EP2033715A1 - Method for suspending or re-suspending particles in a solution and device adapted therefor - Google Patents

Abstract

The method involves the switching on of an effective magnetic field acting in the front end (3) of the mixing bar (1) through a magnetic field generating apparatus (4) while bar is immersed in a mixture (30). Part of the magnetically attractable particles is raised from the bottom of a mixing vessel (10) so that a portion of the particles sticking at the mixing bar is minimized. Repeated mixing movements of the mixing bar is performed without the existence of a magnetic field switched on at the front end of the mixing bar in order to suspend or re-suspend the particles present in the mixture.

Description

The invention relates to a method for suspending particles, in particular magnetically attractable particles and beads such as ferromagnetic and / or paramagnetic particles, in a liquid mixture used for example for diagnostic or analytical purposes.

In the field of sample preparation and sample processing for analytical or diagnostic investigations, methods are increasingly being used which rely on the use of magnetically attractable particles, to which either biological target molecules in particular or contaminants can bind. Magnetically attractable particles can be separated by suitable magnetic fields from the mixture in which they are suspended. This applies in particular to automated methods, since in this way a large number of samples can be analyzed within a short time without expensive centrifugation steps. This allows a large sample throughput and makes it possible to considerably reduce the effort involved in extensive and in particular parallel investigations. Important fields of application are the purification of biological or medical samples, generally the separation and isolation of biological target molecules in particular, medical diagnostics as well as pharmaceutical screening methods for the identification of potential active pharmaceutical ingredients.

Methods for separating magnetically attractable particles are, for example, in DE 44 21 058 . DE 103 31 254 . DE 10 2005 004 664 . WO 94/18565 . WO 99/42832 . WO 02/40173 . WO 2005/044460 . US Pat. No. 5,942,124 and US 6,448,092 disclosed. The basic principle of the methods described therein is based on the fact that a separating device, for example a magnetic bar, is immersed in a usually liquid mixture and, due to the action of the magnetic field, the magnetically attractable particles contained in the mixture are concentrated on the surface of the separating device. The separation device is then guided out of the solution with the adhering particles.

The use of external magnetic fields for mixing and separating magnetic particles is known in WO 2006/010584 described. For this purpose Polschuhè are arranged around a specially designed mixing vessel with which variable magnetic fields can be generated.

For mixing particles, it is also known to use mixing rods which are rotated, such as in US 2006/0118494 described, set the mixture in motion while swirling the particles in the mixture. However, rotational solutions are very expensive, especially in automated parallel processing.

In particular, when in the course of separation and / or purification processes, the magnetic particles come into contact with a plurality of solutions, e.g. in binding or washing processes, yield losses of the target molecules which bind to the magnetic particles or inadequate purification results frequently occur when the particles are not sufficiently suspended in the solutions or mixtures but settle at the bottom. In addition, the particles used in such processes per se have a high tendency for sedimentation. Therefore, it is attempted in the processes described to hold the magnetic particles in suspension at least temporarily by mechanical mixing movements or to resuspend the sedimented particles.

A problem that has been found in the practical application of the processes of the prior art is that the particles having the target molecules, in particular the biological target molecules, or the impurities in generating a magnetic field and direct or indirect collection of the magnetic particles no longer adhere to the magnet as particles on the magnet, but rather as lumps or flakes. As a result, after release of the particles from the magnet, for example for washing the particles or eluting the components bound thereto, they are difficult to suspend and sediment very quickly. This can also lead to poor purification results.

It is therefore the object of the present invention to specify a method with which, in particular, sedimented particles can be suspended or resuspended in a solution as simply as possible.

• Bereitstellen zumindest eines Mischgefäßes, das zumindest teilweise mit einer Mischung gefüllt ist, in welcher sich magnetisch anziehbare Partikel befinden, die zumindest teilweise am Boden des Mischgefäßes sedimentiert sind;
• Bereitstellen zumindest eines Mischstabs mit einem dem Boden des Mischgefäßes zugewandten vorderen Ende, wobei der Mischstab eine Magnetfelderzeugungseinrichtung zum wahlweisen Erzeugen eines Magnetfelds zumindest im Bereich des vorderen Endes aufweist;
• Einschalten eines wirksamen Magnetfeldes, das zumindest im Bereich des vorderen Endes des Mischstabs wirkt, durch die Magnetfelderzeugungseinrichtung während der Mischstab in der Mischung eingetaucht ist;
• Bewegung des Magnetfeldes vom Boden des Mischgefäßes weg, wobei die Bewegung des Magnetfeldes so erfolgt, dass zumindest ein Teil der magnetisch anziehbaren Partikel vom Boden des Mischgefäßes hochgehoben werden und der Anteil der Partikel, die am Mischstab haften, minimiert wird;
• Ausschalten des Magnetfeldes in einer zuvor definierten Entfernung vom Boden, die größer ist als die Entfernung vom Boden bei Einschalten des Magnetfeldes;
• Durchführung wiederholter Mischbewegungen des Mischstabs ohne dass am vorderen Ende des Mischstabs ein eingeschaltetes Magnetfeld vorlieget, um die in der Mischung vorliegenden magnetisch anziehbaren Partikel zu suspendieren bzw. resuspendieren.

This object is achieved by a method for suspending or resuspending magnetically attractable particles. The method comprises the steps:

• Providing at least one mixing vessel which is at least partially filled with a mixture containing magnetically attractable particles at least partially sedimented at the bottom of the mixing vessel;
• Providing at least one mixing bar having a front end facing the bottom of the mixing vessel, the mixing bar having magnetic field generating means for selectively generating a magnetic field at least in the region of the front end;
• Switching on an effective magnetic field acting at least in the region of the front end of the mixing rod by the magnetic field generating means while the mixing rod is immersed in the mixture;
• Moving the magnetic field away from the bottom of the mixing vessel, wherein the movement of the magnetic field is such that at least a portion of the magnetically attractable particles are lifted from the bottom of the mixing vessel and the proportion of particles adhering to the mixing rod is minimized;
• Turning off the magnetic field at a predefined distance from the ground that is greater than the distance from the ground when the magnetic field is turned on;
• Carrying out repeated mixing movements of the mixing rod without a magnetic field being applied to the front end of the mixing rod in order to suspend or resuspend the magnetically attractable particles present in the mixture.

