WO1998058293A2 - Method and device for immobilizing macromolecules - Google Patents

Method and device for immobilizing macromolecules Download PDF

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Publication number
WO1998058293A2
WO1998058293A2 PCT/EP1998/003657 EP9803657W WO9858293A2 WO 1998058293 A2 WO1998058293 A2 WO 1998058293A2 EP 9803657 W EP9803657 W EP 9803657W WO 9858293 A2 WO9858293 A2 WO 9858293A2
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WO
WIPO (PCT)
Prior art keywords
light
solid phase
immobilization
biomacromolecules
field
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PCT/EP1998/003657
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German (de)
French (fr)
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WO1998058293A3 (en
Inventor
Holger Breter
Winfried Jentsch
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Biotools - Institut Für Computerintegriertes Bioengineering Gmbh
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Priority to AU85387/98A priority Critical patent/AU8538798A/en
Publication of WO1998058293A2 publication Critical patent/WO1998058293A2/en
Publication of WO1998058293A3 publication Critical patent/WO1998058293A3/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00373—Hollow needles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00427—Means for dispensing and evacuation of reagents using masks
    • B01J2219/0043—Means for dispensing and evacuation of reagents using masks for direct application of reagents, e.g. through openings in a shutter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
    • 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
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00614—Delimitation of the attachment areas
    • B01J2219/00621—Delimitation of the attachment areas by physical means, e.g. trenches, raised areas
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00709—Type of synthesis
    • B01J2219/00711—Light-directed synthesis
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • the invention relates to a method and a device for immobilizing macromolecules according to the preambles of claims 1 and 19, 20, 21 and 22.
  • Immobilization methods of biomacromolecules on solid phases using light energy to release reactive functional groups are known.
  • DE 34 35 744 AI describes carrier materials for use in immune determinations which have photo-activatable molecules covalently bound on their surface. So-called heterobifunctional or homobifunctional compounds are used here to produce these carrier materials.
  • the compounds also referred to as bridging agents are covalently bound to the solid phase with a functional group, while the second functional group can be coupled to corresponding biomacromolecules by photoactivation.
  • Balls, microtiter plates or tubes made of plastic are used as carrier materials.
  • Plastic surfaces of these moldings or containers are made with the bridging agents such as Lomant's reagent II, N-succinimidyl-6- (4'-azido-2 '-nitrophenylamino) hexanoate treated.
  • Lomant's Reagent II forms a covalent bond with the plastic surface.
  • the second functional group can react with antigens, allergens or antibodies, as is provided here.
  • the carriers described here are not suitable for miniaturized immobilization of biomacromolecules.
  • the invention has for its object to enable light-induced addressable immobilizations of biomacromolecules on extremely small surface elements.
  • the method according to the invention enables biomacromolecules with suitable functional groups to be immobilized on a solid support on the smallest surface elements. For reasons of maintaining native molecular structures, it is important that the immobilization reactions can take place in the liquid phase.
  • biomacromolecules immobilized according to the invention on extremely small surface elements (nanoscale), it is possible to carry out a large number of bioassays such as immunoassays, hybridization assays or Liegand receptor binding assays in an effective manner.
  • the energy supply for the photoactivation of the photoactivatable functional groups of the carrier takes place using methods of scanning probe and optical near-field microscopy. With the local light irradiation that can be achieved in the near field area, it is possible to create extremely miniaturized immobilization areas by directly addressing the areas of the surface of the solid phase intended for molecular immobilization. By means of the method according to the invention, it is possible to achieve packing densities for the discrete planar arrangement that have not yet been achieved for the immobilized biomacromolecules.
  • Fig. 2 is a schematic representation of a die in plan view
  • Fig. 3 is a schematic representation of an immobilization device
  • Fig. 4 is a schematic representation of a
  • FIGS. 6-9 a schematic representation of the production of extremely miniaturized surface elements.
  • a near field light source 6 is directed to an immobilization area 9.
  • the immobilization area 9 is formed by a light exit cone 8, the dimensions of which are in turn defined by an aperture 7.
  • the near-field light source 6 is in a liquid phase 4, in which the biomacromolecules to be immobilized are contained in solution.
