EP1843854B1 - Device and method for the elimination of magnetic or magnetizable particles from a liquid - Google Patents

Abstract

A device (10) is provided for separating magnetic or magnetizable particles from a liquid by using a magnetic field. The device includes a head piece (3) with one or more magnetizable bars (4) which is/are permanently or detachably connected with the head piece (3), as well as one or more permanent magnets (1) whose relative position with respect to the head piece can be changed by a predeterminable movement of the magnet(s) or/and by a predeterminable movement of the head piece.

Description

The invention relates to devices for separating and resuspending magnetic or magnetizable particles from liquids by means of a magnetic field generated by one or more permanent magnets. The invention further relates to methods for separating magnetic or magnetizable particles from liquids and mixing and resuspending magnetic or magnetizable particles in liquids by means of a magnetic field generated by one or more permanent magnets. The devices and methods are, for example, suitable for applications in pharmaceutical development, biochemistry, molecular genetics, microbiology, medical diagnostics and forensic medicine.

Methods based on magnetic separation using specific binding, magnetically attractable particles, or with which it is possible to mix magnetic or magnetizable particles in solutions, are obtained in the field of sample preparation for diagnostic or analytical studies or the performance of diagnostic or analytical studies increasing importance. This is especially true for automated processes, since in this way a large number of samples can be prepared or analyzed within a short time and can be dispensed with laborious Zentrifugationsschritte. This will create the conditions for efficient, cost-effective screening with high sample throughput. This is of enormous importance, for example, for applications in molecular genetic studies or in the field of medical diagnostics, since a purely manual handling of very large numbers of samples is practically unmanageable or payable.

Further important fields of application concern pharmaceutical screening methods for the identification of potential active pharmaceutical ingredients.

The basic principle of the magnetic separation of substances from complex mixtures is based on the fact that magnetic particles (magnetizable or magnetically attractable particles) are specifically functionalized for the intended separation process, d. H. they are provided by chemical treatment with specific binding properties for the target substances to be separated. The size of such magnetic particles is generally in the range of about 0.05 to 500 microns.

Magnetic particles which have specific binding properties for certain substances and are suitable for separating these substances from complex mixtures are described, for example, in US Pat DE 195 28 029 A1 and are commercially available (eg, Chemagen Biopolymer-Technologie AG, DE-52499 Baesweiler).

In known separation processes, the functionalized magnetic particles are added in a first step ("binding step") to a mixture to be purified, which contains the target substance (s) in a liquid which favors the binding of the target substance molecules to the magnetic particles (binding buffer). This results in a selective binding of the target substance (s) present in the mixture to the magnetic particles. Subsequently, these magnetic particles are immobilized by application of magnetic forces or a magnetic field, for example by means of a permanent magnet, at a location of the reaction vessel inner wall ("pellet"). Subsequently, the liquid supernatant is separated and discarded, for example by suction or decantation. Since the magnetic particles are immobilized in the aforementioned manner, it is largely prevented that these particles are separated with the supernatant.

Subsequently, the immobilized magnetic particles are resuspended again. In this case, an elution liquid or an elution buffer is used, which is suitable for dissolving the bond between the target substance (s) and the magnetic particles, so that the target substance molecules are released from the magnetic particles and can be separated with the elution liquid, while the magnetic particles are immobilized by the action of a magnetic field. Before the elution step, one or more washing steps can be carried out.

Functionalized accordingly, the magnetic particles can also be used directly for diagnostic or analytical studies. The functionalization allows specific binding, e.g. of pathogenic substances. However, in order to be able to make as clear a statement as possible regarding, for example, a pathogenic substance, all impurities must be removed in suitable solutions. This requires the most efficient possible mixing (washing) of the analyte-containing particles. Such a process facilitates the present invention, especially if the number of simultaneously processed samples is large or must be worked in small volumes (384 or 1536 format).

Various devices have been described for performing magnetic particle separation processes. DE 296 14 623 U1 discloses a magnetic separator equipped with movable permanent magnets. Alternatively, it is proposed to move the magnetic particles containing reaction vessel by mechanical drive means relative to a permanently installed permanent magnet. In the DE 296 14 623 U1 described device has no magnetizable rods that dip into the sample liquid; rather, the permanent magnets each positioned next to the individual reaction vessels.

After a similar principle also works in DE 100 63 984 A1 described device having a movable magnet holder and a movable reaction vessel holder, wherein the magnets can be positioned laterally from the reaction vessels.

With the above-mentioned devices can be achieved that the magnetic particles are immobilized or collected on the inner wall or at the bottom of a reaction vessel as a "pellet". However, these devices are not suitable for removing the magnetic particles from a reaction vessel. Therefore, to separate the liquid from the magnetic particles in each case the liquid must be sucked from the individual reaction vessels. This is disadvantageous because it involves a high material consumption (disposable pipette tips). Furthermore, it can not be prevented that individual magnetic particles are sucked with; This results in a high error rate. Further errors can be caused by dripping liquids, which lead to cross contamination.

In DE 100 57 396 C1 For example, a magnetic separator is proposed which has a plurality of rotatable rods that can be magnetized by an electromagnetic exciter coil. By immersing the rod in the magnetic particles containing liquid and pulling out the rod in the magnetized state, the magnetic particles can be removed from the liquid and optionally transferred to another reaction vessel. There they can be released by switching off the excitation coil back into a liquid, for example in a washing or elution liquid.

A disadvantage of this device, that the excitation coil has a relatively large amount of space, resulting in constructive limitations. In addition, the geometry and geometry of the electromagnet are constrained in terms of the arrangement and number of rods, which may include limitations on sample processing. The geometry of the electromagnet, however, can not be changed arbitrarily, since then inhomogeneities of the magnetic field must be taken into account.

