WO1994010564A1

WO1994010564A1 - Process for separating substances from dilute solutions and suspensions

Info

Publication number: WO1994010564A1
Application number: PCT/EP1993/003077
Authority: WO
Inventors: Manfred Eigen; Karsten Henco; John Mc Caskill
Original assignee: Evotec Biosystems Gmbh
Priority date: 1992-11-05
Filing date: 1993-11-03
Publication date: 1994-05-11

Abstract

The present invention describes a device and a process for carrying out a multitude of parallel enzymatic of chemical reactions expected to yield different products whose functionality must be determined by simultaneous or successive analysis. The process provides a method of non-cellular molecular cloning, i.e. the separate amplification of defined nucleic acid sequences. It provides for carrying out molecular evolutive optimization experiments with up to 100 million molecular structures that can be examined separately, individually or in dilute mixtures as to their desired function. The process can also be carried out with cells and combined with a number of other analytical and reaction processes.

Description

Process for the separation of substances from dilute solutions or suspensions

The present invention relates to a method for separating substances from a volume or volume element of a dilute solution or suspension by restricting the space available for diffusion of the substances, a device for carrying out the method and the use of a thermostattable capillary.

Evolutive biotechnology requires a very large number of samples to be checked, including even individual molecules and their amplified, identical or mutated structural analogues for functional properties. In experiments of this type, a large number of mutants are generated at the genetic level (genotypes), the functions or functional gene products (phenotypes) of which have to be analyzed, using methods of evolutionary biotechnology (P 41 12 440 "Process for the production of new biopolymers) disposable reaction vessels such as the well-known Eppendorf tubes are unsuitable for processing more than 100 samples simultaneously in an experimental application, using the micro titration systems such as those from Flow are on the market, up to 1000 samples per day can be handled with moderate effort.

P 40 22 792 describes a plate with at least one trough for receiving chemical and / or biochemical

and / or microbiological substances. Although this plate, which is in the form of a film with depressions embossed therein, already enables a large number of samples to be processed, this technology can only be used routinely to a limited extent.

What is required is a system which allows a large number of individual molecules or groups of individual molecules to be distributed inexpensively in such a way that access to the genotype is possible after analysis of these molecules. Ultimately, a volume of a liquid with a larger number of different molecules that do not allow selection of the best molecule should be divided into small volume elements so that an analysis is possible. The quantity of molecules analyzed per volume element must be so small that a certain molecule can also be recognized against the background of several molecules that are not shortlisted. It is often not possible to evaluate the functionality of a single molecule. The corresponding signals must therefore be amplified. This can be done on the one hand using measurement technology, or it can be achieved by cloning the molecules. The molecular amplification can e.g. B. can be realized by repeated reading processes from a single template, as happens when an mRNA is translated into a protein. RNA or DΝA molecules can be directly amplified or replicated. This is achieved using so-called enzymatic amplification systems such as the polymerase chain reaction (PCR) or other amplification strategies such as the ligase chain reaction (LCR) or 3SR. Cellular amplification processes must also be taken into account in the task. To carry out the optimization of biopolymers using evolutionary biotechnology, it is necessary to individualize suitable individual molecules, molecular complexes, cells or a closely related spectrum of mutants and, if necessary, to isolate them subsequently.

Proven methods for separating molecules from homogeneous solutions are pipetting methods in which molecular separation can ultimately be achieved by physically separating smaller volume elements. This is achieved, for example, by serial dilutions. A disadvantage of the pipetting method, however, is its limitation to the manageability of relatively large volumes, which cannot always be present in evolutive biotechnology. On the other hand, changing the collecting vessels is also not desired.

In the application "carrier material for the simultaneous binding of genotypic and phenotypic substance" by the same applicant as the present application, a material is proposed which can bind genotypic and phenotypic substances simultaneously.

Tubes and capillary systems as reaction carriers have long been in use as reaction carriers in routine analysis (Technicon company). Different blood samples are taken sequentially in one and the same tube.

An air bubble is drawn in between the individual sample holders, the surface tension of which in relation to the aqueous phase ensures that there is no disruptive mixing of adjacent samples.

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The present invention is based on the technical problem of specifying a method and creating a device for carrying out a large number of chemical and enzymatic reactions in parallel, in which Different products are expected, the functionality of which must be determined in simultaneous or subsequent analysis. The method is intended to ensure cell-free molecular cloning, ie the separate amplification of defined nucleic acid sequences. In particular, up to 100 million molecular structures should be able to be checked for their functions separately or in limited mixtures per experimental use. The method should also be able to examine cells, how individual molecules can be coupled and with a variety of other reaction methods and analysis methods. At the same time, it should spatially distribute individual molecules or groups of individual molecules such that access to the genotype is possible after analysis of these molecules. The method, which is preferably inexpensive, is intended to ensure that a volume of liquid with a larger number of different molecules, which does not allow selection of the best molecule, is divided into small volume elements, so that an analysis is possible.

According to the invention, the problem is solved by a method with the features of claim 1. Claims 2 to 12 relate to preferred variants of the method according to the invention.

The device according to the invention for carrying out the method according to the invention has the features of claim 13. The subclaims 14 to 30 relate to preferred embodiments of the device according to the invention.

