EP1831690A1 - Supports tridimensionnels a nanostructure et microstructure - Google Patents

Supports tridimensionnels a nanostructure et microstructure

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Publication number
EP1831690A1
EP1831690A1 EP05822614A EP05822614A EP1831690A1 EP 1831690 A1 EP1831690 A1 EP 1831690A1 EP 05822614 A EP05822614 A EP 05822614A EP 05822614 A EP05822614 A EP 05822614A EP 1831690 A1 EP1831690 A1 EP 1831690A1
Authority
EP
European Patent Office
Prior art keywords
functional element
element according
functional
group
nanoparticles
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Withdrawn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
EP05822614A
Other languages
German (de)
English (en)
Inventor
Günter Tovar
Herwig Brunner
Achim Weber
Kirsten Borchers
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.)
Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Universitaet Stuttgart
Original Assignee
Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Universitaet Stuttgart
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 Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV, Universitaet Stuttgart filed Critical Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Publication of EP1831690A1 publication Critical patent/EP1831690A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82B—NANOSTRUCTURES FORMED BY MANIPULATION OF INDIVIDUAL ATOMS, MOLECULES, OR LIMITED COLLECTIONS OF ATOMS OR MOLECULES AS DISCRETE UNITS; MANUFACTURE OR TREATMENT THEREOF
    • B82B3/00—Manufacture or treatment of nanostructures by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements

Definitions

  • the present invention relates to functional elements which comprise microstructures comprising biofunctionalizable or biofunctionalized nanoparticles arranged on a support, processes for producing these functional elements and the use thereof.
  • WO 03/056336 A2 describes the preparation and use of microstructures of biofunctionalized nanoparticles which can be used in a wide variety of detection and analysis methods.
  • microstructures described therein can be improved in terms of their sensitivity, especially in cases where a high detection accuracy is required.
  • the present invention is therefore based on the technical problem, means and methods for the production of miniaturized Develop carrier systems with immobilized biological molecules, such as gene chips and protein chips, in which the disadvantages known in the prior art are eliminated, in particular a further increased detection sensitivity is provided and in which the biomolecules in particular under receipt and Protected their biological activity in high packing density are immobilized on a support or can be immobilized and which are suitable for use in a variety of screening and analysis systems, for example in medical Messr and monitoring technology, and in biocomputers.
  • the present invention solves the underlying technical problem by providing a functional element, comprising a carrier having a surface and at least one arranged on the support surface microstructure, wherein the microstructure of a plurality of three-dimensionally stacked layers of nanoparticles is formed and wherein the nanoparticles molecule-specific recognition sites which allow addressability within the microstructure.
  • the invention thus provides multi-dimensionally constructed microstructures comprising a plurality of nanoparticle layers which, in a preferred embodiment, are formed including at least one biomolecule-stabilizing agent.
  • These multi-dimensionally arranged layers of nanoparticles drastically increase the reaction surfaces of the functional element provided for the desired detection reactions, wherein the inclusion of the protein-stabilizing agent in a preferred embodiment simultaneously achieves that when using proteins or peptides as biological active molecules their natural structure and function is preserved.
  • the several three-dimensionally arranged layers of nanoparticles in particular also in a thickness of 10 nm to 10 .mu.m, preferably 50 nm to 2.5 .mu.m, in particular 100 nm to 1, 5 .mu.m stable on the surface of Stay arranged carrier, even if they are rinsing and / or washing steps also longer duration and intensity exposed.
  • the inventively provided three-dimensionally arranged plurality of layers of nanoparticles surprisingly remains stable on the support surface and thus allows, contrary to expectations, a high detection accuracy even with very small amounts of analyte to be detected.
  • the resistance of the three-dimensional microstructures made of nanoparticles which is made possible in accordance with the invention, makes it possible to produce functional layers which can bind spatially resolved specific analytes.
  • the three-dimensional microstructured nanoparticle layers meet all the conditions for use for the production of biological sensor surfaces.
  • the present invention thus provides a functional element on the surface of which one or more microstructures are arranged, each microstructure consisting of many nanoparticles in multiple layers with identical or non-identical molecule-specific recognition sites.
  • the microstructures of the functional elements may include or be provided with biofunctions. This means that the molecule-specific recognition points of the nanoparticles forming the microstructure can recognize and bind corresponding molecules, in particular organic molecules having a biological function or activity. These molecules may be, for example, nucleic acids or proteins. To the molecules that bind to the molecule-specific If recognition sites of the nanoparticles are bound, then other molecules can be bound, for example molecules of a sample to be analyzed.
  • the present invention does not intend to bind biological molecules directly to a planar surface, but rather to immobilize them to a plurality of three-dimensional nanoparticle surfaces, before or after Immobilization can be used to form a microstructure.
  • the functional elements according to the invention which comprise nanoparticular systems with molecule-specific recognition sequences for binding biological molecules, offer several decisive advantages over conventional systems, for example those in which the biological molecules are immobilized directly on the support.
  • the nanoparticles used according to the invention are extremely flexible and inert systems. They can consist, for example, of a wide variety of cores, for example organic polymers or inorganic materials.
  • Inorganic nanoparticles such as silica particles offer the advantage that they are chemically inert and mechanically stable. While surfmers and molecular imprinted polymers have soft nuclei, nanoparticles with silica or iron cores do not swell in solvents. Non-swellable particles do not change their morphology, even if they are suspended several times in solvents for a long time.
  • Functional elements according to the invention which comprise non-swellable particles can therefore be used without problems in analysis or microstructuring processes which require solvent application without adversely affecting the state of the nanoparticles or immobilized biological molecules.
  • Functional elements which contain such nanoparticles can therefore also be used for purifying the biological molecules to be immobilized from complex mixtures of substances which contain undesired substances such as detergents or salts, the molecules to be immobilized optimally over any desired length of washing process Substance mixtures can be separated.
  • superparamagnetic or ferromagnetic nanoparticles with an iron oxide core can be arranged in a magnetic field along the field lines. This property of iron oxide nanoparticles can be used to directly build microstructures, in particular nanoscopic interconnects.
  • the functional elements according to the invention can be used to immobilize a wide variety of biological molecules while retaining their biological activity.
  • the nanoparticles used to form the microstructures have molecule-specific recognition sites, in particular functional chemical groups, which can bind the molecule to be immobilized in such a way that the molecular regions required for the biological activity are present in a state corresponding to the native molecular state.
  • the biomolecules may be bound covalently and / or noncovalently to the nanoparticles, as required.
  • the nanoparticles can have different functional groups, so that either different biomolecules or biomolecules having different functional groups with preferred orientation can be immobilized.
  • the biomolecules can both non-directionally and directionally immobilized on the nanoparticles, with almost any desired orientation of the biomolecules is possible.
  • the immobilization of the biomolecules on the nanoparticles also stabilizes the biomolecules.
  • At least one biomolecule-stabilizing agent in particular at least one protein-stabilizing agent, is included in the microstructure.
