Patent Application
20090008254
The invention relates to a microfluidic system and an associated control method in accordance with the preamble of the secondary claims.
Such microfluidic systems are known, e.g., from Müller, T. et al.: “A 3D-micro electrode for handling and caging single cells and particles”, Biosensors and Bioelectronics 14, 247-256, 1999 and have a flat carrier flow channel for receiving a carrier flow with particles suspended in it (e.g., biological cells), in which carrier flow channel a dielectrophoretic electrode arrangement is located in order to manipulate the suspended particles. For example, the suspended particles can be centered in the carrier flow by a funnel-shaped electrode arrangement (“funnel”) or fixed by a so-called hook.
However, a disadvantage of the known microfluidic holding systems, e.g., the above-cited dielectrophoretic hooks, is the fact that the particles can be pressed up or down towards the channel wall by the dielectrophoretic electrode arrangement in the carrier flow channel, which is problematic especially in the case of biological cells.
The use of dielectrophoretic field cages for fixing suspended particles in a field minimum inside the field cage is known from Schnelle, T. et al.: “Trapping of Viruses in High Frequency Electric Field Cages”, Naturwissenschaften 83, 172-176 (1996), Springer-Verlag. These field cages have eight cubically arranged cage electrodes and center the suspended particles in all spatial directions, which is necessary here in order to prevent an adhesion of the particles to the channel walls. To this end, an individual electrical supply of the cage electrodes is necessary, which is technically very expensive and makes it difficult, e.g., to achieve the ability to parallelize.
Other exemplary embodiments of such field cages are known from Fuhr, G. et al.: “Positioning and Manipulation of Cells and Microparticles Using Miniaturized Electric Field Traps and Travelling Waves”, Sensors and Materials, Vol. 7, No. 2 (1995) 131-146.
The subsequently published patent application DE 10 2004 017 482 A1 also discloses microfluidic systems with dielectrophoretic elements such as, e.g., the already initially cited field cages, funnels as well as particle switches. However, the field cages center the suspended particles in all spatial directions, which is necessary here in order to prevent an adhesion of the suspended particles on the channel walls of the carrier flow channel.
So-called biochips are known from US2002/0182627 A1 in which suspended particles are manipulated by electrophoresis. Moreover, this patent application also discloses planar dielectrophoretic field cages that position a suspended particle in a bore of a plate. However, this type of fixation results in a purposeful manner in a touching contact between the fixed particle and the channel delimitation, which is especially problematic in the case of biological cells.
Furthermore, DE 199 52 322 C2, DE 103 11 716 A1 and U.S. Pat. No. 5,454,472 disclose methods and devices for separating suspended particles by means of dielectrophoretic elements. However, no measures are known from these patent applications for preventing the adhesion of suspended particles to the channel walls.
It is therefore an object of the invention to appropriately improve and simplify the initially described known microfluidic holding systems, during which it should be prevented that the suspended particles are pressed by the electrode arrangement in the direction of the channel wall.
This object is achieved by a microfluidic system in accordance with the invention and by an associated control method in accordance with the secondary claims.
The invention comprises the general technical teaching of arranging centering electrodes upstream before the manipulation electrode (e.g., a so-called “hook”), which centering electrodes focus the particles suspended in the carrier flow in the central plane of the carrier flow channel, thus preventing the suspended particles from being pressed in the direction of the channel wall.
The concept of centering or focusing used in the framework of the invention preferably means that the suspended particles are centered or focused at a right angle to the direction of flow.
The microfluidic system in accordance with the invention includes an electrode arrangement with at least two manipulation electrodes and at least two centering electrodes arranged upstream before the manipulation electrodes.
The two manipulation electrodes can be, e.g., so-called hooks that are already known from the initially cited publication by Müller, T. et al.: “A 3D-micro electrode for handling and caging single cells and particles”, Biosensors and Bioelectronics 14, 247-256, 1999, so that the content of this publication is to be added to its full extent to the present description as regards the design of the manipulation electrodes.
Furthermore, it should be mentioned that the manipulation electrodes do not necessarily have to be in one piece or continuous, but rather there is also the possibility that individual manipulation electrodes consist of several partial electrodes and that the individual partial electrodes of the manipulation electrodes can be separately controlled. For example, the individual manipulation electrodes can also be interrupted by passivation layers.
