EP1218105B1 - Structured reaction substrate - Google Patents

Abstract

The invention relates to a reaction substrate (20) comprised of a base part (10) and of a flexible compartment layer (21) which is made of a polymer material and which is provided with predetermined compartment structures (30) for forming sample compartments. According to the invention, the polymer material is a viscoelastic polymer composition (e.g. silicone rubber) which has an inherent adhesive property with regard to substrates made of glass, plastic, metal or of semiconductors. The invention also relates to a tool for producing the reaction substrate.

Description

The invention relates to a reaction substrate for recording and / or manipulation of a large number of separate ones Specimens that are used in particular for a structured reaction substrate forms microscopic sample amounts, and uses of the reaction substrate.

In biochemistry, medicine and genetic engineering, there is a broad need for methods for manipulating, observing and / or analyzing a large number of samples. Test methods with high sample throughput (so-called high throughput screening, HTS) have been developed, in which thousands of samples are isolated, cultivated or subjected to specific treatments, for example, in high parallel. These methods are carried out in specially adapted reaction substrates or containers with many sample compartments that have to meet a variety of requirements. The reaction substrates must ensure, for example, rapid and parallel sample loading, observation of the sample during the reaction and further availability of the sample after a reaction, and be inert to the respective reaction. With the advancement of biochemical knowledge and improvements in methods from biotechnology and combinatorial chemistry, there is a need to process as large as possible the smallest possible number of sample volumes in parallel. B. to handle, control and measure. Recently, 10 ³ to 10 ⁵ samples per day with required volumes of, for example, 10 ^-6 to 10 ^-10 1 can be characterized on the basis of modern (fluorescence) screening techniques. In order to increase the sample throughput, reduce the substance consumption and also for reasons of space, miniaturization of the sample compartments is sought. As a result, the requirements for reaction substrates with compartmentalization for individual samples also increase sharply. This applies in particular with regard to the number of compartments available, the miniaturization potential, the ease of use and the cost or reusability.

Sample carriers or reaction substrates with microscopic Structures for use in fluorescence, luminescence or Scintillation measurements, e.g. B. to solve chemical or molecular biological Questions are known per se. In DE-OS 197 12 484, EP 131 934, US 54 17 923 and US 54 87 872 Described reaction substrates in the form of structured microplates, which each have a large number of flat, form sample compartments that are open on one side. A microplate with a filter membrane is described in EP 408 940. This Microplate is because of its complicated structure for both Manufacturing as well as disadvantageous for cleaning. The number available compartments is limited.

Another microstructured reaction substrate is shown in WO 95/01559. On the top of the reaction substrate are made of a semiconductor material or a plastic formed by etching recesses, the bottoms towards the bottom are at least partially porous. These reaction substrates allow Although examinations from both sides, have Disadvantages with regard to the reproducibility of the production of the individual recesses and the manageability of the Reaction substrate. When covering the recesses is provided is, it must be mechanically clamped, glued or be bonded.

DE-OS 197 52 085 describes a reaction substrate that is easier to manufacture for microscopic examinations of a variety of Samples known to be made using injection molding technology and / or hot stamping formed sample compartments. A disadvantage of this reaction substrate is that it is microscopic Examinations only from one side of the substrate which the sample compartments are open can be carried out. In addition, this reaction substrate is not general for HTS process can be used.

The structure of a microsystem is known from WO 99/19717, at the at least one flexible, micro-structured film as a laminate is arranged between solid supports. The film has application-dependent formed microstructures, in which if necessary Electrodes are integrated and in cooperation with form compartments for fluidic samples. This Stacking technology is again disadvantageous because of separate measures to connect the carrier to the film must be taken that affect the handling of the samples or the samples themselves.

A similar structure is described in EP 324 153. Doing so in particular a photopolymer provided with certain microstructures laminated on a solid support. The disadvantage of this technique is that the polymer layer cannot be removed from the carrier without damage. But there is interest in reaction substrates or sample carriers, those for reuse or for further process steps for sample processing, for example, without damage from a Substrate composite are detachable.

A process for the production of microstructures on a metal surface is described in WO 97/29223. The metal surface is through a photolithographically structured polymer layer edited through. With this technique it will However, the problem of covering microstructures has not been solved either. Other structuring techniques for metal materials or semiconductors are described in EP 869 556, WO 97/13633 and WO 98/09745.

A general disadvantage of the conventional reaction substrates for use in microscopy concerns their relatively thick, irregular and / or sagging floors. The floors of the conventional reaction substrates can be made of different materials, z. B. glass. Typical glass thicknesses are around 500 µm. But it can also be pronounced, unreproducible Variations in the soil (e.g. over 400 µm) occur. The focal length of immersion objectives, however, is typical limited to 250 to 300 µm. When subtracting the glass thickness, e.g. B. a cover slip of around 150 microns still remains a permissible Variance of the soil from around 100 to 150 µm to reproducible, continuous measurements on the reaction substrate without constant readjustment of the position of the lens in a direction perpendicular to the plane of the reaction substrate (hereinafter referred to as the z-direction).

In most of the above requirements, but also in terms of reliability and reproducibility the previously available reaction substrates or containers or Sample carriers with the development of the screening technique did not withstand.

Further multi-component sample carriers are known from US-A-4 798 706, US-A-5 738 825, US-A-5 487 872, US-A-5 681 741, WO 86/06488 A. and EP-A-0 983 795.