• ein Kopfstück mit einem oder mehreren magnetisierbaren Stäben aufweist, welche(r) fest oder lösbar mit dem Kopf stück verbunden ist/sind;
• einen oder mehrere Permanentmagnete aufweist, dessen/deren relative Position bezüglich des Kopfstücks durch eine vorherbestimmbare Bewegung des/der Magneten oder/und durch eine vorherbestimmbare Bewegung des Kopfstücks in der Weise veränderbar ist.

The document WO 2005/005049 A , is considered to be the closest prior art. It discloses an apparatus for separating magnetic or magnetizable particles from a liquid using a magnetic field, wherein the apparatus

• a head piece with one or more magnetizable bars, which is (r) fixed or detachably connected to the head piece / is;
• one or more permanent magnets whose relative position with respect to the head piece is variable by a predetermined movement of the / or the magnet and / or by a predetermined movement of the head piece in such a way.

Above all, the known devices are not suitable for the simultaneous treatment of larger numbers of samples, as required for high-throughput applications (eg 364 or 1536 well plates). The design effort would be immense and in addition a significantly higher susceptibility of the mechanics used would have to be accepted.

The known devices are also disadvantageous because they are only suitable for individual sample vessels or only for a specific, fixed predetermined arrangement of the sample vessels, for example in the form of a 96-well microtiter plate. However, for practical purposes it is desirable that such a magnetic separator apparatus be suitable or capable of being adapted for various types of sample vessels or for various types of sample vessels (e.g., 96, 364 or 1536 well plates).

The invention therefore an object of the invention to provide devices and methods with which the separation of magnetic particles from liquids and the transfer of magnetic particles from a liquid is made possible in another liquid, without causing the aforementioned disadvantages occur. In particular, the devices and methods should be suitable for use in high throughput processes. The devices should be multi-sided can be used and in particular suitable for different types of reaction vessels.

These and other objects are surprisingly achieved by a device and method defined in the independent claims, and by the embodiments described in the dependent claims.

The operation of the device is based on the fact that the / the permanent magnet (s) over the head piece (with the rods attached thereto) can be positioned. This causes the magnetization of the rod (s). This state of the device is referred to as "on". When the magnetizable rods are immersed with their lower end or portion, for example, in a sample liquid having magnetic particles therein, the magnetic particles will adhere to the lower end of the rods due to the magnetic forces. The sticks with the adhered magnetic particles can then be dipped in another liquid (e.g., a reagent or wash solution).

When the permanent magnet (s) is now moved out of the position above the head piece, the magnetization of the bars can be canceled out, so that the magnetic particles fall off the bars or can be detached by shaking movements. This state of the device is referred to as "off". The movement of the magnet (s) allows rapid switching between the on state and the off state of the magnetic separator.

According to the invention, the permanent magnets are / are movably arranged relative to the head piece, so that the magnetization of the magnetizable bars attached to the head piece can be alternately switched on and off by movement of the magnet (s). The magnet (s) is / are moved over the head piece or away from the head piece.

Alternatively, the magnetization or demagnetization can also be achieved by moving the head piece under the magnet (s) or away from the magnet, in which case the magnet (s) are preferably arranged stationary.

By the movement of the head piece (with the magnetizable bars attached thereto), it can assume a first position, in which the head piece is located below the area of the permanent magnet (switched-on state), or it can assume a second position in which it is located is outside the specified range (switched off state).

The ability to temporarily magnetize the rods, the device can be used to remove magnetic particles using the magnetizable rods from a first liquid and transferred to a second or more liquid and release there again.

The arrangement of a permanent magnet, which may also be composed of a plurality of individual magnets, a substantially homogeneous magnetic field is generated. Thereby the arrangement of a larger number of rods, for example in several rows, is made possible, wherein the magnetic field at each of the bars is approximately equal; This is particularly advantageous in terms of reproducibility of high throughput processes. The devices according to the invention also have the advantage that the magnetic particles - in the magnetized state - accumulate substantially at the top of the rods whereby the adhering substances to be separated can be absorbed in comparatively small elution volumes. This guarantees - for diagnostic or analytical investigations essentially high concentrations of the substances to be separated.

In principle, all hard magnetic materials known to the person skilled in the art can be used for producing the permanent magnets, in particular ferrites, Al-Ni-Co alloys and rare earth magnets (preferably NdFeB); Such magnetic materials and magnets are commercially available from various manufacturers.

The number of magnetizable rods attached to the header depends on the number of samples or the number of wells in liquid containers that are to be treated simultaneously at most. Microtiter plates, in particular with 96, 384 or 1536 depressions ("wells") are preferably used as containers, so that corresponding numbers of magnetizable bars are provided for these cases. Also suitable as containers are sample tubes or reaction vessels having a volume of, for example, 0.015 to 100 ml, which can be treated individually or in groups, in each case in combination with magnetizable bars adapted thereto.

The magnetizable rods, optionally also the head piece, are preferably made of a soft magnetic material, for example of soft iron (in particular Fe-Ni alloys) or magnetizable steel. Their length and diameter depend on the intended application, in particular on the dimensions of the containers and the volumes of liquid, and can be varied accordingly. If a group of multiple rods (for example 96, 384 or 1536) is used, then these rods each have the same length, thickness and material condition. The rods may optionally be hollow inside, ie be formed as tubes, wherein the lower end is preferably closed. In particular, the rods may be shaped as sleeves, as described below.

In general, the magnetic bars are substantially perpendicular and parallel to each other, and the individual bars of a group or array are preferably equidistant from adjacent bars. Particularly preferred is a grid-like arrangement of the rods, which corresponds to the arrangement of the wells in conventional microtiter plates.