Claim 31 relates to the use of a known thermostattable capillary in the method according to the invention.

The method for separating substances from a volume or volume element of a dilute solution or suspension by restricting the amount available for diffusion of the substances. Bare space is characterized in that at least one degree of freedom of the spatial diffusion of the substances is restricted without substantially changing the connected volume or volume element of this solution or suspension. The principle of separation is based on restricting the three degrees of freedom of spatial diffusion in free solution so that the molecule can either only diffuse quasi-two-dimensionally or, if two degrees of freedom are restricted, can ultimately diffuse one-dimensionally. It is essential that the total volume or the content of the corresponding volume element of the solution or suspension remains essentially unchanged. This means that there is no significant loss of solvent in the solution or suspension, for example by evaporation or other means of removing liquids. An essential prerequisite for using the method according to the invention is that the starting solutions already contain the molecules to be separated in a dilute solution.

Preferably, when only one degree of freedom of the spatial diffusion of the substances is reduced, this is restricted to a thickness <1 mm. This can be done, for example, by pouring the solution onto a flat support which is dimensioned such that the total volume or the volume of the volume element forms a layer thickness of <1 mm. The flat support can be a membrane, filter paper, etc. A further preferred embodiment of the method according to the invention consists in that two degrees of freedom for the spatial diffusion of the substances in the solution or suspension are restricted. This is done in particular by introducing the volume or volume element in which the substances to be separated are located into at least one capillary. The inner diameter of the capillary is preferably 0.1 μm to 1 mm. The substances separated by the process according to the invention can then be analyzed or subjected to a chemical reaction. The method according to the invention is particularly suitable for the evolutionary optimization of biopolymers, in particular of nucleic acids, polypeptides and proteins, using evolutionary biotechnology.

The molecular cloning of nucleic acid fragments in the method according to the invention is preferably carried out using cellular or non-cell-bound, enzymatic replication systems such as PCR, LCR, 3SR and similar systems in which polymerases are used.

The separation method according to the invention prepares the samples in particular for analysis, preferably for the quantifying analysis of different samples taken separately from the capillary on specific nucleic acid sequences, using an enzymatic amplification method. The enzymatic amplification processes are preferably carried out using heating and cooling steps (thermal cycles) which have to be repeated several times.

Nucleic acids from cells can preferably be detected quantitatively and qualitatively, in particular nucleic acids of low multiplicity, in particular certain RNA and / or DNA, mRNA, regulatory RNA, antisense RNA, ribozymes, chromosomes, chromosome fragments, plasmids, mitochondria, chloroplasts and satellites. The nucleic acid sequences which are subjected to the qualitative or quantitative analysis can originate from pathogenically associated nucleic acid sequences, in particular from viruses, bacteria, fungi, from human, animal or vegetable tissue, body fluids and excretions. The method according to the invention allows the group-specific analysis of nucleic acid fragments by focusing the same nucleic acid fragments from neighboring volume elements.

The capillaries which can be used in the method according to the invention and which serve as a device to restrict two degrees of freedom of spatial diffusion of the substances in a volume or volume element do not necessarily have to be arranged regularly in any way. Nonwovens made of pressed fibers or materials such as cellulose fibers, porous glass or porous silica gels are likewise suitable for carrying out the process according to the invention. Such carriers can be loaded, for example, with a solution or cell suspension suitable for enzymatic or cellular amplification, the capillary pores of these materials filling. Then the flat support with the capillaries, e.g. in the device according to the invention, subject to the corresponding reaction and analysis conditions. As in a linear capillary, here too the reaction spaces limit the disruptive diffusion processes and virtually hold the molecule in place. The surface supports can then be brought into contact with a cell suspension or a cell lawn.

If this cell lawn contains competent cells, transformations can take place directly, otherwise electroporation or preferably a laser-induced transformation are possible (G. Weber et al. EJ Selbiol 49, 73 to 79 (1989)). The flat capillary device for carrying out the method according to the invention is also suitable for carrying out evolutionary optimizations at the DNA, RNA or protein or peptide level (P 4112 440). After generating hierarchical mutant spectra and distributing them to the capillary reaction spaces and compartments, the respective mutants can be amplified with increased accuracy in order to arrive at narrower quasi-species distributions, which are described below. be evaluated and isolated. When using the method according to the invention, the separation of individual, observable volume elements is carried out.

The preferred use of capillaries and / or hollow fibers, through which the solution or suspension, which is to be divided into a plurality of individually observable volumes, is passed, enables the implementation of evolutionary optimization methods.

It is not absolutely necessary to divide the corresponding volume elements into different templates, as in the case of pipetting, in such a way that the volume compartments are not in direct contact with one another. A partial mixing of molecules of neighboring elements is not disturbing, as long as the searched molecular species remains detectable and the corresponding phenotype is in the same volume element.

FIG. 1 shows schematically the situation after the separation of individual molecules in a capillary. After the separation, amplification reactions are carried out, with a concentration gradient forming over time as a result of diffusion and turbulence around each starting molecule or cell, which increases by division, the respective profiles being able and permitting to mutually penetrate one another. The efficiency of the method according to the invention can be illustrated using a numerical example.

