DE102009005925B4 - Apparatus and method for handling biomolecules - Google Patents

Abstract

A device for handling biomolecules by means of magnetic particles (60) having an affinity for the biomolecules, comprising: a channel structure (20; 200; 250; 302) adapted to generate a multiphase flow; at least one magnetic force member (10; 212; 270, 274, 276; 300, 300 ') movable relative to the channel structure (20; 200; 250; 302); and means (12; 304, 306, 308) for generating relative movement between said at least one magnetic force member and said channel structure for imparting a time varying magnetic field to said channel structure (20) about a position of said magnetic particles (60) in at least one channel structure portion (30, 40, 50) of the channel structure (20) and to move the magnetic particles across a phase boundary of the multiphase flow.

Description

The present invention relates to devices and methods for handling biomolecules, and more particularly to devices and methods for handling biomolecules by means of magnetic particles having an affinity for the biomolecules.

In biomolecular studies, the handling and processing of biomolecules is indispensable. Many known systems rely on the so-called magnetophoresis, in which the long-range and large forces of magnetic force elements are used to handle biomolecules, see MAM Gijs, "Magnetic bead handling on-chip: new opportunities for analytical applications", Microfluidics Nanofluidics, Oct. , 2004, pp. 22-40.

By means of magnetophoresis, magnetic particles suspended in a continuous flow can be sorted by magnetic susceptibility, Pamme N. et al., "On-chip free-flow magnetophoresis: Continuous flow separation of magnetic particles and agglomerates", Anal. Chem., Vol. 76, No. 24, pp. 7250-7256, Dec. 2004. For this constant magnetic fields are applied, which have at least one component perpendicular to the flow direction and large magnetic field gradients.

If small amounts of a suspension consisting of a buffer solution and magnetic particles are placed on a surface as small drops, the particles can be manipulated by means of appropriate magnetic force elements and transferred from drop to drop. In order to prevent evaporation of the buffer solution, the drop is thereby enclosed with a hydrophobic phase, see J. Pipper et al., "Clockwork PCR including sample preparation", Angewandte Chemie-International Edition, Vol. 47, No. 21, p. 3900-3904, 2008; and U. Lehmann et al., "Droplet-based DNA purification in a magnetic lab-on-a-chip", Angewandte Chemie-International Edition, Vol. 45, No. 19, pp. 3062-3067, 2006.

Within microfluidic channels, magnetic particles are often immobilized by magnetic force elements to overflow them with appropriate buffer solutions. From EP Furiani et al., "A model for predicting magnetic particle capture in a microfluidic bioseparator", Biomedical Microdevices, Vol. 9, No. 4, pp. 450-463, Aug. 2007, the US 2007/0207548 A1 , of the US 2007/0190653 A1 and the US 7,138,269 B2 are known reactions in which particles are immobilized directly on a channel wall. In F. Lacherme et al., "Full on-chip nanoliter immunoassay by geometric magnetic trapping of nanoparticle chains", Anal. Chem., Vol. 80, No. 8, pp. 2905-2910, Apr. 2008 and in the US 6,632,655 B1 are described reactions in which the particles are immobilized in the middle of the channel. In this case, a discontinuous procedure takes place from attaching the magnetic particles to the surface, followed by an overflow with a solution and subsequent detachment of the magnetic particles.

From the US 2007/0207548 A1 It is also known to draw magnetic particles from a sample solution into a buffer solution using a magnet.

The US 7,364,921 B1 describes a system in which gels are used to slow the diffusion of the magnetic particles. Further, here, repeatedly increasing and decreasing the magnetic field in a pulsed manner is used to effect spatial separation of magnetic components. A combination of magnetophoresis and electrophoresis is known from US Pat US 2006/0286596 A1 known while a combination of a magnetophoresis and a dielectrophoresis from the US 7,033,473 B2 is known. However, such combinations are expensive. Furthermore, for example, from the US 2004/0009614 A1 a system is known in which a plurality of magnetic force elements for manipulating magnetic particles is used.

Continuously operating systems for the processing of biomolecules are known from the prior art, see for example the US 7,384,561 B2 and the US 2008/0124779 A1 where either only the buffer solutions can flow continuously through the microchannels or small magnetic strips are inserted into the microchannels, which makes production and flexibility of these systems much more difficult.

Other known systems, see for example the US 2007/0231888 A1 are limited to the use of blood or use multiple variable electrical potentials, see US 6,467,630 B1 , which requires a complex array of electrodes. From the US 6,383,397 B1 For example, a system with many parallel tubes is known, each tube being wrapped with wires to generate magnetic fields. From the US 6,132,607 For example, a system having a plurality of magnetic force elements is known for magnetically separating and treating components of a mixture of chemical entities using a multi-dimensional gradient. From the US 5,705,064 the use of a plurality of magnetic force elements is known, which together form a magnetic tube.

In known systems for magnetophoresis usually superparamagnetic particles are used which have a high susceptibility. As a result, externally applied magnetic fields can easily and rapidly induce magnetic dipoles in the particles. On the other hand, these induced bipoles in the particles and magnetic attraction disappear just as fast as the external magnetic field disappears, leaving no magnetization in the particles. In addition to their magnetic properties, these particles absorb laser radiation, providing further applications such as the removal of a buffer solution, see JG Lee et al. 6, no. 7, pp. 886-895, 2006, or the realization of valves by melting wax See, JM Park et al., Lab., Vol. 7, No. 5, pp. 557-564, 2007, "Multifunctional microvalves control by nanoscale optical illumination and its application in centrifugal microfluidic devices". The surfaces of the superparamagnetic particles can be modified in many ways. Examples of such surface modifications are functional groups, such as amines and epoxides, or biomolecules, such as streptavidin or antibodies.

