WO1999060156A2 - Method and device for photolithographic production of dna, pna and protein chips - Google Patents

Method and device for photolithographic production of dna, pna and protein chips Download PDF

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Publication number
WO1999060156A2
WO1999060156A2 PCT/DE1999/001524 DE9901524W WO9960156A2 WO 1999060156 A2 WO1999060156 A2 WO 1999060156A2 DE 9901524 W DE9901524 W DE 9901524W WO 9960156 A2 WO9960156 A2 WO 9960156A2
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Prior art keywords
dna
mask
pna
production
photolithographic
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PCT/DE1999/001524
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German (de)
French (fr)
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WO1999060156A3 (en
Inventor
Arno Svend Heuermann
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Epigenomics Gmbh
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Priority to AU48968/99A priority Critical patent/AU4896899A/en
Publication of WO1999060156A2 publication Critical patent/WO1999060156A2/en
Publication of WO1999060156A3 publication Critical patent/WO1999060156A3/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • 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
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03F—PHOTOMECHANICAL PRODUCTION OF TEXTURED OR PATTERNED SURFACES, e.g. FOR PRINTING, FOR PROCESSING OF SEMICONDUCTOR DEVICES; MATERIALS THEREFOR; ORIGINALS THEREFOR; APPARATUS SPECIALLY ADAPTED THEREFOR
    • G03F7/00—Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
    • G—PHYSICS
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03F—PHOTOMECHANICAL PRODUCTION OF TEXTURED OR PATTERNED SURFACES, e.g. FOR PRINTING, FOR PROCESSING OF SEMICONDUCTOR DEVICES; MATERIALS THEREFOR; ORIGINALS THEREFOR; APPARATUS SPECIALLY ADAPTED THEREFOR
    • G03F7/00—Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
    • G03F7/20—Exposure; Apparatus therefor
    • G03F7/2002—Exposure; Apparatus therefor with visible light or UV light, through an original having an opaque pattern on a transparent support, e.g. film printing, projection printing; by reflection of visible or UV light from an original such as a printed image
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00427—Means for dispensing and evacuation of reagents using masks
    • B01J2219/00432—Photolithographic masks
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00427—Means for dispensing and evacuation of reagents using masks
    • B01J2219/00434—Liquid crystal masks
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B01J2219/00529—DNA 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
    • 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
    • B01J2219/0059—Sequential 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
    • B01J2219/00608—DNA 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
    • 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
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00686—Automatic
    • B01J2219/00689—Automatic using computers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00709—Type of synthesis
    • B01J2219/00711—Light-directed synthesis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00729—Peptide nucleic acids [PNA]
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • the invention relates to a process for the preparation of oligonucleotides photolithogra ⁇ phical on two-dimensional matrices for the production of so-called DNA, PNA or protein chips, and an apparatus for performing the method.
  • DNA chips Surfaces on which a large number of different DNA molecules are fixed or synthesized in the smallest area are referred to as DNA chips. From any point on such a chip, it is generally known which DNA is on it. An important class of these chips is characterized in that short sequences, so-called oligonucleotides, are synthesized in situ on the chip surface. This considerably limits the number of chemical reaction steps that would otherwise be required to synthesize huge numbers of different sequences.
  • DNA chips can be made in several ways. The simplest but most expensive and most complex is the application of previously synthesized molecules using pipetting systems. Such methods are unlikely to be competitive in the future.
  • nucleotide building systems which carry two types of protective groups.
  • such protective groups which protect the functionalities of the bases
  • a different type of protective group which only allows chain extension by a single building block.
  • the removal of this last protecting group is essential for the m situ synthesis.
  • Protective groups must be split off quantitatively at extremely many points of a matrix without causing such a split-off at the other points. Chemical methods quickly reach the limit of the resolution of the pipetting systems. The individual drops that are applied are too large and overlap from a certain density.
  • Points will be drawn. Two ways are feasible to achieve a high resolution and thus occupancy density on such surfaces.
  • each individual point of a surface is individually controlled with a light beam - for example a laser - and so the protective groups of the nucleotide chains are only deprotected at the illuminated points.
  • the necessary irradiation time is so long that these methods are still too time-consuming.
  • DNA is also destroyed by laser bombardment.
  • the possibly tens of thousands of points have to be addressed one after the other. Even complex ones were possible for example mirror mechanisms which control many points at the same time. Such devices are currently not available.
  • the second and most common method today is to install masks between the chip surface and a light source.
  • the light from the light source is only let through at the points to the chip surface where deprotection is to take place. Therefore, virtually any number of reactions can be carried out in parallel.
  • four different masks must be positioned sequentially over the surface in order to extend all oligonucleotides by one nucleotide.
  • 120 masks have to be produced and successively positioned extremely precisely above the surface.
  • the proposed method according to the invention is intended to enable the future to be able to dispense with the establishment of own factories for the production of DNA and PNA chips.
  • the method is said to make the most complex step of chip division, the production and positioning of the masks superfluous.
  • the object of the present invention is to provide a method which overcomes the disadvantages of the prior art.
  • the object is achieved by a process for the photolithographic production of oligonucleotides on two-dimensional matrices for the production of so-called DNA, PNA or protein chips, in which a dynamically controllable liquid crystal mask is used as the photolithographic mask.