Given is 1 ml of a solution in which there are 10 molecules, among which optimized individual species sought after clonal amplification are to be identified. If this solution is taken up in a capillary with an inner diameter of 400 μm, the filling length of the capillary is approx. 8 m. If there were 10 molecules in a millimeter of homogeneous solution, then this is average distance 8 μm or in a segment of 1 mm length there are 125 molecules. Since large biopolymers, such as nucleic acids, diffuse very slowly, 125 molecules can be analyzed in a 1 mm segment, corresponding to a volume of 0.125 microliters. If the molecule sought can be found on a background of 125 accompanying contaminating molecules and 0.125 microliter volume unit is available for analysis, a filled capillary allows an experiment in which 8000 pipetting steps would otherwise have had to be carried out. With a corresponding dimensioning, a recognition of one molecule in 1000 accompanying molecules can thus be allowed with a corresponding extension of the capillary volume. Will one

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Using a capillary length of approx. 100 m, about 10 molecules could be screened. Capillaries and hollow fibers are meanwhile commercially available in various embodiments, for example from the company Amicon. Glass capillaries can be manufactured with an inner diameter of 1 μm.

Capillaries as a device for separating substances from a volume or volume element, in which reactions can be carried out after the separation, offer a number of further advantages which are both practical and of a fundamental nature.

The setting of defined temperatures of the capillary contents or the rapid change in temperature within the volume of the capillary can be designed in a particularly advantageous manner by arranging the capillary preferably in a space-saving arrangement, for example by flat, parallel guidance, on a temperature-controlled surface. It is particularly simple and efficient to wrap the capillary around a thermostattable support with a column, cone or other geometry. The capillary can be efficiently thermostatted by good heat transfer to the tempered surface and still remains from the opposite Side observable, provided transparent capillary wall materials are used for the corresponding measurement signal. In addition to the possibility of observation, mechanical or other interventions are then possible from the free side.

The selection and identification of individual molecules due to improved properties is very difficult. There are e.g. Techniques to amplify the signals through coupled, enzymatic reactions or, in the case of nucleic acids, to amplify the signals themselves. The polymerase chain reaction, which can be combined in an outstanding manner with the process according to the invention, allows a large number of volume elements located in the capillary to be subjected to an amplification reaction with very little technical effort. Such a section is understood as a volume element of the capillary, which section is also characterized by a suitable one

Process can be resolved by measurement. This volume element is then comparable to a volume that would otherwise only be accessible by micropipetting. The above numerical example assumed a volume of 0.125 microliters, provided that an observation distance of 1 mm was used. In this example, 8000 volume elements were strung together in the capillary.

The cyclic thermostats necessary in the polymerase chain reaction are preferably carried out by the fact that the contents of the capillaries come into contact with different temperatures at time-defined intervals. The capillary content, mediated by a flow through the capillary, can be guided past a static thermostat unit or, in the case of a stationary capillary content, the thermostattable device can be guided past the capillary. If the capillary content is transported relative to the capillary surface, the capillary flow itself can be used to set defined, cyclical temperature levels by fixing the capillaries to surfaces of different temperatures. The dwell time of a certain section of the capillary and thus of the volume therein at a certain temperature is determined via the length of the capillary segment which is in contact with a certain tempered surface. This situation is indicated schematically in Figure 2.

The constructive-mechanical realization of a device for carrying out the method according to the invention is particularly simple by using a cylindrical or conical winding of the capillary, or a winding with a different topology. The dwell times in the specific temperature levels can be defined via the respective geometry of the surface of the geometric body.

Figure 2 shows in a linear representation the basic structure of the heat transfer for capillaries. The respective lengths of the contact points determine the time intervals in which the individual volume element is at the respective temperature. The transition points d are of particular importance. _, d ,, - and d., /. to. A temperature gradient, comparable to a ramp, between the different temperature levels is established via these connecting elements. A preferably linear temperature gradient is built up at a relatively short distance. The thickness of the connecting elements between the different temperature levels also determines here whether a more or less abrupt change in temperature should occur in a very short time or, if it is procedurally necessary, whether the capillary content gradually increases to the other temperature of the following temperature level should be raised or lowered. From FIG. 2, the following total times result for reactions which are preferably carried out cyclically, such as the polymerase chain reaction. For the example of a PCR reaction, the incubation times are:

Dwell time at T. Dwell time at T, dwell time at T.

The "ramp" times are:

Dwell time at ramp T./T ,: t ( _Tl / T ₂ ) = d _{1; 2} / (v _{c +} v _τ ) Dwell time at ramp T ^ / ^' T ,: t (τ ₂ / τ ₃ ) = d _{2 ; 3} / (v _{c +} v _τ ) dwell time at ramp T./T .: t (T ₃ / T ₁ ) = d _{3; 1} / (v _{c +} v _τ )

Cycle time:

t (z) = t (τ _χ ) + t (τ ₂ ) + t (τ ₃ ) + t (τ ₁ / τ ₂ ) + t (τ ₂ / τ ₃ ) + n ^ / τ ^)

Amplification reaction time:

t (total) - n x t (z)

The symbols mean:

Primer hybridization temperature;

Polymerase reaction temperature;

Denaturation temperature;

Length of the respective thermostat segments;

Capillary flow velocity in mm / min;

Speed of the thermostat unit relative to

Capillary surface in mm / min;

V ^T 2 τ ₂ / τ ₃ ₃ / T. Ramping the temperature transitions; t (z) cycle time n number of parallel windings. The device according to the invention for carrying out the method according to the invention therefore preferably has a capillary which is in thermal contact with a thermostattable device, and the capillary is arranged at least partially in a space-saving manner, and this space-saving arrangement likewise the thermostatic device at least partially surrounds and thermal contact between the thermostattable device and the space-saving arrangement is ensured. The space-saving arrangement can be present, for example, in a three-dimensional arrangement in the form of a winding, whereas meandering or spiral arrangements are preferably possible as a flat space-saving arrangement.