However, it is important that the manipulation electrodes are curved in opposition to the direction of flow such as is the case, e.g., with the known so-called hooks. However, there is also the possibility, instead of hook-shaped manipulation electrodes, that the manipulation electrodes are arcuate (e.g., semicircular) or closed in a ring form. However, they can also have the form of a rectangle or of a part of a rectangle, of a hexagon or in general of a polygon. Therefore, the form can be almost as desired, just as in the case of the centering electrodes. For example, the manipulation electrodes can be in the form of a circular ring, which makes it possible to arrange several particles on closed paths.
The invention furthermore provides that the centering electrodes are at least partially surrounded by the manipulation electrodes arranged downstream behind them. In the case of a hook-shaped manipulation electrode this can be readily achieved in that the associated centering electrode is preferably arranged between the two shanks of the hook-shaped manipulation electrode. In the case of a ring-shaped manipulation electrode the associated centering electrode can be arranged inside the manipulation electrode.
In addition, the invention provides that the centering electrodes have a lesser spatial extension transversely to the direction of flow than the manipulation electrodes, which is not the case with the initially mentioned field cages with eight cubically arranged cage electrodes.
The centering electrodes are preferably triangular, rectangular, hexagonal, round, circular or elliptical in form, with the centering electrodes being preferably smaller than the manipulation electrodes transversely to the direction of flow.
It should furthermore be mentioned that the two centering electrodes can be controlled separated electrically from one another in a preferred embodiment in order that the centering electrodes can be controlled in electrical phase opposition.
The same also preferably applies to the two manipulation electrodes that can preferably also be controlled electrically separated from one another in order to make possible a control in phase opposition.
Moreover, even the manipulation electrodes on the one hand and the centering electrodes on the other hand can be controlled in an electrically separated manner since the manipulation electrodes and the centering electrodes associated with them should be controlled in an electrical phase opposition in order to achieve a centering action.
It should furthermore be mentioned that the two manipulation electrodes and/or the two centering electrodes are preferably substantially planar (that is, level), wherein the two manipulation electrodes on the one hand and the two centering electrodes are preferably each arranged in pairs in a substantially coplanar manner. That means that the individual electrodes are arranged in two planes parallel to one another, wherein in each case one manipulation electrode and one associated centering electrode are present in each plane. In comparison to the field cage the suggested arrangement is more robust against shifting, which facilitates the manufacture of the systems.
The centering electrodes and the manipulation electrodes are arranged here in the direction of flow at a distance from each other that is preferably in a range of ⅛ to twice the distance of the electrode planes. For handling animal suspension cells, e.g., blood cells, this is preferably in a range of 5 μm to 80 μm and a distance of approximately 40 μm has proven to be especially advantageous.
In an advantageous variant of the invention the electrode arrangement has several manipulation electrode pairs and centering electrode pairs associated with the latter. The individual manipulation electrode pairs can be arranged relative to the direction of flow adjacent to each other or behind one another in the carrier flow channel. This array arrangement permits, in comparison to traditional dielectrophoretic cages, a simpler and better long-time cultivation of biological cells in microfluidic chips. For example, several so-called hooks can be arranged adjacent to each other in the direction of flow in order to fix suspended particles.
The individual manipulation electrode pairs can be electrically connected to each other, which makes possible a common electrical control in which the individual manipulation electrodes of a manipulation electrode pair are controlled in a traditional manner in phase opposition.
However, there is also the alternative possibility that the individual manipulation electrode pairs are electrically separated from each other at least partially and are separately controlled in an at least partially electrical manner, which makes possible a simple selective detection of the suspended particles.
Furthermore, the scope of the invention includes the goal of minimizing the thermal load of the suspended particles, which is especially important in the case of biological cells. However, the thermal stress of the suspended particles is a function of the electrode width and of the electrode distance, which parameters also influence the force which the electrode arrangement exerts on the suspended particles. The lateral electrode width is preferably in the range of 10% to 50% of the electrode distance between the planes since the ratio of the desired force to the undesired warming of the suspended particles is especially good in this range.
Furthermore, it should be mentioned that the carrier flow channel of the microfluidic system of the invention preferably has a cross section of flow in the range of 0.006 mm2 to 0.6 mm2, which is customary in microfluidic systems. The height of the carrier flow channel here can be, e.g., in the range of 1 μm to 400 μm whereas the width of the carrier flow channel can be, e.g., in the range of 5 μm to 1.5 mm.