  • a device for the photolithography production of oligonucleotides on two-dimensional matrices for the production of so-called DNA, PNA or protein chips is made available, in which a dynamically controllable liquid crystal mask is arranged between the chip and the light source as the photolithographic mask.
  • the method according to the invention achieves the task in a completely new way by combining commercially available components. Compared to modern methods, the synthesis of DNA and PNA chips is therefore cheaper by several orders of magnitude. The monopoly of a few large factories can thus be broken and the manufacture of inexpensive DNA chips made accessible to the general public.
  • the basic concept of the method is the fact known per se that liquid crystal matrices can be used as dynamically controllable photolithographic masks (Bertsch et al., J Photochem. & Photobiol. 107, 275-281 (1997)). However, this technique has never been used or discussed in the field of synthesis of biochemical polymers on surfaces.
  • the method according to the invention eliminates the need to produce a large number of different photolithographic masks.
  • the liquid crystal lattice according to the invention can be driven at every point of the matrix by vapor-deposited transistors.
  • the resolution of the chips to be produced is therefore only dependent on the number of individually controllable cells of the liquid crystal. Instead of a physical arrangement of holes in an opaque surface, each mask is achieved by the purely electronic control of the individual cells.
  • the resolution of the mask - limited by the minimum size of the individual cells - can be increased practically infinitely by using a larger liquid crystal matrix than the final chip.
  • the light that falls through this large dynamic mask can then be telescoped onto the surface through optical lenses.
  • This technology can also prevent the otherwise fading effect of the liquid crystals: less light falls on the liquid crystals per area than is required behind them for deprotection on the chip surface.
  • the control of the liquid crystal is simply changed by the computer in the method according to the invention.
  • Deprotection lies in the synthesis of chemical steps for which no light energy is necessary. This also takes place within the device according to the method, without the movement of the chip or the mask being necessary. Due to the rigid arrangement and extremely precise positioning of the chip, mask and light source, mechanical problems such as the repositioning that is often necessary according to the prior art are avoided.
  • a device according to the method can therefore be designed mechanically in a very simple manner.
  • a device is characterized in that chip blanks are produced which either have chemically activatable surfaces (to which any nucleotide building block can be coupled) or are already covered with protected, photochemically removable molecules.
  • Such molecules can, for example, directly represent individual nucleotides or PNA building blocks which have been applied uniformly to the surface during production.
  • An essential property of these blanks is their packaging. These are manufactured in a clean, pollution-free manner and packaged in such a way that they can be inserted into the device in a manner that enables any contact with an unfiltered “normal” laboratory environment.
  • such a packaging can be sealed with a penetrable film
  • a blank packaged in this way can be inserted into the device by means of a seal, the actual blank being pressed out of the packaging, the film being pierced and then latching into the actual device (FIG. 1), which is the exact and immovable positioning of the blank during all further steps. Furthermore, contamination is excluded.
  • the blank comes to lie below the liquid crystal matrix.
  • the blank thus forms the bottom, the lower electrode plate of the liquid crystal matrix the ceiling of a very small cavity, which is also sealed on the sides. Chemical solutions are introduced into this cavity via several feed lines and this can also be air or gas dried.
  • the ceiling of the cavity can also be directly one of the components of the liquid crystal display due to a surface other than egg. For example, this can consist of a last part of the optics, which focus the light transmitted through the various cells of the liquid crystal.
  • the liquid crystal matrix itself consists in a manner known per se of liquid crystals, which are enclosed between two planar layers of a material which is transparent to the wavelengths essential for deprotection. Wavelengths that cannot specifically contribute to photo-deprotection are absorbed by these layers or other parts of the optics. Particularly short-wave ultraviolet light is absorbed by these layers. This destroys DNA.
  • One layer of material also acts as an electrode. Cables are placed on the other layer in such a way that the entire matrix is defined by cells that can be electrically controlled individually and divided into a narrow grid of points. Electrical excitation leads to an alignment of the liquid crystals only at the defined points. Light is then absorbed there. On the other hand, it is also possible that only the excited points become transparent to light of a certain wavelength.
  • the liquid crystal matrix can have exactly the size of the underlying chip surface. Then light with a relatively parallel beam path must be radiated onto the matrix by a simple light source. However, the liquid crystal matrix can also be of any size than the irradiated surface. In this case, an optical system must be introduced between the matrix and the chip, which bundles the translucent light.
  • the advantages of such an arrangement are that the energy acting on the matrix per area becomes lower than on the chip surface. This avoids the problematic effect which reduces the absorption capacity of the liquid crystals in the event of permanent irradiation.
  • the number of individual points on the chip surface can be increased practically as desired (FIG. 2).
  • a device designed according to the method according to the invention can operate independently by entering one or more sequences which are to be tested by hybridizing a target DNA with the chip.
  • the data processing can independently calculate the oligonucleotides to be synthesized from the sequences entered and calculate the sequence of the masks required for their synthesis. Coordinated with the various chemical reaction steps, these are then implemented fully automatically during the synthesis by the pattern of the voltages applied to the matrix in the individual deprotection steps.
  • a detector for example a CCD camera
  • this can then send signals to the register and evaluate individual points on the chip surface.
  • FIG. 1 shows a schematic illustration of a device according to the invention in the form of a work station
  • Fig. 2 is a schematic representation of a detailed view of the exposure device according to the invention.
