EP1590653A1 - Detection multiparametre dans un microsysteme fluidique - Google Patents

Detection multiparametre dans un microsysteme fluidique

Info

Publication number
EP1590653A1
EP1590653A1 EP04707910A EP04707910A EP1590653A1 EP 1590653 A1 EP1590653 A1 EP 1590653A1 EP 04707910 A EP04707910 A EP 04707910A EP 04707910 A EP04707910 A EP 04707910A EP 1590653 A1 EP1590653 A1 EP 1590653A1
Authority
EP
European Patent Office
Prior art keywords
measuring
measurement
particles
particle
parameter
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.)
Withdrawn
Application number
EP04707910A
Other languages
German (de)
English (en)
Inventor
Thomas Schnelle
Annette Pfennig
Stefan Hummel
Torsten Müller
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
PerkinElmer Cellular Technologies Germany GmbH
Original Assignee
Evotec Technologies GmbH
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from DE10320956A external-priority patent/DE10320956B4/de
Application filed by Evotec Technologies GmbH filed Critical Evotec Technologies GmbH
Publication of EP1590653A1 publication Critical patent/EP1590653A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • 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
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • 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
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • G01N15/12—Investigating individual particles by measuring electrical or magnetic effects by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
    • 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
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1468—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle
    • G01N15/147—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle the analysis being performed on a sample stream
    • 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
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1484—Optical investigation techniques, e.g. flow cytometry microstructural devices
    • 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
    • G01N15/1023—Microstructural devices for non-optical measurement
    • 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
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
    • 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
    • G01N2015/1006—Investigating individual particles for cytology
    • 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
    • G01N2015/1019—Associating Coulter-counter and optical flow cytometer [OFC]
    • 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
    • G01N2015/1028—Sorting particles
    • 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
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1477—Multiparameters
    • 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
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1497—Particle shape
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G01N2035/00099—Characterised by type of test elements
    • G01N2035/00158—Elements containing microarrays, i.e. "biochip"

Definitions

  • the invention relates to methods for measuring properties of particles suspended in a liquid moving through a fluidic microsystem, and to measuring devices and fluidic microsystems that are set up to carry out such methods.
  • flow cytometers In conventional flow cytometry, cells are hydrodynamically focused in so-called flow cytometers, separated into drops and, when passing through a detector, characterized by simultaneous (temporally parallel) recording of fluorescence and scattered light signals.
  • Flow cytometers have a high throughput due to the high drop speeds of 10 m / s, for example.
  • the high hydrodynamic load on the cells is disadvantageous, so that the cell vitality is severely restricted.
  • Further disadvantages of the flow cytometer are that sterile working is not possible and that more complicated measurements, such as kinetic measurements or morphological examinations, in particular of cell components, are difficult or impossible.
  • the cells are then held in a dielectric field cage for a specific detection time at a very low flow rate ( ⁇ 50 ⁇ m / s) for a fluorescence measurement.
  • the measurement of particle properties in fluidic microsystems has advantages in terms of the high specificity and the high resolving power of the measurements, the possibility of working in a sterile manner and the particle-specific procedure after the measurement, for example when cells are deposited in culture vessels.
  • the disadvantage is the significantly lower throughput of the fluidic microsystems, in particular compared to flow cytometry.
  • the object of the invention is to provide improved methods for measuring properties of particles in fluidic microsystems, with which the disadvantages of the conventional measuring methods are overcome and which in particular enable increased throughput and automation of the measurement.
  • the object of the invention is also to provide improved measuring devices and / or fluidic microsystems for implementing the measuring methods.
  • a basic idea of the invention is to provide a method for measuring properties of particles which are suspended in a liquid through a fluidic microsystem, with at least two measurements of a certain particle staggered in time and space and a subsequent joint, correlated evaluation of the measurement results to further develop that the measurement results are characteristic of different properties of the measured particle, and that the evaluation includes a comparison of both measurement results with predetermined expected values in order to effect further measurements or manipulations on the measured particle depending on the result of the comparison. It can be particularly advantageous to sequentially record a morphological (for example geometric) and a material parameter of the particle under consideration. Such a combination enables a classification of the particle with increased safety and reproducibility and a more reliable control of subsequent processes. such as a supplementary precision measurement or sorting.