  • a photoactivatable layer is located on a solid phase 1, the photoactivatable functional groups 3 thereof are directed into the liquid phase 4.
  • Near-field light source 6, liquid phase 4 including the biomacromolecules to be immobilized and the solid phase 1 are arranged in a translucent flow cell, not shown here.
  • This device consists of a transparent plate 22 on which a layer 23 with photoactivatable functional groups is applied in a manner known per se.
  • the transparent plate 22 has continuous hollow channels 24.
  • a die 20 which can be put on and taken off is arranged on the layer 23.
  • This die 20, which is shown in plan view in FIG. 2, has continuous cavities 21.
  • the arrangement pattern of the cavities 21, the die 20, the channels 24 and the plate 22 are identical.
  • the diameter of the channels 24 is very much smaller than the diameter of the cavities 21.
  • the continuous cavities 21 are introduced into a silicon substrate with a thickness of 400-500 ⁇ m using an etching process.
  • the dissolved biomacromolecules to be immobilized are filled into the cavities 21 in a programmed manner.
  • the solution 26 is filled in via a cannula 25.
  • the air is pressed out of the cavities 21 through the channels 24 during filling.
  • the irradiation with light of a certain wavelength and a certain intensity takes place from the lower side of the transparent plate 22.
  • the functional groups of layer 23 activated in this way react with corresponding functional groups of the biomacromolecules in solution 26.
  • the die 20 is then covered with a glass plate from above and tipped over.
  • the plate 22 with the biomacromolecules 27 immobilized thereon, as shown in FIG. 5, is lifted off the matrix 20.
  • a further coated plate 22 with continuous channels 24 is placed on the die 20, turned over, and further solution 26 of biomacromolecules 27 is placed in the continuous cavities 21.
  • the immobilization is repeated on a new plate 22.
  • FIGS. 6 to 9 show the schematic sequence for producing very small three-dimensional elements.
  • a photoresist 12 is applied to a glass substrate 11.
  • a silicon layer 13 with a certain thickness is then evaporated on the photoresist 12.
  • a second photoresist 14 is applied to the silicon layer 13.
  • the photoresist 14 is exposed through a mask 15 with a predetermined pattern and with a specific resolution. Alternatively, this pattern can also be generated by means of an electron beam 16.
  • the corresponding pattern according to FIG. 8 is formed on the surface of the silicon layer 13.
  • the surface is now etched, for example by plasma etching.
  • the spaces 17 to the photoresist 12 are etched.

Abstract

The invention relates to a method and a device for immobilizing macromolecules according to the generic part of claims 1 and 19, 20, 21 and 22. The aim of the invention is to enable light induced, addressable immobilization of bio-macromolecules on extremely small surface elements. This is done by immobilizing macromolecules on a solid phase involving photoactivation of corresponding functional groups of the solid phase and coupling of reaction-capable groups of bio-macromolecules contained in a solution and the solid phase. The invention is characterized in that the immobilization reaction is photoactivated by miniaturizing the effect of the light on the surface of the solid phase, whereby addressable control of the effect of the light on the surface occurs according to a given model.

Description

Verfahren und Vorrichtung zur Immobilisierung von Method and device for immobilizing
MakromolekülenMacromolecules
BesehreibungDescription
Die Erfindung betrifft ein Verfahren und eine Vorrich- tung zur Immobilisierung von Makromolekülen gemäß den Oberbegriffen der Ansprüche 1 und 19, 20, 21 und 22.The invention relates to a method and a device for immobilizing macromolecules according to the preambles of claims 1 and 19, 20, 21 and 22.