The capillary of the device according to the invention preferably has an inner diameter of 0.1 μm - 1 mm. The capillary preferably consists of non-porous materials, such as plastic or mineral material, such as glass, if no exchange with the surrounding medium is to take place. However, if the fluid in the interior of the capillary, with which the medium surrounding the capillary is to have a limited mass exchange, the walls of the capillary consist entirely or partially of material which is permeable to ions. In these cases, hollow fibers, as are commercially available and used in the medical field, are particularly suitable.

The thermostattable device is preferably flat or has the shape of a roller, a cone or related topologies. In a preferred embodiment, the thermostattable device has a heatable, uniform, roller-shaped block. This can be set to certain temperature levels via heating elements and ensures the setting of a certain temperature in the capillary via a thermal contact. Another preferred embodiment of the thermostattable device consists of a preferably cylindrical or cylindrical block, which is made up of at least two thermally insulated segments. The block consists of metals that are thermally conductive and, in the embodiment with different segments, can also have a higher heat capacity, whereas in the embodiment with a uniform temperature level it can be advantageous to use materials with a lower heat capacity in order to achieve a fast To achieve equalization of the temperature level.

One embodiment of the device according to the invention regulates the period of time for which certain volume elements of the solution or suspension enclosed in the capillary are exposed to a certain temperature by the capillary flow. This flow is preferably set and controlled by a conveyor. Pumps which are capable of delivering extremely small volumes come into consideration as the conveying device. For example, this is achieved by dosing systems based on the piston pump principle. However, systems which allow the conveyance of liquid quantities by means of piezoelectric elements are particularly preferred. Similar piezo pumps are already operating extremely successfully with inkjet printers. The functioning of these piezoelectric elements is based on the principle of the deformation of capillary systems to which a piezoelectric element is attached. If a voltage is applied to the electrodes of the piezo ceramic, the element deforms, as a result of which a very rapid rise in pressure is generated in the liquid, which is relatively incompressible. This increase in pressure propagates at the speed of sound through the liquid to a nozzle, where the pressure wave is transformed into a movement of the liquid. A fine column of liquid leaves the nozzle at a very high speed. Capillary forces are used to replace the liquid dispensed from the capillary's storage container, causing the liquid in the capillary to slowly move in the direction of the piezo pump. A further embodiment of the device according to the invention has a thermostatic device which consists of an outer jacket which is in thermal contact with an inner roller which is rotatable about the longitudinal axis. This internal roller is made up of at least two segments which can be thermally decoupled from one another. The capillary is fixed on the outer surface of the thermostattable device. By rotating the inner roller or the inner cylinder, the segments are now guided past the inside of the outer jacket and lead to corresponding heating of the outer jacket, which then transfers its temperature to the capillary fixed to its outer surface and thus heats the capillary contents. The outer jacket of this device can be dispensed with if the friction caused by the rotation of the cylindrical or cylindrical thermostatic device on the capillary wall is reduced by an appropriate lubricant, which can simultaneously serve as a heat transfer medium. Then the thermostattable, movable device is in direct thermal contact with the capillary via a heat transfer medium.

The method according to the invention using the device according to the invention elegantly enables the elimination of troublesome evaporation and condensation problems that otherwise occur when handling open solutions, for example when pipetting. If, for example, the individual volume elements in the capillary are preferably separated from one another by air bubbles, the possible evaporation only occurs between two adjacent volume elements.

The molecules separated and deposited in the individual volume elements can be analyzed, in particular with the aid of temperature gradient gel electrophoresis, as proposed in P 42 34 086. This analytics allows an exact control by internal standardization, whether the corresponding searched molecules are present and their absence suggests contamination or non-functioning enzymatic reactions. An internal standard is preferably added to the reaction mixture, which differs only slightly from the sequence to be detected.

Figure 3 shows an embodiment of the device according to the invention schematically in supervision. The capillary 1 is partially arranged in the form of a winding consisting of n loops and encloses the thermostattable device (3), which has three temperature levels in corresponding segments T1, T2 and T3 (7). The flow through the capillary takes place in the direction of the arrow and is brought about and controlled by the conveying device 4. The thermostattable device 3 can in particular be designed to be rotatable. The sample is drawn into the capillary from the sample holder 20 by means of the suction caused by the conveying device 4. The sample liquid contains the molecules to be separated and analyzed or processed in dilute concentrations as well as all components required for analysis or chemical reaction. The liquid pulls into the capillary in the direction of the arrow through the winding with n loops and cyclically passes through the different temperature segments T1 to T3. In this way, in the event of a PCR reaction to be carried out, the cyclical procedure which is inherent in this methodology is carried out. The winding 2 can be matched to the thermostattable device 4 in such a way that one cycle of the PCR reaction is run through per winding of the winding 2. If twenty cycles are to be run through, the winding 3 has twenty turns.

