WO1999039205A1 - Method for producing test bodies for specific detection of individual reactants of receptor substance-ligand substance complexes - Google Patents

Method for producing test bodies for specific detection of individual reactants of receptor substance-ligand substance complexes Download PDF

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WO1999039205A1
WO1999039205A1 PCT/DE1999/000184 DE9900184W WO9939205A1 WO 1999039205 A1 WO1999039205 A1 WO 1999039205A1 DE 9900184 W DE9900184 W DE 9900184W WO 9939205 A1 WO9939205 A1 WO 9939205A1
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reactants
substance
immobilized
layer
detection
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PCT/DE1999/000184
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German (de)
French (fr)
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Rainer Fislage
Wladimir Teterin
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Rainer Fislage
Wladimir Teterin
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Publication of WO1999039205A1 publication Critical patent/WO1999039205A1/en

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    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • 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
    • 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
    • G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00673—Slice arrays

Definitions

  • the invention relates to a method for producing test bodies (stacked layer chips (SL chips)) for the specific detection of individual reactants of receptor substance-ligand substance complexes according to the preamble of patent claim 1.
  • SL chips stacked layer chips
  • the invention is used in the field of medical diagnostics, food monitoring, environmental analysis, molecular biological analysis, and in the development of pharmaceutical preparations.
  • membrane-based methods such as Southern blots [1], Northern blots [2], Western blots [3,4], dot blots [5,6] or Slot blots or other flat test pieces [10], some of which are referred to as DNA microchips.
  • the latter have a large number of ordered immobilized reactants in the smallest areas, which allow the sequence-specific detection of nucleic acids.
  • the corresponding test specimens are built up on suitable documents.
  • oligonucleotides are synthesized by suitable methods in situ at fixed positions on areal substrates [7] or using physical methods such as e.g. modified inkjet printers [8] applied. Methods for the synthesis of various immobilized peptides on a surface and a subsequent analysis process on this surface are also known [9].
  • REPLACEMENT SHEET / (RULE 26) The orderly immobilization of a large number of nucleic acids, proteins or other bindable substances is currently only possible at high costs and with considerable expenditure on equipment. The invention seeks to remedy this.
  • the invention specified in the claims is based on the problem of creating a simple and inexpensive method for the orderly immobilization of various bindable substances in a very small space.
  • Essential to the invention is the use of a macroscopic arrangement for producing three-dimensional microscopic structures.
  • the immobilizable reactants are each bound to a suitable matrix material and arranged in layers on top of one another.
  • the smallest distance between two immobilized reactants is only determined by the technically achievable minimum layer thickness.
  • the areal expansion of the substance-binding area is not limiting, so that the devices described can be produced in a wide variety of shapes and with variable external dimensions.
  • Binding substances are detected using suitable detection systems after a binding reaction against immobilized reactants on the cut surfaces of the three-dimensional object.
  • the cuts are to be made in such a way that the relevant layers of the three-dimensional arrangement are exposed and accessible to the mixture of substances. Physical or chemical methods can be used to expose the layers.
  • substances to be analyzed can bind not only to the cut surface itself, but also to the surfaces that point into the depth of the three-dimensional object.
  • a particular advantage of the invention is the possibility of immobilizing reactants with different basic chemical structures by varying the test body matrix while maintaining the test body geometry.
  • FIG. 1 shows a structural diagram of the SL chip from the example.
  • the numbers in the drawing have the following meaning:
  • Nylon membranes were either loaded with alkaline denatured DNA from phage lambda or with likewise alkaline denatured genomic DNA from chicken erythrocytes and fixed by UV light. The individual membranes each had an area size of 1 cm x 2 cm. The use of nylon membranes is only one possibility for matrix immobilization of DNA. In general, all immobilization methods can be used which are suitable for the construction of objects according to claim 1 -2 and which allow substances to be bound and detected by suitable detection systems on their cut surfaces.
  • the membranes were glued to one another with contact adhesive according to the manufacturer's instructions, so that a membrane object with the dimensions 2 cm ⁇ 1 cm and a total thickness of 2 mm resulted.
  • the contact adhesive is not absolutely necessary according to the invention.
  • the membrane layers can also be physically fixed.
  • the distinction between matrix and adhesive can also be omitted. In general, all methods can be used which are suitable for the construction of objects according to patent claims 1-2 and which permit the binding and detection of substances on their cut surfaces by means of suitable detection systems.
  • the detection and detection methods are not limited to those described here. Suitable and specific methods must be selected depending on the substance bound.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The analysis of complex mixtures of substances can be considerably accelerated by means of massive parallel testing with the aid of immobilized reactants that specifically bind individual constituents of the mixture of substances. To this end, the reactants are bonded in an appropriate matrix material and arranged layerwise in such a way that a three-dimensional object is obtained. Appropriate cuts in such an object can be used for detecting specific binding ligands on the surfaces of the sections thus obtained. The number of reactants used for detection is limited by the layer thickness of the immobilization matrix used, the spatial resolution of the measuring instrument employed for detection and the sensitivity of the detection system.

