DE19980632B4 - Process for carrying out reactions between at least two reactants in aqueous reaction mixtures - Google Patents

Abstract

method to carry out of reactions between at least two reactants in aqueous reaction mixtures, in which at least one reaction partner spatially separated Passing through compartments with different reaction conditions and immobilized at least one reactant on a surface of a compartment is, wherein the reaction mixture moves magnetohydrodynamically and circularly become.

Description

The The present invention relates to a method and an apparatus to carry out of reactions between at least two reactants, in particular biomolecules wherein at least one reactant reaction areas with different Undergoes reaction conditions and at least one reactant, for example a feature a biochip, is immobilized, and wherein the reaction mixtures magnetohydrodynamic and circular to be moved.

Biochips are typically two-dimensional arrays of arrays (Features) of immobilized, single - stranded biomolecules, im named immobilized probes, for example deoxyribonucleic acid (DNA) molecules with certain Base sequences (sequences), or polypeptides, such as antibodies or other proteins. The sequence of bases (sequences) of the DNA molecules in the individual Features or the sequence of amino acid residues in the polypeptides like proteins or antibodies and / or other characteristics characterizing these are known so that e.g. a feature with DNA of a particular sequence or with a polypeptide with a specific amino acid sequence certain spatial Position coordinates can be assigned.

in the contains the following example a DNA chip 10,000 features. Each feature has a mean diameter of 50 μm. In a grid arrangement is the mean distance between the feature centers is 100 microns. Each feature contains on average, a large one Number of single-stranded DNA molecules (e.g. in the order of magnitude 100,000, 1 million or 10 million) as immobilized probes of each the same or predominantly the same sequence of e.g. 20 or 30 base pairs in length. Generally can determine the geometry, the number, arrangement and geometry of the features, the number and base sequence of immobilized probes in each feature be varied within wide limits.

A essential application of such DNA chips is with their Help statements about the sequence composition of an unknown mixture of nucleic acid molecules, in particular of DNA molecules, to obtain. In many cases contains the mixture to be analyzed a wide variety of different Sequences, so represents a complex mixture. For example contains the mixture of human genomic DNA from a tissue or tumor, optionally by polymerase chain reactions or the like Methods before use was amplified. For an assumed sequence length from 20 to 30 each Base pairs can in this case be the complex mixture several millions Types of DNA molecules each containing different sequences. Any or particular Sequences can thus in the complex mixture in only very small relative Concentrations be represented. In another example, the complex mixture contains several thousand different sequences of complementary DNA, also referred to as cDNA, the information about the genetic expression pattern a tissue included.

In order to obtain information about the sequence composition of the complex mixture, methods of hybridization of nucleic acid molecules can be used. For this purpose, mobile probes are first prepared in a widely used method by first appropriately labeling the DNA molecules to be analyzed, for example by fluorescence labeling of one or more bases of the sequence with fluorochromes thereof or different spectral signature. Currently widely used fluorochromes are, for example, fluorescein isothiocyanate (FITC) or rhodamine Denvate. The spectral signature is given by the optical properties of the substance, such as absorption and emission spectrum, the fluorescence lifetime, or the polarization behavior. In a typical hybridization process, a hybridization mixture is used, consisting of a complex mixture of single-stranded, fluorescently labeled DNA molecules in a buffer solution and optionally other chemical substances. In order to convert the fluorescently labeled DNA molecules into a single-stranded conformation (denaturation) and thus to obtain mobile probes according to the invention, a thermal treatment is usually carried out. The temperature required for denaturing the DNA molecules (denaturation temperature T _D ) depends strongly on the chemical composition of the hybridization mixture. After denaturation, the hybridization mixture at a certain temperature (hybridization temperature T _H ) and for a certain time (hybridization time t _H ) is applied to the DNA chip. In order to fulfill the requirement of single-stranded immobilized probes, the DNA molecules present on the DNA chips already before the start of the hybridization reaction must already be single-stranded either by the method used in the preparation of the DNA chips or, if appropriate, before the actual hybridization reaction of a denaturing Be subjected to treatment. Both denaturing conditions can also be realized, for example, by suitable simultaneous thermal treatment of the object consisting of hybridization mixture and DNA chip.

Instead of a suitable mark the mobi len DNA molecules in the hybridization mixture, instead of or in addition, a labeling of the DNA molecules immobilized on the DNA chips done. Insofar as the method is used only for preparative purposes, for example the depletion of the hybridization mixture of DNA molecules of a specific sequence, labeling of the probes may optionally also be omitted. Another method relates to the detection of the hybridization reaction by non-optical methods, such as electrical conductivity changes.

at appropriate physicochemical conditions (e.g., temperature, Hybridization Time, Ion Composition and pH of the Buffer, Concentration on chaotropic substances such as formamide, dextran sulfate, LiCl in high Concentration, etc.), can be a stringent binding of mobile probes also single-stranded immobilized probes can be achieved in the features.

stringent Bond means that one Collation Between Nucleic Acid Sequences with Complementary Base Sequence comes about. Unbound mobile probes may be used removed by washing steps. The degree of stringency of hybridization immobilized by mobile probes of a specific sequence Probes of complementary Sequence can be determined, for example, by the ratio of the number of complementary base pairs in one of a (before binding) mobile probe and an immobilized one Probe formed hybrid molecule to the total number of bases of the sequence to be hybridized mobile probe can be specified. A stringency of 1.0 means that in one hybrid molecule all bases of a (before binding) mobile probe complementary pairings with Bases of an immobilized probe have been received.

To complete execution of the hybridization process contain the features with immobilized Probes that are complementary Base sequence to mobile probes contained in the hybridization mixture own, an increased Number of specific marker signals, e.g. of fluorescently labeled ds Hybrid molecules. The more, for example, fluorescently labeled mobile probes in one hybridized to certain feature, the higher the number of fluorescence label signals in the feature in question. With suitable detection methods it is then determined which features an increased Number of marker signals included. When using fluorescent labeling techniques suitable detection methods are e.g. Methods of digital epifluorescence microscopy with thermal light sources, laser scanning microscopy, the confocal laser scanning microscopy or multiphoton fluorescence microscopy. Other probes may be used to detect hybrid molecules Procedures come into use.

Provided the base sequence (sequence) of the immobilized probes in a feature is known, is thus due to the method, the (complementary) sequence The mobile probes bound there within the framework of the realized stringency requirements known. The higher the realized stringency, the more accurate the sequence of the there be specified bound mobile probes. If due to the production of the DNA chip method used the assignment of feature position and sequence the immobilized probes are known in the feature allows that Therefore, DNA chip hybridization methods in a relatively short time statements about the sequence composition of complex mixtures of mobile probes hold true; in quantitative analysis of the marker signals and appropriate calibration are also information about the frequencies possible, with the mobile probes are present in the mixture. It is also more preparative Application possible, DNA molecules with such Sequences for to isolate a subsequent use. The higher the number of features with immobilized probes of different sequence, the more more precisely, a complex mixture of mobile probes can be analyzed become. Currently, there are DNA chips with a feature count of several 100,000 to 1,000,000 realized or in production.

The on the example of DNA chips running representation The prior art can be in a manner known to those skilled in the art also transferred to other bio-chips. For example, bio-chips are described in which the features instead of single-stranded DNA molecules immobilized probes polypeptides, e.g. Proteins used become. While in DNA molecules the stringency is essentially the total number of hydrogen bonds between complementary ones Bases in a hybrid molecule dependent is, can the specificity protein-protein binding for example, by the number of hydrogen bonds and Van der Waals bonds between the proteins which in this case are strongly dependent on the spatial conformations of the binding biomolecules dependent is.

Empirical observations indicate that, in the methods known in the art, the secure detection of a particular nucleotide sequence in a complex mixture of DNA molecules as mobile probes by detection by DNA-chip hybridization to the immobilized probes of DNA present in a feature -Molecules complementary sequence is often difficult or impossible. For many users This is to be regarded as a serious disadvantage. For example, the safe detection of a tumor-relevant small deletion in amplified patient DNA requires that in control DNA with the presence of mobile probes of the deletion sequence in the hybridization mixture their safe detection using the DNA-chip hybridization method is possible, while in the Tumor DNA also includes mobile probes with very similar sequences, as those of the deletion can be clearly discriminated. The high stringency requirements, which are often required, can lead to a significant reduction in signal strength, which greatly reduces the need for analysis. Similar problems arise, for example, when a large number of mobile probes with a slightly different base sequence should be analyzed by DNA chip hybridization. Here, the probability of unwanted association processes of the mobile probes can be increased.

As a concrete example, a DNA chip with 10,000 features as described above is assumed, with a DNA chip size of 10 × 10 mm ² . In the hybridization mixture are 1,000,000 different types of single-stranded DNA molecules as mobile probes, each having a length of 20 - 30 bases at a concentration of 100 ng / ul. Such complex mixtures can occur, for example, when using genomic DNA without further prepurification or amplification. Even with the use of complementary DNA (cDNA) of tissues, the complexity of the mixture can be extremely high. The total volume of the hybridization mixture is assumed to be 100 μl. The required amount of mobile probes in this case is 10 μg. Each of the 1,000,000 presumed types of mobile probes thus has a mass of 10 pg in the mixture. Each type of mobile probe is represented in the solution so assuming equal frequencies with about 0.6 billion copies. With a number of 1,000,000 immobilized probes per feature, each with complementary sequence, the ratio V (P / T) {mobile probe number / [number of complementary sequence immobilized probe]} at full denaturation is orders of magnitude higher than 1, ie one immobilized probe come, for example, several hundred mobile probes of the complementary sequence. In addition, the relative concentration of single or multiple types of mobile probes in the hybridization mixture may be very small.

experimental Experiences of DNA chip hybridization with those described in the prior art Method, for example in the case of oligonucleotides as mobile Probes show significant disadvantages.