In the context of the present description, "magnetically attractable particles" are understood as meaning particles and beads which can be attracted by a magnetic field. Examples thereof are particles and beads which have ferro-, ferri-, paramagnetic and / or superparamagnetic materials as well as magnetizable materials. The magnetic or magnetizable particles usually have at least partially a surface of a non-magnetic or magnetizable material, which ultimately causes the binding of the biological target molecules or impurities. The size of such particles can range from about 500 nm to about 25 μm.

The mixing vessel may in particular be any vessel typically used in the field of analysis and diagnostics. For example, it may be a single separate and independent reaction vessel for chemical, biological and / or medical applications or such a reaction vessel, which forms a unit together with one or more further usually similar reaction vessels, for example in the form of a so-called multiwell plate. The reaction vessels can be combined in a stackable plate. Such plates are commonly used in the field of biotechnology for the manual or automated performance of purification of biological samples or isolations of special components, such as nucleic acids or proteins, or for downstream processes such as assays, PCR o. Ä. In this case, each reaction or mixing vessel may have a mixture with magnetically attractable particles contained therein. The mixtures may contain other substances, e.g. dissolved or suspended.

Usually, the magnetic particles are added as a powder or suspension to an untreated or pretreated sample. The particles then usually sink first to the ground. This should also be the case when the magnetic particles are introduced as a suspension and mixed with the sample or a mixture. Typically, the magnetically attractable particles at the time of application of the method according to the invention are therefore predominantly at the bottom of the mixing vessel, d. H. the particles are sedimented. In this case, the particles are resuspended in the mixture again. On the other hand, it is possible that the powdery particles are in the mixing vessel before a sample or mixture is added. In this case, the method serves to suspend the magnetically attractable particles accumulated at the bottom of the mixing vessel.

The mixing rod used for suspending or resuspending has at least one magnetic field generating device. The purpose of this device is to selectively generate an effective magnetic field, in particular in the region of the front end of the mixing rod, ie an effective magnetic field can be switched on and off there. By "turning on" the magnetic field at a location is meant that an effective magnetic field is generated at that location (for example, by switching on an electromagnet located there) or that a magnetic field is transported to that location (for example, by moving a permanent magnet). Under the latter conditions, the magnetic field is considered to be turned on only when the total magnetic field strength is in place, that is, when the magnetic field is still on its way to the location, it is not yet considered turned on. By contrast, the term "switch-off" is understood to mean that no effective magnetic field is generated in the region of the front end (more) or a previously generated magnetic field is removed. A magnetic field is "effective" for the purposes of the present invention, if it moves the particles in the mixture and in particular can be attracted to the mixing rod. "Switching on" and "switching off" therefore refers to the selective generation of a magnetic field, in particular in the region of the front end of the mixing rod. In general, the magnetic field can be generated not only in the region of the front end of the mixing rod, but also extend over the length of the rod. However, it should preferably be avoided that the opposite of the front end of the mixing rod pole of the magnet is also immersed in the mixture. It goes without saying that the strength of the required magnetic field has to be selected as a function of the viscosity of the solution and of the size, weight and magnetic material of the particles.

With the mixing rod, which is already immersed in the solution or is first brought into the solution, the particles located at the bottom of the mixing vessel are first drawn from the bottom in the direction of the front end of the mixing rod. This is done, for example, in that the mixing rod is preferably brought together with the magnetic field generating device with its front end to the bottom of the mixing vessel. It is not only not necessary, but also for reasons of construction and process assurance not desired that the front end of the mixing rod touches the ground. In particular, when the mixing rod with its front end in the vicinity of the soil and thus in the vicinity of the particles located there, a magnetic field is generated in the region of the front end of the magnetic field generating means, with which the particles are attracted to the mixing rod. Alternatively, the mixing rod can also be moved together with the magnetic field generating device, which already generates a magnetic field, to the bottom of the mixing vessel or an already magnetic field generating Magnetic field generating means can be moved in the direction of the front end of the mixing rod, which is already positioned in the vicinity of the bottom of the mixing vessel. The magnetic field generating device is then preferably pulled out of the mixture together with the mixing rod at least partially away from the ground. In particular, the strength of the generated magnetic field and the acceleration and the speed with which the magnetic field is moved out of the mixture should preferably be tuned so that the sedimented magnetic particles move from the ground into the mixture but not necessarily adhere to the mixing rod.

Preferably, this is achieved in that the magnetic field is always in motion and residence times are minimized, especially in the vicinity of the soil. When a permanent magnet is used, this can be achieved by first moving the magnet towards the ground (be it together with the mixing rod or towards the front end of the mixing rod already located there). If the magnet is sufficiently close to the ground to be able to attract the particles via the magnetic field, the magnet reverses and the magnet is now moved away from the ground together with the mixing rod. The residence time of the magnet in the vicinity of the bottom should be just chosen so that the particles, although to move on this, but at least not as completely collect on the mixing rod. The adhesion of a part of the particles to the mixing rod can not normally be completely avoided even with careful coordination of the conditions, but the proportion should be kept as small as possible. The minimum distance of the mixing rod to the bottom is preferably 0.1 to 2 mm, more preferably 0.3 to 1 mm, and most preferably 0.5 to 0.6 mm. The minimum distance of the magnet to the inner tip of the mixing rod prior to the movement reversal of the magnet is preferably> 0 to 10 mm, more preferably 0.3 to 8 mm and most preferably 0.5 to 5 mm. In this case, the above-specified distance ranges from the lowest (distal) end of the magnet to the lowest (distal) inner end of the mixing rod preferably include both the case that both have parallel contours at their lower end and that both have different contours at their lower end ,