Description

Beschreibungdescription
VERFAHREN ZUR HERSTELLUNG VON TESTKÖRPERN ZUM SPEZIFISCHEN NACHWEIS EINZELNER REAKTANDEN VON REZEPTORSUBSTANZ LIG ANDENSUBSTANZ KOMPLEXENMETHOD FOR THE PRODUCTION OF TEST BODIES FOR THE SPECIFIC DETECTION OF INDIVIDUAL REACTORS OF RECEPTOR SUBSTANCE LIG ANDEAN COMPLEX
Die Erfindung betrifft ein Verfahren zur Herstellung von Testkörpern (Stacked-Layer- Chips (SL-Chips)) zum spezifischen Nachweis einzelner Reaktanden von Rezeptorsubstanz-Ligandensubstanz Komplexen gemäß Oberbegriff des Patentanspruchs 1.The invention relates to a method for producing test bodies (stacked layer chips (SL chips)) for the specific detection of individual reactants of receptor substance-ligand substance complexes according to the preamble of patent claim 1.
Die Erfindung findet Anwendung im Bereich der medizinischen Diagnostik, der Lebensmittelüberwachung, der Umweltanalytik, der molekularbiologischen Analytik, sowie bei der Entwicklung pharmazeutischer Präparate.The invention is used in the field of medical diagnostics, food monitoring, environmental analysis, molecular biological analysis, and in the development of pharmaceutical preparations.
Bisher werden bei der parallelen Untersuchung von Substanzgemischen oft filterbasierte Testsysteme angewendet. Im Falle von Gemischen aus verschiedenen DNA-Fragmenten, setzt man zur Analyse häufig membranbasierte Verfahren wie Southern-Blots [1], Northern-Blots [2], Westem-Blots [3,4], Dot-Blots [5,6] bzw. Slot- Blots oder andere flächenhaft ausgeführte Testkörper [10] ein, die teilweise als DNA-Mikrochips bezeichnet werden. Letztere verfügen über eine Vielzahl geordnet immobilisierter Reaktanden auf kleinsten Flächen, die den sequenzspezifischen Nachweis von Nukleinsäuren erlauben. Die entsprechenden Testkörper werden flächenhaft auf geeigneten Unterlagen aufgebaut. Dazu werden Oligonukleotide durch geeignete Verfahren in situ an festgelegten Positionen auf flächenhaften Unterlagen synthetisiert [7] oder mit physikalischen Methoden wie z.B. modifizierten Tintenstrahldruckern [8] aufgebracht. Auch Methoden zur Synthese verschiedener immobilisierter Peptide auf einer Fläche und einem nachfolgenden Analyseprozeß auf dieser Fläche sind bekannt [9].So far, filter-based test systems have often been used for the parallel investigation of substance mixtures. In the case of mixtures of different DNA fragments, membrane-based methods such as Southern blots [1], Northern blots [2], Western blots [3,4], dot blots [5,6] or Slot blots or other flat test pieces [10], some of which are referred to as DNA microchips. The latter have a large number of ordered immobilized reactants in the smallest areas, which allow the sequence-specific detection of nucleic acids. The corresponding test specimens are built up on suitable documents. For this purpose, oligonucleotides are synthesized by suitable methods in situ at fixed positions on areal substrates [7] or using physical methods such as e.g. modified inkjet printers [8] applied. Methods for the synthesis of various immobilized peptides on a surface and a subsequent analysis process on this surface are also known [9].
ERSA7ZELΛTT (RE3EL 26) In den folgenden Literaturstellen ist der zitierte Stand der Technik beschrieben:ERSA7ZELΛTT (RE3EL 26) The cited prior art is described in the following references:
1. Southern, E.M.1. Southern, E.M.
Detection of specific sequences among DNA-fragments separated by gel electrophoresis.Detection of specific sequences among DNA fragments separated by gel electrophoresis.
J. Mol. Biol. (1975); 98: 503J. Mol. Biol. (1975); 98: 503
2. Thomas P.S.2. Thomas P.S.
Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose.Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose.
Proc. Natl. Acad. Sei. USA (1980);77(9):5201-5205Proc. Natl. Acad. Be. USA (1980); 77 (9): 5201-5205
3. Burnette N.W.3. Burnette N.W.