It is the object of the invention to provide an improved reaction substrate provide with the disadvantages of conventional Reaction substrates are avoided, in particular has a simple structure, among those interested Reaction conditions are inert as well as easy with any Structures can be produced and are easy to handle. The new Reaction substrate in particular should also be reusable several times or be recyclable. The object of the invention is in particular to provide an improved reaction substrate, sample handling and analysis when used, z. B. with a Microscope, especially with a confocal microscope, simplified become. The object of the invention is also a Process for producing the reaction substrate and a tool to provide for its implementation.

These tasks are particularly through a reaction substrate solved with the features according to claim 1. advantageous Embodiments and uses of the invention result from the dependent claims.

According to the invention, a structured reaction substrate is provided, that by a composition of one characterized below Sample carrier (compartment layer) with a fixed Bottom part is formed on which the sample holder independently liable. The bottom part is preferably made of glass, plastic, Metal or a semiconductor material. It essentially forms one flat, smooth surface to which the sample carrier adheres is.

A particular advantage of the reaction substrate according to the invention is that the compartment layer is essentially damage free is separable from the bottom part. This means, that the compartment layer from the base plate (e.g. one Cover glass) can be removed in such a way that they can be significant loss of shape, adhesion and / or flexibility can be used again. For reuse the compartment layer with a new or cleaned Base plate by light, e.g. B. manual, print to a new one Reaction substrate connected, its tightness completely the tightness of that previously formed with the compartment layer Reaction substrates corresponds. An essentially damage free one Detachment of the compartment layer means that the Functionality of the compartment layer by the detachment for later applications remain unchanged.

The compartment layer can preferably be lifted off the bottom part by lifting off the compartment layer at one corner from the bottom part, while this at its four corners is held. The compartment layer is on the raised corner bent up and unrolled over the bottom part, whereby the compartment layer is essentially residue-free from the Bottom plate is separated. A particular advantage of the invention is that this takes off and thus reuse as many times as possible. Experimentally, a 50-fold Reuse can be confirmed without loss of functionality.

According to a preferred embodiment of the invention, this is Reaction substrate designed for microscopic examinations. The bottom part is made of a transparent material (e.g. Glass) with an application-specific thickness. It will be the filing the sample holder on a cover glass known per se preferred for microscopy.

The thickness of the cover slip is preferably a few hundred micrometers (µm), particularly preferably around 150 µm. With the one used Microscope is preferably a confocal Microscope. The confocal microscope is preferred in conjunction with detection technologies based on the detection of fluorescence based, combined. Those according to the invention are particularly suitable Reaction substrates for fluorescence correlation spectroscopy, Fluorescence coincidence analysis, fluorescence distribution analysis, Fluorescence lifetime measurements, Fuorescence energy transfer analysis or fluorescence polarization measurements using confocal microscopes. The reaction substrates according to the invention are therefore suitable very good for single molecule detection.

According to an important aspect of the invention, a sample carrier is provided in the form of a flexible compartment layer with recesses to form a predetermined compartment structure, in which the compartment layer consists of a viscoelastic polymer composition which is self-adhesive on glass, plastic, metal or semiconductor substrates. The compartment layer is a dimensionally stable mat which can be produced with the aid of a simple impression process, the material of which has an adhesive connection, for example even with a slight manual contact pressure of a few grams per cm ² . B. by electrostatic forces and / or van der Waals forces, with one of the substrates mentioned. The compartment layer preferably comprises essentially solvent-free natural or synthetic rubbers or compositions composed of these. The polymer composition of the compartment layer is particularly preferably formed from adhesive and solvent-free natural and synthetic rubbers. The compartment layer is preferably free of additives such as resins, plasticizers and / or antioxidants. According to a preferred embodiment, the compartment layer of the reaction substrate according to the invention comprises silicone rubber.

The recesses for the formation of the compartment structures are through holes through the compartment layer or on one side wells worked into the compartment layer. It will closed compartment structures in the form of sample reservoirs or storage pots and / or open compartment structures in the form of those running in the layer plane of the sample carrier Channels formed. The sample reservoirs, storage pots and channels are also referred to below as sample compartments.

The compartment structures form a multitude of matrix-like recesses arranged in straight rows and columns (sample reservoirs), the grid dimension of the matrix arrangement preferably the arrangement of sample reservoirs (so-called wells) from Corresponds to micro and nanotiter plates.

According to a further preferred embodiment of the invention Reaction substrate is the compartment layer with manipulation and examination facilities. To these include in particular fluid lines for feeding the sample compartments formed by the compartment structures or for deriving substances from these, sensor devices for Detection of predetermined sample properties in the sample compartments, Piezo pumps for conveying fluid flows and / or Electrode devices which are used to apply the samples in the sample compartments with electric fields are. A fluid line is, for example, through a in the layer plane of the compartment layer extending capillary, which from the edge of the reaction substrate into one certain sample compartment. Include sensor devices For example, temperature, pH or conductivity sensors. The Electrode devices are preferably made by electrode strips formed on the walls of the sample compartments extend.

The compartment structures in a reaction substrate according to the invention or form sample carriers according to preferred embodiments Microstructures with characteristic dimensions in the area from 500 nm to 1.5 mm.

According to the invention, the stack structure consisting of the bottom part and the sample carrier be modified in such a way that on the sample carrier a cover is attached to the side opposite to the base part which in turn becomes relative through independent attachment is fixed to the sample holder. The cover can be rigid Material like the bottom part or through a flexible. foil be educated. The cover may also have predetermined openings to have access to the compartment structures. The stack construction in sandwich form gives the reaction substrate additional Stability. The cover serves to prevent the Evaporation of liquids introduced.

