WO2004040307A2 - Dispositif et procede pour analyser la structure de glycostructures complexes et detecter ces dernieres - Google Patents

Dispositif et procede pour analyser la structure de glycostructures complexes et detecter ces dernieres Download PDF

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WO2004040307A2
WO2004040307A2 PCT/EP2003/011567 EP0311567W WO2004040307A2 WO 2004040307 A2 WO2004040307 A2 WO 2004040307A2 EP 0311567 W EP0311567 W EP 0311567W WO 2004040307 A2 WO2004040307 A2 WO 2004040307A2
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lectins
microarray
vicia
immobilized
binding assay
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PCT/EP2003/011567
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German (de)
English (en)
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WO2004040307A3 (fr
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Simone Cartellieri
Jürgen Kuballa
Bernd Niemeyer
Heike Helmholz
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Gkss Forschungszentrum
Galab Technologies Gmbh
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Priority to AU2003301693A priority Critical patent/AU2003301693A1/en
Publication of WO2004040307A2 publication Critical patent/WO2004040307A2/fr
Publication of WO2004040307A3 publication Critical patent/WO2004040307A3/fr

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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
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • 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
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    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
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    • B01J2219/00315—Microtiter plates
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    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00385—Printing
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00387—Applications using probes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • 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
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00497—Features relating to the solid phase supports
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    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00603—Making arrays on substantially continuous surfaces
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    • B01J2219/0061—The surface being organic
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
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    • B01J2219/00614—Delimitation of the attachment areas
    • B01J2219/00621—Delimitation of the attachment areas by physical means, e.g. trenches, raised areas
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • 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
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    • 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
    • B01J2219/0063—Other, e.g. van der Waals forces, hydrogen bonding
    • 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
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    • B01J2219/00632—Introduction of reactive groups to the surface
    • B01J2219/00635—Introduction of reactive groups to the surface by reactive plasma treatment
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
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    • 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
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    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00727—Glycopeptides
    • 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 device and a method for determining and analyzing complex glycostructures.
  • Carbohydrates can be converted into monosaccharides, disaccharides. Oligosaccharides and polysaccharides can be divided. The oligo- and polysaccharides can in turn be divided into homoglycans or heteroglycans, the heteroglycans with the representatives of the proteoglycans, peptidoglycans, glycolipids and glycoproteins being among the biologically important structural units.
  • Compound carbohydrates that contain conjugated compounds, such as glycolipids and glycoproteins are also referred to as glycoconjugates. Glycoconjugates are one of the central biomolecules of the ropes and are involved in many physiological processes.
  • glycoconjugates plays a major role in the areas of basic research, diagnostics, pharmaceutical research and for drug development.
  • the term glycoconjugates denotes molecules in which a carbohydrate portion (mono-, oligo- or polysaccharide) is covalently bound to a protein or an ipid.
  • glycoconjugates include, in addition to the glycoproteins and glycolipids, in which the protein or lipid portion predominates, also the lipopolysaccharides and the protoglycans, in which the carbohydrate portion predominates.
  • the carbohydrate content of glycoproteins and glycolipids typically consists of up to 20 monosaccharides; for lipopolysaccharides and protoglycans this can be considerably larger.
  • nature uses a wide variety of linkage and branching options in addition to the selection of different monosaccharide building blocks, so that there is a huge variety of structures within this class of substances.
  • Carbohydrates potentially have the greatest number of possible variations in a short sequence within the biological binding partners of proteins.
  • Using a common library of 20 different monosaccharides theoretically 10 15 different structures result for the formation of a hexasaccharide (including branched isomers). If the oligosaccharide chains are branched, one speaks of oligoantennaren connections, whereby normally di- to pentaantennäräre structures are present.
  • the resulting "multivalency" of the molecules in relation to the interaction with a binding partner is an important aspect for the specificity and strength of the binding.
  • the oligosaccharide structures can vary at each glycosylated site of a protein. The differently glycosylated variants are known as glycoforms of this protein or as micro heterogeneity.
  • glycoproteins are first broken down, that is to say the structure to be examined is first destroyed and the glycans or glycopeptides are characterized by means of mass spectroscopy after chromatographic separation.
  • affinity chromatography saccharides or glycoconjugates are purified with the aid of lectins which are bound to gel particles for the use of further analysis steps.
  • the method of serial lectin affinity chromatography is available, in which the sample components are fractionated in a flow-through method using a combination of lectin adsorbents.
