EP1272667A2 - Procede permettant d'immobiliser un analyte sur une surface solide - Google Patents

Procede permettant d'immobiliser un analyte sur une surface solide

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Publication number
EP1272667A2
EP1272667A2 EP01923375A EP01923375A EP1272667A2 EP 1272667 A2 EP1272667 A2 EP 1272667A2 EP 01923375 A EP01923375 A EP 01923375A EP 01923375 A EP01923375 A EP 01923375A EP 1272667 A2 EP1272667 A2 EP 1272667A2
Authority
EP
European Patent Office
Prior art keywords
cyclodextrin
analyte
solid surface
bound
molecule
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Withdrawn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
EP01923375A
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German (de)
English (en)
Inventor
Claudia Preininger
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.)
Seibersdorf Labor GmbH
Original Assignee
Oesterreichisches Forschungszentrum Seibersdorf 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
Application filed by Oesterreichisches Forschungszentrum Seibersdorf GmbH filed Critical Oesterreichisches Forschungszentrum Seibersdorf GmbH
Publication of EP1272667A2 publication Critical patent/EP1272667A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • 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
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00378—Piezoelectric or ink jet dispensers
    • 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
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00427—Means for dispensing and evacuation of reagents using masks
    • B01J2219/00432—Photolithographic masks
    • 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
    • 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
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/0054—Means for coding or tagging the apparatus or the reagents
    • B01J2219/00572—Chemical means
    • B01J2219/00574—Chemical means radioactive
    • 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
    • B01J2219/0054—Means for coding or tagging the apparatus or the reagents
    • B01J2219/00572—Chemical means
    • B01J2219/00576—Chemical means fluorophore
    • 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
    • B01J2219/00585—Parallel processes
    • 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
    • B01J2219/00596—Solid-phase processes
    • 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
    • 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
    • B01J2219/00659—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
    • 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
    • B01J2219/00677—Ex-situ synthesis followed by deposition on the substrate
    • 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
    • B01J2219/00709—Type of synthesis
    • B01J2219/00711—Light-directed synthesis
    • 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
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • 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
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • the invention relates to a method for immobilizing an analyte on a solid surface and conjugates with analytes bound to solid surfaces.
  • the binding of analytes to solid surfaces is often implemented using linker molecules which connect the surface to the analyte.
  • linker molecules which connect the surface to the analyte.
  • Such crosslinkers are particularly preferred when the analyte to be bound to the solid surface is very small or when an increased free mobility of the analyte is desired for the interaction of the analyte with a ligand that is to be bound to the analyte is.
  • Preferred areas of use for such analyte / solid phase conjugates are, on the one hand, purification processes in which ligands to be isolated from complex mixtures can be bound to the immobilized analyte; on the other hand, such conjugates are used in the analytical / diagnostic field, in particular in the context of screening methods, for example for the detection of rare ligands in biological fluids or for diagnostic methods in the field of DNA technology. In the latter, solid-phase conjugates are used in increasing numbers as biochips.