• (i) Es kann davon ausgegangen werden, daß die Stringenzanforderungen bei DNA-Chip-Hybridisierung zum Teil erheblich größer sind als bei in situ-Hybridisierungsreaktionen, bei denen ein komplexes Gemisch von markierten DNA-Molekülen als mobilen Sonden zur in situ-Hybridisierung an chromosomale Ziel-DNA als "immobilisierte Sonden" verwendet wird. Beispielsweise kann es für die Identifizierung eines Metaphasechromosoms A mit Hilfe einer Fluoreszenz-in-situ-Hybridisierung (FISH) eines komplexen Gemisches mit mobilen Sonden aus einer chromosomenspezifischen DNA-Bibliothek des betreffenden Chromosoms A völlig ausreichend sein, wenn nur die Hälfte der hybridisierten mobilen Sonden an komplementäre "immobilisierte Sonden" im Chromosom A gebunden ist. Bei subchromosomalen in situ-Markierungsorten kann der Wert noch wesentlich kleiner sein. Ist hingegen bei DNA-Chip-Hybridisierungen ein besonders hoher Grad an Stringenz erforderlich, so sind entsprechend stringente Reaktionsbedingungen und Waschungen anzuwenden. Diese können zum Teil auch solche mobile Sonden ablösen, die an vollkommen koplementäre chromosomale DNA-Abschnitte als "immobilisierte Sonden" gebunden sind.
• (ii) Bei in situ-Hybridisierungsreaktionen an chromosomale Ziel-DNA wird aufgrund der Begrenzung der räumlichen Auflösung der Detektionseinheit, z.B. der lichtmikroskopischen, eine scheinbar homogene Bindung der mobilen Sonden an chromosomale Zielorte auch dann beobachtet, wenn in Wirklichkeit nicht alle komplementären DNA-Abschnitte des chromosomalen Zielortes mit (vor der Bindung) mobilen Sonden hybridisiert haben. Beispielsweise läßt sich aus einer Auswertung der Fluoreszenzphotonenstatistik von konfokal registrierten in situ-Hybridisierungssignalen einer insgesamt ca. 100.000 Basen langen chromosomalen Zielsequenz als "immobilisierter Sonde" grob abschätzen, daß insgesamt nur ca. 2.000 effektive Fluoreszenzphotonen registriert wurden. Auf der Objektseite entspricht dies etwa 200.000 – 600.000 insgesamt von der "immobilisierten Sonde" emittierten Photonen bzw. etwa einigen hundert an die "immobilisierte Sonde" gebundenen Fluorochrom-Molekülen; dies würde einer Hybridisierung von nur wenigen der hier z.B. jeweils einige hundert Basen langen mobile Sonden entsprechen. Geht man davon aus, daß in ähnlicher Weise bei vergleichbaren DNA-Chip-Hybridisierungen in einem bestimmten Feature nur wenige der dort befindlichen immobilisierten Sonden mit komplementären (vor der Bindung) mobilen Sonden hybridisiert sind, so wird in vielen Fällen das erhaltene Markierungssignal, z.B. ein Fluoreszenzsignal, erheblich geringer sein als das maximal mögliche, und gegebenenfalls keine Unterscheidung des Signals vom Hintergrundsrauschen gestatten. Ein weiterer Nachteil betrifft eine gegebenenfalls gewünschte Quantifizierung des Signals, z.B. zur Ermittlung der im Hybridisierungsgemisch vertretenen relativen Konzentrationen von DNA-Molekülen bestimmter Sequenz.
• (iii) Verschiedene wichtige Anwendungen der DNA-Chip-Hybridisierung erfordern es, bei hohen Stringenzanforderungen eine Hybridisierung mobiler Sonden aus einem sehr komplexen Hybridisierungsgemisch zu realisieren, das z.B. aus 1.000.000 verschiedener Arten von Oligonukleotiden besteht. Dies kann lange Hybridisierungszeiten zur Folge haben, in denen unter den gewählten Hybridisierungsbedingungen die Wahrscheinlichkeit einer Renaturierung/Assoziation von Oligonukleotiden komplementärer oder teilweise komplementärer Sequenz in Lösung erheblich größer ist als die Wahrscheinlichkeit der Bildung eines Hybridmoleküls aus mobiler Sonde und immobilisierter Sonde mit komplementärer Sequenz.

Some reasons for these results are given below by way of example:

• (i) It may be assumed that the stringency requirements in DNA-chip hybridization are sometimes considerably greater than in in situ hybridization reactions in which a complex mixture of labeled DNA molecules as mobile probes for in situ hybridization to chromosomal Target DNA is used as "immobilized probes". For example, for identification of a metaphase chromosome A by fluorescence in situ hybridization (FISH) of a complex mixture with mobile probes from a chromosome-specific DNA library of the chromosome A in question, it may be sufficient if only half of the hybridized mobile probes is bound to complementary "immobilized probes" in chromosome A. For subchromosomal in situ tags, the value can be much smaller. If, on the other hand, a particularly high degree of stringency is required for DNA-chip hybridizations, suitably stringent reaction conditions and washes must be used. These can also partially replace those mobile probes which are bound to completely coplanar chromosomal DNA sections as "immobilized probes".
• (ii) In situ hybridization reactions to target chromosomal DNA, due to the limitation of the spatial resolution of the detection unit, eg light microscopy, a seemingly homogenous binding of the mobile probes to chromosomal target sites is observed, even if not all complementary DNA segments of the chromosomal target site have hybridized with (before binding) mobile probes. For example, from an evaluation of the fluorescence photon statistics of confocal registered in situ hybridization signals of a total of about 100,000 bases long chromosomal target sequence as "immobilized probe" roughly estimate that only about 2,000 effective fluorescence photons were registered. On the object side, this corresponds to about 200,000-600,000 total photons emitted by the "immobilized probe" or about a few hundred fluorochrome molecules bound to the "immobilized probe"; this would correspond to a hybridization of only a few of the here, for example, each several hundred bases long mobile probes. Assuming that in comparable DNA-chip hybridizations in a certain feature, only a few of the immobilized probes located there are hybridized with complementary (before binding) mobile probes, in many cases the resulting marker signal, eg Fluorescence signal, significantly lower than the maximum possible, and possibly no sub allow the signal to be separated from the background noise. A further disadvantage relates to an optionally desired quantification of the signal, for example for determining the relative concentrations of DNA molecules of specific sequence represented in the hybridization mixture.
• (iii) Several important applications of DNA-chip hybridization require hybridization of mobile probes from a very complex hybridization mixture consisting of, for example, 1,000,000 different types of oligonucleotides, at high stringency requirements. This can result in long hybridization times in which, under the chosen hybridization conditions, the probability of renaturation / association of oligonucleotides of complementary or partially complementary sequence in solution is significantly greater than the probability of formation of a hybrid molecule of mobile probe and immobilized probe with complementary sequence.

When As a consequence, the Mark signal, e.g. a fluorescence signal, in the noise of the detector system goes down.

Similar Problems arise with other organic chips everywhere, where, for example, various desirable ones Reaction conditions can not be optimized simultaneously. Furthermore, can it desirable be in contact with the bio-chip mixture or a other liquid in contact with the biochip, e.g. a washing solution, a controlled, in particular periodic, convection movement to undergo. In the usual small volumes of the mixture or for the inclusion of the / the bio-chip) certain chamber can such convection movements made according to the state of the art, for example, by pipetting, not optimal results to lead, e.g. undesirable Material losses.

DE 39 25 093 C2 relates to a device for the quantitative fractionation of biological particles bound to magnetic particles (beads), by means of the so-called Lorentz force, the electrical conductivity in the magnetic field being used as separation parameter. US 5 685 966 A describes a system for the magnetohydrodynamic movement of liquids. US 5,632,876 A describes the combination of electro-osmotic and electrohydrodynamic pumps in a microchannel, where both polar and non-polar liquids can be moved along this channel. WO 96/04547 describes a microchip laboratory system and method for electrokinetic transport of reaction mixtures using capillary electrophoresis and electrochromatography.

Around the above-mentioned disadvantages of the known in the art Overcome procedures it is required in case of hybridization reactions the measurements of the hybridization signals of the individual features the signal-to-noise ratio (SNR) sustainably without diminishing the stringency requirements have to. Similar Requirements arise when e.g. the association reactions of Proteins or other compounds as mobile probes to polyamino acids or antibodies to be improved.

Generally, this means This can be done using techniques known in the art to happen that way Noise is reduced and / or the signal strength is increased. For example, can using fluorescently labeled mobile probes and given DNA chip after hybridization the SNR ratio can be improved by: Optimized fluorescence excitation by excitation light sources of increased intensity in the absorption maximum of fluorochrome related labels; Reduction of bleaching the fluorochromes by using two or multiphoton excitation; collection of the fluorescence light emitted by the feature through optics higher Numerical aperture; Reduction of optical elements in the detection beam path, which reduce the fluorescence yield; Use of fluorescence detectors increased Quantum yield; chilled Fluorescence detectors; improved tuning of magnification factors of the optical detection system between the feature size in the Object level and the pixel size in the Level of the detection system; improved evaluation algorithms the obtained digital fluorescence images of the DNA chip.

Other Options, In order to overcome the above mentioned disadvantages, the DNA chip is concerned itself and the hybridization of the mobile probes to the immobilized ones Probes in the features. For example, fluorochromes can be used for labeling with elevated photostability be used; the degree of labeling of the mobile probes can be increased; the total number or concentration of immobilized probes in one Feature can be increased be, etc.