  • glycoconjugates it is disadvantageous that purification of the glycoconjugates is necessary because any contamination impairs the analytics. For example, it is necessary to work with large amounts of the purified glycoconjugate - often in the milligram range - which requires a great deal of preparation. So far it has not been possible to detect glycostructures in a uniform process, so that the analysis of glycoconjugates with conventional methods always consists of several sub-steps and is therefore very time-consuming.
  • a central disadvantage of conventional analysis methods is that the glycostructures cannot be analyzed non-destructively, which means that the sample is no longer in its native state, which means that a sample that has been analyzed and that includes glycostructures usually does not can continue to be used.
  • Complex structures such as glycoproteins with more than two or three more or less branched sucker chains (glycans), are generally not covered by the known methods.
  • the object of the invention is therefore to provide a device with which complex carbohydrate structures, such as glycoconjates, can be detected quickly, easily, safely and efficiently and their structure can be determined.
  • the invention solves this technical problem by providing a microarray comprising at least at least two different immobilized lectins directed against the basic structure of a glycoconjugate, the basic structure comprising at least one glycan containing at least two glycosidically linked monosaccharides.
  • the invention is accordingly based on the surprising teaching that complex glycoconjugates, such as glycoproteins or glycolipids, not only interact specifically with one lectin, but that they are able to interact measurably with several lectins in such a way that an analysis of the Structure of the glycoconjugate - the analyte - is possible.
  • This interaction of one and the same analyte, with several different lectins, results in several areas or locations on the array with a special lectin-sucker interaction, which can be detected using various methods known to the person skilled in the art.
  • Immobilization in the sense of the invention is understood to mean all methods for restricting the mobility and solubility of lectins in chemical, biological and / or physical ways.
  • the immobilization can be done by different methods, such as the binding of lectins with each other or to the carrier, by adhering a polymeric matrix in the network 'or immobilization on a membrane.
  • the immobilization becomes a Wash out the lectins in the different washing steps prevented.
  • the biological sample can easily be separated from the immobilized lectin.
  • the lectins can be bound or immobilized on the carrier or microarray by direct carrier connection and by crosslinking.
  • Crosslinking in the sense of the invention is a crosslinking of the detection molecules - that is to say the lectins - with one another or with other polymers.
  • the lectins When immobilized by inclusion, the lectins are enclosed in gel structures or in membranes in such a way that interaction with the glycoconjugates is possible.
  • Lectins e.g. can be immobilized by dropping round spots or by printing lines, whereby planar and non-planar structures such as spheres, cuboids or fibrils and rods can be used as the microarray, which are impregnated or immobilized with different lectins and thus immersed in a spatial arrangement. To be put in order.
  • the immobilization should take place in such a way that (i) that each lectin is assigned a defined position on the support or microarray and (ii) that each position on the microarray or support can be evaluated independently. However, it can also be advantageous that the application sites of different lectins overlap partially or completely or that lectin mixtures are applied in the form of a detection spot.
  • the immobilization can take place, for example, using a method based on semiconductor technology. Printing processes also make it possible to deposit the lectins in defined areas or locations on or on the surface of the microarray, as a result of which stable binding can take place with high coupling efficiency. All immobilization measures of biomolecules known to the person skilled in the art - for example on column materials - can also be used to immobilize the lectins on the array.
  • Selected methods for 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 methods, photoresist lithography, Electroche ical focusing and micro wet printing.
  • comparatively simple sample application devices from thin-layer chromatography or autosamplers for HPLC and pipette and micropipettes (piston stroke pipettes) are used. With the help of these methods, a lectin spot size of approx. 1 ⁇ m to over 6 mm can be generated.
  • the lectin density per cm 2 can be, for example, in the range from approximately 0.01 to 1000 pmol / cm 2 .
  • the methods mentioned enable simple handling and great precision of the volumes created, as well as very homogeneous lectin spots, which are applied with a high degree of parallelization.
  • the methods that enable a direct synthesis of the lectins on the support have in particular a high integration density and a simple combination of immobilization and coupling. It is possible to prepare the lectins, which can also be referred to as probes, with further methods in order to keep the proportion of unspecificly synthesized or bound lectins low.
  • the lectins can also be immobilized on the microarray by a blot method.
  • the lectins or cells or tissues comprising them are separated by means of electrophoresis.
  • the electrophoretic separation can e.g. by means of a one-dimensional or multidimensional electrophoresis, in particular a 2D electrophoresis.