  • conjugates described in the prior art are either extremely complex and costly to produce or have unsatisfactory steric properties, such as insufficient mobility of the analytes, inadequate distance from the surface of the solid phase (which can result in undesired electrostatic interactions with the surface). or badly or not at all controllable arrangement and distribution of the analytes on the solid surface. It is therefore an object of the present invention to provide conjugates which are improved over the known prior art and which in particular allow simple, low-risk production and still provide the analytes in a satisfactory three-dimensional arrangement.
  • the immobilization of the analyte on a solid surface can also be carried out by
  • cyclodextrins are a type of molecule that is widely used in industrial chemistry and are used for complexing a large number of biomolecules, due to the lack of cyclodextrin molecules specifically equipped with functional groups, cyclodextrin molecules have so far been able to do so Uses cannot be tapped. Only with the introduction of chemically definable, with functional ones Cyclodextrin molecules equipped with groups (cf. EP 0 697 415 Al), it was at all possible to conjugate cyclodextrins to solid phases, although they were still used to complex organic substances.
  • conjugates which can be produced by the process according to the invention, that is to say conjugates comprising a solid surface, a cyclodextrin bound thereto and an analyte covalently bound to the cyclodextrin, have various advantages over the conjugates known in the prior art. Due to the increased spacer length, the relatively large cyclodextrin molecule allows a largely unrestricted free mobility of the analyte, which not only significantly improves the interaction between crosslinker and analyte (and thus facilitates the coupling reaction), but also the interaction with the ligand molecule is significantly facilitated. Furthermore, cyclodextrins are biocompatible, non-toxic and temperature-resistant up to 200 ° C. and thus allow easy, non-hazardous work and good stability of the conjugate provided according to the invention.
  • cyclodextrins as a cross-liner between solid phase and analyte allows a high binding capacity, low non-specific adsorption and - for example in the case of fluorescence detection - a low (measurement) background, which can be reduced even further by choosing suitable solid phases.
  • crosslinkers can of course be provided between cyclodextrin and solid phase or cyclodextrin and analytes, e.g. by another crosslinker already sitting on the solid surface or by the analyte having already been modified with another crosslinker. Examples of such further crosslinkers are described in detail in the prior art (e.g. dihydrazides, ...)
  • nucleic acids in particular DNA, peptides, proteins, enzymes, are preferably used as analytes.
  • special oxidoreductases, transferases and hydrolases, antigens, antibodies, receptors, microorganisms (eg prokaryotic or eukaryotic cells, viruses, etc.) or mixtures of these analytes are used.
  • a metal film e.g. thin gold films
  • a plastic surface or glass are preferably used as the solid surface.
  • Biochips especially DNA chips, are used, for example, to analyze pathologically modified gene activity, to elucidate disease mechanisms or to identify new drug candidates, in the diagnosis and resistance analysis of infectious diseases, but also in the environmental field to identify pathogenic germs.
  • DNA carrier molecules are either synthesized in situ using photolithographic techniques using physical masks on the matrix, or pressed on using various methods.
  • the production of printed DNA microarrays is divided into the steps of activating or coating the solid chip matrix, to which biomolecules are fixed using a suitable coupling chemistry.
  • DNA can be immobilized on carrier material by means of adsorption, photolithographic deprotection and covalent and ionic binding.
  • Controlled pore glass (CPG), SiO 2 layers or polymers are used as carrier materials. CPG and SiO 2 surfaces are mostly etched to free OH groups on the surface generate areas that can be reacted directly with the DNA sequences or converted into other functional groups.
  • CPG and SiO 2 surfaces are mostly etched to free OH groups on the surface generate areas that can be reacted directly with the DNA sequences or converted into other functional groups.
  • copolymers which contain functional groups polymers into which functional groups can be introduced by chemical modification, chemically inert polymers such as polysulfones or Teflon which can be activated by radiation (for example UV, Co 60), and chemically inert polymers that are covered with functional copolymers.