Thus, it is an object of the present invention to provide novel systems that are generally useful for carrying out reactions between two reactants, such as biomolecules in features of bio-chips, in which, as described above, several steps, such as in the case of DNA Molecules denaturation / hybridization sequentially and / or peri be traversed odisch, wherein the above-mentioned disadvantages of previously known methods are overcome.

The The above object is achieved by the objects characterized in the claims solved present invention.

The Figures show:

1A is the schematic representation of a horizontal cross section of a microhydrodynamic reaction chamber according to the invention for carrying out reactions with features on bio-chips. The ring-shaped inner electrode (E1, here positive (+) charged) with the outer diameter 2r ₀ , the annular outer electrode (E2, here negative (-) charged) with the inner diameter 2R and the compartments I (I) and II (II ). The arrows indicate the direction of microhydrodynamic transport of the mobile probes.

1B is the schematic representation of a vertical cross section through the same reaction chamber with the compartments I and II as 1A , In addition to the electrodes, the base area F _G and the upper closing surface F _O , which is located at the height H _Z above the base area, can be seen here. A part of the end face of the compartment I is formed as a transparent window Fe. Above the window, a detection device O is indicated, which may for example consist of an excitation and detection optics or other biosensors. The Bio-Chips (BC) are arranged on the base, below these are the microsensors ( 1 . 2 ), eg active elements for example for cooling and / or heating or determination of the ion concentration. The level of the reaction mixture is in the height h _F above the base. The direction of the magnetic field B and the electric field E are indicated.

Especially becomes a procedure to carry out provided by reactions between at least two reactants in aqueous reaction mixtures, in which at least one reaction partner reaction areas with different Undergoes reaction conditions and at least one reactant is immobilized, wherein the Reaction mixtures magnetohydrodynamically and circularly moved become.

Of the Term "reaction conditions" includes all physical conditions such as temperature, pressure, etc. as well as the chemical conditions such as ion concentration, -Strength and composition, pH, etc. of the respective reaction mixtures.

According to one preferred embodiment the reaction areas compartments, which are spatially separated from each other are that yourself the reaction conditions prevailing in them are not mutually exclusive influence.

Of the Term "compartment" generally refers to a reaction area that spatially completed by the environment, but via an opening or a passage o.ä. so that he can, if necessary, such as for the transfer of Reaction mixtures, with the environment or with other reaction areas or compartments can exchange substances. In particular, means the term "compartment" means a spatially delimited reaction area, but over has an exchange area, with an overpass of Reaction mixtures between different compartments is possible.

According to one another preferred embodiment the method according to the invention is at least one reaction partner on one face of a reaction zone or a compartment immobilized, and the reaction between the reactants is a binding reaction.

Of the The term "binding reaction" includes any type of chemical and physical association reaction between two molecules, such as covalent bonding, hydrogen bonds, hydrohobic interactions, van der Waals interactions, etc.

• (a) Halten mindestens eines Reaktionspartners in einem Reaktionsgemisch unter ersten Reaktionsbedingungen in einem Kompartiment I,
• (b) Halten der mobilen und immobilisierten Reaktionspartner in einem Reaktionsgemisch unter zweiten Reaktionsbedingungen im Kompartiment I, wobei eine Bindungsreaktion zwischen den Reaktionspartnern stattfindet,
• (c) Überführen des Reaktionsgemisches mit den nicht gebundenen Molekülen des mobilen Reaktionspartners in ein Kompartiment II, (d) Halten des mobilen Reaktionspartners von Schritt (c) unter dritten Reaktionsbedingungen, die gleich denen von Schritt (a) sein können, im Kompartiment II,
• (e) Überführen des mobilen Reaktionspartners von Schritt (d) in das Kompartiment I und gegebenenfalls
• (f) Wiederholen der Schritte (b) bis (e) bis eine vorgegebene Abbruchsbedingung erfüllt ist.

In a further preferred embodiment, the method according to the invention comprises the steps

• (a) keeping at least one reactant in a reaction mixture under first reaction conditions in a compartment I,
• (b) maintaining the mobile and immobilized reactants in a reaction mixture under second reaction conditions in compartment I, whereby a binding reaction takes place between the reactants,
• (c) transferring the reaction mixture with the unbound molecules of the mobile reactant to a compartment II, (d) maintaining the mobile reactant of step (c) under third reaction conditions which may be the same as those of step (a) in compartment II,
• (e) transferring the mobile reactant from step (d) to compartment I and optionally
• (f) repeating steps (b) through (e) until a predetermined cancellation condition is met.

The term "termination condition" can be any condition associated with the binding reaction, such as the achievement of a particular, related to the bin reaction magnitude to be achieved or value or change of a parameter. Such parameters may include, for example, the number of bound molecules of the mobile and the immobilized reactants, changing or reaching a particular physicochemical parameter such as pH, ionic composition and / or ionic strength, temperature, pressure, etc.

In preferred embodiments of the above method additional steps of detection between steps (c) and (d) and evaluation of the reaction and / or between the steps (b) and (c) additionally one or more washes are performed.

Furthermore can in the method described above, further compartments with the same or other reaction conditions than in the compartments I and II by the mobile and / or immobilized reactants become.

In a further preferred embodiment the method according to the invention the compartments are arranged so that the transfer of the reaction mixture in steps (c) and (e) is on the same trajectory, however the direction of movement is opposite. In a further embodiment can the compartments should be arranged so that the transfer of the reaction mixture in steps (c) and (e) on a circular trajectory in each case the same direction of movement runs.

A preferred embodiment the method according to the invention refers to reactions that take place under micro conditions, where the reaction volume in the compartments is not greater than about 150 μl, more preferably not smaller than 20 μl and not larger than 100 μl, is. Such micro-conditions are for example in another inventive embodiment the method in which the reactants are biomolecules.

preferably, are the biomolecules from the group of nucleic acids and / or polypeptides selected. The term "nucleic acid" means native, semisynthetic or modified nucleic acid molecules from deoxyribonucleotides and / or ribonucleotides and / or modified nucleotides such as Aminonucleotides or [-S] -triphosphate nucleotides. In the case of nucleic acids the binding reaction is a hybridization. In case of hybridization of nucleic acids the corresponding reaction mixtures are called "hybridization mixtures".

Of the Term "hybridization" means the attachment two nucleic acids due to specific hydrogen bonds between the complementary base pairs and unspecific hydrophobic interactions by the so-called. Base stacking within the hybrid molecule. The attachment takes place usually by the fact that the corresponding nucleic acid molecules previously reversibly denatured, for example by increasing the Temperature to a certain value such as over the so-called melting point of the respective nucleic acid species or by change pH, etc., and then renatured, e.g. by lowering the temperature by a certain value below the melting point.

According to one another preferred embodiment the method according to the invention are the polypeptides from the group of proteins, such as Moclonal or polyclonal antibodies selected.

preferably, is in the inventive method at least one reactant, e.g. a nucleic acid or an antibody, provided with a mark.

Of the Term "marking" means suitable, direct or indirectly detectable atoms or molecules that are in the molecules of the reactants installed or connected to it. Suitable markers are for example, those coupled to nucleotides fluorescent dyes and / or for example biotin and / or digoxigenin and / or with radioactive Isotope labeled nucleotides include. In a preferred embodiment the label compound is a fluorescent dye with a for the selection of small amounts of substance sufficient difference in Fluorescence behavior of the emission spectra, e.g. Coumarins and Rodamine and / or the fluorescence lifetime, e.g. Fluoreszenzisothiocyanate (FITC) and europium chelate-labeled and / or porphirin-labeled avidins.

preferred immobilized biomolecules are those in the so-called. Features described above of bio-chips, such as DNA chips are present. In a further preferred Embodiment lie the biomolecules in the form of whole cells or parts thereof, such as chromosomal DNA, especially metaphase chromosomes, before. In the case of whole cells is the hybridization between the nucleic acids in the cells around a "in situ hybridization. "

• (1) Die Konzentration von mobilen Sonden und immobilisierten Sonden vor der Hybridisierungs-Bindung wird soweit als möglich aufrecht erhalten, d.h. die Verminderung der Konzentration von mobilen Sonden sowie gegebenenfalls von immobilisierten Sonden, zum Beispiel durch Renaturierung von DNA-Molekülen komplementärer Sequenz im Hybridisierungsgemisch, wird verhindert bzw. vermindert, ohne die erwünschten (stringenten) Assoziationsreaktionen zwischen mobilen Sonden und immobilisierten Sonden zu beeinträchtigen.
• (2) Eine erfolgte Zusammenlagerung von (vor der Bindung) mobilen Sonden mit immobilisierten Sonden, zum Beispiel von einzelsträngigen DNA-Molekülen als mobilen Sonden mit DNA-Molekülen komplementärer Sequenz als immobilisierte Sonden zu doppelsträngigen Hybridmolekülen wird aufrecht erhalten.

The optimization of the reaction procedure between the reaction partners in the case of bio-chips concerns the association reactions on the bio-chip itself, in particular of hybridization reactions to DNA chips themselves. It starts from Be conditions, in particular of hybridization conditions in which the desired compound of a mobile probe of certain sequence or composition with an immobilized probe of certain complementary sequence or amino acid sequence essentially takes place only between a single-stranded mobile probe and a single-stranded immobilized probe. Accordingly, the method according to the invention achieves several objectives:

• (1) The concentration of mobile probes and immobilized probes prior to hybridization binding is maintained as much as possible, ie the reduction of the concentration of mobile probes and possibly immobilized probes, for example by renaturation of DNA molecules of complementary sequence in the hybridization mixture, is prevented or reduced without affecting the desired (stringent) association reactions between mobile probes and immobilized probes.
• (2) Accumulation of (before binding) mobile probes with immobilized probes, for example, single-stranded DNA molecules as mobile probes with complementary sequence DNA molecules as immobilized probes to double-stranded hybrid molecules is maintained.

in the Following is the solution the task of the invention in a preferred application, the improvement of hybridization reactions to DNA chips.