  • Such agents further enhance the stabilization of biomolecules.
  • the addition of at least one biomolecule-stabilizing additive, in particular at least one protein-stabilizing additive preserves the functionality of nanoparticle-bound biological molecules, in particular peptides or proteins, within the particle layers when they are dried on a substrate, thus guaranteeing the shelf life of nanoparticulate functional layers.
  • the shelf life is up to one year, preferably up to 8 months, especially 3 months.
  • the inclusion according to the invention of at least one biomolecule-stabilizing agent, in particular of at least one protein-stabilizing agent, into the microstructure thus protects the function, in particular the biological function, and the effectiveness of the functional elements according to the invention.
  • the nanoparticles used to form the microstructures have a comparatively very high surface to volume ratio and accordingly can bind a large amount of a biological molecule per mass. Compared to systems in which biological molecules are bound directly to a planar support, a functional element can thus bind a significantly larger amount of the biological molecules per unit area.
  • the menu According to the invention, the size of the molecules bound per unit area, that is to say the packing density, is so great because several particle layers are stacked on top of one another to produce the microstructure on the carrier surface.
  • a further increase in the amount of biological molecules bound per unit area can be achieved by first coating the nanoparticles with hydrogels and then with biological molecules.
  • the preparation according to the invention and the use of a plurality of three-dimensionally arranged layers of nanoparticles increases the reactive surface compared to previously used planar affinity surfaces. Therefore, the microstructures according to the invention can bind more analyte.
  • the nanoparticle multilayers or multilayers bind the more analyte the more nanoparticles are immobilized per surface.
  • the analyte concentrations and the signal intensity achieved are linearly correlated with one another after binding of the analytes to the nanoparticulate surfaces.
  • the nanoparticles used according to the invention have a diameter of 5 nm to 500 nm. Using such nanoparticles, it is therefore possible to produce functional elements which have very small microstructures of any shape in the nanometer to micrometer range. The use of the nanoparticles to generate the microstructures therefore allows a hitherto unattained miniaturization of the functional elements, which is accompanied by significant improvements of significant parameters of the functional elements.
  • the nanoparticles used according to the invention show a very good adhesion to the materials used for the preparation of the carrier or carrier surfaces.
  • the particles As a result, they can easily be used for a large number of carrier systems and thus for a large number of different functional elements with a wide variety of application areas.
  • the microstructures formed using the nanoparticles are very homogeneous, which leads to a location-independent signal intensity.
  • the functional elements according to the invention may have different microstructures on their carrier surface which consist of different nanoparticles with different molecule-specific recognition sites. Accordingly, these different microstructures can also be assigned different biofunctions.
  • the functional elements can therefore contain juxtaposed microstructures which contain or can be provided with different biological molecules.
  • a functional element may therefore contain, for example, several different proteins or several different nucleic acids or at the same time proteins and nucleic acids.
  • the functional elements according to the invention can be prepared in a simple manner using known methods. For example, it is very easy to produce stable suspensions using nanoparticle suspending agents. Nanoparticle suspensions behave like solutions and are thus compatible with microstructuring processes. Nanoparticle suspensions can therefore be deposited directly, for example using conventional methods such as needle-ring printers, lithographic methods, inkjet methods and / or microcontact methods, onto suitable carriers which have been previously pretreated with a bonding agent for firmly adhering the nanoparticles , By a suitable choice of the connection
  • the microstructure formed can be formed such that it can be partially or completely detached from the carrier surface of the functional element at a later time, for example by changing the pH or the temperature, and optionally transferred to the carrier surface of another functional element.
  • the functional elements according to the invention can be embodied in various forms and can therefore also be used in very different areas.
  • the functional elements according to the invention may be biochips, for example gene or protein arrays, which are used in medical analysis or diagnostics.
  • the functional elements according to the invention can also be used as an electronic component, for example as a molecular circuit, in medical measurement and monitoring technology or in a biocomputer.
  • a “functional element” is understood to mean an element which, either alone or as part of a more complex device, that is to say in connection with other similar or different functional elements, performs at least one defined function
  • the individual constituents of a functional element can perform different functions within a functional element and can contribute to the overall function of the element to varying degrees or in different ways
  • a functional element comprises a Carrier with a carrier surface on which defined layers of nanoparticles are arranged three-dimensionally as microstructure (s), the nanoparticles being provided with biological functions, for example biological molecules such as nucleic acids, proteins and / or PNA molecules, and / or being provided and these are preferably protected by the inclusion of at least one biomolecule-stabilizing agent, in particular a protein-stabilizing agent.
  • biomolecule-stabilizing agents in particular “protein-unstabilizing agents” are meant according to the invention agents which stabilize the three-dimensional structure of proteins, ie secondary, tertiary, quaternary, under drought stress and thus the functionality of the proteins in the dry state, the is called after evaporation of the solvent.
  • the protein-stabilizing agent is a saccharide, in particular sucrose, lactose, glucose, trehalose or maltose, a polyalcohol, in particular inositol, ethylene glycol, glycerol, sorbitol, xylithol, mannitol or 2-methyl-2,4-diene pentanediol, an amino acid, in particular sodium glutamate, proline, alpha-alanine, beta-alanine, glycine, lysine HCl or 4-hydroxyproline, a polymer, in particular polyethylene glycol, dextran, polyvinylpyrrolidone, an inorganic salt, in particular sodium sulfate, ammonium sulfate, potassium phosphate , Magnesium sulfate or sodium fluoride, an organic salt, in particular sodium acetate, sodium polyethylenes, sodium caprylate, propionate, lactate or succinate, or trimethylamine N
  • a “carrier” is understood to mean that component of the functional element which primarily determines the volume and the external shape of the functional element.
  • the term “carrier” means in particular a solid matrix.
  • the carrier may be of any size and shape, such as a ball, cylinder, rod, wire, plate or foil.
  • the carrier may be both a hollow body and a solid body.
  • a solid body is meant, in particular, a body which essentially has no cavities and can consist entirely of a material or a combination of materials.
  • the solid body can also consist of a layer sequence of the same or different materials.
  • the carrier of the functional element in particular the carrier surface, consists of a metal, a metal oxide, a polymer, glass, a semiconductor material or ceramic.
  • the support or its surface is at least about 60%, preferably about 70%, more preferably about 80%, and most preferably about 100% of any of the foregoing materials or a combination of such materials.
  • the support of the functional element consists of materials such as transparent glass, silica, metals, metal oxides, polymers and copolymers of dextranes or amides, for example acrylamide derivatives, cellulose, nylon, or polymeric materials such as polyethylene terephthalate, cellulose acetate, polystyrene or polymethyl methacrylate or a polycarbonate of bisphenol A.
  • the surface of the functional element carrier is planar or also prestructured, for example contains supply and discharge lines.
  • the surface of the carrier and the carrier itself can be impermeable and / or porous.
  • Such carriers are for example membranes or filters.