  • the inventive combination of at least two spatially separated measuring stations arranged in a common, flow-through channel for carrying out the correctly evaluated measurements directed at different parameters (properties) of the particle can advantageously overcome the disadvantages of the conventional measurement in fluidic microsystems.
  • the throughput can be increased by making the measurements faster while the flow is running. While a particle is completely measured and the parameters determined for this particle are evaluated, the next particle following in the flow through the microsystem can already be subjected to the first measurement. Due to the spatial separation of the measuring stations, mutual interference in the detection processes, for example due to unwanted scattered light, can be avoided. The absence of interference enables a reproducible and objectifiable measurement independent of an operator and thus an automation of the measurement. Finally, compared to measurements in flow cytometers, undesirable loads on the particles by mechanical or hydrodynamic forces are avoided.
  • a morphological parameter of the particle is determined in a first measurement.
  • Morphological parameters or geometric properties generally include information about the shape, shape or structure of the particle or about the position of components of the particle.
  • the morphological parameter can advantageously be used to determine whether the particle should be subjected to further processing steps after the measurements.
  • the first measurement preferably comprises a transmitted light measurement or an impedance measurement. These measurements can be quick and beneficial possess exact recording of the morphological parameter. It can also be determined whether particles are present individually or combined to form aggregates. The latter would be undesirable in cloning experiments, for example. However, an electrical or magnetic measurement can also be carried out during the first measurement.
  • a material parameter of the particle which is characteristic of the chemical composition of the particle, is determined in a second measurement.
  • Material parameters or properties generally include information about the chemical composition of the particle or its components. In biological applications, it can advantageously be determined whether the particle, e.g. B. a cell contains certain metabolic products or genetically generated substances.
  • the second measurement preferably comprises a fluorescence measurement, which can have advantages with regard to a high specificity of the detected substances in the particle.
  • an electrical measurement e.g. impedance measurement
  • a magnetic measurement can also be carried out as part of the second measurement.
  • the first measurement is carried out before the second measurement.
  • the morphological parameter is measured before the material parameter.
  • the measurement conditions of the second measurement can advantageously be adapted to the morphological properties of the particle under consideration. For example, a relatively small intensity of the fluorescence excitation can be set for a large particle due to an expected high fluorescence signal.
  • the second measurement is carried out before the first measurement.
  • the material parameter occurs before the morphological parameters are measured, there can be advantages in terms of the throughput of the measurements.
  • the method according to the invention can be carried out with different types of particles, in particular synthetic or biological particles.
  • the particles comprise biological materials, for example biological cells, cell groups, cell components or biologically relevant macromolecules, each in combination with other biological particles or synthetic carrier particles.
  • Synthetic particles can comprise solid particles, liquid particles delimited from the suspension medium or multi-phase particles which form a separate phase in relation to the suspension medium in the channel.
  • manipulation elements in particular dielectric and / or optical elements in the microsystem, are actuated in order to subject the particles in question to a further measurement and / or deflection into a specific area or exit of the microsystem.
  • At least one dielectric cage, at least one dielectric switch and / or at least one optical manipulator are preferably used as dielectric or optical elements, which are arranged downstream relative to the measuring stations.
  • the manipulation elements can also be a porator or generally fluidic components, such as a particle deposition unit.
  • the at least one further measurement carried out as a result of the comparison comprises a measurement of the stationary particle held in the liquid, for example with a dielectric field cage.
  • the downstream manipulation elements can be controlled with greater reliability at the times when the particle under consideration arrives at these elements with the flow.
  • This tracking of the particle movement is also referred to as "cell tracking" and can also take place between the measuring stations. The "cell tracking" can thus take place within the entire channel of the microsystem.
  • the measured variables include the cell speed or the movement speed of the particles, the particle or cell spacing or the flow state.
  • FIG. 1 Further objects of the invention are a measuring device that is adapted to carry out the method according to the invention and a fluidic microsystem that is equipped or connected with such a measuring device.
  • the measuring device is characterized in particular by at least two measuring stations for measuring particle parameters, which are characteristic of different properties of the respective particle, and an evaluation device with a comparator device for comparing the parameters with predetermined expected values, with which, depending on the result of the comparison, a signal can be generated for further measurements or manipulations on the particle.
  • the measuring stations can generally be set up for optical or electrical measurements.
