EP0765470A2

EP0765470A2 - Process and device for selectively extracting components from complex mixtures

Info

Publication number: EP0765470A2
Application number: EP95923338A
Authority: EP
Inventors: Manfred Eigen; Rudolf Rigler; Karsten Henco
Original assignee: Evotec Biosystems GmbH
Current assignee: Evotec OAI AG
Priority date: 1994-06-17
Filing date: 1995-06-16
Publication date: 1997-04-02
Also published as: WO1995035492A3; AU2793295A; US20020073787A1; WO1995035492A2; US6849461B2; US20050250157A1; US20090194706A1

Abstract

The process of the invention facilitates the selective extraction of one or a few molecularly dispersed or cellular components of a system, like molecules, molecule complexes, vesicles, micells, cells, possibly with a relevant volume element V of size 10?-9 1 » 10-18¿ 1, from a larger sample volume. The required component is selectively transferred to another environment by setting the place and time of the extraction by means of a signal correlating the small component to be extracted. The process is particularly suitable for the extraction of rare components, the existence of which can be detected in a previous stage by means of a scanning process. The process is also suitable for the extraction of unidentified components.

Description

Method and device for the targeted removal of components from complex mixtures

The present invention relates to a method for removing one or a few components of a system, an apparatus for carrying out the method and its applications.

The functional characterization of individual molecules or molecular complexes, viruses or individual cells is carried out with the help of the in Rigler et al. (PCT / EP 94/00117) described methods possible. With the method described, statements can be made as to whether in a complex mixture of a solution or suspension or in a two-dimensional layer, individual molecules or molecular complexes are contained in very small volume elements (<10 ^"12 1) that contain certain interactions enter into defined target molecules.

For many analytical and synthetic questions, it is already of great advantage to know that a desired molecule is in an analyzed mixture. Often, however, the problem to be solved according to the invention is to remove the molecule once recognized as desired from the mixture in the sense of a preparative approach.

ORIGINAL DOCUMENTS Enrichment, for example to avoid or simplify cloning steps. Another problem with particularly dilute solutions of a concentration of 10 ^"12 M is to quickly find a volume element as part of the sample volume in which a representative of the substance sought is located. The substance sought does not have to be known. In the search after unknown pathogens or active substances, only interactions with known substances may be known, or interactions with detection molecules that may be present can be postulated.

Methods and devices for removing whole cells are described. The corresponding devices are known as line sorters. It can be based on certain parameters of light scattering or fluorescence such. B. identify certain cell types of a blood count and count the cells of a defined specification. The cells can be stained with fluorescence-labeled antibodies and differentiated on the basis of their surface antigens or classified by their nucleic acid content using hybridization methods (in situ). After being separated into droplets, the cells are analyzed in the flow and can be selectively separated or fractionated by targeted electrostatic deflection of individual drops. Appropriate devices are commercially available and are of great importance for clinical diagnostics and research. PCT / EP 93/03077 discloses how individual, linearly separated volume elements can be separated from a capillary flow.

The object on which the invention is based is to provide a method which makes it possible to take a small volume element of the solution or suspension from a larger volume element of a sample volume of a solution or suspension at defined spatial coordinates, this volume element containing a target substance. Such a small volume element can be procedures are defined, as described in the Rigler et al. (PCT / EP 94/00117) are described. Here, extremely small measurement volumes are analyzed as part of a larger, surrounding sample volume, in that the presence of certain constituents is inferred by confocal illumination of a volume element, excitation by the light used for illumination and / or registration of specific fluorescence signals. The illumination can alternatively also take place, for example, by the method of near-field spectroscopy, in which diaphragms with openings are used which are smaller than the wavelength of the radiated electromagnetic radiation. The object is also to be achieved, based on the spectral properties of a sought-after, detected substance, of a specific molecule, a molecular complex or a cell, to selectively remove these.

The method according to the invention can be used in a particularly advantageous manner for the detection of previously unidentified unknown pathogens or immunogens. (Unknown) pathogens or immunogens can be characterized and, if necessary, prepared. A particularly advantageous procedure in the sense of the invention makes use of the fact that pathogens initially multiply after infection of a host organism without being prevented by a present immune defense. Only after a certain period has elapsed does the immune system establish a humoral immune response, e.g. B. by the synthesis of various immunoglobulins (primarily IgM, followed by IgG) and later a cellular immune response. Patients with suspected infection or contact with a previously unknown pathogen or immunogen without detectable known antigen properties with regard to a reaction and detectability with known antisera / antibodies represent the starting material for the pathogen isolation later, in the phase of the chronic stage of the disease, it is assumed that Antibodies against the supposed pathogen, such as a virus, have now formed, but the virus titer has now been greatly reduced as a result. Sera from this stage of the disease are used to prepare the immunoglobulins.

