WO2001062962A2

WO2001062962A2 - Component for interaction analysis comprising probe molecule species which create co-operation effects

Info

Publication number: WO2001062962A2
Application number: PCT/DE2001/000807
Authority: WO
Inventors: Derya ÖZKAN
Original assignee: Variom Biotechnology Ag
Priority date: 2000-02-28
Filing date: 2001-02-27
Publication date: 2001-08-30
Also published as: EP1305605A2; WO2001062962A3; AU2001256101A1

Abstract

The invention relates to a component comprising a support and probe molecule fields that are arranged thereon in a defined configuration. Each probe molecule field carries probe molecules of at least one probe molecule species and an association exists between the geometric positions of the probe molecule fields within the component and the species of the probe molecule fields. The invention is characterized in that each probe molecule field carries probe molecules of a respectively different, selected species group, whereby group elements of each species group bind mutually to a defined target molecule by creating co-operative effects.

Description

Component for interaction analysis with cooperative • ^• effects forming sample molecule species.

Field of the Invention.

The invention relates to a component with a carrier and with at least one sample molecule field arranged on the carrier in a defined arrangement, the sample molecule field carrying sample molecules of at least one sample molecule species, a method for producing such a component and the use of such a component.

Background of the Invention and Prior Art.

Components of the structure mentioned at the beginning, for example biochips, serve, among other things. the rapid analysis of samples for the presence or absence of target molecules. At its core, this is a parallel method, since a sample is contacted simultaneously with several or all elements of the component and target molecules react with those elements that carry sample molecules specific for the target molecule.

Biochips are known in a wide variety of embodiments. For example, the literature reference US-A-5, 744, 305 describes a biochip with a planar structure, on the surface of which in each case selected and assigned sample molecules are applied in defined areas, the so-called spots. Such biochips can with respect to the specificity of Binding is not accurate enough. In particular, for example in the case of nucleic acids as sample and target molecules, there is a risk that a singular mismatch in the sequence will nevertheless result in one; Binding and consequently leads to a (faulty) signal. For example, this is particularly disturbing in cases in which it is necessary to examine for point mutations.

A biochip of another construction is known from the literature US-A-6, 037, 186. Accordingly, a plurality of porous rods are produced, each of which is, as it were, impregnated with a solution containing a selected sample molecule species. After bundling the impregnated rods, slices are cut from the bundle in a plane orthogonal to the longitudinal extent of the bundle, which form the biochip. The cut surfaces form the spots. A biochip with yet another structure is known from the literature US-A-5, 837, 196. In the biochip known in this respect, it is formed from a bundle of optical fiber elements, the end faces of which are arranged in one plane and carry the sample molecules. A reading takes place by taking optical signals at the opposite end of the fiber elements carrying the sample molecules. These biochips also have the problem of incorrect signals, as described above.

From the literature US-A-5, 770, 365 it is known to carry out a hybridization of nucleic acids with the formation of a stacking effect. The sample molecules are each one Element of a sample molecule species group, namely a partially double-stranded hairpin oligonucleotide. The stacking effect occurs between the (singular) sample molecule and the target molecule.

Technical problem of the invention.

The invention is based on the technical problem of specifying a component which works in a simple manner with increased selectivity and, consequently, increased reliability, and which allows the detection of a comparatively large number of target molecules with a smaller number of sample molecule species.

Basics of the invention.

To solve this technical problem, the invention teaches that the sample molecule field carries at least two sample molecule species of a sample molecule species group, wherein group elements of the sample molecule species group bind together to a defined target molecule with the formation of cooperative effects. Several sample molecule fields can be provided, in which case an assignment is made between the geometric positions of the sample molecule fields within the component and the sample molecule species (or the respectively different sample molecule species group) of the sample molecule. The cooperative effect occurs when a target molecule is bound, preferably between the group elements of the sample molecule species group of a sample molecule field. With the cooperative effect, an additional energetic gain is obtained in the event of a bond, with the consequence of increased specificity of the bond events. In contrast, binding events that do not trigger the cooperative effect are energetically destabilized.

Furthermore, the invention teaches a method for producing a component according to the invention, with the following process steps: a) a carrier, preferably a planar carrier, is produced, b) sample molecule fields are set up in a defined arrangement on the carrier, c) sample molecules become on each sample molecule field a respectively selected, different sample molecule species group is immobilized, group elements of each sample molecule species group binding together to form a defined target molecule, with the formation of cooperative effects, d) optionally the product from stage c) is fed to a washing process stage, e) the sample molecule species group immobilized in stage c) is added assigned, and a method for producing a component according to the invention, comprising the following steps: a) there is produced at least one endless fiber, b) the endless fiber is formed by a fluid sample containing ^as' molecules to at least two elements of a selected sample molecule species group are directed, c) the sample molecules of the sample molecule species group are immobilized on the continuous fiber, d) optionally, the continuous fiber is fed to at least one washing process stage, e) the continuous fiber is the sample molecule species group immobilized on the fiber in stage c) , assigned, f) a fiber element is cut off from different continuous fibers or from different sections of an endless fiber and the fiber elements of different continuous fibers or sections are bundled and fixed.