  • electrophoretically separated or separated lectins are then either placed on the support or Transfer microarray or on a membrane, which is applied in a next step to the carrier or microarray.
  • the microarray comprises at least two different immobilized lectins, the lectins being isolable, selected from the group consisting of Abrus precatorius, Adenia digitata, Agaricus bisporus, Aleuria aurantia, Amaranthus caudatus, Amphicarpaea biacteata, Anguilla anguilla, Arachis hypogaea, Artocarpus integrifolia, Bauhinia purpurea alba, Brachypodium sylvaticum, Canavalia ensiformis, Carcino scorpius rotunda canda, Cicer arietinum, Codium fragile, Crotalaria juncea, Cytisus sessilifolius, Datura stramonium, Dioclea grandifhrinausiallichristinauria, dendella erifone, Erythema, dolomized erythematosus, dolomized
  • lectins which are suitable for binding complex glycostructures or glycoconjugates in such a way that detection and structural analysis are possible can advantageously be isolated from the organisms mentioned.
  • the lectin ABA from Agaricus bisporus, from Aleuria aurantia AAL, from Amaranthus caudatus ACL (ACA), from Anguilla (Eel) anguilla AAA, from Arachis hypogaea PNA, from Artocarpus integrifolia AIL (Jacalin), from Griffonia
  • DB A 'from Erythrina coralldendron Ecor A, from Erythrina cristagalli ECL (ECA), from Euonymos europaeus EEL, from Galanthus nivalis GNL, from Glycine ⁇ tax SBA, from Helix aspersa HAA, from Helix po atia HPA, from Hippeastrum hybrid HHL (AL ), from Lens culinaris LCA, LcH (Lentil), from Lotus tetragonolobus LTL, from Lycopersicon esculentum LEA (Tomato), from Maackia amurensis MAA, from Maclura pomifera MPA, from Narcissus pseudonarcissus NPL, NPA, from Phaseolus coccineus PCA, from Pha ⁇ eolu ⁇ limensis LBA, from Phaseolus vulgaris PHA, from Phytasuumum s PSA, from Pseudomonas aeruginos
  • the microarray comprises at least two different immobilized lectins, the lectins having a binding affinity to the glycostructures to be detected of Kd 10 10 ⁇ s mol / 1.
  • the immobilized lectins comprise a binding linker.
  • Binding linkers in the sense of the invention are all structures which are associated with the lectins in such a way that they can be immobilized covalently or complexly on a support of the microarray or on the microarray itself.
  • the linkers can be, for example, peptide fragments in the range from 0 to 50 amino acids.
  • the lectins comprising linkers are accordingly advantageously not immobilized on the microarray via non-specific adsorption.
  • linkers can also be designed in such a way that an interaction between the individual lectins or lectins and the carriers is excluded in such a way that disturbances or non-specific interactions do not occur or only to a very small extent.
  • the linkers can be used as spacers, for example.
  • linkers in the sense of the invention can also be short synthetic DNA double strands. They can also be used to first immobilize certain lectins in order to then split them off again with the help of specific enzymes, for example those that break down the DNA double strand.
  • the linker is in particular streptavidin and / or biotin.
  • Streptavidin in the sense of the invention is a protein from Streptomyci avidinii which binds to biotin with an affinity of 10 "15 M " 1 and which has a molecular weight of approximately 60,000 Da. It advantageously has a lower unspecific P-action than avidin.
  • Biotin in the sense of the invention are coenzymes of the enzymes that catalyze carboxylations. The very high affinity of biotin for streptavidin as well as for avidin can advantageously be used to immobilize biotinylated reagents.
  • the lectins are immobilized with a packing density of> 50 ng / cm 2 .
  • the resulting surface coverage of> 10% enables an optimal interaction with the glycostructures.
  • the variants of the possible functional The base materials are of course diverse. A requirement for the binding of lectins is the presence of appropriate functional groups on the support material with which the lectin can react.
  • the following variants can be used, among other things: functionalization with carboxyl groups, functionalization with amino groups, functionalization with aldehyde groups, functionalization with phenyl groups, functionalization with thiol groups, functionalization with divinylsulphones (resulting bond: ether, thioether, secondary amines), Functional ionization with carbonyldiimidazole, functionalization with epichlorohydrins (resulting bond: ether, thioether, secondary amines), functionalization with
  • the functionalization is advantageously possible in a rapid reaction, the immobilization of the lectins using the methods described being particularly advantageous since a covalent bond is formed here. This prevents the leaching of the lectins and thereby a loss of binding activity or, if the sample is detached, contamination of these by lectin molecules.
