EP3134505A1

EP3134505A1 - Method and apparatus for purifying biological molecules

Info

Publication number: EP3134505A1
Application number: EP15717482.2A
Authority: EP
Inventors: Bernd Faltin; Christian Dorrer
Original assignee: Robert Bosch GmbH
Current assignee: Robert Bosch GmbH
Priority date: 2014-04-25
Filing date: 2015-04-17
Publication date: 2017-03-01
Also published as: US20170044483A1; DE102014207774A1; KR20160145610A; WO2015162059A1; JP2017515500A; DE102014207774B4; CN106232799A

Abstract

In a method and apparatus for purifying biological molecules, especially nucleic acids or proteins, at least one filter (50) is used. At least some of the fluids needed for the operation are pumped in a circuit (51) via the filter (50). First a fluid with biological cells is pumped via the filter (50). The cells retained on the filter are digested. To bind the biological molecules to the filter, binding buffer is pumped in the circuit (51) via the filter (50). A washing buffer for cleaning the biological molecules bound to the filter is pumped via the filter (50), and so the biological molecules bound to the filter are available for further use.

Description

description

Method and device for purifying biological molecules

The present invention relates to a method and an apparatus for the purification of biological molecules, in particular of nucleic acids or proteins, wherein at least one filter is used in the method.

State of the art

For the purification of biological molecules and in particular of

Nucleic acids or proteins exist in many different ways. As a rule, the biological molecules are obtained from cell material, ie from prokaryotic or eukaryotic cells. Before intracellular material can be further processed, it is usually necessary to open the cells themselves. This cell disruption is commonly referred to as cell lysis. After separation of cell debris, for example

Nucleic acids, proteins or peptides are further purified, processed and analyzed. When referring to proteins in the following, they also mean peptides. For example, to detect a particular nucleic acid, the purified nucleic acids can be amplified selectively by PCR (Polymerase Chain Reaction) so that the particular nucleic acid sequence is made detectable.

The digestion of the cells can take place in different ways. An enzymatic digestion of the cells is widespread, for example, a treatment with the enzyme proteinase K or lysozyme is performed. A thermal cell disruption by heating and / or freezing the sample or a cell disruption with chemical reagents is also possible. Furthermore, the Cell disruption done mechanically, for example by a

Sonication. A common method for further purification of, for example, nucleic acids provides that the so-called lysate resulting from cell disruption is mixed with a binding buffer and brought into contact with a solid matrix, for example a silica filter or a silica membrane. In this case, the nucleic acids adsorb to the filter and can then be washed with a washing buffer and then eluted from the solid matrix and used further. Various commercially available kits and laboratory equipment work according to this principle.

Often it is necessary to concentrate the cell material prior to further treatment or to accumulate the cells. For this purpose, for example, a centrifugation of the sample can be carried out with the cells. German laid-open specification DE 10 2005 009 479 A1 describes a method in which the cells are accumulated by means of filtration. With such

For example, it is also possible to quantify the accumulated cells, for example bacteria, as described in the paper by Dufour, Alfred P., et al. (Applied and Environmental Microbiology, May 1981, pages 1152-1158).

German Offenlegungsschrift DE 10 2010 030 962 A1 describes a method for the hybridization of nucleic acids in a microarray, in which the sample is first pumped through a denaturation unit and then through a separate reaction area with the microarray with the immobilized probes. Here, the pumping section can be configured as a circuit.

The German patent application DE 10 2010 043 015 AI discloses a

Method of amplifying nucleic acids on a filter, i. be duplicated. In advance, a concentration and a lysis of the

Nucleic acid containing cells take place on the filter.

Disclosure of the invention Advantages of the invention

With the method according to the invention, biological molecules, in particular nucleic acids or proteins or other biological molecules, can be concentrated and purified. The purification is carried out in principle by a non-specific adsorption of the biological molecules to a matrix, in particular to a membrane. In the following, it is generally referred to as a filter, which hereby means the matrix, in particular in the form of a membrane or, for example, in the form of a bed. The core of the invention is that at least some of the required for the process management

Liquids are pumped through the filter in a cycle. In detail, the steps of the method according to the invention initially comprise pumping a liquid with biological cells, that is to say a sample liquid, via the filter. The term "biological cells" is generally understood to mean cells from which biological molecules, such as, for example, nucleic acids or proteins, are to be treated or purified, for example pathogenic microorganisms such as bacteria or fungi is also suitable for human cells or other cells and can be used in general for the purification of proteins or nucleic acids from prokaryotic or eukaryotic cells The term "sample liquid" is generally understood to mean the liquid containing the corresponding cells, for example one

Cell suspension or a patient sample, for example, blood, lavage, urine, cerebrospinal fluid, sputum or a flushed Swab or smear. Depending on the application, the volume of the sample may be different, for example, between a few μΙ to 10 ml. After the sample application, the cells retained on the filter are digested, whereby in principle different methods can be used for cell disruption. The biological molecules contained in the cell lysate are bound to the filter by means of a binding buffer, whereby the binding buffer is pumped in circulation via the filter. The biological molecules bound to the filter are cleaned in the subsequent step with wash buffer, which is pumped over the filter. As a result, the biological molecules to be purified are reversible

immobilized form on the filter. For further processing or analysis For example, the bound biological molecules may be eluted from the filter in a conventional manner, or the filter having the reversibly immobilized biological molecules may be used directly as such.