  • the first measuring station preferably comprises a transmitted light detector or an impedance detector, which is combined with a fluorescence detector as the second measuring station.
  • the microsystem according to the invention contains the measuring device as a system component, there may be advantages with regard to the compactness of the structure.
  • the measuring device can be provided as an external device, for example with a microscope structure.
  • the microsystem upstream of the arrangement of the measurement stations is equipped with focusing electrodes for lining up flowing, suspended particles along a straight row parallel to the direction of flow.
  • the row is aimed at the measuring stations, so that the measurements are carried out under essentially identical geometric conditions for all particles.
  • the microsystem downstream of the arrangement of the measuring stations is equipped with further measuring stations, for example at least one dielectric cage, at least one dielectric switch and / or at least one optical and / or magnetic manipulator
  • further measuring stations for example at least one dielectric cage, at least one dielectric switch and / or at least one optical and / or magnetic manipulator
  • the comparator device is connected directly to the further measuring stations, the dielectric cage, the dielectric switch and / or the optical manipulator in order to actuate them with the signal of the comparator device.
  • the carrier flow channel branches into at least two output lines, one of the two output lines serving to discharge negatively selected particles, whereas the other output line serves to continue passing positively selected particles.
  • the particles suspended in the carrier flow channel are focused off-center, for which purpose a focusing device arranged off-center is preferably provided. This is advantageous because the particles suspended in the carrier flow channel and focussed off-center by the focusing device then automatically and precisely defined reach a certain output line if there is no active activation of a switch or switch arranged in the region of the branching point.
  • the particles suspended in the carrier stream on the side of the outlet can be focused off-center for the positively selected particles if, for example, the focusing device is arranged off-center on this side.
  • the particles suspended in the carrier stream automatically get into the output line for the positively selected particles without active control of the switch or switch, whereas active control of the switch or switch is necessary to move the particles into to convey the output line for the negatively selected particles.
  • This arrangement is therefore particularly suitable for investigations in which a large percentage of the particles are selected positively.
  • the electrical cage fulfills two functions, namely on the one hand the fixation of the particles suspended in the carrier stream and on the other hand the function of a switch or a switch to one of the particles suspended in the carrier stream to feed several output lines.
  • the field cage is arranged in the area of the branching point of the output lines.
  • branch point used in the context of the present description is to be understood generally and is not restricted to the geometric intersection of the output lines. Rather, it is also possible for the cage or the switch to be arranged upstream of the intersection of the output lines.
  • branching point also includes the so-called "separatrix". This is the dividing line of the laminar flow in the carrier flow channel.
  • a focusing device can be arranged in at least one of the output lines in order to prevent the particles in the output lines from sinking. countries. This is advantageous since the flow velocity in the output lines decreases from the center of the output lines to the walls, so that the suspended particles could deposit near the wall if the output lines sank, which is prevented by the focusing device.
  • the carrier flow channel is fed from at least two input lines, each of which feeds a carrier flow with suspended particles.
  • the two partial flows with the suspended particles can in this case first be separated from one another in the upstream region of the carrier flow channel by a partition and / or separately from each other by a measuring station.
  • the particles of the individual partial flows can then be combined or derived.
  • FIG. 1 to 4 sections of fluidic invention
  • FIGS. 1 to 4 illustrate various embodiments of fluidic microsystems according to the invention in a schematic partial view.
  • Fluidic microsystems in particular for manipulating biological cells, are known per se and are therefore not described here with further details here. The invention is explained below with reference to the measurement and manipulation of biological cells, without being restricted to this exemplary embodiment.
  • FIG. 1 shows a channel 30 (or a compartment) of the microsystem 100 in a schematic plan view.
  • the channel 30 is delimited by the side walls 31, 32, a bottom 33 and a top surface (not shown).
  • the distance between the side surfaces 31, 32 is preferably in the range from 50 ⁇ m to 5 mm, in particular in the range from 100 ⁇ m to 1 mm, and especially in the range from 200 to 800 ⁇ m (width of the channel), while the distance between the floor 33 and the top surface preferably around 5 microns to 200 microns, for. B. 20 to 100 microns (height of the channel).
  • the microsystem 100 preferably consists of a transparent material, for example glass or plastic, walls with optical quality (such as, for example, microscope cover glasses) being used at least in the area of the measuring stations in the detection direction.