  • Immobilization takes place under individual reaction conditions and is often to be carried out using other chemicals.
  • the immobilization on epoxy groups requires, for example, 8-48 h at pH 8.5-12.
  • the binding of proteins to matrices activated with carboxyl groups takes place at pH 4-6 in 24-48 h.
  • the use of carbodiimide for the reaction is advantageous. Proteins are also immobilized on supports substituted with amino groups using other chemicals such as carbodiimide or NaCNBH 3.
  • tresyl chloride (2,2,2-trifluoroethane sulfonyl chloride) is advantageously used.
  • the functionalization of carrier materials with tresyl chloride is particularly suitable for the binding of proteins, since the reaction takes place under mild conditions (pH 7-9) and does not damage the proteins.
  • the binding takes place via primary amino groups or thiol groups of the amino acids of the protein.
  • the reaction is comparatively quick (a few hours) and takes place without the use of additional chemicals.
  • Tresyl activation is stable for a few months with dry material or when stored in dilute hydrochloric acid.
  • spacer increases the distance between the support surface and the ligand and can therefore contribute to better accessibility of the ligand.
  • Usual spacers consist of two terminal functional groups and a length-selectable C chain, e.g. 6-aminohexanoic acid.
  • the linker used that is to say the carrier functionalization used, can itself already be a spacer.
  • the microarray comprises metal, polypropylene, Teflon, silicon, polyethylene, polyester, polystyrene, nitride, ceramic, quartz and / or glass.
  • 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 blurred ßig, so that the elements Ce, Sn, As and Sb are 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 meta-stable, 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 thermoplastic polymers of propylene. Polypropylenes are characterized in particular by high hardness, R ückstellêt, stiffness and heat resistance of.
  • the microarray can also include Teflon. Teflon according to the invention are polytetrafluoroethylene, which advantageously have good thermoplastic properties.
  • Polyethylenes are produced in particular by polymerizing ethylene using essentially two different methods, the high-pressure and the low-pressure process. Polyethylenes which are produced in the high-pressure process advantageously have a low density.
  • microarrays comprising polypropylene are essentially determined by the character of the polyethylene as a partially crystalline hydrocarbon.
  • Polyethylenes are advantageously up to 60 ° in all customary solutions. practically insoluble.
  • polar liquids such as alcohol, esters and ketones hardly cause polyethylene to swell at room temperature.
  • Polyethylenes are advantageously completely indifferent to water, alkalis and salt solutions as well as inorganic acids.
  • microarrays or carriers that include polyethylenes have very low water vapor permeability.
  • the microarray can expediently also comprise polyester.
  • polyesters are compounds 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, polyester imides, 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. However, it can also be provided that the microarray comprises ceramics.
  • Ceramic in the sense of the invention is a collective name for a class of substances composed of in particular inorganic and predominantly non-metallic compounds and elements and in particular comprising more than 30% by volume of crystalline materials.
  • the specialist are various ceramics or ceramic materials known that he can use as a microarray. It can be, for example, so-called pottery, earthenware, split plates, laboratory porcelain, tableware, bone china, 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.
  • microarray 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 that the glass state in the sense of the invention can be understood 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. In the context of the invention, crystals, quartz, melts and supercooled melts are of course also to be understood as glasses.
  • the glasses can be, for example, flat glass, container glass, industrial glass, laboratory equipment glass, lead crystal glass, fiber glass, optical glass fibers and others. Of course it is also possible that silicate-free glasses, for example phosphate glasses, are used.
  • the microarray can also be designed such that optical glasses, that is to say, glasses with special optical refractive indices, are used.
  • the invention also relates to a binding assay for determining glycostructures in a sample, the sample being brought into contact with at least two different immobilized lectins to form lectin-sugar complexes and the lectin-sugar complexes in a manner known per se are detected and the lectins can be isolated, selected from the group comprising Abrus precatorius, Adenia digitata, Agaricus bisporu ⁇ , Aleuria aurantia, Amaranthus caudatus, Amphicarpaea biacteata, Anguilla anguilla, Arachis hypogaea, Artocarpus integrifolia, Bauhinia purpuriformum alba, bavarian purpuriform alba, Carcino scorpius rotunda canda, Cicer arietinum, Codium fragile, Crotalaria juncea, Cytisus sessilifolius, Datura stramonium, Dioclea grandiflora, Dolichos
  • Determination of glycostructures by the binding assay according to the invention relates on the one hand to the detection, that is to say the qualitative detection of glycostructures in a sample, and also to the detection of certain patterns on the microarray, which allow conclusions to be drawn as to which immobilized different lectins the same glycostructure interacts in order to determine the structure of the complex oligosaccharide or polysaccharide or the glycoconjugate.
