg fluorescent, electroluminescent or electrically conductive polymers (polyaniline, polypyrrole) compounds.
  • catalytic for example photocatalytically active substances such as porphyrins and their derivatives or catalysts for the stereoselective transformation of organic or inorganic substrates
  • optical or electrical properties eg fluorescent, electroluminescent or electrically conductive polymers (polyaniline, polypyrrole) compounds.
  • Substance libraries which are accessible, for example, by combinatorial solid-phase synthesis, are immobilized on the article in order to screen the functionality of these bound substances, for example their inhibitory action on biological enzymes or receptors.
  • the basis of the present invention is, among other things, the surprising observation that nucleic acid-functionalized glass surfaces, which were produced by the method according to the invention, not only have a drastically improved detection limit in the detection of complementary nucleic acids, but also show a significantly increased physicochemical stability that the regenerability of the nucleic acid-modified supports could be carried out many times by treatment with alkaline washing solutions without loss of activity on the surface. It was found that the articles produced by the process according to the invention have a significantly improved physicochemical stability and thus the loss-free regenerability of the carriers modified with the bioorganic macromolecules is possible.
  • the chemical nature of the dendrimeric basic units means that the linker system for the connection of the bioorganic macromolecules is highly flexible, which results in additional advantages over linear linker systems in terms of the efficiency of the heterogeneous affinity reaction.
  • a cross-linked dendrimeric framework with free coupling groups as a macromolecular (intermediate) layer on a substrate surface (cf. FIG. 1).
  • the attachment of the macromolecular layer is achieved, for example, by treating a glass surface (for example amino-, epoxy- or carboxyl-modified (as substrate surface) produced by standard processes) first with a dendrimeric macromolecule and then with a homo- or heterobifunctional linker reagent.
  • a glass surface for example amino-, epoxy- or carboxyl-modified (as substrate surface) produced by standard processes
  • a homo- or heterobifunctional linker reagent By connecting the dendrimeric component to the modified glass surface, the number of binding sites for the immobilization of bioorganic macromolecules (eg certain nucleic acids) is increased.
  • the dendrimer units linked to the substrate surface are used for the immobilization of the activated bioorganic macromolecules, ie equipped with free coupling groups and (b) covalently cross-linked the dendrimer units.
  • Bioorganic macromolecules can be immobilized very stably on the surface of an article produced in this way.
  • a glass substrate with a surface based on silicon dioxide was provided.
  • the glass surface was cleaned thoroughly (CH2CI2 - H2O2 / H2SO4 + ultrasound ⁇ Bidest).
  • the glass surface was then silylated (cf. FIG. 1; step 1) with 3-aminopropyltriethoxysilane in ethanol / water (95: 5) using a method described in the literature (Maskos, U .; Southern EM; Nucleic Acids Res ., 20 (7), 1679-1684 (1992) (other silylation processes are also applicable).
  • the aminosilylated glass surface prepared according to 1.1 was carboxy-functionalized with a 10 mM solution of disuccinimidyl glutarate in CH2Cl2 / n-ethyldiisopropylamine (100: 1) for 2 h at RT under argon (cf. Fig. 1, step 2). The surface was then thoroughly washed several times with CH2CI2. The carboxy group introduced in this way could then be used as an initiator group
  • the surface was covered with a 10% solution of an aminodendrimer (Fa. Aldrich Chem. Co, sold under the trade name Starburst (PAMAM), see also Yamakawa, Y et al. J. of Polymer Science: Part A 37 3638-3645 (1999) ) wetted in methanol (see Fig. 1, step 3).
  • PAMAM Starburst
  • This step can be carried out, for example, using the advantageous sandwich method already described.
  • After a reaction time of 30 minutes reaction of the carboxy groups with the amino groups of the dendrimer), the excess of dendrimer was removed by washing and the (glass carrier) surface now provided with dendrimer was dried in a stream of nitrogen.
  • the glass supports equipped with dendrimer in accordance with 1.3 were transferred into a 20 mM solution of a homobifunctional linker molecule, for example phenylene-1,4-diisothiocyanate, in CH2Cl2 / pyridine (100: 1) (cf. FIG. 1, step 4). After a reaction time of 30 minutes, the mixture was washed thoroughly with CH2Cl2.