Superparamagnetic particles are available in commercial DNA extraction kits. The buffer solutions provided with such kits provide optimal conditions for individual steps of a complete DNA extraction process. After binding the DNA molecules to the particles, contaminants are removed by washing steps and the purified DNA is eluted from the particles. However, commercial DNA extraction kits do not allow for continuous operation and require a variety of individual operations. Every step of the process poses the risk of contamination.

Furthermore, pipetting robots are known which can reduce the risk of contamination by automating DNA extraction kits. Again, however, a continuous operation is not possible.

A system that works quasi-continuously is in the WO 2006/056579 A2 described. The particles are held in solution by periodic polarity reversal of electromagnets and flowed through with a buffer solution. By keeping the particles locally confined to a particular region, the required buffers must be sequentially passed through the particles. Although this allows a purification of the particles, but is discontinuous.

Also known from the prior art are lab-on-a-chip systems for the purification of biomolecules, see M. Matsunaga et al., "Microfabricated devices for DNA extraction toward realization of deep-sea in situ gene analysis", Oceans, Vol. 1, pp. 89-94, 2004, and the US 5,834,303 , However, such systems do not work continuously and are limited to one class of biomolecules. Furthermore, a single cartridge usually performs only one operation, such as cell lysis, DNA purification, DNA amplification, or DNA detection. Thus, for successful extraction of DNA on cells, at least two cartridges are required, one for cell lysis and one for DNA purification.

Furthermore, lab-on-a-chip systems are known in which biological essays can be carried out in a continuous flow and with the use of multiple buffer solutions. Such systems rely on magnetic particles for the transport of a target substance into the different buffer solutions in a laminar flow by means of magnetophoresis with a permanent magnet, see SA Peyman et al., "Rapid, multi-step bioassays on the surface of mobile magnetic particles in continuous flow", μTas 2008, pp. 1114-1116, or several permanent magnets, LA Sasso et al., "Continuous microfluidic immunosensing with antibody conjugated paramagnetic beads", μTas 2008, pp. 77-79. In these systems, the permanent magnets are mounted at fixed positions on the cartridge, whereby this position must be calibrated precisely, so that the magnetic particles are attracted on the one hand and moved across the phase boundary in the individual buffer solutions, but on the other hand not immobilized on the channel wall.

From the DE10355460 A1 is a microfluid system with a base body is known in which at least one microchannel is formed, which is traversed by a particle laden with particles, in particular bioparticles, fluid flow. Adjacent to the at least one microchannel, magnetic force elements are arranged, wherein the magnetic force elements comprise at least one magnetic force element, which is arranged outside of a plane defined by other of the magnetic force elements along the at least one microchannel in the base body.

From the WO 2007/044642 A2 For example, a microfluidic separation device is known in which material bound to magnetic particles is drawn from one laminar flow path to another by applying a local magnetic field gradient.

From the DE 10 2004 040 785 A1 a microfluidic system is known in which two stationary electromagnets 6 and 7 are provided. An electromagnet is activated to subject immunomagnetic particles to an electromagnetic field to sweep them across a liquid flow boundary. By means of a second Electromagnets are then withdrawn the immunomagnetic particles.

The US 6,297,061 B1 discloses reagents immobilized on beads, which beads may be magnetic in order to use a magnetic field for control.

The EP 1 327 473 A1 describes a method for carrying out reaction solutions, for which purpose magnetic field fluctuations are used to move magnetic beads, these field fluctuations being produced by sequentially exciting a plurality of electromagnets or by moving permanent magnets arranged outside the reaction vessel.

The US 2001/0017158 A1 deals with magnets that are used to move liquid droplets through a channel structure. Magnets used to control fluid movement may be individual magnets or one or more arrays of magnets, the elements of which may be individually controlled for this purpose.

Shikida, M. [et al.]: Magnetic handling of droplets in micro chemical analysis system surface tension and resistance. In: Proceedings of the 17 ^th IEEE International conference on micro electromechanical systems (MEMS 2004), Maastricht, The Netherlands, January 2004, pages 359-362, disclose a way to handle magnetic drops without them sticking to a glass surface. For this purpose, the surface of a glass plate is provided with silicone oil. Further, this document describes moving magnetic beads within a buffer solution using a moving magnet.

The object of the present invention is to provide devices and methods for handling biomolecules that enable simple construction and continuous operation.

This object is achieved by a device according to claim 1 and a method according to claim 13.

Embodiments of the invention provide a device for handling biomolecules by means of magnetic particles which have an affinity for the biomolecules, with the following features:
a channel structure adapted to generate a multi-phase flow;
at least one magnetic force element that is movable relative to the channel structure; and
means for generating relative movement between the at least one magnetic force element and the channel structure, for imparting the magnetic field to a position of the magnetic particles in at least one channel structure portion of the channel structure and for imparting the magnetic particles across a phase boundary of the multiphase flow move.

In embodiments of the invention, the magnetic force element movable relative to the channel structure comprises at least one rotatable magnetic force element configured to generate a rotating magnetic field. In embodiments of the invention, one or more magnetic force elements on a conveyor, such. As a conveyor belt, be mounted to cause a translational movement of the magnetic force or the elements to act on the channel structure with a time varying magnetic field. In embodiments of the invention, the one or more magnetic force elements may comprise permanent magnets or electromagnets.

In embodiments, the means for generating a relative movement may be configured to move the at least one magnetic force element while the channel structure remains stationary. In alternative embodiments, the means for generating a relative movement may be configured to move the channel structure while the at least one magnetic force element remains stationary. Again alternatively, the device may be configured to move the at least one magnetic force element and the channel structure.