Immobilisierungsverfahren von Biomakromolekülen an festen Phasen unter Ausnutzung von Lichtenergie zur Frei- setzung reaktiver funktioneller Gruppen sind bekannt. So werden in der DE 34 35 744 AI Trägermaterialien zur Verwendung für Immunbestimmungen beschrieben, die auf ihrer Oberfläche kovalent gebundene photoaktivierbare Moleküle aufweisen. Zur Herstellung dieser Trägermate- rialien werden hier sogenannte heterobifunktionelle oder homobifunktionelle Verbindungen eingesetzt. Die auch als Brückenbildner bezeichneten Verbindungen sind mit einer funktioneilen Gruppe kovalent an der festen Phase gebunden, während die zweite funktionelle Gruppe durch Photoaktivierung mit entsprechenden Biomakromolekülen gekoppelt werden kann.Immobilization methods of biomacromolecules on solid phases using light energy to release reactive functional groups are known. DE 34 35 744 AI describes carrier materials for use in immune determinations which have photo-activatable molecules covalently bound on their surface. So-called heterobifunctional or homobifunctional compounds are used here to produce these carrier materials. The compounds also referred to as bridging agents are covalently bound to the solid phase with a functional group, while the second functional group can be coupled to corresponding biomacromolecules by photoactivation.
Als Trägermaterialien werden Kugeln, Mikrotiterplatten oder Röhrchen aus Kunststoff verwendet. Kunststoffober- flächen dieser Formkörper bzw. Behältnisse werden mit den Brückenbildnern wie zum Beispiel Lomant's Reagenz II, N-Succinimidyl-6- (4' -azido-2' -nitrophenylamino) - hexanoat behandelt. Das Lomant's Reagenz II geht mit der KunststoffOberfläche eine kovalente Bindung ein. Nach entsprechender Aktivierung kann die zweite funk- tionelle Gruppe mit, wie hier vorgesehen ist, Antige- nen, Allergenen oder Antikörpern reagieren. Die hier beschriebenen Träger eignen sich nicht zur miniaturisierten Immobilisierung von Biomakromolekülen.Balls, microtiter plates or tubes made of plastic are used as carrier materials. Plastic surfaces of these moldings or containers are made with the bridging agents such as Lomant's reagent II, N-succinimidyl-6- (4'-azido-2 '-nitrophenylamino) hexanoate treated. Lomant's Reagent II forms a covalent bond with the plastic surface. After appropriate activation, the second functional group can react with antigens, allergens or antibodies, as is provided here. The carriers described here are not suitable for miniaturized immobilization of biomacromolecules.
Der Erfindung liegt die Aufgabe zugrunde, lichtinduzierte adressierbare Immobilisierungen von Biomakromolekülen auf extrem kleinen Oberflächenelementen zu ermöglichen.The invention has for its object to enable light-induced addressable immobilizations of biomacromolecules on extremely small surface elements.
Die Lösung der Aufgabe erfolgt mit den kennzeichnenden Merkmalen der Ansprüche 1, 19, 20, 21 und 22.The object is achieved with the characterizing features of claims 1, 19, 20, 21 and 22.
Vorteilhafte Weiterbildungen sind in den Unteransprüchen angegeben.Advantageous further developments are specified in the subclaims.
Das erfindungsgemäße Verfahren ermöglicht Biomakromoleküle mit geeigneten funktioneilen Gruppen an einen festen Träger auf kleinsten Oberflächenelementen zu immobilisieren. Hierbei ist es aus Gründen des Erhaltes na- tiver Molekülstrukturen wichtig, daß die Immobilisierungsreaktionen in flüssiger Phase stattfinden können.The method according to the invention enables biomacromolecules with suitable functional groups to be immobilized on a solid support on the smallest surface elements. For reasons of maintaining native molecular structures, it is important that the immobilization reactions can take place in the liquid phase.