In general, the cross section of the carrier flow channel can be different, thus it can be, e.g., rectangular or trapezoidal.
Furthermore, there is the possibility that the two manipulation electrodes of the electrode arrangement of the invention, which are arranged in a coplanar manner, are arranged offset to one another in the direction of flow. In the same manner even the two centering electrodes of the electrode arrangement are offset to one another in the direction of flow. The offset in the direction of flow in relation to the distance between the manipulation electrodes and the centering electrodes can be in a range of 5% to 95%, 10% to 90%, 20% to 80% or 30% to 70%. The possibility of offsetting the electrodes has the advantage that as a result not so high requirements have to be placed on the manufacturing process as, e.g., in the case of the already known field cages, in which an exact alignment of the decoupling layers is basic for the functionality.
However, the invention comprises not only the microfluidic system in accordance with the invention but also a biological apparatus (e.g., a cell sorter) with such a microfluidic system.
Furthermore, the invention comprises an associated control method for such a microfluidic system. In it the manipulation electrodes on the one hand and the centering electrodes associated with them on the other hand are preferably electrically controlled in phase opposition in order to achieve the desired centering effect.
Alternatively, the arrangement can also be operated in a single-phase manner. The control takes place as was described above, wherein the second phase is replaced by earth or free potential. This represents a significant simplification in comparison to the known field cage (2- or 4-phase control). Not only the chip and the control electronics are simplified but even the requirements on the interface (capacitances, inductivities) are reduced since phase shifts and runtime delays become less important.
Furthermore there is the possibility that the centering electrodes are cut out when a particle has been fixed by the associated manipulation electrodes. The trapped particles then remain nevertheless in spite of the cutting out of the centering electrodes in the hydrodynamic flow in the central plane before the manipulation electrodes located downstream. As a result thereof, the thermal as well as the electrical load of the trapped particles is reduced, which is especially significant for biological cells.
The cutting out of the centering electrodes can take place selectively in that the centering electrodes are switched to earth or in a potential-free manner, the centering electrodes having a floating electrical potential in the case of a potential-free switching.
There is furthermore the possibility that the centering electrodes are briefly controlled with an elevated electrical voltage before they are cut out.
Moreover, the flow rate in the carrier flow channel can be briefly elevated shortly before the centering electrodes are cut out.
In an advantageous variant of the invention the centering electrodes serve not only to center the suspended particles in the carrier flow channel but also to examine the suspended particles. For example, the centering electrode can first bring about the centering of the suspended particles trapped by the manipulation electrodes arranged downstream. During this centering phase the manipulation electrodes and the centering electrodes are electrically controlled in phase opposition, as explained above. After the suspended particles have been trapped by the manipulation electrodes the centering electrodes can then be used as measuring electrodes. To this end, the centering electrodes are separated from the electrical control and connected to an appropriate measuring apparatus. For example, the centering electrodes can be used as impedance-measuring electrodes and carry out an impedance-spectroscopic examination of the trapped particles. This has the advantage of a good signal-to-noise ratio since the centering electrodes and/or measuring electrodes have a small size and the particles to be examined are fixed close to the centering electrodes and/or measuring electrodes.
In a variant of the invention the electrode arrangement has ring-shaped manipulation electrodes on the upper channel wall of the carrier flow channel as well as on the lower channel wall of the carrier flow channel. The manipulation electrodes on the upper channel wall on the one hand and on the lower channel wall on the other hand are preferably electrically controlled in phase opposition. There is, however, also the alternative possibility that the manipulation electrodes on the lower channel wall are on earth and only the manipulation electrodes on the upper channel wall are electrically controlled. Furthermore, there is also the possibility that the manipulation electrodes on the upper channel wall are on earth and only the manipulation electrodes on the lower channel wall are electrically controlled. Furthermore, there is the possibility that the manipulation electrodes on the upper channel wall on the one hand and on the lower channel wall on the other hand are electrically controlled with a phase difference of 900 in order, e.g., to generate rotation fields.
In the previously described exemplary embodiments with ring-shaped manipulation electrodes the centering electrodes are preferably located in the middle of the ring-shaped manipulation electrodes. The centering electrodes can be electrically on earth here. Alternatively, there is the possibility that only the centering electrodes on the upper channel wall or only the centering electrodes on the lower channel wall are on earth whereas the particular other centering electrodes are electrically controlled. In a variant of the invention the electrical control of the centering electrodes on the upper channel wall on the one hand and on the lower channel wall on the other hand takes place electrically in phase opposition.