In previously known methods, the accumulation of cells from a sample by centrifugation takes place. An implementation of this method in a microfluidic system and thus a cost-effective automation is not possible. Also known are methods in which the accumulation of the cells from a sample is carried out by rinsing through a filter, wherein the lysis then takes place by applying a lysis buffer to the filter. These

However, methods for integration into a microfluidic system have the disadvantage that the exact positioning of the lysis buffer on the filter is difficult or associated with great expense. This must be done manually or it will be e.g. Cameras or photocells needed. Furthermore, there is a risk when displacing the lysis buffer on the filter to displace cells and already released nucleic acids from the filter, which are then no longer available for further purification and thus reduce the efficiency of the purification. Furthermore, in these methods, the diffusion of lysis reagents, e.g. Enzymes on the filter, which reduces the effectiveness of lysis, especially in cells difficult to lyse, e.g. Mushrooms. The invention solves these problems and thereby enables the simple implementation of the described method in an automated microfluidic system (lab-on-chip system).

The particular effectiveness of the purification process according to the invention is achieved in that the substances are rinsed several times through the filter by the circulation of the liquids, in particular during the binding process of the biological molecules to the filter. By guiding the fluids in a circular fluidic path, the filter material is repeatedly contacted with the fluids. It turns

Saturation equilibrium, in which the maximum binding capacity of the membrane is exhausted. In conventional processes, it is often the case that only a portion of the molecules of interest are actually adsorbed. In the method according to the invention by the circular fluid guide ensures, for example, that all nucleic acids released during cell disruption are effectively pumped through the filter in the binding step. In a sense, no nucleic acids are lost, so that the yield is increased. In addition, the circulation in the pump ensures that an optimal mixture of the reagents, for example the binding buffer and the cell lysate, takes place. The mixing of reagents is often a problem, especially in microfluidic systems. The circular fluid guide provided according to the invention achieves thorough mixing of the various reagents and buffers, which makes the method of the invention particularly suitable for implementation within a microfluidic system. Irrespective of this, the mixing efficiency, in particular in a microfluidic system, can also be increased by further measures, in particular by means of mixer structures or mixing chambers known per se, in a microfluidic system.

In the first method step, an accumulation of the biological cells takes place, wherein the cells are retained on the filter according to the size exclusion method and / or by electrostatic interactions when the sample liquid is pumped over the filter. The more sample liquid is pumped through the filter, the higher the number of accumulated cells. Depending on the dimension of the device according to the invention, the diameter of such a known filter may be different, for example between 1 and 25 mm. Suitable filters are, for example, fiber filters, fabric filters and / or membrane filters, in particular of silica.

Also suitable are particle beds, in particular

Microparticle beds, e.g. from silica particles. The pore diameter of the materials is preferably below 100 μm. When applying the sample liquid on the filter, it may be provided that the sample is pumped circular over the filter several times. This has the advantage of having cells that pass the first time the filter

may not have been retained when the filter passes again. Since also electrostatic forces act and the Size distribution of the pores in a silica filter is generally not considered to be homogeneous, it may happen that cells are not retained in a first passage. In addition, cells can become trapped in "dead ends" of the system, which can increase efficiency by providing guidance during sample application.

The digestion of the cells can take place in various ways, for example mechanically or by heat. Advantageously, e.g. be provided an ultrasonic treatment, which can be carried out with relatively little expenditure on equipment. In this case, no additional reagents for lysis or cell disruption are required. The ultrasound can be entered directly into the filter. For this example, the wall of a corresponding filter chamber be designed as a membrane into which the

Ultrasound is coupled by means of a horn. During the

Ultrasonic treatment, the filter chamber should be filled with liquid or with a buffer or water. The ultrasonic treatment may cause the filter material to be partially disassembled. As a result, on the one hand, the cells accumulated in the filter are at least partially released and made accessible to the lysis effect by the ultrasound. On the other hand, the resulting particles can produce an additional grinding effect and thereby further support cell disruption, with the particles being caught again by the intact areas of the filter in the further course of the processing.

