Magnetic Tissue Grinding

BACKGROUND OF THE INVENTION

Field of the invention

The present invention relates to a method and device for grinding by mechanical action, a sample in a container having a container wall with the sample adhered at a site of the container wall.

Description of the prior art

Sample preparation plays a crucial role in particular in preparing samples for sensitive diagnostic analyses. For example, nucleic acids for use in diagnostics often have to be isolated from various tissues. The isolation is frequently followed by amplification reactions, such as the so-called polymerase chain reaction (PCR). It is preferred that the methods for isolation and in particular also the initial step of sampling from tissue can be automated. For example, a container comprising a sample is introduced in a centrifuge, wherein, due to the centrifugal forces action on the sample, a solid fraction of the sample is adhered at a site of the container wall. This solid fraction of the sample has to be treated further in accordance with procedures known in the art. Up to now, one of the initial steps for obtaining nucleic acids is work-up of tissue for treating disrupted tissues or tissue cells by a mechanical and/or chemical treatment for example by enzymes such as proteinases. Then the nucleic acid e. g. genomic DNA is adsorbed on carriers such as e.g. magnetic beads. After washing steps often a rigid solid body remains, from which the DNA can often only be removed in a cumbersome way.

The quality of the isolation of nucleic acids for PCR is often determined by the skill of the technician and thus not reproducible when changing from one laboratory to another.

It is basically known to provide sample preparation by means of steering a sample within a liquid. For these purposes, it is also known to use magnetic steering methods and apparatus. One example of such a steering apparatus is disclosed in WO-A-2004/004874. However, this known steering apparatus cannot be used e.g. for disintegrating or grinding a sample adhered to a container wall and to be dissolved in a liquid.

An object of the present invention is to provide a method which improves sample preparation. SUMMARY OF THE INVENTION

According to one aspect of the present invention, there is provided a method for grinding, by mechanically action, a sample in a container, wherein the container comprises a container wall with the sample adhered at a site of a container wall, and wherein the method comprises the steps of

placing a magnetic grinding element in the container and

subjecting the container to a magnetic field having a direction which is time- variant,

wherein the magnetic grinding element, under the influence of the time- variant magnetic field, impinges on an area of the container wall comprising the site where the sample adheres at the container wall thereby grinding the sample. Moreover, in a further aspect of the present invention, there is provided a device for grinding, by mechanical action, a sample in a container, comprising

a support for supporting the container,

a magnetic field generating means for creating a magnetic field having a direction which is time-variant, with the magnetic field extending through the container, and

a magnetic grinding element for placing into the container,

wherein the magnetic grinding element is capable of being moved in diverse time-variant directions under the influence of the changing magnetic field so as to mechanically interact with the sample for grinding the same.

One of the main features of the present invention is the use of a time-variant magnetic field and at least one magnetic grinding element subjected to the magnetic field. Since the magnetic field is time-variant (in its direction and optionally in its magnetic force distribution), the magnetic grinding element is moved within the container in a plurality of directions in space and, moreover, is rotated, wherein both movements are highly inhomogeneous and irregular. This effect can be used according to the invention by impinging on the sample for grinding the same.

In the context of the present invention, grinding means the result of each kind of mechanical action of the grinding element on the sample. Accordingly, grinding means crushing, fragmenting, comminuting, breaking, disintegrating, mincing, smashing or the like.

For generating the magnetic field having a time-variant direction and orientation and, optionally, a time-variant magnetic force distribution, a magnetic field generating means is used which comprises a permanent magnet or an electromagnet both provided with a north pole region and a south pole region. When using a permanent magnet, this magnet preferably is formed of an NbFeB alloy. In case of providing an electromagnet, the magnetic field generating means further comprises a control unit so as to control the current through the electromagnet for generating a magnetic field having a timed-variant direction/orientation and, optionally, a time-variant magnet force distribution. Basically, the grinding element may have an arbitrary shape. However, experiments have shown that forming the grinding element as a substantially elongated body is preferred for the grinding process. In case of a substantially elongated grinding element, the same is divided along its longitudinal direction into a north pole region and a south pole region wherein each of these regions extend along a lateral side of the substantially elongated body.