The fraction of immunoglobulins normally contains only a minor percentage of antibodies that are directed against the unknown pathogen. The majority of the antibodies are directed against a large number of antigen structures which are not related to the specific pathogen / immunogen sought. Therefore, it is more difficult to characterize the pathogen through the immune complex using sera from a single patient.

The sought immune complex can, however, be isolated by including some expected characteristics:

The immune complex contains the major part of a pathogen, the mobility of which lies between that of a small RNA virus and that of a bacterium.

The pathogen has several antigenic binding sites that are occupied by more than one dye-labeled antibody.

In a preferred procedure (FIG. 5), the antibodies from two patients in the state of the hypothetically chronic phase can be prepared separately and labeled with different dyes or those which can be distinguished by cross-correlation. The at least two patients are selected so that there is a high probability that both patients have been infected with the same pathogen. Since pathogens generally carry a large number of the antigenic determinant on the surface / virus envelope, it is likely that the immune complexes formed after the reaction with a mixture of different labeled antisera simultaneously have the different labeling dyes.

The experiment is carried out as shown in FIG. 5.

Detection of individual bacteria via the binding specificities of surface-expressing bacteria

For a large number of important applications in modern biotechnological research, it would be extremely advantageous and efficient if, instead of detecting a functional biomolecule in a given sample volume, a single bacterium or virus with functional surface proteins could be detected in one method. The decisive advantage lies in the interesting coupling of a phenotypic expression product, e.g. a natural or a recombinant surface protein on its genetic blueprint. The method is to be seen in particular in connection with a preparative use according to the invention, by means of which cells or molecular complexes determined as desired can be separated from an environment.

Determination and preparation of the immunogenic epitopes of microorganisms

The genome of microorganisms comprises approximately 10 ⁷ nucleotides. Shotgun expression can be used to express subgenic fragments with an average length of 100 amino acids using the described method. Taking into account the reading frame variation (factor 3) and an assumed non-coding counter strand, 10 ⁸ recombinant bacterial clones contain about 100 times each segment. 10 ⁸ recombinant bacterial clones are contained in 1 ml of a suspension of 1 OD, which can be separated individually in about 24 hours using the method according to the invention, for example on their binding agents. have properties examined with IgE or IgE-bearing cells from an allergy patient. According to the invention, the correspondingly characterized bacteria are separated out, but at least highly enriched, biologically expanded or the corresponding genome segment is amplified and characterized by enzymatic amplification methods.

If a corresponding bacterium is detected in the measuring volume element, it can be removed according to the invention directly from the mixture by sucking the volume element surrounding the bacterium / molecular complex through a capillary or by separating the molecular complex by electrophoresis, electroosmosis or dipole induction (FIGS. 1 and 2).

One idea of the invention on which the invention is based is that an electrically, optically or mechanically controlled suction device is used, the opening dimension of which is large compared to the measurement volume but small compared to the dimensioning of the sample volume, in order to solve the technical problem on which the invention is based. According to the invention, the electrical, optical or mechanical pump system is controlled by an FCS-controlled pulse generator, so that a small proportion of the sample volume is separated from the total volume in such a way that back diffusion can be largely ruled out. This is done either by electrophoresis, induced dipole moments, electroosmosis, mechanically induced pressure jumps or by light pressure.

The method allows the removal of one or a few components of a system such as molecules, molecular complexes, vesicles, micelles, cells, optionally embedded in an associated volume element (removal volume) V (10 ^{~ 9} 1 ≥ V ≥ 10 ^"18 1). This volume element is Part of a larger volume of an environment that contains the small components to be removed (sample volume). The removal is carried out by transferring the component or the components to another environment, the place and time of the removal. is determined by a signal of an analysis that correlates with the small component to be removed. Methods which can be analyzed are the methods in which the molecular content of the smallest volume elements (10 ^"14 1) can be analyzed, as described by the international application Rigler et al. PCT / EP 94/00117. The sample volume is connected to the recording device by a pore of a capillary or a pore of a membrane wall, the narrowest opening D of which is given by 100 μm D D 0,1 0.1 μm.

In a preferred embodiment, a capillary is used for this purpose, as described in Neher et al. , Methods in Enzymology, Vol. 207, 3-14. This capillary is e.g. connected to a conveyor, such as a pump, which is capable of withdrawing the volume of solution surrounding the molecular complex. According to the invention, preference is given to using a mechanically, light pressure or electrically controlled suction system or a (piezo / solenoid) pump-controlled dispenser system.

Several withdrawals can be carried out in one or more steps in one and the same pick-up device, the individual withdrawal processes being able to take place independently of one another in the sense of a collection process.