Mittels der erfindungsgemäß immobilisierten Biomakromoleküle auf extrem kleinen Oberflächenelementen (Nanobereich) ist die gleichzeitige Durchfürhung einer großen Zahl von Bioassays wie I munoassays, Hybridisie- rungsassays oder Liegand-Rezeptor-Bindungsassays auf effektive Weise möglich. In einer Weiterbildung der Erfindung erfolgt die Energiezufuhr für die Photoaktivierung der photoaktivierba- ren funktionellen Gruppen des Trägers mit Verfahren der Rastersonden- und optischen Nahfeldmikroskopie. Mit den damit erreichbaren lokalen Lichteinstrahlungen im Nahfeldbereich ist es möglich, durch die nun erreichbare direkte Adressierung der zu Molekülimmobilisierung vorgesehene Bereiche der Oberfläche der festen Phase extrem miniaturisierte Immobilisierungsbereiche zu schaf- fen. Mittels des erfindungsgemäßen Verfahrens ist es möglich, für die immobilisierten Biomakromoleküle bisher noch nicht erreichte Packungsdichten für die diskrete flächenmäßige Anordnung zu erreichen.Using the biomacromolecules immobilized according to the invention on extremely small surface elements (nanoscale), it is possible to carry out a large number of bioassays such as immunoassays, hybridization assays or Liegand receptor binding assays in an effective manner. In a further development of the invention, the energy supply for the photoactivation of the photoactivatable functional groups of the carrier takes place using methods of scanning probe and optical near-field microscopy. With the local light irradiation that can be achieved in the near field area, it is possible to create extremely miniaturized immobilization areas by directly addressing the areas of the surface of the solid phase intended for molecular immobilization. By means of the method according to the invention, it is possible to achieve packing densities for the discrete planar arrangement that have not yet been achieved for the immobilized biomacromolecules.
Anhand von beispielhaften Zeichnungen wird die Erfindung näher erläutert, ohne sie darauf zu beschränken. Es zeigenThe invention is explained in more detail on the basis of exemplary drawings, without being restricted thereto. Show it
Fig. 1 eine Vorrichtung zur Immobilisierung im Nahfeldbereich,1 shows a device for immobilization in the near field,
Fig. 2 eine schematische Darstellung einer Matrize in Draufsicht, Fig. 3 eine schematische Darstellung einer Immobilisierungsvorrichtung, Fig. 4 eine schematische Darstellung einerFig. 2 is a schematic representation of a die in plan view, Fig. 3 is a schematic representation of an immobilization device, Fig. 4 is a schematic representation of a
Probenzufuhr, Fig. 5 eine schematische Darstellung einer lichtinduzierten Immobilisierung unter Verwendung der Matrize gemäß Fig. 2, Fig. 6 - 9 eine schematische Darstellung der Herstellung extrem miniaturisierter Oberflächenelemente . In Fig. 1 ist eine Nahfeldlichtquelle 6 auf einen Immobilisierungsbereich 9 gerichtet. Der Immobilisierungs- bereich 9 wird gebildet durch einen Lichtaustrittskegel 8, dessen Abmaße wiederum durch eine Apertur 7 festge- legt ist. Die Nahfeldlichtquelle 6 befindet sich in einer flüssigen Phase 4, in der die zu immobilisierenden Biomakromoleküle gelöst enthalten sind. Auf einer festen Phase 1 befindet sich eine photoaktivierbare Schicht, deren photoaktivierbare funktionelle Gruppen 3 in die flüssige Phase 4 gerichtet sind. Nahfeldlichtquelle 6, flüssige Phase 4 einschließlich der zu immobilisierende Biomakromoleküle sowie die feste Phase 1 sind in eienr lichtdurchlässigen, hier nicht dargestellten Durchflußküvette angeordnet .5, a schematic representation of a light-induced immobilization using the die according to FIG. 2, FIGS. 6-9 a schematic representation of the production of extremely miniaturized surface elements. In Fig. 1, a near field light source 6 is directed to an immobilization area 9. The immobilization area 9 is formed by a light exit cone 8, the dimensions of which are in turn defined by an aperture 7. The near-field light source 6 is in a liquid phase 4, in which the biomacromolecules to be immobilized are contained in solution. A photoactivatable layer is located on a solid phase 1, the photoactivatable functional groups 3 thereof are directed into the liquid phase 4. Near-field light source 6, liquid phase 4 including the biomacromolecules to be immobilized and the solid phase 1 are arranged in a translucent flow cell, not shown here.