  • the DNA or PNA chip 4 is inserted in the processing station 9 and supplied with the chemicals necessary for the reaction from the reagent storage 11.
  • the actual exposure station consists of the light source 1 and the LCD mask 2.
  • the other components shown, namely the polarizer 3, the focusing 7 and the barrier 8 serve to optically process the light. All functions are controlled by means of the computer 10, in particular the LCD mask 2 is controlled accordingly.
  • FIG. 2 shows a detailed view of a further embodiment of the device according to the invention.
  • the LCD mask 2 is again shown.
  • the chip 4 is arranged below the LCD mask 2.
  • the other optical elements namely a diffuser 5, a polarization 3 and a lens 6 are shown.

Abstract

Disclosed is a method and a device for photolithographic production of oligonucleotides on two-dimensional matrices for the production of so-called DNA, PNA or protein chips characterized in that a dynamically controlled liquid crystal mask is used as a photolithographic mask. The invention also relates to a device for implementing said method. In the device according to the invention, the DNA or PNA chip (4) is placed in the processing station (9) and provided with the chemicals required for reaction from the reagent storage (11). The actual exposure station is composed of the light source (1) and the LCD mask (2). The remaining components described, i.e. the polarizer (3), the focussing (7) and the interlock (8), serve to optically process the light. All functions are controlled by the computer (10), especially, the LCD mask (12) is correspondingly controlled.

Description

Verfahren und Vorrichtung zur photolithographischen Herstellung von DNA, PNA und Protein Chips Method and device for the photolithographic production of DNA, PNA and protein chips
Die Erfindung betrifft ein Verfahren zur photolithogra¬ phischen Herstellung von Oligonukleotiden auf zweidimen- sionalen Matrizen zur Produktion von sogenannten DNA-, PNA- oder Protein-Chips, sowie eine Vorrichtung zur Durchführung des Verfahrens.The invention relates to a process for the preparation of oligonucleotides photolithogra ¬ phical on two-dimensional matrices for the production of so-called DNA, PNA or protein chips, and an apparatus for performing the method.
Als DNA-Chips werden Oberflächen bezeichnet, auf denen auf kleinster Fläche eine große Anzahl verschiedener DNA Moleküle fixiert oder synthetisiert werden. Von jedem beliebigen Punkt eines solchen Chips ist in der Regel be- kannt, welche DNA sich an diesem befindet. Eine wichtige Klasse dieser Chips ist dadurch gekennzeichnet, daß kurze Sequenzen, sogenannte Oligonukleotide in situ auf der Chip-Oberfläche synthetisiert werden. Dadurch wird die Zahl der notwendigen chemischen Reaktionsschritte, die anderenfalls zur Synthese riesiger Zahlen von verschiedenen Sequenzen notwendig wären erheblich eingeschränkt.Surfaces on which a large number of different DNA molecules are fixed or synthesized in the smallest area are referred to as DNA chips. From any point on such a chip, it is generally known which DNA is on it. An important class of these chips is characterized in that short sequences, so-called oligonucleotides, are synthesized in situ on the chip surface. This considerably limits the number of chemical reaction steps that would otherwise be required to synthesize huge numbers of different sequences.
DNA-Chips können auf mehrere Arten hergestellt werden. Die einfachste aber teuerste und aufwendigste ist das Aufbringen vorher synthetisierter Moleküle mittels Pipet- tieranlagen. Solche Methoden werden in Zukunft wahrscheinlich nicht konkurrenzfähig sein.DNA chips can be made in several ways. The simplest but most expensive and most complex is the application of previously synthesized molecules using pipetting systems. Such methods are unlikely to be competitive in the future.
Methoden, die sich die oben genannten Vorteile der in Si- tu Synthese zunutze machen lassen sich in rein chemische und photolithographische Verfahren unterteilen.Methods that take advantage of the above-mentioned advantages of site synthesis can be divided into purely chemical and photolithographic processes.
Chemische Verfahren beruhen auf dem Aufbringen der zur Oligonukleotidsynthese notwendigen Lösungen mittels auf- wendiger Pipettieranlagen. Daher sind diese zwar einer konventionellen (nicht in situ) Synthese hinsichtlich Ge- schwmdigkeit und Kosteneffizienz überlegen, können aber bei weitem nicht mit den Möglichkeiten der photolithogra- phischen Synthesen konkurrieren.Chemical processes are based on the application of the solutions necessary for oligonucleotide synthesis by means of complex pipetting systems. Therefore, they are a conventional (not in situ) synthesis in terms of superior to fatigue and cost efficiency, but far from competing with the possibilities of photolithographic synthesis.