  • a sample in the sense of the invention is the designation for a biological sample taken by sampling or chemical good or a part or a small amount of such, the nature of which is to be tested chemically, biologically, clinically or similarly. Sampling takes place in such a way that the partial quantity taken corresponds to an average of the total quantity.
  • the characteristics determined by examining the sample are used to assess the amount contained in the sample, which allows RUCKSuefsen to the total amount, such as drinking water, food, tissue, cells, cell cultures, sell culture supernatants, transplanted organs and others.
  • the samples can be pretreated by mixing, separating, separating, prefractioning, crushing, adding enzymes or markers - or otherwise.
  • pretreating the samples are known to the person skilled in the art.
  • the sample is taken in such a way that it does not correspond to an average of the total amount.
  • a sample can be all biological and non-biological materials, such as biological tissues and fluids, e.g. blood, lymph, urine, brain fluid and others, as well as environmental waste water, bioreactor fluids, food ingredients, hazardous substances, aerosols, lipid substances and much more.
  • the lectins are physically, chemically and / or biologically by in situ synthesis or by Placing previously synthesized lectins immobilized on the microarray.
  • the carriers or microarrays can be produced in two different ways:
  • Spotting is understood to mean processes in which liquid drops are deposited, whereby essentially round spots are created by surface interaction and drying. Other printing processes allow the substrate to be placed on the surface in defined areas.
  • the lectins are used by Contacc Tip Printing, Ring and Pin Printing and Nanopipetting, Buble Jet Printing, TopSpot Printing, Micro Contact Printing, Micro Fluidic Networks Methods, Photolithographic Activation Methods, Photoresist Lithography, Electro- mical focusing and / or micro wet printing immobilized.
  • the surface of the microarray is activated with poly-L-lysines, aminosilanes, aldehyde silanes, epoxy groups, gold, streptavidin, polylysines, silanes, reactive groups, polyacrylamide pads, immobilized nitrocellulose Aldehydes, agarose aldehyde groups and / or tresyl groups coated.
  • substrate surface treatments advantageously make it possible to improve the durability and the binding capacity of the surface of the microarrays in such a way that the lectins can remain immobilized very well over a relatively long period of time.
  • the surface of the carrier can be made very generally, in particular by the action of biological, physical and / or chemical influences.
  • Physical effects include, 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, the 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 that the detection molecules or lectins adhere particularly well to the support or adhere such that their activity is not disadvantageously modified.
  • a surface modification of the carrier naturally also includes a treatment which leads to increased stability and breaking strength, in particular of the microarray. It is of course also possible to make classic surface modifications from histology, such as coating with, for example, protein glycerol, polylysine, activated dextrans and / or chromium gelatin.
  • a labeled or unlabeled lectin, a labeled or unlabeled glycostructure and / or a labeled or unlabeled antibody are added before or after the formation of the sugar-lectin complexes.
  • sample molecules themselves are provided with a marker (enzyme (A), fluorescent or Vis dye (B), biotin (C) ).
  • binding event and washing either: be read out directly (B) or after converting an enzyme substrate and reading out the resulting color signal (A) or (C) after adding another marker (enzyme, fluorescent or Vis dye) which is coupled with avidin / streptavidin.
  • Sandwich After adsorption of the sample molecules, they are provided with the same lectin that is immobilized on the surface (see example). This lectin binds specifically to the glycoconjugate to be determined from the opposite side and is itself marked. After binding, the event can be read out.
  • the same sugar structure is advantageously recognized and detected "twice”. It can be the one sugar spec be determined. Only complex systems with at least two parts of glycosylation are recorded. Only the lectins already present are required, no further antibodies or the like.
  • model adsorptive After adsorption of the sample molecules, the wells are provided with a labeled model adsorptive. As is known, this binds the immobilized lectin.