Furthermore, there is the possibility that the ring-shaped manipulation electrodes are interrupted and consist of several electrode segments with the form of a segment of a circle that are, however, electrically connected to each other. The interruptions between the individual electrode segments advantageously make it possible for particles to enter into the electrode arrangement and for particles to exit from the electrode arrangement.
Furthermore, there is the possibility here that the individual electrode segments have outwardly projecting shanks, wherein the outwardly projecting shanks of adjacent electrode segments form a funnel-shaped electrode arrangement like the one initially explained in the description of the state of the art. These funnel-shaped electrode arrangements facilitate the introduction of particles into the electrode arrangement.
Furthermore, there is the possibility that particle switches like the ones known from the initially mentioned publication of Müller, T. et al.: “A 3D-micro electrode for handling and caging single cells and particles”, Biosensors and Bioelectronics 247-256, 1999 are arranged downstream behind the electrode arrangements. The particles exiting from the field cages can then be selectively transported into a further electrode arrangement by the particle switches located behind them or deflected laterally.
In an advantageous exemplary embodiment of the invention a plurality of electrode arrangements is provided arranged in a matrix shape that each have at least one centering electrode and at least one manipulation electrode. The individual electrode arrangements can be constructed in accordance with the previously described variants. Preferably two row control lines are provided here for each row of the electrode arrangements, the one row control line of which being connected to the centering electrodes of the electrode arrangements of the particular row whereas the other row control line is connected to the manipulation electrodes of the electrode arrangements of the particular row. In addition, two column control lines are provided for each column and the one column control line is connected to the centering electrodes of all electrode arrangements of the particular column whereas the other column control line is connected to the manipulation electrodes of all electrode arrangements of the particular column. Each centering electrode and manipulation electrode is therefore connected to a row control line and to a column control line. In this manner certain electrode arrangements can be cut out in a purposeful manner in that the two associated row and column control lines are switched to earth or free potential. However, the other electrode arrangements then remain cut in.
Other advantageous further developments of the invention are characterized in the subclaims or are explained in detail in the following together with the description of the preferred exemplary embodiments of the invention with reference made to the figures, in which:
The perspective view in
The carrier flow channel 1 has a rectangular cross section here with a height of 40 μm and a width of 150 μm and conducts a carrier flow with particles suspended in it, where only one biological cell is schematically represented for the sake of simplification.
The carrier flow with the biological cells 2 suspended in it flows in the carrier flow channel 1 in direction x, as is illustrated by the arrows.
An electrode arrangement 3 is arranged in the carrier flow channel 1 and consists of two hook-shaped manipulation electrodes 4, 5 and two circular centering electrodes 6, 7.
The two manipulation electrodes 4, 5 are designed in a traditional manner and are correspondingly controlled, which is known from the publication of Müller, T. et al.: “A 3D-micro electrode for handling and caging single cells and particles”, Biosensors and Bioelectronics 14, 247-256, 1999 that was already mentioned initially, so that in order to avoid repetitions this publication is referred to, whose content is to be added to its full extent to the present description. It needs only be briefly mentioned at this point that the two manipulation electrodes 4, 5 are each designed in a planar manner and are aligned in a coplanar manner with one another, the manipulation electrode 4 being arranged on the upper channel wall of the carrier flow channel 1 whereas the other manipulation electrode 5 is arranged on the lower channel wall of the carrier flow channel 1.
The two centering electrodes 6, 7 are also designed in a planar manner and aligned in a coplanar manner with one another, the centering electrode 6 being arranged on the upper channel wall of the carrier flow channel 1 whereas the centering electrode 7 is arranged on the lower channel wall of the carrier flow channel 1. Therefore, the centering electrode 6 is located in a plane with the manipulation electrode 4 whereas the centering electrodes 7 is located in a plane with the manipulation electrode 5.
A distance of approximately 40-50 μm is present here in the direction of flow between the centering electrode 6 or 7, respectively and the associated manipulation electrode 4 or 5, respectively, which makes a good centering effect of the centering electrode 6, 7 possible.