Für die chemische Synthese von Oligonukleotiden werden Nukleotidbausteme eingesetzt, welche zwei Arten von Schutzgruppen tragen. Einerseits solche Schutzgruppen, die Funktionalitäten der Basen schützen, und andererseits eine anders geartete Schutzgruppe, welche lediglich die Kettenverlangerung um einen einzigen Baustein zulaßt. Die Abspaltung dieser letzten Schutzgruppe ist wesentlich für die m Situ Synthese. Es müssen an extrem vielen Punkten einer Matrix spezifisch Schutzgruppen quantitativ abgespalten werden, ohne an den anderen Punkten eine solche Abspaltung zu verursachen. Chemische Methoden stoßen dabei sehr schnell an die Grenze der Auflosung der Pipet- tiersysteme. Die einzelnen Tropfen, die aufgetragen werden sind zu groß und überlappen ab einer bestimmten Dichte.For the chemical synthesis of oligonucleotides, nucleotide building systems are used which carry two types of protective groups. On the one hand, such protective groups, which protect the functionalities of the bases, and on the other hand, a different type of protective group which only allows chain extension by a single building block. The removal of this last protecting group is essential for the m situ synthesis. Protective groups must be split off quantitatively at extremely many points of a matrix without causing such a split-off at the other points. Chemical methods quickly reach the limit of the resolution of the pipetting systems. The individual drops that are applied are too large and overlap from a certain density.
Daher werden für DNA-Chips hoher Dichte heute photolitho- graphische Synthesewege gewählt. Dabei sind die Schutzgruppen der Nukleotidbausteme photochemisch abspaltbar. Durch Bestrahlung einzelner Punkte der Syntheseoberflache kann die Kettenverlangerung spezifisch nur an diesenFor this reason, photolithographic synthetic routes are now chosen for high-density DNA chips. The protective groups of the nucleotide building systems can be removed photochemically. By irradiating individual points on the synthesis surface, the chain extension can only be specific to these
Punkten ausgelost werden. Zwei Wege sind gangbar, eine große Auflosung und damit Belegungsdichte auf solchen Oberflachen zu erreichen. Zum einen wird jeder einzelne Punkt einer Oberflache einzeln mit einem Lichtstrahl - zum Beispiel einem Laser - angesteuert und so werden die Schutzgruppen der Nukleotidketten nur an den angestrahlten Punkten entschutzt. Die notwendige Bestrahlungsdauer ist aber so lang, daß diese Verfahren noch zu Zeitaufwendig sind. Außerdem wird DNA durch Laserbeschuß zerstört. Die möglicherweise Zehntausende von Punkten müssen nacheinander angesteuert werden. Möglich waren auch komplexe zum Beispiel Spiegelmechanismen, welche viele Punkte gleichzeitig ansteuern. Solche Vorrichtungen sind aber zur Zeit nicht erhältlich.Points will be drawn. Two ways are feasible to achieve a high resolution and thus occupancy density on such surfaces. On the one hand, each individual point of a surface is individually controlled with a light beam - for example a laser - and so the protective groups of the nucleotide chains are only deprotected at the illuminated points. However, the necessary irradiation time is so long that these methods are still too time-consuming. DNA is also destroyed by laser bombardment. The possibly tens of thousands of points have to be addressed one after the other. Even complex ones were possible for example mirror mechanisms which control many points at the same time. Such devices are currently not available.
Die zweite und heute gebrauchlichste Methode ist es, Masken zwischen der Chip-Oberflache und einer Lichtquelle zu installieren. In jedem Syntheseschritt wird so das Licht der Lichtquelle nur an den Punkten zur Chip-Oberflache durchgelassen, an denen eine Entschutzung stattfinden soll. Daher können praktisch beliebig viele Reaktionen parallel durchgeführt werden. Bei vier Nukleotidbaustei- nen müssen also für eine Verlängerung aller Oligonukleo- tide um ein Nukleotid vier verschiedene Masken sequentiell über der Oberflache positioniert werden. Um also eine Lange aller Oligonukleotide von zum Beispiel 30 Nukleoti- deinheiten zu erreichen müssen 120 Masken hergestellt werden und nacheinander extrem genau über der Oberflache positioniert werden.The second and most common method today is to install masks between the chip surface and a light source. In each synthesis step, the light from the light source is only let through at the points to the chip surface where deprotection is to take place. Therefore, virtually any number of reactions can be carried out in parallel. In the case of four nucleotide building blocks, four different masks must be positioned sequentially over the surface in order to extend all oligonucleotides by one nucleotide. In order to achieve a length of all oligonucleotides of, for example, 30 nucleotide units, 120 masks have to be produced and successively positioned extremely precisely above the surface.
Je großer die Auflosung des Chips, desto schwieriger ist die Positionierung der Masken über der Oberflache. Extrem aufwendige Technologie ist dafür erforderlich. Die Herstellungskosten von DNA-Chips liegen daher bei einigen Hunderttausend Mark. Außerdem, ηe mehr einzelne Punkte auf einem solchen Chip synthetisiert werden sollen, desto aufwendiger wird die Herstellung der Masken. Im Prinzip kann heute ein solcher Chip deswegen nur in eigens konstruierten Fabriken hergestellt werden. Voraussetzung zur Herstellung solcher Chips ist auch die Installation aller dafür notwendigen Gerate m staubfreien Remraumen. Es besteht aber ein erheblicher Markt an solchen Chips, die von Firmen und Laboratorien auf ad hoc Basis selber entworfen und hergestellt werden könne. Dies verbietet sich nach dem Stand der Technik. Das vorgeschlagene erfmdungsgemaße Verfahren soll es ermöglichen Zukunft auf die Etablierung eigener Fabriken für die Herstellung von DNA und PNA-Chips verzichten zu können. Das Verfahren soll den aufwendigsten Schritt der Chiphersteilung, die Herstellung und Positionierung der Masken überflüssig machen. Außerdem soll auf Reinraume verzichtet werden können, die Synthese also in jedem Labor möglich werden.The greater the resolution of the chip, the more difficult it is to position the masks over the surface. Extremely complex technology is required for this. The production costs of DNA chips are therefore a few hundred thousand marks. In addition, ηe more individual points are to be synthesized on such a chip, the more complex it is to manufacture the masks. In principle, such a chip can only be manufactured in specially designed factories today. A prerequisite for the production of such chips is the installation of all the necessary equipment in dust-free remainders. However, there is a significant market for such chips, which companies and laboratories can design and manufacture themselves on an ad hoc basis. This is prohibited according to the state of the art. The proposed method according to the invention is intended to enable the future to be able to dispense with the establishment of own factories for the production of DNA and PNA chips. The method is said to make the most complex step of chip division, the production and positioning of the masks superfluous. In addition, it should be possible to dispense with clean rooms, so that synthesis can be carried out in any laboratory.