Particularly preferred is cell disruption using enzymes or other lysing reagents, for example chemical reagents. For this cell disruption with lysing reagents can be provided with particular advantage also a circulation of the fluids. For this purpose, a suitable lysis buffer is fed into the circular fluidic path and pumped in a circle over the filter. It is pumped in particular in the direction in which the sample was pumped through the filter. This avoids the loss of cells or already released nucleic acids when the lysis buffer is initially placed on the filter. Furthermore, air bubbles, which possibly reach the filter, in the course again removed from this. In the previously known methods, such air bubbles remain on the filter and locally prevent lysis. Furthermore, the leaching of the lysis reagents on the filter is thereby avoided by the continuous introduction of lysis buffer on the cells, thus achieving a particularly effective and complete lysis of the cells on the filter.

Surprisingly, it has been shown that when using enzymes for lysis their activity is not reduced by the constant pumping and the resulting shear forces, so that an enzymatic lysis can be carried out according to the scheme described with advantage. For example, depending on the type of biological cells and the reagents used, lysis may require a period of between 2 and 30 minutes. A lysis buffer is to be understood as meaning a buffer which is suitable for cell disruption or lysis of the target cells. The buffer may contain, for example, lysis enzymes known per se, such as, for example, lysozyme and / or

Proteinases. Alternatively or additionally, chaotropic salts, detergents and / or basic ingredients such as e.g. NaOH be included. Further, buffering agents (e.g., Tris-HCl), nuclease inhibitors (e.g., EDTA or EGTA) and / or reducing agents (e.g., β-mercaptoethanol) may be included.

In the subsequent binding step, the binding buffer is fed into the circular fluidic path and pumped in a circle. In this case, the binding buffer mixes with the lysate and, for example, the nucleic acids bind to the filter under the conditions set in the process.

Before the binding step, especially in the purification of nucleic acids, a further denaturation step can be carried out. A buffer suitable for this purpose may in particular contain chaotropic reagents, for example GIT

(Guanidinium isothiocyanate). Through such reagents, the proteins contained in the lysate are "salted" so to speak, so that the proteins can be washed out more easily. The denaturation buffer is also preferably pumped in the circulation to further increase the effectiveness of the denaturation step. Before the binding step, an additional digestion step can be carried out, especially in the purification of nucleic acids. A suitable one

Verdaupuffer can, for example, various enzymes, in particular

Proteinases that contain a digestion of the liberated in the lysis step

Cause proteins. As a result, the effectiveness of a nucleic acid purification and the purity of the nucleic acids obtained can be further improved. For this step, the appropriate digestion buffer is fed into the path and advantageously also pumped in a circle. Here too

Surprisingly, it has been shown that the activity of the enzymes used for the digestion is not reduced by the constant pumping and the resulting shear forces.

Following binding of the biological molecules to the filter, one or more wash steps are performed, with wash buffer passed over the filter. The composition of suitable wash buffers is chosen such that in this step, for example, the nucleic acids remain bound to the filter while other molecules, especially proteins, are not adsorbed and removed. As the washing buffer, for example, an alcohol-containing washing buffer, e.g. 70% EtOH, are used.

For many applications, it is advantageous if the filter is dried after treatment with washing buffer. This can be done, for example, by passing air or nitrogen through the filter. Subsequently, elution of the adsorbed target molecules from the filter can be carried out using water or a suitable elution buffer for this purpose. It can also be provided that the filter with the target molecules adsorbed on it is used as such. For example, a PCR can be carried out with the reversibly immobilized on the filter nucleic acids, as is known per se from the prior art.

When using certain samples, it may be advantageous to pre-treat the sample before applying it to the filter. For example, in the filtration of blood there may be a problem that the filter becomes clogged with blood cells and thus clogged, making further filtration impossible. For this purpose, it has proven to be advantageous to first lyse the blood cells selectively. By "selectively lyse" it is meant that the blood cells, also called human cells, are disrupted while other cells contained in the sample, especially pathogens, remain intact, for example, by treating the sample with chaotropic reagents or

Detergents or by osmotic shock and has the advantage that clogging of the filter is avoided. A commercially available kit

(Molzym MolYsis Completeö) additionally performs a digestion of the liberated human nucleic acids by means of a DNase in such a selective lysis. Such a digestion can be incorporated into the method of the invention and have the advantage that the filterability of the sample is further improved and a human nucleic acid background is partially removed from the sample. For example, the sample is first mixed with a chaotropic buffer and then incubated with a DNase, e.g. for a period of 10 minutes before the sample is applied to the filter.

In a preferred embodiment of the method according to the invention, one or more of the method steps may be at least partially under

Heat supply done. In particular for the cell disruption and / or the additional digestion step and / or for the drying of the filter, it may be advantageous to increase the temperature. For example, the enzymes used for enzymatic cell disruption may be elevated

Have optimum temperature, so that with an increase in temperature, for example, to temperatures between 35 and 60 ° C, especially between 35 and 45 ° C, the lysis of the cells can run faster and more effective. The same applies to a digestion or denaturation step. Also, the drying of the filter can be accelerated by increasing the temperature, for example by increasing the temperature to a temperature between 40 and 60 °. In general, for the heat supply, in particular the filter can be heated directly, for example via a per se known Peltier element or a film heater, which is brought into contact with the device containing the filter. In addition, it is also possible to work with tempered liquids, for example it can be provided, a Vorlagerungsgefäß for the liquids used and / or the At least partially to heat the pipeline system. Depending on the application, a cooling or generally a temperature control may be advantageous.