When electromagnets are used, they can already be switched on a considerable distance from the ground. Under these circumstances, the first one runs Process step of raising the particles preferably analogous to when using the permanent magnet. If the electromagnet is only activated when it is close to the ground, then the movement of the magnetic field should take place together with the mixing rod away from the ground directly after generation of the magnetic field and acceleration of the particles in the direction of the mixing rod.

wobei a₁ die Beschleunigung des Magneten bzw. des Mischstabes, t₁ die Zeit, die benötigt wird, um die Verfahrgeschwindigkeit des Magneten bzw. des Mischstabes in Richtung des Gefäßbodens zu erreichen, t₃ die Zeit, während der eine konstante Verfahrgeschwindigkeit in Richtung des Gefäßbodens vorliegt, t₂ die Zeit, die benötigt wird, um die Verfahrgeschwindigkeit des Magneten bzw. des Mischstabes in Richtung des Gefäßbodens auf 0 zu reduzieren, und s die zurückgelegte Strecke darstellen und wobei vorzugsweise a₁ = -a₂ und t₁=t₂ ist. Der Verfahrweg des Mischstabs kann dabei parallel zu dem des Magneten oder aber verschieden zu diesem ablaufen. Die Funktion, die dem Verfahrweg des Mischstabs zu Grunde liegt, sollte dabei der des Magneten entsprechen, wobei die einzelnen Parameter für den Magneten und den Mischstab unterschiedliche Werte aufweisen können. Wenn die Verfahrwege für Magnet und Mischstab unterschiedlich verlaufen, sollte zumindest gewährleistet sein, dass, wenn der Magnet seine Position in minimalem Abstand zum Gefäßboden mit der Geschwindigkeit 0 erreicht hat, auch der Mischstab sich in minimaler Entfernung zum Gefäßboden befindet und die Geschwindigkeit 0 aufweist.Preferably, the residence time of the magnet turned on with a field strength in the range of 0.5 to 1.5 T at the location where the distance of the integrated-magnet mixing rod to the ground is minimum (preferably 0.1 to 2 mm, more preferably 0) , 3 to 1 mm, and most preferably 0.5 to 0.6 mm) 0.02 to 5 s, more preferably 0.04 to 3 s, even more preferably 0.1 to 0.5 s, and most preferably 0, 2 s. When using a permanent magnet, the travel of the magnet should first have an acceleration of the stationary magnet to a travel speed (preferably a ₁ * t ₁ ) in the direction of the vessel bottom, the magnet either together with the mixing rod or on the already closer to the vessel bottom located mixing rod can be accelerated. Optionally, the magnet may then further have a directed in the direction of the vessel bottom constant travel speed a ₁ * t ₁ , wherein the magnet in turn can move simultaneously with the mixing rod or on this. Subsequently, the magnet is accelerated to a speed of 0 with a negative acceleration (preferably a ₂ * t ₂ ). This negative acceleration can also follow directly after the positive acceleration. The mixing bar may accordingly experience the same negative acceleration or have been previously accelerated to a speed of zero. After passing through this travel path, the magnet together with the mixing rod should preferably be at a speed 0 at the position at which the distance between magnet and mixing rod to the bottom of the vessel is always minimal. This travel path is preferably based on the following function: $$\mathrm{s}\left(\mathrm{t}\right)\mathrm{=}{\mathrm{<figure-callout\; id="1"\; label="1⁄2a"\; filenames="imgf0003.png,imgf0004.png"\; state="\{\{state\}\}">½a</figure-callout>}}_{\mathrm{1}}\mathrm{*}{{\mathrm{<figure-callout\; id="1"\; label="t"\; filenames="imgf0003.png,imgf0004.png"\; state="\{\{state\}\}">t</figure-callout>}}_{\mathrm{1}}}^{\mathrm{2}}\mathrm{+}{\mathrm{a}}_{\mathrm{1}}\mathrm{*}{\mathrm{<figure-callout\; id="1"\; label="t"\; filenames="imgf0003.png,imgf0004.png"\; state="\{\{state\}\}">t</figure-callout>}}_{\mathrm{1}}\mathrm{*}{\mathrm{<figure-callout\; id="3"\; label="t"\; filenames="imgf0003.png,imgf0004.png"\; state="\{\{state\}\}">t</figure-callout>}}_{\mathrm{3}}\mathrm{+}{\mathrm{<figure-callout\; id="2"\; label="1⁄2a"\; filenames="imgf0003.png,imgf0004.png"\; state="\{\{state\}\}">½a</figure-callout>}}_{\mathrm{2}}\mathrm{*}{{\mathrm{<figure-callout\; id="2"\; label="t"\; filenames="imgf0003.png,imgf0004.png"\; state="\{\{state\}\}">t</figure-callout>}}_{\mathrm{2}}}^{\mathrm{2}}\mathrm{.}$$

where a _{1 is} the acceleration of the magnet or of the mixing rod, t ₁ the time required to reach the travel speed of the magnet or the mixing rod in the direction of the vessel bottom, t ₃ the time during which a constant travel speed in the direction of the If the bottom of the vessel is present, t ₂ the time that is required to the speed of movement of the magnet or the mixing rod in the direction of the vessel bottom 0 and s represent the distance traveled and preferably wherein a ₁ = -a ₂ and t ₁ = t ₂ . The travel of the mixing rod can be parallel to that of the magnet or different from this run. The function underlying the travel of the mixing rod should correspond to that of the magnet, whereby the individual parameters for the magnet and the mixing rod can have different values. If the travel distances for magnet and mixing rod are different, it should at least be ensured that when the magnet has reached its position at a minimum distance from the bottom of the vessel at zero speed, the mixing rod is also at a minimum distance from the bottom of the vessel and the speed is zero.

This is followed by the above-specified residence time of the magnet, preferably at a minimum distance from the ground, whereupon the magnet, together with the mixing rod, preferably passes through a path of travel analogous to the above, but in the direction of the vessel opening. The durations t ₁ and t _{2 of} the accelerations are preferably in the range from 0.02 to 5 s, more preferably from 0.04 to 3 s and even more preferably from 0.1 to 0.5 s.