Finally, the invention teaches the use of a component according to the invention in a method for the detection of target molecules, with the following process steps: a) a solution with prospective target molecules is supplied to the component under conditions in which target molecules bind to sample molecules, b) simultaneously with fed to step a) or subsequently thereto, the component of detection methods will drive stage in which binding events of step a) are detected, where ^'is carried out a detection with lateral spatial resolution, and wherein the spatial resolution is so high, at least as the distance between two adjacent Probenmolekulfelder, c) at the same time as stage b) or thereafter, there is an assignment of detected signals to the sample molecule fields from which the signals are obtained, and the use of a component according to the invention in a method for the detection of target molecules, with optically contactable end faces de r Fiber elements are optically connected, for example, to a CCD array or via a micromirror system to a photomultiplier, which are sensitive to optical radiation of a detection wavelength, and sensor elements of the CCD array or micromirror or micromirror positions are each assigned to the fiber elements, with the following process stages: a) the component is a solution with prospective Target molecules supplied under conditions in which target molecules bind to sample molecules, b) simultaneously with stage a) or thereafter the component is irradiated with primary radiation that excites a detection wavelength, c) simultaneously with stage b) or thereafter the signals of the sensor elements are read out of the CCD array or photo ultiplier and processing and storage of the signals. Alternatively or additionally, the fiber elements can, for example, be electrically contacted and / or contacted, for the purpose of evaluation by measuring the impedance or the changes in impedance. Evaluations by detection of surface plasmon resonances or scattering processes are also possible. Above all, luminescence detection is also possible. In the context of the invention, the expression of binding also includes interactions in the broadest sense.

Definitions.

An assembly or biochip is an arrangement which carries discrete and defined surface areas, the sample molecule fields, sample molecules of a sample molecule species or sample molecule species group. As a rule, each sample molecule field of a component will carry a different sample molecule species / group. The sample molecule fields are addressable in the sense that an assignment is / is made between each sample molecule field or its geometric position within the component and the sample molecule species group carried by the sample molecule field. The assignment can be direct or indirect his. In the latter case, an assignment between sample molecules or sample molecule groups and (different) markings of the fields takes place first, for example in the course of production. In essence, a marker is initially assigned to the sample molecules or sample molecule groups. After completion of the component, a detection of the markings and assignment of the markings and consequently the sample molecule species or sample molecule species groups to the spatial arrangement of the fields in the component is then carried out. Marking can be done, for example, by inserting or applying quantum spots on or in a field. Any other type of marking, for example color pigment coding, is also possible. The final assignment between sample molecule species / group to their spatial arrangement can be made by the manufacturer or only by the user. As a result, it is not necessary to maintain an exact spatial arrangement in the actual manufacturing process; rather, after the component has been put together, a calibration takes place, as it were, by spatially resolved detection of the markings.

The expression of the component also includes the terms of the biochip and the "composite analysis system made up of a large number of independent individual elements.

Sample molecules are molecules that can interact with target molecules in a specific way. Examples of such interactions are: antibody-antigen, lectin carbohydrate, protein Aptamer, _t nucleic acid nucleic acid, nucleic acid ribozyme, biotin avidin, etc.

Target molecules are molecules on which a sample to be analyzed, which is placed on the component, is examined. Target molecules can also be molecules that are to be removed from a sample (e.g. to be analyzed with other methods or with the same method). 0

A sample molecule species contains sample molecules exclusively of one structure, for example a sequence in the case of nucleic acids or proteins or peptides. 5

A sample molecule species group contains at least two sample molecule species as group elements. The sample molecule species can be of the same or different types of sample molecules. Nucleic acids, DNA, RNA, PNA, peptides, proteins and saccharides are designated as sample molecule types.

Cooperative effects between molecules of several sample molecule species or elements of a sample molecule group and a target molecule species are characterized in that the energetic gain through simultaneous interaction between the molecules of the different sample molecule species on the one hand and between the different sample molecule species and the target molecule on the other hand is greater than the sum of the energetic gains of the interactions of a molecule in each case a sample molecule species with a molecule of the target molecule species. The additional energy gain thus comes from the cooperation between the sample molecules of different sample molecule species in the case of binding of the sample molecules with the target molecule. In the case of nucleic acids as sample molecules and target molecules, stacking effects are to be mentioned as examples. The stacking effect is an energy gain through interactions, namely delocalization of the π electrons of the hydrophobic ring structures of neighboring bases in double-stranded nucleic acids. The stacking effect occurs between the ends of the sample nucleic acids when binding to a target molecule takes place in such a way that the ends of the sample nucleic acids are bound adjacent to one another. In the case of proteins, cooperative effects can result from special secondary structures of interacting proteins. In general, a higher specificity and binding energy of a bond between sample molecules and a target molecule is achieved with cooperative effects.

A hybridization area is a sequence area of a sample nucleic acid which can hybridize with a target nucleic acid. A spacer region is a group attached to one end of the sample nucleic acid, which is attached to the sample molecular field with a second binding site. A spacer area is expediently designed in such a way that binding or hybridization with a target molecule cannot take place. A spacer region can be formed, for example, from a non-hybridizing oligonucleotide. With a spacer area it is achieved that on the one hand the hybridization area is arranged at a sufficient distance from the surface of the sample molecule field and on the other hand the hybridization area is made more flexible and can consequently bind to a target molecule without steric or conformational restrictions.

Pieces cut from an endless fiber are referred to as fiber elements. As a rule, the cut will be made in a plane orthogonal to the longitudinal extension of the endless fibers, but it is of course also possible to have a cutting plane that is angled less than 90 °.