In a particularly preferred embodiment of the method according to the invention, the pumping direction in one or more of the

Procedural steps are reversed once or several times. In this way, in particular clogging of the filter or clogging of the filter can be avoided or optionally reversed. Furthermore, the mixing of liquids in circulation can be improved and optionally precipitated solids can be brought back into solution. By reversing the pumping direction after binding of the target molecules to the filter, there is generally no detachment of the target molecules from the filter since the adsorption of the molecules to the filter is independent of the pumping direction. A reversal of the pumping direction is particularly advantageous during the binding step and / or during the washing step and / or during the elution step. During the sample application or during the accumulation of the cells on the filter, it may also be advantageous to repeatedly reverse the pumping direction repeatedly, in order to avoid clogging of the filter with cells and thus clogging or, if appropriate, reversing it.

With particular advantage, the method according to the invention is carried out in a microfluidic device. In general, microfluidic devices have the advantage that they are particularly suitable for automated processes. Automation reduces the time and cost of analysis and reduces the risk of contamination. Furthermore, an automated system does not necessarily have to be operated by qualified personnel, since the operation is generally easy to learn. The

inventive method in conjunction with a microfluidic device has the particular advantage that a particularly good mixing of the liquids is achieved by the circulation of the fluids. Often, therefore, can be dispensed with further structures and active components such as stirrers for mixing. Nevertheless, however, additional mixer structures or mixing chambers known per se can also be used be provided in a corresponding device in order to further increase the mixing efficiency.

The invention further comprises an apparatus for carrying out a purification of biological molecules, in particular of nucleic acids or

Proteins. The device has at least one pump for pumping liquids. In addition, the device comprises at least one device for fixing at least one filter. The purification protocols that can be carried out here are based on the fact that the biological molecules to be purified can adsorb to the filter. According to the invention, the

Device a conduit system for the circular pumping of liquids through the filter. Above all, the described method according to the invention can advantageously be carried out with this device. An essential aspect of the invention is that by the circular pumping of liquids through the filter, the efficiency of the cleaning process can be significantly improved.

The device for fixing the filter is in particular a filter chamber for receiving the filter material. The term "filter chamber" is to be understood in particular as meaning a fluidic cavity with a filter, for example, the filter chamber may be designed as a tube or, in a particularly preferred manner, as a microfluidic element

Filter chamber preferably has a multilayer structure. In this case, for example, two or more structured plates, in particular polymer plates, can be provided. In one of the plates can be a flat

Recess may be provided in which the filter, e.g. a membrane, or other filter material, e.g. a microparticle bed, can be inserted. It has proven to be advantageous, in particular with relatively large filter diameters (> 3 mm), to pass the filter through a support structure, e.g. a porous polymer carrier (frit), to aid in sagging or

Sagging of the filter to avoid. For the passage of liquids, one or more inlet and outlet channels are provided. Furthermore, it can be provided that an additional membrane is inserted between the two plates, realized by the additional functionalities of the filter chamber can be, for example, a pneumatic actuation of

Diaphragm valves and / or a diaphragm pump. Preferably cover films or cover membranes or other polymer layers are provided as lateral outer terminations of the system. A cover film can also be used for coupling ultrasound. In particular, in embodiments of the device, which are set up for an ultrasound treatment during the cell lysis, it can be provided that the filter chamber has an extension for introducing the ultrasound. Here is the extension that

may be circular or part-circular, for example, realized as a breakthrough to the outside, which may be closed in particular with a cover membrane. This extension of the filter chamber can also take over the function of ventilation for the system, wherein

Ventilation channels may be provided, which lead to the extension.

In a particularly preferred embodiment, the device according to the invention has at least one ventilated vessel, with which liquids can be introduced into the system. In particular, an upwardly vented vessel may be incorporated into the fluidic path or conduit system for circular pumping of liquids across the filter. The installation of a ventilated vessel has the advantage that thereby the

increasing volume of the liquids in the circuit added and the pressure in the circuit can be kept constant, whereby a microfluidic realization is possible in a particularly advantageous manner. The liquids may e.g. be introduced by hand, in particular by pipette, or by pumping with a second pump integrated in the system into the vessel. A ventilated vessel also has the advantage that air bubbles which have entered the fluidic path can rise in the vessel and thus leave the system. This will avoid that

Air bubbles remain in the circulation and lead to foaming. Such a vessel may for example be designed as a tube, chamber or other fluidic element having a volume, for example, between 100 μΙ and 10 ml. With particular advantage, moreover, a plurality of ventilated vessels can be provided in the system, which can serve primarily as displacement chambers for reagents. The pump may be, for example, a peristaltic pump or a micromembrane pump. With particular advantage, the pump is more or less directly upstream or downstream of the filter, so that the liquids can be pumped with very high positive or negative pressure through the filter. Conveniently, the channel piece between the pump and the filter is relatively short. Furthermore, it can be advantageously provided that an inlet channel for the sample liquids opens more or less directly in front of the pump or the filter. This has the advantage that other displacement vessels for buffer solutions are not contaminated by the sample liquid. It can also be provided that several

Inlet channels are provided for different liquids.