Electrophoretic transfer of proteins from sodium dodecylsulfate- polyacrylamide gels to unmodified nitrocellulose and radiographic detection witn antibody and radioiodinated protein A. Anal. Biochem. (1981 ); 112: 195-203Electrophoretic transfer of proteins from sodium dodecylsulfate- polyacrylamide gels to unmodified nitrocellulose and radiographic detection witn antibody and radioiodinated protein A. Anal. Biochem. (1981); 112: 195-203
4. Beisiegel U, Schneider W.J., Brown M.S, Goldstein J.L4. Beisiegel U, Schneider W.J., Brown M.S, Goldstein J.L
Immunoblot analysis of Iow density lipoprotein receptors in fibroblasts from subjeets with familial hypercholesterolemia. J. Biol. Chem. (1982); 257(21 ):13150-13156Immunoblot analysis of Iow density lipoprotein receptors in fibroblasts from subjeets with familial hypercholesterolemia. J. Biol. Chem. (1982); 257 (21): 13150-13156
5. Kafatos F.C., Jones C.W., Efstratiadis A.5. Kafatos F.C., Jones C.W., Efstratiadis A.
Determination of nucleic aeid sequence homologies and relative concentrations by a dot hybridization procedure Nucleic Acids Res. (1979); 7: 1541 -1552Determination of nucleic aeid sequence homologies and relative concentrations by a dot hybridization procedure Nucleic Acids Res. (1979); 7: 1541-1552
6. Dyson N.J.6. Dyson N.J.
Immobilization of nucleic acids and hybridization analysis. In: Essential molecular biology: A practical approach. Vol. 2 (T.A. Brown, ed.), 111-156, IRL Press, OxfordImmobilization of nucleic acids and hybridization analysis. In: Essential molecular biology: A practical approach. Vol. 2 (T.A. Brown, ed.), 111-156, IRL Press, Oxford
7. Hubbell E.A., Lipshutz R.J., Morris M., Winkler J.L. Computer-aided engineering System for design of sequence arrays and lithographic masks7. Hubbell E.A., Lipshutz R.J., Morris M., Winkler J.L. Computer-aided engineering system for design of sequence arrays and lithographic masks
Patent Nummer: 5593839 Offenlegungstag: 1995 06 02 Land: USPatent Number: 5593839 Publication Date: 1995 06 02 Country: US
8. Wallace R.W.8. Wallace R.W.
DNA on a chip: serving up the genome for diagnostics and research Molecular Medicine Today (1997), 3(9): 384-389DNA on a chip: serving up the genome for diagnostics and research Molecular Medicine Today (1997), 3 (9): 384-389
9. Hudson D., Johnson C.R., Giebel L.9. Hudson D., Johnson C.R., Giebel L.
Method and apparatus for peptide synthesis and screening Patent Nummer: 5591646 Offenlegungstag: 1997 01 07 Land: USMethod and apparatus for peptide synthesis and screening Patent number: 5591646 Publication date: 1997 01 07 Country: US
ERSÄTZBLATT (REGEL 26) 10. Wölfl S.REPLACEMENT BLADE (RULE 26) 10. Wölfl S.
Optischer Nachweis von Hybridisierungssignalen Patent Nummer: 196 12 356 A1 Offenlegungstag: 02.10.1997 Land: DeutschlandOptical detection of hybridization signals Patent number: 196 12 356 A1 Date of disclosure: 02.10.1997 Country: Germany
ERSATZBLATT/(REGEL 26) Die geordnete Immobilisierung einer großen Zahl von Nukleinsäuren, Proteinen oder anderen bindungsfähigen Substanzen ist derzeit nur unter hohen Kosten und erheblichem apparativen Aufwand möglich. Hier will die Erfindung Abhilfe schaffen.REPLACEMENT SHEET / (RULE 26) The orderly immobilization of a large number of nucleic acids, proteins or other bindable substances is currently only possible at high costs and with considerable expenditure on equipment. The invention seeks to remedy this.
Der in den Patentansprüchen angegebenen Erfindung liegt das Problem zugrunde, ein einfaches und kostengünstiges Verfahren zur geordneten Immobilisierung verschiedener bindungsfähiger Substanzen auf engstem Raum zu schaffen.The invention specified in the claims is based on the problem of creating a simple and inexpensive method for the orderly immobilization of various bindable substances in a very small space.
Erfindungsgemäß wird das Problem durch die in den Patentansprüchen dargelegten Merkmale gelöst.According to the invention, the problem is solved by the features set out in the patent claims.
