EP1495326A1 - Procede pour immobiliser des molecules sur des surfaces - Google Patents

Procede pour immobiliser des molecules sur des surfaces

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Publication number
EP1495326A1
EP1495326A1 EP03722450A EP03722450A EP1495326A1 EP 1495326 A1 EP1495326 A1 EP 1495326A1 EP 03722450 A EP03722450 A EP 03722450A EP 03722450 A EP03722450 A EP 03722450A EP 1495326 A1 EP1495326 A1 EP 1495326A1
Authority
EP
European Patent Office
Prior art keywords
molecules
polymer
polymer layer
biomolecules
immobilization
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Withdrawn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
EP03722450A
Other languages
German (de)
English (en)
Inventor
Holger Klapproth
Ulrich Sieben
Ingo Freund
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.)
TDK Micronas GmbH
Original Assignee
TDK Micronas GmbH
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
Priority claimed from DE10236925A external-priority patent/DE10236925A1/de
Application filed by TDK Micronas GmbH filed Critical TDK Micronas GmbH
Publication of EP1495326A1 publication Critical patent/EP1495326A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3204—Inorganic carriers, supports or 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
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
    • B01J20/103—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate comprising silica
    • 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
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28011—Other properties, e.g. density, crush strength
    • 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
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3257—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one of the heteroatoms nitrogen, oxygen or sulfur together with at least one silicon atom, these atoms not being part of the carrier as such
    • 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
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/327—Polymers obtained by reactions involving only carbon to carbon unsaturated bonds
    • 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
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • 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
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
    • 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
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3276—Copolymers
    • 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
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3289—Coatings involving more than one layer of same or different nature
    • 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
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Definitions