  • the area sections, not covered by the microstructure, of the surface of the carrier contain functionalities or chemical compounds which prevent non-specific attachment of biomolecules to these surface sections.
  • these chemical compounds are polyethylene glycols, oligoethylene glycols, dextran or a mixture thereof.
  • the surface of the functional element carrier contains an ethylene oxide layer.
  • At least one layer of a connecting means is arranged between the carrier surface and the microstructure.
  • the connecting agent serves for the firm binding of the nanoparticles to the carrier surface of the functional element.
  • the choice of the bonding agent depends on the surface of the support material and the nanoparticles to be bound.
  • the connection means they are preferably charged or uncharged polymers.
  • the bonding agents are preferably weak or strong polyelectrolyte, ie their charge density is pH-dependent or pH-independent.
  • the bonding agent consists of poly (diallyldimethylammonium chloride), a sodium salt of poly (styrenesulfonic acid), a sodium salt of poly (vinylsulfonic acid), poly (allylamine hydrochloride), linear / branched poly (ethyleneimine), poly ( acrylic acid), poly (methacrylic acid) or a mixture of these.
  • the polymer may also be a hydrogel.
  • the bonding agent may also be a charged-gas plasma layer, such as a polyelectrolyte, or a plasma layer having chemically reactive groups.
  • the compound can also be a self-assembled monolayer based on silane, thiol, phosphate or fatty acid.
  • the bonding agent layer consists of at least one layer of a bonding agent.
  • the bonding agent layer can also consist of several layers of different bonding agents, for example of an anionic plasma layer and a cationic polymer layer or of a plurality of polymer layers which are alternately anionic and cationic.
  • Another preferred embodiment of the invention relates to bonding agents whose properties, for example their cohesive properties, can be changed by an external stimulus and which can therefore be switched from the outside.
  • the cohesion properties of the connecting agent can be reduced to such an extent by changing the pH, the ion concentration and / or the temperature that the microstructures bound to the carrier surface of the functional element can be detached and, if appropriate, transferred to the carrier surface of another functional element ,
  • the support in particular the support surface, is pretreated with a surface-activating agent prior to the application of the bonding layers and microstructures in order to improve the bonding of the bonding layers and microstructures to the support or to its surface
  • the surfaces of the support can be activated, for example, by means of chemical processes, for example using primers or an acid or a base.
  • the surface activation can also be done using a plasma.
  • the surface activation may also include the application of a self-assembled monolayer.
  • microstructure in the range of individual micrometers or nanometers are understood as meaning a "microstructure.”
  • microstructure is understood to mean a structure consisting of at least two individual components in the form of several three-dimensionally arranged layers of nanoparticles with molecule-specific recognition sites is arranged on the surface of a carrier, wherein a certain surface portion of the surface of the carrier is covered, which has a defined shape and a defined surface area and which is smaller than the carrier surface.
  • the microstructure is in the shape of a circle, the diameter of the circle is in the micrometer range. If the microstructure is designed as a rectangle, for example, the width of this rectangle is in the micrometer range. According to the invention, provision is made in particular for the at least one surface-length parameter, which determines the surface section covered by the microstructure, to be smaller than 999 ⁇ m. Since the microstructure according to the invention consists of at least two nanoparticles, the lower limit of this surface-length parameter is 10 nm.
  • the three-dimensionally arranged layers of nanoparticles, which form the microstructure in their entirety have a total thickness of 10 nanometers to 10 micrometers.
  • three-dimensional support means a support which is suitable for the attachment of functional groups or biomolecules the surface of a, possibly microstructured, nanoparticle aggregate, where one aggregate means two, three or many layers of nanoparticles which are arranged one above the other, in particular the explicit expansion into the third te dimension by the aforementioned application of, optionally microstructured, nanoparticle multiple or multilayer reactive surface per occupied footprint.
  • the area section covered by the microstructure according to the invention can have any desired geometric shape, for example that of a circle, an ellipse, a square, a rectangle or a line.
  • the microstructure can also be composed of a plurality of regular and / or irregular geometric shapes.
  • the functional element according to the invention is, for example, a gene chip or a protein array, the microstructure preferably has a circular or elliptical-like shape.
  • the functional element according to the invention is an electronic component for use in a biocomputer, the microstructure may also have a circuit-like shape. According to the invention it is also provided that a plurality of microstructures of the same or different shape are arranged at a distance from one another on the carrier surface of a functional element.
  • the microstructures are applied to the surface of the functional element carrier by means of a ring / pin by means of lithographic processes, such as photolithography or micro-pen lithography, ink-jet techniques or micro-contact printing processes, for example.
  • lithographic processes such as photolithography or micro-pen lithography, ink-jet techniques or micro-contact printing processes, for example.
  • the selection of the method by means of which the microstructure or microstructures are applied to the surface of the functional element depends on the surface of the carrier material, the nanoparticles containing the microparticles. Ro Design should form, and the subsequent application of the functional element.
  • nanoparticle is understood as meaning a particulate binding matrix which has first molecule-specific recognition sites comprising functional chemical groups
  • the nanoparticles used according to the invention comprise a core with a surface on which the first functional groups are arranged, which are located in the By virtue of the interaction between the first and second functional groups, the biomolecule is immobilized on the nanoparticle and thus on the microstructure of the functional element and / or can be immobilized thereon.
  • the nanoparticles used according to the invention for forming the microstructures have a size of less than 500 nm, preferably less than 150 nm.
  • "addressable” means that the microstructure is retrievable and / or detectable after application of the nanoparticles to the carrier surface If the microstructure is applied to the carrier surface using, for example, a mask or a stamp, the address of the On the one hand, microstructure results from the coordinates x and y of the region of the carrier surface on which the microstructure is applied, on the other hand, the address of the microstructure results from the molecule-specific recognition sites on the surface of the nanoparticles, which retrieves or retrieves If the microstructure is biologically is functionalizable, ie nanoparticles with molecule-specific recognition sites to which no biomolecules are bound, the microstructure can be rediscovered and / or demonstrated that one or more biomolecules bind specifically to the molecule-specific recognition sites of the nanoparticles forming the microstructure however, to the surface portions of the support surface that are not covered by the microstructure.
  • the immobilized molecule is labeled with detection markers such as fluorophores, spin labels, gold particles, radioactive labels, etc.
  • detection of the microstructure can be accomplished using appropriate detection methods.
  • the microstructure is biofunctionalized, that is to say comprises nanoparticles to whose molecule-specific recognition sites one or more biomolecules are already bound, "addressable” means that these biomolecules can be found and / or detected by interaction with complementary structures of further molecules or by metrological methods, only the microstructure consisting of nanoparticles shows corresponding signals, but not the surface sections of the carrier surface, which are not covered by the microstructure.
  • the matrix-assisted laser desorption / ionization time-of-flight mass spectrometry MALDI-TOF
  • MS matrix-assisted laser desorption / ionization time-of-flight mass spectrometry
  • MS which has become an important method for the analysis of a wide variety of substances, in particular proteins, waveguide spectroscopy, fluorescence, impedance
  • the biological molecule while maintaining its biological activity on the surface of the ro Quilt forming nanoparticles is bound or immobilized or can be bound or immobilized, wherein the molecule is preferably directionally bound or becomes.