In many cases, the electrically controlled removal via a directed transport of the volume element by at least one electrical voltage or field pulse is preferred. Other embodiments work mechanically by means of at least one pressure difference pulse and / or by means of at least one light pressure pulse in the direction of the pore opening using methods such as those described by Weber and Greulich, Int. Rev. Cytol. , 1992, 133, pp. 1 - 41, have been published. The receiving device can be a capillary, the lumen of which is preferably larger than the diameter of the pore or Capillary tip, the opening of which is in direct contact with the sample volume. Electro-osmosis can be carried out in consistently thin capillaries, as is customary in capillary electrophoresis. Otherwise, the flow resistance could be disadvantageously high in the case of mechanical volume transport.

In the case of an electrically mediated removal, the desired constituent in the measurement volume is deliberately made more electrically neutral via an electrical field pulse via the at least one, brief application of an electrical field, for the purpose of electrophoresis of electrically charged components and / or for the purpose of electroosmosis with coupled transport Molecules transferred to the receiving device. For this purpose, one electrode can be brought into an electrically conductive contact with the solution on the side of the sample volume, the other electrode can be brought into an electrically conductive contact with the solution on the side of the receiving device, and the conductive contact between the two compartments can be established via the pore. In the case of removal via a targeted pressure pulse, the desired transport is brought about by at least one short-term increase in the pressure in the volume outside the receiving compartment in comparison to the pressure within the receiving compartment and / or by a brief pressure reduction within the receiving compartment. In this case, known dispensers (microdrop) or pump / suction devices (solenoid pumps, stepper motor-controlled pumps) have proven themselves. This means that small volume elements of ≤ 1 nl can be transferred from a larger volume of a solution or suspension by transferring the small volume element through a pore with a diameter of ≤ 100 μm into a receiving compartment, the time and the spatial coordinate of the removal process using a correlated analysis system such as is controlled by fluorescence correlation spectroscopy (FCS). In particular, by means of a vacuum pulse via a piezo-controlled dispensing module, the filling volume of which is located within the receiving compartment and / or by means of a pressure pulse and / or negative pressure pulse, the volume of the receiving device is increased or the sample volume is reduced in favor of the receiving device. According to the invention, the transport into the receiving volume follows. The size of the recorded volume is controlled via the number of drops dispensed or the step motor steps or via the length and intensity of the light pressure pulse.

The hardware / software-coupled on-line analysis system triggers the sampling time via the recorded, correlating signal. The removal takes place at the moment when the particle or particles to be removed are with a high probability within the removal volume. This does not necessarily have to occur approximately at the same time as the positive analysis result obtained. If a molecule, molecular complex, virus or cell has been identified by FCS as a component to be removed, there is the possibility of direct removal or a later removal. It is based on the assumption that the respective component to be removed

either shortly after the measurement has been carried out by means of forced transport as described or electrophoretic migration is at a specific point in time at a specific point in time and since electrophoretic or mechanical preparation can be carried out according to FIGS. 1 and 2, or

is in the vicinity of the detection volume from which the component can be separated electrophoretically, optically by light printing or mechanically without forced translation. Commercial cell sorters are equipped with a system for separating cells after prior analysis, which according to the invention can be coupled with the analytical method of fluorescence correlation spectroscopy. Usually, cells in a liquid-coated, capillary flow are passed through a cuvette which is coupled to a conventional fluorescence measuring device and / or a light scattering measuring device. At a defined spatial and temporal distance after the measuring device, a standardized and continuous division of the thin liquid jet into individual emerging droplets is ensured at the end of the capillary by an applied continuous sound frequency. In the event that a cell of a desired cell type passes the cuvette, this cell is in one of these droplets after a defined time, which can be deflected from its trajectory after the separation by a coupled signal, for example by means of a pulse in the electrostatic field, and so on can be separated in a separate receiving device. The disadvantage of the method lies in the fact that only an integral fluorescence signal in the sense of a pure intensity measurement can be obtained in the flowing flow. In contrast to this, the coupling according to the invention using the method of fluorescence correlation spectroscopy in small volume elements enables the fluorescence signals obtained to be differentiated according to their belonging to different molecular sizes and molecular mobilities. This is necessary in order to distinguish, for example, receptor-bound ligands from free ligands present. According to the invention, the method of fluorescence correlation spectroscopic detection technology of fluorescent particles such as molecules, vesicles, cells or molecular complexes is used in the mechanically induced, capillary flow with a targeted sorting device through a pore.

The actual separation process can take place after the passage of individual volume elements through the pore by after a defined spatial and temporal correlation, the positively registered measurement volumes with an associated volume element are transferred to separate recording devices.