Erfindungswesentlich ist die Verwendung einer makroskopischen Anordnung zur Herstellung dreidimensionaler mikroskopischer Strukturen. Dazu werden die immobilisierbaren Reaktanden jeweils an geeignetes Matrixmaterial gebunden und jeweils schichtweise aufeinander angeordnet. Der geringste Abstand zwischen zwei immobilisierten Reaktanden wird nur durch die technisch erreichbare minimale Schichtdicke bestimmt. Im Gegensatz zu den bisher bekannten Verfahren ist die flächenhafte Ausdehnung des substanzbindenden Bereiches nicht limitierend, so daß die beschriebenen Vorrichtungen in großer Formenvielfalt und mit variablen äußeren Abmessungen hergestellt werden können.Essential to the invention is the use of a macroscopic arrangement for producing three-dimensional microscopic structures. For this purpose, the immobilizable reactants are each bound to a suitable matrix material and arranged in layers on top of one another. The smallest distance between two immobilized reactants is only determined by the technically achievable minimum layer thickness. In contrast to the previously known methods, the areal expansion of the substance-binding area is not limiting, so that the devices described can be produced in a wide variety of shapes and with variable external dimensions.
Der Nachweis bindender Substanzen erfolgt unter Verwendung geeigneter Nachweissysteme nach einer Bindungsreaktion gegen immobilisierte Reaktanden auf den Schnittflächen des dreidimensionalen Objektes. Die Schnitte sind dabei so zu führen, daß die nachweisrelevanten Schichten der dreidimensionalen Anordnung freigelegt und für das Substanzgemisch zugänglich werden. Zur Freilegung der Schichten können physikalische oder chemische Verfahren zur Anwendung kommen. Im Gegensatz zu den bekannten flächenhaft ausgeführten Anordnungen können zu analysierende Substanzen nicht nur an die Schnittfläche selbst, sondern auch an die Flächen binden, die in die Tiefe des dreidimensionalen Objektes weisen.Binding substances are detected using suitable detection systems after a binding reaction against immobilized reactants on the cut surfaces of the three-dimensional object. The cuts are to be made in such a way that the relevant layers of the three-dimensional arrangement are exposed and accessible to the mixture of substances. Physical or chemical methods can be used to expose the layers. In contrast to the known two-dimensional arrangements, substances to be analyzed can bind not only to the cut surface itself, but also to the surfaces that point into the depth of the three-dimensional object.
Ein besonderer Vorteil der Erfindung ist die Möglichkeit zur Immobilisierung von Reaktanden mit unterschiedlicher chemischer Grundstruktur durch die Variation der Testkörpermatrix unter Beibehaltung der Testkörpergeometrie.A particular advantage of the invention is the possibility of immobilizing reactants with different basic chemical structures by varying the test body matrix while maintaining the test body geometry.
Die Erfindung wird an einem Beispiel in den nachfolgenden Schritten näher erläutert. Die beigefügte Zeichnung (Abb. 1 ) zeigt ein Strukturbild des SL-Chips aus dem Beispiel. Die Ziffern in der Zeichnung haben folgende Bedeutung:The invention is explained in more detail using an example in the following steps. The attached drawing (Fig. 1) shows a structural diagram of the SL chip from the example. The numbers in the drawing have the following meaning:
1. Nylonmembran beschichtet mit DNA des Bakteriophagen λ1. Nylon membrane coated with DNA of the bacteriophage λ
2. Nylonmembran beschichtet mit genomischer DNA aus Hühnererythrozyten2. Nylon membrane coated with chicken erythrocyte genomic DNA
3. Nylonmembran unbeschichtet (negativ Kontrolle)3. nylon membrane uncoated (negative control)
4. Abgeschnittener Stacked-layer Chip4. Cut stacked-layer chip
Beispielexample