The invention therefore also extends to arrangements of magnetizable bars in which a plurality of magnetizable bars (4) are mounted on a base plate (9), the bars being substantially parallel to one another and preferably arranged in one, two or more rows are, each row comprising two or more rods.

The magnetizable rods, whether fixed or detachably connected to the head piece, preferably have a thickness of 0.5 mm to 10 mm, in particular 1 to 5 mm. The length of the rods is preferably 1 to 20 cm, in particular 5 to 10 cm. In order to firmly connect the rods, as mentioned, with the head piece, conventional means may be used (eg glued connection, screw connection, welding).

According to a further embodiment, the head piece (without rods) may be wholly or partly made of a non-magnetic or non-magnetizable material.

For releasable attachment of the magnetizable rods, these are preferably connected by means of a clamping connection to the head plate. For example, the head piece may be provided on its underside with corresponding recesses or holes into which the rods can be inserted. Alternatively, rods may be used which are tubular or at least have a recess at their upper end and which can be plugged onto corresponding pins or protrusions provided on the underside of the header.

Preferably, the head piece is provided with a mechanism which releases the clamping connection between the bars and the head piece, thus causing the bars to be pushed off or thrown off when they are to be replaced by unused bars after use. This may preferably be accomplished by electromotive drive, or by pneumatic, electromagnetic or hydraulic means, or by a combination thereof.

According to a preferred embodiment, the magnetizable rods are attached to a base plate and form a unit with this. In this case, it is preferable that the base plate can be detachably connected to the header. The base plate and the rods located thereon can optionally also be made in one piece.

The production of base plates with rods attached thereto can be carried out by means of known materials and processes for the production of molded parts, for example by deep-drawing, extrusion, welding, gluing, etc. Preferably, the units consisting of Base plate and connected rods, manufactured and used as Disposables.

According to another preferred embodiment, the base plate is provided with a plurality of magnetizable bars arranged in one or more rows, each row containing a plurality of bars. The rods are preferably arranged in a regular matrix, for example in accordance with the arrangement of the wells of a microtiter plate (in particular a microtiter plate with 96, 384 or 1536 wells). The base plate generally has a rectangular or square outline.

By using a releasably secured baseplate (with rods thereon) it is possible to easily convert the device so that it is suitable, for example, for different types of microtiter plates.

The releasable connection between said baseplate and the header may be effected in a manner known to those skilled in the art, e.g. by clamping, clamping or clamping devices, levers, springs, etc ..

According to a particularly advantageous embodiment, the device has means by which the rods, or the base plate with the rods located thereon, can be detachably connected to the head piece and / or removed from the head piece. Said means will preferably be actuated by electromotive drive, or by pneumatic, electromagnetic or hydraulic means, or by a combination thereof. In this way, the picking up, attaching and dropping of the base plate can be done automatically, and these operations can be controlled by, for example, a program.

According to a further, particularly preferred embodiment of the invention it is provided that the said rod (s) - regardless of whether they are fixed or detachable, with or without base plate, are connected to the head piece - each with a strippable, replaceable Sleeve are provided. This has the advantage that the sleeve between the individual working cycles can be replaced and renewed, so that cross-contamination between different samples or the carryover of sample material can be prevented.

Preferably, the sleeves are - depending on the dimensions of the rods - dimensioned so that they can be fixed by means of clamping connection to the rods. In order to facilitate the insertion of the sleeves, it is advantageous if the group of sleeves is provided on a dispensing device, wherein the arrangement of the sleeves on the dispenser device corresponds to the respective arrangement of the magnetizable bars (eg arrangement corresponding to the distances between the individual Wells of a microtiter plate).

It when several sleeves are connected to each other and form a common unit is particularly advantageous. In this way, the replacement of the sleeves is much easier. Preferably, the number and arrangement of the sleeves on such a unit corresponds to the respective number and arrangement of the magnetizable bars.

The invention also extends to arrays of sleeves suitable for use with any of the devices of the invention; in particular to arrangements with a plurality of sleeves (8) which are attachable to the magnetizable bars of said device and are arranged substantially parallel to each other. Preferably, the sleeves are in one, two or more rows arranged, wherein each row comprises two or more rods.

The sleeves mentioned can be made of known materials, for. As plastics such as polyethylene, polypropylene, Teflon, polyethylene terephthalate, nylon, polyvinyl chloride, etc., or from metallic materials such as stainless steel, tinplate, aluminum foils, etc., or combinations of such materials, in a manner known in the art are made (in particular by injection molding or deep drawing).

Further, it is also possible to make the sleeves or the sleeves connected to a common unit of a magnetizable material (as mentioned above).
In this case, the magnetizable sleeves or the unitized magnetizable sleeves assume the function of the above-described magnetizable rods or the magnetizable rods connected to a base plate.
Preferably, the units formed from groups of sleeves are manufactured as disposables and used to eliminate contamination.

According to a further advantageous embodiment, the device according to the invention comprises means by which the interchangeable sleeves or the sleeves forming a common unit on the rods - or at the head of the device - recorded, held and / or by the rods (or of the head piece ) can be removed or discarded. Said means are preferably operated by electromotive drive, or by pneumatic, electromagnetic or hydraulic means, or by a combination thereof. In this way, the recording, attaching and dropping the sleeves can be done automatically or automatically, in particular programmatically.

The apparatus may further comprise means by which individual envelopes, arrays of envelopes, or interconnected envelopes are automatically or programmatically provided (e.g., disposed in a stand or dispenser) so that they can be received by the bars.

In order to enable the replacement of the sleeves, or the sleeves forming a common unit, they can - as mentioned - be clamped on the rods; alternatively or additionally, the sleeves on the specialist z. B. by clamping, clamping or clamping devices, levers, springs, etc. are attached to the rods and / or with the top plate or other parts of the device.