  • the biological activity of a molecule is understood to mean all functions that it performs in an organism in its natural cellular environment.
  • the molecule is a protein, it may be specific catalytic or enzymatic functions, immune defense functions, transport and storage function, regulatory function, transcriptional and translational functions, and the like.
  • the biological function may be, for example, the coding of a gene product or that the nucleic acid is useful as a template for the synthesis of other nucleic acid molecules or as a binding motif for regulatory proteins.
  • Retention of biological activity means that a biological molecule, after immobilization on the surface of a nanoparticle, can exert the same or nearly the same biological functions to at least similar extent as the same molecule in a non-immobilized state under suitable in vitro conditions the same molecule in its natural cellular environment.
  • the term "directionally immobilized” or “directed immobilization” means that a molecule is bound at defined positions within the molecule to the molecule-specific recognition sequences of a nanoparticle such that the three-dimensional structure is for biological activity required domain (s) is not changed from the non-immobilized state and that this domain (s), for example for cellular reactants, is / are freely accessible to them upon contact with other native cellular reactants.
  • Directed immobilized also means that when immobilizing a molecular species, all or nearly all of the individual molecules, but at least more than 80%, preferably more than 85% of all molecules on the surface of the nanoparticles forming the microstructure have an identical or nearly identical orientation reproducibly take.
  • the biological molecule immobilized or immobilizable on the microstructure of the functional element according to the invention is in particular a nucleic acid, a protein, a protein complex, a PNA molecule, a fragment thereof or a mixture thereof.
  • nucleic acid is understood as meaning a molecule which consists of at least two nucleotides connected via a phosphodiester bond.
  • Nucleic acids may be both deoxyribonucleic acid and ribonucleic acid.
  • the nucleic acid can be both single-stranded and double-stranded. In the context of the present invention, therefore, a nucleic acid can also be an oligonucleotide.
  • the nucleic acid bound to the microstructure of the functional element according to the invention preferably has a length of at least 10 bases.
  • a nucleic acid may be of natural or synthetic origin.
  • the nucleic acid may be modified by genetic engineering methods against the wild-type nucleic acid and / or unnatural and / or unusual nucleic acid. Contain linoleic acid components.
  • the nucleic acid may be linked to other types of molecules, such as proteins.
  • a "protein” is understood to mean a molecule which comprises at least two amino acids which are linked to one another via an amide bond
  • a protein may therefore also be a peptide, for example an oligopeptide, a polypeptide or, for example Such a protein may be of natural or synthetic origin
  • the protein may be modified by genetic engineering methods from the wild-type protein and / or may contain unnatural and / or unusual amino acids
  • the protein may be derivatized from the wild-type form.
  • a protein may in particular be an enzyme, a receptor, a cytokine, a structural protein Antigen or an antibody be.
  • Antibody means a polypeptide that is substantially encoded by one or more immunoglobulin genes, or fragments thereof that specifically bind / bind and detect an analyte (antigen) Antibodies are, for example, as intact immunoglobulins or as one Series of fragments generated by cleavage with various peptidases. “Antibody” also means modified antibodies (eg, oligomeric, reduced, oxidized and labeled antibodies). “Antibody” also includes antibody fragments which are either modified by whole antibody modification or by de novo synthesis using DNA Recombination techniques have been generated. The term “antibody” includes both intact molecules and fragments thereof, such as Fab, F (ab ') 2 and Fv 1 which can bind the epitope determinant.
  • protein complex means an aggregating unit of at least two proteins
  • a “protein complex” may also include nucleic acids, metal ions and other substances.
  • PNA Protein Nucleic Acid or Polyamide Nucleic Acid
  • PNA Peptide Nucleic Acid or Polyamide Nucleic Acid
  • DNA DNA
  • PNA sequences comprise a polyamide backbone of N- (2-aminoethyl) glycine units and have no glucose units and no phosphate groups.
  • molecule-specific recognition sites are understood as meaning regions of the nanoparticle which allow a specific interaction between the nanoparticle and biological molecules as target molecules.
  • the interaction can be based on targeted attractive interaction between one or more pairs of first functional molecules Groups of the nanoparticle and the first functional groups binding, complementary second functional groups of the target molecules, so the biological molecules are based.
  • Single interacting pairs of functional groups between nanoparticles and biological molecule are each spatially fixed to the nanoparticle and the biological molecule arranged Fixation need not be a rigid arrangement, but rather can be carried out quite flexible.
  • the attractive interaction between the functional groups of the nanoparticles and the biological molecules can be carried out in the form of non-covalent bonds such as van der Waals bonds, hydrogen bonds, ⁇ - ⁇ bonds, electrostatic interactions or hydrophobic interactions. Also conceivable are reversible covalent bonds as well as mechanisms based on complementarity of shape or form.
  • the inventively provided interactions between the molecule-specific recognition sites of the nanoparticles and the target molecule thus based on directed interactions between the pairs of functional groups and on the spatial arrangement of these pairs forming groups to each other on the nanoparticle and the target molecule. This interaction results in a covalent or non-covalent affinity bond between the two binding partners, such that the biological molecule is immobilized on the surface of the nanoparticles forming the microstructure.
  • the first functional groups which are part of or form the molecule-specific recognition sites on the surface of the nanoparticle are selected from the group consisting of active ester, alkyl ketone group, aldehyde group, amino group, carboxy group, epoxy group, maleimido group, Hydrazine group, hydrazide group, thiol group, thioester group, oligohistidine group, strep-tag I 1 strep-tag II, desthiobiotin, biotin, chitin, chitin derivatives, chitin binding domain, metal chelate complex, streptavidin, streptactin, avidin and neutravidin.
  • the molecule-specific recognition site is a larger molecule such as a protein, an antibody, etc., which contains the first functional groups.
  • the molecule-specific recognition site may also be a molecular complex consisting of several proteins and / or antibodies and / or nucleic acids, at least one of these molecules containing the first functional groups.
  • a protein may comprise, as a molecule-specific recognition sequence, an antibody and an associated protein.
  • the antibody may also comprise a streptavidin group or a biotin group.
  • the antibody linked to the antibody may be a receptor, for example, an MHC protein, cytokine, a T cell receptor such as the CD-8 protein, and others capable of binding a ligand.
  • a molecular complex can also comprise a plurality of proteins and / or peptides, for example a biotinylated protein which binds a further protein in a complex and additionally a peptide.
  • the second functional group ie the functional group of the biomolecule to be immobilized, is selected according to the invention from the group consisting of the group consisting of active ester, alkyl ketone group, aldehyde group, amino group, carboxy group, epoxy group, maleimido group, hydrazine group, hydrazide group, thiol group , Thioester group, oligohistidine group, Strep-Tag I, Strep-Tag II, desthiobiotin, biotin, chitin, chitin derivatives, chitin binding domain, metal chelate complex, streptavidin, streptactin, avidin and neutravidin.