  • the glass slide with a dendrimer surface could now be used directly for the immobilization of bioorganic macromolecules described in more detail below, or alternatively it could be kept under argon until use.
  • the surface prepared according to 1.4 was wetted with drops of an aqueous solution which contained a bio-organic macromolecule to be immobilized in a typical concentration range of 1 - 100 ⁇ M.
  • the bio-organic component was, for example, 5'-amino modified oligonucleotides, such as those available from a variety of commercial suppliers.
  • the wetted surfaces were incubated in a humidity chamber for several hours.
  • the optimal incubation time was dependent on the type and concentration of the component to be immobilized.
  • the glass carrier element was, for example, transferred to a 6-aminohexanol solution (100 mM in dimethylformamide (DMF)) in order to inactivate still active isothiocyanate groups of a phenylene diisothiocyanate linker (see 1.4 above).
  • the glass carrier surface was then freed of non-covalently bound bio-organic macromolecules with detergent-containing solutions, for example sodium dodecyl sulfate, then rinsed several times in bidistilled water and dried in a stream of N2.
  • the loaded sensors were stored at - 20 ° C until use.
  • Dendrimer-based, isothiocyanate-functionalized surfaces which are loaded, for example, with amino-functionalized oligonucleotides, are of particular interest as sensor surfaces for DNA chip technology, cf. Niemeyer, CM .; Blohm, D. Angew. Chem. 111/19 3039-3043 (1999). See also Fig.2 and the associated explanations in this context.
  • Example 1 The amination of a glass-based carrier was carried out as described in Example 1 under 1.1.
  • a carboxy-terminated dendrimer (trade name Starburst (PAMAM) dendrimer; Aldrich Chem. Co) was first used by known methods, for example as described by Johnsson, B .; Löfas, S .; Lindquist, G. Anal. Biochem. 198 268-277 (1991), esterified in the presence of dicyclohexylcarbodiimide / N-hydroxysuccinimd.
  • the activated dendrimer was placed in DMF on the aminosilylated surface and after a reaction time of 30 minutes the excess dendrimer was washed off with DMF.
  • the surfaces could then be used directly, as described in Example 1, for the immobilization of bioorganic macromolecules.
  • the activated surfaces could be stored under argon.
  • Example 1, 1.1 The glass surfaces are cleaned as described in Example 1, 1.1.
  • hydrolysis-stable silanes such as 3-carboxypropyltrialkoxysilane
  • the silanization is carried out as indicated in Example 1, 1.1.
  • it is a hydrolysis-sensitive silane, such as, for example, 3-glycidoxypryopyltrimethoxysilane, 2- (3,4-epoxycyclohexyl) ethyltriethoxysilane, 3-iodopropyltrimethoxysilane, 3-isothiocyanatotrialkoxysilane and the corresponding trihalosilanes
  • the silanization of the literature is preferably described under dry conditions such as in the literature and dry conditions (Southern, EM et al .; Nucleic Acids Res., 22 (8), 1368-1373 (1994); other methods are also conceivable.
  • Epoxy, isothiocyanato and iodo-terminated silane surfaces are used directly to bind the amino-functionalized dendrimer while carboxy-functionalized silane surfaces are first activated by reaction with DCC / NHS, as described in Example 7, 7.2
  • the further reaction of the dendrimer surface with a homo- or heterobifunctional spacer to produce the first coupling group and the macromolecules are attached to it as follows under Example 1, 1.4 - 1.5, see above as described in Example 7, 7.4.
  • plastic carriers made of, for example, polyethylene, polypropylene, polystyrene, polycarbonate, polyacrylonitrile or their copolymers were produced by known methods, for example by the method of Hartwig, A. et al. Advances in Colloid and Interface Science 52, 65-78 (1994) aminated by radio frequency plasma discharge in an ammonia atmosphere.
  • Gold layers were applied in a conventional manner to various supports and aminated by known methods, for example by producing an amino-terminated SAM on gold, as described by Glodde, M .; Hartwig, A .; Hennemann, O.-D .; Stohrer, W.-D. Intern. J. of Ahesion & Adhesivs 18 359-364 (1998).