Basically, the shape of the container within which the at least one grinding element is arranged is not relevant for the invention. However, substantially cylindrical containers or other test tubes are preferred experiments carried out in which the container was an Eppendorf test tube having a substantially conically- shaped bottom. In such a container (either substantially cylindrical and, optionally, provided with a substantially conically-shaped bottom) it is preferred if the grinding element is a substantially elongated body oriented in a substantially upright direction within the container so as to impinge onto the inner side wall of the container if the grinding element is subjected to the time- variant magnetic field.

The method and device according to the invention can be applied to a plurality of containers positioned in a rack and arranged in columns and rows of a two- dimensional area or arranged along a circle of an annular rack, wherein at least one grinding element is arranged in each container.

The present invention can be used for e.g. dissolving samples of any kind in a liquid. For example, the sample can be tissue of various origins such as plant, animal or human tissue and/or wherein the sample comprises biopolymers such as proteins, carbohydrates, lipids, nucleic acids, in particular DNA, RNA, and/or wherein the sample is a lysate from cells prepared for the isolation of nucleic acids, in particular genomic DNA.

BRIEF DESCRIPTION OF THE DRAWINGS

A full and enabling disclosure of the present invention, including the best mode thereof, enabling one of ordinary skill in the art to carry out the invention, is set forth in greater detail in the following description, including reference to the accompanying drawing in which Figs. 1 to 4 schematically show the interaction of a displaceable magnetic field and a magnetic grinding element arranged within a test tube for grinding a sample adhered at a site of the inner wall of the test tube, and Fig. 5 shows a section of a rack supporting a plurality of test tubes with a magnetic field generating means arranged between the test tubes for generating a time-variant magnetic field subjected to adjacent test tubes. Fig. 6 shows comparative results of qualitative PCR results. Fig. 7 shows an electrophoretic gel of conventionally prepared DNA and the one according to the invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

Figs. 1 to 4 show schematically the principles of the present invention. A container 10 comprises a substantially conically-shaped bottom 12 with a sample 14 to be dissolved in a liquid 16 within the container 10 is adhered at a site 18 at the inner side of the container wall 20. A magnetic field generating means 22 is arranged outside of the container 10 and comprises a permanent magnet 24 which is displaceable. The permanent magnet 24 has a north pole region 26 and a south pole region 28 and generates a magnetic field 30 having magnetic field lines 32 extending through the container wall 20 and the sample 14. The permanent magnet 24 in this embodiment is rotatable in the direction of arrow 34. However, in the sense of the invention it would be sufficient if the permanent magnet 24 would oscillate about an oscillation access extending between the north and south pole regions 26 and 28, respectively.

It is important that the magnetic field generating means generates a time- variant changing magnetic field within the container 10. Again, it is to be noted that this can be provided by any kind of movement of the permanent magnet 24. Also it is to be noted that the permanent magnet 24 could be replaced by an electromagnet. Within the container 10 is arranged a grinding element 36 which in this embodiment comprises an elongated body. The magnetic grinding element also comprises a north pole region 38 and a south pole region 40 which under the influence of the magnetic field established by the magnetic field lines 32 is oriented accordingly. Therefore, when changing the magnetic field of the magnetic field generating means 22, the magnetic grinding element 36 will be moved by tilting, rocking, turning and the like movements within the container 10 as shown in Figs. 1 to 4. The result of these movements of the magnetic grinding element 36 in diverse directions in space and around the longitudinal axis of the magnetic grinding element 36 is that the magnetic grinding element 36 impinges and acts on the sample 14 for grinding the same. This process is very efficient as experiments have shown.