In a further preferred embodiment, the FCS measurement process can also take place before it emerges from the pore of a capillary tip, which is coupled to a microdispensing device and where, by means of various mechanical or field-induced deflections, small measurement volumes with an associated volume element of the environment as Droplets can be collected in different receptacles.

In particular, the temporal and / or location-specific correlation takes place between an analysis of a sub-volume of the sample volume (measurement volume) within the volume element

V and a removal of a desired component by removal of the volume element V hardware / software supported. It is carried out, with at least one component that was identified positively in the volume element V being located in the recorded volume element V during the removal process. The pore of the receiving device is mechanically brought up to the volume element and / or the volume element

V or components thereof with a predetermined temporal correlation are transported via a flow in the river or via electrostatic or magnetic field gradients or components thereof to the pore of the receiving compartment with a temporal correlation. The analysis can also be carried out directly in front of the pore of the receiving compartment. The sub-volume (measurement volume) is preferably smaller than the volume element V. This increases the probability that the positively identified component is removed before it has moved too far from its measurement volume.

The analysis process must meet certain requirements in order to be used in the process. The signal correlating to the removal process is e.g. B. about a optical analysis system that can analyze specific molecular properties in small volume elements of <10 ^"14 1. This is preferably done by analysis systems based on confocal laser correlation spectroscopy or fluorescence correlation analysis based on near field spectroscopy, whose signal is on line Coupled analysis and removal processes can be cascaded together in accordance with the invention, the recorded sample volumes with or without a dilution step being subsequently subjected to an analysis and again in an enriched form by a second and / or further removal unit after the removal Analysis can be taken.

Components can be removed which form a complex with at least one reaction partner which can be detected spectroscopically (indirectly) or which themselves have sufficient fluorescence properties (direct).

The method is of particular interest in combination with the procedure according to the invention for the removal of as yet unidentified (unknown) pathogens. The aim is the multiplication of an isolated pathogen in vivo or the multiplication of the genetic material of the pathogen or parts thereof in vitro by amplification of the nucleic acid contained in the sense of a shot-gun method and characterization via sequencing.

Also important is the targeted removal of components that are not yet known in terms of their molecular nature, such as molecules, cells, vesicles, and molecular complexes that are functional z. B. identify via an enzymatic effect or complex formation.

As sketched in FIG. 5, unknown particles, such as pathogens or immunogens, are removed according to the invention by Sera can be obtained from at least one organism infected with the unidentified pathogen. At least one serum (serum 1) is obtained from the acute infection phase by the previously unknown pathogen or immunogen and at least one further serum (serum 2) from the same or at least one other organism with the same or homologous infection from the chronic infection phase won. The unknown pathogen or immunogen from serum 1 is caused to complex with indirectly or directly fluorescent dye-labeled antibodies from serum 2 and the complex formed is measured. In this context, cross-correlation is important, as described in PCT / EP 94/00117, by means of which, for example, the simultaneous binding of different fluorescent ligands such as antibodies from different organisms can be measured. Antibodies can be labeled directly via at least one reaction with couplable dyes or indirectly by reaction with dye-labeled antibody binding domains, in particular protein A derivatives or protein G derivatives. The non-identified pathogenic components can turn out to be known microorganisms. As a characteristic, specific interactions or enzymatic activities with fluorescence-labeled target molecules to form surface-expressed or cytosolic-expressed structural elements of natural or genetically recombined proteins or peptides are detected.

The method according to the invention can cause the detection of molecules in a very low concentration. For example, fetal cells can be scanned in maternal blood. With the method according to the invention, very low concentrations (<10 ^"12 M) of fluorescent molecules can be determined. However, the method can become impractical if one has to wait too long with unchanged spatial coordinates to determine one or more measurement volumes until a wanted molecule the space element of the measuring volume happens. This problem also arises at higher concentrations (> 10 ^"12 M) if the diffusion times are very short, as is the case, for example, with cells and cell-bound molecules. In such cases, the method according to the invention can be modified so that the actual one Measuring method is preceded by a scanning process, in which the spatial coordinates are varied in temporally continuous measuring technique or in temporally discontinuous measuring technique until a signal of the desired quality is detected, which can occur, for example, when a correlated fluorescence occurs together two different emissions when using the cross-correlation method. If a signal is detected, the FCS measurement process is started. The duration of a scanning process can be less than one millisecond per measurement process. It can then be determined that the "scanned" measurement volume or the measuring volumes measured in parallel do not contain the component sought b When doing this, it should be noted that the average characteristic diffusion times are influenced in a calculable manner in their absolute size. This happens, for example, in such a way that fixed molecules (for example on fixed bacterial cells) directly and exclusively reflect the temporal variation of the relative change in the spatial coordinates of the measurement volume in comparison to the coordinates of the sample volume, or in the case of movable small molecules and a sudden change in the Space coordinates approximately half the average length of stay, since the molecules are already within the measurement volume at the start of the measurement process.