Depending on the application, it can be provided that the compartment layer is formed from several separate parts that are on a common bottom part to form an inventive Reaction substrates are arranged. You can also have several Compartment layers bonded together as a stack to build a three-dimensional fluidic microsystem.

It will also be a procedure for the preparation of the sample carrier described above. For this purpose, an impression tool with the desired polymer material of the compartment layer in solution State filled and then the solvent by annealing and / or drying, preferably at room temperature, from the filling withdrawn or a crosslinking of the polymer composition brought about. The impression tool consists in particular of a structured base plate and a counter plate, 'the liquid-tight be held together. The base plate carries protruding structures according to the desired compartment structures in the sample holder. These protruding structures protrude from the base plate to or in depending on the application the counter plate (formation of through holes) or up to a height at a distance from the counter plate (formation of depressions). For the reproducible production of structures in The counter plate is preferably in the form of through holes a coating facing the base plate, e.g. B. made of PTFE, Mistake. The individual components of the impression device are assembled via detachable plug or screw connections. After solvent removal or crosslinking of the polymer composition these connections are loosened and the dried one firm, dimensionally stable compartment layer as a sample carrier Impression tool removed.

The sample carrier or the reaction substrate according to the invention are for the manipulation and / or examination of any liquid Samples with characteristic sample volumes e.g. B. in the area from 1 nl to 10 µl. The liquid samples can in particular solutions of predetermined reaction partners and / or Suspensions include those that are synthetic in a suspension liquid or contain biological objects. To the one Reaction substrate manipulated objects count in particular Solid particles (so-called beads) as synthetic objects and biological cells or cell components, microorganisms, Viruses and biologically relevant macromolecules as biological objects.

The invention has the following advantages Reaction substrates or sample carriers can be used with simple Averaging with an essentially pressure-free tool be mass-produced. About the design of the Mask or imprint form of the tool is any format design sample compartments from macro to nano sizes possible. For the production of masks for microscopic Compartment structures are known processing techniques for glass or semiconductors, such as. B. the LIGA process or conventional etching. The compartment structures can be precisely positioned over the entire thickness of the compartment layer. The structures can be in the Layer-level characteristic dimensions in the sub-micrometer range and perpendicular to it in the mm range.

The compartment structures can be of any format, e.g. B. round, square, rectangular or with more complicated geometric Shapes to be trained. The production of the compartment layer made of a viscoelastic polymer has several Benefits. On the one hand, the attachment of the sample holder is on compared to a base part by simply pressing it conventional sandwich constructions with mechanical clamping devices or laminate connections simplified. on the other hand stands out the material of the compartment layer, in particular when using silicone rubber, due to its excellent properties in the form that unspecific adsorption does not occur. This is particularly important for miniaturized samples. The sample carrier is under the reaction conditions of interest for applications in medicine, biochemistry and molecular Biotechnology inert. The biologically inert material enables biological dressing, cultivation and measurement Samples or substrates in the reaction substrates or sample carriers. Finally, the sample carrier material also allows after the actual use, cleaning in a bath or a dishwasher with conventional cleaning agents or solvents, without the shape or stability of the sample carrier be adversely affected. The sample carrier is essentially without loss of shape and without influencing its adhesive properties autoclavable and sterilizable. By simply pulling it off from the bottom part the sample carriers are reusable.

The reaction substrate according to the invention from the bottom part with attached Sample holder has special advantages with regard to the Structure of the reaction substrate, the sealing of the sample compartments and the mutual alignment of the sample compartments. The sample holder is pressed evenly with a defined, e.g. B. manually applied pressure with the bottom part connected. The sample holder can be used without a frame nevertheless allows, when applying alignment marks, a exact spatial orientation and positioning, e.g. in relation on a microscope or a sample feeder. The Sealing of the sample compartments by continuous recesses are formed in the compartment layer, opposite the bottom part is made without additional sealants or adhesives. Influencing the biochemical reactions in the compartments is excluded by such means.

The adhesive bond between the sample holder and the bottom part and the cover allows the planarity of larger areas Reaction substrates or sample carriers with characteristic Dimensions up to 118 mm • 82 mm. Over the whole Surface area of the bottom portion can vary sample chamber positions in the z direction (perpendicular to the sample carrier plane) preferably to values less than 250 μm, particularly preferred are kept smaller than 150 µm, in particular smaller than 100 µm. This is of particular advantage for microscopic examinations. It is during the measurement of a reaction substrate not required, the z position of the microscope objective is running readjust. The reaction substrates according to the invention are therefore very suitable for use in test procedures with high Sample throughput (so-called high throughput screening, HTS) in biotechnical and / or chemical research and development, since the time-consuming readjustment, e.g. B. microscope objectives, in the z direction, not applicable.

The stability of the reaction substrate is as high as in conventional sample chamber structures, but according to the invention dispense with additional adhesives or clamps can be. The stability is when the cover is applied still significantly increased.

The reaction substrate has a wide range of uses, depending on the requirements, a suitable floor part can be used as a base for the sample holder. The Bottom part can be freely varied in terms of material and thickness. Glass of any kind is preferably used as the transparent base part Starch, e.g. B. with cover glass thickness, for use in of microscopy. The bottom part can be made of UV-permeable Quartz glass exist. It has excellent optical properties and is neither chemically modified by the sample carrier still physically stressed.

The impression tool has the advantage of one simple, modular structure. The tool can go through Change the mask or impression form to the desired one Requirements are adjusted. It is alike for applications in the laboratory or in mass production suitable. With the method according to the invention, any Structures can be produced without any special effort. This is a special advantage over the conventional ones Techniques for structuring glass or semiconductors.