After passing the area of the space-saving arrangement of the capillary, the liquid flow is conveyed further through the conveying device in the direction of the sample outlet 30 and caught there. Appropriate templates on the sample receiving side 20 can be used, for example, to introduce air bubbles to separate the volume elements or further templates and washing solutions into the capillary using appropriate mixing systems (two-way or reusable valves).

FIG. 4 shows a side view of the space-saving arrangement 2 as it is wound around the thermostattable device 3.

If the optimized nucleic acids are to be expressed in the corresponding protein products by means of evolutionary biotechnology, a large number of reproducible transformations of bacterial or eukaryotic cells are required. Electroporation has proven to be an extremely efficient process for transformation. Competent cells are mixed with DNA. This mixture must be dialyzed against water and then subjected to electroporation. In the device according to the invention this can be achieved in that the DNA / cell mixture is passed from the capillary into a hollow fiber or a hollow fiber bundle or is immediately placed in a hollow fiber. The exchange liquid, such as water, for example, flows around this hollow fiber over a certain length. The length of the hollow fiber or the hollow fiber bundle is preferably chosen so that the desired exchange effect / dilution factor with respect to the previous buffer system is achieved. In a subsequent section, which can also be in the form of a further hollow fiber bundle, there are electrodes for electroporation of the cells. When leaving the electroporation device, the cells are either in a transient, transformed or already in a stably transformed form.

If cell growth is required after the transformation has taken place, in a further area in which, for example, hollow fibers are arranged, the medium can be Suitable medium for growth. The cells are then incubated at a suitable growth temperature. This is preferably done in a reactor, for example in that the capillaries or hollow fibers are arranged in a space-saving manner, eg. B. by a cylinder winding, this arrangement having the required to grow the cells, preferably optimal temperature. It is sufficient that the corresponding thermostattable device only has a uniform temperature level. The number of turns and flow adjustment controls the time the cells are exposed to the optimal growth conditions. If it must subsequently be induced chemically, for example by IPTG, the introduction of this substance can also be done by a further dialysis with the hollow fiber system or hollow fiber bundle system. An expression unit can then be connected to this unit, which is constructed similarly to the growth unit. If the expression system does not secrete the corresponding phenotypic substances (such as proteins, etc.), a device must be provided in which these cells are lysed so that the cell constituents can be released. This is preferably done by introducing lytic substances and removing the substances exchanged by dialysis. In the case of secreting systems such as B. subtilis or some E. coli systems, this lysis device can already be used to examine the functionality sought.

Methods that are based on luminescence phenomena are particularly suitable as the detection system for the desired products. If a positive measurement signal is recorded, the corresponding volume element can be separated. For example, small amounts of the corresponding DNA and RNA of interest or coding or the functional DNA or RNA can be discharged by arranging hollow fibers or a hollow fiber itself in a cruciform manner with the hollow fiber transporting the substances. sersystem is arranged. Some of the corresponding substances are exchanged, the usual capillary content is deposited in individual samples, which in turn is preferably carried out using a piezo pipetting system, and the solution or suspension is deposited on a suitable carrier in the form of small, individual drops becomes. This reservoir can serve, for example, as a reserve sample or for documentation purposes. The discharged DNA or RNA can then be amplified in the process according to DE 4112 440 C2.

The device according to the invention in the form of the capillary system enables simple and direct access to individual volume elements. This means that they can be measured non-destructively using optical systems, provided the capillary walls are transparent to the measurement signal used for analysis. In particular, the proven luminescence detection methods can be used to analyze the molecules separated by the method according to the invention (McCaskill, 1989).

Once a particular volume element has been located, that element can be mechanically accessed directly. It can be cut out or melted. Larger sub-segments of the capillary can e.g. mechanically broken down into segments of smaller defined length. In principle, this process then corresponds to pipetting as it can also be achieved by piezo pipetting for smaller portions.

FIG. 5 schematically describes a preferred embodiment of the device according to the invention. Cells can be fed into the capillary 1 on a sample receiving side 20. In a mixing system 11, a certain amount of DNA is fed in for transformation and, together with the cells, is brought into a device 15, in which, if necessary, the liquid is used for electroporation suitable liquid such as water is exchanged. This dialysis device 15 can consist, for example, of a storage vessel with inlet and outlet, which is constantly filled with the liquid to be introduced. The exchanged, diluted solution is then removed through the drain. The capillary 1 is at least partially permeable to the corresponding liquids in the area of the dialysis device. The transforming DNA is then brought into the cells in the electroporation device 16. An exchange of the medium with a medium suitable for the growth of the transformed cells then takes place in the medium dialysis device 17. After leaving the medium dialysis device 17, the cells are cultivated in an incubation device 18 which has the configuration described above. This growth device consists, for example, of a capillary arranged in a space-saving manner. Thereafter, the multiplied cells leave the growth device 18 and optionally pass a device 19 with which the chemicals required for any induction are preferably introduced into the capillary by dialysis. The capillary contents then pass through an induction device 25, which can have a configuration similar to that of the growth device 18. After the induction device 25, a lysis device 26 can optionally be arranged, in which chemicals for lysing the transformed and induced cells are introduced, for example by dialysis become. As already described above, this is necessary if the substances are not secreted by the cells into the culture medium. Otherwise, a chamber can already be arranged at this point, in which the functional tests for identifying the desired proteins or nucleic acids are carried out. The capillary flow is then preferably passed through a device 28 which selects the volume elements of interest on the basis of optical signals. The selected volume elements are then led through the capillary 29 to the thermostattable device 3, where further mutagenization and amplification steps are optionally carried out for further optimization of the nucleic acids. The capillary flow is then conveyed to the sample outlet side and passes through a mixing unit 31, in which at least a portion of the sample exposed to further evolutionary optimization can be returned to the process already described via the capillary 32. The respective non-branched samples can be collected at the sample outlet 30 and fractionated.