An endless fiber is a rod-like or thread-like structure with a longitudinal extension that is large in relation to the length of fiber elements, which can typically be produced by means of drawing technologies, blowing technologies and / or extrusion technologies and wound up and stored on drums or the like.

An endless fiber and / or a fiber element can have a wide variety of cross-sectional shapes in a cross-sectional plane orthogonal to the longitudinal extent. An essentially circular cross section is only preferred. In this respect, the term envelope surface in the context of the invention includes not only cylinder jacket surfaces, but also jacket surfaces in the case of non-circular cross sections. In this respect, the term radial direction in the context of the invention denotes all directions in a cross-sectional plane. In this respect, finally, the diameter in the context of the invention d = 2 * (F / 2π) ^0.5 , where F is the cross-sectional area (any shape).

The end face of a fiber element is formed by a cut through an endless fiber.

Spacing the fiber elements means that the lateral surfaces of adjacent fiber elements do not touch each other. A bundling of the fiber elements without line contact between individual fiber elements of the bundle is then created. Line contact means that the (mechanical) contact does not exist in areas of mutually parallel surfaces of adjacent fiber elements. A spacing of the fiber elements and / or a line contact between adjacent fiber elements is equivalent to the establishment of lugs extending in the radial direction in the area of the outer surface of a fiber element, as a result of which adjacent fiber elements except for point, line or area contact in the area of the lugs kept apart from each other.

A fiber element bundle generally has coplanar end faces of the bundled fiber elements. But it is also possible within one

To form fiber element bundles groups of fiber elements, each with coplanar end faces, the string surfaces of fiber elements of different groups being non-coplanar with one another.

Optical fiber elements are optically transparent to electromagnetic radiation, at least in a partial area, the area IR, visible light and / or UV. Optically transparent means that the attenuation of the electromagnetic radiation is sufficiently low to allow detection of electromagnetic radiation generated at one end of a fiber element at the opposite end of the fiber element by means of conventional detection technologies.

The term “optical contactability” denotes preparation of a partial area of a fiber element, which allows the emission of electromagnetic radiation from the fiber element through the partial area. Strong scatter should be avoided if possible. Possibly. the partial surfaces can be processed in a suitable manner, for example smoothed or polished. It is also possible to polish or apply microlenses to focus on existing radiation.

Functional groups of a polymer material are those chemical groups of the polymer structure which allow an unspecific binding between target molecules and the polymer material. In the case of nucleic acids and / or proteins as target molecules, functional groups would be, for example, amino groups, hydroxyl groups, thiol groups and carboxyl groups. It goes without saying that what has been said also applies to any auxiliaries added to the polymer material.

Preferred embodiments of the invention. The sample molecules of the sample molecule species groups can be selected from the group consisting of "nucleic acids, DNA, RNA, PNA, aptamers, proteins, peptides, saccharides and mixtures of these sample molecules". It is only essential that the sample molecules of different sample molecule species arranged on a sample molecule field can bind to a target molecule with the formation of cooperative effects. In this respect, it is fundamentally not absolutely necessary for the elements of a sample molecule species group to belong to a common sample molecule type. In principle, sample molecules and target molecules can also belong to different types of molecules.

In a special embodiment of the invention, the sample molecule species are nucleic acids, the cooperation effects being the stacking effects of the (sample) nucleic acids when a target molecule, in particular a target nucleic acid, is attached. The sample nucleic acids can have a hybridization area and a spacer area, the hybridization area being immobilized on a sample molecular field via the spacer area. The hybridization area can in principle be of any length, but it is advisable to keep it as small as possible; it preferably comprises 3 to 25 nucleic acid bases, in particular 3 to 10 nucleic acid bases. The spacer area is preferably formed from nucleic acid bases, in particular thymidine, the spacer area comprising 5 to 80, preferably 25 to 40, nucleic acid bases. It is also possible to use synthetic organic oligomers or polymers as To use spacer areas, in whose polymer chain, for example, carbamate groups can be incorporated.

Each sample molecule field can carry sample molecules of at least two respectively selected, different sample molecule species. In other words, each sample molecule field carries sample molecules of two different structures. In view of the cooperative effects used, it will be advisable to keep the respective molar amounts of the sample molecules of different structures on a sample molecule field within deviations of up to + 50%, better still up to + 20%, of the same size. The sample molecules of different structures can advantageously be stochastically distributed on a sample molecule field with regard to the spatial arrangement of the binding sites on the sample molecule field with a view to economical production.

The invention can in principle be used in components of any structure, for example the structure as described at the outset as prior art. It is preferred if each sample molecule field is in each case formed on a fiber element of a plurality of fiber elements, the sample molecules being immobilized on lateral surfaces of the fiber elements, and the fiber elements being fixed or spaced apart from one another by means of a support element in the radial direction with respect to the fiber elements are bundled in line contact with each other. A fiber element can also carry several sample fields. In principle, the fiber elements can have any shape in the direction of the longitudinal extent. With regard to a securely spaced fixation over the entire longitudinal extent of the fiber elements, it may be advisable to design the fiber elements to be straight and arranged parallel or radially to one another. It goes without saying that the spacing of the fiber elements is coordinated in such a way that, taking into account possible lateral mechanical loading forces of the fiber elements and the

Modulus of elasticity of the material of the fibers and their length, the cylinder jacket surfaces of adjacent fiber elements cannot touch under normal operating conditions of the component. However, the alternatives already mentioned also apply.