According to a further preferred embodiment it is provided that the head piece of the device is arranged to be movable and can be set in motion by a drive device. Electromotive, pneumatic, electromagnetic or hydraulic drive means, or a combination thereof, are particularly suitable for the drive device.

• Translationsbewegungen in einer horizontalen Ebene;
• Bewegungen entlang einer Kreisbahn, einer elliptischen Bahn oder unregelmäßigen Bahn, jeweils in einer horizontalen Ebene;
• Bewegungen in vertikaler Richtung.

Preferably, the head piece is movably arranged in such a way that it can perform one or more of the following types of movement:

• Translation movements in a horizontal plane;
• Movements along a circular path, an elliptical path or an irregular path, each in a horizontal plane;
• Movements in the vertical direction.

The vertical direction corresponds substantially to the longitudinal direction of the substantially vertically oriented magnetizable rods.

In particular, the vertical movements serve to effect the immersion of the rods into the sample liquid and the withdrawal of the rods from the liquid. The horizontal movements may be used in particular for performing shaking or vibrating movements (e.g., circular or orbital shaker movements). Suitable mechanisms by which the mentioned types of movement can be effected are known to the person skilled in the art.

For the separation of magnetic particles liquids containing such particles, below the magnetizable rods, introduced, wherein containers of the type mentioned above can be used. Preferably, for this purpose, at least one holding device is provided which can be positioned below the bars, so that the bars are aligned with the openings of the container. This holding device can be configured for example in the form of a holding plate.

The holding device is preferably arranged to be movable and can be set in motion by a drive device so that the sample containers can be alternately positioned in a region below the bars or outside this region.

• Translationsbewegungen in einer horizontalen Ebene;
• Bewegungen entlang einer Kreisbahn, einer elliptischen Bahn oder unregelmäßigen geschlossenen Bahn, jeweils in einer horizontalen Ebene;
• Bewegungen in vertikaler Richtung.

Dabei entspricht die vertikale Richtung im wesentlichen der Längsrichtung der magnetisierbaren Stäbe (4).In particular, the invention includes embodiments in which the holding device is movable in a substantially horizontal plane in one or more directions; Alternatively or additionally, the holding device may be movable in the vertical direction.
Preferably, the holding device is movably arranged in such a way that it can perform one or more of the following types of movement:

• Translation movements in a horizontal plane;
• Movements along a circular path, an elliptical path or an irregular closed path, each in a horizontal plane;
• Movements in the vertical direction.

The vertical direction substantially corresponds to the longitudinal direction of the magnetizable rods (4).

Electromotive, pneumatic, electromagnetic or hydraulic drive means, or combinations thereof, are preferably used as drive means of the holding device.

In particular, the holding devices and their drive means may also be designed in such a way that they are suitable for carrying out shaking or vibrating movements. The design measures required for this purpose are generally known to the person skilled in the art.

Furthermore, it is provided according to a further embodiment, that both the head piece and the holding device is movable and in particular are capable of carrying out shaking movements. In this way, a particularly effective mixing of the sample liquid is possible if the rods are immersed therein.
According to a further embodiment of the invention, the device is equipped with a movable holding device, the head piece, however, arranged immovable.

According to a further, particularly advantageous embodiment of the invention, the holding device is part of a program-controlled laboratory robot system. Preferably, it is arranged so that a plurality of individual ones of the said containers or groups of such containers, in particular microtiter plates, are alternately brought into a position below said bars and then returned to a position after a predetermined time interval, which is outside the below Bars located area lies. In this way, a high sample throughput is possible.

Furthermore, in connection with the embodiment described above, it is preferred that there is a control or regulating device by means of which the vertical movement of the holding device (s) is adjustable or controllable in such a way that the rods are immersed in the liquid-filled upon upward movement Container is effected.

As mentioned above, the operation of the device according to a preferred embodiment is based on the fact that the / the permanent magnet (s) can be positioned over the head piece, and then can be moved out of this position again / can.

In order to allow movement of the permanent magnet (s) for turning on and off the magnetic field, the magnet, or a group of a plurality of magnets, may be slidably mounted, rotatable or tiltable in a dedicated device. By moving, turning or tilting the magnet can be brought into a position in which its poles or its magnetic field point in the direction of the bars (switched-on state, maximum field strength on the bars), or the magnet can be moved to another position, in which the magnetic field emitted by it does not magnetize the rods of the head piece (switched-off state). The magnet (s) may also be slid, rotated or tilted in intermediate positions to cause the field strength at the magnetizable bars to be less than the maximum value.

The movement of the permanent magnet (s) is made possible according to a preferred embodiment in that the / the permanent magnet (s) is slidably arranged so that the / the magnet (s) from the outside by shifting (or by pulling forces) into the region lying above the head piece and can be moved out of this area again / can. In order to enable the displacement, the permanent magnet can be mounted for example on rails, rollers or racks.

According to a further preferred embodiment, the movement of the / the permanent magnet (s) is made possible by the / the permanent magnet (s) (1) on a rotatable or tiltable device is / are arranged, by means of which the / the permanent magnet (s) via the head piece and can be moved away from this again / can.

The movement (eg, tilting, turning, shifting) of the permanent magnet (s) can be done either directly or indirectly by hand or by means of a drive device, which preferably has electromotive, pneumatic, electromagnetic or hydraulic drive means, or a combination thereof. These drive means are generally known to those skilled in the art, as well as other components (e.g., gears, linkages) that may be needed for the drive means.

Furthermore, a device according to the invention is preferably equipped in such a way that the extent of the movement of the permanent magnet (s) is predictable (eg turning angle or tilting angle, displacement distance).