Fig. 1

eine schematische Perspektivansicht eines Reaktionssubstrates mit einem Probenträger gemäß der Erfindung,

Fig. 2

eine Draufsicht auf eine erste Ausführungsform einer erfindungsgemäßen Kompartimentschicht,

Fig. 3, 4 und 5

Illustrationen eines Abdruckwerkzeugs im zusammengesetzten bzw. auseinandergenommenen Zustand,

Fig. 6

eine Draufsicht auf ein erfindungsgemäßes Reaktionssubstrat in Form eines Mikroprobenträgers und auf eine herkömmliche Halbleiterstruktur,

Fig. 7

eine vergrößerte Ausschnittsansicht eines Mikroprobenträgers gemäß Fig. 6,

Fig. 8

eine Illustration von Einzelheiten der Kompartimentstrukturen bei einem Reaktionssubstrat gemäß den Figuren 6 und 7,

Fig. 9

eine Draufsicht auf weitere Ausführungsformen eines erfindungsgemäßen Reaktionssubstrates mit Mikrokanälen,

Fig. 10

ein erfindungsgemäßes Reaktionssubstrat in Form einer schichtförmigen Fluoreszenzküvette,

Fig. 11

experimentelle Ergebnisse zur Illustration der hervorragenden Planarität erfindungsgemäßer Reaktionssubstrate, und

Fig. 12

experimentelle Ergebnisse zur Illustration der Dichtheit von Kompartimenten erfindungsgemäßer Reaktionssubstrate.

Further details and advantages of the invention will become apparent from the description of the accompanying drawings. Show it:

Fig. 1

2 shows a schematic perspective view of a reaction substrate with a sample carrier according to the invention,

Fig. 2

2 shows a top view of a first embodiment of a compartment layer according to the invention,

3, 4 and 5

Illustrations of an impression tool in the assembled or disassembled state,

Fig. 6

2 shows a plan view of a reaction substrate according to the invention in the form of a micro sample carrier and onto a conventional semiconductor structure,

Fig. 7

6 shows an enlarged detail view of a micro sample carrier according to FIG. 6,

Fig. 8

an illustration of details of the compartment structures in a reaction substrate according to FIGS. 6 and 7,

Fig. 9

2 shows a top view of further embodiments of a reaction substrate according to the invention with microchannels,

Fig. 10

a reaction substrate according to the invention in the form of a layered fluorescence cuvette,

Fig. 11

experimental results to illustrate the excellent planarity of reaction substrates according to the invention, and

Fig. 12

experimental results to illustrate the tightness of compartments of reaction substrates according to the invention.

The invention is described below with reference to a reaction substrate with a microstructured sample holder for Handling of biological samples described. The invention is but not limited to applications where microscopic small sample amounts are manipulated in microstructures. Furthermore, the invention is not limited to those illustrated Forms of sample compartments limited. Depending on the application can also use any other shapes with straight or curved walls of the sample compartments can be realized.

Figure 1 illustrates in a schematic perspective view Reaction substrate with a sample carrier according to the invention. Various compartment structures are on the sample carrier and additional devices shown that are application-dependent individually or can be provided simultaneously. The reaction substrate 100 comprises the base part 10 and the sample carrier 20.

The base part 10 is, for example, a flat glass plate with a Thickness corresponding to the thickness of coverslips for use in microscopy (around 150 µm) and an area of around 120 mm • 70 mm. The bottom part 10 can also by any other body with a substantially smooth, flat or curved surface are formed. Preferably owns the bottom part has an essentially smooth, flat surface.

The sample carrier 20 comprises a compartment layer 21 (mat) with compartment structures 30. The compartment layer 21 is preferably made of silicone rubber and has one Thickness from around 0.5 mm to 4 mm. On one or more sides the mat can be a tab 22 for pulling the sample carrier 20 from the bottom part 10 and / or alignment marks 23 for Position the sample carrier 20 relative to a measuring or Sample loading device may be provided. The adjustment marks 23 are, for example, punctiform or cross-shaped recesses in the surface of the sample carrier 20, which if necessary with an additional marker substance (e.g. Fluorescent dye) are provided. The adjustment marks have characteristic dimensions that are significantly smaller than the dimensions of the compartment structures 30 can.

The silicone rubber is, for example, polydimethylsiloxsan (PDMS, Manufacturer Wacker-Chemie GmbH, designation M 4600). Generally elastic plastics (elastomers) can be used, that remain elastic at different temperatures. In the The molecular chains (carbon chains) are loose elastomers cross-linked so that the elastomers are rubber-elastic. That preferred The silicone used is a plastic from the group of elastomers and consists mainly of silicon and Oxygen. When uncrosslinked, the silicones are oil-like, water-clear and heat-resistant. Form in a networked state the silicones a silicone rubber.

The compartment structures 30 include closed ones in detail Sample reservoirs 31 in the form of through holes 31a or depressions recessed in the surface of the sample carrier 31b (diameter e.g. around 200 µm to 1.5 mm) or in the layer plane of the sample carrier is straight, curved or branching channels 32. The reference symbol 33 refers to so-called stock pots, which are like the sample reservoirs 31 for sample collection and delivery, but with larger volumes are designed.

The manipulation and examination devices 40 comprise For example, a fluid line in the form of at least one capillary 41, at least one electrode 42 and / or at least one Sensor 43, in the layer plane of the sample carrier 20 the walls of the compartment structures 30 or in the compartment structures 30 are arranged. The capillary 41 can e.g. with a sample or reagent delivery system (not shown). It is made during manufacture of the sample carrier 20 (see below) embedded in this or subsequently inserted into the sample holder 20. The electrodes are built in the way it is from microsystems technology of microelectrodes for electroosmotic pumping processes, Manipulation of particles using negative Dielectrophoresis or particle processing, such as. B. Electroporation on biological cells is known. The Electrodes or their leads are preferably used during the manufacture of the sample carrier 20 embedded in this or on its inner surfaces (walls of the compartments) arranged.