  • the first and second functional groups may have been generated by, for example, molecular imprinting.
  • the first and second functional groups may also be active esters, such as the so-called surfmers.
  • a nanoparticle used according to the invention therefore has on its surface a first functional group which is covalently or non-covalently linked to a second functional group of a biomolecule to be immobilized, the first functional group being a group other than the second functional group.
  • the two binding groups must be complementary to one another, ie be able to form a covalent or non-covalent bond with one another.
  • the second functional group is a hydrazine or hydrazide group.
  • the second functional group is an alkyl ketone, in particular methyl ketone or aldehyde group.
  • the second complementary functional group is a thioester group. If the first functional group used is a thioester group, according to the invention the second functional group is a thiol group.
  • the second functional complementary group is an oligohistidine group.
  • the second functional complementary group is a metal ion chelate complex.
  • Strep-Tag I 1 Strep-Tag II, Biotin or Desthiobiotin is used as the first functional group
  • Streptavidin, Streptactin, Avidin or Neutravidin is used as the second complementary functional group.
  • streptavidin, streptactin, avidin or neutravidin is used as the first functional group
  • strep-tag I 1 Strep-Tag II, biotin or desthiobiotin is used as the second complementary functional group.
  • a chitin binding domain is used as the second functional complementary group.
  • a chitin binding domain is used as the first functional group
  • chitin or a chitin derivative is used as the second functional complementary group.
  • first and / or second functional groups may be connected to the biomolecule or nanoparticle core to be immobilized by means of a spacer or may be introduced by means of a spacer to the nanoparticle core or into the biomolecule.
  • the spacer serves on the one hand as a spacer of the functional group to the core or biomolecule, on the other hand as a support for the functional group.
  • such a spacer can be alkylene groups or ethylene oxide oligomers having from 2 to 50 carbon atoms, which in a preferred embodiment is substituted and has heteroatoms.
  • the second functional groups are a natural constituent of the biomolecule, in particular of a protein.
  • a protein of medium size ie a size of about 50 kDa with about 500 amino acids
  • reactive amino groups there are about 20 to 30 reactive amino groups, which in principle come into question as a functional group for immobilization.
  • it is the amino group at the N-terminal end of a protein. All other free amino groups, in particular those of the lysine residues, are also suitable for the immobilization.
  • Arginine with its guanidium group is also considered as a functional group.
  • the second functional groups into the biomolecule by means of genetic engineering, biochemical, enzymatic and / or chemical derivatization or chemical synthesis methods.
  • the derivatization should be such that the biological activity is retained after immobilization.
  • unnatural amino acids can be inserted into the protein molecule by genetic engineering or during chemical protein synthesis, for example together with spacers or linkers.
  • Such unnatural amino acids are compounds which have an amino acid function and a radical R and are not defined by a naturally occurring genetic code, these amino acids most preferably having a thiol group.
  • It can also be provided according to the invention to modify a naturally occurring amino acid, for example lysine, for example by derivatization of its side chain, in particular its primary amino group, with the carboxylic acid function of levulinic acid.
  • tags can be introduced into the protein by modification of a protein, wherein tags, ie labels, are added to the protein, preferably to the C-terminus or the N-terminus.
  • tags can also be arranged intramolecularly.
  • a protein is modified by adding at least one strep tag, for example a strep tag I or strep tag II or biotin.
  • a Strep tag is also understood as meaning functional and / or structural equivalents, provided that they can bind streptavidin groups and / or their equivalents.
  • a protein is also provided according to the invention by adding a His tag containing at least three histidine residues, but preferably one oligohistidine.
  • the His tag introduced into the protein can then bind to a metal chelating complex-comprising molecule-specific recognition site.
  • proteins which are modified for example, with unnatural amino acids, natural, but unnaturally derivatized amino acids or specific strep tags, or bind antibody-bound proteins with nanoparticle surfaces which are complementary to them, that a suitable specific, in particular non-covalent binding of the proteins and thus a directed immobilization of the proteins to the surfaces takes place.
  • these molecules can additionally be covalently bound, in For example, with a crosslinker such as glutaric dialdehyde. This will make the protein surfaces more stable.
  • the nanoparticles deposited to form a microstructure on the carrier surface of the functional element have, in addition to the surface with the molecule-specific recognition sites, a nucleus.
  • a "core" of a nanoparticle is understood as meaning a chemically inert substance which serves as a carrier for the molecule to be immobilized
  • the core is a compact or hollow particle with a size of 5 nm to 500 nm ,
  • the core of the nanoparticles used according to the invention consists of an inorganic material such as a metal, for example Au, Ag or Ni, silicon, SiO 2 , SiO, a silicate, Al 2 O 3 , SiO 2 ⁇ Al 2 O 3 , Fe 2 O 3 , Ag 2 O, TiO 2 , ZrO 2 , Zr 2 O 3 , Ta 2 O 5 , zeolite, glass, indium tin oxide, hydroxyapatite, a Q-dot or a mixture thereof or contains this.
  • a metal for example Au, Ag or Ni, silicon, SiO 2 , SiO, a silicate, Al 2 O 3 , SiO 2 ⁇ Al 2 O 3 , Fe 2 O 3 , Ag 2 O, TiO 2 , ZrO 2 , Zr 2 O 3 , Ta 2 O 5 , zeolite, glass, indium tin oxide, hydroxyapatite, a Q-dot or a mixture thereof or contains this.
  • the core consists of or contains an organic material.
  • the organic polymer is polypropylene, polystyrene, polyacrylate, a polyester of lactic acid, or a mixture thereof.
  • the cores of the nanoparticles used according to the invention can be prepared using conventional methods known in the art, such as sol-gel synthesis, emulsion polymerization, suspension polymerization, and the like. After preparation of the cores, the surfaces of the cores are transformed by chemical modification reactions with the specific first functional For example, using conventional methods such as graft polymerization, silanization, chemical derivatization, etc. One way to produce surface-modified nanoparticles in one step, the use of Surfmeren in the emulsion polymerization. Another possibility is molecular imprinting.
  • molecular embossing is meant the polymerization of monomers in the presence of templates, which can form a relatively stable complex with the monomer during the polymerization.
  • the materials thus prepared can specifically bind template molecules to the template molecules structurally related molecular species or molecules that structurally related or identical groups have the template molecules or parts thereof.
  • a template is therefore a substance present in the monomer mixture during the polymerization to which the polymer formed has an affinity.
  • reactive surfmers possess functionalizable end groups that can be reacted under mild conditions with nucleophiles such as primary amines (amino acids, peptides, proteins), thiols, or alcohols. In this way, a variety of biologically active polymeric nanoparticles accessible.
  • the density of the first functional groups and the distance between these groups can be optimized according to the invention for each molecule to be immobilized.