According to Fig. 5, several containers 10 can be supported in a rack 42 wherein within each container 10 a sample 14 and a grinding element 36 are arranged. A magnetic field generating means 22 is arranged close to the containers 10 for generating a time-variant magnetic field (with regard to the direction of the magnetic field lines and/or the magnetic force distribution) . Under the influence of this time-variant magnetic field, the magnetic grinding elements 36 act on the respective sample 14 for grinding them. In one embodiment of the invention it can be used in the field of molecular biology and chemistry and is applied in laboratory diagnostics, in particular for sample preparation for PCR analysis, for the extraction of DNA from biological preparations and for the elimination or neutralization of contaminants to obtain DNA having a purity grade that is suitable for the amplification reaction. The invention relates also to a process for grinding up inhomogeneities of concentration as formed during mixing of a blood sample with magnetic iron oxide nanoparticles coated with Si0 ₂ adsorbent in a lysing solution.

The use of magnetic particles is not fundamentally different from the use of other kinds of sorbents (latexes, polymer sorbents or glass), but enables the application of magnetic separation instead of centrifugation and filtration. The magnetic particles are based on iron oxide nanoparticles having superparamagnetic properties. Particles based on iron oxide having superparamagnetic properties possess a number of advantages, in particular: - The particles have virtually zero residual magnetization, i.e., after the external magnetic field has been removed (transfer from a magnetic stand to a usual stand), they will not agglutinate and can be easily resuspended.

The particles are chemically inert and can be stored for a long time in aqueous solutions, do not inhibit enzymatic reactions and can be used in both in vitro and in vivo experiments without undesirable consequences.

The particles offer many possibilities for the automatization of processes for the isolation of nucleic acids, proteins and cells.

Processes for obtaining nucleic acids using magnetic particles are known. For example the process for the purification of a substance follows a protocol wherein the material containing the substance and magnetic particles coated or treated with a reagent which binds the particles to the substance are dispensed in a first medium, and upon binding of the substance to the particles a magnetic probe is added to the medium, whereby the particles adhere to the probe, and the probe together with the particles and the substance bound to them is transferred to a second medium, and if desired, separated from the second medium and transferred to a third medium for further working-up. One of the main drawbacks of this purification process is the fact that the magnetic probe with the particles adhering thereto must be transferred between the individual media, which highly increases the risk of contaminating the sample to be examined.

Another method of prior art uses magnetic polymer particles based on polyvinyl alcohol. Magnetic granular supports are used which were obtained by suspending the polymer phase containing magnetic colloids of polyvinyl alcohol in an organic phase containing a mixture of specific emulsifiers. In this way, particles having a diameter of 1 to 8 pm that can chemically bind ligands are obtained. The supports can be used for the isolation and detection of biomolecules, cells, antibodies and nucleic acids.

In a further method of prior art a substance is detected and quantified by the use of magnetic colloidal particles functionalized at the surface with a specific ligand that is to be detected or subject to the quantification of the analyte. For the magnetic separation, strong magnets based on rare earth elements are used. The complete collection of the magnetic particles in a magnetic stand usually takes several seconds to several minutes. The separation time depends on the volume and viscosity of the liquid from which the magnetic particles are collected.

A problem arising when magnetic particles are used is as follows : After the adsorption, i.e., the "adhesion" of the DNA to the surface of the magnetic particles and the collection thereof by a magnetic field on one of the walls of the test tube, the problem is the formation of a "bundle" of magnetic particles, i.e., a "DNA bundle", which complicates the implementation of the further washing steps and prevents the PCR reaction from being performed during a period of time realistic for DNA analysis.

According to another embodiment of the present invention to develop a process for the homogenization, i.e., resuspension, of a solution of DNA adsorbed to magnetic particles by partial defragmentation thereof and reduction of supercoiling, the effective washing and elution or separation of the DNA from the magnetic particles is performable without transfer of the magnetic probe, which would involve the risk of contamination between the individual volumes, and for ensuring optimum conditions for the fluorescence analysis of the DNA isolated and purified with "PCR RealTime" type equipment, and thus for the automatization of the whole DNA analysis cycle.