• Bewegung des/der Permanentmagneten, insbesondere die Zeitintervalle, innerhalb welcher der/die Magnete über den magnetisierbaren Stäben positioniert sind;
• Bewegung des Kopfstückes in horizontaler oder/und vertikaler Richtung, insbesondere Dauer, Frequenz und Amplitude einer Schüttel- oder Vibrationsbewegung;
• Betätigung der Mittel zum lösbaren Befestigen der Grundplatte am Kopfstück und zum Entfernen der Grundplatte vom Kopfstück;
• Betätigung der Mittel zum Festhalten der Hülsen an den Stäben und zum Entfernen der Hülsen von den Stäben;
• Bewegung der Haltevorrichtung, um Behälter oder Gruppen von Behältern abwechselnd unterhalb der Stäbe zu positionieren und anschließend wieder aus dieser Position zu entfernen, insbesondere Geschwindigkeit und Frequenz der Bewegungen, sowie Verweildauer der Haltevorrichtung unterhalb der Stäbe;
• vertikale Bewegung der Haltevorrichtung, um den Stab/ die Stäbe in die Flüssigkeit des/der Behälter einzutauchen und wieder daraus zu entfernen; insbesondere Eintauchtiefe, Dauer und Frequenz;
• sofern vorgesehen, Rotations-, Schüttel- oder Vibrationsbewegung der Haltevorrichtung, insbesondere Rotationsgeschwindigkeit, -Amplitude und Intervalle zwischen einzelnen Arbeitsphasen.

According to a further preferred embodiment of the invention it is provided that the device is associated with a program-controlled computer and connected to it. This program-controlled computer makes it possible to control or regulate at least one of the following functions of the device, or to coordinate or to synchronize at least two of the following functions:

• Movement of the permanent magnet (s), in particular the time intervals within which the magnet (s) are positioned over the magnetizable bars;
• Movement of the head piece in the horizontal and / or vertical direction, in particular duration, frequency and amplitude of a shaking or vibrating motion;
• Actuating the means for releasably securing the base plate to the header and removing the base plate from the header;
• Actuating the means for retaining the sleeves on the rods and for removing the sleeves from the rods;
• Movement of the holding device to position containers and groups of containers alternately below the bars and then to remove again from this position, in particular speed and frequency of the movements, as well as residence time of the holding device below the bars;
• vertical movement of the fixture to dip and remove the rod (s) into the liquid of the container (s); in particular immersion depth, duration and frequency;
• if provided, rotational, shaking or vibrating movement of the holding device, in particular rotational speed, amplitude and intervals between individual working phases.

The devices of the invention may be advantageously combined with other devices for the automated treatment of sample material. Furthermore, two or more of the devices according to the invention can be arranged side by side and combined.

• eine oder mehrere thermostatisierbare Heiz- oder Kühleinrichtungen;
• eine oder mehrere Pipettierstationen zum Hinzudosieren von Flüssigkeiten, insbesondere Reagenzien;
• eine oder mehrere Saugeinrichtungen zum Absaugen von Flüssigkeit aus den Behältern;
• eine oder mehrere Einrichtungen zum Schütteln oder Durchmischen der in den Behältern enthaltenen Flüssigkeiten;
• analytische Geräte, insbesondere für photometrische Messungen oder Lumineszenzdetektion.

The invention therefore also extends to devices of the type described above, to which one or more of the following devices are assigned, their functions are preferably coordinated by a common control with the functions of the device:

• one or more thermostatable heating or cooling devices;
• one or more pipetting stations for metering in liquids, in particular reagents;
• one or more suction means for sucking liquid from the containers;
• one or more means for shaking or mixing the liquids contained in the containers;
• analytical apparatus, in particular for photometric measurements or luminescence detection.

1. a) Eintauchen zumindest eines magnetisierbaren Stabes der Vorrichtung in die partikelhaltige Flüssigkeit;
2. b) Einschalten eines magnetischen Feldes durch Veränderung der Position eines Dauermagneten relativ zu dem magnetisierbaren Stab, wodurch der Stab magnetisiert wird und die Partikel sich im wesentlichen am unteren Ende des Stabes ansammeln;
3. c) Entfernen des Stabes mit den anhaftenden Partikeln aus der Flüssigkeit.

The invention further includes methods for separating magnetic or magnetizable particles from a liquid using a magnetic field; These methods are preferably carried out using one of the devices described above. The processes according to the invention have the following steps according to a preferred embodiment:

1. a) immersing at least one magnetizable rod of the device in the particle-containing liquid;
2. b) turning on a magnetic field by changing the position of a permanent magnet relative to the magnetizable rod, thereby magnetizing the rod and allowing the particles to collect substantially at the lower end of the rod;
3. c) removing the rod with the adhering particles from the liquid.

The devices and methods of the invention may be advantageously used to separate and / or mix a target substance from / in a liquid mixture or solution. In this case, the magnetizable rods are immersed in a liquid which contains a target substance which is specific but reversible the particle is bound. The target substances may be, for example, antibodies, enzymes, receptors, ligands, pharmaceutical agents, nucleic acids. These can also be present in the form of complex mixtures with other substances, the target substances, depending on the binding properties of the magnetizable particles, are specifically bound to them.

• d) Eintauchen des Stabes mit den anhaftenden Partikeln in ein vorgegebenes Volumen einer Waschflüssigkeit;
• e) Ausschalten des magnetischen Feldes durch entgegengesetzte Veränderung der Position des Dauermagneten, wodurch die Partikel in die Flüssigkeit freigesetzt werden;
• f) Mischen;
• g) Magnetisieren der Stäbe durch Veränderung der Position des/der Dauermagneten, wodurch sich die Partikel im wesentlichen am unteren Ende des Stabes ansammeln;
• h) Herausheben des Stabes aus der Waschflüssigkeit.