  • a flow of liquid flows through the channel 30 in the direction of the arrow.
  • the liquid flow is typically a laminar flow with a flow rate in the range of e.g. B. 20 ⁇ m / s to 20 mm / s.
  • Cells 20, 21, 22 ... which are to be detected with the method according to the invention are suspended in the liquid flow.
  • the cells move in the direction of flow at the same speed as the liquid.
  • a measuring device 10 according to the invention is provided in the channel 30, which generally comprises two schematically illustrated measuring stations 11, 12.
  • the measuring stations generally set up for optical measurements are constructed as is known per se for scattered light, transmitted light, phase contrast or fluorescence measurements. They include an illumination device and a detector device (not shown in detail).
  • the lighting device is used depending on the measurement would be pure lighting or excitation of fluorescent light and includes, for example, a laser light source.
  • Each detector device is set up to detect the light transmitted, shadowed, scattered or emitted by a cell and, depending on the measurement task, comprises one or more light-sensitive elements, e.g. B. photodiodes or CCD lines or arrays. The detection can be provided directly or after optical magnification with a microscope from the top or bottom surface.
  • the lighting device can be designed for local fluorescence excitation. This can be associated with the advantages that, in the case of local excitation, there is no interference with the first optical detection (phase contrast), several local excitation spots can be realized and the cell load is reduced. These advantages have not been achieved with conventional detection techniques.
  • microsystem 100 can be covered with a shielding scattered light mask (dashed line) on the top or bottom surface drawn).
  • the mask is positioned opposite to the fluorescence detector device.
  • the mask is located, for example, on the upper side (top surface) of the microsystem. This embodiment is particularly advantageous for improving the signal-to-noise ratio in weakly fluorescent cells.
  • the mask is made of an opaque material to hide the transmitted light that is fed in from the other side. al, in particular an absorbent layer.
  • the mask can comprise, for example, a highly reflective metal layer or a highly absorbent material, for example blackened Pt.
  • a metal layer may require an appropriately tuned dichroic mirror so that the reflected fluorescence excitation light does not interfere with the fluorescence emission measurement.
  • a Pt layer can easily be processed electrolytically from "normal" Pt in the channel. It would reflect the transmitted light and absorb the fluorescence (excitation and emission). This variant is particularly advantageous since the fluorescence excitation (eg 488 nm) then does not the fluorescence emission measurement and transmitted light measurement.
  • the measuring device 10 is connected to an evaluation device 40 which is only illustrated schematically in FIG. 1 and which serves to evaluate the detector signals and in particular contains a comparator device 41, the function of which is explained below.
  • a focusing device 50 is provided upstream of the measuring stations 11, 12.
  • the funnel-shaped focusing device 50 is used to line up the cells in a straight line along a pass-through area with a width corresponding to the width of the detectors of the measuring device 10.
  • the focusing device 50 comprises focusing electrodes in the manner known per se in the form of straight electrode strips, each on the top surface and / or on the Bottom 33 are directed from the channel edge to the center of the channel.
  • the ends of the focusing electrodes are spaced apart from one another so that the passband is formed.
  • the focusing electrodes are each connected to a control device (with a high-frequency voltage source) via a connecting line (not shown).
  • a dielectric field cage 60 Downstream after the measuring stations 11, 12 is a dielectric field cage 60 with a group of eight strip-shaped ones Electrodes are provided in which a cell 25 can be held and measured in a manner known per se under the action of high-frequency fields.
  • the measurement in the field cage 60 includes, for example, an electrorotation measurement or a high-resolution fluorescence measurement.
  • the implementation of the method according to the invention comprises the following steps. First, the cells 21 with the liquid flow in a disordered manner through the channel 30 until they reach the focusing device 50. A funnel-shaped field barrier is formed on this with the focusing electrodes, which narrows in the direction of flow. In the lineup thus formed, the cells 20 pass the measuring stations 11, 12 on a common trajectory.
  • a transmitted light measurement is carried out as the first measurement, for example using the phase contrast method.
  • a first positive signal is generated which represents the time of the passage.
  • the transmitted light measurement provides an image or, when the detector device is masked, a partial image of the cell 24 which, as morphological parameters, provides information about, for example, the type of the object, the object size or shape or the object state (for example living / dead cell).