  • Metal and semiconductor surfaces were aminated using known methods, for example by silylation with amino, epoxy, carboxy or thiolsilanes (see Chrisey, L .; O'Ferrall, CE; Spargo, BJ; Duicey, CS; Calvert, JM Nucleic Acids Research 24/15 3040-3047 (1996) and Bhatia, SK et al. Anal. Biochem. 178, 408-413 (1989)) or by hydrosilylation with ⁇ -undecenecarboxylic acid (Sieval, AB et al .; Langmuir 14 (7) 1759-1768 (1998).
  • Biorganic macromolecules were immobilized as described in Example 1 under 1.5.
  • the aminosilylated glass surface prepared according to 1.1 was carboxy-functionalized with a saturated solution of glutaric anhydride in DMF for 4 h at RT under argon. The surface was then thoroughly washed several times with DMF and water. The free carboxyl groups were then activated with dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS). For this purpose, the surface was wetted with a solution of 1 M DCC and 1 M NHS in DMF. After a reaction time of 4 hours, the supports were washed thoroughly with DMF and acetone.
  • DCC dicyclohexylcarbodiimide
  • NHS N-hydroxysuccinimide
  • the glass supports equipped with dendrimeric basic units according to 1.3 were transferred into a saturated solution of glutaric anhydride in DMF. After a reaction time of 4 hours, the mixture was washed thoroughly with DMF and water. The free carboxyl groups were then activated with dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS). For this purpose, the surface was wetted with a solution of 1 M DCC and 1 M NHS in DMF. After a reaction time of 4 hours, the supports were washed thoroughly with DMF and acetone.
  • the glass slide with a dendrimer surface could now be used directly for the immobilization of bioorganic macromolecules described in more detail below, or alternatively it could be kept under argon until use.
  • FIGS. 1 and 2 which are explained in more detail below. They represent:
  • Figure 1 Surface modification to produce dendrimer-based, macromolecular sensor surfaces.
  • FIG. 2 attachment of biomolecules to the surfaces according to FIG. 1
  • FIG. 1 shows the surface modification of a glass carrier, plastic carrier or gold-coated carrier (as a substrate), cf. especially Examples 1, 4, 5 and 7.
  • Step 1 shows the amino activation of the surface by silylation, RFPD or ⁇ -aminoalkylthiol-SAM, cf.
  • Example 1 1.1.
  • Step 2 leads to a carboxylated surface by binding a dicarboxylic acid, cf.
  • step 3 a polyfunctionalized amino dendrimer is fixed on this surface.
  • a 4th generation dendrimer with 64 amino groups is typically used for this, cf. Example 1, 1.3.
  • step 4 The final end group activation and crosslinking of the fixed dendrimers can be seen in step 4.
  • the intramolecular crosslinking of amino end groups is not shown, cf. Example 1, 1.4.
  • FIG. 2 shows a dendrimer-based, isothiocyanate-functionalized surface which is loaded with bioorganic macromolecules.
  • the macromolecules are amino-functionalized oligonucleotides.
  • Such surfaces are of particular interest as sensor surfaces for DNA chip technology, cf. Niemeyer, CM .; Blohm, D. Angew. Chem. 111/19 3039-3043 (1999).
  • the oligonucleotides are bound as described in Example 1 under 1.5; regeneration can take place, for example, in an alkaline medium (e.g. under the following conditions: use of 50 mM NaOH, room temperature, 2 x 3 min).
  • Figure 3-1 shows a conventional DNA array carrying 5 ' amino modified capture oligonucleotides which are surface-fixed via a conventional linear linker.
  • the surface was activated as follows: After silylation with 3-aminopropyltriethoxysilane as described in Example 1 under 1.1, 1,4-phenylenediisothiocyanate was bound.
  • the individual spots were applied to the conventional surface with a piezoceramic pipette to form the oligonucleotide array.
  • the concentration of the spotted oligomer solution was 10 ⁇ mol / l in bidest.
  • FIG. 3-2 shows a DNA array according to the invention with a (sensor) surface according to the invention.