In Fig. 3 ist eine weitere Vorrichtung zur miniaturisierten Immobilisierung von Biomakromolekülen dargestellt. Diese Vorrichtung besteht aus einer durchsichtigen Platte 22, auf der auf an sich bekannte Weise ei- ne Schicht 23 mit photoaktivierbaren funktionellen Gruppen aufgebracht ist. Die durchsichtige Platte 22 weist durchgehende hohle Kanäle 24 auf. Auf der Schicht 23 ist eine auf- und absetzbare Matrize 20 angeordnet. Diese Matrize 20, die in Fig. 2 in Draufsicht darge- stellt ist, weist durchgehende Hohlräume 21 auf. Das Anordnungsmuster der Hohlräume 21, der Matrize 20, der Kanäle 24 und der Platte 22 sind identisch. Der Durchmesser der Kanäle 24 ist sehr viel kleiner als der Durchmesser der Hohlräume 21. Zur Herstellung der Ma- trize 20 werden in ein Siliciumsubstrat der Dicke 400- 500 μm mit Hilfe eines Ätzverfahrens die durchgehenden Hohlräume 21 eingebracht. In die Hohlräume 21 werden mit Hilfe eines Überdruckes die gelösten zu immobilisierenden Biomakromoleküle programmiert eingefüllt. Das Einfüllen der Lösung 26 erfolgt über eine Kanüle 25. Durch Anwendung des Überdruckes wird beim Einfüllen die Luft aus den Hohlräumen 21 durch die Kanäle 24 herausgedrückt. Von der unteren Seite der lichtdurchlässigen Platte 22 erfolgt die Bestrahlung mit Licht bestimmter Wellenlänge und bestimmter Intensität. Die dadurch aktivierten funktionellen Gruppen der Schicht 23 reagieren mit entsprechenden funktionellen Gruppen der sich in der Lösung 26 befindlichen Biomakromolekülen. Danach wird die Matrize 20 von oben mit einer Glasplatte be- deckt und umgekippt. Die Platte 22 mit den darauf immobilisierten Biomakromolekülen 27 wie in Fig. 5 dargestellt, wird von der Matrize 20 abgehoben. Eine weitere beschichtete Platte 22 mit durchgehenden Kanälen 24 wird auf die Matrize 20 aufgesetzt, umgedreht und wei- tere Lösung 26 von Biomakromolekülen 27 wird in die durchgehenden Hohlräume 21 gegeben. Die Immobilisierung wird auf einer neuen Platte 22 wiederholt.3 shows a further device for miniaturized immobilization of biomacromolecules. This device consists of a transparent plate 22 on which a layer 23 with photoactivatable functional groups is applied in a manner known per se. The transparent plate 22 has continuous hollow channels 24. A die 20 which can be put on and taken off is arranged on the layer 23. This die 20, which is shown in plan view in FIG. 2, has continuous cavities 21. The arrangement pattern of the cavities 21, the die 20, the channels 24 and the plate 22 are identical. The diameter of the channels 24 is very much smaller than the diameter of the cavities 21. To produce the die 20, the continuous cavities 21 are introduced into a silicon substrate with a thickness of 400-500 μm using an etching process. The dissolved biomacromolecules to be immobilized are filled into the cavities 21 in a programmed manner. The solution 26 is filled in via a cannula 25. By applying the overpressure, the air is pressed out of the cavities 21 through the channels 24 during filling. The irradiation with light of a certain wavelength and a certain intensity takes place from the lower side of the transparent plate 22. The functional groups of layer 23 activated in this way react with corresponding functional groups of the biomacromolecules in solution 26. The die 20 is then covered with a glass plate from above and tipped over. The plate 22 with the biomacromolecules 27 immobilized thereon, as shown in FIG. 5, is lifted off the matrix 20. A further coated plate 22 with continuous channels 24 is placed on the die 20, turned over, and further solution 26 of biomacromolecules 27 is placed in the continuous cavities 21. The immobilization is repeated on a new plate 22.