It is preferred if the fiber elements are optical fiber elements. With this embodiment, signals, for example, fluorescence signals from corresponding marker groups of molecules bound to the fibers (optically) can be excited and guided for detection by means of optical sensors to optically contactable locations of the fiber elements. Such locations can be, in particular, the end faces of the fiber elements. For example, optical fibers can be made at least partially of glass. However, it is preferred that the fiber elements are formed from a polymer material, which is preferably selected from the group consisting of "polyethylene (PE), polycarbonate (PC), polyvinyl chloride (PVC), polystyrene (PS), polytherephthalate (PETP)," Polyether sulfone (PES), polyether ether ketone (PEEK), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polybutylene terephthalate (PBT, - polyomethylene (POM), polysulfone (PSU), polyetherimide (PEI), polyamide (PA) and mixtures and copolymers of the monomers of such polymers ", in particular selected from the group consisting of" polycarbonate (PC ), Polyvinyl chloride (PVC), polystyrene and mixtures as well as copolymers of the monomers of such polymers ". The only thing that is important in the selection of material is that the material is sufficiently (permanently) temperature-resistant to the temperatures that occur during processing, processing or use of the component. In this sense, all polymer materials are designated as resistant to high temperatures, which have been found to be resistant for 1000 hours when exposed to at least 90 ° C., preferably at least 120 ° C., in particular at least 140 ° C. Typically, this is achieved if the glass temperature is above the stated one For example, polycarbonate with a glass transition temperature of 150 ° C. is particularly temperature-resistant It is clear that the polymer material in plastics technology can contain auxiliaries customary in the art, such as plasticizers, light stabilizers, in particular UV stabilizers, and the like. Additives influencing the (wavelength-dependent) dielectric constant can also be introduced for the purpose of optimizing the optical properties in a wavelength range of interest.

A fiber element can also be made of several materials in a composite. In particular,

Area of the cylinder jacket surface from the material of the core different materials can be used, for example, to reflectivity in the Modify fiber element of running light at the solid / liquid or solid / gas interface, if necessary, wavelength-selective. For example, the core can also consist of a mechanically rigid material, for example metal, glass or polycarbonate, while the optically transparent material surrounding the core can then be less rigid.

Geometrically, the fiber elements can be designed and arranged as follows. The fiber elements preferably have a diameter in the range from 0.01 μm to 1000 μm and a length in the range from 0.1 μm to 100 mm. The ratio of diameter to length can be in the range 100 to 10 ^"4. Furthermore, it is preferred if the fiber elements are packed in a density of 1 to 10 ⁷ fibers / cm ² , based on a radial cross-sectional plane of the fiber elements.

Various embodiments are possible with regard to the support element. The support element can be arranged at one end of the fiber elements and the ends of the fiber elements can be of extensive design, the end faces of the enclosed fiber elements being optically or directly contactable. In the case of indirect contactability, the support element must be optically transparent at least in the area of one of the ends of the fiber. Such a support element is typically Plattenför ig and its main surfaces are orthogonal to the longitudinal extension of the

Fiber elements. Such a component can be produced, for example, in that the support element, which is designed as a perforated plate, is introduced by introducing the lϊ

Fiber elements or the endless fiber in the holes of the perforated plate and subsequent fixation of the fiber elements in the holes with the fiber elements. In the case of the introduction of continuous fibers, a cutting process step must of course take place before or after the fixation. Likewise the ends is ^"possible to immerse a held by a holding device bundle of fiber elements (or ends of endless fibers) in a non-cured material and then to perform the curing process. Also, the support member may be formed out of the fiber elements, for example by contacting fixing in a In the field of fiber elements and fusing, welding, gluing etc. The same materials as those mentioned above in connection with the fiber elements are basically suitable as the material for such support elements. However, it is generally recommended that the support element not be optically transparent to carry out, for example by adding pigments, and to completely form the fiber elements through the support element, thereby reducing cross-talking optical signals.

The support element can also be made from a winding support tape. This is a long, band-shaped elastic construct, for example made of a thermoplastic elastomer, such as thermoplastic polyurethane (TPU), on or in which one end of the fiber elements is applied or embedded. The fiber elements are orthogonal to the longitudinal extent of the winding support tape. The winding carrier tape is then, for example, rolled up in a spiral or folded in a meandering manner in the zigzag, as a result of which the fiber elements are arranged in a 2-dimensional grid.

A component can only have one support element. In principle, this support element can be arranged at any point in relation to the longitudinal extent of the fiber elements, for example at one end of or in the middle of the fiber elements. However, it is also possible to set up two or more support elements within one component. This is particularly recommended in the case of very long and / or flexible fiber elements. If several support elements are provided, it is still advisable to provide 2 support elements at the opposite ends of the fiber elements.

With a view to reducing non-specific binding of target molecules directly to the surfaces of the sample molecule fields, it is advantageous if the polymer material does not have any functional groups on its surface, and if the sample molecules are nucleic acids, in particular oligonucleotides with less than 300 bases, that the nucleic acids are bound to the surface of the polymer material from an aqueous solution under irradiation with UV light.