With particular advantage, one or more elements of the device can be heated. For example, the pump and / or the line system or parts thereof and / or the filter and / or optionally a vessel which is provided for the pre-storage or for the introduction of liquids, be heated. In this way, individual process steps can be tempered

be performed. For example, the lysis step may be performed at an elevated temperature by preheating the lysis buffer and / or heating the filter itself.

Preferably, the device according to the invention is designed as a microfluidic system. With regard to the advantages of the device according to the invention in microfluidic design, reference is made to the advantages already mentioned above. The method according to the invention and the device according to the invention can be implemented, for example, with particular advantage in molecular diagnostics and / or, for example, in a lab-on-a-chip system.

Other features and advantages of the invention will become apparent from the

following description of embodiments in conjunction with the drawings. In this case, the individual features can be implemented individually or in combination with each other. Brief description of the drawings

In the drawings show:

Fig. 1 is a schematic representation of the principle of the circular

Fluid management;

Fig. 2 is a schematic representation of the components of an exemplary

Embodiment of an apparatus for carrying out the method according to the invention;

Fig. 3 is a schematic representation of another exemplary

Fig. 4 is a schematic representation of another exemplary

Fig. 5 is a schematic representation of another exemplary

Fig. 6 is a schematic representation of a multilayer filter chamber as

Component of the device according to the invention;

Fig. 7 microfluidic device according to the invention in a plan view; Fig. 8 side view of the multilayer structure of the microfluidic

Apparatus of Fig. 7;

FIG. 9 is an oblique view of the microfluidic device of FIG. 7; FIG.

10/11 detail views of an exemplary filter chamber a

According to the invention microfluidic device in side view (Fig. 10) and in plan view (Fig. 11) and

12/13 detail view of another exemplary filter chamber of a microfluidic device according to the invention in side view

(FIG. 12) and in plan view (FIG. 13).

Description of exemplary embodiments The schematic representation in FIG. 1 illustrates the principle of a circular fluidic path 11, which extends over a filter 10. The fluid in the fluidic connections 11 is driven by a pump 12. The fluidic connections 11 can be formed, for example, by hoses or channels. The pump 12 is a

Liquid pump, such as a peristaltic pump or a

Diaphragm pump. Conventional integrable microfluidic pumps can be used for a microfluidic configuration of the device. The filter 10 shown here is realized in the form of a filter chamber. The following description of a filter is to be understood in many cases as a synonym for a filter chamber. The filter chamber can be called microfluidic

Be configured component. The type of inflow and outflow on the filter 10 can optionally be set exactly. The filter chamber itself can be realized, for example, in a multilayer structure of a plurality of structured polymer layers. By this construction, a particularly cost-effective production is possible. When the pump 12 is connected via hoses to the inlet and outlet of the filter chamber 10, the feeding into the fluidic path can be done, for example, by opening the hose connection and pipetting.

Fig. 2 shows a preferred variant for the schematic structure of an apparatus for carrying out the method. In addition to the filter or the filter chamber 20 and the pump 22 and the fluidic connections 21, an upwardly ventilated vessel 24 is integrated into the fluidic path. Via this vessel 24 liquids can be introduced into the circular fluidic path. The required solutions or buffers can be pipetted into the vessel 24, for example. In this way, it is advantageously possible to feed buffer or other solutions into the fluidic path during the process. In addition, this embodiment has the advantage that air bubbles that have entered the fluidic path, rise in the vessel 24 up and so can leave the system. Depending on the configuration of the arrangement, the volume of the vessel 24 can be selected accordingly, for example between 100 .mu.l and 10 ml. The vessel 24 can be designed, for example, as a tube or as a microfluidic element. advantageously, The bottom of the vessel is the outlet channel of the system. By "bottom" is meant the part of the vessel that is at the lowest point in gravity

Liquids can be completely removed from the vessel.

Advantageously, the vessel 24 is designed so that it tapers downwards.