However, it is also conceivable that the travel described above for both the mixing rod and for the magnet is represented independently by other functions, as by the above example given, as long as the expiration of downward movement, stopping at a minimum distance to the vessel bottom, residence time and Upward movement generally corresponds to the above.

When using an electromagnet that is turned on before it has reached the minimum distance to the ground, the travel should be analogous to that for the permanent magnet. When using an electromagnet, which is turned on only when it has taken the minimum distance to the ground, the path should correspond to the above-described travel of the permanent magnet in the direction of the vessel opening.

If the particles have been lifted sufficiently far, for example to a defined height, they are released, ie the direction of movement of the particles is no longer influenced by the magnetic field. This is preferably done by switching off the magnetic field or removing the magnetic field generating device from the mixing rod. Simultaneously with the release of the particles or shortly thereafter, the mixing rod is in a Mixed movement, with which he distributes the particles as homogeneously as possible in the solution. The mixing movement is typically a repeated up and down movement of the mixing rod, ie a vertical movement of the mixing rod. In general, a rotating movement or a combination of vertical and rotating movement of the mixing rod is conceivable. The number of mixing operations is not fixed and is usually determined by the user according to what degree of homogeneous distribution of the particles in the mixture is desired. The particles are therefore preferably sufficiently suspended or resuspended if the degree of suspension or resuspension meets the requirements of the user or the best possible suspension or resuspension of the particles in the present system. In most cases, the particles will then be sufficiently suspended if the proportion of resuspended particles after lifting and suspending is still relatively small.

Experiments have shown that the sedimented particles can be effectively removed from the soil with the aid of the method according to the invention and suspended or resuspended in the solution. It is therefore preferably not a separation process in the true sense, in which the particles are held as quantitatively as possible on the magnet or on a surrounding sleeve and removed from the mixing vessel, but the particles should only be brought back into suspension, in particular order to achieve the best possible binding, washing action, elution or the like. The magnetic field is preferably used only for lifting the sedimented particles, while the distribution of the particles in the solution takes place by the mixing movement of the mixing rod with the magnetic field switched off.

The inventive method has the advantage that the mere distribution of the already raised from the ground particles can be done by comparatively gentle mixing movements. A stirring up of the sedimented particles only with strong mixing movements, as it would be necessary without the use of a magnetic field, is not necessary. In the method according to the invention, therefore, the solution does not have to be moved very strongly, whereby the risk of cross-contamination of adjacent mixing vessels in automated parallel processing is considerably reduced.

In addition, the mixing rod for lifting the sedimented particles need not be completely led to the ground in the process according to the invention, but only be brought close to the ground. As a result, impacts of the mixing rod are avoided on the bottom of the mixing vessel. When fluidizing the sedimented particles exclusively by a mixing movement of the mixing rod and without the use of a magnetic field, the mixing rod must be brought directly to the ground, otherwise there is a risk that a large part of the particles is not stirred up. In particular, in vessels that have no flat bottom, a purely mechanical mixing can cause the particles are not suspended or resuspended, but rather are pressed to the ground. In addition, such a purely mechanical Resuspendierungsverfahren requires a high design effort to exclude or minimize collisions of soil and mixing vessel and related damage to the soil and leakage of the mixture.

• Bereitstellen zumindest eines Mischgefäßes, das zumindest teilweise mit einer Lösung gefüllt ist, in welcher magnetisch anziehbare Partikel am Boden des Mischgefäßes sedimentiert sind;
• Bereitstellen zumindest eines Mischstabs mit einem dem Boden des Mischgefäßes zugewandten vorderen Ende, wobei der Mischstab eine Magnetfelderzeugungseinrichtung zum wahlweisen Erzeugen eines Magnetfelds im Bereich des vorderen Endes aufweist;
• wobei durch das am vorderen Ende des in die Lösung eingetauchten Mischstabs erzeugte Magnetfeld zumindest ein Teil der magnetisch anziehbaren Partikel vom Boden des Mischgefäßes hochgehoben und nachfolgend durch wiederholte Mischbewegungen des Mischstabs ohne am vorderen Ende des Mischstabs erzeugtes Magnetfeld in der Lösung suspendiert bzw. resuspendiert werden.

The above object can be achieved by a method for suspending magnetically attractable particles according to another embodiment. The method comprises:

• Providing at least one mixing vessel which is at least partially filled with a solution in which magnetically attractable particles are sedimented at the bottom of the mixing vessel;
• Providing at least one mixing bar having a front end facing the bottom of the mixing vessel, the mixing bar having magnetic field generating means for selectively generating a magnetic field in the region of the forward end;
• wherein at least a portion of the magnetically attractable particles are lifted from the bottom of the mixing vessel by the magnetic field generated at the front end of the dipstick immersed in the solution and subsequently resuspended in the solution by repeated mixing movements of the mixing bar without magnetic field generated at the front end of the mixing bar.

This embodiment can be suitably combined with individual aspects and features of the embodiments described above and below, in particular with regard to the structure of the mixing rod, the type of magnetic field generation and the timing of the mixing movement and the magnetic field generation.

• zumindest einen Mischstab mit einem vorderen Ende, wobei der Mischstab eine Magnetfelderzeugungseinrichtung zum wahlweisen Erzeugen eines Magnetfelds im Bereich des vorderen Endes aufweist;
• wobei die Vorrichtung zur Durchführung des Verfahrens gemäß einer der hierin beschriebenen Ausführungsformen eingerichtet ist.

According to another embodiment, an apparatus for suspending or resuspending magnetically attractable particles is provided. The apparatus comprises:

• at least one mixing bar having a front end, the mixing bar having magnetic field generating means for selectively generating a magnetic field in the region of the front end;
• wherein the apparatus is arranged to perform the method according to one of the embodiments described herein.

In the following, the invention will be described with reference to embodiments shown in the attached figures, from which further advantages and modifications result. However, the invention is not limited to the specific embodiments described, but may be modified and modified as appropriate. It is within the scope of the invention to suitably combine individual features and feature combinations of one embodiment with features and feature combinations of another embodiment in order to arrive at further embodiments according to the invention.