  • the model adsorptive can only dock onto free binding parts or competes with the sample molecule for these binding sites. A positive signal is obtained if no or only a few sample molecules are attached or according to the ratio that arises in this competitive situation.
  • the detection of systems with only a complex sugar structure is advantageously possible.
  • Known model systems are used.
  • the competitive situation also provides information about the binding constants of the complex with the sample molecule.
  • an enzyme, a dye, a fluorescence marker, a radioisotope, a metal colloid and / or a chelator is used for labeling.
  • Methods are known to the person skilled in the art how he can make markers more sensitive, in particular by means of amplification mechanisms.
  • the lectins are immobilized on micro test plates, glass plates, membranes, braids, fibrils, in particular made of polypropylene, nitrocellulose, glass and / or PVDF.
  • microtiter plates can be used as microtest plates. Microtest plates advantageously have dimensions that allow their use in numerous laboratory routines.
  • microtest plates can be used as standard.
  • the immobilization of the molecules on special laboratory vessels for example microtiter plates, petri dishes, multi-dishes, multi-dishes and other culture vessels and slides, therefore advantageously also enables the use of the existing laboratory means and devices for incubating, freezing, lyophilizing and similar laboratory devices in clinical or research laboratories.
  • microtiter plates with a transparent, non-fluorescent flat bottom or white or black, opaque flat bottom can preferably be used as the micro test plates.
  • a largely planar surface of the microarray is coated with a polymer and subsequently lectins are immobilized on the surface by means of the polymer.
  • the lectins are advantageously immobilized directly via the polymer.
  • the polymer is a polyimide or polystyrene. If the polymer is selected appropriately, for example polyimides, further microarrays or even additional microsystems with the same inorganic surface, for example semiconductor formwork, can also be industrially combined to form a system.
  • Polyimides are in particular high-temperature-resistant polymers; they advantageously have excellent mechanical, thermal and electrical properties.
  • the applications of the polyimide include in particular buffer layers, passivation layers, binding layers and dielectric intermediate layers on the carrier. Polyimides are applied in particular in liquid form and then cured. In this curing step, the polyimide advantageously obtains the desired properties. For the applications, the polyimide can be structured graphically.
  • 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. Silicon can be applied to the micro array in interconnects.
  • the polyimide must be cured in particular under very uniform temperature conditions in order to prevent the formation of cracks in the polyimide and color irregularities. Low oxygen values are advantageous, for example, in order to achieve good adhesion.
  • Polystyrene or polystyrene is a thermoplastic that is primarily obtained by radical polymerization of styrene.
  • UV-reactive molecules are preferably covalently immobilized by irradiation with UV light. It is also preferred that the polymer is only applied to the surface of the microarray in predefined areas. In this way, advantageously, similar to circuits, certain Structures, a defined arrangement of the lectin spots or patterns of immobilization of the lectins are predefined. Thus, certain reactions or interactions between lectins and G are lyko Modellen controllable.
  • the surface on which the lectins are to be bound directly is positively and / or negatively charged electrically by plasma treatment.
  • 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.
  • the invention also relates to the use of the microarray and / or the binding assay of the invention for detecting an analyte and / or to a nalysis Pattern A.
  • ellipsometry can also be used.
  • adsorbed layers are measured from one surface using a polarized laser beam.
  • the reflected laser beam with changed polarization can be measured.
  • SPR Surface Plasmon Resonance
  • the to be tested glycostructures can, for example, in production ben be contained include lyko Wegner Natural or synthetic G and derivatives and breakdown products, defined mixtures, synthetic and natural multicomponent mixtures such as, for example, cells, cell supernatants, broths, extracts and / or sera.
  • the screening function of the described microarray and the binding assay is based in particular on the parallel use of many different, or at least two, lectins or lectin systems.
  • the targeted selection of carbohydrate-binding lectins advantageously offers a broad spectrum of binding affinities for defined sugar structures.
  • the use of the microarray and the binding assay thus makes it possible to use lectins in the form of an array for the analysis, in particular structural analysis, of glycoconjugates.
  • glycoconjugates In contrast to the known screening methods, it is advantageously also possible to detect and analyze glycoconjugates based on their glycosylation.
  • the co- and post-translational modes are fications, such as glycosylation, which takes into account and detects proteins that have a decisive influence on the physiological properties of the substances. Due to the high specificity of the selected lectins, even slightly altered sugar structures can be distinguished from one another, and structural information can thus be obtained, above all about unknown glycoconjugates.