Scanning processes, which are upstream of the actual measurement, become important when e.g. B. cell populations are to be analyzed, with only a fraction of the cells carrying molecules or molecular complexes that determine the removal properties. This is the case, for example, when analyzing evolutionarily produced mutant populations of recombinant cells, but also when analyzing maternal blood for the Presence of childhood, nucleated erythrocytes, which are to be analyzed for certain genetic makeup or chromosomal abnormalities.

The method is suitable for a method which is referred to below as functional gene extraction. What is meant is the production of genetic probes for the detection / detection / cloning of specific functions which are encoded on an entire genome or in a cDNA library. Examples of applications are functional genome analysis using phage or bacterial display systems, as well as corresponding applications in evolutionary biotechnology. Both examples are concerned with the detection and targeted selection of cells or phages with specific binding properties to specific ligands against a background of unreacted phages or bacteria.

The number of patterned volume elements increases significantly. In combination with the cross-correlation, many volume elements can be screened in the μs to nanosecond range in single and multi-array operation. The shift is only interrupted if e.g. Have differently colored signals correlated with each other. If this situation exists, material parameters of the components, e.g. the translation diffusion can be determined. This time is statistically shorter by a time element to be calculated (approx. 50%), compared to the case in which a particle has to penetrate the volume element by itself or by forced diffusion. Once a particle has been detected, it can also be detected a second time by scanning the immediate surroundings.

According to the invention, the measurement volume can be composed of sub-measurement volumes illuminated in parallel, in that the simultaneous illumination of several measurement volumes by at least one radiation source for electromagnetic radiation At least one holographic grating is used to generate several volume elements.

The parallel illumination of several volume elements with confocal optics is described in DE 40 35 799. Parallel illumination of measurement volumes, the relative distances of which lie in the sub-μm range, is not possible or is unsatisfactory using the devices described. The illuminations to be provided in the method according to the invention with dimensions in the lower μm range and below are achieved by using holographic gratings.

Extensive arrays of small volume elements can be illuminated using holographic gratings. According to the invention, the measurement volumes are measured confocally either by using a plurality of pinhole diaphragms in the object plane, by positioning multiarray detector elements in the object plane or by using optical fiber bundles with coupling the light into the object plane and transmission to photon detectors for fluorescence properties of the molecules contained therein.

In the case of the highly parallelized illumination of small volume elements, the problem of registering the emitted fluorescence signals from the individual volume elements arises. In the patent application PCT / EP 94/00117 it is described that it is possible to illuminate small spatial elements in parallel and to map the respective fluorescence signals individually on multi-array detectors by using confocal pinhole systems in the object plane or the signals at the position and instead of the pinhole to couple into light guides and to guide on detector elements or to position the multiarray detectors instead of and in the position of the pinhole itself. It also describes the possibility of illuminating a larger volume element and using the confocal, to combine parallel images of small sub-volume elements.

With high parallelization, however, the requirements regarding the number of detector arrays and the computing effort associated with the parallel processing become significant. According to the invention, these problems are solved in that, in a further form of coupling small, parallel illuminated room elements with a registration device, the signals are recorded in an integrated manner over several room elements. This procedure is particularly useful in those applications in which

a large number of volume elements are to be screened, the computational effort in favor of the computing capacity used and the computing time is to be minimized, the number of volume elements recorded per measurement and thus the total volume measured is to be maximized,

Signals of molecules, molecular complexes or cells are to be analyzed in high dilution, the precision of the spatially resolved detection is of secondary importance, the number of light quanta emitted during diffusion through a single spatial element is sufficient for a correlation.

According to the invention, at least two measurement volumes are confocally imaged on the signal plane in the object plane together or combined in groups on at least one detector element of a photon-registering measurement system.

To detect fluorescent molecules in a very low concentration, the sample volume is inventively according to the invention before the actual measurement and removal of at least one partly subjected to a scanning process, the time for acquiring a sought-after particle being shortened by continuous or discontinuous variation in time of the spatial coordinates of the measurement volume, relative to the spatial coordinates of the sample volume. The time interval δt for the measurement of one or more volume elements with defined spatial coordinates before a sought-after molecule is detected via its fluorescence measurement signal is shorter than the average duration of stay of the sought-after molecule within a measurement volume.

The device for carrying out the method is characterized in that a pore of a porous receiving device is brought close to the optical measurement volume and the receiving device is connected to a mechanically, optically or electrically controllable removal device. It comprises the arrangement of a closed or open container for receiving a sample volume, coupled with a measuring device for illuminating and / or measuring a small volume element (measuring volume) by electromagnetic radiation and at least one connection to at least one second volume element in a receiving device, the is in direct contact with the sample volume through an opening via a liquid phase, the opening preferably being spatially immediately adjacent to the measuring volume.