In embodiments of the invention, the channel structure has a plurality of separate inlets for generating the multiphase flow. The multiphase flow may have substantially laminar flows of various liquids. In embodiments of the invention, means for generating the multiphase flow comprises injection means, for example pumps, for injecting different liquids into the inlets. The multiphase flow may be in a channel or in a plurality of channels that are fluidly connected.

According to the invention, a relative movement between a magnetic force element and a channel structure, such. B. a rotation of a magnetic force element, such as. As a permanent magnet or an electromagnet, generate a time-varying magnetic field, are controlled by the magnetic particles in a channel structure, which may be part of a microfluidic system. Through the generated, time-varying magnetic field can magnetic particles are always effectively attracted in phases of strong magnetic fields, while they can be released in phases of weak fields, so that they are not retained on a channel wall of the channel structure. Embodiments of the invention thus allow without a complex adjustment simply by a continuous change of the magnetic field strength due to the relative movement of the magnetic force or elements to the channel structure, a transfer of magnetic particles across phase boundaries of liquids, a steering of magnetic particles in certain output branches of channel branches and / or a transport along one, at an angle to or against a flow direction of buffer solutions in which the magnetic particles are located. This is not possible with fixed magnets and in the case of stationary electromagnets only by a targeted control achievable.

In embodiments of the invention, the channel structure is formed in a channel structure body, for example a chip, and has microchannels through which different liquids can flow. In embodiments, the invention allows for continuous processing of biomolecules, wherein buffer solutions needed for a particular assay can flow continuously through the microchannels of the channel structure. The biomolecules are coupled to a mobile solid phase consisting of magnetic particles, convective or non-convective, and can then be sequentially exposed to different buffer solutions. For this purpose, the magnetic particles in the microchannels can be deflected by the relative movement and transported into a desired buffer solution. In a last step, the biomolecules can be released from the magnetic particles, which can finally be removed from the buffer solution.

Embodiments of the present invention enable continuous and automated processing of biomolecules using a variety of different buffer solutions as required in extensive processing of biomolecules. As a result of the time-varying magnetic fields generated by the relative movement, particles can be separated from a buffer solution in a microchannel on the one hand, while on the other hand they can not immobilize on the channel wall, which allows a continuous operation. The magnetic force element moving relative to the channel structure further enables control of the position of magnetic particles at a plurality of positions distributed, for example, radially along a rotational path of a rotating magnetic force element. Thus, it is possible to control at a plurality of different positions using only a magnetic force element, such. As a permanent magnet, which allows a significantly reduced complexity. Embodiments of the invention thus make it possible to expose magnetic particles of a plurality of different chemical solutions in a very simple structure with only one external magnetic force element.

In embodiments of the invention, the microchannel structure may have closed channels, wherein for the required liquids, such as. B. the sample solution and required buffer solutions, each separate inlets can be provided. Thus, contamination can be largely excluded, which can lead to high reproducibility of extraction results.

In embodiments of the invention, the device may provide multiple inlets into the channel structure through which different buffers may be injected in parallel so that magnetic particles can be continuously operated by a suitable channel design using one or more magnetic force elements moving relative to the channel structure, e.g. B. a single rotating permanent magnet, can be transferred from one buffer to the next. Thus, in embodiments of the invention, a true continuous operation can be achieved since both the magnetic particles and the liquids or solutions can be transferred continuously.

In embodiments of the invention, the device can be used for processing different biomolecules. The channel structure may be designed to accommodate all steps required to successfully purify biomolecules on a cartridge, i. H. in a channel body or chip. Embodiments of the invention may be configured to perform an immunoassay, a nucleic acid extraction, or a continuous DNA amplification.

Embodiments of the invention are explained below with reference to the accompanying drawings. Show it:

1 a schematic representation of an embodiment of a device according to the invention;

2 a schematic representation of an alternative embodiment of a device according to the invention;

3a and 3b enlarged sections of 2 ;

4a and 4b a schematic plan view and a schematic side view of an embodiment of the invention;

5a and 5b a schematic plan view and a schematic side view of another embodiment of the invention;

6a and 6b a schematic plan view and a schematic side view of another embodiment of the invention;

7 a schematic representation for explaining a further embodiment of the invention;

8a to 8c schematic representations for explaining how the position of magnetic particles can be controlled in embodiments of the invention; and

9a and 9b schematic representations for explaining how the position of magnetic particles can be controlled in embodiments of the invention.

Based on 1 An embodiment of the present invention in the form of a microfluidic system for a purification of DNA will be explained in more detail below.

An embodiment of a device according to the invention has for this purpose a magnetic force element in the form of a rotatable permanent magnet 10 and a channel structure 20 on. A rotary encoder 12 , such as B. a stepper motor, is provided to the permanent magnet 10 to apply a rotation. The channel structure 20 may be formed in a suitable channel structure body or chip (not shown). The channel structure 20 includes a first inlet 22 , a second inlet 24 , a third inlet 26 and a fourth inlet 28 , The channel structure comprises a first reaction stage 30 , a second reaction stage 40 and a third reaction stage 50 , The inlet 22 flows into an entrance 31 the first reaction stage 30 which have a first and a second output 32 and 33 having. The second exit 33 is fluidly connected to a first input via a microchannel 41 the second reaction stage 40 connected. The second inlet 24 is via a microchannel with a second input 42 connected to the second reaction stage. The second reaction stage 40 also has a first exit 43 and a second exit 44 up, with a first entrance 51 the third reaction stage 50 connected is. The third reaction stage 50 also has a second input 52 on that with the third inlet 28 is fluidically coupled, and a third input 53 that with the fourth inlet 28 is fluidically coupled. The third reaction stage 50 further includes a first output 54 and a second exit 55 ,

In the illustrated embodiment, three reaction stages are integrated into a microfluidic system, wherein the reaction stages each consist of a cavity with at least one inlet and at least two outlets. As in 1 As can be seen, the cavities of the reaction stages to a larger channel diameter than the inputs and outputs thereof, so that there the flow velocity of a flow can be reduced, whereby magnetic particles can be drawn easily to one side of the cavity. As a result, a concentration of magnetic particles can be achieved, which then leave the reaction stage through one of the outlets, which is located closer to the rotatable permanent magnet, namely the second outlet, while the majority of each buffer solution is contaminated by the other outlet, that of the rotatable permanent magnet is further away, namely in each case the first output, leaves the reaction stages.