Figures 1A and 1B show a first and a second embodiment of a mixing rod.

FIG. 2 shows a third embodiment of a mixing rod.

FIGS. 3A to 3E show individual sequences of the sequence of an embodiment of the method according to the invention.

FIGS. 4A to 4E show individual sequences of the sequence of another embodiment of the inventive method

FIG. 5 shows a lift diagram of a mixing rod with a movable permanent magnet according to another embodiment of the method according to the invention.

The embodiments shown in the figures are not to scale, but are merely illustrative of the respective embodiments. In this case, individual features can be enlarged or reduced displayed. Identical elements are provided in the figures with the same reference numerals.

Figure 1A shows a first embodiment of a mixing rod 101. The mixing rod may for example have an elongated cylindrical shape. The mixing rod 101 has an example cylindrical or rotationally symmetrical outer sleeve 102 made of a typically non-magnetic material. The material of the sleeve 102 should preferably be chosen so that it does not or only slightly attenuates magnetic fields. For example, the sleeve 102 may consist of an inert plastic, which is for example largely dimensionally stable. To achieve the dimensional stability, the material thickness of the sleeve 102 can be selected accordingly. It is also possible to reinforce the sleeve by further structures, for example on the inside of the sleeve 102, wherein the structures may then consist of a material other than the sleeve 102. Composite materials are also possible. Furthermore, the sleeve 102 may be structured on its outer side. At its front end 103, the sleeve 102 is typically closed. This end simultaneously forms the front end 103 of the mixing rod 101.

In the mixing rod 101 according to the first embodiment, a permanent magnet 104 is movable within the sleeve 102, in particular in the longitudinal direction of the sleeve 102, respectively. The permanent magnet 104 can be moved in the sleeve 102 by a rod 105 in the longitudinal direction, i. in particular out of the region of the front end 103 and again brought into the region of the front end 103 into it. This is done for example by means of a corresponding actuator, which is not shown here. The mixing rod 101 is also movable, for example, in the longitudinal direction. In this case, mixing rod 101 and permanent magnet 104 can be moved independently of each other. The movable permanent magnet 104 forms the magnetic field generating means in this embodiment.

The mixing rod 101 may, as in Figure 1A shown inserted into a mixing vessel 110. For example, the mixing vessel 110 may be made of a dimensionally stable, soft material, which may be partially flexible. For example, a plastic can be used for the mixing vessel. In this case, the material of the mixing vessel 110 or softer be as the material of the sleeve 102. Typically, a plurality of mixing vessels 110 may be grouped next to each other to a plate, not shown here.

Figure 1A shows a mixing vessel 110 with a tapered, for example, approximately conical bottom 111. The front end 103 of the mixing rod 101 may be adapted to the shape of the mixing vessel 110 and may also taper pointed, for example approximately conical. Other shapes for the bottom 111 of the mixing vessel and the front end 103 of the mixing rod are also possible, for example approximately concave-shaped, conical, flat or round. In general, free-form surfaces are also conceivable as forms for the bottom 111 of the mixing vessel and the front end 103 of the mixing rod, although these are less preferred for reasons of construction, production and the course of the process. It is favorable if the permanent magnet 104 has a vertical extension which is dimensioned so that its upper end (its north pole N in the exemplary embodiment shown) is always above the liquid level even when the mixing rod 103 is fully immersed.

The permanent magnet 104 generates according to the in Figure 1A In the embodiment shown, a magnetic field that runs essentially in the longitudinal direction of the mixing rod 110. This is in Figure 1A indicated by the arrangement of the poles (north and south). It is also possible for the magnetic field to have a different orientation, for example a lateral alignment with respect to the longitudinal extent of the mixing rod 101. The permanent magnet 104 is in Figure 1A shown comparatively short in the longitudinal direction of the mixing rod 101. It is also possible for the permanent magnet 104 to have a different extension in the longitudinal direction, for example significantly longer. Likewise, the permanent magnet 104 may be formed by two or more permanent magnets.

The spatial position of the magnetic field generated by the permanent magnet 104 with respect to the front end 103 of the mixing rod 101 can be changed by moving the permanent magnet 104. If the permanent magnet 104 is pushed to the front end 103 of the mixing rod 101, the magnetic field generated by the permanent magnet 104 acts there. An "effective" magnetic field is therefore "turned on" in the front region of the mixing rod 101. If, however, the permanent magnet 104 sufficiently far away from the front end 103 of the mixing rod 101, the effect of the magnetic field generated by the permanent magnet 104 at the front end 103 is so far weakened that there is no to Lifting of magnetically attractable particles effective magnetic field is more present. The magnetic field is therefore "turned off" at the front end 103 of the mixing rod 101.

Another embodiment for turning on and off the magnetic field shows FIG. 1B , This comprises one in comparison to the permanent magnet 104 Figure 1A in the longitudinal direction comparatively long permanent magnet 106, which is surrounded by a shielding 107 of, for example, ferromagnetic material. Both the permanent magnet 106 and the shielding sleeve 107 may be arranged to be movable in the longitudinal direction of the mixing rod 101 and be moved independently of one another by corresponding actuating devices, not shown here. For "switching on" of the magnetic field, for example, the shielding sleeve 107 can be withdrawn from the front end 103 in order to expose the south pole of the permanent magnet 106 shown here. As a result, the field lines can penetrate through the sleeve 102 and extend outside the mixing rod 101. To "turn off" the magnetic field, the shielding sleeve 107 is pushed back over the permanent magnet 106 and thereby shields the magnetic field generated by the permanent magnet to the outside. Alternatively, the permanent magnet 106 may be withdrawn from the front end 103. The permanent magnet 106 together with the shielding sleeve 107 in this embodiment forms the magnetic field generating means.

