EP0765470A2 - Verfahren und vorrichtung zur gezielten entnahme von komponenten aus komplexen mischungen - Google Patents
Verfahren und vorrichtung zur gezielten entnahme von komponenten aus komplexen mischungenInfo
- Publication number
- EP0765470A2 EP0765470A2 EP95923338A EP95923338A EP0765470A2 EP 0765470 A2 EP0765470 A2 EP 0765470A2 EP 95923338 A EP95923338 A EP 95923338A EP 95923338 A EP95923338 A EP 95923338A EP 0765470 A2 EP0765470 A2 EP 0765470A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- volume
- removal
- measurement
- measuring
- analysis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
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Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/02—Burettes; Pipettes
- B01L3/0241—Drop counters; Drop formers
- B01L3/0268—Drop counters; Drop formers using pulse dispensing or spraying, eg. inkjet type, piezo actuated ejection of droplets from capillaries
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
- C12M33/07—Dosage or metering devices therefore
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
- Y10T436/2575—Volumetric liquid transfer
Definitions
- 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 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.
- a small volume element can be procedures are defined, as described in the Rigler et al. (PCT / EP 94/00117) are described.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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).
- 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.
- 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.
- 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 ⁇ m D D 0,1 0.1 ⁇ m.
- 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.
- a conveyor such as a pump
- preference is given to using a mechanically, light pressure or electrically controlled suction system or a (piezo / solenoid) pump-controlled dispenser system.
- 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.
- 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.
- 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.
- 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.
- 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.
- known dispensers microdrop
- pump / suction devices solenoid pumps, stepper motor-controlled pumps
- FCS fluorescence correlation spectroscopy
- the volume of the receiving device is increased or the sample volume is reduced in favor of the receiving device.
- 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
- 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
- this cell 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.
- 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.
- 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.
- 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.
- 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
- 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.
- unknown particles such as pathogens or immunogens
- 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.
- 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.
- 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.
- 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.
- 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.
- volume elements increases significantly.
- many volume elements can be screened in the ⁇ s 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.
- 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- ⁇ m 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 ⁇ m range and below are achieved by using holographic gratings.
- Extensive arrays of small volume elements can be illuminated using holographic gratings.
- 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.
- 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,
- 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.
- 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 ⁇ t 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.
- genomic and / or subgenomic segments from extensive collectives in their function, e.g. determine their functional binding behavior to target molecules.
- allergens e.g. Aspergillus, milk protein, alpha-amylase
- allergens e.g. Aspergillus, milk protein, alpha-amylase
- 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.
- extracellular molecules e.g. protease inhibitors
- surface membrane receptors e.g. insulin
- soluble receptors as mediators steroid hormone binding receptors
- cellular soluble structural proteins or enzymes e.g. 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:
- 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 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 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.
- 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.
- a stepper motor-controlled syringe nl holder / step
- 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.
- 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.
- 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.
- IPTG is supplied as an expression-inducing reagent.
- 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.
- 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.
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Abstract
Description
Claims
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE4422313 | 1994-06-17 | ||
DE4422313 | 1994-06-17 | ||
DE4422290 | 1994-06-25 | ||
DE4422290 | 1994-06-25 | ||