  • the spot volume was varied within the respective rows 1-16: upper 7 spots: 2 nl; lower 8 spots: 4 nl.
  • the concentration of the spotted oligomer solution was 10 ⁇ mol / l in bidest.
  • the DNA array shown in FIGS. 4-1 to 4-3 comprises a conventional linear linker, while the array shown in FIGS. 4-4 to 4-8 has a (sensor) surface according to the invention.
  • the surface modification and hybridization of the arrays was carried out as described in Example 7. Quadrupoles with varying volumes of 1.4 - 5.6 nl were scoffed at. Regeneration was carried out in each case 2 ⁇ 3 min with 50 mM NaOH at RT.
  • Fig. 4-1 shows the array with linear linker after the first hybridization
  • Fig. 4- 2 shows the array after the regeneration
  • Fig. 4-3 shows the array after the second hybridization. It can be clearly seen that after the first regeneration step, the signal intensity is greatly reduced. The cause is Detachment of the capture oligonucleotide during the regeneration process due to the inherent instability of the conventional linker.
  • 4-4 to 4-8 show the regeneration properties improved by the method according to the invention. Even after a repeated regeneration process, no decrease in the signal intensity can advantageously be observed.
  • FIG. 5 is a highly schematic illustration of the preferred sandwich preparation technique, see above.
  • FIG. 6 is a highly schematic illustration of a preferred method according to the invention for producing an article with an activated surface for immobilizing macromolecules or other compounds.

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Abstract

L'invention concerne des articles à surface activée pour l'immobilisation de macromolécules bio-organiques, caractérisés en ce qu'ils comprennent un substrat doté d'une surface, une structure dendrimère liée à la surface du substrat, une pluralité de premiers groupes de couplage liés à la structure dendrimère pour l'immobilisation des macromolécules bio-organiques. Les articles sont fabriqués suivant un procédé consistant à préparer un substrat doté d'une surface, à lier la surface de ce substrat avec une structure dendrimère, et à munir la structure dendrimère d'une pluralité de premiers groupes de couplage en vue d'immobiliser les macromolécules bio-organiques.
EP01925501A 2000-03-22 2001-03-22 Article a surface activee pour l'immobilisation de macromolecules, et procede de fabrication d'un tel article Withdrawn EP1230213A1 (fr)

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DE2000113993 DE10013993A1 (de) 2000-03-22 2000-03-22 Verfahren zur Erzeugung von aktivierten Sensoroberflächen zur hocheffizienten kovalenten Immobilisierung bioorganischer Makromoleküle
DE10013993 2000-03-22
PCT/EP2001/003295 WO2001070681A1 (fr) 2000-03-22 2001-03-22 Article a surface activee pour l'immobilisation de macromolecules, et procede de fabrication d'un tel article

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US8623822B2 (en) 2002-03-01 2014-01-07 Bracco Suisse Sa KDR and VEGF/KDR binding peptides and their use in diagnosis and therapy
US7794693B2 (en) 2002-03-01 2010-09-14 Bracco International B.V. Targeting vector-phospholipid conjugates
WO2004065621A1 (fr) 2002-03-01 2004-08-05 Dyax Corp. Peptides de liaison kdr et vegf/kdr et leur utilisation a des fins diagnostiques et therapeutiques
US7261876B2 (en) 2002-03-01 2007-08-28 Bracco International Bv Multivalent constructs for therapeutic and diagnostic applications
DE10311163A1 (de) * 2003-03-12 2004-09-23 Albert-Ludwigs-Universität Freiburg, vertreten durch den Rektor Proteinabweisende Oberflächen auf der Basis dendritischer Polymerfilme
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US5561043A (en) * 1994-01-31 1996-10-01 Trustees Of Boston University Self-assembling multimeric nucleic acid constructs
US6117631A (en) * 1996-10-29 2000-09-12 Polyprobe, Inc. Detection of antigens via oligonucleotide antibody conjugates
EP1102634B1 (fr) * 1998-07-10 2005-02-09 Dupont Canada Inc. Catalyseur sur support a base de dendrimeres et son utilisation en hydroformylation et dans les reactions de couplage c-c

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