In further steps, it may be useful to wash magnetic particles together with adhering target substances in suitable washing solutions. Such a washing process can be carried out, for example, as follows:

• d) immersing the rod with the adhering particles in a predetermined volume of a washing liquid;
• e) switching off the magnetic field by oppositely changing the position of the permanent magnet, thereby releasing the particles into the liquid;
• f) mixing;
• g) magnetizing the rods by altering the position of the permanent magnet (s), whereby the particles accumulate substantially at the lower end of the rod;
• h) lifting the rod out of the washing liquid.

• i) Eintauchen des Stabes mit den anhaftenden Partikeln in ein vorgegebenes Volumen einer Elutions-Flüssigkeit, welche die Elution der Zielsubstanz von den Partikeln bewirkt;
• k) Herausheben des Stabes aus der Elutionsflüssigkeit, wobei die Partikel an dem Stab haften bleiben und so aus der Flüssigkeit abgetrennt werden.

In many cases, it is desirable to elute the target substances after binding after separating the magnetic particles therefrom. Therefore, according to a further embodiment, it is provided that the method has the following additional steps:

• i) immersing the rod with the adhering particles in a given volume of an elution liquid which causes the elution of the target substance from the particles;
• k) lifting the rod out of the elution liquid, the particles sticking to the rod and thus being separated from the liquid.

To improve the purity and yield, it may be advantageous to release the particles following the step b) or d) by switching off the magnetic field in the liquid to mix them, and then collect again by turning on the magnetic field on the rods. The mixing can be effected, for example, by shaking the holding device and / or the head part.

By using one of the devices according to the invention described above, the said methods can be carried out in a particularly simple and rapid manner. The devices and methods of the invention are particularly suitable for the application areas mentioned above, in particular for high-throughput methods.

• l) eine erste Gruppe von magnetisierbaren Stäben, oder mehrere zu einer gemeinsamen Einheit verbundenen Stäbe, wird/werden lösbar an einer Vorrichtung befestigt, die einen oder mehrere angeordnete Permanentmagnete aufweist, dessen/deren relative Position bezüglich der magnetisierbaren Stäbe veränderbar ist;
• m) die erste Gruppe von magnetisierbaren Stäben wird von der Vorrichtung abgetrennt oder abgeworfen und durch eine zweite Gruppe von magnetisierbaren Stäben ersetzt, die lösbar an der Vorrichtung befestigt wird.

According to a further preferred embodiment, it is provided in the method according to the invention that the magnetizable rods are exchanged and renewed between two working cycles or between two method steps, for example to avoid cross-contamination. Preferably, such a method therefore additionally has at least one of the following steps:

• l) a first group of magnetizable bars, or a plurality of bars connected to a common unit, is / are releasably secured to a device having one or more arranged permanent magnets whose relative position with respect to the magnetizable bars is changeable;
• m) the first group of magnetizable rods is separated or discarded by the device and replaced by a second group of magnetizable rods which is releasably secured to the device.

• n) eine erste Gruppe von Hülsen, oder zu einer gemeinsamen Einheit verbundene Hülsen, wird/werden auf die magnetisierbaren Stäbe einer Vorrichtung nach Anspruch 1 aufgesteckt;
• o) die erste Gruppe von Hülsen wird von den magnetisierbaren Stäben der Vorrichtung abgestreift oder abgeworfen und durch eine zweite Gruppe von Hülsen ersetzt, die auf die Stäbe aufgesteckt werden.

As an alternative to this measure, or in addition to this, according to a further embodiment of the method according to the invention, the magnetizable rods are provided with sleeves which are exchanged and renewed between two working cycles or between two method steps in order to avoid carryover of reagents or cross contamination ,
Preferably, such a method therefore additionally has at least one of the following steps:

• n) a first group of sleeves, or sleeves connected to a common unit, is / are attached to the magnetizable rods of a device according to claim 1;
• o) the first group of sleeves is stripped or discarded from the magnetizable rods of the device and replaced by a second group of sleeves which are slipped onto the rods.

The invention will now be described by way of example with reference to the accompanying diagrammatic drawings. The reference numbers used have the same meaning in all drawings unless otherwise indicated. Since these are only schematic representations, the actual size ratios may differ therefrom.

Fig. 1A and 1B show an embodiment of a device (10) according to the invention in a side view, wherein Fig. 1A the switched on, Fig. 1B illustrates the off state. The device (10) comprises a permanent magnet (1) which is slidably mounted on rails (2) and can be moved in the direction of the arrow (a) in a horizontal plane. A fixed frame of the device (not shown) carries a head piece (3) with magnetizable bars or rods (4) attached thereto. Below the bars is a vertically displaceable holding device (6). On the holding device is a sample container (7) arranged with a plurality of wells for receiving liquid samples, for example, removably fixed on the holding device (6).

The head piece (3) is connected to a drive unit (5), which makes it possible to move the head piece with the rods attached thereto in movement, preferably in a shaking movement in the horizontal plane, as indicated by arrow (b). The holding device (6) is equipped with a drive unit (not shown), which allows an upward and downward movement of the holding device (arrow c). On the rods (4) sleeves (8) are attached or clamped.
The rods are firmly connected to the head piece; Alternatively, they can also be releasably connected to the head piece.

As can be seen, the magnet (1) is in Fig. 1A in a position substantially above the header or magnetizable bars so that the bars can be magnetized by the magnet. As a result, a magnetic field is generated at the ends of the rods (7) which can be used to attract magnetic particles. In Fig. 1B (off state), the magnet has been moved out of this position and is no longer above the bars (4).