In operation, a sample solution is introduced into the inlet 22 flushed. The sample solution has biomolecules coupled to the magnetic particles in a first buffer solution. Injecting causes a flow of the sample solution into the first reaction stage 30 causes where the magnetic particles 60 through the rotating permanent magnet 10 radially inwards and thus into the second exit 33 to get distracted. From there, the particle-bound sample becomes the entrance 41 the second reaction stage 40 directed, as by an arrow 62 is indicated. The remainder of the first buffer solution and impurities leave the first reaction stage 30 over the first exit 32 , See arrow 64 which may be connected to a waste container (not shown).

A second buffer solution 24 gets into the second inlet 24 washed in, see arrow 66 , so that in the second reaction stage 40 a laminar flow over the first inlet 41 inflowing suspension with the particulate bound sample and via the second inlet 42 inflowing second buffer solution, as shown by the dashed line 68 is indicated. By the rotating central permanent magnet 10 become the magnetic particles 60 pulled across the phase boundary in the second buffer solution and in the second output 44 directed. The particle-bound sample thus leaves the second reaction stage 40 in the second buffer solution via the second outlet 44 , The first buffer solution as well as possible impurities leave the second reaction stage via the first outlet 43 as if by an arrow 70 in 1 is indicated.

It should be noted at this point that slight diffusion mixing may occur at the phase boundary between the two buffer solutions.

The particulate bound sample in the second buffer solution flows along the microchannel, which is the second exit 44 the second reaction stage with the first input 51 the third reaction stage connects. In the third inlet 26 a third buffer solution is flushed in and into the fourth inlet 28 a fourth buffer solution is purged so that the flow of the particle-bound sample in the second buffer solution is between the flow of the third buffer solution and the fourth buffer solution. The second buffer solution may include ethanol which, due to the hygroscopic properties of the ethanol, may rapidly mix with the third and fourth buffer solutions and may again form a laminar flow, such as a dashed line 82 is indicated. The fourth buffer solution, which is flushed in from the side opposite the third buffer solution, serves to take up ethanol from the second buffer solution and thus the ethanol concentration in the eluate, which is the third reaction stage 50 over the second exit 55 of the same leaves, decrease. In the third reaction stage 50 in turn causes the rotation of the permanent magnet 10 in that the magnetic particles from the second buffer solution are brought into a buffer solution mixture comprising the third buffer solution and into the second exit 55 the third reaction stage 50 is steered. The exit 55 is with a microchannel 80 fluidly coupled, via which the purified sample can be supplied to the magnetic particles of a further real-time analysis.

The second buffer solution and the fourth buffer solution may be the third reaction stage 50 over the second exit 54 leave as if by an arrow 84 is indicated.

Thus, in the illustrated embodiment, control of the position of the magnetic particles at a plurality of portions of the channel structure 20 radially along a rotation path of the rotating magnet 10 distributed. In other words, the position of the magnetic particles in each of the first, second, and third reaction stages becomes the single rotating permanent magnet 10 controlled.

The first exits 32 . 43 and 54 The first, second and third reaction stages may be connected via respective channels or microchannels, which may be in 1 are indicated to be fluidly coupled to a waste container or more waste containers.

In the described embodiment, a separate inlet is provided for each required buffer solution, wherein the reaction stages may be formed so that laminar flows form, so that the individual buffer solutions do not mix or only in a defined and very small extent. About the phase boundary of this laminar flow can then be the magnetic particles by means of magnetophoresis by the time-varying magnetic field by the rotating permanent magnet 10 is generated, pulled. In this way, the magnetic particles can each be brought into the optimum environment for the respective steps to be carried out, namely for example binding of the biomolecules, removal of impurities and / or release of the biomolecules from the magnetic particles.

The rotating permanent magnet generates a regular change of strong and weak magnetic field at the locations of the magnetic particles, so that they can be effectively moved on the one hand by the strong fields over the phase boundary and on the other hand are not immobilized in the channel due to the weak fields. Thus, an exact vote of the magnetic field to one in which the magnetic particles are not immobilized on the wall of the microchannel, but still be deflected sufficiently to be transferred over the phase boundary in the next buffer solution, not required because of the rotating permanent magnet is applied a time-varying magnetic field.

To generate the periodically varying magnetic field, for example, a rotating NdFeB permanent magnet can be used, which is set in a uniform rotation by means of a stepping motor. As a result, the magnetic field in the microchannels can change periodically, which on the one hand draws the purged magnetic particles to the channel wall during the phases of strong fields and, on the other hand, does not immobilize the magnetic particles on the wall during phase-weak fields. The flow of the various liquids, for example buffer solutions, through the microchannels can be achieved by appropriately pumping the liquids into the respective inlets.

In embodiments of the invention, a rotating body may be formed entirely as a permanent magnet. In alternative embodiments, a rotating body may include magnetic portions and non-magnetic portions. For example, an elongated body of revolution at one or both ends of the same permanent magnet. Again, alternatively, a rotationally symmetrical rotation body could be provided, for example in the form of a A disc having portion-wise magnetic portions for generating a time-varying magnetic field in fluidic structures in which magnetic particles are to be controlled. In alternative embodiments, the magnetic force element could include one or more rotating electromagnets.