  • the environment of the first functional groups on the surface can also be prepared in a corresponding manner with regard to the most specific possible immobilization of a biomolecule.
  • additional functions are anchored in the core, which enable a simple detection of the nanoparticle cores and thus of the microstructures using suitable detection methods.
  • These functions may, for example, be fluorescent labels, UV / Vis labels, superparamagnetic functions, ferromagnetic functions and / or radioactive labels.
  • Suitable methods for detecting nanoparticles include, for example, fluorescence or UV-Vis spectroscopy, fluorescence or light microscopy, MALDI mass spectrometry, waveguide spectroscopy, impedance spectroscopy, electrical and radiometric methods. A combination of the methods can be used for the detection of nanoparticles.
  • the core surface can be obtained by applying additional functions, such as fluorescence labeling. ments, UV / Vis markings, superparamagnetic functions, ferromagnetic functions and / or radioactive markings.
  • additional functions such as fluorescence labeling. ments, UV / Vis markings, superparamagnetic functions, ferromagnetic functions and / or radioactive markings.
  • the core of the nanoparticles be surface-modified with an organic or inorganic layer having the first functional groups and the additional functions described above.
  • the core surface has chemical compounds which serve for steric stabilization and / or for preventing a conformational change of the immobilized molecules and / or for preventing the addition of further biologically active compounds to the core surface.
  • chemical compounds are preferably polyethylene glycols, oligoethylene glycols, dextran or a mixture thereof.
  • Nanoparticles with ion-exchange functions are particularly suitable for optimizing the MALDI analysis, since it can bind interfering ions.
  • the biological molecule immobilized on the microstructure of the functional element has markers which enable a simple detection of the biological molecules immobilized on the microstructure using suitable detection methods.
  • These labels may be, for example, a fluorescent label, a UV / Vis label, a superparamagnetic label, act table function, a ferromagnetic function and / or a radioactive marker.
  • suitable detection methods for these labels are, for example, fluorescence or UV-VIS spectroscopy, MALDI mass spectrometry, waveguide spectroscopy, impedance spectroscopy, electrical and radiometric methods or a combination of these methods.
  • a further embodiment of the invention relates to a functional element having at least one biological molecule immobilized on the microstructure, to which immobilized molecule at least one further biological molecule is covalently or noncovalently bound.
  • the molecule immobilized on the microstructure is a protein, for example, a second protein or an antibody may be bound thereto, for example by protein-protein interaction or by antibody-antigen binding.
  • the molecule immobilized on the microstructure is a nucleic acid, for example, a protein may be bound thereto.
  • a further preferred embodiment of the invention relates to a functional element whose microstructure (s) consists of several superimposed layers of the same nanoparticles, each individual layer being firmly bound to the underlying layer via the already described bonding layers of suitable polymers.
  • Yet another preferred embodiment of the invention relates to a functional element, on the support surface of which a plurality of different microstructures are arranged side by side, which are made of
  • Such functional elements therefore contain side by side microstructures to which different biological molecules are immobilized or can be immobilized.
  • the support surface of such functional elements may therefore for example comprise at the same time microstructures on which proteins are or may be immobilized, and microstructures on which nucleic acids are or may be immobilized.
  • the functional element can also have microstructures to which different proteins or different nucleic acids are or can be immobilized simultaneously.
  • a further embodiment of the invention relates to a functional element in which the surface portions of the support surface, which are not covered by the microstructure, are modified by applying additional functionalities or chemical compounds.
  • These may in particular be functionalities or chemical compounds which prevent a non-specific attachment of biomolecules to the regions of the carrier surface which are not covered by the microstructure.
  • these chemical compounds are polyethylene glycols, oligoethylene glycols, dextran or a mixture thereof.
  • the surface of the functional element carrier contains an ethylene oxide layer.
  • the present invention likewise relates to a method for producing a functional test according to the invention, wherein at least one layer of a bonding agent and then at least one microstructure consisting of nanoparticles with molecule-specific recognition sequences are applied to the surface of a suitable substrate.
  • the surface of a functional element is pre-structured before the application of the bonding agent layer.
  • a layer of a compound can then be applied to the prestructured carrier surface, which prevents non-specific attachment of biological molecules to the carrier surface.
  • This is preferably an ethylene oxide layer.
  • the surface of the support of the functional element according to the invention is activated after the pre-structuring and before the application of the bonding agent layer. Activation may also include purification.
  • the activation of the surface of the support of the functional element can be carried out according to the invention using a chemical process, in particular using primers or acids or bases. However, according to the invention it is also possible to activate the surface of the carrier using a plasma. The activation may also include the application of a SeIf-Assembled monolayer.
  • the bonding agent in a structured manner to the surface of the carrier.
  • Structured means in context of the invention in that a bonding agent layer defined in terms of shape and area is applied to the carrier surface, the bonding agent layer thus applied defining the surface portion of the carrier surface to be covered later by the microstructure.
  • the microstructure is then applied by dipping the functional element support in a nanoparticle suspension, wherein the nanoparticles adhere only to the structured applied bonding agent layer, but not to the surface portions of the support surface, which have no bonding agent layer. In this way, a microstructure defined in terms of shape and area is produced.
  • the net surface charge of functional nanoparticles depends on the surface modification and the suspension medium. A surface charge different from 0 stabilizes the particle suspension (electro-static repulsion).
  • aqueous solutions as suspending medium are well suited for microstructured nanoparticle layers to be applied to polyelectrolyte-coated surfaces (for example silicon or glass).
  • the particles generally carry a net negative charge.
  • the zeta potentials of functionalized nanoparticles are
  • the structured bonding agent layer is applied, for example, by means of a needle-ring printer, an ink-jet method, for example a piezoelectric or thermo-process, or a microcontact printing method.
  • a lithographic process in particular the photolithography or the micropen lithography process, the carrier surface is covered with the bonding agent and then the bonding agent layer thus produced is patterned by means of the lithographic process.
  • the microstructure to be applied can be designed such that the microstructure or parts thereof can be switched from the outside, for example by changing the pH, the ion concentration or the temperature, at a later time again (debond on command). This allows, for example, a microstructure to be transferred from one functional element to another.
  • Stable nanoparticle suspensions can be prepared simply by suspending the nanoparticles in liquids, in particular aqueous media, if appropriate using additional constituents, for example pH agents, suspending aids, etc.
  • the carrier In the second embodiment of the method according to the invention for producing a functional element, it is provided to first provide the carrier with a bonding agent layer covering the entire carrier surface. This can be done for example by immersing the carrier in a suspension or solution of the bonding agent.
  • the microstructure is subsequently produced by, for example, using a nanoparticle suspension using a needle-ring printer, an ink-jet printer. driving, for example, a piezoelectric or thermal process, or a MikroWallet horrbacters structured applied and thus a shape and area defined microstructure is generated.
  • a lithographic process in particular the photolithography or micropen lithography process
  • the carrier surface is covered with the nanoparticle suspension and then the nanoparticle layer thus produced is patterned by means of the lithographic process.