According to the present invention partial defragmentation of the DNA molecules is achieved by grinding up any inhomogeneities of the concentration of magnetic particles jammed, by a strong inhomogeneous magnetic field, between the interior surface of the test tube and the magnetic probe rotating about its axis. The rotation of the magnetic probe, which is simultaneously pressed against the interior wall of the test tube, is caused by the rotating superparamagnetic (e.g. 1.2-1.3 T) beads of the magnetic stand. The following example illustrates this embodiment.

To a 1.5 ml test tube as used in a standard procedure of DNA isolation, the following was added :

1. 5 μΙ of the standard concentration of magnetic particles;

2. 50 μΙ of blood for the isolation of the DNA;

3. 150 μΙ of lysing solution;

4. a magnetic cylindrical probe. The rotation of the magnetic probe about its own axis, which is collinear with the test tube axis and generates microturbulent flows, favored maximum adsorption, i.e., the "sticking" of the DNA to the surface of the magnetic particles.

After the rotational movement of the probe has stopped and the magnetic nanoparticles with the DNA have been collected on the interior surface of the test tube by the strong inhomogeneous magnetic field of the beads of the stand and in part on the surface of the magnetic probe, the magnetic particles, i.e., the "entangled" DNA, form a "bundle", which is a very elastic porous medium of high tear strength, which was the major obstacle in the use of the standard method for isolating DNA using magnetic particles and a magnetic field.

For the elimination (disentanglement) of the agglomeration of the magnetic particles, different attempts have been made, such as repeated pipetting, comminuting by means of the blades of a mixer at a frequency of above about 50 Hz and comminuting by means of larger ferromagnetic particles according to the functional principle of ball mills. However, all these attempts failed to succeed. Thus, it was not possible to perform the further standard steps of sample processing, i.e., the 3 washing steps and the elution of the DNA from the magnetic particles, nor was it possible to perform the isolation of pure DNA samples suitable for PCR analysis. A positive result, i.e., the production of a homogeneous solution of the magnetic particles with the DNA adhering thereto, was obtained only by applying the principle of grinding up microinhomogeneities jammed between the two surfaces.

The microinhomogeneities with the magnetic particles, i.e., the "agglomerates", are attracted into the range of a strong magnetic field and at the same time pressed against the walls of the test tube. This is done in a standard magnetic stand with a stationary magnetic field. As the magnets for the stand, we used superparamagnetic beads of Nd-Fe-B having a diameter of 5 mm (B _reS | _d uai » 1-2- 1.3 T). They were positioned on the outside of the test tube and were free to rotate about any axis. According to the method proposed by us, the second surface causing the pressing effect is the magnetic probe introduced into the test tube, i.e., a small cylinder with a diameter of 1.5-2.0 mm and a length of 6-8 mm that is magnetized transversely to the cylinder axis (in parallel with the test tube axis) and has a value of B _residU ai « 0.2-0.3 T on the surface. As the material for preparing the magnetic cylinder, magnetoplast prepared by casting (silicone with an addition of 80% quickly cured magnetic powder made of a neodymium alloy) is used, followed by magnetization by means of an inductor.

The magnetic cylindrical probe attracted by a strong magnet jammed the "agglomerates" between itself and the interior wall of the test tube. By rotating about its own axis while remaining pressed against the interior wall of the test tube, the magnetic probe caused the "agglomerates" of the magnetic particles of the "entangled DNA" to be ground up, which also in part defragments the DNA as seen from a subsequent electrophoresis. In the test tube, a suspension of magnetic particles with DNA adsorbed thereto is formed.