PCT/EP1995/002344 WO1995035492A2 (de) | 1994-06-17 | 1995-06-16 | Verfahren und vorrichtung zur gezielten entnahme von komponenten aus komplexen mischungen |
Publications (1)
Publication Number | Publication Date |
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EP0765470A2 true EP0765470A2 (de) | 1997-04-02 |
Family
ID=25937730
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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EP95923338A Ceased EP0765470A2 (de) | 1994-06-17 | 1995-06-16 | Verfahren und vorrichtung zur gezielten entnahme von komponenten aus komplexen mischungen |
Country Status (4)
Country | Link |
---|---|
US (3) | US6849461B2 (de) |
EP (1) | EP0765470A2 (de) |
AU (1) | AU2793295A (de) |
WO (1) | WO1995035492A2 (de) |
Families Citing this family (13)
Publication number | Priority date | Publication date | Assignee | Title |
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EP0765470A2 (de) * | 1994-06-17 | 1997-04-02 | Evotec BioSystems GmbH | Verfahren und vorrichtung zur gezielten entnahme von komponenten aus komplexen mischungen |
DK1145066T3 (da) | 1998-12-21 | 2005-07-04 | Evotec Ag | Positionering af målevolumen i en scanningsmikroskopisk fremgangsmåde |
US6943036B2 (en) * | 2001-04-30 | 2005-09-13 | Agilent Technologies, Inc. | Error detection in chemical array fabrication |
EP1262545A1 (de) * | 2001-05-31 | 2002-12-04 | Direvo Biotech AG | Mikrostrukturen und deren Verwendung für die gerichtete Evolution von Biomolekülen |
US9476856B2 (en) | 2006-04-13 | 2016-10-25 | Advanced Liquid Logic, Inc. | Droplet-based affinity assays |
US7439014B2 (en) | 2006-04-18 | 2008-10-21 | Advanced Liquid Logic, Inc. | Droplet-based surface modification and washing |
US8809068B2 (en) | 2006-04-18 | 2014-08-19 | Advanced Liquid Logic, Inc. | Manipulation of beads in droplets and methods for manipulating droplets |
US7901947B2 (en) | 2006-04-18 | 2011-03-08 | Advanced Liquid Logic, Inc. | Droplet-based particle sorting |
US10078078B2 (en) | 2006-04-18 | 2018-09-18 | Advanced Liquid Logic, Inc. | Bead incubation and washing on a droplet actuator |
US8716015B2 (en) * | 2006-04-18 | 2014-05-06 | Advanced Liquid Logic, Inc. | Manipulation of cells on a droplet actuator |
US9008884B2 (en) | 2010-12-15 | 2015-04-14 | Symbotic Llc | Bot position sensing |
TWI622540B (zh) | 2011-09-09 | 2018-05-01 | 辛波提克有限責任公司 | 自動化儲存及取放系統 |
EP3921085B1 (de) * | 2019-02-07 | 2023-10-04 | University of Washington | Vorrichtungen und systeme zur tröpfchenerzeugung und verfahren zur erzeugung von tröpfchen |
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DE2408646A1 (de) * | 1974-02-22 | 1975-08-28 | Max Planck Gesellschaft | Reaktionskinetisches messgeraet |
DE2716095C2 (de) * | 1977-04-12 | 1987-02-19 | Becton, Dickinson and Co., Paramus, N.J. | Verfahren zur Steuerung der Bewegungsbahn von in einem Elektrolyten suspendierten Teilchen und Vorrichtung zur Durchführung dieses Verfahrens |
DE3574617D1 (de) | 1984-09-11 | 1990-01-11 | Partec Ag | Verfahren und vorrichtung zur sortierung von mikroskopischen partikeln. |
US4784737A (en) | 1986-04-18 | 1988-11-15 | The United States Department Of Energy | Electromicroinjection of particles into living cells |
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DE3707111A1 (de) * | 1987-03-05 | 1988-09-22 | Max Planck Gesellschaft | Verfahren zum einbringen von genmaterial in organellen, insbesondere chloroplasten |
US4765737A (en) * | 1987-03-30 | 1988-08-23 | Cornell Research Foundation | Cell size measurements using light in flow cytometry and cell sorting |
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DE3841507C1 (de) * | 1988-01-22 | 1989-06-29 | Danfoss A/S, Nordborg, Dk | |
US5000000A (en) * | 1988-08-31 | 1991-03-19 | University Of Florida | Ethanol production by Escherichia coli strains co-expressing Zymomonas PDC and ADH genes |
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WO1994010564A1 (de) * | 1992-11-05 | 1994-05-11 | Evotec Biosystems Gmbh | Verfahren zur trennung von substanzen aus verdünnten lösungen oder suspensionen |
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EP0765470A2 (de) * | 1994-06-17 | 1997-04-02 | Evotec BioSystems GmbH | Verfahren und vorrichtung zur gezielten entnahme von komponenten aus komplexen mischungen |
US5571398A (en) * | 1994-12-23 | 1996-11-05 | Northeastern University | Precise capillary electrophoretic interface for sample collection or analysis |
DE102004047953A1 (de) * | 2004-10-01 | 2006-04-20 | Rudolf Rigler | Selektion von Partikeln im laminaren Fluss |
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1995
- 1995-06-16 EP EP95923338A patent/EP0765470A2/de not_active Ceased
- 1995-06-16 AU AU27932/95A patent/AU2793295A/en not_active Abandoned
- 1995-06-16 US US08/750,715 patent/US6849461B2/en not_active Expired - Fee Related
- 1995-06-16 WO PCT/EP1995/002344 patent/WO1995035492A2/de not_active Application Discontinuation
-
2004
- 2004-12-20 US US11/015,179 patent/US20050250157A1/en not_active Abandoned
-
2009
- 2009-04-08 US US12/385,440 patent/US20090194706A1/en not_active Abandoned
Non-Patent Citations (1)
Title |
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See references of WO9535492A2 * |
Also Published As
Publication number | Publication date |
---|---|
WO1995035492A3 (de) | 1996-02-08 |
US6849461B2 (en) | 2005-02-01 |
US20090194706A1 (en) | 2009-08-06 |
AU2793295A (en) | 1996-01-15 |
WO1995035492A2 (de) | 1995-12-28 |
US20050250157A1 (en) | 2005-11-10 |
US20020073787A1 (en) | 2002-06-20 |
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