The method according to the invention is used in particular for the preparative extraction of unknown pathogens, immunogens or organisms which functionally express parts of a genome, and for the analysis and preparative extraction of fetal cells containing nuclei from maternal blood.

Such problems are associated with methods for the evolutionary optimization of peptides and / or proteins through the use of mutagenesis methods and selection methods, as described, for example, in the international patent application PCT / EP 94/00117 are proposed. About 10 ⁹ bacteria can be screened in their binding properties to specific dye-labeled substances within 24 hours, for example for the presence of a bacterium which expresses a surface protein / peptide which has the ability to interact with the target molecule of a given concentration to step. The corresponding bacterium can be cloned from such a reaction batch using conventional methods or directly isolated using the method according to the invention.

Another important field of application results from the so-called genome project for the functional mapping of gene segments from genomic banks, cDNA banks or banks of subgenic structural elements (shape space). In this way, genomic and / or subgenomic segments from extensive collectives in their function, e.g. determine their functional binding behavior to target molecules.

The use of the methodology described for the functional assignment of genetically coded peptide segments can be of great importance, in particular in allergological research. The assignment of immunodominant epitopes to allergens (e.g. Aspergillus, milk protein, alpha-amylase) is extremely important and has so far been a difficult problem to solve. Typical problems in practice are:

Determination of the IgE-binding molecules from a mostly undefined mixture of substances. It is important, for example, to answer the question of which constituents of soy lecithin are immunogenic, the pure substance alone, the pure substance in its interaction with impurities in the preparation or the interaction with structures of the recipient organism. According to the invention, the different immunogenic sub- differentiate from the patient in the mixture with labeled IgE.

By expression of subgenic gene segments, the above-mentioned method can be used to narrow down, characterize, and prepare the immunopre- dominant epitopes. With these results,

Using the methods described in DE 41 12 440 C2, generate evolutionarily analog, functional molecules that lack the corresponding immunodominant regions, e.g. a weakened immunogenic alpha-amylase or detergent proteases which simply represent specific epitopes using standard methods using genetic engineering and which are used as pure detection reagents or used for desensitization.

Certain tasks can be solved by the method according to the invention, which until now could not be solved or could only be solved with disproportionately great effort.

Screening of pharmacologically active substances via the binding of known fluorescence-labeled ligands to receptors which are unknown per se and which may be located on cells or on natural or artificial vesicle structures.

There are natural and chemically synthesized active ingredients with pharmacological activity, the target molecules of which are unknown. The target molecules can be extracellular molecules (e.g. protease inhibitors), surface membrane receptors (e.g. insulin), soluble receptors as mediators (steroid hormone binding receptors) or cellular soluble structural proteins or enzymes. According to the invention, the extremely important task of finding, characterizing and, if necessary, preparing the pharmacologically important target molecule for a known active ingredient can thus be achieved:

Orphan receptor search

Clarification of pharmacological mechanisms of action Search for analog active substances

Search and differentiation of different receptor molecules, preferably in differentiable biological targets (different cell differentiation, tumor / non-tumor), disease-related - not disease-related etc.

Legend of the figures

Figure 1

(Molecule collector)

The schematic drawing describes the principle of the device in which the contents of a small volume element from the compartment A in by a pore, which represents the open connection between the compartments A and B, a pressure or vacuum pulse or an electrical pulse a compartment B can be transferred. Part of this volume element is the darkly symbolized measuring volume of <10 ^"14 1 directly in front of the pore in which the FCS analysis takes place. A cell or a macromolecular complex can also be achieved by means of a correspondingly directed light pressure using a laser pulse that is perpendicular to the Pore diameter takes place and can be guided into the receiving device of compartment B for the transport of a complex recognized as desired. Figure 2

(FCS selection of individual microorganisms with targeted fractionation)

The figure shows the FCS selection according to the invention of individual microorganisms with targeted fractionation from a continuously or discontinuously moving sample volume. The FCS measurement volume is located directly in front of the opening of a capillary pore and is identified by the hatched column in the focusing cone of the FCS laser illumination or near-field illumination. In each case in cosecutive steps, computer-controlled measurement volumes can be transported as positively identified measurement volumes together with a surrounding volume from the sample device to the receiving device. This works z. B. by connecting a microdrop dispenser device or e.g. in a simple version by coupling a stepper motor-controlled syringe (nl holder / step) or an electrical pulse. Thus, several volume elements can be accumulated in the receiving device during a measurement.