During operation the manipulation electrodes 4, 5 are electrically controlled in phase opposition to one another just as the centering electrodes 6, 7 are also electrically controlled in phase opposition to one another. Moreover, the centering electrode 6 is also controlled in phase opposition to the associated manipulation electrode 4 just as the centering electrode 7 is also controlled in phase opposition to the associated manipulation electrode 5. In this manner, the suspended biological cells 2 in the carrier flow channel 1 are focused in the central plane, which prevents a touching contact of the biological cells 2 with the channel walls of the carrier flow channel 1.
However, the centering electrodes 6, 7 and the manipulation electrodes 4, 5 do not have to be controlled exactly in phase opposition (that is, with a phase shift of 180°) but rather other phase shifts are also possible within the scope of the invention. This phase shift can be as desired between the electrodes on the upper channel wall and those on the lower channel wall, which shift is generally between 90° and 270°. For electrodes in one plane, e.g., manipulation electrode and centering electrode on the upper channel wall, the shift is generally in the range of 135°-225° (180°±45°).
Moreover, the centering electrodes 6, 7 and the manipulation electrodes 4, 5 can also be controlled with different frequencies and voltages, as will be described in detail later.
Finally, the
The difference between the exemplary embodiments according to
Thus, the manipulation electrodes 18-21 of the electrode arrangement 16 according to
On the other hand, in the electrode arrangement 16 according to
Even several of the electrode arrangements 16 or 17, respectively, represented in
Several electrode arrangements in accordance with FIG. 7A/B can be arranged in series in the direction of flow in order to store particles in defined arrays.
Finally,
A curve 26 reproduces here the dependency of the warming ΔT of the suspended biological cells 2 as a function of the ratio between electrode width and electrode distance between the planes at constant voltage. It is apparent from the course of the curve 26 that the warming ΔT of the suspended biological cells 2 increases with the electrode width and decreases with the electrode distance. It needs to be mentioned here that the warming of the biological cells 2 by the dielectrophoretic electrode arrangement can be damaging to the biological cells 2 and is therefore undesirable.
On the other hand, a further curve 27 shows the dependency of the force F exerted by the dielectrophoretic electrode arrangement on the biological cell 2 as a function of the ratio of the electrode width to the electrode distance. It is apparent from the course of the curve 27 that the exerted force F increases with the electrode width and decreases with the electrode distance.
Finally, a further curve 28 shows the ratio of the desired force F to the undesirable warming ΔT of the suspended cells as a function of the ratio of electrode width to electrode distance. It is apparent from the course of the curve 28 that a certain operating range is especially advantageous in which the ratio of electrode width to electrode distance is approximately between 0.15 to 0.5. In this range the force exerted by the electrode arrangement on the suspended particles is relatively large in proportion to the undesirable warming ΔT.
The individual electrode arrangements each consist of a centering electrode 29 and of a manipulation electrode 30. The centering electrode 29 would thus take the place in the exemplary embodiment according to
The different electrode arrangements according to
Thus, the centering electrode 29 can be rectangular, triangular, drop-shaped, angular or box-shaped, as is apparent from the various figures.
Furthermore,
This exemplary embodiment has the particularity that the upper manipulation electrode 4 is offset in the direction of flow compared with the lower manipulation electrode 5.
In the same manner, the upper centering electrode 6 is also arranged offset in the direction of flow compared with the lower centering electrode 7.
The offset corresponds here to one half of the distance between the manipulation electrodes 4, 5 and the associated centering electrodes 6, 7.
The images on the left side of
On the other hand, field distributions are represented on the right side in
Furthermore,
This exemplary embodiment has the particularity that there is a phase shift of 90° between the manipulation electrode 4 and the centering electrode 6 on the upper channel wall on the one hand and the manipulation electrode 5 and the centering electrode 7 on the lower channel wall on the other hand.
Moreover, the manipulation electrodes 4, 5 on the one hand and the centering electrodes 6, 7 on the other hand are also controlled with a phase shift of 90°.
This is represented in the left column of
Furthermore,
This exemplary embodiment has the particularity that the manipulation electrodes 4, 5 on the one hand and the centering electrodes 6, 7 on the other hand are controlled with different frequencies.
The images in the left column show the field distribution for the case that the manipulation electrodes 4, 5 and the centering electrodes 6, 7 are controlled with the same voltage values, with the control frequency F1 respectively F2 being selected in such a manner that the cells 2 experience an equally strong polarization in both fields.