Aufgabe der vorliegenden Erfindung ist es, ein Verfahren zur schaffen, welches die Nachteile des Standes der Technik überwindet.The object of the present invention is to provide a method which overcomes the disadvantages of the prior art.
Die Aufgabe wird durch ein Verfahren zur photolithogra- phischen Herstellung von Oligonukleotiden auf zweidimen- sionalen Matrizen zur Produktion von sogenannten DNA-, PNA- oder Protein-Chips gelost, bei dem man als photoli- thographische Maske eine dynamisch ansteuerbare Flussig- kristallmaske verwendet.The object is achieved by a process for the photolithographic production of oligonucleotides on two-dimensional matrices for the production of so-called DNA, PNA or protein chips, in which a dynamically controllable liquid crystal mask is used as the photolithographic mask.
Ferner wird erfmdungsgemaße eine Vorrichtung zur photo- lithographischen Herstellung von Oligonukleotiden auf zweidimensionalen Matrizen zur Produktion von sogenannten DNA-, PNA- oder Protem-Chips zur Verfugung gestellt, bei der als photolithographische Maske eine dynamisch ansteuerbare Flussigkristallmaske zwischen dem Chip und der Lichtquelle angeordnet ist.Furthermore, according to the invention, a device for the photolithography production of oligonucleotides on two-dimensional matrices for the production of so-called DNA, PNA or protein chips is made available, in which a dynamically controllable liquid crystal mask is arranged between the chip and the light source as the photolithographic mask.
Das erfmdungsgemaße Verfahren lost die gestellte Aufgabe auf völlig neuartige Art und Weise durch Kombination kommerziell erhaltlicher Komponenten. Im Vergleich zu heute modernen Verfahren verbilligt sich daher die Synthese von DNA- und PNA-Chips um mehrere Größenordnungen. Die Monopolstellung einiger weniger großer Fabriken kann so ge- brochen werden und die Herstellung von kostengünstigen DNA-Chips der Allgemeinheit zuganglich gemacht werden. Das grundlegende Konzept des Verfahrens ist die an sich bekannte Tatsache, daß Flüssigkristall-Matrixen als dynamisch ansteuerbare photolithographische Masken verwendet werden können (Bertsch et al., J Photochem. & Photobiol. 107, 275-281 (1997)). Diese Technik ist allerdings noch nie auf dem Gebiet der Synthese von biochemischen Polymeren auf Oberflächen eingesetzt oder diskutiert worden.The method according to the invention achieves the task in a completely new way by combining commercially available components. Compared to modern methods, the synthesis of DNA and PNA chips is therefore cheaper by several orders of magnitude. The monopoly of a few large factories can thus be broken and the manufacture of inexpensive DNA chips made accessible to the general public. The basic concept of the method is the fact known per se that liquid crystal matrices can be used as dynamically controllable photolithographic masks (Bertsch et al., J Photochem. & Photobiol. 107, 275-281 (1997)). However, this technique has never been used or discussed in the field of synthesis of biochemical polymers on surfaces.
Das erfindungsgemäße Verfahren eliminiert die Notwendigkeit sehr viele verschiedene photolithographische Masken herzustellen. Das erfindungsgemäß Flüssigkristallgitter ist durch aufgedampfte Transistoren an jedem Punkt der Matrix ansteuerbar. Die Auflösung der herzustellenden Chips ist daher nur durch die Zahl der einzeln ansteuerbaren Zellen des Flüssigkristalls abhängig. Jede Maske wird also anstelle einer physikalischen Anordnung von Löchern in einer lichtundurchlässigen Oberfläche durch die rein elektronische Ansteuerung der einzelnen Zellen er- reicht. Die Auflösung der Maske - durch die minimale Größe der einzelnen Zellen limitiert - kann dadurch praktisch unendlich gesteigert werden, daß eine größere Flüssigkristallmatrix verwendet wird als der letztendliche Chip. Das Licht, welches durch diese große dynamische Maske fällt kann dann hinter dieser durch optische Linsen auf die Oberfläche teleskopiert werden. Durch diese Technik kann auch der sonst erfolgende Ausbleicheffekt der Flüssigkristalle verhindert werden: Weniger Licht fällt pro Fläche auf die Flüssigkristalle, als hinter dieser für die Entschützung auf der Chip-Oberfläche benötigt wird.The method according to the invention eliminates the need to produce a large number of different photolithographic masks. The liquid crystal lattice according to the invention can be driven at every point of the matrix by vapor-deposited transistors. The resolution of the chips to be produced is therefore only dependent on the number of individually controllable cells of the liquid crystal. Instead of a physical arrangement of holes in an opaque surface, each mask is achieved by the purely electronic control of the individual cells. The resolution of the mask - limited by the minimum size of the individual cells - can be increased practically infinitely by using a larger liquid crystal matrix than the final chip. The light that falls through this large dynamic mask can then be telescoped onto the surface through optical lenses. This technology can also prevent the otherwise fading effect of the liquid crystals: less light falls on the liquid crystals per area than is required behind them for deprotection on the chip surface.