The in the Figures 1A and 1B embodiments shown cause switching on and off of the magnetic field by moving permanent magnets or shielding sleeves. In contrast, shows FIG. 2 an embodiment in which the magnetic field is generated by an electromagnet 120. The electromagnet 120 has a core 121 with, for example, a thick front end 122. The core 121 is surrounded by a coil 123 through which a current for generating a magnetic field can flow. The switching on and off of the magnetic field takes place here by corresponding switching on and off of the current. Mechanical actuating means for moving a permanent magnet or a shielding sleeve can be dispensed with in the embodiment shown here. The magnetic field generating device is formed by the electromagnet 120 in this embodiment. In general, any type of magnetic field generating device for use in the method according to the invention is conceivable, as long as this allows the switching on and off of a magnetic field.

With reference to the FIGS. 3A to 3E will be described below an embodiment of the method according to the invention. This is an in Figure 1A shown mixing rod, but with a long permanent magnet used. However, it is also possible the others in Figures 1B and 2 shown to use mixing rods or otherwise constructed mixing rods. It is only necessary to ensure that the mixing rod allows selective generation of a magnetic field at least at its front end.

First, we provided a mixing vessel 10. The mixing vessel 10 may contain a predominantly liquid mixture 30 with magnetically attractable particles 40 therein. In the following, we only talk about particles. For example, the particles 40 may be particles 40 sedimented from the mixture. The particles 40 have accumulated on the bottom 11 of the mixing vessel 10. Alternatively, it is possible for the mixing vessel 10 to be provided without mixture 30, but only with the particles 40 located at the bottom 11, either as a powder or in suspension, and subsequently then the mixture 30 is transferred into the mixing vessel 10.

The particles 40 may be particles or beads that are attracted to a magnetic field, ie that have, for example, a ferro-, ferri-, para- or superparamagnetic material and at least partially have a surface that is capable of For example, to bind contaminants or biological target molecules such as nucleic acids or proteins. The bondable surface may be formed by the magnetic material itself or at least partially often completely by a non-magnetic material such as a polymer or a SiO ₂ -containing material, which may also be functionalized. The particles have a typical particle diameter of about 500 nm to 25 microns, preferably from about 1 to 20 microns and more preferably from about 4 to 16 microns. It goes without saying that the particles have a certain size distribution. In some cases, the surfaces of the particles 40 are functionalized, the functionalization depending on the specific analytical or diagnostic application and is irrelevant to the inventive method. Such magnetic particles are already known in various embodiments and for various applications from the prior art.

The mixture 30 may be any homogeneous or heterogeneous mixture that may be present in the applications described, and one has sufficient low viscosity in order to carry out the inventive method can. In particular, they are mixtures which have a considerable proportion of liquid constituents. For example, it is a lysis, binding, washing or elution solution or a mixture containing specific or biological, substances or impurities to be examined or separated. If the mixture is a biological sample, it may be untreated or pretreated, for example as a lysate, and may also contain solid components such as cell residues. The nature of the mixture is irrelevant to the performance of the process.

Into the mixture 30 is a mixing rod 1 with its front end 3 ahead, which faces the bottom 11 of the mixing vessel 10, immersed. This is done, for example, by lowering the mixing rod 1 along its longitudinal extent. The downward movement of the mixing rod 1 is in FIG. 3A indicated by an arrow. However, the front end 3 of the mixing rod 1 may already be immersed in the mixture 30 and is then only lowered.

Simultaneously with the lowering of the mixing rod 1, the permanent magnet 4 can be pushed by actuation of the rod 5 to the front end 3 of the mixing rod 1, so that there is a sufficiently strong magnetic field is built up. However, the permanent magnet 3 may already be located at the front end 3 of the mixing rod 1 when it is lowered. Regardless of the way in which the permanent magnet 3 is guided to the front end 3 of the mixing rod 1, this is at least temporarily at the front end 3 when the mixing rod 1 is close to the bottom 11 of the mixing vessel 10. This situation is in FIG. 3B shown. As indicated there, the front end 3 of the mixing rod 1 preferably does not touch the bottom 11 of the mixing vessel, but is somewhat spaced therefrom, typically defined. As a result, on the one hand it can be ensured that there is a certain freedom in the relative vertical arrangement of the mixing vessel 10 to the mixing rod 1. On the other hand, in the case of parallel processing of a plurality of mixing vessels 10, which are composed, for example, into multiwell plates, manufacturing tolerances of the individual mixing vessels, in particular in the case of plastic plates with integrally formed mixing vessels, can be compensated. Finally, it can be avoided that the mixing rod abuts the ground and thus damaging the mixing vessel 10 and possibly leads to leakage of the mixture. For example, the mixing rod can be up about 0.5 to 2 mm are brought to the bottom 11 of the mixing vessel. This distance has been found in most applications to be sufficient to avoid collisions of the mixing rod with the bottom of the mixing vessel. Preferably, the distance to the bottom is preferably 0.1 to 2 mm, more preferably 0.3 to 1 mm, and most preferably 0.5 to 0.6 mm.

As FIG. 3B can be seen, the particles 40 are attracted to the magnetic field generated by the permanent magnet 4 in the region of the front end 3 of the mixing rod 1 and move away from the ground into the mixture, but adhere only to a slight extent on the outer surface of the mixing rod. 1 , or the sleeve 2. As a result, the particles 40 are lifted from the bottom 11 and can be pulled away from the ground by the mixing rod 1. For this purpose, the mixing rod 1 is pulled upwards together with the permanent magnet 4 located at the front end 3, as in FIG FIG. 3C indicated by an arrow. This upward movement can take place comparatively slowly, so that the adhering particles 40 do not detach from the mixing rod 1. However, the movement should not be too slow, since otherwise the proportion of the particles adhering to the mixing rod can become too large.