Fig. 1C shows a modification of the in Fig. 1A and 1B shown apparatus (10), wherein the sleeves (8 ') are connected together at their upper ends and form a common unit.

Fig. 2A to 2C show a further embodiment of the device (10), in which a plurality of bars (4) are connected in a regular arrangement with a base plate (9) and form a unit with this. Preferably, the plate (9) is releasably attached to the head piece (3).

The header is then preferably equipped with a holding and dropping device which allows automatic picking and dropping of the plate through the header (not shown). - Fig. 2A shows the switched on, Fig. 2B the off state.

Fig. 2C shows a further advantageous embodiment of the invention (in the on state), wherein the entire unit comprising the head piece and the magnet, can be moved up and down, preferably by drive means of the type mentioned above. In this case, can on a vertical mobility the holding device (6) are dispensed with.

Fig. 2D shows a further advantageous embodiment of the invention, in which instead of a base plate (9) with bars (4) attached thereto, a group of sleeves or hollow bars (8 ") joined together in one unit is used, in which case the sleeves are made of a magnetizable one Material produced.

Fig. 3 shows a further embodiment of the device according to the invention, in which the holding device (6) for the sample vessels with a drive unit (5 ') in the horizontal direction (arrow d) can be moved.

Fig. 4 shows a further embodiment of the invention, wherein two units, each with a head piece (3), magnetizable bars (4), holding device (6), are combined, wherein the magnet (1) via a rail (2) alternately over the one or the other header (3) can be positioned.

The apparatus and methods described above can be advantageously used in the methods and techniques mentioned in the opening paragraph, and they can be easily adapted to different requirements.

Claims (31)

1. Device for separating magnetic or magnetizable particles from a liquid by using a magnetic field, wherein said device (10) comprises

   - a head piece (3) with one or more magnetizable bars (4) which is/are connected in a fixed or detachable manner with said head piece (3);

   - one or more permanent magnets (1) whose relative position with respect to the head piece can be changed by a predeterminable movement of the magnet (s) or/and by a predeterminable movement of the head piece such that the permanent magnet(s) assume(s) a first position which is above the head piece, or a second position which is outside said first position.
2. Device according to claim 1, characterised in that the magnetizable bars (4) are attached to a base plate (9) and form a unit therewith, it being preferred for the base plate to be detachably connected with the head piece (3).
3. Device according to claim 2, characterised in that the plate (9) is provided with a plurality of magnetizable bars (4) which are arranged in one or more rows, each row containing a plurality of bars (4).
4. Device according to claim 2 or 3, characterised in that it has means by which the bars (4), or the base plate (9) together with the bars (4) attached thereto, can be detachably connected with the head piece and/or removed therefrom, said means preferably being actuated by an electromotive drive or by pneumatic, electromagnetic or hydraulic means, or by a combination thereof.
5. Device according to any one of the preceding claims, characterised in that the said bar(s) (4) are each provided with a strippable, replaceable shell (8).
6. Device according to claim 5, characterised in that it has a plurality of shells which form a common unit (8').
7. Device according to claim 5 or 6, characterised in that it has means by which the replaceable shells (8), or the shells (8') forming a common unit, can be retained at the bars (4), or at the head piece (3), or removed from the bars (4), or the head piece (3), said means preferably being actuated by an electromotive drive or by pneumatic, electromagnetic or hydraulic means, or by a combination thereof.
8. Device according to any one of the preceding claims, characterized in that the head piece is arranged so as to be moveable and can be set into motion by a drive device, said drive device preferably comprising electromotive, pneumatic, electromagnetic or hydraulic drive means or a combination thereof.
9. Device according to claim 8, characterised in that the head piece (3) is moveably arranged such that it is able to perform one or more of the types of motion indicated below:

   - translatory movements in a horizontal plane;

   - movements along a circular path, an elliptic path or an irregular path, in each case within a horizontal plane;

   - movements in a vertical direction;

   said vertical direction corresponding substantially to the longitudinal direction of the magnetizable bars (4').
10. Device according to any one of the preceding claims, characterized in that it has a holding device (6) for one or more sample vessels (7).
11. Device according to claim 10, characterised in that the holding device (6) is arranged so as to be moveable and that said holding device (6) can be set in motion by a drive device such that the sample vessels (7) can be positioned within a region lying below the bars (4) or outside said region, said drive device of the holding device (6) preferably comprising electromotive, pneumatic, electromagnetic or hydraulic drive means, or a combination thereof.
12. Device according to claim 10 or 11, characterised in that the holding device (6) is moveably arranged in such a manner that it is able to perform one or more of the types of motion set out below:

    - translatory movements in a horizontal plane;

    - movements along a circular path, an elliptic path or an irregular closed path, in each case within a horizontal plane;

    - movements in a vertical direction;