  • the present invention relates to a method for immobilizing compounds, in particular molecules, such as biomolecules, on surfaces or solid supports.
  • Chip packaging techniques in which multiple ICs (integrated circuit) are housed in one package are known in the art.
  • ICs integrated circuit
  • intermediate layers of polymers are often used, which establish the connection of the stacked ICs and at the same time help to mechanically protect the sensitive top side of the lower chip.
  • the production of such layers is part of the series production process in some factories and in particular the thicknesses of such layers can be set precisely in the range of a few ⁇ and even below.
  • EP 1132739 B1 discloses a method which serves to bind molecules to inorganic substrates via cross-linkers during bioconjugation, such cross-linkers being silanes, for example.
  • EP 1132739 B1 also proposes a linker system which can be used for the detection and isolation of biomolecules and as a component of a sensor or biochip or as a diagnostic instrument.
  • an immobilized enzyme can be used several times in biochemical processes.
  • the immobilization of enzymes and other biomolecules is one Key technology in the development of bio-compatible implants.
  • a total of several methods are known for binding molecules - for example biomolecules - to surfaces of supports.
  • polystyrene surfaces such as PolySorp and MaxiSorp are used to bind or conjugate biomolecules to surfaces.
  • a method for attaching biomolecules to surfaces using linker-like groups is described in DE 100 04 884.
  • the method involves contacting the polymer having the linker-like groups with a source of hydroxide ions, whereby biomolecules, e.g. Heparin, can be applied to substrate surfaces.
  • biomolecules e.g. Heparin
  • a disadvantage of the known methods for bioconjugation is that linkers are used to bind the molecules to the carrier. The presence of the linkers adversely reduces the activity of the bound molecules.
  • the known methods are still time-consuming and expensive due to the use of the linkers or the corresponding analogs.
  • the technical problem on which the present invention is based is to provide a method which enables simple and inexpensive conjugation or immobilization of biomolecules on a surface and in which the activity of the bound molecules is largely retained.
  • hydrophobic polymers are, for example, polyimide or polystyrene.
  • the surface to which the polymer layer is applied preferably consists of an inorganic material, such as, for example, a semiconductor material, in particular silicon, a semiconductor oxide, in particular silicon dioxide, glass, nitride or ceramic.
  • Hydrophobic polymers such as polyimide or polystyrene
  • Hydrophobic polymers have the advantage that they can be applied to the surface of an inorganic carrier by means of conventional processes known in semiconductor technology.
  • they electrically isolate the carrier from the molecules applied to the surface of the polymer layer or from substances related to these molecules.
  • Electrical sensors and evaluation circuits can thus be integrated in the carrier, which consists for example of a semiconductor material, without negatively influencing their function by the molecules and substances applied to the surface of the polymer layer.
  • the surface of the carrier can be completely or only partially covered with the organic hydrophobic polymer, parts of the surface being able to be left out with the aid of a mask process customary in the semiconductor industry.
  • electrical connection contacts can later be attached to the carrier, for example a chip. It is also possible to leave out parts of the surface for other reasons and thus leave areas of the possibly inorganic surface uncovered, or vice versa to coat only those specific * areas of the surface with which molecules, for example biomolecules, can later adhere.
  • the polymer layer is brought into contact with organic molecules which can form a connection with it.
  • the contacting takes place in such a way that the molecules are bound site-specifically.
  • Sensor elements are preferably integrated in the carrier below the surface to which the polymer layer is applied, in order to be able to carry out measurements on the molecules immobilized on the surface of the polymer layer. These measurements can be used, for example, to characterize the properties of the biomolecules or chemical reactions that take place in their environment.
  • antibodies can be well bound to surfaces of hydrophobic polymer layers, for example made of a polyimide or a polystyrene, with which semiconductor bodies or semiconductor layers serving as supports can be easily coated, so that classic detection reactions such as ELISA reactions can subsequently be carried out.
  • Molecules in connection with the present invention are in particular peptides, proteins, genes and their fragments, nucleic acids, carbohydrate structures such as sugar, cells and their fragments, cell membrane components and / or hormones.
  • microorganisms, cell extracts, ligands, antigens, antibodies, receptors, lectins, glycopeptides and / or lipids can also be immobilized as a molecule on the surface of the polymer layer.
  • the microorganisms can be living or dead microorganisms, the living microorganisms in the sense of the invention relating to both growing and resting cells.
  • the microorganisms can be pre-immobilized on the carrier by intracellular crosslinking of the cells, pre-immobilization being understood to mean all methods which, before immobilization according to the method according to the invention, can lead to fixation of the molecules or cells.
  • biopolymers are expediently used, such as, for example, polysaccharides or proteins, or else synthetic polymers.
  • Ligands in the sense of the invention are, for example, molecules, such as proteins or ions, which can be grouped around a central structure. Ligands can be monodentate. However, ligands can also be understood to mean molecules which are bound to specific sites of macromolecules, for example substrates or coenzymes to a protein.
  • molecules or biomolecules also include antigens and / or antibodies.
  • antigens are all substances which can trigger an immune response.
  • An antigen according to the invention manure can consist of a high-molecular part, which serves as a substrate for usually several small-molecule groups, which are decisive for the specificity of the immune response and the reaction of the antigens with the corresponding immunoglobulins.
  • the antigens can be polyvalent and monovalent and thus interact with one or more types of antibodies.
  • antibodies particular glycoproteins to be understood that specifically mi * t an antigen interact. The interaction leads to the formation of antigen-antibody complexes.
  • the antibodies can be, for example, different groups of the immunoglobulins.
  • the antibodies can be immobilized as intact antibodies or as different fragments, which can be generated, for example, by cleaving different peptidases.
  • the antibodies can be modified before, during or after immobilization on the support, for example by reduction, oxidation or by oligomerization. It is also possible to use receptors as biomolecules.
  • Receptors are, for example, proteins which interact with an extracellular signaling molecule, for example a ligand, and which activate or initiate certain functions, in particular via secondary messenger substances, through changes in conformation.
  • receptors in the sense of the invention can also be special cells that absorb stimuli and pass on the corresponding information; Examples would be photo, chemo, thermo and baroreceptors.