1. Nylonmembranen wurden entweder mit alkalisch denaturierter DNA des Phagen Lambda oder mit ebenfalls alkalisch denaturierter genomischer DNA aus Hühnererythrozyten beschickt und durch UV-Licht fixiert. Die einzelnen Membranen hatten jeweils eine Flächengröße von 1 cm x 2 cm. Die Verwendung von Nylonmembranen stellt nur eine Möglichkeit zur Matriximmobilisierung von DNA dar. Generell sind alle Immobilisierungsmethoden verwendbar, die zur Konstruktion von Objekten nach Patentanspruch 1 -2 geeignet sind und die an ihren Schnittflächen die Bindung und Detektion von Substanzen durch geeignete Nachweissysteme erlauben.1. Nylon membranes were either loaded with alkaline denatured DNA from phage lambda or with likewise alkaline denatured genomic DNA from chicken erythrocytes and fixed by UV light. The individual membranes each had an area size of 1 cm x 2 cm. The use of nylon membranes is only one possibility for matrix immobilization of DNA. In general, all immobilization methods can be used which are suitable for the construction of objects according to claim 1 -2 and which allow substances to be bound and detected by suitable detection systems on their cut surfaces.
2. Die Membranen wurden mit Kontaktkleber nach Angaben des Herstellers aufeinander verklebt, so daß sich ein Membranobjekt mit den Maßen 2 cm x 1 cm und einer Dicke von insgesamt 2 mm ergab.2. The membranes were glued to one another with contact adhesive according to the manufacturer's instructions, so that a membrane object with the dimensions 2 cm × 1 cm and a total thickness of 2 mm resulted.
Die Verwendung des Kontaktklebstoffes ist erfindungsgemäß nicht zwingend notwendig. Neben der Verwendung anderer Klebstoffe kann auch eine physikalische Fixierung der Membranschichten erfolgen. Auch kann die Unterscheidung zwischen Matrix und Klebstoff entfallen. Generell sind alle Methoden verwendbar, die zur Konstruktion von Objekten nach Patentanspruch 1 - 2 geeignet sind und die an ihren Schnittflächen die Bindung und Detektion von Substanzen durch geeignete Nachweissysteme erlauben.The use of the contact adhesive is not absolutely necessary according to the invention. In addition to using other adhesives, the membrane layers can also be physically fixed. The distinction between matrix and adhesive can also be omitted. In general, all methods can be used which are suitable for the construction of objects according to patent claims 1-2 and which permit the binding and detection of substances on their cut surfaces by means of suitable detection systems.
3. Vom Membranobjekt wurden 1 cm lange und 2 mm breite Stücke abgeschnitten. Diese Objektstücke enthielten jeweils Schnittflächen aller verklebten Membranen. Die angegebenen geometrischen Formen und Maße sind nicht bindend. Die Parameter können je nach Anwendungszweck und dem zur Verfügung stehenden Auswertegerät angepaßt werden.3. 1 cm long and 2 mm wide pieces were cut off from the membrane object. These object pieces each contained cut surfaces of all glued membranes. The specified geometric shapes and dimensions are not binding. The parameters can be adjusted depending on the application and the evaluation device available.
4. Ein Objektstück wurde über Nacht mit einer Lambda-spezifischen DNA-Sonde hybridisiert, die zuvor mit Fluorescein-12-dGTP markiert worden war. Nach dem Waschen fluoresziierte nur die mit Lambda-DNA beschickte Membranschicht des Objektstückes. Zur Beobachtung diente ein Fluoreszenzmikroskop unter Verwendung geeigneter Objektive und eines geeigneten Filtersatzes.4. An object piece was hybridized overnight with a lambda-specific DNA probe which had previously been labeled with fluorescein-12-dGTP. After washing, only the membrane layer of the object piece loaded with lambda DNA fluoresced. A fluorescence microscope was used for the observation using suitable lenses and a suitable filter set.
Die Nachweis- und Detektionsmethoden beschränken sich nicht auf die hier beschriebene. Je nach gebundener Substanz müssen geeignete und spezifische Methoden ausgewählt werden.The detection and detection methods are not limited to those described here. Suitable and specific methods must be selected depending on the substance bound.
ERSAΪZBLATT (REGEL 26) SUBSTITUTE SHEET (RULE 26)