On the other hand, in the images in the right column the polarization of the particle relative to the medium at the frequency F2 is only ¼ the polarization at the frequency F1.
Even when controlling with two different (not necessarily consumerable) frequencies F1, F2 the cells 2 are focused in the Z direction. However, in the case of moderate voltages they are not centrally held in the horizontal X-Y central plain but rather can be held in two positions according to the equilibrium with the hydrodynamic force. This makes two further types of operation possible:
On the one hand, two cells 2 or particles can be conducted to one another or separated from one another by the inverse process if the bond is not too strong by switching to control with a unified frequency or by absolute or relative weakening of the manipulation electrodes (lower voltage, frequency change, increase of the flow rate). This can be utilized to determine bonding constants and/or to purposefully actuate and/or to influence cells, especially immune cells.
On the other hand, a conclusion can be made by variation of one of the two frequencies F1, F2 from the positional change of the cells 2 about their dielectrical properties (in the images in the right column the two cells 2 are further apart from one another). This makes the dielectrophoresis spectrum readily accessible.
The exemplary embodiment according to the
A particularity of this exemplary embodiment is the diameter of the manipulation electrode 10, which is smaller in comparison to the exemplary embodiment according to
Furthermore, it needs to be mentioned that the manipulation electrodes 10, 11 on the one hand and the centering electrode 12 on the other hand are controlled in phase opposition in this exemplary embodiment, as is apparent from the phase indication in
Moreover, there is also a phase-opposition control in this exemplary embodiment of the electrodes on the upper channel wall on the one hand and on the lower channel wall on the other hand. Thus, the manipulation electrode 10 on the upper channel wall on the one hand and the manipulation electrode 10 on the lower channel wall are controlled in phase opposition. In the same manner the centering electrodes 12 on the upper channel wall are also controlled in phase opposition to the centering electrodes 12 on the lower channel wall.
The electrical fields are formed in this electrode structure in such a manner that the cells 2 are centrally trapped and not on a ring as
The exemplary embodiment according to
This exemplary embodiment has the particularity that the centering electrodes 12 are connected to earth. As a result thereof, the trapped cells 2 are brought in the Z direction into the vicinity of manipulation electrode 10 and therewith into zones with a calmer flow. This has the advantage that a stable holding can be realized in free solution with reduced electrical (heating) power.
The exemplary embodiment according to
A particularity of this exemplary embodiment is the electrical control of the centering electrodes 12 on the top and on the bottom of the carrier flow channel. Thus, the control of the centering electrode 12 on the upper channel wall takes place with earth whereas the centering electrode 12 on the lower channel wall is controlled in phase opposition to the upper manipulation electrode 10, as is apparent from the phase indication in
The exemplary embodiment according to
This exemplary embodiment has the particularity that the manipulation electrode 10 consists of four circular ring-shaped electrode segments 31A, 31B, 31C and 31D. The individual electrode segments 31A-31D are electrically connected to each other and are only spatially separated from each other in order that the trapped cells 2 can enter more readily in the X direction and Y direction into the field cage and leave it.
The exemplary embodiment according to
A particularity of this exemplary embodiment is the electrical control of the manipulation electrode 10 and of the centering electrodes 12.
On the other hand, in this exemplary embodiment the manipulation electrode 10 on the top of the channel wall and the manipulation electrode 10 on the bottom of the channel wall are controlled with a phase difference of 90°.
Furthermore, in this exemplary embodiment even the two centering electrodes 12 on the top or on the bottom of the carrier flow channel, respectively, are controlled in phase opposition. In this manner rotation fields are generated in the field cage.
In addition, the manipulation electrodes 36 also have funnel-shaped electrode arrangements as initially explained in the description of the state of the art. These funnel-shaped electrode arrangements facilitate the entering of particles into the electrode structure.
Finally,
A particularity of this exemplary embodiment is that particles switches 37 are arranged downstream behind the individual electrode arrangements and make it possible to transport the exiting particles selectively into the electrode structure located downstream behind them or laterally deflect them.
The invention is not limited to the previously described exemplary embodiments but rather a plurality of variants and modifications are possible that also make use of the inventive concept and therefore fall into the scope of protection.
List of reference numerals:
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2005 012 128.4 | Mar 2005 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2006/002431 | 3/16/2006 | WO | 00 | 4/30/2008 |