  • the speed of the object can be determined with a suitable mask shape.
  • a fluorescence measurement is carried out at the second measuring station 11 as a second measurement, offset in time and space. As soon as the cell appears above the second measuring station 12, a second positive signal is generated. Furthermore, the fluorescence measurement provides information as to whether the cell is loaded with a specific marker, for example, or contains a specific substance (e.g. expression of genes by means of GFP).
  • the fluorescence measurement can be carried out like the transmitted light measurement, a detector mask known per se (see above) can be provided.
  • the reverse order of detection can be provided by first measuring the fluorescence and then the phase contrast. This can be advantageous if the fluorescence events are very rare.
  • the positive signals and the detector signals of the measuring stations are supplied to the evaluation device 40.
  • the correlation of the positive signals allows the determination of the object speed and thus the triggering for switching further dielectric and / or optical elements, for example the dielectric field cage 60.
  • the detector signals are also compared with the comparator device 41 with certain expected values stored in the evaluation device in order to obtain control signals for downstream dielectric and / or optical elements.
  • the detector signals of the measuring stations are compared with certain expected values in relation to the cells (living / dead) and the intensity during the fluorescence detection (threshold value comparison). If both prerequisites for a particular cell are met, the field cage 60 is automatically timed, so that the cell is caught, and the liquid flow in the channel 30 is stopped for the duration of the high-resolution microscopic examination. For this, the lighting device of the second measuring station 12 can advantageously be moved to the field cage 60.
  • Dead or non-fluorescent cells would be registered, but would not be found in the field cage 60. be.
  • the throughput is advantageously increased thereby, which has an effect particularly in the "rare event sorting", in which rare events are searched for in a large number of samples (for example 1: 100).
  • downstream dielectric switches can be actuated in order to deflect certain cells into an adjacent channel or to couple them out of the microsystem into a cultivation device.
  • the deflection into an adjacent channel takes place in reverse to the principle shown in FIG. 4.
  • the first measuring station 11 comprises an impedance detector 13.
  • the impedance detector 13 is arranged in the flow direction at a distance in the range of, for example, 10 ⁇ m to 2 mm from the focusing device 50.
  • the impedance detector comprises, in a manner known per se, for example two or four detector electrodes which are arranged on the top surface and / or on the bottom 33 of the channel 30 and are subjected to alternating voltages for measuring the impedance in the channel 30.
  • a measurement signal is obtained, from which the size of the cell and possibly the vitality state can be derived analogously to the optical measurement. Disruptive stray light effects between the measuring stations can advantageously be avoided.
  • the interaction with the second measuring station 12 and the evaluation device takes place as described above.
  • FIG. 3 shows an embodiment of the invention in which further measuring stations 14, 15,... Are arranged after the first and second measuring stations 11, 12.
  • the measuring stations 14, 15, ... like the measuring station 12, are set up for a fluorescence measurement and are also connected to the evaluation device (not shown).
  • This design enables that initially a first cell parameter (for example the size) at the measuring station 11 and then a second cell parameter (for example the fluorescence intensity) is repeatedly measured at the measuring stations 12 and 14, 15, ...
  • a sequence of fluorescence intensities is determined, which is characteristic of the kinetics of a change in the cells as they pass through the measuring device 10.
  • cells 20 suspended in a fluorescent dye solution are flowed through the channel 30 and lined up with the focusing device 50.
  • dye increasingly accumulates in the cells.
  • the degree of enrichment can be quantified at the measuring stations (e.g. recording of calcium kinetics or progressive loading with a fluorescent substance, e.g. CalceinAM).
  • the fluorescence can be measured as a function of time specifically for the individual cells.
  • FIG. 4 illustrates an application of the invention in a two-channel system.
  • the microsystem 100 comprises two channels 34, 35, which are connected via a passage opening 36 in a partition 37.
  • a measuring device 10 and further measuring stations are located analogously to FIG. 3 in channel 35.
  • Cells 20 suspended in a carrier liquid are flushed into channel 34.
  • a combined dielectric element 50 which combines a deflection function and the above-mentioned funnel function, can be used to transfer the cells into the second channel 35 and to arrange them there dielectrically.
  • In the channel 35 is generally a different chemical environment or z.