Combinatorial synthesis on the fibers is possible for nucleic acids and other groups of substances. A component according to the invention can of course not only be used analytically but also preparatively, for example for the targeted separation of Substances from complex mixtures, such as blood. A component according to the invention can, for example, be provided with a self-wetting surface in the area of the lateral surfaces by means of detergents. Then solutions with target molecules automatically wet the outer surfaces. It is also possible to form "integrated devices" by means of a combination of components, for example for sample preparation, for analysis and for reading. As part of the production of the continuous fibers and / or the fiber elements, the polymerization or shaping of the polymer material can also be carried out in the presence or with the admixture of the sample molecules, so that the sample molecules are incorporated in volume, but are only accessible on the surface of a fiber element] Furthermore, the formation of cartridges containing at least one component according to the invention is possible, such cartridges being connectable in series, for example. Auxiliaries, such as enzymes for PCR or ligation, but also interaction-imparting molecules can be located on the surface of the components or in a cartridge.

A component according to the invention can be introduced and fixed in a fluidic component, for example a pipette tip or nozzle. Several components according to the invention can be connected fluidically in series or in parallel. Installation in integrated analysis systems is possible.

For components that exploit cooperative effects, such as stacking effects, Förster energy transfer (FRET) can be used by one the. two immobilized sample molecule group members, for example oligonucleotides, is labeled with a donor fluorescent dye and the other with an acceptor dye. With spatial proximity, for example stacking, Förster transfer now occurs, corresponding to a modulation of the emission wavelength. The use of FRET makes it possible that target molecules need not be labeled. The electrical conductivity can also be changed, for example by means of impedance measurement.

Detection by means of solid and possibly metallic particles, which are bound to target molecules, is also possible. A measurement is then carried out, for example, by means of scattering, evanescent waves, or by metallic precipitation on a fiber element (for example, if silver nanospheres are bound to the target molecules and photographic emulsions are used. If necessary, SERS (Surface Enhanced Raman Spectroscopy) can be used.

Due to the fact that the component is composed of different individual elements, each individual can also be addressed. Complete spectra can be run and a more detailed statement can be made about the composition of the specific binding partner or also freely resident molecules in the solution. For example, Raman, SERS, NIR can be used for this. Components can be read out by exposing the entire component or by controlling individual fiber elements (electrical, optomechanical via x-y-stage and, for example, fiber optics or optoelectric, by coupling or recording focused light via deflectable 'micromirrors.

Fiber elements can be pretreated and / or post-treated by coplanar grinding of fiber ends, grinding of fiber ends to microlenses, metallic coating of fibers for the generation of evanescent waves, roughening of fibers, mirroring of fibers with one end and thus the emission of the coupled light again the same (not mirrored) end is removed and measured, optionally using the same optical device. The signal coupling or modulation can be strengthened by introducing a modulating or reflecting substance into the fiber interspaces in solution. However, this substance can also be present as a solid.

All of the above statements apply accordingly to the methods and uses according to the invention.

The invention is explained in more detail below on the basis of examples which merely illustrate embodiments. Example 1:. Immobilization of two different nucleic acid species on a continuous fiber.

The basic procedure for immobilization is as follows. There ^'is a ^{Oligonukleot'} idpaar introduced into a crosslink-solution containing 1 M NaCl, 0.2 M MgCl _2, and 0.3 M Tris, pH 8, in a (total) concentration of 10 nM. In a device, a continuous PS fiber of 80 μm in diameter, which was extruded in the usual way, is passed from a supply roll through a quartz glass tube with an inside diameter of 1. mm and a length of 1 m, the ends of which are open and bent upwards , with an additional drain pipe and an additional inlet pipe attached to one end of the pipe. The inlet connection is connected to a peristaltic pump which is connected to a storage container in which the oligonucleotide solution is stored. The continuous fiber is wound at the exit end of the quartz glass tube onto a collecting roller which is driven by a stepping motor. The oligonucleotide solution is introduced into the quartz glass tube and the quartz glass tube is irradiated with a UV lamp attached above with a main emission at 254 nm. The speed of the collecting spool is adjusted so that a dwell time of each area of the endless fibers in the oligonucleotide solution in the quartz glass tube of approx. 5 min. is set up. At the same time, the volume flow of the oligonucleotide solution through the quartz glass tube is adjusted so that within about 5 minutes. a complete exchange of the oligonucleotide solution in the quartz glass tube takes place. Different continuous fibers are coated with different pairs of oligonucleotides in different quartz glass tubes or reactors. Alternatively, different sections of a single continuous fiber, for example each 10 m long, can be coated with different pairs of oligonucleotides by exchanging the oligonucleotide solution in the quartz tube in accordance with the desired section length and taking into account the feed speed of the continuous fiber through the quartz glass tube. This means that many different specificities can be generated on a single continuous fiber.

The following sample pairs are used, for example: pair a) wt-oligo (T) 35-CTACCC (left arm) and t-oligo CTACAG- (T) 35 (right arm);

Pair b) mt-oligo (T) 35-CTACCA (left arm) and wt-oligo CTACAG- (T) 35 (right arm). Always from 5 'to 3'. In general, the procedure is advantageously such that one oligonucleotide of a pair is immobilized at the 5 'end and the other at the 3' end.