An exemplary embodiment of a device according to the invention may be a microfluidic system in which a DNA extraction assay is implemented on a polymer chip as a lab-on-a-chip system. As buffer solutions, it is possible to use those of a commercially available DNA extraction kit, three reaction stages and at least three different buffer solutions being required for carrying out this assay, as is the case for example with reference to FIG 1 has been described.

An exemplary implementation in which corresponding channel structures have been milled by a microfiller into polycarbonate is in FIG 2 shown. In the 2 shown structures provide substantially the same functionality as described above 1 has been described. As in 2 are microchannels of the channel structure of the microfluidic system shown circular around a central magnetic force element, which in turn is a rotating permanent magnet 10 acts arranged. The rotating permanent magnet 10 deflects the magnetic particles toward the center, respectively, to control their position in the channel structure.

Via a microchannel 102 For example, the sample, magnetic particles, lysis and a binding buffer which is a first buffer solution are supplied as indicated by an arrow 100 is indicated. In a first reaction stage, the magnetic particles become due to the action of the permanent magnet 10 concentrated and into a channel 108 distracted while the rest of the binding buffer as well as impurities in a waste channel 110 flow. The magnetic particles are in the 3a and 3b , the magnifications of sections X and Y in 2 represent, shown schematically hatched and with the reference numeral 60 designated.

A wash buffer, which is a second buffer solution, is passed through a microchannel 106 fed, as by an arrow 104 is indicated. At an estuary 112 the canals open 108 and 106 into a canal 114 where the over the channels 106 and 108 supplied buffer laminar flow in the channel 114 form. By virtue of the time-varying magnetic field generated by the rotating permanent magnets, the magnetic particles, together with the sample adhering to them, are drawn across the phase boundary between the binding buffer and the wash buffer into the wash buffer. The channel 114 then branches again into a waste channel 116 and a sample channel 118 in which the magnetic particles are deflected due to the time-varying magnetic field. The transfer of the magnetic particles and thus also the sample to the second reaction stage thus takes place with a minimal amount of the first buffer solution, which forms a laminar flow with the second buffer solution before the second reaction stage.

As has been described, in the second reaction stage, the magnetic particles re-enter the channel 118 distracted and concentrated. In the channel 118 the magnetic particles enter the third reaction stage, as indicated by an arrow 120 in 3b is indicated. In the third reaction stage, in turn, both a third buffer solution via a microchannel 122 as well as a fourth buffer solution via a microchannel 124 fed by arrows 123 and 125 in the 2 and 3b is indicated. The third and fourth buffer solutions are again added from opposite sides. The ethanol contained in the washing buffer mixes quickly with these two buffers and it forms a laminar flow. By mixing the ethanol with the fourth buffer solution, the ethanol concentration on the side of the third buffer solution decreases, so that subsequently, a real-time PCR analysis of the eluate containing the third reaction stage via a microchannel 130 leaves, can take place. The third buffer represents an elution buffer, while the fourth buffer represents a dilution buffer. The magnetic particles are generated by the time-varying magnetic field generated by the rotating permanent magnet 10 is generated, drawn in the third reaction stage from the second buffer solution across the phase boundary in the fourth buffer solution and into the microchannel 130 directed. The remaining buffer solutions leave the third reaction stage via a microchannel 132 for example, in a waste container.

Using a microfluidic system, as referenced in U.S. Pat 2 . 3a and 3b was described, an extraction of genomic E. coli DNA was successfully performed. For this purpose, the DNA was purified with a commercial DNA extraction kit from E. coli DH5αZ1 using the microfluidic system shown. Kit buffer solutions were injected sequentially through the individual inlets into the microfluidic system at a flow rate of 2 μl / s in order of protocol. As a magnetic force element, an NdFeB permanent magnet was positioned below the microfluidic system and rotated by a stepping motor at a frequency of one hertz. The eluate was collected and subsequently analyzed successfully outside the microfluidic system using real-time PCR. The Analysis showed that PCR inhibitors were sufficiently diluted or completely removed from the eluate.

For the production of the channel structure, all methods that allow to form channels or micro channels, such as. As milling, embossing, injection molding, deep drawing and / or etching. Suitable materials can be used for the preparation, such as. As polycarbonates, polymers, plastics, ceramics, semiconductor materials, glasses, metals and the like. The microchannels formed in the carrier body can then be closed, i. H. be capped. Again, there are a variety of options available, such. As solvent bonding, thermal bonding, adhesive bonds or sticking a self-adhesive film. The advantage of such methods lies in the potential to be able to manufacture microfluidic systems, for example in the form of chips, as cost-effective disposable items in large numbers.

An alternative embodiment of the invention in which the position of magnetic particles in buffer solution flows between two plates is controlled is shown in FIGS 4a and 4b shown. In this embodiment, a channel structure 201 through two plates 201 and 202 defined, which are secured together with a distance between them, as in 4b can be seen. Between the two plates 201 and 202 Thus, there is a fluid region which may be closed inwardly and may have external outlets. In the upper plate 201 There are four inlets for four different liquids, two of which are in 4b with the reference numerals 206 and 208 are provided. Liquids, such. For example, buffer solutions can be injected into the inlets so that laminar flows out to the outer outlets, two of which are in 4b with the reference numerals 207 and 209 are indicated, as indicated by arrows 210 is indicated. Four different liquids or buffer solutions P1, P2, P3, P4 are in 4a shown.

For example, one of the liquids may be a suspension with a buffer solution and magnetic particles 60 be inserted in one of the inlets. A rotatable magnetic force element 212 is provided, for example in the form of a disc 214 , at the outer ends of which one or more permanent magnets 216 are arranged. The magnetic force element rotates below the channel structure, so that the channel structure is subjected to a time-varying magnetic field to the position of the magnetic particles 60 in the canal structure 200 to control. As a result, the magnetic particles can be sequentially moved through the four buffer solutions, such as an arrow 218 in 4a is indicated.