The Currently used hybridization methods based on the fact that the hybridization mixture with the denatured DNA molecules serving as mobile probes the DNA chip with the there denatured or already single immobilized DNA molecules is applied as immobilized probes. In a variant of this Procedure, the hybridization mixture already before denaturation brought together with the immobilized probes fixed on the DNA chip. Subsequently becomes the hybridization mixture and DNA chip system subjected to a denaturing thermal treatment, and then cooled to a temperature allowing hybridization. This Procedure fulfilled the above condition (ii). Due to the renaturation / association of DNA molecules complementary or partially complementary Sequence in the hybridization mixture itself, however, decreases the number the for a hybridization available standing mobile probes constantly from. As the example calculation above shows, the ratio of mobile probes in the hybridization mixture to immobilized probes complementary Sequence in the features take high values, i. the probability a renaturation already in the hybridization mixture between mobile Probes with complementary Sequence can be much larger as the probability of hybridization between mobile Probes and immobilized probes. Renatured in the hybridization mixture DNA molecules however, are no longer considered mobile probes for the process of hybridization to immobilized Probes available, with the possible (not relevant here) exception of the formation of triple DNA strands.

When The consequence of these physicochemical reactions is the both tasks mentioned above (1) and (2) in the hybridization method not optimized at the same time according to the state of the art: Thus, maintaining the concentration of mobile probes requires i.e. on (typically labeled) single-stranded DNA molecules in the hybridization mixture or the concentration of immobilized probes, i. (where relevant) the immobilized single-stranded DNA molecules on the features, an increased Temperature or a corresponding chemical composition of Hybridization mixture. The maintenance of the double-stranded hybrid molecules, however requires lower temperatures or a different chemical composition of the hybridization mixture.

• (A) Das Hybridisierungsgemisch und gegebenenfalls die in einem Kompartiment I befindlichen DNA-Chip-Features zunächst einer stark erhöhten Denaturierungstemperatur T_D auszusetzen [gemäß obigem Schritt (a)], so daß eine Denaturierung der im Hybridisierungsgemisch befindlichen DNA-Moleküle und damit die Bildung der mobilen Sonden bewirkt wird; dasselbe Verfahren führt, soweit noch erforderlich, zur Denaturierung der auf den Features befindlichen DNA-Moleküle, d.h. zu der Bildung der immobilisierten Sonden.
• (B) Nach Abschluß der ersten Denaturierungsphase das Hybridisierungsgemisch und die DNA-Chip Features in dem Kompartiment I bei einer niedrigeren Hybridisierungstemperatur T_H gemäß obigem Schritt (b) miteinander reagieren zu lassen, so daß Hybridmoleküle von immobilisierten Sonden mit (vor der Bindung) mobilen Sonden komplementärer Sequenz mit der gewünschten Stringenz gebildet werden.
• (C) Das Hybridisierungsgemisch mit den nicht gebundenen DNA-Molekülen vom Hybridisierungsort gemäß obigem Schritt (c) zu entfernen und in einem räumlich getrennten Kompartiment II wiederum einer denaturierenden thermischen Einwirkung auszusetzen [gemäß obigem Schritt (d)], z.B. bei der oben genannten Temperatur T_D. Statt thermischen Veränderungen oder zusätzlich zu diesen können auch lokale chemische oder physikalische Einwirkungen, z.B. durch lokale Änderungen von pH, Ionen-Milieu, Ladungsverhältnissen etc., erfolgen.
• (D) Das neu denaturierte Hybridisierungsgemisch von Schritt (C) aus dem Kompartiment II wiederum an den Ort der DNA-Chip Features in Kompartiment I gemäß obigem Schritt (e) zu verbringen und dort unter Hybridisierungsbedingungen wiederum reagieren zu lassen, z.B. unter den Bedingungen von Schritt (B) bei der Hybridisierungstemperatur T_H. Zulässig sind alle Bedingungen, die eine Hybridisierungsreaktion mit der gewünschten Stringenz ermöglichen. Die beiden Kompartimente können sich demnach anstatt oder zusätzlich zu thermischen Unterschieden auch in anderen lokalen physikalisch-chemischen Parametern unterscheiden.
• (E) Das Verfahren gemäß obigem Schritt (f) solange zu wiederholen, bis eine optimale oder vorgesehene Abbruchsbedingung erfüllt ist. Eine solche Abbruchbedingung kann z.B. durch eine bestimmte Signalstärke von einzelnen oder mehreren Features, z.B. mit einer oder mehreren Kontrollsequenz(en), realisiert werden, durch eine bestimmte Zeitvorgabe für den Gesamtprozeß, durch bestimmte Mikrosensorwerte, etc.

The solution according to the invention of this problem is to carry out the method according to the invention in the form of the following steps under the microcircuits typical of DNA chip hybridizations (reaction volumes of the order of, for example, 50 to 100 μl):

• (A) The hybridization mixture and optionally the present in a compartment I DNA chip features initially exposed to a greatly increased denaturation temperature T _D [according to step (a)] above, so that a denaturation of the DNA molecules present in the hybridization mixture and thus the formation the mobile probe is effected; The same procedure leads, if still necessary, to the denaturation of the DNA molecules located on the features, ie to the formation of the immobilized probes.
• (B) After completion of the first denaturation phase, allowing the hybridization mixture and the DNA chip features in the compartment I to react with one another at a lower hybridization temperature T _H according to step (b) above, such that hybrid molecules of immobilized probes with (before binding) mobile Probes complementary sequence can be formed with the desired stringency.
• (C) to remove the hybridization mixture with the unbound DNA molecules from the hybridization site according to step (c) above and in a spatially separated compartment II such as in order to suspend a denaturing thermal action [according to step (d) above], eg at the above-mentioned temperature T _D. Instead of thermal changes or in addition to these also local chemical or physical effects, eg by local changes of pH, ion environment, charge ratios, etc., take place.
• (D) to spend the newly denatured hybridization mixture of step (C) from the compartment II again to the location of the DNA chip features in compartment I according to step (e) above and react there again under hybridization conditions, for example under the conditions of Step (B) at the hybridization temperature T _H. All conditions that allow a hybridization reaction with the desired stringency are permissible. Accordingly, the two compartments may differ in other local physico-chemical parameters instead of or in addition to thermal differences.
• (E) Repeating the procedure of the above step (f) until an optimal or intended termination condition is satisfied. Such a termination condition can be realized, for example, by a specific signal strength of individual or multiple features, eg with one or more control sequences, by a specific time specification for the overall process, by specific microsensor values, etc.

To Termination of the periodic procedure and, if necessary, one or several washes to remove unbound mobile Probes and / or to increase the stringency, the resulting hybridization signals are detected, the method combines with any detection methods can be.

As the examples known in the prior art show, it is particularly important for economic reasons (amount of labeled DNA required) and diagnostic reasons (such as the limited availability of DNA / RNA at low cell levels), with small amounts of substance or volumes get along the hybridization mixture. The examples mentioned in the prior art show that in the solution of the problem according to the invention special micro-conditions are to be fulfilled, such as a total reaction volume of only 100 μl. The distance between the compartments I and II D (I-II) is chosen so that sufficient for the purposes of the hybridization process thermal or other physical-chemical isolation of the compartments I and II can be achieved, ie that prevailing in compartment II denaturation T _D does not lead to an undesirable increase in temperature T _H in compartment I. The same applies to the sufficient isolation of the compartments when using other physical-chemical parameters. In order to achieve this and the desired temperatures in compartment I and compartment II, actively cooling (eg Peltier elements) or heating elements (eg ohmic conductors) are placed in the compartments or in a suitable spatial relationship with them. If necessary, the heating can also be effected by a focused laser beam. The temperature and other parameters important for the hybridization reaction, such as ion concentration, pH, electrical charge of the features, can be measured by microsensor systems and used as controls. A preferred device is to mount the microsensor systems either just above or just below the DNA chip level to use the other side for the detection of hybridization sites on the features. Corresponding local definitions are also possible for other physical-chemical parameters, for example by ion-releasing or withdrawing locally bound storage molecules in the compartments.

• 1. Wird die Bewegungsrichtung des Hybridisierungsgemisches von Kompartiment I nach Kompartiment II mit "I nach II" bezeichnet, so erfolgt die Bewegung des Hybridisierungsgemisches von "II nach I" grundsätzlich auf derselben Bahn, die Bewegungsrichtung ist jedoch entgegengesetzt.
• 2. Eine weitere erfindungsgemäße Anordnung sieht vor, daß der Materialtransport zwischen den Kompartimenten I und II auch nur in einer Richtung stattfinden kann. Dies wird z.B. durch eine Anordnung der Kompartimente erreicht, die eine kreisförmige Bewegung des Hybridisierungsgemisches erlaubt. Zum Beispiel besteht die Hybridisierungsvorrichtung aus einem Zylinder mit einem Durchmesser 2R und einer Höhe h; ein Innenbereich mit dem Durchmesser 2r ist für das Hybridisierungsgemisch nicht zugänglich. Das Kompartiment I mit dem DNA-Chip sowie den zugehörigen Kühl-/Heizelementen/Mikrosensoren befindet sich mit dem Schwerpunkt z.B. in Uhrzeigerposition "12". Das Kompartiment II mit den zugehörigen Kühl/Heizelementen befindet sich z.B. in Uhrzeigerposition "6". Die Bewegung des Hybridisierungsgemisches erfolgt z.B. im Uhrzeigersinn, also von Kompartiment I nach Kompartiment II und in gleicher Richtung von Kompartiment II nach Kompartiment I.