Anstelle des nach dem Stand der Technik notwendigen Aus- tauschens von Masken nach jedem Syntheseschritt, wird im erfindungsgemäßen Verfahren einfach nur die Ansteuerung des Flüssigkristalls vom Computer geändert. Zwischen den Entschützungen liegen bei der Synthese chemische Schritte, für welche keine Lichtenergie notwendig ist. Dies findet auch innerhalb der verfahrensgemäßen Vorrichtung statt, ohne daß die Bewegung des Chips oder der Maske notwendig wäre. Durch die starre Anordnung und extrem präzise Positionierung von Chip, Maske und Lichtquelle, werden mechanische Probleme wie die nach dem Stand der Technik oft notwendige Neupositionierung vermieden. Eine verfahrensgemäße Vorrichtung kann also mechanisch sehr einfach ausgelegt sein.Instead of the replacement of masks after each synthesis step, which is necessary according to the prior art, the control of the liquid crystal is simply changed by the computer in the method according to the invention. Between Deprotection lies in the synthesis of chemical steps for which no light energy is necessary. This also takes place within the device according to the method, without the movement of the chip or the mask being necessary. Due to the rigid arrangement and extremely precise positioning of the chip, mask and light source, mechanical problems such as the repositioning that is often necessary according to the prior art are avoided. A device according to the method can therefore be designed mechanically in a very simple manner.
Technisch zeichnet sich eine erfindungsgemäße Vorrichtung dadurch aus, das Chip-Rohlinge hergestellt werden, welche entweder chemisch aktivierbare Oberflächen (an welche ein beliebiger Nukleotidbaustein gekoppelt werden kann) aufweisen oder schon mit geschützten, photochemisch abspaltbaren Molekülen belegt sind. Solche Moleküle können zum Beispiel direkt einzelne Nukleotide oder PNA Bausteine darstellen, welche in der Produktion gleichmäßig auf der Oberfläche angebracht worden sind. Eine wesentliche Eigenschaft dieser Rohlinge ist deren Verpackung. Diese werden unter Reinbedingungen verschmutzungsfrei hergestellt und so verpackt, daß sie auf eine Art und Weise in die Vorrichtung eingeführt werden können, die jeden Kon- takt mit einer nicht gefilterten „normalen" Laborumgebung ermöglicht. Zum Beispiel kann eine solche Verpackung mit einer durchstoßbaren Folie versiegelt werden. Eine derart verpackter Rohling kann durch eine Dichtung in die Vorrichtung eingeschoben werden, wobei der eigentliche Roh- ling aus der Verpackung herausgedrückt wird, die Folie durchstößt und dann in der Eigentlichen Vorrichtung einrastet (Figur 1) . Damit ist die genaue und unverrückbare Positionierung des Rohlings während allen weiteren Schritten gesichert. Außerdem wird Verschmutzung ausge- schlössen. Nach dem Einrasten kommt ein solcher Rohling unterhalb der Flüssigkristallmatrix zu liegen. Der Rohling bildet so den Boden, die untere Elektrodenplatte der Flüssigkristallmatrix die Decke eines sehr kleinen Hohlraumes, der auch an den Seiten abgedichtet ist. Über mehrere Zuleitungen werden chemische Lösungen in diesen Hohlraum eingeführt und dieser kann auch luft- oder gasgetrocknet werden. Die Decke des Hohlraumes kann aber auch durch ei¬ ne andere Fläche als direkt einer der Bestandteile der Flüssigkristallanzeige sein. Zum Beispiel kann dieser aus einem letzten Teil der Optik bestehen, welche das durch die verschiedenen Zellen des Flüssigkristalls durchgelassene Licht fokussieren.Technically, a device according to the invention is characterized in that chip blanks are produced which either have chemically activatable surfaces (to which any nucleotide building block can be coupled) or are already covered with protected, photochemically removable molecules. Such molecules can, for example, directly represent individual nucleotides or PNA building blocks which have been applied uniformly to the surface during production. An essential property of these blanks is their packaging. These are manufactured in a clean, pollution-free manner and packaged in such a way that they can be inserted into the device in a manner that enables any contact with an unfiltered “normal” laboratory environment. For example, such a packaging can be sealed with a penetrable film A blank packaged in this way can be inserted into the device by means of a seal, the actual blank being pressed out of the packaging, the film being pierced and then latching into the actual device (FIG. 1), which is the exact and immovable positioning of the blank during all further steps. Furthermore, contamination is excluded. After snapping in, such a blank comes to lie below the liquid crystal matrix. The blank thus forms the bottom, the lower electrode plate of the liquid crystal matrix the ceiling of a very small cavity, which is also sealed on the sides. Chemical solutions are introduced into this cavity via several feed lines and this can also be air or gas dried. The ceiling of the cavity can also be directly one of the components of the liquid crystal display due to a surface other than egg. For example, this can consist of a last part of the optics, which focus the light transmitted through the various cells of the liquid crystal.