  • the dye accumulates in the transferred cells over time.
  • a fluorescence readout and a measurement of the kinetics of the dye enrichment take place at predetermined measuring locations.
  • the cells are stimulated with area lighting (object field of view). To reduce bleaching, the coupling of local excitation and detection is advantageous.
  • the deflection function of the combined dielectric element 50 can be set in such a way that certain cells pass the field barrier in the channel 34 (for example small cells, which only a small dielectrophoretic force acts in comparison to the hydrodynamic force), while the other cells enter the channel 35 are distracted (large cells).
  • the transfer into the channel 34 can also be controlled in dependence on the detector signals of the measuring device 10 in such a way that it is prevented that there are too many cells in the area of the measuring device 10 at the same time.
  • an examination can also be carried out in the channel 34, wherein the examination methods described above can be used.
  • a dielectric element can be provided with which incoming particles, in particular cells, are lined up separately on several trajectories and fed to separate measuring devices which are arranged in or on the microsystem.
  • FIG. 5 largely corresponds to the exemplary embodiment described above and shown in FIG. 1, so that in order to avoid repetition, reference is made to the above description and only the special features of this exemplary embodiment are described below.
  • the same reference numerals are used in the following for corresponding components as in the description of FIG. 1 in order to facilitate the assignment.
  • a special feature of this exemplary embodiment is that in the channel 30 between the focusing device 50 and the field cage 60 upstream in front of the measuring station 11 conical electrode arrangement 70 is arranged, which makes it possible to hold particles suspended in the carrier stream and to park them in front of the measuring station.
  • the structure and the mode of operation of the electrode arrangement 70 is described, for example, in Müller, T. et al .: “Life Cells in Cell Processors" in Bioworld 2, 2002, so that a detailed description of the electrode arrangement 70 can be dispensed with and the content of this document of this description is fully attributable.
  • a further special feature of this exemplary embodiment is that the channel 30 branches downstream behind the field cage 60 into two output lines 71, 72 in order to select the particles suspended in the carrier stream depending on the test result of the measuring stations 11, 12.
  • the output line 71 is used to receive and discharge negatively selected particles, whereas the output line 72 is used to receive and continue positively selected particles.
  • the distribution of the particles to the two output lines 71, 72 takes place here by a further electrode arrangement 73, which is arranged in the region of the branching point of the two output lines 71, 72 and drives the positively selected particles into the output line 72 when electrically controlled.
  • the structure and mode of operation of the electrode arrangement 73 is described, for example, in Müller, T. et al .: “A 3-D microelectrode system for handling and caging single cells and particles” in Biosensors & Bioelectronics 14 (1999) 247-256, see above that a detailed description of the electrode arrangement 73 can be dispensed with and the content of the above-mentioned document is to be fully attributed to the present description.
  • the focusing device 50, the electrode arrangement 70, the two measuring stations 11, 12, the field cage 60 and the electrode arrangement 73 are arranged eccentrically in the channel 30 on the side of the output line 71.
  • the result of this is that the particle stream that is eccentrically focused by the focusing device 50 in the channel 30 automatically reaches the output line 71 for negatively selected particles without any external influence, whereas active control of the electrode arrangement 73 is required to transfer the particles into the output line 72 for to transport positively selected particles.
  • This eccentric arrangement is therefore particularly suitable for examinations in which only a few particles are selected positively, since the electrode arrangement 73 is then rarely activated.
  • a further focusing device 74 is arranged in the output line 72 for the positively selected particles, which corresponds in structure and function to the focusing device 50 in the channel 30.
  • the purpose of the focusing device 74 is to prevent the particles in the outlet line 72 from sinking and to keep the particles in the outlet line 72 in the middle in the region of high flow velocities. This is advantageous since the flow velocity in the outlet line 72 decreases from the center outwards, so that the particles could deposit near the wall when the outlet line 72 sinks, which is prevented by the focusing device 74.
  • this exemplary embodiment has two sheath flow feed lines 75, 76 which open downstream of the focusing device 74 into the output line 72 and feed a sheath flow, in order to ensure, for example, a quick and safe sample storage.
  • the exemplary embodiment shown in FIG. 6 largely corresponds to the exemplary embodiment described above and shown in FIG. 5, so that in order to avoid repetition, reference is largely made to the above description, while only the special features of this exemplary embodiment are described below.