Figure 1 also shows a cover 50. The cover 50 is no mandatory feature of the reaction substrate according to the invention. It is intended depending on the application and consists of how the base part 10 made of a solid plate (e.g. made of glass) or from a flexible cover film. It can be provided be that the cover 50 has openings 51 corresponding to the positions which has compartment structures 30. The openings 51 serve to load sample reservoirs 31 or Storage pots 33 or the sample entry in the channels 32. You can with an additional (not shown) film as Evaporation protection must be closed.

An interesting one for practical applications in biochemistry An embodiment of a compartment layer 21 is shown in FIG 2 shown. The compartment layer 21 is a flexible one Mat made of silicone rubber (e.g. Elastosil M 4600 A + B, Manufacturer Wacker-Chemie GmbH, Germany). She has one Area of 118 mm x 82 mm and a thickness of 4 mm. The sample reservoirs 31 (partially shown) are matrix-like in arranged even rows and columns in the format 48 · 32 and each have a center-to-center distance of 2.25 mm. This corresponds to the standard format for microtiter plates with 1536 Wells. The diameter of each sample reservoir 31 is 1.5 mm. The reference numeral 23 refers to an alignment mark, which also in this embodiment by a Recess how the sample reservoir is formed and a Can record reference sample.

The sample carrier 20 illustrated in FIG. 2 or the compartment layer 21 is with a bottom part (not shown) connected, preferably the same area dimensions as the Has compartment layer 21. The bottom part is preferred a cover slip with a thickness of around 150 µm.

The following is the production with reference to Figures 3 to 5 of a reaction substrate or sample carrier according to the invention by casting the compartment layer in an impression tool explained. The figures show the impression tool in a perspective phantom view or pulled apart in Perspective or side view. The impression tool 200 exists basically from a closed container with an inner one Cavity according to the outer shape of the desired compartment layer or with inner surfaces, the projections according to the desired compartment structures. The container is modular for universal use of a base plate 60, an intermediate plate 70 and one Counter plate 80 constructed, which are connected to each other in a liquid-tight manner can be. The basic, intermediate, and counter plates releasably connected together.

The base plate 60 carries on the inside of the impression tool 200 facing side protrusions for structure formation in the compartment layer. Aside from the tabs the surface of this inner side is uniform and smooth educated. In the example shown, the projections include Pins 61 arranged in a matrix (partially shown) with a diameter corresponding to the desired one Diameter of the sample reservoirs 31 (see Fig. 2). The pencils 61 are in corresponding recesses on the inside the base plate 60 inserted. The base plate and the pins are preferably made of metal (e.g. stainless steel or Aluminum). For the ledges to structure formation can but also other materials such. As silicon or glass be used. These materials can be used in themselves known, special molding techniques (e.g. LIGA process or etching) with high precision down to the sub-micrometer range edit, with the resulting projections heights can have up to 1 mm. To hold the projections (Metal pins or other structures) can be the base plate 60 have a separate mask insert. Figure 4 shows also the metal pin 61a, which is used to form the alignment mark 23 (see Fig. 2) is provided.

The intermediate plate 70 is a spacer which is the thickness the compartment layer (silicone mat) determined and its Internal dimensions, the outer dimensions of the compartment layer. The intermediate plate 70 has a filling opening 71 which interacts with the filler neck 90 (see below), and Outlet openings 72 equipped. The outlet openings 72 serve to discharge displaced air or excess Layer material from the impression tool 200. The intermediate plate 70 is not a mandatory feature of an invention Footprint tool. The function of the spacer can alternatively also through appropriate structures (surrounding steps) on the base plate and / or the counter plate.

The counter plate 80 is the end of the impression tool 200 compared to the base plate 60. It is also one Metal plate. Pointing to the inside of the impression tool 200 is a frame 81 with a plastic insert in the counter plate 80 82 arranged. The plastic insert 82 is one Layer of elastically deformable plastic with a thickness of around 10 mm. It is preferably made of PTFE. The plastic insert 82 has recesses 83 that lead to the projections on the base plate 60 are complementary. In the illustrated Examples are 82 1536 holes in the plastic insert (partially shown) for receiving the metal pins 61 in Assembled state of the impression tool 200 provided. The introduction of the complementary recesses is not mandatory. If the ledges on the Base plate 60 is sufficiently stable or the plastic insert 82 is sufficiently easily deformable so that when assembled Condition of the impression tool 200 the projections are not damaged can be on separate recesses in the plastic insert 82 can be dispensed with.

The reference numeral 20 refers to the finished sample carrier (According to Figure 2), which with an impression tool 200 according to the Figures 3 to 5 is produced.

It can be provided that the recesses 83 completely drilled through in the plastic insert 82 are and also in corresponding recesses 84 in continue the counterplate 80. These openings serve the Escape of displaced air or excess layer material.

The filler neck 90 is on the outside of the composite impression tool 200 attached to the fill opening 71. He serves that Introducing the dissolved polymer material into the composite Mold.

The impression tool 200 is fastened with mounting pins 62, 63, 64, 65 held together by appropriate holes the corners of the base, intermediate and counter plates. to Fixing the parts is a screw connection (in detail not shown) provided. Alternatively, outer Clamping devices or a separate frame to hold together the plates may be provided.