Fig. 3 shows a further variant of the device for carrying out the

Process. This embodiment is particularly suitable as a microfluidic system. In general, a microfluidic system has the advantage that the dead volume of the structure can be kept very low and the risk of foaming is low. The circular fluid guide in the fluidic connections 31 is driven by the pump 32. Upstream of the pump is the filter chamber 30. Further, a vessel 34th

provided that can be vented through an opening or a vent passage 35. The opening or the venting channel 35 is advantageously at the respect of the gravitation upper end of the vessel. As a result, inadvertent leakage of reagents can be avoided.

Upstream of the vessel 34, an inlet channel 36 is provided, wherein the inlet channel 36 can also open directly into the vessel 34. Furthermore, a plurality of inlet channels 36 may be present. Downstream of the pump is an outlet channel 37. The flow of liquids is controlled by integrated valves 38 located at various locations in the system. This can be, for example, rotary or

Diaphragm valves act.

When using such a device, the method according to the invention can be carried out as follows: The sample, ie the liquid with the biological cells, is introduced through the opening 35 into the vessel 34 via the inlet channel 36 or by introduction (eg pipetting). The vessel 34 may be designed, for example, as a microfluidic cavity. The air contained in the vessel 34 is released through the opening or the venting channel 35, so that the vessel 34 is vented. The pump 32 then pumps the sample via the filter 30 in the direction of the outlet channel 37. Es it can be provided that the venting channel 35 is closed, so that the pump 32 can suck in the sample directly via the inlet channel 36. The cells contained in the sample accumulate on the filter 30. The cells are then disrupted by, for example, treating them with a suitable lysis buffer. The lysis buffer is first introduced into the vessel 34, for example via the inlet channel 36.

Subsequently, the lysis buffer is pumped by the pump 32 via the circular fluidic path 31 in the circuit via the filter 30. Alternatively, the cell disruption can also be done in other ways, for example with ultrasound. In this case, the filter 30 is sonicated accordingly. This is followed by a binding step in which the target molecules adsorb to the filter 30. For this purpose, suitable binding buffer is introduced into the vessel 34 and pumped in the circuit 31. For the subsequent washing step, the washing buffer is first introduced into the vessel 34 and pumped by the pump 32 via the filter 30 in the direction of the outlet channel 37. When drying of the filter 30 is provided, for example, air or nitrogen is pumped from the inlet channel 36 via the filter 30 in the direction of the outlet channel 37. It is also possible to use the pump 32 for drying. Finally, an elution step can be carried out, wherein suitable elution buffer is introduced into the vessel 34 and pumped by the pump 32 via the filter 30 in the direction of the outlet channel.

In general, the introduction of the sample and the buffer into the vessel 34 can take place, for example, by means of a further pump or manually by pipetting or the like. For this purpose, a resealable opening in the vessel 34 may be provided.

4 shows a further variant of the system, wherein in addition to the vessel 34 one or more further vessels 44, for example storage vessels, are provided. These vessels 44 are equipped with an opening or a vent channel 45. The contents of the vessel 44 can be introduced via a further valve 48 in the rest of the pipe system. Incidentally, the system substantially corresponds to the device shown in FIG. 3. The corresponding elements are therefore provided with the same reference numerals. Between the vessel 34 and the supply line from the other vessel 44, another valve 49 is provided. Various buffers, for example the lysis, digestion, denaturing, binding, washing or elution buffers, can be stored upstream in the vessel or vessels 44. This variant has the advantage that an automatic implementation is simplified because the buffers no longer have to be individually introduced into the vessel 34. The method can be carried out in such a way that, in particular, the sample is manually introduced into the vessel 34 before the filter 30 is subjected to it. The various required buffers in the subsequent process steps can be automatically introduced from the vessel or vessels 44.

FIG. 5 illustrates a further preferred example of a device for carrying out the method according to the invention, which can be realized, for example, in a microfluidic system. In this system, the pump 52 is located upstream of the filter chamber 50. This has the advantage that liquids can be pumped through the filter 50 at very high pressure. For this purpose, it is particularly advantageous if the channel piece or piece of tubing between the pump 52 and the filter 50 is relatively short. The inlet channel 56 opens directly in front of the pump 52. This has the advantage that the pumped from the inlet channel 56 via the filter 50

Liquids, especially the sample with the cell material, not the

Pre-storage vessel 54 pass, so that contamination can be avoided. Advantageously, the system can be heated partially or in sections, for example, the filter 50 and / or the pump 52 and / or the vessel 54 can be heated. A heating may conveniently take place during the lysis step, so that the cell digestion can proceed even more efficiently. Conveniently, an optimum temperature for the lysis enzymes used can be set, which may for example be in a temperature range between 35 and 55 ° Celsius, for example at 45 ° Celsius. The process control in the Lyseschritt preferably takes place in a circular shape over the circular fluidic path 51, comparable to the other described embodiments. As a variant, further inlet channels may be present, via which the required reagents can be pumped into the system. For example, the vessel or vessels 54 have a volume of 2 ml and the filter has a diameter of between 2 and 10 mm, for example. The fluid flow is controlled via the valves 58. The fluids can leave the system via the outlet channel 57.