  • a pattern analysis of the sample is also advantageously possible. This also enables the comparison of samples - such as cell lines - with each other, which is a common problem in basic research, medicine and clinic.
  • the invention thus enables the detection and analysis of new, unknown glycostructures in complex multi-substance mixtures.
  • Fig. 1 shows a schematic representation of an exemplary embodiment of the lectin microarray
  • 2 shows an arrangement of immobilized lectins on a 96-well microtiter plate in array format
  • FIG. 1 shows the schematic representation of an exemplary embodiment of the lectin microarray.
  • a lectin is immobilized on the surface of a microtiter well by the high affinity blomplex streptavidin-biotin being formed. Complex glycans of glycoproteins can then bind to the saccharide binding sites of the lectin.
  • FIG. 2 shows the arrangement of immobilized lectins on the surface in array format.
  • FIG. 3 shows the advantage of this application example, which lies in the information that only complex glycoconjugates with at least two glycans are detected, which can interact with both the immobilized lectin and the detection lectin at the same time.
  • This demonstration in the form of a sandwich technique makes the analytical result particularly specific and meaningful. example
  • biotinylated RCA (lectin from F.icinus communis)
  • biotinylated WGA (lectin from Triticum vulgaris)
  • Buffer solution A- BisTris buffer pH 7.0; 0.15 mol / L NaCl, 1 mmol / L calcium, manganese and magnesium ions
  • Buffer solution B Buffer A in which 250 ⁇ g / mL BS A (P-Indian serum albumin) are dissolved.
  • Concanavalin A (lectin from Canavalia ensiform), labeled with FITC (fluorescein-5-isothiocyanate, fluorescent dye).
  • biotinylated lectins Con A, RCA. and WGA are each dissolved in buffer A at the following concentrations: Con A: 5.5 ⁇ g / mL
  • the plate is then washed three times with buffer A. Then incubate with 100 ⁇ L buffer B per well for a further 4 h at S ° C. The plate is then washed three times with buffer A.
  • the device produced in this way consists of arranging immobilized lectins on a surface in array format (FIG. 2).
  • the lectins Con A, RCA and WGA labeled with FITC are each dissolved in buffer A at the following concentrations: Con A: 10 ⁇ g / mL RCA: 10 ⁇ g / mL WGA: 10 ⁇ g / mL
  • the plate is then washed three times with buffer A.
  • a positive fluorescence activity is measured in well A 2 and A 3.
  • Well A 1 shows no fluorescence.
  • the glycostructure (s) in the serum each have two glycans (two different glycosylation sites), the glycans have ⁇ -D-galactoses (corresponding to the affinity of RCA see below) and terminal N-acetylglucosamines or sialic acids (according to the affinity of WGA see below).
  • - Mannose-containing structures corresponding to the affinity of Con A do not appear.
  • the properties mentioned apply to the glycoprotein fetuin present in the serum.
  • P-inder-Fetuin has a carbohydrate content of around 30%. It has a total of 6 glycans, one of which
  • glycans are bound with terminal galactoses.
  • the glycostructure ß-D-Gal-1, 4-ß-D-GlcNAc-1, 0-R (terminal lactosamine) is a prerequisite for the binding of a glycan, whereby solid bonds are highly specific only with branched, multivalent N-glycans occurrence.
  • WGA is a chitin-binding lectin that has binding sites for N-acetylglucosamine (GlcNAc) and N-acetyl-neuraminic acid (NeuNAc).
  • GlcNAc N-acetylglucosamine
  • NeuroNAc N-acetyl-neuraminic acid
  • the binding strength for oligosaccharides increases strongly with the number of GlcNAc residues.
  • the disaccharide N, N'-diacetyl-cliitobiose is bound 131 times more than the monomer and the trisaccharide N, N ', N' '-triacetyl-chitotriose is 3700 times stronger.
  • WGA binds glycoproteins containing sialic acid.

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Abstract

L'invention concerne un microréseau et un essai de liaison servant à détecter des glycoconjugués complexes et à analyser la structure de ces derniers, chaque glycoconjugué interagissant avec au moins deux lectines différentes.
PCT/EP2003/011567 2002-10-18 2003-10-17 Dispositif et procede pour analyser la structure de glycostructures complexes et detecter ces dernieres WO2004040307A2 (fr)

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EP2037274A1 (fr) * 2007-09-11 2009-03-18 GALAB Technologies GmbH Investigation de glycane à base de lectine

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