In den Figuren 6 bis 9 ist der schematische Ablauf der Herstellung sehr kleiner dreidimmensioanler Elemente dargestellt. Auf einem Glassubstrat 11 wird ein Photo- resist 12 aufgebracht. Auf dem Photoresist 12 wird anschließend eine Siliciumschicht 13 mit bestimmter Dicke aufgedampft. Auf die Siliciumschicht 13 wiederum wird ein zweiter Photoresist 14 aufgetragen. Durch eine Maske 15 mit vorgegebenem Muster und mit bestimmter Auflösung wird der Photoresist 14 belichtet. Alternativ kann dieses Muster auch mittels eines Elektronenstrahles 16 erzeugt werden. Nach dem Abspülen des Photoresist 14 entsteht auf der Oberfläche der Siliciumschicht 13 das entsprechende Muster gemäß Fig. 8. Die Oberfläche wird nun geätzt, zum Beispiel durch Plasmaätzung. Geätzt werden die Zwischenräume 17 bis zum Photoresist 12. Hierbei entstehen dreidimensionale Siliciumformkörper, die nach Beschichtung mit homo- bzw. heterobifunktio- nellen Verbindungen für Immobilisierungreaktion vorbereitet werden können. Die auf diese Weise hergestellten Oberflächenelemente 18 sind von extremer Kleinheit. Sie lassen sich durch Lösen des Photoresist 12 vom Glassubstrat 11 entfernen. Immobilisierungen können vor dem Ablösen erfolgen. Sie können auch anschließend durchgeführt werden, nachdem die vereinzelten Oberflächenelemente 18 zu neuen adressierbaren Strukturen zusammengesetzt worden sind. FIGS. 6 to 9 show the schematic sequence for producing very small three-dimensional elements. A photoresist 12 is applied to a glass substrate 11. A silicon layer 13 with a certain thickness is then evaporated on the photoresist 12. In turn, a second photoresist 14 is applied to the silicon layer 13. The photoresist 14 is exposed through a mask 15 with a predetermined pattern and with a specific resolution. Alternatively, this pattern can also be generated by means of an electron beam 16. After the photoresist 14 has been rinsed off, the corresponding pattern according to FIG. 8 is formed on the surface of the silicon layer 13. The surface is now etched, for example by plasma etching. The spaces 17 to the photoresist 12 are etched. This results in three-dimensional shaped silicon bodies which, after coating with homo- or heterobifunctional can be prepared for immobilization reaction. The surface elements 18 produced in this way are extremely small. They can be removed from the glass substrate 11 by detaching the photoresist 12. Immobilization can take place before detachment. They can also be carried out after the isolated surface elements 18 have been put together to form new addressable structures.
BezugszeichenlisteReference list
16 Lichtstrahlung feste Phase16 light radiation solid phase
17 Zwischenräume photoaktivierbare17 spaces can be photoactivated
Schicht 18 OberflächenelementLayer 18 surface element
photoaktivierbare funk- 19 tioneile Gruppenphotoactivatable functional groups
20 Matrize flüssige Phase20 liquid phase matrix
21 Hohlräume Flüssig/Gas-Phasengrenze21 cavities liquid / gas phase boundary
22 Platte Nahfeldlichtquelle22 Plate near-field light source
23 Schicht Apertur23 layer aperture
24 Kanal Lichtaustrittskugel24 channel light outlet ball
25 Kanüle Immobilisierungsbereich25 cannula immobilization area
26 Lösung Gasphase26 Gas phase solution
27 Biomakromoleküle Glassubstrat27 biomacromolecules glass substrate
PhotoresistPhotoresist
SiliciumschichtSilicon layer
PhotoresistPhotoresist
Maske mask

Claims

Patentansprüche claims
1. Verfahren zur Immobilisierung von Makromolekülen an einer festen Phase durch Photoaktivierung entsprechender funktioneller Gruppen der festen Phase und Kopplung an reaktionsfähige Gruppen der in Lösung befindlichen Biomakromoleküle an die feste Phase, dadurch gekennzeichnet, daß die Photoaktivierung der Immobilisierungsreaktion durch Miniaturisierung der Lichteinwirkung auf die Oberfläche der festen Phase durchgeführt wird, wobei eine adressierbare Steuerung der Lichteinwir- kung auf die Oberfläche nach vorgegebenem Muster erfolgt .1. A method for immobilizing macromolecules on a solid phase by photoactivating appropriate functional groups of the solid phase and coupling to reactive groups of the biomacromolecules in solution to the solid phase, characterized in that the photoactivation of the immobilization reaction by miniaturizing the action of light on the surface of the solid phase is carried out, an addressable control of the light effect on the surface taking place according to a predetermined pattern.
2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß die Miniaturisierung der Lichteinwirkung durch Miniaturisierung der Einwirkfläche der Lichtstrahlung erfolgt .2. The method according to claim 1, characterized in that the miniaturization of the exposure to light is carried out by miniaturizing the surface of the light radiation.