The impression tool 200 can be modified as follows. in the A metal frame can additionally be provided inside the intermediate plate 70 be attached, the desired external dimensions of the compartment layer owns and with this also for later use stays connected. The pins 61 can be at their ends for relief in the introduction to the corresponding recesses be rounded in the base or counter plate. to Integration of the manipulation mentioned with reference to FIG. and examination devices in the sample carrier 20 can be provided with the intermediate plate 70 accordingly To provide brackets for these additional facilities.

These brackets include passage openings in the frame formed by the intermediate plate 70 from the inside of the Impression tool 200 to the outside, each with fixations (e.g. terminals) for the respective additional devices are equipped. After all, it is not imperative that all structures of the desired compartment layer actually as protrusions on the base plate 60 are trained. The finished sample holder can easily be used can be provided with additional structures (e.g. drilling of the stock pots 33).

The impression tool is used to manufacture the sample holder 200 composed. The pins 61 are in the Base plate 60 inserted. The base, intermediate and counter plates are assembled so that the pins 61 into the recesses 83 protrude into the plastic insert 82. In this way there is an essentially closed on all sides Container, between the side plates (base and counter plates) the pins 61 extend. The guide pins 62 to 65 are e.g. B. tightened with wing nuts. The composite Tool is aligned with vertically Plates placed upright. The filling opening 71 has up.

The impression tool 200 is then passed through the filling opening 71 with a solution of the desired polymer composition filled. This is preferably done directly in with a syringe the fill opening 71 or using the filler neck 90. The filling takes place as slow running in avoiding of splashes or swirls, so that the inside of the impression tool 200 is filled as uniformly as possible. Preferably the polymer composition is essentially pressure-free in the Impression tool filled. The filling takes place until the dissolved polymer composition from the outlet openings 72 oozes out. These are then closed, for example, with an adhesive tape. After closing, there will be a little more Refilled material.

The polymer composition is then dried or crosslinked preferably at room temperature. This can, for example, take around 8 to 12 hours. The solvent withdrawal or the crosslinking of the polymer composition be accelerated by tempering. Finally be the connections of the plates via the guide pins 62 to 65 solved, the plates separated and the elastic Compartment layer pulled from the mask or impression form. A particular advantage of using silicone rubber here is that this peeling off with no problems and can be done without damaging the sample holder.

Crosslinking takes place when the polymer Elastosil M is used 4600 preferably at room temperature, but can also be used at higher ones Temperatures are carried out in a drying cabinet or an oven become. Crosslinking is essentially chemical Networking, where necessary in the presence of a Catalyst carried out a polymerization reaction becomes. In the case of other polymers, the crosslinking takes place in the specified cross-linking temperature.

There can be a final treatment for subsequent application of compartment structures (e.g. storage pots) or to adjust or align the additional manipulation and connect examination facilities. Also one chemical post-treatment of the surface of the sample carrier possible. The finished sample holder is then placed on a bottom part applied and with this by simple manual pressing connected.

Another embodiment of the invention is based on the example a micro sample holder for the smallest amounts of liquid in Figures 6 to 8 illustrated. Figure 6 first shows one Size comparison between a reaction substrate according to the invention or a sample carrier 20 (left part of the figure) and a conventional sample carrier 20 'made of silicon is made. On a base area of around 10 mm • The sample carrier 20 carries a matrix arrangement of 15 mm overall around 600 funnel-shaped compartments (see below). Each compartment has a characteristic Cross-sectional dimension of around 0.5 mm. The conventional one Silicon sample carrier 20 ', on the other hand, has a considerably coarser one Raster, which also with complex processing techniques was produced.

Figure 7 shows an enlarged section of the sample holder 20. This image was taken with an inverted microscope recorded on a CCD camera. The sample carrier 20 carries the compartments 34 arranged in straight rows and columns. These are located on the surface of the sample carrier 20 cross-sectional shape tapering into the compartment layer like an inverted, truncated pyramid. At the Soil the compartments have a characteristic side length, which is approximately 1/3 of the top edge length. The floor, which is shown brightly, is created by the common floor part 10 (see Fig. 1) formed. The compartment layer 21 of the sample carrier 20 becomes complete from the compartments punctured.

A sample carrier according to Figures 6 to 8 is with a correspondingly adapted impression tool analogous to that under Produced with reference to Figures 3 to 5 described method. The protrusions on the base plate are in the impression tool then not through inserted pins, but pyramid-shaped formed by mechanical milling. After manufacture the compartment layer 21 is on a glass bottom part adhered. Then the compartments filled and then optionally with another Glass as cover or closed with a film. The microscopic measurement of the samples in the compartments takes place from the side of the bottom part 10 through the lower smaller openings in the compartment layer 21. The edge length the lower openings are each around 150 µm.

Figure 8 shows details of the between the compartments formed webs. As can also be seen from FIG. 7, the Compartment layer shaped so that the walls between the Compartments 34 in the row direction continuous webs 35 and Form webs 36 interrupted in the column direction. Between the ends of the interrupted webs 36 and the adjacent one continuous web 35 forms an overflow 37. The Overflow 37 allows a fluid connection to be made between neighboring compartments without trespassing over the top surface of the sample carrier 20. The arrangement the overflows can be modified depending on the application his.