A component comprising the two above pairs of samples was tested by incubating the component with a solution containing a wt oligonucleotide target of the sequence biotin-CTGTAGGGGTAG. After streptavidin-peroxidase conjugate formation, colorimetric analysis was carried out. Hybridization was shown only on the fiber element with the pair of samples A. In contrast, an mt oligonucleotide target of the sequence biotin-CTGTAGTGGTAG bound only on the fiber element with the Sample pair B. Control experiments with wt left arm, mt ^• left arm or wt right arm alone were negative, ie hybridization can only be determined when the two oligonucleotides of a pair of samples interact with the target. Furthermore, no signal is produced when a gap of 1 or more ^'nucleotides between right and left arm present when hybridized to the target sequence.

Example 2: Immobilization of nucleic acids on a glass of continuous fiber.

An endless fiber made of glass is first passed through a silanization solution and thereby aminosilanized. Otherwise, the procedure is as in Example 1, with the difference that the solution in the reactor contains an amino-specific chemical crosslinker, for example FDC, and the oligonucleotides are amino-modified. Irradiation is not necessary.

Example 3: Joining fiber elements into a component by meandering laying.

An endless fiber with a diameter of 200 μm and a length of 1000 m, which is coated at 1000 equidistant different lengths (of 1 m in length) with different pairs of oligonucleotides, is wound in opposite directions across two rows with 100 guide hooks each, so that the transition points between the different lengths come to rest on the guide hooks. The In the end, fiber is laid in a meandering shape, with the various length sections being arranged parallel to one another. The various length sections are then brought to bear against one another in the direction of the meandering plane, and the resulting dense layer of length sections is then covered with a cover film. A layer of 20 mm width and 1 m length is created. This is repeated 99 times with further lengths of the continuous fiber or another continuous fiber, the layers being produced being stacked on top of one another with the interposition of a cover film. A 100 x 100 grid is created, viewed in a plane orthogonal to the longitudinal extension of the longitudinal sections. The cover foils are pulled out lengthways, without shifting the length sections and a compressing pressure is applied. The result is an essentially square 100 x 100 grid in a dense packing. Then, to mechanically stabilize the positions of the length sections relative to one another, a support sleeve, for example as a band, is attached around the grid and the regions on the guide hooks are removed by two cuts in planes orthogonal to the longitudinal extension of the length sections. The position of a length segment and the pair of oligonucleotides assigned to it are determined by means of location-selective, selective coupling of light on a length segment thus created and measurement of the signal obtained at the other end thus obtained. Finally, blocks with a height of 1 mm are cut off in the direction orthogonal to the longitudinal extent, whereby not quite 1000 biochips of the same structure are obtained. Example 4: Joining fiber elements together

Web technology. ^;

Endless fibers, as used in Example 3, are woven in a meandering fashion between warp threads in accordance with a weft thread. Fibers not coated with nucleic acids serve as warp threads. A flat textile with length sections arranged parallel to each other is created. A plurality of such flat textiles are stacked and held on top of one another, the warp threads then being pulled out. During or after this, compression is carried out and a raster is produced according to example 3. In this embodiment of the invention, fibers provided with nucleic acids can also be used as warp threads (then fibers of different continuous fibers, each with only one pair of oligonucleotides, will be involved) ) and the weft threads are formed by fiber not coated with oligonucleotides.

Example 5: Joining fiber elements using

Supporting member.

An endless fiber according to Example 3 is threaded back and forth through a plurality of thermoplastic perforated plates, the transition points of the different length sections coming to lie at the reversal points of the first and last perforated plate. Between the first and the last perforated plate Enlarged perforated plates are fixed in the direction of the longitudinal extension of the longitudinal sections, for example by means of spacers, pairs of perforated plates being formed in each case ((shorter) spacers can also be arranged between the pairs of perforated plates, but the perforated plates of adjacent pairs can also abut one another directly ). Due to the action of heat and / or mechanical compressive forces on the perforated plates in the directions of the main plane of the perforated plates, the length sections in each perforated plate are, as it were, fixed therein. Then cuts are made between the perforated plates of adjacent pairs. The outer surfaces of the perforated plates of the constructs thus obtained are polished so that the ends of the fiber elements are coplanar with the outer surfaces of the perforated plates. The perforated plates can consist of an opaque material. The construct obtained can be equipped with an opaque sleeve. The perforated plates can have inlet and / or outlet openings for fluids, for example analytes.

Example 6: Production of a planar biochip with two different nucleic acid species in one field.

The procedure is basically the same as in Example 1, with the difference that 5 .mu.l of different solutions containing different pairs of nucleotides are dripped into different fields of an essentially planar support. Other solution components as well as UV radiation also correspond Example 1. Hybridization with biotinylated target then takes place. The biotin target is detected via enzyme conjugate formation and chemiluminescent substrate and CCD measurement or an insoluble reaction product of TMB and violet color precipitate.

Example 7: Stabilization of stacking, interaction.

The stacking interaction can be further stabilized if the right arm is 5 '-phosphorylated and the contact with the target takes place in the presence of ligase. The two oligos of a pair of samples are linked together.

Example 8: Carrying out a measurement with a component according to the invention.