Das erfindungsgemäße Verfahren soll aber auch andere technische Ausführungen einschließen.However, the method according to the invention is also intended to include other technical designs.
Die Flüssigkristallmatrix selber besteht in an sich bekannter Art und Weise aus Flüssigkristallen, welcher zwi- sehen zwei planen Schichten eines solchen Materials eingeschlossen werden, welches für die für die Entschützung wesentlichen Wellenlängen durchlässig ist. Wellenlängen, welche nicht spezifisch zur Photoentschützung beitragen können durch diese Schichten oder andere Teile der Optik absorbiert werden. Besonders kurzwelliges ultraviolettes Licht wird durch diese Schichten absorbiert. Dieses zerstört DNA. Die eine Schicht Material wirkt dabei auch als Elektrode. Auf die andere Schicht werden in der Art Leitungen gelegt, daß die gesamte Matrix definiert durch Zellen, die einzeln elektrisch ansteuerbar sind in ein enges Gitter von Punkten unterteilt ist. Elektrische Anregung führt zu einer Ausrichtung der Flüssigkristalle nur an den definierten Punkten. Dort wird dann Licht absorbiert. Andererseits ist es aber auch möglich, das nur die angeregten Punkte für Licht einer bestimmten Wellenlänge durchlässig werden. Beide Varianten sollen also ge- schützt werden. Im Prinzip kann die Flussigkristallmatπx genau die Große der darunterliegenden Chip-Oberflache haben. Dann muß Licht mit einem verhältnismäßig parallelen Strahlengang durch eine einfache Lichtquelle auf die Ma- tπx gestrahlt werden. Die Flussigkristallmatrix kann aber auch beliebig großer sein, als die bestrahlte Oberflache. In diesem Fall muß zwischen Matrix und Chip eine Optik eingef hrt werden, welche das durchscheinende Licht bunαelt. Die Vorteile einer solchen Anordnung sind, daß die pro Flache auf die Matrix einwirkende Energie geringer wird als auf der Chip-Oberflache. Damit kann der problematische Effekt vermieden werden, welcher bei dauerhafter Bestrahlung die Absorptionsfähigkeit der Flussig- kristalle schmälert. Außerdem kann die Zahl der einzelnen Punkte auf der Chip-Oberflache praktisch beliebig gesteigert werden (Figur 2) .The liquid crystal matrix itself consists in a manner known per se of liquid crystals, which are enclosed between two planar layers of a material which is transparent to the wavelengths essential for deprotection. Wavelengths that cannot specifically contribute to photo-deprotection are absorbed by these layers or other parts of the optics. Particularly short-wave ultraviolet light is absorbed by these layers. This destroys DNA. One layer of material also acts as an electrode. Cables are placed on the other layer in such a way that the entire matrix is defined by cells that can be electrically controlled individually and divided into a narrow grid of points. Electrical excitation leads to an alignment of the liquid crystals only at the defined points. Light is then absorbed there. On the other hand, it is also possible that only the excited points become transparent to light of a certain wavelength. Both variants should therefore be protected. In principle, the liquid crystal matrix can have exactly the size of the underlying chip surface. Then light with a relatively parallel beam path must be radiated onto the matrix by a simple light source. However, the liquid crystal matrix can also be of any size than the irradiated surface. In this case, an optical system must be introduced between the matrix and the chip, which bundles the translucent light. The advantages of such an arrangement are that the energy acting on the matrix per area becomes lower than on the chip surface. This avoids the problematic effect which reduces the absorption capacity of the liquid crystals in the event of permanent irradiation. In addition, the number of individual points on the chip surface can be increased practically as desired (FIG. 2).
Die letzte wesentliche Eigenschaft die beschriebenen Verfahrensweise liegt m den Algorithmen, welche zur An- Steuerung des Flussigkristallmatrix verwendet werden. Eine nach dem erfmdungsgemaßen Verfahren ausgelegte Vorrichtung kann von der Eingabe einer oder vieler Sequenzen, welche durch Hybridisierung einer Ziel-DNA mit dem Chip getestet werden sollen, selbständig operieren. Die Datenverarbeitung kann aus den eingegebenen Sequenzen selbständig die zu synthetisierenden Oligonukleotide errechnen und die Abfolge der zu deren Synthese notwendigen Masken berechnen. Diese werden dann, koordiniert mit den verschiedenen chemischen Reaktionsschritten vollautoma- tisch wahrend der Synthese durch das Muster der den einzelnen Entschutzungsschritten an die Matrix angelegten Spannungen umgesetzt.The last essential property of the procedure described lies in the algorithms that are used to control the liquid crystal matrix. A device designed according to the method according to the invention can operate independently by entering one or more sequences which are to be tested by hybridizing a target DNA with the chip. The data processing can independently calculate the oligonucleotides to be synthesized from the sequences entered and calculate the sequence of the masks required for their synthesis. Coordinated with the various chemical reaction steps, these are then implemented fully automatically during the synthesis by the pattern of the voltages applied to the matrix in the individual deprotection steps.