  • a special feature of this exemplary embodiment is that the field cage 60 is arranged in the branching region of the two output lines 71, 72 and in this case fulfills two functions, namely on the one hand the fixing of the particles for examination by the measuring station 12 and on the other hand the distribution of the particles over the two Output lines 71, 72.
  • FIG. 7 largely corresponds to the exemplary embodiment described above and shown in FIG. 6, so that in order to avoid repetition, reference is largely made to the above description, while only the special features of this exemplary embodiment are described below.
  • a special feature of this exemplary embodiment is the structural design of the field cage 60, which here consists of six electrodes arranged in a spatially distributed manner.
  • the field cage 60 is also bifunctional here and enables both the fixing of particles in the carrier stream for examination by the measuring station 12 and the distribution of the particles on both output lines 71, 72.
  • FIG. 8 largely corresponds to the exemplary embodiment described above and shown in FIG. 7, so that in order to avoid repetition, reference is largely made to the above description, while only the special features of this exemplary embodiment are described below.
  • a special feature of this exemplary embodiment is that a sorting element 77 in the form of an electrode arrangement is arranged in the channel 30 at the branching point of the two output lines 71, 72, in order to sort the particles suspended in the particle stream depending on the examination by the measuring station 11, 12 to supply the two output lines 71, 72.
  • the sorting device is also referred to as an "Ultra-Fast Sorter” (UFS) and enables the suspended particles to be sorted quickly.
  • UFS Ultra-Fast Sorter
  • the arrow electrodes lying one above the other are permanently activated, while the deflection into the output line 71 or into the output line 72 takes place by switching the lower or upper pair of input electrodes of the sorting device 77. It should be mentioned here that the lateral distance between the electrodes is less than the vertical distance.
  • FIG. 9 shows a further exemplary embodiment, in which the channel 30 has two input lines 78, 79, which open into the channel 30 and in each case feed a carrier stream with suspended particles.
  • a focusing device 80, 81 is arranged in each of the two input lines 78, 79 in order to focus the particles contained in the two carrier streams of the input lines 78, 79.
  • a dividing wall 82 is arranged in the channel 30 upstream in the region of the junction of the input lines 78, 79. before the carrier streams supplied via the two input lines 78, 79 initially separate from one another in the upstream region of the channel 30.
  • the partition 82 is only optional, so that the partition 82 can also be dispensed with.
  • a measuring station 83, 84 is arranged on each side of the partition 82 in the channel 30 in order to examine the particles suspended in the two partial flows.
  • the particles are either focused in the center by a focusing device 85 and fed to the field cage 60, or they flow past the field cage 60 laterally and reach two output lines 86, 87 for negatively selected particles.
  • a further measuring station 88 is located in the channel 30, which makes an examination of the suspended particles in the field cage 60, i.e. in the braked state.
  • the particles are then fed through a sorting device 89 either to one of the two output lines 86, 87 for negatively selected particles or to another output line 90 for positively selected particles.
  • FIG. 10 largely corresponds to the exemplary embodiment described above and shown in FIG. 5, so that reference is largely made to the above description and only the special features of this exemplary embodiment are described below.
  • a special feature of this exemplary embodiment is that no field cage is arranged in the channel 30 around which the To fix carrier flow suspended particles. The examination of the particles suspended in the carrier flow takes place during their flow movement.
  • Another special feature of this exemplary embodiment is the constructive design of the electrode arrangement 73, which is designed here as a double electrode.
  • the focusing device 50, the electrode arrangement 70, the two measuring stations 11, 12 and the electrode arrangement 73 are likewise arranged eccentrically on the side of the output line 71 for the negatively selected particles, so that the electrode arrangement 73 must be actively controlled in order to avoid the in the carrier current to convey suspended particles into the outlet line 72 for positively selected particles.
  • This eccentric arrangement is therefore particularly suitable for examinations in which only a small percentage of particles are positively selected.
  • FIG. 11 largely corresponds to the exemplary embodiment described above and shown in FIG. 10, so that in order to avoid repetition, reference is made to the above description and only the special features of this exemplary embodiment are explained below.