A component according to the invention is contacted with an analyte containing a mixture of fluorescence-labeled oligonucleotides under hybridization conditions. Some of the oligonucleotides of the analyte have complementarity with some oligonucleotide pairs immobilized on the component. The analyte distributes itself automatically within the component due to capillary forces, whereby oligonucleotides of the analyte, which are complementary to immobilized oligonucleotide pairs, are bound to the component or the respective fiber elements by means of hybridization. A washing process stage follows Wash buffer, wherein non-hybridized oligonucleotides of the analyte are washed out. An end face of the component is then irradiated with a wavelength suitable for exciting the fluorescent dye. On the opposite side, a detection 'of signals in' is performed emission wavelength of the fluorescent dye ^■, with spatial resolution in the directions of the plane of the end face by means of a CCD element. The use of a scanner and / or a photomultiplier is also possible. An evaluation takes place taking into account the positions of the fiber elements and the signals emitted therefrom.

Example 9: cooperative protein binding

The antigen binding site of an antibody molecule (equivalent to a Fab fragment) is formed by the terminal parts of a light and heavy protein chain. These cooperatively bind an antigen in that both chains interact with each other as well as with the antigen. The binding only arises when both chains are in a certain conformation and can interact with each other, whereby the antigen binding is further strengthened by a change of conformation and interaction of both chains. The same applies to protein chains, for example in enzymes, which are folded into structures so that, for example, a sub pocket is created. This pocket is often formed by different protein chains. The substrate often only occurs with low interaction forces the protein in contact. This first contact is so far strengthened by cooperative effects and change of conformation ("induced fit") that it is strongly bound and possibly implemented. The reverse of the 5 separation of the end products is likewise often - achieved "cts by repealing the Kooperationseffe ^'-.

Cooperation effects can occur in analysis systems whose sensor elements consist of proteins or peptides.

10 can be exploited by immobilizing one or more amino acid chains in a sensor element array, e.g. when using two chains, one light and one heavy chain of an antibody molecule. Through various combinations of inputs

Is zelketten 15 a high number of specificities with a relatively low number of function SINGLE ^'sors achieved.

20 Example 10: dynamic addressing

Different continuous fibers are extruded from a polymer granulate, whereby different quantum dots (with different wavelength characteristics) are mixed in different templates of granulate mass. In this way, the respective continuous fibers produced are uniquely coded. As described above, the continuous fibers are each coated with different sample molecule species or sample molecule species groups, with an association between coding and sample molecule species / groups taking place. Then it is made in the same manner as described above a component made up of several different continuous fibers. By means of the manufacturer or before the component is used by the user, a spatial assignment of the local position of the respective one is made by means of spectral analysis of the individual fiber elements

Fiber elements hit with their respective coding. With the known assignment of the codes and the sample molecule species / groups, they are ultimately assigned to spatial positions. As a result, the exact positioning of the fiber elements need not be taken into account in the manufacturing process.

Example 11: Detection using FRET

A fiber element is coated with sample molecules, which enter into a cooperative interaction upon contact with the specified target molecule. The cooperation partners are each equipped with a donor and an acceptor for FRET. When the target molecule binds, there is a spatial proximity of the donor / acceptor pair, which leads to FRET upon optical contact with the excitation wavelength. This structure makes labeling the target molecules, for example with dyes, and a washing step unnecessary.

Claims

claims:

1. component with a carrier and with at least one sample molecular field arranged on the carrier in a defined arrangement, the sample molecule field carrying sample molecules of at least one sample molecule species,

characterized,

that the sample molecule field carries at least two sample molecule species of a sample molecule species group, wherein group elements of the sample molecule species group bind together to form a defined target molecule with the formation of cooperative effects.

2. Component according to claim 1, characterized in that a plurality of sample molecule fields is set up, an assignment being made between the geometric positions of the sample molecule fields within the component and the sample molecule species of the sample molecule fields,

3. Component according to claim 1 or 2, characterized in that the sample molecules of the sample molecule groups are selected from the group consisting of "nucleic acids, DNA, RNA, PNA, aptamers, proteins, peptides, saccharides and mixtures of these sample molecules".

4. Component according to one of claims 1 to 3, characterized in that the sample molecule species are nucleic acids, and in that the cooperation effects are stacking effects. Of the nucleic acids upon attachment of a target molecule, in particular a target nucleic acid, *, stacking effects being optional Addition of ligase to a solution containing target nucleic acids can also be stabilized.

5. Component according to claim 4, characterized in that the sample nucleic acids have a hybridization area and a spacer area, the hybridization area being immobilized on the sample molecular field via the spacer area.

6. Component according to claim 4 * or 5, characterized in that sample nucleic acids of a sample molecule species group are assigned to one another in pairs and are connected to one another via a single spacer area, the sample nucleic acids being immobilized on the sample molecule field via a binding site in the spacer area.

7. Component according to one of claims 4 to 6, characterized in that the hybridization region comprises 3 to 25 nucleic acid bases.

8. Component according to one of claims 4 to 7, characterized in that the spacer area Nucleic acid bases, in particular thymidine, is formed, and that the spacer region comprises 5 to 80, preferably 25 to 40, nucleic acid bases.

9. Component according to one of claims 1 to 8, characterized in that each sample molecule field is formed in each case from a fiber element of a plurality of fiber elements, wherein the sample molecules are immobilized on the outer surfaces of the fiber elements, and wherein the fiber elements by means of a support element in the radial direction , based on the fiber elements, fixed at a distance from one another or bundled in line contact with one another.

10. The component according to claim 9, characterized in that the fiber elements are arranged parallel to one another as a fiber element bundle.