According to the 4a and 4b Thus, flows flow between two parallel plates, wherein the magnetic particles are guided by a magnetic field perpendicular to the currents.

Alternatively, the streams may also flow between two cylinder jackets, as in FIGS 5a and 5b is shown. A channel structure 250 is through an inner cylinder jacket 251 and an outer cylinder jacket 252 educated. Four inlets, two of which are in 5b with the reference numerals 254 and 256 are designated are provided by the four buffer solutions P1 to P4 in the channel structure 250 can be introduced so that in the channel structure four laminar streams (indicated by arrows 262 ) from the inlets 254 . 256 to outlets, two of which are denoted by the reference numerals 258 and 260 are designated result. With one of the buffer solutions can magnetic particles 60 be introduced into the channel structure, which can move with the laminar flows through the channel structure. The position of the magnetic particles may be defined by at least one rotatable magnetic force element within the inner cylinder shell 251 is arranged to be controlled, so that the magnetic particles in turn can be moved through more of the buffer solutions. Alternatively or additionally, one or more outer magnetic force elements 272 . 274 be provided, which is around the outer cylinder jacket 252 can be moved, as by arrows 276 is indicated. Also in this embodiment, the magnetic particles can be guided perpendicular to the flows, as by an arrow 278 is indicated.

With reference to the 4a . 4b . 5a and 5b described embodiments, the magnetic particles are guided radially by the time varying magnetic field.

The 6a and 6b show an embodiment in which a plurality of magnetic force elements 300 on a conveyor belt 302 is arranged, translational relative to a channel structure 302 is movable to cause a time-varying magnetic field in the channel structure. For this purpose, the plurality of magnetic force elements on a conveyor belt 304 arranged, over two axes 304 and 306 running, of which at least one can be driven to the conveyor belt 304 with the magnetic force elements 300 along a canal structure 310 to move, as by an arrow 312 is indicated. An identical structure with magnetic force elements 300 ' , a conveyor belt 304 ' and axes 306 ' and 308 ' is below the channel structure in the embodiment shown 302 intended. Alternatively, only one of the structures could be provided.

The channel structure 302 has an inlet 320 and an outlet 322 on. Different buffer solutions can move to different areas of the channel structure 302 be introduced. There are four inlet channels for this purpose 324 . 326 . 328 and 330 provided, which branch into respective Einlasssteilkanäle with smaller flow cross-sections. The Einlasssteilkanäle then open into different areas of the channel structure. In contrast to the inlet part channels, outlet part channels open into the channel structure 302 , The outlet part channels unite, as in 6a is shown, to exhaust ducts 332 . 334 . 336 and 338 ,

Different buffer solutions are introduced via the inlet channels, as indicated by arrows 340 is indicated. The buffer solutions are applied via the outlet channels, as indicated by arrows 342 is indicated. Through these inlet and outlet channel structures, laminar flows of the respective buffer solutions form in the different regions of the channel structure 302 from the inlet part passages to the outlet part passages.

About the inlet 320 can be a suspension containing a buffer solution and magnetic particles 60 has to be introduced into the channel structure, as indicated by an arrow 344 is indicated. By the movement of the magnetic force elements 300 and 300 ' relative to the channel structure 302 In this embodiment, the magnetic particles 60 defined to be moved from left to right and thus be exposed sequentially to the various buffer solutions.

7 shows a schematic representation for explaining a further embodiment. A channel structure 400 has inlets 402 . 404 . 406 . 408 . 410 . 412 and 414 and outlets 416 . 418 . 420 and 422 on. The channel structure 400 also has a first channel 424 , a second channel 426 , a third channel 428 and a fourth channel 430 on. The first channel 424 and the second channel 426 are via a first connection channel 432 connected, the second channel 426 and the third channel 428 are via a second connection channel 434 connected, and the third channel 428 and the fourth channel 430 are via a fourth connection channel 436 connected. The connection channels 432 . 434 and 436 are, as shown, offset from one another. A magnetic force element and means for generating a relative movement between the magnetic force element and the channel structure 400 are intended to make a relative movement, as indicated by an arrow 440 in 7 is shown to produce. The magnetic force element and the device for generating a relative movement are in 7 for the sake of simplicity not shown.

In operation, over the inlet 402 a sample 450 fed to a flow 451 in the channel 424 to effect. About the inlet 404 becomes a lysis buffer 452 fed. The sample flows along the channel 424 , wherein by the lysis buffer lysis of the sample is carried out by heating 454 can be supported. Through the inlet 406 which is optional, can be a cleaning buffer 456 be supplied. Through the inlet 408 become magnetic particles 60 and a binding buffer so that biomolecules contained in the lysed sample are bound to the magnetic particles. The magnetic particles flow through the channel 424 and are caused by the magnetic field caused by the relative movement 440 is generated in the connection channel 432 distracted. eluate 490 leaves the channel 424 over the outlet 416 ,

In the inlet 410 becomes a wash buffer 460 introduced to a flow 462 in the channel 426 to effect. Due to the generated magnetic field, the magnetic particles enter the wash buffer and are moved counter to the flow direction of the wash buffer along the channel 426 and in the connection channel 434 distracted. Waste leaves the canal 426 over the outlet 418 , About the inlet 412 becomes a dry buffer 470 fed to a flow 472 in the channel 428 to effect. The magnetic field causes the magnetic particles to enter the dry buffer 470 and are against the flow direction of the dry buffer along the channel 428 and in the connection channel 436 distracted. waste 459 leaves the channel 428 over the outlet 420 ,

Above the inlet 414 becomes an elution buffer 480 fed to a flow 482 in the channel 430 to effect. Due to the generated magnetic field, the magnetic particles enter the elution buffer and become in the flow direction of the elution buffer and in an outlet channel 484 distracted. waste 459 leaves the channel 430 over the outlet 422 ,

The 8a to 8c show schematically, as in embodiments of the invention by a relative movement between the magnetic force element 500 and channel structure 502 the position of magnetic particles 60 in the canal structure 502 can be controlled. The magnetic force element after a rotation is denoted by the reference numeral 500 ' designated, while the particles at the position thereby effected by the reference numeral 60 ' are designated. The magnetic particles 60 arrange along the magnetic field lines caused by the magnetic force element, thereby forming particle chains or particle sticks. By a rotation of the magnetic force element 500 also move the particle chain or particle sticks, as in the 8a to 8c is shown.