  • Polymers that already have functional groups, the activation and conversion of which into other functional groups have been described, are, for example, polyamide, polyacrylamide and polyester.
  • Unreactive polymers e.g. Polyethylene
  • a reactive monomer such as e.g. Glycidyl methacrylate or N-vinylformamide
  • a very elegant method for introducing functional groups is surface modification using plasma treatment.
  • plasma treatment In the case of polypropylene, various sub-types of plasma treatment make it possible to introduce amino, hydroxy or thiol groups.
  • slides are etched and amino- or epoxysilanized.
  • Oligonucleotide arrays e.g. Filter materials such as nitrocellulose or nylon (Clontech, USA) with polylysine or with glass slides derivatized with various silanes, carboxymethylated dextrans (Biacore AB, Sweden) or polyacrylamide gel pads (Packard / Motorola, USA) are used.
  • Filter materials such as nitrocellulose or nylon (Clontech, USA) with polylysine or with glass slides derivatized with various silanes, carboxymethylated dextrans (Biacore AB, Sweden) or polyacrylamide gel pads (Packard / Motorola, USA) are used.
  • nylon membranes are characterized by a high binding capacity, but with fluorescent detection they have a higher background than glass.
  • Polyacrylamide and dextran are three-dimensional hydrogels which, in contrast to flat surfaces such as glass, have a very high binding capacity and a low non-specific binding.
  • the choice of the specific cyclodextrin molecule or of the functional groups is generally not critical, so that in particular ⁇ -, ⁇ - or ⁇ -cyclodextrins can be used.
  • the cyclodextrin molecule are selected from halogen, amine, thiol, isothiocyanate and sulfonic acid groups, aromatic groups, preferably aromatics with heteroatoms, in particular with the above functional groups, or combinations thereof.
  • a suitable cyclodextrin for use in the context of the present invention is a monochlorotriazinyl, substituted ⁇ -cyclodextrin, which is already known as a crosslinking agent or surface-modifying agent, for example on textiles or papers. This ⁇ -cyclodextrin derivative can easily be prepared, for example by treating cyanuric chloride with ⁇ -cyclodextrin in water.
  • a cyclodextrin molecule used according to the invention preferably contains 2 to 4 functional groups, in particular identical functional groups.
  • the binding to the solid phase can thus also preferably take place covalently.
  • more than two functional groups per cyclodextrin molecule several analytes can also be bound to one cyclodextrin molecule.
  • the present invention relates to a conjugate comprising a solid surface, a cyclodextrin bound to it and an analyte covalently bound to the cyclodextrin.
  • This conjugate is obtainable according to the inventive method described above.
  • the conjugate according to the invention is preferably designed as a biochip, ie the solid surface and the analytes are designed in accordance with the established methods known for biochips and are incorporated in methods adapted for such biochips, in particular as regards the software and hardware detection of the reactions at the solid surfaces (see the already established biochip products from Affimetrix Inc. and Incyte).
  • the conjugate according to the invention preferably comprises, especially in practical use, a ligand molecule specifically bound to the analyte, for example a complementary nucleic acid, an antibody, an antigen, a receptor ligand, a receptor, and the like.
  • a ligand molecule specifically bound to the analyte for example a complementary nucleic acid, an antibody, an antigen, a receptor ligand, a receptor, and the like.
  • the conjugate according to the invention is a biochip.
  • the conjugate according to the invention comprises a whole series (a library) of analytes, the analyte library preferably being applied to the solid surface in such a way that the localization of the different analytes is spatially exactly possible.
  • the present invention relates to a method for the specific detection and possibly isolation of a ligand molecule from a sample, which is characterized in that a sample containing the ligand molecule to be detected or isolated (or a sample , which may contain such a ligand molecule) is contacted with a conjugate according to the invention, the ligand molecule is specifically bound to the bound analyte and this specific binding is verified by measures known per se, whereupon the ligand molecule is optionally detached from the conjugate and is isolated.