If the mixing rod is pulled far enough up, with the front end 3 of the mixing rod with the adhering particles 40 remain immersed in the mixture 3, the permanent magnet 4 is pulled by the rod 5 relative to the sleeve 2 also upwards, ie from removed front end 3 of the mixing rod. The permanent magnet 4 can be pulled up comparatively quickly, for example, jerky. By jerk, it is preferably meant that the magnet has a velocity at which it has a time of from 0.05 to 1 s, more preferably from 0.2 to 0.4 s, and most preferably from 0.25 to 0.3 s travels a distance of 100 mm. Since the above information only serves to describe the speed, the path can therefore also be n * 100 mm, where n> 0 and where the associated travel times in this case are also to be multiplied by n. The aim of this measure is to reduce the effect of the magnetic field at the front end 3 of the mixing rod 1 sufficiently fast or off, so that the particles are no longer attracted to the mixing rod 1. The fact that the permanent magnet 4 is removed from the front end 3, the magnetic field is attenuated there and is no longer strong enough to attract the particles 40. The particles 40 are thereby released, ie the direction of movement of the particles is no longer determined by the magnetic field.

In order to avoid that during the pulling up of the permanent magnet 4, the particles 40, which were still in suspension or belong to the part of the particles that adhere to the mixing rod, along the outer surface of the mixing rod 1 with upward, the permanent magnet 4 can be withdrawn sufficiently quickly from the front end 3 of the mixing rod 1, so that the particles 40 can not follow the movement due to friction and the viscosity of the mixture 30. The preferably cone-shaped front end of the mixing rod 3 also counteracts a "walking" of the particles 40. The comparatively rapid pulling up of the permanent magnet 4 is in Figure 3D indicated by a long arrow. Typically, the permanent magnet 4 is brought to a position above the mixture 30, so that in the mixture 30 no effective magnetic field is formed.

In analytical and diagnostic studies, comparatively small amounts of liquid or solution, for example a few milliliters, are typically used. For example, the mixing vessel 10, calculated from the bottom 11, be filled to a height of, for example, about 15 mm. The particles 40 can then be brought to a height of, for example, about 10 mm and released there.

The pulling up of the mixing rod 1 and the permanent magnet 4 need not be done exactly in the manner described above. It is also possible to retract the permanent magnet 4 at least partially and slightly offset in time already when pulling up the mixing rod 1. Regardless of the specific way chosen, the goal is to pick up the particles 40 from the bottom 11 and continue to "up", i. from the bottom of the mixing vessel, so that they can then be better suspended in the mixture 30. It should be recorded by the mixing rod 1 largely all particles sedimented at the bottom 11 40.

The particles 40 are preferably not or only in small quantities adhere to the mixing rod 1. For the best possible suspension of the particles, it is sufficient if they are raised sufficiently far from the ground 11 by the action of the magnetic field. It is still sufficient if the particles 40 are lifted so far that they afterwards can be easily distributed by the subsequent onset of mixing movement of the mixing rod 1 in the mixture.

The mixing movement of the mixing rod 1, which follows the "switching off" of the magnetic field at the front end 3 of the mixing rod, is in FIG. 3E shown. In this embodiment of the method, the mixing rod 1 repeatedly moves up and down, distributing the high-particulate matter 40 in the mixture 30. The stroke of the mixing movement and the frequency are adjusted so that on the one hand sufficient mixing is ensured and on the other hand a " Spilling over "the mixture from a mixing vessel into an adjacent mixing vessel is safely avoided. For example, the mixing movement can take place at a frequency of about 1 Hz to about 20 Hz. The mixing movement of the mixing rod 1 is particularly effective when the mixing rod displaces a not inconsiderable part of the solution volume, as this causes the liquid level to migrate. The change in the liquid level is, for example, from the comparison of FIGS. 3A and 3B clearly. The mixing movement can in particular also be gentler compared to those mixing devices in which no magnetic field-assisted picking up of the particles 40 takes place and which require more vigorous mixing movements in order to stir up the sedimented particles. The lifting height of the mixing rod 1 during the mixing process can be, for example, 30 to 100% of the liquid column.

Other mixing movements, for example a rotation of the mixing rod 1, are also possible. However, rotational movements, especially in parallel processing of multiple mixing vessels, each with associated mixing rod require a higher mechanical effort than strokes. Therefore, in the case of corresponding devices or robots with a large number of, for example, arrayed mixing rods, these are preferably movable only along their longitudinal extent, especially since such a movement is already required for the insertion of the mixing rods and thus no additional mechanism is required.

As a result, the particles 40 are removed by the method according to the invention, as in FIG FIG. 3E indicated, largely uniformly suspended or resuspended in the entire volume of the mixture 30 to above including above the front end 3. This can make better use of resources.

If, despite the mixing movement, a renewed partial sedimentation of the particles 40 occurs, the sedimented particles 40 can be taken up again by the permanent magnet 4. Partial sedimentation is in FIG. 4A indicated. Regardless of whether any Teiledimentation occurs, by re-switching the magnetic field after a certain time or at regular intervals, the particles 40 are again raised sufficiently far, whereby a safe suspensions or resuspension of the particles 40 is ensured.

For the eventual picking up of the particles 40, the permanent magnet 4, for example during a downward movement of the mixing rod 1, is moved towards the front end 3 of the mixing rod 1, so as to build up a sufficiently strong magnetic field there. The movement of the permanent magnet 4, driven by the rod 5 and an actuator, not shown here, is in FIG. 4B indicated by a long arrow. Whose length is to represent in the embodiment shown there, the speed and the stroke of the downward movement, which in the event that the mixing rod 1 does not move simultaneously with the permanent magnet 4, but the permanent magnet 4 moves to the mixing rod 1, higher than that Speed or greater than the stroke of the downward movement of the mixing rod 1, so that preferably permanent magnet 4 and mixing rod 1 arrive at the same time at the bottom of the vessel.

FIG. 4C shows that the particles 40 are drawn again from the front end 3 of the mixing rod 1 from the ground into the mixture. By a in Figure 4D indicated pulling up of the mixing rod 1 with subsequent rapid startup of the permanent magnet 4, the particles 40 moved up from the front end 3 are again brought to a defined height and released there. This is followed by a renewed mixing movement of the mixing rod 1. This is in Figure 4E indicated.