Sixteen test tubes with magnetic cylindrical disposable probes were uniformly arranged on the inside at the circumference of the magnetic stand. Superparamagnetic (1.2-1.3 T) beads having a diameter of 5 mm were arranged between each of the eight pairs of test tubes in the cylindrical cavities (diameter 5.1 mm) of the stand 8 for a total of eight beads, each of which was free to rotate about any axis. Figure 1 shows a model of the device without test tubes. The rotation of the eight superparamagnetic beads of the stand was brought about by generating an induction vector of the magnetic field in the plane of the circumference of their arrangement that rotates within the horizontal plane. According to our model, the generation of a magnetic field rotating at a frequency of 10 to 30 Hz was effected by the rotation in the radial plane of a bar magnet arranged along the diameter of the circumference of the test tubes.

Figure 2, which shows the model from underneath, shows the arrangement of the bar magnet that generates the rotating magnetic field. An electric motor supplied with power by batteries (3-6 V) and having a rotation frequency of 30-50 Hz served as a drive. The cavities in the stand for the test tubes were surrounded by an elastic heating element (nichrome in a carbon tape, power consumption about 10 W) for heating at 50-60 °C after the third washing step and for the drying (5 minutes) as well as for heating at 60-65 °C (10 minutes) in the elution solution for separating the DNA from the sorbent on the surface of the magnetic particles. The elastic carbon heating tape with two nichrome filaments is shown in Figure 1 and Figure 2.

The last step of the sample processing is the collection of the magnetic particles on the test tube wall and on the magnetic probe. The isolated and purified DNA will then remain dissolved.

Steps of the isolation of the DNA from a biological sample using the invention

The DNA is isolated from blood samples using a standard reagent kit. For a 1.5 ml test tube, the following steps apply: First step

50 μΙ of blood + 150 μΙ of lysing solution + 5 μΙ of standard concentration of magnetic particles;

washing;

holding the magnetic particles to the test tube wall;

- removing the liquid phase from the test tube.

Second step

400 μΙ of standard washing solution No. 1;

washing;

holding the magnetic particles to the test tube wall;

- removing the liquid phase from the test tube. Third step

200 μΙ of standard washing solution No. 2;

washing;

holding the magnetic particles to the test tube wall;

- removing the liquid phase from the test tube.

Fourth step

200 pi of standard washing solution No. 3;

washing;

holding the magnetic particles to the test tube wall;

- removing the liquid phase from the test tube;

drying the content of the test tube for 5 minutes at a temperature of 50- 65 °C.

Fifth step

100 μΙ of standard elution solution for 10 minutes at a temperature of 50- 65 °C;

holding the magnetic particles to the test tube wall;

collecting the isolated and purified DNA from the test tube for the subsequent analysis.

Concrete Example

For isolating the DNA from blood cells, sixteen 1.5 ml test tubes were arranged in the model of the device. One test tube served to survey the temperature conditions on steps 4 and 5 of the sample processing. For this purpose, a digital temperature sensor was inserted into the sixteenth test tube. In the remaining 15 test tubes, the magnetic cylindrical probe was arranged.

First step: After pouring a solution containing 50 μΙ of blood + 150 pi of lysing solution into the test tube, 5 μΙ of a standard concentration of magnetic particles was added. By switching on a rotating magnetic field for 5 to 10 seconds, the rotation of the magnetic probe caused a uniform distribution of the magnetic particles in the solution, wherein optimum conditions for the adsorption of the DNA to their surface were achieved. As experiments have shown, the rotation frequency of the magnetic probe must not exceed 50 Hz, because otherwise the solution would foam up and spray onto the test tube walls.

After the rotational movement was stopped, the superparamagnetic beads of the stand then collected the magnetic particles at the wall of the test tube at a level of about 100 μΙ. At the same time, the magnetic cylindrical probe was also pressed against the test tube wall. Part of the magnetic particles settled on the surface of the magnetic probe. The drifting of the magnetic particles from the solution and the settling thereof on the test tube wall and the probe surface took about 1 minute. Thereafter, the liquid phase was removed from the test tube using a usual pipette with a disposable spout.