  • the surface to which the polymer layer is applied is preferably largely planar, ie it is a surface with low roughness, such as surfaces of semiconductor layers or semiconductor bodies with integrated circuits (IC surfaces), but which is lo kale microscopic structures that would be suitable, for example, for the absorption of biomolecules.
  • Immobilization or pre-immobilization in the sense of the invention is to be understood to mean all methods for restricting the mobility and solubility of molecules in chemical, biological and / or physical ways, with pre-immobilization relating to all methods for fixing the molecules which, according to the invention, prior to immobilization procedure.
  • the immobilization and / or pre-immobilization can be carried out by different methods, such as the binding of the molecules to one another or to supports, by being held in the network of a polymer matrix or enclosed by membranes.
  • the immobilization not only makes the molecules reusable, but can also be used the process of interaction with the sample can be easily separated off, and can be used in much higher local concentrations and in continuous flow systems.
  • the binding or immobilization of the molecules to the support can be carried out by direct support connection and by crosslinking
  • carrier binding takes place in particular by ionic, adsorptive or by covalent binding.
  • the crosslinking in the sense of the invention is a crosslinking of the molecules with one another or with other polymers.
  • the molecules are in gel structures or in M membranes are enclosed before being immobilized on the surface of the support.
  • the immobilization should take place in such a way that each probe or molecule can be assigned a defined position on the support and that each position on the support can be evaluated independently. However, it may also be desirable for the application sites of different molecules or probes to overlap partially or completely, or for biomolecule mixtures to be worn.
  • the immobilization can take place, for example, using a method based on semiconductor technology.
  • the molecules or biomolecules can be immobilized on the support in two fundamentally different ways: (a) on the one hand, the in situ synthesis of the molecules at defined positions on the support is possible by successive coupling of monomeric synthesis building blocks, (b) on the other others, it is possible to deposit and immobilize previously synthesized or library-derived biomolecules or other molecules at defined positions of the, in particular, functionalized carrier material. Both spotting and printing processes can be used for this. Spotting is understood to mean processes in which liquid drops in which the molecules are located are deposited on the support, essentially round spots being formed by surface interaction and drying.
  • Selected methods for immobilization or pre-immobilization are, for example, contact tip printing, ring and pin printing, nanoelectric printing and nanopipetting, bubble jet printing, top spot printing, micro contact printing, micro fluidic networks methods, photolithographic activation.
  • inorganic surfaces comprising metal, polypropylene, Teflon, polyethylene, polyester, polystyrene, nitride, ceramic and / or glass can be used as supports. be set, or IC (integrated circuit) surfaces, silicon, silicon dioxide or others.
  • Metals in the sense of the invention are all compounds whose cohesion is created by a crystal lattice. The boundary between metals and non-metals is fluid, so that the elements Ce, Sn, As and Sb are also metals in the sense of the invention.
  • the metals according to the invention also include the metallic glasses, that is to say materials which are in a metastable, largely amorphous state.
  • metallically conductive polymers are metals in the sense of the invention.
  • metals advantageously have, in particular, good strength, good hardness and wear resistance, high toughness and good electrical and thermal conductivity.
  • Polypropylenes in the sense of the invention are thermoplastics
  • Polymers of propylene Polypropylenes are particularly characterized by their high hardness, resilience, rigidity and heat resistance. Teflon according to the invention are polytetrafluoroethylenes which advantageously have good thermoplastic properties. Polyethylenes are created in particular by polymerizing ethylene using essentially two different methods, the high-pressure and low-pressure processes. Polyethylenes which are produced in the high-pressure process advantageously have a low density. The properties of carriers comprising polypropylene are essentially determined by the character of the polyethylene as a partially crystalline hydrocarbon. Advantageously, polyethylenes of up to 60 ° are practically insoluble in all common solvents.
  • polar liquids such as alcohol, esters and ketones hardly cause polyethylene to swell at room temperature and thus cause the carrier coating to swell.
  • Polyethylenes are advantageously completely indifferent to water, alkalis and salt solutions as well as inorganic acids.
  • Carriers comprising polyethylenes for example, have a very low water vapor permeability.
  • the carrier can expediently also comprise polyester.
  • polyester For the purposes of the invention, compounds are those which are prepared by ring-opening polymerization of lactones or by polycondensation of hydroxycarboxylic acids or of diols and dicarboxylic acids or dicarboxylic acid derivatives.
  • polyesters also include polyester resins, polyesterimides, polyester rubbers, polyester polyols and polyester polyuretanes.
  • Polyesters are advantageously thermoplastics and have a distinct material character. For example, they are characterized by high thermal stability and can be processed into alloys with metals such as copper, aluminum and magnesium.
  • the carrier comprises ceramic.
  • Ceramic in the sense of the invention is a collective name for in particular inorganic and predominantly non-metallic compounds that contain more than 30 vol. % comprise crystalline materials.
  • Various ceramics or ceramic materials are known to the person skilled in the art, which he can use as a carrier. It can be, for example, so-called earthenware, split plates, laboratory porcelain, aluminum oxide ceramics, permanent magnet materials, silica stones and magnesia stones.
  • clay ceramic materials a distinction is made between coarse and fine materials in the sense of the invention, fine clay ceramic materials comprising earthenware, earthenware, stoneware and porcelain.
  • the carrier can preferably also comprise glass.
  • Glass in the sense of the invention are substances in the amorphous, non-crystalline solid state, that is to say the glass state can be understood in the sense of the invention as frozen, supercooled liquid or melt. Glasses are therefore inorganic or organic, mostly oxidic melt products, which have been converted into a solid state by an insertion process without crystallization of the melt phase components.
  • crystals, melts and supercooled melts are also glasses in the sense of the invention specific.
  • the glasses can be, for example, flat glass, laboratory equipment glass, lead crystal glass, fiber glass, optical glass fibers and others.
  • silicate-free glasses for example phosphate glasses.
  • the support can also be designed in such a way that optical glasses, that is to say, for example glasses with special optical refractive indices, are used.
  • the surface of the carrier can be modified.
  • the carriers can be modified in particular by the action of biological, physical and / or chemical influences. Physical action would be, for example, polishing, etching, pickling, sandblasting, but also physical processes that lead to hardening, coating, tempering, covering with protective skins and the like.