  • the verification of the specific binding can be adapted to the respective ligand / analyte system or can be carried out by generally accepted methods, such as (secondary) antibody reactions, dye reactions, signaling methods via the solid phase (biochip), radioactivity or fluorescence labeling, etc ,
  • Biomolecules such as enzymes, antibodies, microorganisms and oligonucleotides (DNA, RNA) can be adsorbed or embedded in polymers.
  • lyvinyl alcohol PVA
  • Polyvinyl alcohol has a large, porous surface in which biomolecules can be embedded. The pore size can be determined by basic or acid catalysis during gel crosslinking. Three-dimensional networks are created that are mechanically stable and have excellent swelling behavior in water.
  • PVA gels can be cross-linked covalently with glutaraldehyde, which leads to an increase in gel hardness.
  • functional, reactive groups can easily be introduced into polyvinyl alcohol by acetylation or acylation.
  • PVA with styrylpyridinium groups for example, is photosensitive and includes biomolecules in its pores when irradiated by cyclodimerization.
  • PVA gels crosslinked with polyallylamine and polyacrylic acid are also used for biosensors. Due to the large pores of PVA and the property of swelling into a three-dimensional network in water, a high immobilization capacity is achieved. However, the large-pore matrix also means that immobilized biomolecules are washed out very easily.
  • MCT monochlorotriazinyl- ⁇ -cyclodextrin, Na salt
  • the advantages of these methods are:
  • the PVA gels described are stable, hydrophilic and porous. They swell on contact with an aqueous solution. This increases the surface area and increases the immobilization capacity.
  • the hydrophilic character of the gel enables easy and quick access of the biomolecules to the polymer surface and reduces non-specific adsorption.
  • Biomolecules can be immobilized in PVA in a hydrophilic, three-dimensional, porous matrix in a solution-like state. Due to the resulting greater freedom of movement, biomolecules immobilized in PVA are more reactive than on planar surfaces. Due to the covalent cross-linking of the gel and the covalent binding of the biomolecules to the gel (see 2.), the washing out of the biomolecules can be prevented.
  • PVA gels which is not only suitable for immobilizing oligonucleotides (DNA, RNA), but also for immobilizing antibodies, enzymes and microorganisms, was demonstrated using a 16S rRNA chip.
  • the oligonucleotides spotted on PVA / Palam or PVA / Palam / MCT remained after washing in hybridization solution (20 mM Tris, pH 7.4, 0.01% lauryl sulfate, 0.9 M NaCl and 35% formamide) bound.
  • PVA Polyvinyl alcohol
  • PVA-1 to PVA-5 PVA gels were used (PVA-1 to PVA-5), which hydrolyzed 5 g of a 10% aqueous PVA (99 +%, MW 85,000-146,000, Aldrich, AT), 0.1 g Palam (Aldrich, AT), 0.1 g monochlorotriazinyl-ß-cyclodextrin (ß-CD)), Cavasol W7 MCT (Wacker, DE) in 5 ml of distilled water and contained pH's of 4 (PVA-1), pH 6.8 (PVA-2), pH 8 (PVA-3) and pH 9 (PVA-4) (with the addition of Na2C0 3 ).
  • PVA-5 and PVA-1 were identical except for the addition of ß-CD (PVA-5 contained no ß-CD).
  • the thickness of the PVA films was approximately 8 ⁇ m (with a resolution of 10 nm).
  • the PVA gels on the chips were polymerized by 6 freezing (-18 ° C) and drying (25 ° C) steps.
  • Unmodified and amino-modified EUB338 (5'-GCT GCC TCC CGT AGG AGT-3 '), ALFlb (5' -CGT TCG (CT) TC TGA GCC AG-3 ') and BET42a (5' -GCC TTC CCA CTT CGT TT-3 ') (with and without Cy5 label) were dissolved in 0.05 M phosphate buffer, pH 8 and spotted onto the chips by a piezoelectric biochip array.
  • the oligonucleotides were lined up in blocks of 5 x 3 spots from 0.35 to 1 nl. The distance between the spots was 300 ⁇ m.
  • the ⁇ -CD-containing chips produced according to the invention were compared with commercially available products such as CMT-GAPS TM, FAST TM, Silane-Prep TM and Hybond N +.
  • the results are shown in FIG. 3.
  • the fluorescence after spotting ti the fluorescence after blocking and t2 the fluorescence after hybridization of the complementary DNA on the chip.
  • immobilization capacity the ß-cyclodextrin chips (except for PVA-5) were well above all tested, commercially available products. Immobilization capacities between 85 and 120% after hybridization were significantly better than the comparison products (below 40%).
  • the chips according to the invention accordingly have a immobilization capacity which is far better than that of all other products.