A repeated (periodic) execution of the method steps (a) to (f) [or (C) to (E)] using microhydrodynamic transport techniques is basically possible in two ways:

• 1. If the direction of movement of the hybridization mixture from compartment I to compartment II is referred to as "I to II", then the movement of the hybridization mixture from "II to I" always takes place on the same path, but the direction of movement is opposite.
• 2. A further arrangement according to the invention provides that the material transport between the compartments I and II can take place only in one direction. This is achieved, for example, by an arrangement of the compartments which allows a circular movement of the hybridization mixture. For example, the hybridization device consists of a cylinder with a diameter 2R and a height h; an interior area with the diameter 2r is not accessible to the hybridization mixture. The compartment I with the DNA chip and the associated cooling / heating elements / microsensors is located with the center of gravity eg in the clockwise position "12". The compartment II with the associated cooling / heating elements is eg in clockwise position "6". The movement of the hybridization mixture takes place, for example, in a clockwise direction, ie from compartment I to compartment II and in the same direction from compartment II to compartment I.

In general, any arrangement permitting periodic microhydrodynamic movement of the hybridization mixture of compartment I in FIG Compartment II and back again from compartment II to compartment I allows it, as in point 1, both in one direction and in the other direction possible, or unidirectionally directed (movement only in the clockwise direction as in point 2).

At the inventive method it is further as above in a preferred embodiment described, possible, to make a suitable increase in the number of compartments. For example a compartment I.A may be equipped to contain DNA chips contains features whose immobilized probes in their sequence are complementary to Sequences of such mobile probes relevant to the type of analysis not wanted are. For example, contains a compartment I.A one or more DNA chips for repetitive Sequences of a complex sample to be analyzed. compartment I.B contains one or more DNA chips with immobilized probes, their sequences complementary are to sequences of such mobile probes that are desired for the analysis in question, e.g. of unique DNA sequences of interest. By repeated passage of the hybridization mixture through the compartment I.A becomes a depletion of the hybridization mixture mobile probes of "unwanted" sequence (e.g., repetitive Sequences) reached; across from a depletion of the mixture of mobile probes before the beginning of the DNA chip hybridization is the advantage of the invention in the saving of this required separate steps. This is e.g. of importance in many performed diagnostic Applications in clinical laboratory investigations, in clinical or biomedical research, or in pharmaceutical development. This approach, with appropriate modification in accordance with the invention also to a positive selection of certain mobile probes be used the hybridization mixture.

In a further example, further compartments I.A1, I.A2, I.A3, etc. may be introduced, each binding "unwanted" mobile probes, optionally at individually determined temperature conditions T _H1 , T _H2 , T _H3 , etc. (or Other physical-chemical parameters) or bind those molecules whose immobilization is desired for further processing. The number of compartments IB can also be extended in a similar manner. The advantage according to the invention consists, for example, in an improved adaptation of the temperature to the respective hybridization conditions. For example, in a compartment I.B1, features of immobilized probes are housed with all those sequences that optimally hybridize at a temperature T _H1 . In a compartment I.B2, features of immobilized probes are housed with all those sequences optimally hybridizing at a temperature T _H2 , etc. An additional compartment III can serve as a storage compartment for mobile probes. An implementation according to the invention of such a storage compartment can consist, for example, of elements of a greatly enlarged surface, which have a reversible affinity for the mobile probes, such as a positive / negative electrical surface potential, or a temperature-dependent chemical affinity.

The optimum number, size and distribution of the compartments for a given DNA chip hybridization also depends in a particular manner on the available amount of hybridization mixture (reaction mixture). For example, a cylindrical hybridization device having an outer diameter of 2R = 30 mm and an inner (inaccessible to the hybridization mixture) diameter of 2r ₀ = 10 mm has a base area of 630 mm ² free for hybridization reactions. With an assumed size of a single compartment of 100 mm ² , therefore, a maximum of 6 compartments with this base area can be accommodated in a hybridization device dimensioned in this way. With an assumed liquid height h _F = 0.1 mm of the hybridization mixture in the hybridization device, a volume of the hybridization mixture of 63 mm ³ = 63 μl is thus needed in this example. For example, with a concentration of mobile probes of 20 ng / μl, there is a need for mobile probes of 1.3 μg. At higher / lower concentrations, correspondingly higher / lower values result. In the same way, the volumes of hybridization mixtures required for other desired numbers, geometries and sizes of compartments can be estimated. For rapid diagnostic decisions using patient DNA from low starting amounts, for example, an arrangement which minimizes the required amounts of mobile probes is desirable. This is possible, for example, by reducing the number of compartments, feature count, feature sizes, and so on.

For the movement according to the invention of the usually small volumes of hybridization mixture, microhydrodynamic processes are provided. According to the invention, methods are particularly suitable which are based on the combined effect of an electrical and a magnetic field configuration. In general, such magnetomotive methods allow to realize in principle any flow structure desired by the user in the case of incompressible, conductive liquids. The aqueous hybridization mixture is to be regarded as a very good approximation as incompressible liquid. The ionic strength of hybridization mixtures (typical ion concentrations in the range from 150 to 800 mmol / l (150 to 800 mM)) is also sufficient for the movement of the hybridization mixture by means of electromagnetic fields. However, the application of the general principles of liquid transport to the microhydrodynamic movement, for example of a hybridization mixture as in DNA chip hybridization, requires the solution of specific inventive tasks.

• – mindestens zwei Kompartimenten, die zur Aufnahme der wäßrigen Reaktionsgemische ausgelegt sind,
• – einer eine Flüssigkeitsverbindung zwischen den Kompartimenten bereitstellenden Flüssigkeitsverbindungseinrichtung,
• – mindestens einer Immobilisierungseinrichtung, die in mindestens einem der Kompartimente angeordnet ist und zum Immobilisieren mindestens eines Teils mindestens eines der Reaktionspartner ausgelegt ist, und
• – einer Flüssigkeitsbewegungseinrichtung, die zum magnetohydrodynamischen, kreisförmigen Bewegen der wäßrigen Reaktionsgemische zwischen den Kompartimenten über die Flüssigkeitsbewegungseinrichtung ausgelegt ist.

Another object of the present invention therefore relates to a reaction feedthrough apparatus for carrying out reactions between at least two reactants in aqueous reaction mixtures

• At least two compartments designed to receive the aqueous reaction mixtures,
• A liquid connection device providing fluid communication between the compartments,
• At least one immobilization device arranged in at least one of the compartments and designed to immobilize at least one part of at least one of the reactants, and
• - A liquid movement device, which is designed for magnetohydrodynamic, circular moving the aqueous reaction mixtures between the compartments via the liquid movement device.

Of the The term "fluid communication device" according to the present invention Invention any reversibly controllable compound, such as a reversibly closable opening or an adjustable passage or other connection, between the compartments.

Of the The term "immobilizer" includes, for example those that absorb biomolecules, especially bio-chips and / or whole cells or parts thereof are suitable.

Under a "magnetohydrodynamic and circular Movement "is in accordance with the present For example, a movement of the aqueous reaction mixtures to understand that of ionic friction in the reaction mixtures is caused. Such a movement can for example by Influence of a force on the dissolved in the aqueous reaction mixtures present Ions by means of a combination of electric and magnetic fields be achieved. Such a caused movement of reaction mixtures becomes hereinafter referred to as "magnetohydrodynamic material transport".

In a preferred embodiment the reaction feedthrough device according to the invention comprises the fluid movement device at least one first field generating device for generating a electric field and a second field generating device for Generation of a magnetic field, wherein at least one field control device for controlling the first and second field generating devices is provided, and wherein the electrical and the magnetic field enforce the compartments. Preferably, run thereby the field lines of the electric field in each space point of the compartments and the liquid connection device vertically to field lines of the magnetic field.

In a further preferred embodiment of the reaction feedthrough device according to the invention, the first field generating device comprises a circular hollow cylinder with inner diameter 2R and a concentric inner cylindrical cylinder having outer diameter 2r ₀ , the inner cylinder further comprises a first electrode E1 and the outer cylinder a second electrode E2 arranged in such a way in that at least in a region between the hollow cylinder and the inner cylinder, a radially symmetrical electric field can be generated by applying an electrical potential difference between the electrodes by means of the field control device. Furthermore, it is preferred that the compartments are arranged between the hollow cylinder and the inner cylinder.

at a further preferred embodiment the reaction feedthrough device according to the invention is the second field generating device at one end of the Cylinder arranged such that the magnetic field lines, which enforce the compartments, in essentially parallel to the cylinder axis. Such an arrangement corresponds for example to a permanent magnet or electromagnet, in a suitable orientation to and suitable distance from the Compartments, e.g. below a cylinder at the bottom flush final Base plate on which the compartments are arranged attached.

The Description of the invention relevant changes of the general method of magnetohydrodynamic material transport will be discussed below For the sake of clarity, by way of example, as defined above Cylinder geometric arrangement shown using the example of a hybridization device become. Modifications of a cylinder geometry arrangement also considered inventive, provided by these changes not a basically different method is described.