An experimental procedure for the accumulation and lysis of cells and a DNA purification in a microfluidic system according to the invention can be carried out, for example, as follows: 10 ⁵ Staphylococci in 1 ml of physiological saline solution are introduced by pumping via the inlet channel 56 or by pipetting into the vessel 54 in the system and pumped by the pump 52 through the filter 50. For lysis 100 μΙ lysis buffer are pipetted into the vessel 54 and with the pump 52 for 10 minutes with simultaneous

Temperature control pumped to 45 ° C via the filter 50 and the channel system 51 in a circle. Subsequently, a digestion buffer and a binding buffer are successively pipetted into the vessel 54 and likewise circulated. This has the advantage that a very good mixing of the reagents takes place and

Nucleic acids are effectively bound to the filter 50. The filter 50 is then washed by pipetting a wash buffer into the vessel and pumping it via the filter 50 into the outlet channel 57. The bound DNA is eluted with water by pipetting water into the vessel 54 and pumping it via the filter 50 into the outlet channel 57. The eluate is collected. In parallel, a reference is processed: 10 ⁵ staphylococci in 1 ml of physiological saline are collected by centrifugation at 13,000 g and the supernatant is pipetted off. 100 μl lysis buffer are added by pipette, mixed and incubated for 10 min at 45 ° C. The resulting lysate is successively mixed with digestion buffer and binding buffer and applied to a commercially available column. Subsequently, the column is washed with washing buffer and the DNA is eluted with 100 .mu.l of water. An analysis of the samples is carried out by means of a quantitative PCR. The test results show that comparable results can be achieved with the method according to the invention as in the reference, wherein in the method according to the invention a considerable labor savings by the possibilities of simple

Automation can be achieved.

6 shows a section through an exemplary microfluidic filter chamber 60 based on a multilayer construction. Two structured polymer plates 61, an intermediate polymer membrane 62 and externally overlying

Lid films 63 form the multi-layer structure. The filter 64 is in one

Recess of one of the polymer plates 61 inserted. The liquid supply and the liquid discharge via the inlet channel 65 and the outlet channel 66. The upstream of the filter 64 membrane 62 can perform additional functions, such as a valve function. This structure is particularly suitable for microfluidic embodiments.

FIGS. 7 to 9 show an exemplary embodiment of a microfluidic system 700 for carrying out the method according to the invention. Fig. 7 shows a top view from the front, Fig. 8 is a side view and Fig. 9 is an oblique view from the front. The microfluidic system is implemented as a multilayer structure consisting of two structured polymer plates 750 and 760 (FIG. 8) and lid films arranged on the right and left or other polymer layers (not shown) for covering the structures. The system comprises a pump 702, a vessel 704 with a ventilation opening 724 and a filter device 710 and a plurality of valves 708, 718, 728. The fluid is guided in the direction of the arrow (FIGS. 7, 9), whereby the direction of flow can also be reversed. The

Filter device 710 is provided by a recess 713 (filter chamber) in the

Polymer plate 750, a frit 711 for the mechanical support of the filter and the actual filter 712 formed (Fig. 8). On the outsides of the system run channels 701 which are capped by cover sheets or other polymer layers (not shown). The valves 708, 718, 728 are designed as microfluidic diaphragm valves. The pump 702 is as

microfluidic diaphragm pump having a pumping chamber and an inlet valve 708 and two outlet valves 718, 728 and located downstream of the filter device 710. The outlet valves 718, 728 form a T-junction and allow switching of the fluid path between the circuit 701 (valve 728) and the outlet channel 707 (valve 718). Between the polymer plates 750 and 760 is a polymer membrane, not shown, which is used for pneumatic actuation of the valves and the pump. The vessel 704 is shaped so that even the smallest amounts of liquid converge at the lowest point of the vessel and from there into the channel system 701 can enter. The vessel 704 has a taper towards the bottom.

The illustrated system 700 may be part of a larger microfluidic system that includes other functionalities, e.g. more pumps and

Mixing chambers, chambers for pre-storage of reagents, chambers for further processing of the biological molecules, e.g. for the amplification of the recovered nucleic acids, for example by means of PCR, and components for the detection of biological molecules, e.g. of nucleic acids.