FIG. 9 shows different designs of channel structures shown enlarged in a sample carrier according to the invention. The channels 32 are generally open in the layer plane Sample compartments or compartment structures whose Expansion significantly larger in one direction than in one are perpendicular to this. Channels are in the sample holder molded by using a mask shape to make it web-shaped projections on the base plate of the impression tool be used. The channels can be straight or curved individually or branching or with each other run together. Depending on the design of the sample holder closed channels can even be formed, if the channel floor itself is part of the sample holder, the corresponding compartment structures are not complete go through the compartment layer.

FIG. 9A shows a channel structure with a plurality of channels 32a to 32c, which are connected at a mixing cross 32d. On there are storage pots 33a to the channel ends 33d. The reference symbol 32e indicates a constriction point. The constriction 32e can be flow mechanically through barriers (arched channel wall) or electrically through electrical field barriers are formed, for example around the fluid flow to delay before this area and take measurements there on suspended particles in the fluid flow.

A modification is shown in Figure 9B. Two sub-channels 32a, 32b connect in a common channel 32c. This Structure is used to mix two fluid flows into one single fluid flow. The angle α between the subchannels 32a, 32b is application dependent to achieve uniformity Flow set at the mixing point 32d. A further modification of structures for mixing the fluid flows is in Figure 9C as a double cross arrangement with several Subchannels illustrated in two mix points 32d flow out.

The meander shape 32f according to FIG. 9D serves to create a particularly long measuring distance. Between the storage pots 33a to 33c on the one hand and the storage pot 33d extends long, sinuous channel in an area that, for example, forms a target for illumination for fluorescence measurements.

The reaction substrates or sample carriers according to the invention have particular advantages in terms of training the Channel structures. For the production of the mask for the impression tool can with conventional precision mechanical tools (e.g. CNC machines) made of common material, preferably Aluminum or other metallic materials, any Channel profiles are prepared. You can in particular Terms of length, relative orientation (angle), the Bends and twists, mix structures and sub-channels be designed depending on the application in a predetermined manner. Channels of this type can be down to channel widths of around 6 µm with conventional precision mechanical tools and produce reproducibly. Projections can be made in the channels or edges are incorporated, which is an improved Mixing several fluid flows when merging allow multiple channels. The channels can be with Electrodes for measuring the properties of fluid flows or to manipulate it on the basis of electroosmosis, with sensors or temperature control elements and also with Blocking or valve elements as well as piezo pumps his.

Another embodiment of the invention with a macroscopic Compartment structure is in Figure 10 in top and bottom Illustrated sectional view.

An inventive reaction substrate or an inventive one Sample carrier 20 can also be used with a single chamber compartment 38 be equipped. The compartment layer 21 is just a ring of the polymer composition used in each case, z. B. silicone rubber. This ring sticks between a bottom part 10, e.g. B. a glass plate, and one Cover 50 so that a closed, layered Cuvette e.g. B. is formed for fluorescence spectroscopy. Because of the liquid-tight adherence of the sample carrier 20 on the glass materials of the bottom part 10 or the cover 50 this cuvette can permanently with solvents or sample solutions loaded and like a solid layer sample fluorescence measurements be subjected.

Figures 11 and 12 illustrate particular advantages of the invention Reaction substrates with regard to the planarity of the Sample arrangement, which is important for microscopic examinations and the well-to-well tightness of the compartment structures. The variation was used to demonstrate planarity the z position over the entire area of the floor area of the reaction substrate with a confocal microscope assembly (Reflection of the laser beam on the glass surface of the floor, recorded with a CCD camera) in a conventional, commercially available reaction substrate or sample carrier (left Partial image in Figure 11) and for a reaction substrate according to the invention (right drawing in Figure 11) measured.

With conventional, commercially available sample carriers or reaction substrates one results for confocal fluorimetric Applications no longer tolerable variation of the slab floor in the z direction by up to 300 µm from the edge of the plate to its Center. It is a clear course in one direction ("sagging" of the reaction substrate in the middle). The reaction substrate which is the subject of this application shows a variation of the plate bottom in the z-direction from far less than 100 µm. This deviation is significantly below the tolerance of around 150 µm for confocal fluorimetric Applications. Furthermore, there is only a statistical fluctuation the z deviation around the mean upwards or downwards to recognize, there is no tendency in the deviation (e.g. no "sagging" of the reaction substrate in the middle).

In Figure 12 are the 1536 wells of a reaction substrate according to the invention with the results of each in the wells performed measurements shown. For the investigation a reaction substrate alternately in the form of a "checkerboard pattern" with suspensions of so-called active and so-called filled with inactive bacteria (330 nl per well). To After an incubation period of 21 h, all wells were evenly mixed with 1 µl assay added after a further incubation period from 30 min all wells of the reaction substrate with Measure using CFCA measurements (1 s measurement time per well).

In wells that contain active bacteria, the two-tone labeled assay molecules cleaved so that the CFCA signal becomes small (black fields in the plot). In samples with non-active bacteria are added, they become two-colored labeled assay molecules are not cleaved, so the CFCA signal remains large (white fields in the plot). Only in total Six out of 1536 wells encountered an "error" that resulted from a leak between individual wells. It can but these are also errors that are already evident when pipetting of the bacterial suspensions have arisen. The upper Limit for errors caused by leaks from well to well occur therefore is at most 0.4%.

This excellent result is advantageously also time-stable. The reaction substrate is also after at least 48 hours (Time from sample preparation to incubation to completion of measurements) is still stable in such a way that the wells are completed against each other and measurements in the plate can be carried out (without gluing the floor glass, the can be removed again after the end of the measurements).

The result illustrated in FIG. 12 also shows that the Bacterial growth from the compartment layer is not prevented becomes (biocompatibility).