  • a surface treatment by biological action can include, for example, overgrowth by microorganisms.
  • a chemical modification of the surface of the carrier includes, for example, treatment with acids, bases, metal oxides and others.
  • the surface of the carrier can be modified in such a way that the molecules adhere particularly well to the carrier or in such a way that their activity is not disadvantageously modified.
  • the surface modification also includes coating with poly-L-lysines, aminosilanes, aldehyde silanes, epoxy groups, gold, streptavidin, reactive groups, polyacrylamide pads, immobilized nitrocellulose and / or activated aldehydes or agarose aldehyde groups, which means that in particular are bound: DNA, COO ⁇ groups, NH 2 groups, biotin, thiol groups and others.
  • a surface modification of the carrier naturally also includes a treatment which leads to increased stability and breaking strength. Of course, especially when immobilizing biomolecules, classic surface modifications from histology can also be carried out.
  • further semiconductor bodies or semiconductor layers with integrated circuits or additional microsystems are provided in certain sections of the surface of the polymer layer.
  • a polyimide which is known for such applications in semiconductor technology is particularly suitable as the polymer layer.
  • Polyimides are particularly high-temperature resistant polymers; they advantageously have excellent mechanical, thermal and electrical properties.
  • Previously known applications of the polyimide in semiconductor technology include in particular buffer layers, passivation layers, bonding layers and dielectric intermediate layers on the carrier.
  • Polyimides are especially applied in liquid form and then cured. In this curing step, the polyimide is advantageously given the desired properties.
  • the polyimide can be structured lithographically for the applications.
  • polyimide can also be used as an adhesion promoter for potting material and as a buffer layer.
  • the polyimide layer for example, reduces the stress in silicon caused by the encapsulation and prevents cracks on the edges.
  • the polyimide must be cured in particular under very uniform temperature conditions in order to prevent cracking in the polyimide and color irregularities. Low oxygen values are advantageous, for example, in order to achieve good adhesion.
  • the polystyrene which can also be used as a polymer layer for immobilizing molecules according to the invention is a thermoplastic which is obtained above all by free-radical polymerization of styrene.
  • the radical end of a growing polymer chain never attacks a double bond in the ring because the benzene ring is an extremely stable structure.
  • This has several advantages when using polystyrene for example polystyrene is resistant to acids, alkalis and alcohol.
  • the hydrophobic polymer is applied to the surface only in predefined areas.
  • the surface is positively and / or negatively charged electrically by plasma treatment, i.e. the surfaces are charged differently at the most varied of locations.
  • Polymer materials are particularly available in various forms. The individual forms place different demands on the machining process. Depending on the shape of the surface, this is e.g. accessible to the plasmas in different ways.
  • a plasma treatment of the polymer surface can advantageously greatly increase the surface energy and enable other processing methods.
  • the ions and radicals of the plasma in particular react with the polymer surface and generate functional groups there which advantageously determine the surface properties of the polymer.
  • the positive or negative charge in particular improves the wettability and / or the binding of the biomolecules.
  • UV reactive molecules are covalently immobilized by the irradiation with UV light.
  • it can be provided to activate photolabile protective groups on glass by means of light which radiates selectively through a photolithographic mask for the oligosynthesis in a location-specific manner.
  • the glass is then flooded with photolabile molecules, for example DNA bases, which bind to the defined, previously illuminated array locations.
  • Other photolithographic masks are then used accordingly for the next oligo bases in the sequences and the process is repeated. For each base in the sample oligo (per position), 4 masks are required.
  • Advantages can thus be produced directly from known sequence databases, with uniform standardization being achieved. If hydrophobic molecules, in particular biomolecules, are applied to the surface of the hydrophobic polymer layer by means of one of the abovementioned processes, for example a printing process, these molecules adhere to this surface on account of a well-known interaction.
  • one embodiment of the method according to the invention provides for the surface of the polymer layer to be activated at least in sections, for example using a conventional mask technique, in an oxygen plasma.
  • aldehyde groups, carboxy groups or hydroxide groups are formed on the surface of the polymer layer.
  • These groups are hydrophilic and enable covalent bonds with biomolecules which are applied to these activated areas, for example by printing with a solution containing the molecules.
  • These covalent bonds are so stable that the polymer layer with the molecules immobilized on it can then be boiled in soap without destroying the bonds.
  • the surface is preferably only activated island-like by oxygen plasma treatment, the areas of the polymer layer which remain hydrophobic and which surround the "island” limit the running of the applied solution on the surface.
  • Silicon sensor chips with CMOS photodiodes are coated with polystyrene in a spin coater with a layer of approx. 100 to 200 nm Polystyrene covered.
  • the chips are coated with 200 ⁇ l of a 0.1% (w / v) polystyrene solution in toluene for one minute at 3000 rpm in a spin coater.
  • the sensor areas (photodiodes) are then printed with a protein solution in a grid-like arrangement.
  • Antibodies in PBS buffer are used. The antibodies are used in a concentration of 5 ⁇ g / ml. Part of the grid is printed with antibodies that are conjugated with fluorescent dyes.
  • the antibodies are incubated in a moist chamber at 4 ° C. overnight and the unbound antibodies are then rinsed off with PBS buffer. After washing with aqua dest. the success of the immobilization is checked with the aid of a fluorescence measuring device. The successful binding of the antibodies to the sensor areas is demonstrated by the fluorescence of the antibodies.
  • the chip is then sealed by applying a PMMA reaction chamber. The reaction chamber is applied by connecting the PMMA to the polystyrene layer. The finished structure is stabilized by the use of a commercially available stabilizing reagent for proteins and is ready for use.
  • Silicon sensor chips with CMOS photodiodes are already coated on the waver with a 5 ⁇ m layer of polyimide.
  • the polyimide is then coated with a copolymer of benzophenone methacrylate and acrylic acid.
  • the carriers can then be easily printed with biomolecules such as DNA (5 ⁇ M oligonucleotide in PBS buffer).
  • the immobilization is carried out by UV exposure at 300 nm for about 10 minutes.
  • the benzophenone of the copolymer forms radicals that create a covalent bond to the polyimide coating and to the DNA.
  • the same process can also be carried out with all other biomolecules such as proteins, in particular antibodies, peptides, sugars, lipids and triglycerides, as well as complex structures thereof.