Figure 3

(Cascade enrichment)

The separating performance can be increased by connecting separating devices as described in FIG. 2 in series. This is important, for example, if a highly complex mixture (10 ¹² particles) of e.g. B. recombinant bacteria or phages in high concentration to separate individual particles as background-free as possible.

Figure 4

(FCS selection of individual microorganisms) The figure describes a device with which bacteria can be separated from mixtures that express certain properties to be measured by FCS. An initially uninduced bacterial mixture is fed to a capillary flow system. In a mixing chamber, for example, IPTG is supplied as an expression-inducing reagent. After a sufficiently long flow time, the expression product is supplied with an assay system with marker molecules, which can then be measured at a defined position in their interaction with any expression product. The figure is also intended to indicate that a positively identified measurement volume can also be taken at a position that is spatially and temporally distant, provided that the space / time coordinates are in a known and defined relationship to one another.

Figure 5

(Detection and preparation of new pathogens)

The figure demonstrates the procedure according to the invention in the selection of unknown pathogens which can be detected by cross-correlation using FCS, the differently labeled antibodies which are directed against a specific pathogen preferably being able to come from different patients.

Claims

Expectations

1. Process for the removal of one or a few constituents of a system, such as molecules, molecular complexes, vesicles, micelles, cells, optionally with an associated volume element (removal volume). V, 10 ^" ⁹ ≥ V ≥ 10 ^{~ 18} 1, from a larger volume of an environment containing the small components to be removed (sample volume) by transferring the component or components into another environment, the place and time of removal by a correlating signal is fixed with the small component to be removed.

2. The method according to claim 1, characterized in that the sample volume is connected to the other environment in the form of a receiving device by a pore of a capillary or membrane wall, the narrowest opening D of which is given by 100 μm ≤ D ≤ 0.1 μm.

3. The method according to claims 1 and / or, characterized gekenn¬ characterized in that at least one removal in one or more steps takes place in one and the same receiving device, the individual removal operations taking place independently of one another in the manner of a collecting process.

4. The method according to at least one of claims 1-3, characterized in that the removal takes place via a directional transport of the volume element by at least one electrical voltage or field pulse and / or mechanically by at least one pressure difference pulse and / or by at least one light pressure pulse .

5. The method according to at least one of claims 2-4, characterized in that the recording device is a capillary whose lumen is larger than the diameter of the pore or capillary tip, the opening of which is in direct contact with the sample volume.

6. The method according to at least one of claims 1-5, characterized in that the removal targeted via an electrical field pulse via the at least one, brief application of an electrical field for the purpose of electrophoresis of electrically charged components and / or for the purpose of electro-osmosis with coupled Electrically neutral molecules are transported in that one electrode is in electrically conductive contact with the solution on the side of the sample volume and the other electrode on the side of the receiving device is in electrically conductive contact with the solution and the conductive contact between the two compartments is established via the pore becomes.

7. The method according to at least one of claims 1-6, characterized in that the removal occurs specifically via a mechanical pressure pulse by at least one-time brief increase in pressure in the volume outside the receiving compartment compared to the pressure within the receiving compartment and / or by one brief pressure reduction within the receiving compartment.

8. The method according to claim 7, characterized in that the negative pressure pulse is generated by a piezo-controlled dispensing module, the filling volume of which is located within the receiving compartment and / or that the pressure pulse and / or negative pressure pulse occurs in that a preferably stepper motor-controlled stroke change of a coupled piston syringe device the volume of the receiving device is increased or the sample volume is reduced in favor of the receiving device.

9. The method according to at least one of claims 6-8, characterized in that the size of the volume taken up is controlled via the number of dispensed drops or the steps of the stepping motor.

10. The method according to at least one of claims 1-9, characterized in that the correlating signal triggers the time of removal, which occurs at the moment when the particle or particles to be removed is with a high probability within the removal volume.

11. The method according to at least one of claims 2-10, characterized in that the temporal and / or location-specific correlation between an analysis of a subvolume of the sample volume (measurement volume) within the volume element V and a removal of a desired component by removal of the volume element V Computer / software-supported, at least one component that was analyzed in the volume element V being in the picked-up volume element V during the removal process, the pore of the receiving device being mechanically guided to the volume element and / or the volume element V or Components thereof with a predetermined temporal correlation via a transport in the river or via electrostatic or magnetic field gradients or components thereof are transported in a time-correlated manner to the pore of the receiving compartment and / or the analysis is geometrically directly in front of the pore of the receiving com partimentes takes place.

12. The method according to claim 11, characterized in that the sub-volume (measuring volume) is smaller than the volume element V.

13. The method according to at least one of claims 1-12, characterized in that the correlating signal is determined via an optical analysis system which can analyze specific molecular properties in small Vplumen elements of <10 ^"14 1, in particular analysis systems based on the confocal Laser correlation spectroscopy or on the basis of near-field spectroscopy, the signal of which is controlled on-line and software controls a targeted removal process in a timed manner.