Verfahren nach Anspruch 1 oder 2 , dadurch gekennzeichnet, daß die Lichteinwirkung örtlich und/oder zeitlich versetzt durchgeführt wird.A method according to claim 1 or 2, characterized in that the exposure to light is carried out locally and / or at different times.
Verfahren nach einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, daß die Lichteinwirkung mittels einer Nahfeldlichtquelle im Nahfeldbereich erfolgt.Method according to one of claims 1 to 3, characterized in that the exposure to light takes place in the near field by means of a near field light source.
5. Verfahren nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, daß als Nahfeldlichtquelle an sich bekannte spitze lichtemittierende Sonden eingesetzt werden.5. The method according to any one of claims 1 to 4, characterized in that known light-emitting probes are used as a near-field light source.
6. Verfahren nach einem der Ansprüche 1 bis 5, dadurch gekennzeichnet, daß Sonden aus Quarzglas verwendet werden.6. The method according to any one of claims 1 to 5, characterized in that probes made of quartz glass are used.
7. Verfahren nach einem der Ansprüche 1 bis 6, dadurch gekennzeichnet, daß die Sondenspitzen im Nahfeldbereich angeordnet wer- den .7. The method according to any one of claims 1 to 6, characterized in that the probe tips are arranged in the near field.
8. Verfahren nach einem der Ansprüche 1 bis 7, dadurch gekennzeichnet, daß die feste Phase mittels der Z-Bewegung eines Scanners im konstanten Abstand zur Sondenspitze gehalten wird.8. The method according to any one of claims 1 to 7, characterized in that the solid phase is kept at a constant distance from the probe tip by means of the Z movement of a scanner.
9. Verfahren nach einem der Ansprüche 1 bis 8, dadurch gekennzeichnet, daß mittels der X-Y-Bewegung des Scanners adressierbare Immobilisierungsbereiche erzeugt werden. 9. The method according to any one of claims 1 to 8, characterized in that addressable immobilization areas are generated by means of the XY movement of the scanner.
10. Verfahren nach einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, daß die Miniaturisierung der Einwirkfläche der Lichtstrahlung auf der Oberfläche der festen Phase durch Anwendung von Matrizen (20) erfolgt.10. The method according to any one of claims 1 to 3, characterized in that the miniaturization of the surface of the light radiation on the surface of the solid phase is carried out by using matrices (20).
11. Verfahren nach einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, daß die Miniaturisierung der Einwirkfläche der Lichtstrahlung durch Herstellung extrem kleiner Oberflächenelemente (19) einer festen Phase erfolgt, die getrennt und adressierbar neu zusammengesetzt werden, wobei die Belichtung vor der Trennung und/oder danach erfolgt.11. The method according to any one of claims 1 to 3, characterized in that the miniaturization of the surface of the light radiation takes place by producing extremely small surface elements (19) of a solid phase, which are reassembled separately and addressably, the exposure before the separation and / or afterwards.
12. Verfahren nach einem der Ansprüche 1 bis 11, dadurch gekennzeichnet, daß als Lichtstrahlung monochromatisches Licht einge- setzt wird.12. The method according to any one of claims 1 to 11, characterized in that monochromatic light is used as the light radiation.
13. Verfahren nach einem der Ansprüche 1 bis 12, dadurch gekennzeichnet, daß als Lichtstrahlung UV-Licht eingesetzt wird.13. The method according to any one of claims 1 to 12, characterized in that UV light is used as light radiation.
14. Verfahren nach einem der Ansprüche 1 bis 13, dadurch gekennzeichnet, daß als Lichtstrahlung monochromatisches Licht mit einer Wellenlänge zwischen 100 und 500 nm eingesetzt wird. 14. The method according to any one of claims 1 to 13, characterized in that monochromatic light with a wavelength between 100 and 500 nm is used as light radiation.