Abstract

La présente invention concerne un procédé pour immobiliser des molécules sur des surfaces, une surface plane dans une large mesure étant recouverte avec un polymère, puis les molécules étant immobilisées sur la surface au moyen dudit polymère.
EP03722450A 2002-04-12 2003-04-10 Procede pour immobiliser des molecules sur des surfaces Withdrawn EP1495326A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE10216446 2002-04-12
DE10216446 2002-04-12
DE10236925A DE10236925A1 (de) 2002-04-12 2002-08-12 Verfahren zum Immobilisieren von Molekülen auf Oberflächen
DE10236925 2002-08-12
PCT/EP2003/003782 WO2003087823A1 (fr) 2002-04-12 2003-04-10 Procede pour immobiliser des molecules sur des surfaces

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EP1495326A1 true EP1495326A1 (fr) 2005-01-12

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US (1) US20050158848A1 (fr)
EP (1) EP1495326A1 (fr)
JP (1) JP2005527807A (fr)
CN (1) CN1650166A (fr)
AU (1) AU2003229645A1 (fr)
WO (1) WO2003087823A1 (fr)

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US20150136690A1 (en) * 2012-06-01 2015-05-21 National Unibersity of Singapore Method of making a membrane and a membrane for water filtration
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CN104952839B (zh) * 2014-03-28 2018-05-04 恒劲科技股份有限公司 封装装置及其制作方法
CN108469515B (zh) * 2018-03-12 2020-11-13 黔南民族师范学院 一种热稳定性生物芯片及其制备方法

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Also Published As

Publication number Publication date
AU2003229645A8 (en) 2003-10-27
WO2003087823A1 (fr) 2003-10-23
CN1650166A (zh) 2005-08-03
US20050158848A1 (en) 2005-07-21
AU2003229645A1 (en) 2003-10-27
JP2005527807A (ja) 2005-09-15

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