Claims

Patentansprüche claims
1. Verfahren zur Herstellung von Testkörpern (Stacked-Layer-Chips (SL-Chips)) zum spezifischen Nachweis einzelner Reaktanden von Rezeptorsubstanz- Ligandensubstanz Komplexen, dadurch gekennzeichnet, a) daß immobilisierte Reaktanden jeweils an geeignetes Matrixmaterial gebunden und dreidimensional, schichtweise aufeinander angeordnet sind, b) daß der geringste Abstand zwischen zwei immobilisierten Reaktanden durch die technisch erreichbare minimale Schichtdicke bestimmt ist, c) daß geeignete Schnittflächen durch die dreidimensionale Anordnung der Reaktandenschichten zum Nachweis von Bindungspartnern dienen und d) daß weitere Schichten verwendet werden, deren Schnittflächen nicht der Bindung von Bindungspartnern dienen.1. A method for producing test bodies (stacked-layer chips (SL chips)) for the specific detection of individual reactants of receptor substance-ligand substance complexes, characterized in that a) that immobilized reactants are each bound to a suitable matrix material and arranged three-dimensionally, layer by layer , b) that the smallest distance between two immobilized reactants is determined by the technically achievable minimum layer thickness, c) that suitable cut surfaces serve to detect binding partners through the three-dimensional arrangement of the reactant layers, and d) that further layers are used, the cut surfaces of which are not binding serve by attachment partners.
2. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, a) daß Nukleinsäuren oder Proteine, b) daß Desoxyribonukleinsäuren, Ribonukleinsäuren sowie ihre natürlich vorkommenden oder synthetischen Derivate oder Analoga jeweils in einzelsträngiger oder doppelsträngiger Form, c) daß natürlich vorkommende oder synthetische Derivate oder Analoga von Proteinen und Peptiden verwendet werden,2. The method according to claim 1, characterized in that a) that nucleic acids or proteins, b) that deoxyribonucleic acids, ribonucleic acids and their naturally occurring or synthetic derivatives or analogs in each case in single-stranded or double-stranded form, c) that naturally occurring or synthetic derivatives or analogs of Proteins and peptides are used
d) daß Reaktanden immobilisiert werden, deren chemische Grundstruktur sich von Proteinen oder Nukleinsäuren unterscheidet.d) that reactants are immobilized whose basic chemical structure differs from proteins or nucleic acids.
ERSÄTZBLATT (REGEL 26) REPLACEMENT BLADE (RULE 26)
PCT/DE1999/000184 1998-01-28 1999-01-26 Method for producing test bodies for specific detection of individual reactants of receptor substance-ligand substance complexes WO1999039205A1 (en)

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DE1998103077 DE19803077C1 (en) 1998-01-28 1998-01-28 Stacked-layer chip carrying immobilized reactants for receptor-ligand assays
DE19803077.0980128 1998-01-28

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996017246A1 (en) * 1994-11-30 1996-06-06 Pharmacia Biotech Ab Multifunctional surfaces
DE19612356A1 (en) * 1996-03-28 1997-10-02 Knoell Hans Forschung Ev Nucleic acid hybridisation assay
WO1997046313A1 (en) * 1996-06-07 1997-12-11 Eos Biotechnology, Inc. Immobilised linear oligonucleotide arrays

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996017246A1 (en) * 1994-11-30 1996-06-06 Pharmacia Biotech Ab Multifunctional surfaces
DE19612356A1 (en) * 1996-03-28 1997-10-02 Knoell Hans Forschung Ev Nucleic acid hybridisation assay
WO1997046313A1 (en) * 1996-06-07 1997-12-11 Eos Biotechnology, Inc. Immobilised linear oligonucleotide arrays

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