In such a cylinder geometry Arrangement consists of the geometry of the hybridization device of a cylinder of diameter 2R and the height H _Z h _F , where h _{F represents} the height of the hybridization mixture in the filled hybridization device. The hybridization mixture, compartments I, II, etc., the cooling and heating elements, and the elements used for microsensor measurements are suitably located on the bottom surface F _G ("bottom") in an outer region of the cylinder of radius rr ₀ and r R , The axis of rotation of the cylinder is perpendicular to the base. DNA chips, cooling and heating elements, as well as microsensor elements in the compartments are arranged so that the DNA chips can be fully wetted by the hybridization mixture. A serving arrangement for this purpose is, for example, an arrangement of said elements directly on the base F _G , wherein the electrical leads are made sideways or downwards. Above that, the DNA chips are placed at a small distance. The hybridization device is terminated from the surface F _O at a height H _Z ("above"). At or above the surface F _O are the devices required for the detection of the hybridization signal, eg electrical biosensors, or optical elements, such as the front lens of an epifluorescence microscope system, laser scanning microscope system etc. For this purpose, the surface F _{O is provided} with the suitable properties for optical Measurements eg transparent at suitable places.

• (a) Die dem Gemisch mit den mobilen Sonden, z.B. dem Hybridisierungsgemisch, zugewandte Oberfläche (Größe z.B. gleich 2 r₀ h, h h_F) des dem Gemisch nicht zugänglichen Innenraumes von 2 r₀ Durchmesser ist als eine Elektrode E1 ausgebildet, während die dem Gemisch zugewandte Oberfläche des Außenraumes (Größe z.B. gleich 2 R h_F) der Reaktionsvorrichtung, zum Beispiel der Hybridisierungsvorrichtung, von 2R Durchmesser als Elektrode E2 mit entgegengesetzter Polung ausgestaltet ist. Bei einer Potentialdifferenz von beispielsweise 2 V zwischen E1 und E2 und einem Elektrodenabstand von 10 mm wird hierdurch in dem Gemisch ein elektrisches Feld E von 200 V/m an gelegt, dessen Richtung jeweils radial und horizontal zu der Oberfläche des Gemisches verläuft.
• (b) Senkrecht zu dem elektrischen Feld wird ein das Gemisch durchsetzendes Magnetfeld angelegt. Grundsätzlich ist es z.B. denkbar, dies mit Hilfe von Helmholtzspulen zu realisieren, deren Achse mit der Rotationsachse der Außen- und Innenzylinder der Reaktionsvorrichtung zusammenfällt. Derartige Anordnungen haben sich bei der magnetomotorischen Bewegung größerer Flüssigkeitsmengen, z.B. 100 ml und mehr bewährt. Unter den mikrohydrodynamischen Bedingungen der Bio-Chip-Reaktionen, z.B. DNA-Chip Hybridisierung, können jedoch z.B. wegen der hier bedeutsamen Kapillarkräfte für eine erfindungsgemäße mikrohydrodynamische Bewegung des Gemisches, z.B. des Hybridisierungsgemisches, erheblich größere Produkte von Feldstärke E zwischen den Elektroden E1 und E2 und magnetischer Flußdichte B erforderlich sein. Eine wesentliche Erhöhung von E kann jedoch zu einem wesentlichen Anstieg der elektrischen Stromstärke mit einer unerwünschten Ohmschen Wärmeentwicklung führen. Große magnetische Flußdichten mit Hilfe von Helmholtzspulen in nicht-magnetischen Medien zu erreichen, ist zwar möglich, kann jedoch mit erheblichem technischem Aufwand verbunden sein. Daher wird in einer bevorzugten Anordnung eine auch bei geringer elektrischer Potentialdifferenz (z.B. einige Volt) ausreichende magnetische Flußdichte mit Hilfe eines Elektromagneten/Permanentmagneten realisiert, der sich in dem Raum unterhalb der Grundfläche F_G befindet und dessen wirksame Polfläche zum Beispiel parallel zur Ebene der Grundfläche F_G und damit parallel zur Ebene der gewünschten mikrohydrodynamischen Bewegung des Gemisches und in möglichst geringem Abstand von dieser angeordnet ist. In einer erfindungsgemäßen Ausführung wird durch eine geeignete Geometrie der wirksamen Polfläche erreicht, daß die magnetische Flußdichte B das Gemisch senkrecht oder fast senkrecht zur Richtung des elektrischen Feldes E durchsetzt. Bei einer derartigen erfindungsgemäßen Ausführung kann der Abstand der wirksamen Polfläche zum Beispiel einige 100 μm bis 1 mm betragen. Der Elektromagnet besteht zum Beispiel aus einer Spule mit einem fer romagnetischen Kern. Sofern die Verringerung des Abstandes zum Gemisch es erfordert, kann die wirksame Polfläche mit einer geeigneten Geometrie des ferromagnetischen Materials ausgestattet werden, die z.B. Vertiefungen für die Aufnahme von Heiz- und Kühlelementen sowie Mikrosensoren und deren elektrischen Zu- und Ableitungen enthält. Eine hierbei auftretende Inhomogenität der magnetischen Flußdichte und/oder Richtungsänderung ist ebenfalls erfindungsgemäß möglich, sofern sie so konfiguriert wird, daß die gewünschte mikrohydrodynamische Bewegung des Gemisches zwischen den Kompartimenten ermöglicht wird.

The micro-hydrodynamic movement of the mixture according to the invention with the mobile probes, for example a hybridization mixture, includes the following features:

• (a) The surface facing the mixture with the mobile probes, eg the hybridization mixture (size eg equal to 2 r ₀ h, hh _F ) of the interior of 2 r ₀ diameter, which is not accessible to the mixture, is designed as an electrode E1, whereas the one Mixture facing surface of the outer space (size, for example equal to 2 R h _F ) of the reaction device, for example, the hybridization device, of 2R diameter is designed as an electrode E2 with opposite polarity. In the case of a potential difference of, for example, 2 V between E1 and E2 and an electrode spacing of 10 mm, an electric field E of 200 V / m is applied in the mixture, the direction of which in each case runs radially and horizontally to the surface of the mixture.
• (b) A magnetic field passing through the mixture is applied perpendicular to the electric field. In principle, it is conceivable, for example, to realize this with the aid of Helmholtz coils whose axis coincides with the axis of rotation of the outer and inner cylinders of the reaction device. Such arrangements have been proven in the magnetomotive movement of larger amounts of liquid, eg 100 ml and more. However, under the micro-hydrodynamic conditions of the biochip reactions, eg DNA chip hybridization, considerably larger products of field strength E between the electrodes E1 and E2 and, for example, due to the capillary forces important here for a microhydrodynamic movement of the mixture according to the invention, for example the hybridization mixture magnetic flux density B may be required. However, a substantial increase in E can result in a significant increase in electrical current with undesirable ohmic heat generation. Although it is possible to achieve large magnetic flux densities with the aid of Helmholtz coils in non-magnetic media, this can be associated with considerable technical effort. Therefore, in a preferred arrangement, even at low electrical potential difference (eg, a few volts), sufficient magnetic flux density is realized by means of an electromagnet / permanent magnet located in the space below the base F _G and its effective pole area parallel to the plane of the base, for example F _G and thus is arranged parallel to the desired micro-hydrodynamic movement of the mixture and in the smallest possible distance from this. In one embodiment of the invention is achieved by a suitable geometry of the effective pole face that the magnetic flux density B passes through the mixture perpendicular or almost perpendicular to the direction of the electric field E. In such an embodiment according to the invention, the distance of the effective pole face may be for example a few 100 μm to 1 mm. The electromagnet consists for example of a coil with a ferromagnetic core. If the reduction of the distance to the mixture requires it, the effective pole face can be equipped with a suitable geometry of the ferromagnetic material, which contains, for example, recesses for receiving heating and cooling elements as well as microsensors and their electrical inlets and outlets. An inhomogeneity of the magnetic flux density and / or change of direction occurring in this case is likewise possible according to the invention, provided that it is configured in such a way that the desired microhydrodynamic movement of the mixture between the compartments is made possible.

• (c) Die Stärke von elektrischem Feld und Magnetfeld können durch geeignete Schaltungsvorrichtungen nach dem Stand der Technik geregelt werden. Dies ermöglicht es, eine Bewegung des Gemisches, z.B. des Hybridisierungsgemisches, je nach Erfordernis in Gang zu setzen, zu beschleunigen, zu verlangsamen, anzuhalten, sowie in ihrer Richtung zu verändern. Insoweit zuverlässige Messungen in der Reaktionsvorrichtung, z.B. der Hybridisierungsvorrichtung, z.B. mit Hilfe der Mikrosensorelemente, nur bei abgeschalteten elektrischen/magnetischen Feldern möglich sind, können derartige Messungen bei Pausen in der mikrohydrodynamischen Bewegung des Hybridisierungsgemisches durchgeführt werden.
• (d) Durch geeignete Wahl von Feldstärke E und magnetischer Flußdichte B kann das Gemisch, z.B. das Hybridisierungsgemisch, in dem genannten Beispiel z.B. kreisförmig um die innere Elektrode E1 herum bewegt werden. Eine Ver minderung z.B. der Feldstärke E und/oder der magnetischen Kraftflußdichte B vermindert die Bewegungsgeschwindigkeit, eine Erhöhung erhöht sie. Richtungsänderungen der Bewegung des Gemisches können entweder durch eine Umpolung des elektrischen Feldes oder des magnetischen Feldes bewirkt werden. Damit kann das Gemisch je nach Bedarf von einem Kompartiment ins andere bewegt und seine Verweildauer in den Kompartimenten gesteuert werden.
• (e) Aufgrund einer geringen Flüssigkeithöhe des Gemisches (typische Volumina liegen im Bereich um 50 bis 100 μl, gegebenenfalls auch weniger) in der Hybridisierungsvorrichtung besteht weitgehend eine laminare Strömung. Dies vereinfacht die Steuerung der mikrohydrodynamischen Bewegung.