Second step: After pouring 400 μΙ of washing solution No. 1 into the test tube, the model of the device was switched on for about 1 minute. The rotating magnetic probe, which was pressed against the test tube wall by the superparamagnetic beads of the stand, ground up any inhomogeneities contained in the magnetic particles at the interior wall of the test tube to produce a suspension of magnetic particles. The microturbulent rotational movement of the liquid in the test tube favored a thorough washing of the DNA. After the rotational movement was stopped, the superparamagnetic beads of the stand collected the magnetic particles at the test tube wall at a level of about 100 μΙ. At the same time, the cylindrical magnetic probe was also pressed against the test tube wall. Part of the magnetic particles settled on the surface of the magnetic probe. The drifting of the magnetic particles from the solution and the settling thereof on the test tube wall and the probe surface took about 1 minute. Thereafter, the liquid phase was removed from the test tube using a conventional pipette with a disposable spout.

Third step: After pouring 200 μΙ of washing solution No. 2 into the test tube, the same procedure as above was applied.

Fourth step: After pouring 200 μΙ of washing solution No. 3 into the test tube, the same procedure as above was applied. However, after the liquid phase had been drawn off, the contents of the test tube had to be dried for 5 minutes at a temperature of 50-65 °C. For this purpose, the elastic heating element that surrounded all the 16 test tubes at the positions where they were fitted into the stand was turned on. The heating element was supplied with power by a traditional type HY3005C power source with a voltage of about 15 V. The temperature monitoring was effected by means of a digital temperature sensor arranged in the sixteenth test tube. The drying process had to be performed thoroughly, since the washing solution No. 3 blocked the reaction of DNA amplification.

Fifth step: After 100 μΙ of elution solution separating the DNA from the magnetic particles had been poured into the test tube, the elastic heating element was again turned on, and a temperature of 50-65 °C was set in the test tubes for 10 minutes. During this time, the rotation of the magnetic probe was effected periodically for 10 to 15 seconds once a minute. This enabled the homogenization of the solution on the one hand and prevented the DNA molecules from too strongly defragmenting on the other.

After the device had been turned off, the magnetic particles were collected for one minute on the side walls of the test tube and on the disposable magnetic probe under the action of the inhomogeneous magnetic field of the beads.

The purified DNA remained in the solution and was ready for further analysis. For a comparison of the amount of DNA isolated by the conventional method with the corresponding amount obtained using the model of the device, including in different test tubes, amplification reactions were performed with samples of all kinds. The amplification was effected by means of a type DT-96 detection amplificator (registration certificate of the Federal Regulatory Agency in the area of public health and social development No. FSR 2007/01250 of November 20, 2007). The results of the amplification performed are shown in Figure 6. The identification number of the cycle for the beginning of the reaction is 27 in both cases.

The degree of defragmentation of the DNA isolated by means of the model of the device can be established by means of the electrophorogram shown in Figure 7.

Figure 6 shows the results of a comparison of the qualitative PCR analysis of the DNA using the conventional manual method of DNA isolation with the method using the model of the device for sample processing. The coincidence of the upper curves demonstrates the high performance of the method of the invention. The two curves shifted to a higher number of PCR cycles (x-axis: No of cylces) have been prepared conventionally.

Figure 7 shows the results of a comparison of the qualitative PCR analysis of the DNA obtained by conventional sample processing on the one hand with the PCR analysis of the DNA obtained by the process of homogenization using the model of the device for sample processing. The results of the electrophoresis show some defragmentation of the DNA isolated using the model of the device (see the first 6 columns) as compared to the conventional manual method (see columns 7 and 8).

Although the invention has been described and illustrated with reference to specific illustrative embodiments thereof, it is not intended that the invention be limited to those illustrative embodiments. Those skilled in the art will recognize that variations and modifications can be made without departing from the true scope of the invention as defined by the claims that follow. It is therefore intended to include within the invention all such variations and modifications as fall within the scope of the appended claims and equivalents thereof.