15. Verfahren nach einem der Ansprüche 1 bis 14, dadurch gekennzeichnet, daß als feste Phase Substrate wie Glimmer, Glimmer mit Goldschicht, weitere Goldoberflächen, Siliciu wa- fer, Glasoberflächen oder Polymeroberflächen verwendet werden, die chemisch und/oder adsorptiv gebundene Moleküle mit photoaktivierbaren funktionellen Gruppen aufweisen.15. The method according to any one of claims 1 to 14, characterized in that substrates such as mica, mica with a gold layer, further gold surfaces, silicon wafers, glass surfaces or polymer surfaces are used as the solid phase, the chemically and / or adsorptively bound molecules with photoactivatable have functional groups.
16. Verfahren nach einem der Ansprüche 1 bis 15, dadurch gekennzeichnet, daß zur Kopplung photoaktivierbarer funtioneller Grup- pen an die feste Phase bifunktionelle Moleküle eingesetzt werden.16. The method according to any one of claims 1 to 15, characterized in that bifunctional molecules are used for coupling photoactivatable functional groups to the solid phase.
17. Verfahren nach Anspruch 1 oder 16, dadurch gekennzeichnet, daß zwischen den örtlich und/oder zeitlich versetzten Lichteinwirkungen zur Immobilisierung von Biomakromolekülen weitere Bindungsreaktionen von Biomakromolekülen unter Zwischenschaltung von Waεchschrit- ten durchgeführt werden.17. The method according to claim 1 or 16, characterized in that between the locally and / or temporally offset light effects for the immobilization of biomacromolecules further binding reactions of biomacromolecules are carried out with the interposition of washing steps.
18. Verfahren nach einem der Ansprüche 1 bis 9 und 12 bis 17, dadurch gekennzeichnet, daß als Nahfeldlichtquelle ein optisches Nahfeldmikroskop eingesetzt wird. 18. The method according to any one of claims 1 to 9 and 12 to 17, characterized in that an optical near-field microscope is used as the near-field light source.
19. Vorrichtung zur Immobilisierung von Makromolekülen auf sehr kleine Oberflächenelemente einer festen Phase, bestehend aus einer Nahfeldlichtquelle (6) , deren Lichtaustrittsöffnung (7) im Nahfeldbereich (8) einer festen Phase (1) angeordnet ist, die auf einen Scanner dreidimensional bewegbar gelagert ist, wobei Nahfeldlichtquelle (6) und feste Phase (1) in einer 1ichundurchlässigen Durchflußküvette angeordnet sind.19. Device for immobilizing macromolecules on very small surface elements of a solid phase, consisting of a near-field light source (6), the light exit opening (7) of which is arranged in the near-field region (8) of a solid phase (1), which is movably mounted on a scanner in three dimensions The near-field light source (6) and the solid phase (1) are arranged in an impervious flow cell.
20. Vorrichtung zur Immobilisierung von Makromolekülen auf sehr kleinen Oberflächenelementen einer festen Phase, bestehend aus einer Kombination einer lichtdurchlässigen Platte (22) mit durchgehenden Kanälen (24) , die auf einer Oberfläche eine Beschichtung mit photoaktivierbaren funktionellen Gruppen (23) aufweist und einer Matrize (20) mit durchgehenden Hohlräumen (21) , wobei je ein durchgehender Kanal (24) je einem durchgehenden Hohlraum (21) zugeordnet ist.20. Device for immobilizing macromolecules on very small surface elements of a solid phase, consisting of a combination of a translucent plate (22) with continuous channels (24), which has a coating with photoactivatable functional groups (23) on one surface and a matrix ( 20) with continuous cavities (21), one continuous channel (24) each being assigned to one continuous cavity (21).
21. Träger für eine photoaktivierbare Immobilisierung von Biomakromolekülen, bestehend aus mindestens zwei, extrem miniaturisierten, adressierbaren Bereichen lichtreduziert immobilisierter Moleküle.21. Carrier for a photoactivatable immobilization of biomacromolecules, consisting of at least two, extremely miniaturized, addressable areas of light-reduced immobilized molecules.
22. Verwendung des Trägers gemäß Anspruch 21 für die Durchführung von Immunoassays oder Hybridisierungs- reaktionen. 22. Use of the carrier according to claim 21 for carrying out immunoassays or hybridization reactions.
PCT/EP1998/003657 1997-06-18 1998-06-18 Method and device for immobilizing macromolecules WO1998058293A2 (en)

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