Using materials with a high magnetic permeability can be realized in this way with reasonable technical effort in the hybridization mixture magnetic flux density of 0.1 to 1 Tesla. Since the elements for generating the magnetic field are located below the base area F _G , the Reaction space and the detection of the marker signal, such as the hybridization signal, thereby not affected.

• (c) The strength of electric field and magnetic field can be controlled by suitable switching devices of the prior art. This makes it possible to initiate, accelerate, slow down, stop, as well as to change in direction, a movement of the mixture, for example of the hybridization mixture, as required. Insofar as reliable measurements in the reaction device, for example the hybridization device, for example with the aid of the microsensor elements, are possible only when the electrical / magnetic fields are switched off, such measurements can be carried out during pauses in the microhydrodynamic movement of the hybridization mixture.
• (d) By a suitable choice of field strength E and magnetic flux density B, the mixture, for example the hybridization mixture, in the example mentioned, for example, can be moved in a circle around the inner electrode E1. A reduction of, for example, the field strength E and / or the magnetic flux density B reduces the speed of movement, an increase increases it. Directional changes of the movement of the mixture can be effected either by a reversal of the electric field or the magnetic field. Thus, the mixture can be moved from one compartment to another as required and its residence time in the compartments can be controlled.
• (e) Due to a low liquid level of the mixture (typical volumes are in the range of 50 to 100 .mu.l, possibly less) in the hybridization device is largely a laminar flow. This simplifies the control of microhydrodynamic motion.

The mentioned above Examples serve to illustrate the process. As inventive solutions of Problems of microhydrodynamic motion are all geometries combined electric and magnetic fields, the the microhydrodynamic transport of the mixture between the at least two compartments in a reaction feedthrough device such as e.g. effect a hybridization device.

According to the invention, in order to reduce the transport of the capillary braking resistors, the surface F _{O of} the reaction device located on the detection side parallel to the plane of the biochip, eg of the DNA chip, may be rotated in the same way as the mixture due to magnetomotive effect. If the detection of the marking signal with an optical detection system, for example by detection of the fluorescence emission, the surface F _{O is made} transparent at several suitable locations for the fluorescent light, for example by suitable window, so that an optical detection at different angles of rotation is possible. Since, in the case of optical detection of the marking signal, excitation of the fluorochromes preferably takes place in the epifluorescence mode, a transmission of the excitation light must additionally be taken into account. If another detection of the marker signal, eg the hybridization signal, in addition to or instead of the optical registration provided, the detection side can be configured so that despite rotation at least in certain positions, a detection of the marker signal (in a Meßkompartiment) or the marker signals ( in several Meßkompartimenten) is possible.

Elementary calculations show that at a field strength of a few 100 V / m, a salt concentration of several 100 mmol / l (100 mM) (corresponding to that in a typical hybridization mixture or mixture of proteins), an ion mobility of some 10 ^-8 m ² V ^-1 s ^-1 (corresponding to those of monovalent ions in an aqueous solution), a viscosity of the mixture of about 0.001N sm ^-2 (corresponding to that of an aqueous solution), and a magnetic flux density of 1 Tesla (corresponding to that in the immediate vicinity 500 μm / s or more, which is sufficient to allow a full cycle at a diameter of the reaction device within a few minutes, eg of the hybridization device, of 30 mm, with appropriate and technically possible Ver Smaller dimensioning or, for example, increasing B will result in considerably shorter times. At lower B and / or lower E, the movement is correspondingly slower, so it can be modified depending on the requirements of the association reaction, eg the hybridization reaction.

In an elementary experimental setup (s. 1 ) the technical realization of the above mentioned estimations was confirmed. In a reaction chamber consisting of a nonconductive, optically transparent material, a cylindrical inner electrode E1 (diameter 2r ₀ = 2 mm) and an annular outer electrode E2 (hollow cylinder, free inner diameter 2R = 25 mm) were used. To generate the magnetic field (Kraftflußdichte B) was a permanent magnet of a standard radio speaker, whose front side just below the base F _G and parallel to this was arranged. For the realization of a reaction mixture with a usual in DNA hybridization Ionenkon centering an aqueous solution with 600 mmol / l (600 mM) NaCl, 60 mmol / l (60 mM) sodium citrate, pH 7.0 (adjusted with NaOH) was used.

The Liquid level was 0.8 mm. The micro-hydrodynamic generated by magnetomotive forces Movement of the liquid was detected in two different ways: a) A 2 μl drop of bromophenol blue was in the initially colorless liquid brought in. b) The liquid was stained with bromophenol blue in total and using the movement of an added drop of fat detected.

at Applying an electrical potential difference U (DC voltage) of 1.5 V (standard battery) between the electrodes E1 and E2 became a moving speed the liquid of approx. 300 μm / s observed.

Should the movement of the mixture can not be unanimous, it is possible this by reversing the electrical polarity between the electrodes Reach E1 and E2 or the direction of magnetic flux density, e.g. by Reversal of the current direction in the B-field generating electromagnet.

It is also considered to be in accordance with the invention if, in the case of special requirement profiles of convection, such as repetitive circular motion of the mixture or generally of the mixture containing the mobile probes, the microhydrodynamic motion is entirely or predominantly by means of a mechanical rotational movement of the surface F _O , For this purpose it is according to the invention that the surface F _{O has} a wetting contact with the mixture or another liquid which is located in the hybridization device or the biochip-containing chamber.

Becomes are (in the described reaction device DNA chips) by a preparation with chromosomal immobilized Probes "replaces how e.g. Cell nuclei or metaphase chromosomes, so can the otherwise identical or almost identical hybridization device as well for one Improvement of in situ hybridization reactions on chromosomal immobilized Probes "are used where the "immobilized Probes "still in situ in the cells. As for the DNA chip hybridization described process principle is substantially identical in this application, Such applications are also considered to be according to the invention.

The economic prospects of success of improvement procedures the microhydrodynamic optimization of association reactions Organic chips are given the enormous importance of organic chips extraordinarily Good. As an example, in particular the application to hybridization reactions cited.

The DNA chip hybridization using a microhydrodynamic motion of the hybridization mixture between at least two compartments avoids with different physical-chemical conditions the disadvantages of the prior art through improved use of the in the complex sample contained DNA molecules for hybridization to the immobilized probes in the features of DNA chips. For the economic Usage is expected to improve hybridization conditions everywhere Of importance is where DNA chips in clinical diagnostics, in biomedical Research, as well as used in pharmaceutical developments. About the Already mentioned advantages can be different under otherwise Conditions of use can be reduced to quantity of analysis material.

This applies to an application of the method for improving in situ hybridization in particular complex mobile probes to chromosomal "immobilized probes". Such improvements are everywhere of interest, where in clinical diagnostics, in biomedical / pharmaceutical Research and development the occurrence and location of certain DNA sequences detected in situ in single cells / chromosomes should be.

The Hybridization device according to the invention For example, as an accessory to existing devices provided by the DNA chip analysis.

The The present invention is intended to be illustrated by the following non-limiting Examples are explained:

example 1

The object underlying this example is to improve the hybridization to a DNA chip for the mapping of gene expression. This is to be carried out with the aid of a DNA chip measuring 5 mm × 5 mm, which contains at intervals of 100 μm in a matrix arrangement a total of 2500 features with DNA sequences which correspond to the sequences of cDNA (complementary DNA) to be analyzed. in the reaction are complementary. Such a number of features is sufficient for a large number of clinically relevant diagnostic examinations. In order to optimize the hybridization reaction, repeated denaturation of the reaction mixture with the cDNA molecules as mobile probes should be carried out without detaching the already immobilized probes, ie the cDNA molecules bound by hybridization to the complementary sequences in the features.

The for the Investigation needed Amount of reaction mixture is 150 μl, with a cDNA concentration of 20 ng / μl, an ion concentration of 600 mmol / l (600 mM) NaCl, 60 mmol / l (60 mM) sodium citrate, and a pH of 7.0 (adjusted with NaOH). The mobile probes will be directly with a commercially available DNA fluorescent dye coupled, e.g. with FITC (fluorescein isothiocyanate, optimal excitation with argon ion laser at 488 nm), TexasRed (trade name; optimal excitation with He-Ne laser at 594 nm; CY5.5 (brand name; optimal excitation with diode laser at 675 nm), or IRD 800 (trade name; optimal excitation with diode laser at 775 nm).

To carry out the task, a microhydrodynamic reaction chamber ( 1 ) realized with the following special conditions: A cylindrical inner electrode E1 with an outer diameter of 2 r ₀ = 2 mm is inserted into a reaction chamber made of non-conductive material which is heat-resistant up to about 100 ° C. The outer electrode E2 is formed as a hollow cylinder with the same axis of symmetry as E1 and an inner diameter 2R = 15 mm. In order to avoid corrosion, the electrodes are gold-plated, platinum-plated or provided with another corrosion-reducing protective layer. Just below the reaction chamber, a permanent magnet with a magnetic flux density B is attached, which is for example 0.5 Tesla. The end face of the permanent magnet is aligned parallel to the base surface F _{G of} the reaction chamber, so that the direction of B is perpendicular to the base surface and parallel to the axis of symmetry of the electrodes E1, E2.

In The bottom of the chamber will be miniaturized in the prior art known heating elements (ohmic resistors) and cooling elements (Peltier elements) so embedded and distributed that the reaction mixture be influenced in its temperature can. For example, such elements (according to the clock positions) are in the positions "12, 1,2,3, ... 11". To the reaction control become in the positions "12, 7 "(or even others) in addition Detectors for introduced the temperature measurement. The heating, cooling and detector elements are with the help of pipes through the bottom of the reaction chamber with connected to the control and detector electronics.