    said vertical direction corresponding substantially to the longitudinal direction of the magnetizable bars (4).
13. Device according to any one of claims 10 to 12, characterised in that the holding device (6) is a component of a programme-controlled laboratory robot system and is adapted such that a plurality of individual ones of the said containers or of groups of such containers (7), particularly microtitre plates, is alternately moved into a position below the said bars (4) and subsequently, after a predeterminable time interval, again into a position which is outside the region below the bars.
14. Device according to any one of claims 10 to 13, characterised in that the vertical movement of the holding device (6) can be adjusted or controlled by an open-loop control unit or a closed-loop control unit such that an upward movement of the holding device (6) causes the bars (4) to be immersed in the containers (7), which are filled with liquid.
15. Device according to any one of the preceding claims, characterised in that the head piece or/and the holding device is capable of performing shaking or vibrating movements.
16. Device according to any one of claims 1 to 14, characterised in that the head piece is permanently attached.
17. Device according to any one of the preceding claims, characterised in that the permanent magnet(s) (1) is/are displaceably arranged such that said magnet(s) can be moved from outside into the region located above the head piece (3), and then again out of the said region.
18. Device according to any one of the preceding claims, characterized in that the permanent magnet(s) (1) are arranged so as to be rotatable or tiltable.
19. Device according to any one of the preceding claims, characterized in that the movement of the permanent magnet(s) is accomplished by means of a drive device which preferably comprises electromotive, pneumatic, electromagnetic or hydraulic drive means, or a combination thereof.
20. Device according to any one of the preceding claims, characterized in that a program-controlled processor is associated to said device and is connected therewith, by means of which at least one of the following functions of the device can be open-loop controlled or closed-loop controlled, or by means of which at least two of the functions mentioned below can be coordinated with one another;

    - movement of the permanent magnet(s) (1), particularly the time intervals within which the magnet(s) are positioned above the magnetizable bars (4);

    - movement of the head piece (3) in horizontal or/and vertical direction, particularly duration, frequency and amplitude of a shaking or vibrating motion;

    - actuating the means for detachable attachment of the base plate (9) to the head piece (3) and for removal of the base plate from the head piece;

    - actuating the means for retaining the shells (8) at the bars (4) and for removing the shells (8) from the bars (4);

    - movement of the holding device (6) in order to position containers (7) or groups of containers alternately below the bars (4) and subsequently to remove them from that position, particularly velocity and frequency of the movements, as well as the dwell time of the holding device below the bars;

    - vertical movement of the holding device (6) to immerse the bar/the bars (4) into the liquid of the container(s) (7) and remove the same therefrom; particularly immersion depth, duration and frequency;

    - if provided, rotation, shaking or vibrating motion of the holding device (6), particularly rotation speed, rotation amplitude and intervals between the individual operation phases.
21. Device according to one or more of the preceding claims, characterized in that one or more of the below-mentioned means are associated to the said device, the functions of said means being coordinated with the functions of the said device by means of a common control:

    - one or more thermostattable heating or cooling means;

    - one or more pipetting stations for metered addition of liquids, especially reagents;

    - one or more suction means for exhausting liquid from the containers by suction;

    - one or more means for shaking or intermixing the liquids contained in the containers;

    - analytic apparatuses, particularly for photometric measuring or luminescence detection.
22. Arrangement of magnetizable bars for use in a device for separating magnetic or magnetizable particles according to any one of claims 1 to 21, characterised in that a plurality of magnetizable bars (4) is arranged on a base plate (9), said bars (4) being oriented substantially parallel to one another and preferably being arranged in one, two or more rows, each row comprising two or more bars.
23. Arrangement according to claim 22, characterised in that the magnetizable bars (4) form a unit with the base plate (9) that is preferably made in one piece, especially by deep drawing.
24. Arrangement of shells for use in a device for separating magnetic or magnetizable particles from a liquid according to any one of claims 1 to 21, characterised in that a plurality of shells (8), which can be slipped onto the magnetizable bars of the said device, is arranged substantially parallel to one another, preferably in one, two or more rows, each row comprising two or more bars.
25. Arrangement according to claim 24, characterised in that the shells (8) are connected so as to form a common unit (8') that is preferably made in one piece, especially by deep drawing.
26. Method for separating magnetic or magnetizable particles from a liquid by using a magnetic field, which method can be performed by means of a device according to any one of claims 1-21, said method comprising the following steps:

    a) immersing at least one magnetizable bar of the device into the liquid containing the particles;

    b) activating a magnetic field by changing the position of a permanent magnet relative to the magnetizable bar such that the permanent magnet assumes a position above the head piece, whereby the bar is magnetized and the particles accumulate substantially at the lower end of the bar;

    c) removing the bar, along with the adhering particles, from the liquid.
27. Method according to claim 26, characterised in that the liquid used in step (a) contains a target substance which is bound specifically, but reversibly, to the particles.
28. Method according to claim 27, characterised by the following additional steps:

    d) immersing the bar, along with the particles adhering thereto, in a predetermined volume of a wash liquid;

    e) deactivating the magnetic field by an opposite change of the position of the permanent magnet such that the permanent magnet assumes a position which is not above the head piece, whereby the particles are released into the liquid;

    f) mixing;

    g) magnetizing the bars by changing the position of the permanent magnet(s) such that the permanent magnet assumes a position above the head piece, whereby the particles accumulate substantially at the lower end of the bar;

    h) lifting the bar out of the wash liquid.
29. Method according to claim 27 or 28, characterised by the following additional steps:

    i) immersing the bar, along with the particles adhering thereto, into a predetermined volume of an elution liquid that causes the elution of the target substance from the particles;

    k) lifting the bar out of the elution liquid, during which process the particles remain adhering to the bar and are thereby separated from the liquid.
30. Method according to any one of the claim 26-29, said method additionally comprising at least one of the below-mentioned steps:

    l) a first group of magnetizable bars, or a plurality of bars connected to form a common unit, is detachably attached to a device comprising one or more arranged permanent magnets (1) whose relative position with respect to the magnetizable bars can be changed;

    m) the first group of magnetizable bars is separated from the device or discarded therefrom, and replaced by a second group of magnetizable bars which is detachably attached to the device.
31. Method according to any one of the claims 26-30, said method additionally comprising at least one of the below-mentioned steps:

    n) a first group of shells, or shells connected to form a common unit, is/are slipped on the magnetizable bars of a device according to claim 1;

    o) the first group of shells is stripped off or discarded from the magnetizable bars of the device and replaced by a second group of shells which are slipped onto the bars.

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