The system according to the invention consisting of device and method also offers the possibility of surface-mediated reactions and assays or binding processes by using chemically modifiable capillaries or hollow fibers.

Reversible methods for surface-bound interactions are particularly suitable for the continuous management of evolutionary processes. If nucleic acid fragments are to be bound reversibly, anion exchangers such as the commercially available Qiagen are preferably used. The system of peptide ligand-chelate binding, such as the 6 x His / Ni / NTA affinity system, has proven itself for the reversible binding of proteins.

The individual components of the device according to the invention can also be used for specific tasks in individual parts. The capillary separation unit, the enzymatic amplification unit for purely analytical or analytical-preparative purposes, the dialysis units, the induction and growth units, and the conveyor in the form of the piezo pump can also be used individually for specific problem solutions.

With the method for the determination of in vitro amplified nucleic acids according to P 42 03178, the Copy number of certain DNA or RNA sequences are determined. The device according to the invention now enables simple determination of very low copy numbers of certain DNA or RNA sequences (1 to 100). Furthermore, the possibility is provided to provide information about the transcription activity of individual cells in certain development phases or under certain living or induction conditions.

Single cell analysis is of increasing importance for the branch of somatic genetics. The somatic occurrence of physiologically induced or nonphysiologically induced mutations in individual cells is the subject of several research directions. Also in the analysis of infectious diseases such as in the case of HIV / AIDS or Mycobacterium tuberculosum, reliable quantitative detection methods with very low copy numbers are required. For use in medicine, forensic medicine or disciplines in biology, the concentrations of a nucleic acid to be detected are often not relatively small, but the amount of the material available is the limiting factor of an analysis. This allows minimal amounts a fine needle biopsy or a few cells of an amniotic fluid biopsy for genetic defects such as chromosomal aberration or trisomy 21.

The device according to the invention with the method according to the invention allows the amplification of certain sequences, with a few copies of this sequence being distributed over different volume elements in such a way that they can be multiplied separately during the amplification and thus can be counted individually. The correct amplicons can be recognized by the fact that they are all amplified with practically comparable kinetics, while the contaminations involved across all volume elements are amplified with a kinetics that is different from them. The method according to the invention also allows different sequences to be to quantify each other. This is achieved, for example, by using several pairs of primers in an amplification approach, for example one primer each being equipped with a differentiable dye which, for example, indicates amplifications by changing the spectral properties of the dyes, which relate both to the frequency of the emitted light can also change the life of the excited state. A particular advantage offered by the method according to the invention is that the amplification product is produced in a small volume element of the total volume and essentially remains there. There is therefore a local, high concentration of these products at this point. Concentration-dependent processes, such as reactions with other molecules or photons, are therefore favored.

The method according to the invention particularly advantageously combines a preparative advantage associated with quantification. In the case of dilute initial concentrations of the matrices, amplificates from a volume element contain descendants of a single defined sequence. The corresponding elements can be prepared and, for example, sequenced as described above. In this way, coexisting variants can be analyzed very quickly and quantitatively recorded in their respective concentrations. It is easy to obtain information about the sequence distribution and the nature of a quasi-species.

The restriction of the possibility of diffusion of the molecules or cells or particles in question to virtually one dimension by using the capillary can also be obtained by a further arrangement of the capillaries, as is described schematically in FIG. 6. Here, capillaries form a flat pattern and are arranged in a larger, spatial complex, in which, however, the substances in the individual volume elements in turn only have one one-dimensional diffusion is allowed. If the length of these capillaries, which are preferably glued to one another, is very short, a macroporous surface is finally obtained which is composed of individual capillary units.

Capillaries with an outer diameter of 1 mm and an inner diameter of 0.5 mm can be arranged in a density of 114,000 per dm ² . These surface elements can be used on both sides open, open on one side or closed on both sides, wherein the closing base surfaces C can be porous membranes which allow the exchange of molecules of certain sizes. One square meter of such a flat arrangement with a thickness of 3 mm contains a 3,430 m long capillary. The production takes place, for example, by bundling long capillaries, the bundling according to Figure 6 being carried out in optimal packaging. The gaps are filled with a suitable adhesive. After it has solidified, the surface segments can be cut perpendicular to the capillary axis. In this special embodiment, capillaries can be understood as a flat arrangement of capillary volume elements which only consist of linearly arranged volume units in contact with one another within the short subsegments. However, the subsegments do not allow any exchange with one another by diffusion of the macromolecular complexes of interest.