Of the DNA chip (here with the dimensions 5 mm × 5 mm) is in the position range "11 - 1" (compartment I) used. Optionally, the heating, cooling and detector elements become instantaneous integrated into the bottom of the DNA chip itself.

The height H _{Z of} the reaction chamber is chosen so that the surface F _O can wet the reaction mixture. For example, for an amount of 150 μ1, H _Z = h _F = h = 0.9mm.

After filling the reaction mixture in the reaction chamber (eg at ambient temperature such as 20 ° C) it is closed. With the help of the wetting surface F _O a uniform distribution of the reaction mixture in the chamber is brought about, for example, supported by mechanical rotational movements.

To initiate the hybridization reaction, the nominal sizes of the heating and cooling elements in the reaction chamber are adjusted so that the mobile probes (eg double-stranded cDNA molecules), possibly also the immobilized probes in the features, denatured, ie made single-stranded. A typical denaturation temperature in the absence of denaturing chemical agents is T _D = 90 to 95 ° C. After, for example, 2 minutes at this temperature, the hybridization temperature T _{H is} cooled. In the absence of denaturing chemical agents, T _{H is} typically 70 to 75 ° C. Additional use of denaturing chemical agents results in lower T _D and T _H values, respectively, which are typically lower by about 20 ° C and 25 ° C, respectively.

After a period of, for example, 30 minutes at the temperature T _H , a potential difference U = 3 V is applied to the electrodes. The magnetomotive force induced thereby in combination with the permanent magnetic field leads to a microhydrodynamic movement of the reaction mixture. After 1 to 2 minutes, the portion (compartment I, position "11-1") of the reaction mixture prior to the start of the movement has been transferred to the denaturation region (compartment II, position "6 - 8"). By switching off the voltage between the electrodes E1 and E2, the micro-hydrodynamic movement is interrupted. In the denaturation region (compartment II), the heating power is adjusted so that the denaturation temperature T _{D is} realized there, in the case mentioned here 90 ° C to 95 ° C. The heating and cooling elements located in the other positions (positions "2, 3, 4, 9, 10, 11") are set so that the best possible maintenance of the temperature T _H on the DNA chip and the temperature T _D in the denaturation area is reached. After eg 30 min In turn, the potential difference of 3 V is applied for 1 to 2 minutes. As a result, the part of the reaction mixture in the denaturation region is transported into the hybridization region above the DNA chip (compartment I). Conversely, the portion of the reaction mixture above the hybridization region is transported further to the denaturation region.

Instead of a discontinuous movement of the reaction mixture can also a continuous movement will be beneficial. For example, on the electrodes E1 and E2 a continuous potential difference of 0.03 V applied. This will be a constant slow circular motion the liquid of a few μm / s through which the mobile probes pass through the hybridization region, respectively (Compartment I) and through the denaturation area (compartment II) are moved through.

at a reaction chamber in the selected small dimensions can already in the absence of electric and magnetic fields Diffusion processes and convection currents for an exchange between Hybridization region (compartment I) and denaturation region (Compartment II) lead. A simple calculation, however, shows that the transport of mobile Probes between the two compartments by magnetomotive personnel performed more effectively and can be regulated. The additional equipment required for this purpose are of the economic one Side relatively low.

The effectiveness of the hybridization process is e.g. by optical stimulation and Detection controlled at the site of hybridization. To this end is over the hybridization region (compartment I) an optical window provided, e.g. from a cover glass of 170 microns, above which an optionally with immersion oil connected to the window microscope objective of the prior art known epifluorescence microscope or a confocal Auflicht laser scanning microscope is located. An abort condition for the process is e.g. then given if in the individual features detected fluorescence intensity the immobilized probes does not change in time. Then you can in the same optical way the result (fluorescence distribution of immobilized probes in the DNA chip) are analyzed, if necessary after removal of the hybridization mixture and appropriate washes.

Example 2:

The underlying problem of Example 2 is to hybridize as mobile probes fluorescently labeled genomic DNA (eg from a patient) to a DNA chip. Here we search for the presence or absence of certain DNA sequences in the sample to be analyzed. Optionally, the method of comparative genome hybridization (CGH) known to those skilled in the art is used. For the object to be solved according to the invention, it is important to introduce, in addition to a change between a hybridization region (compartment I) and a denaturation region (compartment II), a third compartment in which a depletion of the hybridization mixture of "unwanted" sequences interfering with the analysis (eg repetitive sequences). For this purpose, a DNA chip with features whose sequences bind at a temperature T _DEPL these "unwanted" sequences and thus reduce their unwanted immobilization in the analysis area in compartment I in the compartment III.

In the example 2 treated here, the DNA chip with the sequences to be tested in compartment I (hybridization region at temperature T _H ) again consists of 2500 features, while the "depletion chip" in compartment III likewise has, for example, 2500 features suitable for depletion Contains sequences. The geometric dimensions of the chips are each 5 mm × 5 mm. The chamber construction is the same as in Example 1. In addition, however, the depletion chip is placed in the position "3-5" or in another position between compartment I and compartment II.

The temperature in compartment III is adjusted by means of the heating and cooling elements so that there prevails the temperature T _DEPL , while in compartment I the temperature T _H and in compartment II the temperature T _D prevails. The microhydrodynamic movement of the liquid and the control and analysis are otherwise carried out as in Example 1.

Claims (25)

Process for carrying out reactions between at least two reactants in aqueous reaction mixtures, in which at least one reaction partner spatially separated Passing through compartments with different reaction conditions and immobilized at least one reactant on a surface of a compartment is, wherein the reaction mixture moves magnetohydrodynamically and circularly become. The process of claim 1 comprising the steps of (a) maintaining at least one reactant in a reaction mixture under first reaction conditions in a compartment I, (b) maintaining the mobile and immobilized reactants in a reaction mixture under second Reaction conditions in compartment I, wherein a binding reaction between the reactants takes place, (c) transferring the reaction mixture with the unbound molecules of the mobile reactant in a compartment II, (d) maintaining the mobile reactant of step (c) under third reaction conditions, the same in compartment II, (e) transferring the mobile reactant from step (d) to compartment I and (f) repeating steps (b) to (e) until a predetermined termination condition is met. The method of claim 2, wherein between the steps (c) and (d) additionally Steps of detection and evaluation of the reaction are carried out. Method according to claim 2 or 3, wherein between in addition to steps (b) and (c) one or more washes are performed. The method of claim 2 to 4, wherein further compartments with the same or different reaction conditions than in the compartments I and II from the mobile and / or immobilized reactants to go through. Method according to one of claims 2 to 5, wherein the compartments are arranged so that the transfer of the Reaction mixture in steps (c) and (e) runs on the same trajectory, however the direction of movement is opposite. Method according to one of claims 2 to 6, wherein the compartments are arranged so that the transfer of the Reaction mixture in steps (c) and (e) on a circular trajectory in each case in the same direction of movement runs. Method according to one of claims 1 to 7, wherein the reaction volume not larger than in the compartments 150 μl. The method of claim 8, wherein the reaction volume not less than 20 μl and not bigger than 100 μl. Process according to any one of claims 1 to 9, wherein the reaction is a binding reaction. Method according to one of claims 1 to 10, wherein the reactants biomolecules are. The method of claim 11, wherein the immobilized biomolecules in the form of features of bio-chips or in the form of cells or Parts of it are available. A method according to claim 11 or 12, wherein the. Biomolecules from the Group of nucleic acids and / or Polypeptides selected are. The method of claim 13, wherein the nucleic acids are deoxyribonucleotides and / or ribonucleotides and / or modified nucleotides. The method of claim 14, wherein the binding reaction a hybridization, in particular an in situ hybridization. The method of claim 13, wherein the polypeptides are selected from the group of proteins. The method of claim 16, wherein the proteins are monoclonal or polyclonal antibodies are. Method according to one of claims 1 to 17, wherein at least a reaction partner is marked. The method of claim 18, wherein the label one or more fluorescent dyes. Reaction through device to carry out of reactions between at least two reactants in aqueous reaction mixtures With - at least two compartments designed to receive the aqueous reaction mixtures are, - one a fluid connection between the compartments providing fluid connection means, - at least an immobilization device located in at least one of the compartments arranged and for immobilizing at least one part at least one of the reactants is designed, and - one Fluid moving device, for magnetohydrodynamic, circular moving the aqueous reaction mixtures between the compartments over the fluid movement device is designed. A reaction feed-through device according to claim 20, wherein said liquid moving means comprises at least first electric field generation field generating means and at least one field controller for controlling said first and second electric field generating means, and said field generating means is arranged that the electri and the magnetic field enforce the compartments. Reaction through device according to claim 21, wherein field lines of the electric field in each Spatial point of the compartments and the liquid connection device perpendicular to field lines of the magnetic field. A reaction feedthrough device according to claim 21 or 22, wherein said first field generating means comprises a circular hollow cylinder having an inner diameter 2R and a circular inner cylinder having an outer diameter 2r ₀ concentrically disposed therein, said inner cylinder comprises a first electrode E1 and said outer cylinder comprises a second electrode E2 arranged so that at least a radially symmetrical electric field can be generated by applying an electrical potential difference between the electrodes by means of the field control device in a region between the hollow cylinder and the inner cylinder. Reaction through device according to one of the claims 20 to 23, wherein the compartments between the hollow cylinder and the inner cylinder are arranged. Reaction through device according to one of the claims 20 to 24, wherein the second field generating means on an end face the cylinder is arranged such that the magnetic field lines, which enforce the compartments, substantially parallel to the cylinder axis run.