11. Component according to claim 9 or 10, characterized in that the fiber elements are optical fiber elements.

12. Component according to one of claims 9 to 11, characterized in that the fiber elements are formed from a polymer material, which is preferably selected from the group consisting of "polyethylene (PE), polycarbonate (PC), polyvinyl chloride (PVC), Polystyrene (PS), polytherephthalate (PETP), polyether sulfone (PES), polyether ether ketone (PEEK), Polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polybutylene terephthalate (PBT, polyoxymethylene (POM), polysulfone (PSU), polyetherimide (PEI), polyamide (PA) and mixtures and copolymers of the monomers of such polymers ", in particular selected from the group consisting of "polycarbonate (PC), polyvinyl chloride (PVC), polystyrene and mixtures and copolymers of the monomers of such polymers".

13. Component according to one of claims 9 to 12, characterized in that the end faces of at least one end of the fiber elements, preferably both ends, are optically contactable.

14. Component according to one of claims 9 to 13, characterized in that the fiber elements have a diameter in the range of 0.01 microns to 1000 microns.

15. Component according to one of claims 9 to 14, characterized in that the fiber elements have a length in the range of 0.1 microns to 100 mm.

16. Component according to one of claims 9 to 15, characterized in that the fiber elements are packed in a density of 1 to 10 ⁷ fibers / cm ² , based on a radial cross-sectional plane of the fiber elements.

17. Component according to one of claims 9- to 16, characterized in that the support element is arranged at one end of the fiber elements and the ends of the fiber elements are formed comprehensively, the end faces of the 5-encompassed fiber elements being directly or indirectly optically contactable.

18. Component according to one of claims 9 to 17, characterized 10 in that a support element is arranged at both ends of the fiber elements.

19. Component according to one of claims 9 to 18, characterized 15 in that the support element between the two ends of the fiber elements, for example in the middle, is arranged.

20 20. Component according to one of claims 9 to 19, characterized in that the support element is designed as a perforated plate.

25 21. Component according to one of claims 9 to 20, characterized in that the support element is designed as a winding support tape.

30 22. Component according to one of claims 9 to 21, characterized in that the polymer material has no functional groups on its surface, that the sample molecules are nucleic acids, and that the Nucleic acids from an aqueous solution are bound to the surface of the polymer material under irradiation with UV light.

5

23. A method for producing a component according to one of claims 1 to 22, with the following process stages:

10 a) a carrier, preferably a planar carrier, is produced,

b) sample molecule fields are set up on the carrier in a defined arrangement,

15 c) Sample molecules of a respectively selected, different sample molecule species group are immobilized on each sample molecule field, group elements of each sample molecule species group being shared under

20 binding cooperative effects to a defined target molecule,

d) optionally the product from stage c) is fed to a washing process stage,

25 e) the sample molecule species group immobilized in stage c) is assigned to each sample molecule field.

24. The method for producing a component according to one of claims 1 to 22, with the following process stages: a) at least one continuous fiber is produced,

b) the continuous fiber is passed through in each case a fluid containing as sample molecules at least two elements of a selected sample molecule species group,

c) the sample molecules of the sample molecule species group are immobilized on the continuous fiber.

d) optionally, the continuous fiber is fed to at least one washing process stage,

e) the continuous fiber is assigned to the sample molecule species group immobilized on the fiber in stage c),

f) a fiber element is cut off from different continuous fibers or from different sections of a continuous fiber and the fiber elements of different continuous fibers or sections are bundled and fixed.

25. The method of claim 23 or 24, wherein the number of elements of the sample molecule species group is 2.

26. Use of a component according to one of claims 1 to 22 in a method for the detection of target molecules, with the following method steps: a) a solution with prospective target molecules is added to the component under conditions in which target molecules bind to sample molecules,

b) at the same time as stage a) or thereafter, the component is fed to a detection method stage, in which binding events from stage a) can be detected, and wherein a detection takes place with lateral spatial resolution, and the spatial resolution is at least as high as the distance between two neighboring sample molecule fields,

c) at the same time as stage b) or thereafter, the signals detected are assigned to the sample molecule fields from which the signals are obtained.

27. Use of a component according to one of claims 1 to 22 in a method for the detection of target molecules, wherein optically contactable end faces of the fiber elements are optically connected to a detector which is sensitive to optical radiation of a detection wavelength, and wherein signals from the detector in each case are assigned to the fiber elements with the following process stages:

a) a solution with prospective target molecules is added to the component under conditions in which target molecules bind to sample molecules, b) simultaneously with stage a) or thereafter, the component is irradiated with primary radiation that excites a detection wavelength,

c), simultaneously with stage b) or thereafter, the signals from the detector are read out and the signals are processed and stored.

28. Use of a component according to one of claims 1 to 22 in a method for the preparative preparation of a solution containing a mixture of substances, the component carrying sample molecules which bind to target molecules to be separated from the mixture of substances, with the following process stages:

a) the solution is fed to the component, the target molecules to be separated being bound to sample molecules and thus being immobilized,

b) then the solution freed from target molecules to be separated is removed from the component and, if necessary, fed to a further processing, for example a method according to one of claims 26 or 27.

29. A method for interaction analysis in which at least one sample molecule is immobilized on a solid and in the presence of a target molecule which is complementary to the sample molecule in a solution brought into contact with the solid, a cooperation effect involving various parts of a Sample molecule or different sample molecules takes place in the course of the interaction between the sample molecule and the target molecule and the interaction is stabilized energetically.