The re-collection or suspending of the particles 40 by the mixing rod can take place, for example, during a downward and upward movement of the mixing movement. It is also possible for the mixing movement to be interrupted or slowed down in order not to hinder the suspension by the mixing movement.

To clarify this situation will open FIG. 5 referenced, which represents a lift diagram for the lifting movement of the mixing rod 1 and the permanent magnet 4. In this case, curve 50 shows the stroke movement of the mixing rod or the sleeve 2 and curve 51, the lifting movement of the permanent magnet 4 as a function of time t. The lifting heights h are shown relative to a separate reference point, for example the bottom 11 of the mixing vessel 10.

In a first phase 61, the sleeve 2 and the permanent magnet 3 are moved together downwards and then together again up to a predefined height, wherein the permanent magnet 4 is in the region of the front end 3 of the mixing rod. This lifting movement can take place comparatively slowly and serves to raise the sedimented particles 40, which are brought to the predefined height. Then, in a second phase 62, a rapid movement away of the permanent magnet 4 from the front end 3 of the sleeve 2 and the mixing rod 1, while the sleeve 2 can also be pulled up a little. Due to the rapid pulling up of the permanent magnet 4 from the front end 3, the particles are released. This is followed by a third phase 63, in which essentially only the sleeve 2 is moved to produce a mixing movement. It is also possible to move along the permanent magnet 4, wherein this should have a sufficient distance to the liquid surface of the mixture 30. The mixing movement is in FIG. 5 represented by periodic or oscillating strokes.

Optionally, re-raising and suspending the particles 40 may follow. This is indicated by the phase 64, in which a slower compared to the mixing movements stroke movement takes place and the permanent magnet 4 can be moved asymmetrically to the lifting movement of the sleeve 2 and the mixing rod 1. In this case, the permanent magnet 4 is moved, for example, very quickly towards the front end 3, when the front end 3 of the mixing rod 1 in the vicinity of the bottom 11 of the mixing vessel 10 is located. This is to prevent that still suspended particles are pulled down again. Then, the upward movement of the sleeve 2 takes place together with the permanent magnet 4, which is withdrawn quickly at a defined height again from the front end 3 of the mixing rod. This is followed in phase 65 by a re-mixing without magnetic field.

In the FIG. 5 Phases shown can also merge into each other. For example, it is possible to perform the magnetic field-assisted picking up of the particles during the mixing movement.

The invention is not limited to the embodiments described above, but includes suitable modification within the scope indicated by the claims. The appended claims are intended as a first, non-binding attempt to describe the invention in general terms.

LIST OF REFERENCE NUMBERS

1, 101

Mischstab

2, 102

shell

3, 103

front end of the mixing bar

4, 104

permanent magnet

5, 105

Rod

106

permanent magnet

107

shielding

10, 110

mixing vessel

11, 111

Bottom of the mixing vessel

30

mixture

40

particle

50

Lifting curve of the mixing rod

51

Lifting curve of the permanent magnet

61

first phase

62

second phase

63

third phase

64

fourth phase

65

fifth phase

120

electromagnet

121

core

122

End of the electromagnet

123

Kitchen sink

Claims (10)

A method of suspending or resuspending magnetically attractable particles comprising the steps of: - Providing at least one mixing vessel (10) which is at least partially filled with a mixture (30) in which magnetically attractable particles (40) are at least partially sedimented at the bottom (11) of the mixing vessel (10); Providing at least one mixing rod (1) with a front end (3) facing the bottom (11) of the mixing vessel (10), wherein the mixing rod (1) comprises magnetic field generating means (4) for selectively generating a magnetic field at least in the region of the front end ( 3); - Switching on an effective magnetic field, which acts at least in the region of the front end (3) of the mixing rod (1), with the aid of the magnetic field generating means (4) while the mixing rod (1) in the mixture (30) is immersed; - Movement of the magnetic field together with the mixing rod from the bottom (11) of the mixing vessel (10) away, wherein the movement of the magnetic field together with the mixing rod is such that at least a portion of the magnetically attractable particles (40) from the bottom (11) of the mixing vessel (10) are lifted and the proportion of particles adhered to the mixing bar is minimized; - switching off the magnetic field at a previously defined distance from the ground, which is greater than the distance from the ground when switching on the magnetic field; - Perform repeated mixing movements of the mixing rod (1) without the front end (3) of the mixing rod (1) is a switched magnetic field to suspend the present in the mixture (30) magnetically attractable particles or resuspend. The method of claim 1, wherein after repeated mixing movements, the magnetic field at the front end (3) of the mixing rod (1) is generated again to raise again magnetically attractable particles (40). A method according to claim 1 or 2, wherein for mixing, the mixing rod (1) is reciprocated along its longitudinal direction. Method according to one of the preceding claims, wherein the magnetic field at the front end (3) of the mixing rod (1) is switched on at least when the mixing rod (1) with its front end (3) at a defined minimum distance from the bottom (11). of the mixing vessel (10) is located. Method according to one of the preceding claims, wherein the mixing rod (1) has at least one permanent magnet (4) which is movable in the longitudinal direction of the mixing rod (1). Method according to Claim 5, in which the permanent magnet (4) is moved towards the front end (3) of the mixing rod (1) in order to switch on the magnetic field in the region of the front end (3) of the mixing rod (1) and to switch off the magnetic field from the front end (3). 3) is moved away. Method according to claim 5 or 6, wherein the permanent magnet (4) is moved abruptly away from the front end (3) of the mixing rod (1) when the magnetic field is switched off. Method according to one of claims 1 to 4, wherein the mixing rod (100) at least one permanent magnet (106) in the region of its front end (103) and at least one of the permanent magnet (106) surrounding and in the longitudinal direction of the mixing rod (100) displaceable shielding sleeve (107 ) having. Method according to one of claims 1 to 4, wherein the mixing rod (100) comprises an electromagnet (120) for generating the magnetic field. Method according to one of the preceding claims, wherein the magnetically attractable particles (40) are ferro-, ferri-, paramagnetic and / or superparamagnetic particles.

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