The sample carriers or reaction substrates according to the invention can be common in all areas of biochemistry, biology or molecular biotechnology, where one or more samples held, manipulated in a defined form or should be changed. Preferred applications are in the processing of suspensions with certain Particle mixtures. With reaction substrates according to the invention can for example cell sorters, molecular sorters or other Cell manipulators are built. They are all applications of fluidic microsystem technology can be implemented.

The reaction substrates according to the invention are special Benefit usable in synthetic processes based on combinatorial Chemistry based. In particular, the inventive Reaction substrates for the identification and validation of targets, d. H. specific biological molecules, such as enzymes, Receptors or ion channels can be used. Furthermore, you can they are very good for identifying biologically active substances and / or active pharmaceutical ingredients are used. The possibility of using the reaction substrates according to the invention in test procedures with high sample throughput (so-called high throughput screening (HTS) can do much more Substances within a short time in terms of their biological Activity and / or pharmaceutical efficacy are examined. This is of particular importance in order to be combinatorial Substance banks obtained in relation to their chemistry biological activity and / or pharmaceutical effectiveness investigate. It is with the reaction substrates according to the invention possible to achieve a high sample throughput and between investigate several thousand up to 100,000 substances per day.

The reaction substrates according to the invention are still very good suitable for performing assay procedures. With these Assay methods are used to target and chemical compounds Investigation of chemical and / or biological interactions combined. It is thus easily possible to create a model system establish that allows substances to be identified, that affect the target in the desired way. The reaction substrates according to the invention can be used both for biochemical as well as cellular assay methods can be used. This also includes assay procedures based on use of vesicular particles or solid particles (so-called Beads).

The reaction substrates according to the invention are also suitable very good for performing assay procedures based on use based on simplified model systems that represent the physiology reproduce in humans or animals. This means that Assay systems can include: a. used to information about the solubility of biologically active and / or pharmaceutical active substances in blood plasma, their penetration properties, their liver toxicity, their bioavailability, their Stability in the blood or its breakdown profiles after passage through the liver to obtain.

The chemical and biotechnical investigations can, for example. i) for the identification and characterization of synthetic or biological objects, ii) for identification and Characterization of chemical compounds, iii) for identification and / or validation of targets, iv) for searching for biologically active substances and / or pharmaceutical Active substances, v) for the identification of lead structures, vi) for genome analysis, vii) for proteome analysis, viii) for purification and concentration of substrates, or ix) for evolutionary Optimization of biologically relevant macromolecules used become.

Claims (16)

1. Reaction substrate (100) having a compartment structure (30) by which sample reservoirs (31) are formed which are arranged in matrix fashion in straight rows and columns, and a base portion (10), which includes a glass, plastics material, metal or semiconductor substrate and has a substantially flat, smooth surface, and said reaction substrate having a flexible compartment layer (21) made of a polymer material in which the sample reservoirs (31) are formed,
   characterised in that
   the polymer material is a viscoelastic polymer composition which is fixed with respect to the surface of the glass, plastics material, metal or semiconductor substrate by an inherent adhesiveness relative to the base portion (10) and is separable from the base portion (10) in a substantially damage-free manner without sacrificing shape, adhesiveness and flexibility, the compartment layer (21) being completely pierced by the sample reservoirs (31) such that the surface of the base portion is exposed at the bases of the sample reservoirs (31).
2. Reaction substrate according to claim 1, wherein the base portion consists of a transparent material.
3. Reaction substrate according to claim 2, wherein the base portion (10) is a substantially flat, smooth glass plate.
4. Reaction substrate according to claim 3, wherein the glass plate has the thickness of a cover glass used in microscopy.
5. Reaction substrate according to claim 4, wherein the thickness of the cover glass is about 150 mm.
6. Reaction substrate according to one of the preceding claims, wherein the polymer composition includes natural or synthetic rubbers which are free of adhesive and solvent.
7. Reaction substrate according to claim 6, wherein the polymer composition includes silicon rubber.
8. Reaction substrate according to claim 6, wherein the polymer composition adheres to the base portion (10) without any adhesive.
9. Reaction substrate according to one of the preceding claims, wherein the compartment layer (21) is separable from the base portion (10) in a substantially damage-free manner without sacrificing shape, adhesiveness and flexibility.
10. Reaction substrate according to one of the preceding claims, wherein the compartment layer (21) bears a covering (50) on the opposite side to the base portion (10).
11. Reaction substrate according to claim 10, wherein the covering (50) has through apertures (51) for charging the compartment structures (30) with liquid samples or for removing such samples.
12. Reaction substrate according to one of the preceding claims, wherein the compartment structures (30) comprise ducts (32) and/or storage jars (33) in addition to the sample reservoirs (31).
13. Reaction substrate according to one of the preceding claims, wherein manipulating and testing devices (40) are provided in the compartment layer (21) which include fluid lines (41), electrodes (42) and/or sensors (43).
14. Reaction substrate according to one of the preceding claims which forms a micro- or nano-titration plate.
15. Reaction substrate according to one of the preceding claims, wherein variations of the sample reservoir positions in a direction perpendicular to the plane of the reaction substrate over the entire area of the base portion are less than 250 mm, preferably less than 150 mm, by particular preference less than 100 mm.
16. Use of a reaction substrate according to one of claims 1 to 15:

    to identify and characterise synthetic or biological objects,

    to identify and characterise chemical compounds,

    to identify and/or validate targets,

    to search for biologically active substances and/or active pharmaceutical agents,

    to identify conducting structures,

    for genome analysis,

    for proteome analysis,

    to clean and concentrate substrates, or

    for the evolutive optimisation of biologically relevant macromolecules.

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