  • a special feature of this exemplary embodiment is that the focusing device 50, the electrode arrangement 70, the two measuring stations 11, 12 and the electrode arrangement 73 are arranged eccentrically in the channel 30 on the side of the output line 72 for positively selected particles. Without active activation of the electrode arrangement 73, the particles suspended in the carrier stream and focused off-center by the focusing device 50 in the channel 30 thus automatically reach the output line 72 for positively selected parameters. particles, whereas the electrode arrangement 73 must be actively controlled in order to convey the particles suspended in the carrier stream into the outlet line 71 for negatively selected particles.
  • This eccentric arrangement is therefore particularly suitable for examinations in which only a small percentage of the suspended particles are selected negatively.
  • FIG. 12 largely corresponds to the exemplary embodiment described above and shown in FIG. 10, so that in order to avoid repetition reference is made to the above description and only the special features of this exemplary embodiment are explained below.
  • a special feature of this exemplary embodiment is that the channel 30 branches downstream under the two measuring stations 11, 12 into three output lines 91, 92, 93, so that the particles suspended in the carrier stream can be divided into three classes.
  • FIG. 13 largely corresponds to the exemplary embodiment described above and shown in FIG. 12, so that in order to avoid repetition, reference is made to the above description of FIG. 12 and only the special features of this exemplary embodiment are explained below.
  • a special feature of this exemplary embodiment is that the two measuring stations 11, 12 do not lie one behind the other in the direction of flow, but are laterally offset from one another with respect to the direction of flow, a deflection device 94 being arranged between the two measuring stations 11, 12, which deflects the side in the Carrier current suspending particles. Without active activation of the deflection device 94, the particles suspended in the carrier stream and focused by the focusing device therefore only pass the measuring station 11 and flow past the measuring station 12 laterally.
  • the deflection device 94 When the deflection device 94 is actuated, on the other hand, the particles suspended in the carrier stream are deflected laterally to the measuring station 12 after leaving the measuring station 11 and also pass through this.
  • the deflection device 94 is activated as a function of the result of the examination by the measuring station 11.

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Abstract

L'invention concerne un procédé pour mesurer des propriétés de particules qui se déplacent à travers un microsystème fluidique en suspension dans un liquide. Le procédé selon l'invention comprend les étapes suivantes : une première mesure d'un premier paramètre d'une particule définie au niveau d'une première station de mesure (11), une deuxième mesure d'un deuxième paramètre de la particule après un intervalle de temps par rapport à la première mesure au niveau d'une deuxième station de mesure (12) séparée dans l'espace de la première station de mesure (11), et une évaluation corrélée commune du premier et du deuxième paramètre. Le procédé selon l'invention est caractérisé en ce que le premier et le deuxième paramètre sont caractéristiques de différentes propriétés de la particule mesurée et en ce que l'évaluation comprend une comparaison du premier et du deuxième paramètre avec des valeurs attendues prédéfinies, une autre mesure ou des manipulations sur la particule mesurée étant effectuées en fonction du résultat de la comparaison. L'invention concerne également un microsystème fluidique servant à mettre en oeuvre ce procédé.
EP04707910A 2003-02-05 2004-02-04 Detection multiparametre dans un microsysteme fluidique Withdrawn EP1590653A1 (fr)

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DE10304653 2003-02-05
DE10304653A DE10304653B4 (de) 2003-02-05 2003-02-05 Mehrparametrige Detektion in einem fluidischen Mikrosystem
DE10320956 2003-05-09
DE10320956A DE10320956B4 (de) 2003-02-05 2003-05-09 Untersuchungsverfahren für biologische Zellen und zugehörige Untersuchungseinrichtung
PCT/EP2004/001031 WO2004070361A1 (fr) 2003-02-05 2004-02-04 Detection multiparametre dans un microsysteme fluidique

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EP04707905A Withdrawn EP1590652A1 (fr) 2003-02-05 2004-02-04 Procede de triage et d'identification de cellules a plusieurs parametres et dispositif correspondant

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WO2004070361A1 (fr) 2004-08-19
US20060139638A1 (en) 2006-06-29
US20060152708A1 (en) 2006-07-13
EP1590652A1 (fr) 2005-11-02
JP2006517292A (ja) 2006-07-20
DE10304653B4 (de) 2005-01-27
WO2004070362A1 (fr) 2004-08-19
DE10304653A1 (de) 2004-08-19

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