As in 8a is shown migrating without external flow in the channel structure 502 the magnetic particles along the wall to the left, see arrow 506 , in a clockwise rotation of the magnetic force element. Due to the course of the magnetic field lines, tilting of the particle chain takes place, arrow 507 ,

As in 8b is shown migrating without external flow in the channel structure 502 the magnetic particles along the wall to the right, see arrows 508 during a rotation of the magnetic force element counterclockwise. Again, there is a tilt 509 instead of.

As in 8c is shown, remain at an outer flow to the right, arrow 510 , the magnetic particles at their position in the channel during rotation of the magnetic force element 500 clockwise, with only a tilt 511 occurs. In the case of an external flow from left to right, the magnetic particles would rapidly move to the right in the case of a rotation of the magnetic force element in the counterclockwise direction, since they would additionally be carried along by the flow.

The 9a and 9b show an alternative embodiment in which the position of smaller secondary superparamagnetic particles 600 which have an affinity for biomolecules, involving larger primary superparamagnetic particles 602 which does not necessarily have to have an affinity for biomolecules is controlled. It is characterized by a relative movement between a magnetic force element and a channel structure (both in 9a and 9b not shown) causes a movement and magnetization of the large superparamagnetic particle, which in turn causes the position of the secondary particles 600 which can be attracted by the primary particles can be controlled.

According to 9a causes the magnetic force element a slow movement of the primary particle 602 so that the secondary particles 600 are attracted and there is no suspension of the secondary particles. According to 9b is a fast movement, z. B. Rotation 604 , causes the primary particle by the magnetic force element, so that takes place a suspension of the secondary particles.

An approach, as regards the 9a and 9b may be advantageous because the smaller secondary particles provide a larger specific surface area for the biomolecules. Furthermore, an advantage can be achieved if smaller particles can not be guided so well perpendicular to a flow. Larger particles are easier to guide perpendicular to a flow or even against a flow. Thus, smaller particles can be transported in correspondence with larger particles, whereby by a strong movement of the primary particles, the small particles can be brought into suspension, for example in order to come into better contact with biomolecules.

In addition to rotating magnetic force elements, such. As permanent magnets or electromagnets, embodiments of the invention can thus cause a movement of one or more magnetic force elements using a magnetic conveyor belt. This allows the realization of arbitrarily shaped separation sections. In alternative embodiments of the invention, any means may be provided which allow relative movement between one or more magnetic force elements and a channel structure, along or perpendicular to the channel structure, to impose a time varying magnetic field on them.

Embodiments of the present invention have been described above with reference to DNA extraction. However, it will be appreciated that the present application is also useful for handling biomolecules in other applications, wherein a greater or lesser number of reaction stages in which the position of magnetic particles in a channel structure is controlled may be provided.

Embodiments of the invention advantageously enable a continuous and automated operation of a microfluidic system. So far, there is no system that can continuously purify biomolecules by means of magnetophoresis. Furthermore, embodiments of the present invention allow versatility since, in contrast to known systems, embodiments of the invention need not be limited to a particular application. By different functionalization of the magnetic particles, different biomolecules can be purified without the structure having to be changed. Likewise, different buffers can be injected through the individual inlets, which allows the implementation of different assays with the corresponding functionalization of the magnetic particles.

Embodiments of the present invention may be used for the continuous purification of DNA, RNA or proteins. Furthermore, immuno and cell based assays and assays can be performed. In fact, using the present invention, all biological assays can be realized in which the target substance can be bound or transferred to magnetic particles. This results in many applications, such. These include monitoring the growth of cell cultures in bioreactors, the biosafety of drinking water, and generally continuous controls at the molecular level.

Claims (22)