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  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne un procédé permettant d'immobiliser un analyte sur une surface solide. Ce procédé est caractérisé en ce qu'il consiste à fixer une molécule de cyclodextrine ayant au moins deux groupes fonctionnels sur une surface solide, de façon qu'encore au moins un groupe fonctionnel de la molécule de cyclodextrine puisse être lié de manière covalente à un analyte, puis à lier de manière covalente l'analyte à la molécule de cyclodextrine fixée sur la surface. En variante, l'analyte peut d'abord être lié de manière covalente à la molécule de cyclodextrine, puis la molécule de cyclodextrine peut ensuite être reliée à la surface solide.
EP01923375A 2000-04-14 2001-04-12 Procede permettant d'immobiliser un analyte sur une surface solide Withdrawn EP1272667A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AT656002000 2000-04-14
AT0065600A AT408150B (de) 2000-04-14 2000-04-14 Verfahren zur immobilisierung eines analyten an einer festen oberfläche
PCT/AT2001/000108 WO2001079533A2 (fr) 2000-04-14 2001-04-12 Procede permettant d'immobiliser un analyte sur une surface solide

Publications (1)

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EP1272667A2 true EP1272667A2 (fr) 2003-01-08

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EP01923375A Withdrawn EP1272667A2 (fr) 2000-04-14 2001-04-12 Procede permettant d'immobiliser un analyte sur une surface solide

Country Status (6)

Country Link
US (1) US20030040008A1 (fr)
EP (1) EP1272667A2 (fr)
AT (1) AT408150B (fr)
AU (1) AU2001250146A1 (fr)
CA (1) CA2406091A1 (fr)
WO (1) WO2001079533A2 (fr)

Families Citing this family (3)

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Publication number Priority date Publication date Assignee Title
EP1486561B1 (fr) * 2001-12-26 2013-12-04 Canon Kabushiki Kaisha Milieu de sonde
US20050053954A1 (en) * 2002-11-01 2005-03-10 Brennan John D. Multicomponent protein microarrays
GB2414479A (en) * 2004-05-27 2005-11-30 Croda Int Plc Reactive cyclodextrins derivatised with proteins

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US5068227A (en) * 1989-01-18 1991-11-26 Cyclex, Inc. Cyclodextrins as carriers
US5661040A (en) * 1993-07-13 1997-08-26 Abbott Laboratories Fluorescent polymer labeled conjugates and intermediates
WO1995002700A1 (fr) * 1993-07-13 1995-01-26 Abbott Laboratories Conjugues marques fluorescents polymeres et intermediaires
DE4401618A1 (de) * 1994-01-20 1995-07-27 Consortium Elektrochem Ind Verfahren zur Herstellung von aminofunktionellen Cyclodextrin Derivaten
DE4418513A1 (de) * 1994-05-27 1995-11-30 Bayer Ag Immuntest zum Nachweis hochmolekularer Antigene
DE4429229A1 (de) * 1994-08-18 1996-02-22 Consortium Elektrochem Ind Cyclodextrinderivate mit mindestens einem stickstoffhaltigen Heterozyklus, ihre Herstellung und Verwendung
BE1008978A5 (fr) * 1994-12-27 1996-10-01 Solvay Adjuvants pour vaccins.
DE19520989A1 (de) * 1995-06-08 1996-12-12 Consortium Elektrochem Ind Cyclodextrinmodifizierte Polymere
US5877310A (en) * 1997-04-25 1999-03-02 Carnegie Mellon University Glycoconjugated fluorescent labeling reagents
US6582583B1 (en) * 1998-11-30 2003-06-24 The United States Of America As Represented By The Department Of Health And Human Services Amperometric biomimetic enzyme sensors based on modified cyclodextrin as electrocatalysts
DE19925475B4 (de) * 1999-06-03 2004-12-30 Gkss-Forschungszentrum Geesthacht Gmbh Kompositmembran aus einer porösen Trägermembran, Verfahren zu ihrer Herstellung und deren Verwendung
WO2001004631A1 (fr) * 1999-07-08 2001-01-18 Radiometer Medical A/S Detecteur comportant une matiere matricielle hydrophile

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See references of WO0179533A2 *

Also Published As

Publication number Publication date
AT408150B (de) 2001-09-25
WO2001079533A3 (fr) 2002-04-04
CA2406091A1 (fr) 2002-10-11
ATA6562000A (de) 2001-01-15
US20030040008A1 (en) 2003-02-27
WO2001079533A2 (fr) 2001-10-25
AU2001250146A1 (en) 2001-10-30

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