14. The method according to at least one of claims 1-13, characterized in that the coupled analysis and removal processes are cascaded in a row, the recorded sample volumes with or without a dilution step are subsequently subjected to an analysis again and again in enriched form by a second and / or further removal unit can be removed after the analysis has been carried out.

15. The method according to at least one of claims 1-14, characterized in that components are removed which form a complex with at least one reagent which can be detected by spectroscopy.

16. The method according to at least one of claims 1-15, characterized in that components are removed that are not yet known with regard to their molecular nature, molecules, cells, vesicles, molecule complexes, which are based on an interaction with known structures or identify effects such as enzymatic activity or complex formation.

17. The method according to claim 16, characterized in that the unknown particles are pathogens or immunogens which are specifically removed by collecting sera from at least one organism, at least one serum (serum 1), from the phase of an acute infection by also previously unidentified (unknown) pathogen or immunogen is obtained and at least one serum (serum 2) is obtained from the same or at least one other organism with the same or homologous infection from the phase of the chronic infection, the pathogen also being unknown or immunogen from serum 1 with indirectly or directly fluorescence-labeled antibodies from serum 2 for measurable complex formation.

18. The method according to at least one of claims 16 - 17, characterized in that the simultaneous binding of ligands with various fluorescence signals, z. B. labeled antibodies from different organisms is determined.

19. The method according to at least one of claims 16 - 18, characterized in that the labeling of the antibodies directly via at least one reaction with couplable dyes or indirectly by reaction with dye-labeled, antibody-binding domains, in particular protein A derivatives or protein G derivatives.

20. The method according to at least one of claims 1-19, characterized in that the also unknown particles are microorganisms or vesicles known per se, wherein as a feature specific interactions or enzymatic activities with fluorescence-labeled target molecules to surface-expressed or cytosolic- expressed structural elements more naturally or genetically recombined proteins or peptides are detected.

21. The method according to at least one of claims 1-20, characterized in that the measuring volume is composed of parallel illuminated sub-measuring volumes, the simultaneous illumination of a plurality of measuring volumes by at least one radiation source for electromagnetic radiation using at least one holographic grating.

22. The method according to claim 21, characterized in that the registration of fluorescence signals from at least one measurement volume by confocal imaging by using a plurality of confocal pinhole in the object plane or coupling of the signals in light guide in the object plane or by multiarray detectors in the object plane.

23. The method according to at least one of claims 21-22, characterized in that to carry out parallelized measurements on at least two measurement volumes during signal registration in the object plane, at least two measurement volumes are mapped together or in groups onto at least one detector element of a photon-registering measurement system become.

24. The method according to at least one of claims 1-23, characterized in that for the detection of fluorescent molecules in a very low concentration, the sample volume is subjected to a scanning process before the actual measurement and removal of at least one component, the time for the detection of a the particle sought is shortened by varying the spatial coordinates of the measuring volume relative to the spatial coordinates of the sample volume in a discontinuous or continuous manner. / 35492 PCI7EP95 / 02344

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25. The method according to at least one of claims 1-24, characterized in that the time interval δt for the measurement of one or more volume elements with defined spatial coordinates before a desired molecule is detected via its fluorescence measurement signal is shorter than the average length of stay of the searched molecule within of a measuring volume.

26. Device for carrying out the method according to at least one of claims 2-25, characterized gekenn¬ characterized in that the pore of a receiving device is brought close to the measuring volume, the receiving device is connected to a mechanically, optically or electrically controllable removal device.

27. The device according to claim 26, comprising an arrangement of a closed or open container for holding a sample volume, coupled with a measuring device for illuminating and / or measuring a small volume element (measuring volume) by means of electromagnetic radiation and at least one connection to at least one a second volume element which is in direct contact with the sample volume through an opening via a liquid phase, the opening preferably being spatially immediately adjacent to the measuring volume.

28. Use of the method according to at least one of claims 1-27, for the preparative extraction of unidentified pathogens, immunogens or organisms which functionally express parts of a genome.

29. Use of the method according to at least one of claims 1-25 for the production of genetic probes for the detection / detection / cloning of functional elements of an entire genome and / or diagnostics and / or therapeutics derived therefrom.

30. Use of the method according to at least one of claims 1-25, for the analysis and preparative extraction of nucleated fetal cells from maternal blood.

31. Use of the method according to at least one of claims 1-25, for the detection and preparation of at least one specific gene. Microorganism, the at least one gene product of which is presented on the inner or outer cell membrane or virus envelope.

32. Use of the method according to at least one of claims 1-25, for determining the function of gene products of defined gene segments.