  • the nanoparticles applied to the microstructure on the carrier surface of a functional element according to the invention may be biofunctionalizable nanoparticles, ie nanoparticles which have only molecule-specific recognition sites but to which no biological molecules are yet bound.
  • biofunctionalized nanoparticles for structuring the carrier surface ie nanoparticles at whose molecule-specific recognition sites biological molecules have already been immobilized while retaining their biological activity.
  • the immobilized biological molecule is in particular a protein, a PNA molecule or a nucleic acid.
  • the nanoparticles After the nanoparticles have been applied to the carrier surface of the functional element, it is possible in accordance with the invention to subsequently further convert the particles.
  • the particles contain reactive esters, they can be used to directly bind proteins.
  • the nanoparticles can also be implemented in order to provide them with additional functions.
  • the present invention also relates to the use of the functional element according to the invention for the examination of an analyte in a sample and / or for its isolation and / or purification therefrom, wherein the functional element according to the invention is used, for example, as a gene array or gene chip or as a protein chip. Array is executed.
  • an "analyte” is understood as meaning a substance in which the type and amount of its individual constituents are determined and / or which is to be separated off from mixtures the analyte to proteins, nucleic acid, carbohydrates and the like.
  • the analyte is a protein, peptide, drug, contaminant, toxin, pesticide, antigen or nucleic acid.
  • a sample is meant an aqueous or organic solution, emulsion, dispersion or suspension containing an analyte as defined above in isolated and purified form or as part of a complex mixture of different substances
  • a sample may also be a culture medium, such as a fermentation medium, in which organisms, for example, a biological fluid, such as blood, lymph, tissue fluid, etc., are taken
  • a sample in the sense of the invention may also be an aqueous solution, emulsion, dispersion or suspension of an isolated and purified analyte
  • a sample may already have been subjected to purification steps. but can also un have been purified.
  • the present invention therefore also relates to the use of the functional element according to the invention for carrying out analysis and / or detection methods, these methods being MALDI mass spectrometry, fluorescence or UV-VIS spectroscopy, fluorescence or light microscopy, waveguide spectroscopy, an electrical method such as impedance spectroscopy or a combination of these methods.
  • the present invention also relates to the use of a functional element according to the invention for controlling cell adhesion or cell growth.
  • the present invention also relates to the use of a functional element according to the invention for the detection and / or isolation of biological molecules.
  • a functional element according to the invention to whose microstructures one, preferably single-stranded, nucleic acid is immobilized, can be used to detect a complementary nucleic acid in a sample and / or to isolate this complementary nucleic acid.
  • a functional element according to the invention, to whose microstructures a protein is immobilized can be used for example for the detection and / or isolation of a protein interacting with the immobilized protein from a sample.
  • the present invention also relates to the use of a functional element according to the invention for the development of pharmaceutical preparations.
  • the invention also relates to the use of the functional elements according to the invention for the investigation of the effects and / or side effects of pharmaceutical preparations.
  • the functional elements according to the invention can likewise be used for the diagnosis of diseases, for example for the identification of pathogens and for the identification of mutated genes which lead to the development of diseases.
  • Another possible use of the functional elements according to the invention consists in the investigation of microbiological contamination of surface waters, groundwater and soil. Likewise, the functional elements of the invention can be to investigate the microbiological contamination of food or animal feed.
  • a further preferred use of the functional elements according to the invention is the use of the functional element according to the invention as an electronic component, for example as a molecular circuit, etc., in medical technology or in a biocomputer.
  • the use of the functional element according to the invention as optical memory in optical information processing is particularly preferred, the functional element according to the invention in particular comprising photoreceptor proteins immobilized on microstructures, which can convert light directly into a signal.
  • the present invention also relates to a method for the identification and / or detection of analytes in a solution or suspension.
  • a functional element according to the invention is provided in a first process step, which is brought into contact with an analyte-containing solution or suspension in a second process step.
  • unbound analyte is removed from the functional element according to the invention by washing with a biocompatible washing liquid.
  • a detection method is carried out.
  • the biocompatible washing liquid is water and / or buffer, e.g. B. Phosphate buffered saline: PBS, and / or buffer with the addition of a detergent, eg. TritonX-100.
  • the support may be sequentially stored in water and buffer at room temperature, if appropriate, with a detergent or buffer, if appropriate with a detergent, and water, for example, for 30 min each.
  • a fluorescence detection method or a MALDI mass spectrometry method is used as the detection method for analytes in a solution or suspension.
  • a method of the aforementioned type for identifying and / or detecting analytes in a solution or suspension is particularly preferred, after performing the aforementioned first three method steps in a fourth method step, according to which in a preferred embodiment a fluorescence detection method is used fluorescence-labeled analyte and / or fluorescently labeled biologically active bound to the nanoparticle molecule is excited with light and read with light.
  • the analyte and / or the biological molecule bound to the molecule-specific recognition sites of the nanoparticles is preferably fluorescence-labeled.
  • FIG. 1 shows scanning electron micrographs of a three-dimensional nanoparticle microstructure (detail of a microspot, diameter -150 ⁇ m), produced by 5 times the application of a 16% nanoparticle suspension in aqueous trehalose solution (5% w / v).
  • FIG. 2 shows scanning electron micrographs of nanoparticle layers (sections of microspots, diameter -150 ⁇ m), produced by 5 times the application of nanoparticle suspension (1%, 2%, 4%, 8%, 16% and 32%). ) in aqueous trehalose solution (5% w / v) on polyelectrolyte-coated silicon surfaces. The particle surfaces were incubated for 2 h in PBS buffer and washed for 30 min each in PBS / 0.1% TritonX-100, PBS and MilliQ water.
  • FIG. 3 shows scanning force micrographs (100 ⁇ 100 ⁇ m 2 ) of three-dimensional nanoparticle microstructures (sections of microspots, diameter -150 ⁇ m), produced by 5-fold application of 2%, 16% and 32% nanoparticle suspension in aqueous trehalose solution (5% (w / v)) on polyelectrolyte-coated silicon surfaces.
  • FIG. 4 shows the fluorescence scan of a nanoparticle microarray. Hase IgG nanoparticles were spotted, incubated with Cy5- labeled anti-rabbit IgG. The bound amount of analyte per spot increases with the amount of particles transferred per spot.
  • FIG. 5 shows the linear relationship between signal intensity and analyte concentration.
  • Goat IgG or rabbit IgG nanoparticles were spotted. Incubation was carried out with Cy5-labeled anti-goat or Cy3-labeled anti-rabbit IgG in different concentrations. Sample concentrations left: 0.04 - 4 pM, right: 4 - 400 pM. Left: The sensitivity (slope) increases for higher particle amounts / spot.
  • FIG. 6 shows the fluorescence scan of a nanoparticle microarray.
  • Goat IgG or hare IgG nanoparticles were spotted in suspensions with different solids contents. Incubation was carried out with a solution of Cy5-labeled anti-goat or Cy3-labeled anti-rabbit IgG.