Erfmdungsgemaß kann auch ein Detektor (zum Beispiel eine CCD Kamera) m die Vorrichtung eingebaut werden. Diese kann dann nach einer Hybridisierung Signale an den ein- zelnen Punkten der Chip-Oberfläche registrieren und auswerten.According to the invention, a detector (for example a CCD camera) can also be installed in the device. After hybridization, this can then send signals to the register and evaluate individual points on the chip surface.
Mit dem erfindungsgemäßen Verfahren ist zum ersten Mal eine Methode geschaffen worden, welche ein billiges und schnelles Synthetisieren von DNA- oder PNA-Chips ermög¬ licht, deren Belegung mit Sequenzen vom eigentlichen Bediener individuell festgelegt werden kann. Unser Verfahren revolutioniert die Technologie der DNA-Chips von Grund auf.With the inventive method for the first time a method has been provided which allowed a cheap and rapid synthesizing DNA or PNA chips ¬ light whose layout can be set individually with sequences from the actual operator. Our process revolutionizes the technology of DNA chips from scratch.
Die beigefügten Zeichnungen erläutern die Erfindung: Es zeigen:The accompanying drawings illustrate the invention:
Fig. 1 eine schematische Darstellung einer erfindungsge- mäßen Vorrichtung in Form einer Arbeitsstation und1 shows a schematic illustration of a device according to the invention in the form of a work station and
Fig. 2 eine schematische Darstellung einer Detailansicht der erfindungsgemäßen Belichtungsvorrichtung.Fig. 2 is a schematic representation of a detailed view of the exposure device according to the invention.
In Figur 1 wird in der Bearbeitungsstation 9 der DNA- oder PNA-Chip 4 eingelegt und aus dem Reagenzienspeicher 11 mit den für die Reaktion notwendigen Chemikalien versorgt. Die eigentliche Belichtungsstation besteht aus der Lichtquelle 1 und der LCD-Maske 2. Die übrigen dargestellten Bestandteile, nämlich der Polarisator 3, die Fo- kussierung 7 und die Sperre 8 dienen der optischen Aufbereitung des Lichts. Mittels des Computer 10 werden alle Funktionen gesteuert, insbesondere die LCD-Maske 2 entsprechend angesteuert.In FIG. 1, the DNA or PNA chip 4 is inserted in the processing station 9 and supplied with the chemicals necessary for the reaction from the reagent storage 11. The actual exposure station consists of the light source 1 and the LCD mask 2. The other components shown, namely the polarizer 3, the focusing 7 and the barrier 8 serve to optically process the light. All functions are controlled by means of the computer 10, in particular the LCD mask 2 is controlled accordingly.
Figur 2 zeigt eine Detailansicht einer weiteren Ausführungsform der erfindungsgemäßen Vorrichtung. Neben der Lichtquelle 1 ist wiederum die LCD-Maske 2 gezeigt. Unterhalb der LCD-Maske 2 ist der Chip 4 angeordnet. Ferner sind die übrigen optischen Elemente, nämlich ein Diffusor 5, eine Polarisation 3 und eine Linse 6 dargestellt. Bezugs zeichenlisteFIG. 2 shows a detailed view of a further embodiment of the device according to the invention. In addition to the light source 1, the LCD mask 2 is again shown. The chip 4 is arranged below the LCD mask 2. Furthermore, the other optical elements, namely a diffuser 5, a polarization 3 and a lens 6 are shown. Reference character list
LichtquelleLight source
LCD-MaskeLCD mask
PolarisatorPolarizer
Chipchip
DiffusorDiffuser
Linselens
Fokussierung (BündelungFocusing (bundling
SperreLock
BearbeitungsStationMachining station
Computercomputer
Reagenzienspeicher Reagent storage

Claims

Patentansprüche claims
1. Verfahren zur photolithographischen Herstellung von Oligonukleotiden auf zweidimensionalen Matrizen zur Produktion von sogenannten DNA-, PNA- oder Protein- Chips, dadurch gekennzeichnet, daß man als photolithographische Maske eine dynamisch ansteuerbare Flüssigkristallmaske verwendet.1. A process for the photolithographic production of oligonucleotides on two-dimensional matrices for the production of so-called DNA, PNA or protein chips, characterized in that a dynamically controllable liquid crystal mask is used as the photolithographic mask.
2. Vorrichtung zur photolithographischen Herstellung von Oligonukleotiden auf zweidimensionalen Matrizen zur Produktion von sogenannten DNA-, PNA- oder Protein- Chips, dadurch gekennzeichnet, daß als photolithogra- phische Maske eine dynamisch ansteuerbare Flüssigkristallmaske zwischen dem Chip und der Lichtquelle angeordnet ist. 2. Device for the photolithographic production of oligonucleotides on two-dimensional matrices for the production of so-called DNA, PNA or protein chips, characterized in that a dynamically controllable liquid crystal mask is arranged between the chip and the light source as the photolithographic mask.
PCT/DE1999/001524 1998-05-18 1999-05-17 Method and device for photolithographic production of dna, pna and protein chips WO1999060156A2 (en)

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