In this way, separate, capillary reaction spaces can be accommodated over a large area. In this embodiment, the material exchange that may be desired as well as the optical observation take place parallel to the orientation of the capillary, ie perpendicular to the surface that is spanned by the capillaries. The capillaries are accessible both individually and in their entirety for experimental access. Leave molecule complexes are transferred locally to affinity membranes, for example by blotting.

By superimposing an electric field in the direction parallel to the direction of flow of the capillary contents in the sense of a capillary-countercurrent gradient electrophoresis, analytically determined substance mixtures can be separated from one another and concentrated in sharp bands in an autofocusing manner. High-molecular substances are separated and focused, which undergo a reversible structural transformation through an externally applied gradient, which causes a change in the electrophoretic mobility.

With the aid of the present invention, specific sequences can be concentrated at a fixed point using temperature gradient gel electrophoresis. With the introduction of denaturing gradient gel electrophoresis, it is possible to separate macromolecules from one another in a matrix, which differ only in a single building block, e.g. a point mutation in nucleic acids.

According to the invention, the desired molecules can be separated and fixed at a specific location, which is only dependent on the structure of the respective molecules and is not dependent on the duration of the action of the electric field. At the same time, disruptive molecules are removed. Due to the ability of autofocusing, large separation effects can be made visible with small differences in the thermodynamic stability of the molecules in question.

The method is particularly advantageous when the biopolymers sought are present in a low concentration or are covered by a high concentration of foreign molecules. Molecules of low electrophoretic mobility are mechanically removed from the observation compartment by the flowing medium. Foreign molecules higher electrophoretic mobility is removed from the capillary observation compartment by the electric field. Only the molecules that have an electrophoretic mobility within the gradient that is identical to the flow rate of the matrix are fixed at a defined position. This situation is shown schematically in FIG. 7.

The method is not limited to nucleic acids, but is equally suitable for separating other biopolymers, such as proteins, which can carry out reversible structural changes which are accompanied by a change in electrophoretic mobility.

Macromolecules from several volume elements can be focused according to the invention by applying a rectified electric field over a certain distance of the capillary, for example using hollow fiber elements. The accelerating direction which the electric field exerts on the molecules is opposite to the direction of flow, and at the same time a thermal gradient is superimposed on the electric field in space or time. In this way, in particular identical nucleic acids can be focused which undergo reversible structural changes through the application of a denaturing gradient. The liquid content of the capillary volume elements is in a mechanical flow during the process and is overlaid by a voltage gradient of the electrophoresis matrix. The biopolymers to be analyzed concentrate auto-focussing at the position at which the electrophoretic mobility coincides with the opposite flow velocity.

The method according to the invention and the device according to the invention can be used to divide a solution or suspension with the aim of analyzing and / or evaluating functional biopolymers with different ones Functions. In particular, this involves the evolutionary in vitro optimization of biopolymers, in particular nucleic acids, polypeptides and proteins. The method according to the invention is particularly suitable for the quantifying analysis of different samples taken separately from the capillary on specific nucleic acid sequences using an enzymatic amplification method, preferably using repetitive thermal cycles. The qualitative and quantitative detection of nucleic acids from cells, in particular nucleic acids of low multiplicity, in particular certain RNA and / or DNA, such as rnRNA, regulatory RNA, antisense RNA, ribozymes, chromosomes, chromosome fragments, plastids, mitochondria, chloroplasts, satellites and Plasmids are of interest. The quantitative and / or qualitative detection of pathogen-associated nucleic acid sequences, in particular viruses, bacteria, fungi, from human, animal or vegetable tissue, body fluids and excretions can also be examined.

The method according to the invention can be used with the device according to the invention for the preparative production of nucleic acid fragments by molecular cloning, using cellular or non-cell-bound, enzymatic replication systems. PCR, LCR, 3SR and similar systems which use polymerases are particularly suitable as enzymatic replication systems. The combination with superimposed electrical fields allows the focus of identical nucleic acid fragments from adjacent, arbitrary volume elements.

Claims

P atent claims

1. A method for separating substances from a volume or volume element of a dilute solution or suspension, by limiting the space available for the diffusion of the substances, characterized in that at least one degree of freedom of the spatial diffusion of the substances is restricted, without the contiguous volume or volume element of these Significantly change the solution or suspension.

2. The method according to claim 1, characterized in that one of the degrees of freedom of the spatial diffusion of the substances is limited to a thickness <1 mm.

3. The method according to claim 1 and / or 2, characterized in that a degree of freedom of the spatial diffusion of the substances in the solution or suspension is applied by applying the volume or volume element to a flat support, such as a membrane, filter paper, where the layer thickness is <1 mm.

4. The method according to claim 1 and / or 2, characterized in that two degrees of freedom of the spatial diffusion of the substances in the solution or suspension are restricted by introducing the volume or volume element into at least one capillary, the inner diameter of the capillary being 0.1 mm to 1 mm.