  • the nanoparticulate microstructures selectively bind only the specific interaction partners of the immobilized capture molecules.
  • FIG. 7 shows the dependence of the functional integrity of particle-bound capture proteins on the trehalose concentration in the spotting suspension.
  • FIG. 8 shows the dependence of the functional integrity of particle-bound catcher proteins on the trehalose concentration in FIG.
  • IgG nanoparticles or rabbit IgG nanoparticles were incubated after 5 weeks of storage with Cy5-labeled anti-goat IgG and Cy3-labeled anti-hase IgG.
  • FIG. 9 shows a light micrograph of the gold substrate before nanoparticles were immobilized thereon (25 ⁇ ) and a photograph of the streptavidin particle-coated surface (1000 ⁇ , dark field).
  • the upper spectrum (blue) is a MALDI-TOF spec- trum of the analyte solution with which the nanoparticle surface was incubated (biotinylated and non-biotinylated insulin).
  • the lower spectrum is the MALDI-TOF spectrum of nanoparticle-bound molecules after incubation.
  • Example 1 Preparation of nanoparticle-based protein biochips for fluorescence readout
  • nanoparticle-based protein biochips which are suitable for fluorescence readout
  • glass substrates are used.
  • the adhesion of nanoparticles to surfaces is largely mediated by electrostatic interaction.
  • For adsorption of protein-coated nanoparticles on the substrate are mostly
  • the substrates are incubated at RT for 20 min in an aqueous polycation solution (0.02 M poly (allylamine) (based on the monomer), pH 8.5), for 5 min in MiIIiQ- H 2 O and then centrifuged dry.
  • aqueous polycation solution 0.2 M poly (allylamine) (based on the monomer), pH 8.5
  • a 1 wt .-% aqueous suspension of the silica particles is 10
  • 1 mg carboxy-functionalised silica particles is mixed with 4 .mu.l of a solution of IgG (11, 3 mg / ml) and 10 .mu.l of a solution of EDC (N- (3-dimethylaminopropyl) -N '- ethylcarbodiimide hydrochloride, 3.8 mg / mL ) and made up to 1 ml with MES buffer (pH 4.5).
  • the particles are used to coat in 5% (w / v) aqueous trehalose solution ( Figures 7, 8).
  • hare and / or goat IgG-coated nanoparticles are transferred onto the pretreated glass substrate with the aid of a pin-ring spotter.
  • concentration of the suspensions used is 0.5% - 50% (w / v) ( Figure 1-6).
  • Per pin contact with the surface about 50 pl of suspension are transferred, it is printed five times per spot.
  • the resulting nanoparticle layers are 100 nm - 1500 nm thick.
  • the Spof diameter is about 150 microns.
  • the placement of the individual spots on the substrate is freely programmable.
  • the nanoparticle surfaces are first blocked for one hour with a 3% (w / v) solution of BSA in PBS buffer, then incubated for 1.5 h in the dark at RT with the analyte solution and then each 30 min in PBS / 0.1% TritonX 100, washed in PBS and MilliQ water. All steps are performed in glass slide racks.
  • the analyte solution consists of fluorescence-labeled IgG molecules dissolved in PBS buffer (Figure 4: 40 pM Cy5-labeled anti-hase IgG 1 Figure 5: 0.04 pM, 0.4 pM and 4 pM and 4 pM, 40 pM and 400 pM Cy5-labeled anti-goat IgG, Figure 6: 400 pM Cy3-labeled anti-rabbit antibody and 400 pM Cy5-labeled anti-goat antibody). Chip selection:
  • the fluorescence signal of the bound analyte molecules is detected in a commercial chip reader system from ArrayWorx.
  • the exposure times are between 0.1s and 2s and are kept constant within an experiment.
  • the signal intensities are stored in the form of gray scale graduations.
  • the evaluation of the data takes place with the help of the program Aida of the company Raytest, Berlin.
  • Gold surfaces in this case the MALDI target itself, are rubbed off with acetone, sonicated for 3 minutes in 1: 1 isopropanol / HCl (0.2 M), rinsed with isopropanol and blown dry with nitrogen. They are then incubated for 20 min at RT in aqueous polyanion solution (0.02 M (based on the monomer) poly (acrylic acid) in MiIIiQ-H 2 O, pH 8.5), washed for 5 min in MiIIiQ-H 2 O. , 20 min in polycation solution (see Example 1) incubated, washed for a further 5 min and blown dry with nitrogen.
  • aqueous polyanion solution 0.02 M (based on the monomer) poly (acrylic acid) in MiIIiQ-H 2 O, pH 8.5
  • Silica particles are synthesized as in Example 1 and subsequently amino- and carboxy-functionalized.
  • Streptavidin binding to silica particles 2.68 nmol of streptavidin are introduced into 10 ml of 0.1 M MES buffer (pH 5). 5 mg of the carboxy particles are added. For this purpose, 2 ⁇ mol of EDC are added. After shaking at RT for 3 hours, the particles are washed once with 10 ml of MES buffer (pH 5) and once with 10 ml of phosphate buffer (pH 7).
  • streptavidin particles Approximately 250 ⁇ g streptavidin particles are suspended in 10 ⁇ l MiIIiQ-H 2 O + 5% trehalose and dried ⁇ L-wise on the substrate surface (about 2 mm 2 ).
  • the nanoparticle surfaces are first blocked for one hour with a 3% (w / v) solution of BSA in PBS buffer, then incubated for 1.5 h at RT with the analyte solution and then for 30 min in PBS / 0.1% TritonX 100, washed in PBS and MiIIiQ water.
  • the analyte solution is a mixture of one to three times biotinylated and unbiotinylated insulin dissolved in PBS buffer (about 3: 1, total concentration about 250 nM).
  • Matrix A saturated solution of 3,5-dimethoxy-4-hydroxy cinnamic acid in 6: 4 (v / v) 0.1% trifluoroacetic acid (pA) and acetonitrile (HPLC grade) was dissolved, applied to the particle layer and air dried.
  • a mass spectrometer HP G 2025A LD-TOF modified with a time lag focusing (TLF) unit (Future, GSG company) was used.
  • the data was collected using a Le Croy 500 MHz oscilloscope.

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Abstract

La présente invention concerne des éléments fonctionnels qui comprennent des microstructures contenant des nanoparticules biofonctionnalisées, situées sur un support, ainsi que des procédés pour produire ces éléments fonctionnels et leur utilisation.
EP05822614A 2004-12-24 2005-12-03 Supports tridimensionnels a nanostructure et microstructure Withdrawn EP1831690A1 (fr)

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JP2008525763A (ja) 2008-07-17
WO2006072306A1 (fr) 2006-07-13
DE102004062573A1 (de) 2006-07-13
US20080044830A1 (en) 2008-02-21
KR20070099597A (ko) 2007-10-09
AU2005324159A1 (en) 2006-07-13

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