Device for handling biomolecules by means of magnetic particles ( 60 ) which have an affinity for the biomolecules, having the following features: a channel structure ( 20 ; 200 ; 250 ; 302 ) designed to generate a multiphase flow; at least one magnetic force element ( 10 ; 212 ; 270 . 274 . 276 ; 300 . 300 ' ), which is relative to the channel structure ( 20 ; 200 ; 250 ; 302 ) is movable; and a facility ( 12 ; 304 . 306 . 308 ) for generating a relative movement between the at least one magnetic force element and the channel structure for acting on the channel structure ( 20 ) with a time-varying magnetic field to a position of the magnetic particles ( 60 ) in at least one channel structure section ( 30 . 40 . 50 ) of the channel structure ( 20 ) and to move the magnetic particles across a phase boundary of the multiphase flow. The apparatus of claim 1, wherein the channel structure comprises a plurality of separate inlets for generating the multiphase flow. Apparatus according to claim 1 or 2, wherein the magnetic force element ( 10 ; 212 ; 270 . 274 . 276 ) is a rotatable magnetic force element for generating a rotating magnetic field and in which the means for generating a relative movement, a rotary encoder ( 12 ) having. Apparatus according to claim 3, wherein the channel structure ( 20 ) a plurality of channel structure sections ( 30 . 40 . 50 ) which extends radially along a rotational path of the rotatable magnetic force element ( 10 ) are distributed to the position of the magnetic particles ( 60 ) in the plurality of channel structure sections ( 30 . 40 . 50 ) to control. Device according to claim 1 or 2, comprising a plurality of magnetic force elements ( 300 ; 300 ' ), wherein the means for generating a relative movement a conveyor belt ( 304 ), which has axes ( 306 . 308 ), on which the plurality of magnetic force elements is mounted. Device according to one of claims 1 to 5, wherein the at least one magnetic force element comprises a permanent magnet or an electromagnet. Device according to one of claims 1 to 6, wherein at least one channel structure section ( 30 . 40 . 50 ) at least one input ( 31 . 41 . 42 . 51 . 52 ) and at least two outputs ( 32 . 33 . 43 . 44 . 54 . 55 ), wherein the magnetic particles ( 60 ) by the time-varying magnetic field in one of the outputs ( 33 . 44 . 55 ) to be controlled. Apparatus according to claim 7, wherein between the at least one input and at least one output, a channel region is arranged with a larger channel cross-section. Device according to one of Claims 1 to 8, in which at least one channel structure section has a first input ( 41 . 51 ) for a first liquid containing the magnetic particles ( 60 ) and a second input ( 42 . 52 ) for a second liquid, wherein the magnetic particles are brought by the time varying magnetic field from the first liquid into the second liquid or a mixture comprising the second liquid. Device according to one of Claims 1 to 9, in which the microchannel ( 20 ) comprises the following features: a first channel structure section ( 30 ) with an input ( 31 ) in fluid communication with a first inlet ( 22 ) for a first liquid containing the magnetic particles ( 60 ) and a first and second output ( 32 . 33 ), the magnetic particles ( 60 ) by the time-varying magnetic field in the second output ( 33 ) of the first channel structure section ( 30 ) to be controlled; a second channel structure section ( 40 ) with a first input ( 41 ) in fluid communication with the second output ( 33 ) of the first channel structure section ( 30 ), a second input ( 42 ) in fluid communication with a second inlet ( 24 ) for a second liquid, a first outlet ( 43 ) and a second output ( 44 ), the magnetic particles ( 60 ) by the time-varying magnetic field from the first liquid in the second liquid and in the second output ( 44 ) of the second channel structure section; and a third channel structure section ( 50 ) with a first input ( 51 ) in fluid communication with the second output ( 44 ) of the second channel structure section ( 40 ), a second input in fluid communication with a third inlet ( 26 ) for a third fluid, a first outlet ( 54 ) and a second output ( 55 ), the magnetic particles ( 60 ) by the time-varying magnetic field from the second liquid in the third liquid or a mixture comprising the third liquid, and the second output ( 55 ) of the third channel structure section ( 50 ) to be controlled. Apparatus according to claim 10, wherein the third channel structure section ( 50 ) a third input ( 53 ) in fluid communication with a fourth Inlet ( 28 ) for a fourth liquid, wherein the first input ( 51 ) of the third channel structure section ( 50 ) between the second and third inputs ( 52 . 53 ) of the third channel structure section ( 50 ) is arranged. Apparatus according to claim 10 or 11, wherein the first outputs ( 32 . 43 . 54 ) of the first, second and third channel structure sections ( 30 . 40 . 50 ) are in fluid communication with one or more waste structures. Method for operating a device according to one of Claims 1 to 12, with a step of generating a relative movement between the at least one magnetic force element ( 10 ; 212 ; 270 . 274 . 276 ; 300 . 300 ' ) and the channel structure ( 20 ; 200 ; 250 ; 302 ) for controlling the position of the magnetic particles ( 60 ) in the channel structure to move the magnetic particles across a phase boundary of a multiphase flow. Method according to claim 13, comprising a step of generating a flow of a liquid containing the magnetic particles ( 60 ) and a parallel flow of a second liquid in the channel structure, wherein the magnetic particles ( 60 ) by moving the at least one magnetic force element ( 10 ; 212 ; 270 . 274 . 276 ; 300 . 300 ' ) are brought from the first liquid into the second liquid. The method of claim 14 including a step of generating a flow of a third liquid parallel to a flow of the second liquid having the magnetic particles in the channel structure ( 20 ), the magnetic particles ( 60 ) by moving the at least one magnetic force element ( 10 ; 212 ; 270 . 274 . 276 ; 300 . 300 ' ) from the second liquid into the third liquid or a mixture comprising the third liquid. The method of claim 15 including a step of generating a flow of a fourth liquid parallel to the flow of the second liquid containing the magnetic particles (10). 60 ), and parallel to the flow of the third liquid, the flow of the second liquid containing the magnetic particles ( 60 ) contains, between the flows of the third and fourth liquids. A method according to any one of claims 13 to 16, wherein the generated flows are at least partially laminar. Process according to one of Claims 13 to 17, in which flows of different buffer solutions are produced in the channel structure, the magnetic particles ( 60 ) are deflected by the time varying magnetic field to expose them to the different buffer solutions. The method of claim 18, including a step of providing a separate inlet ( 22 . 24 . 26 . 28 ) for each buffer solution, whereby the flows of different buffer solutions are generated continuously. A method according to any one of claims 13 to 19, wherein the magnetic force member is rotated in a rotational direction so that the magnetic particles which align along the generated magnetic field lines move in a direction opposite to the direction of rotation. A method according to any one of claims 13 to 19, wherein the temporally varying magnetic field generated by the relative movement controls the position of a primary superparamagnetic body, whereby the primary superparamagnetic body is the position of secondary supermagnetic particles having an affinity for biomolecules , controls. Use of a device according to any one of claims 1 to 12 for performing an immunoassay, a nucleic acid extraction or a continuous DNA amplification.

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