5. The method according to at least one of claims 1 to 4, characterized in that the separated substances are analyzed.

6. Process for the production and/or qualitative and/or quantitative analysis of biopolymers, whereby in addition Next, the biopolymers are separated according to at least one of claims 1 to 4 and are subjected to at least one chemical reaction at the same time or in a close temporal connection.

7. Method according to claim 6 for the evolutionary optimization of biopolymers, in particular nucleic acids, polypeptides and proteins.

8. Method according to claim 6 and/or 7 for the molecular cloning of nucleic acid fragments using cellular or non-cell-bound enzymatic replication systems, in particular PCR, LCR, 3SR and similar systems in which polymerases are used.

9. The method according to at least one of claims 6 to 8, for analysis, preferably for quantifying analysis, of different samples taken separately from the capillary for specific nucleic acid sequences using an enzymatic amplification process, preferably using repetitive thermal cycles.

10. The method according to at least one of claims 6 to 9, wherein nucleic acids from cells are recorded quantitatively and qualitatively, in particular nucleic acids of low multiplicity, in particular certain RNA and / or DNA, such as mRNA, regulatory RNA, antisense RNA, ribozymes, chromosomes, Chromosome fragments, plasmids, mitochondria, chloroplasts and satellite DNA.

11. The method according to at least one of claims 6 to 10, wherein pathogen-associated nucleic acid sequences are recorded quantitatively and / or qualitatively, in particular from viruses, bacteria, fungi, from human, animal or plant tissue, body fluids and excretions.

12. The method according to at least one of claims 6 to 11, wherein nucleic acid fragments are analyzed in a group-specific manner by focusing on the same nucleic acid fragments from neighboring volume elements.

13. Device for carrying out the method according to at least one of claims 1 to 12 with a capillary (1) which is in thermal contact with a thermostatable device (3), characterized in that the capillary (l) is at least partially is arranged to save space, this arrangement (2) at least partially surrounding the thermostatable device (3) and thermal contact between the thermostatable device (3) and the space-saving arrangement (2) is ensured.

14. The device according to claim 13, characterized in that the capillary (1) has an inner diameter of 0.1 μm to 1 mm.

15. The method according to claim 13 and / or 14, characterized in that the capillary (1) consists of a non-porous material, such as plastic, or mineral material, such as glass.

16. Device according to at least one of claims 13 to

15, characterized in that the walls of the capillary (1) are made entirely or partially of ion-permeable material

Material exists that allows a limited exchange of material between the fluid inside the capillary (1) and the medium surrounding the capillary (1).

17. Device according to at least one of claims 13 to

16, characterized in that the thermostatable Device (3) is flat or has the shape of a roller, a cone or related topologies.

18. Device according to at least one of claims 13 to 17, characterized in that the thermostatable device (3) consists of a heatable, uniform, roller-shaped block.

19. Device according to at least one of claims 13 to 17, characterized in that the thermostatable device (3) consists of a heatable, uniform, roller-shaped block which is constructed from at least two segments (7) which can be thermally insulated from one another.

20. Device according to at least one of claims 13 to 20, characterized in that the flow of the compartments arranged in the capillary is controlled by a conveyor device (4).

21. Device according to at least one of claims 13 to

20, characterized in that the flow rate of the solution or suspension through the capillary (1) is controlled by a device in such a way that the solution or suspension in the capillary (1) is subject to thermal action for a determinable period of time averaged by the thermostatable device (3), is exposed.

22. Device according to at least one of claims 13 to

21, characterized in that the thermostatable device (3) consists of an outer jacket (5) which is in thermal contact with an internal roller (6) which can be rotated about the longitudinal axis and which is made up of at least two segments (7), which can be thermally decoupled from one another or via the thermostatable, movable device (1). a heat-transferring medium that is in direct thermal contact with the capillary (1).

23. Device according to at least one of claims 6 to

22, characterized in that the conveying device (4) is arranged at the end of the capillary (1).

2. Device according to at least one of claims 6 to

23, characterized in that the conveying device can be used to convey (4) even small volumes.

25. Device according to at least one of claims 6 to

24, characterized in that, by means of a device, an electric field can be applied parallel to the direction of the capillary (1).

26. The device according to claim 25, characterized in that the effect of the electric field on the substances located in the compartments, such as nucleic acids, is opposite to the effect of the force exerted by the conveying device on the solution or suspension in the capillary (1 ) is exercised.

27. Device according to at least one of claims 13 to

26, characterized in that the thermostatable device (3) allows the provision of a spatial and/or temporal temperature gradient.

28. Device according to at least one of claims 16 to

27, characterized in that an electroporation device (16) is arranged on the porous capillary (1).

29. The device according to claim 28, characterized in that the electrodes for electroporation are arranged outside the porous capillary.

30. Device according to at least one of claims 13 to 29, characterized in that the inner walls of the capillary (1) at least partially carry a coating with ligands such as ionic ligands, in particular cationic ligands for anion exchange chromatography or affinity ligands for specific interactions with proteins, Peptides or nucleic acids, such as antibodies, antibody fragments, biotin, avidin or streptavidin, as well as intercalating dyes or nitrilo-triacetic acid derivatives (NTA) or imino-diacetic acid derivatives (IDA).

31. Use of a thermostatable capillary in a method according to at least one of claims 1 to 12.