Using the microfluidic device 700, the method of the invention may be performed as follows: The sample is first introduced into the vented vessel 704, e.g. by pipetting and pumping, and

then pumped from below via the filter device 710 in the outlet channel 707. Lysis buffer is then introduced into the vented vessel 704 and circulated through the filter device 710 during lysis with the pump 702. Alternatively, the lysis buffer may also be circulated only briefly to safely fill the filter chamber 713 with liquid, after which, e.g. Heat or ultrasound is applied to the filter 712. Subsequently, a binding buffer is placed in the vented vessel 704 and circulated by the pump 702 via the filter device 710. This results in the mixing of lysate and binding buffer and nucleic acids (as an example of molecules to be purified) bind to the filter 712. The mixture is then pumped into the outlet channel 707. Subsequently, a washing buffer is introduced into the ventilated vessel 704 and pumped via the filter device 710 into the outlet channel 707. Finally, an elution buffer is introduced into the vented vessel 704 and pumped via the filter device 710 into the outlet channel 707. Alternatively, the elution buffer can also be sucked in via the outlet channel 707 and pumped in the reverse direction via the filter device 710 into the ventilated vessel 704.

In a variant of this method, human cells initially present in the sample can be selectively lysed. In a further variant of the method, a digestion of proteins can be carried out after the lysis. In a further variant of the method, a denaturation step can be carried out before the binding of the DNA. In a further variant of the method, the filter before the Elution dried. In a further variant of the method, the pumping direction can be reversed briefly, for example, for 5 to 60 seconds.

10 is a side view and FIG. 11 is a plan view of an exemplary one

Embodiment of a filter chamber 813 for microfluidic devices. In Fig. 10, the multilayer structure of two polymer plates 850 and 860 can be seen. The circular recess 814 in the polymer plate 850 is provided as a blind hole for inserting a filter membrane or a filter material bed and optionally a frit. Immediately above the filter to be inserted, a circular extension 815 is provided. In the other polymer plate 860, a circular aperture 816 is provided, which is covered in the assembled state by a cover membrane (not shown). Via the channel 817, a supply or discharge of liquids can take place. The circular aperture 816 may, for example, have a diameter between 5 and 50 mm and may be arranged approximately concentrically with the filter, but may also be displaced, in particular with respect to the direction of gravity upward, e.g. by the difference between the radii of the filter and the circular area. Through the opening 816 and the extension 815, ultrasound can be introduced into the interior of the filter chamber 813 by means of a sonotrode, as a result of which ultrasound lysis can be carried out. This variant has the advantage that a particularly efficient ultrasonic lysis is possible. Alternatively, the area provided for introducing ultrasound can also be oval, square or oblong with comparable dimensions. Furthermore, this variant has the advantage that the extension 815 of the filter chamber 813 can simultaneously fulfill the function that air bubbles rise upwards in the extension and are thus eliminated from the fluid circuit and that a volume of liquid rising during processing is absorbed, and thus optionally replace other vessel of the system. For this purpose, an additional vent channel 819 may be provided. It is useful if the

Extension, for example, has a volume between 500 μΙ and 5 ml. For this purpose, an additional extension 818 can be provided. As a filter here, for example, silica membranes, but also beds of microparticles can be used. In one variant, the filter or the bed of microparticles extends into the extension 815 and the opening 816, so that there is contact with the cover membrane on this side. This has the advantage that when coupling of Ultrasound in the lid memb ran the filter or the bed of microparticles is particularly efficiently vibrated, whereby the lysis of accumulated cells can take place with greater yield.

FIGS. 12 and 13 show a further embodiment of a filter chamber 913 which, comparable to the filter chamber 813, has a blind-hole-shaped recess 914 in the polymer plate 950 for receiving a filter or a filter material bed and possibly a frit. About the channel 919 can a

Liquid supply or removal take place. In the polymer plate 950 a part-circular extension 915 of the blind hole 914 is provided. The other

Polymeric plate 960 has in these areas an aperture 916 which, when assembled, makes the connection between the blind hole 914 and the extension 915 and which is covered with a cover membrane (not shown). The extension 915 and the aperture 916, similarly to the embodiment 813, are also suitable for ultrasonic coupling.

Claims

claims

1. A process for the purification of biological molecules, in particular of

Nucleic acids or proteins, using at least one filter (10; 20; 30; 50; 64; 710), characterized in that at least some of the liquids required for a process control in a circuit (11; 21; 31; 51; 701) the filter (10; 20; 30; 50; 64; 710) are pumped, wherein at least the following process steps are carried out:

- Biological cell fluid is passed through a filter (10; 20; 30; 50; 64;

710),

the cells retained on the filter (10; 20; 30; 50; 64; 710) are disrupted,

Binding buffer for binding the biological molecules to the filter is pumped through the filter (10; 20; 30; 50; 64; 710) in the circuit (11; 21; 31; 51; 701),

Washing buffer for purification of the biological molecules bound to the filter is pumped through the filter (10; 20; 30; 50; 64; 710).

2. The method according to claim 1, characterized in that for the digestion of the cells lysis buffer in the circulation (11; 21; 31; 51; 701) via the filter (10; 20; 30; 50; 64; 710) is pumped or that the Cells are mechanically disrupted, in particular by ultrasonic treatment.

3. The method according to any one of the preceding claims, characterized in that a denaturation step is carried out before the treatment with binding buffer.

4. The method according to any one of the preceding claims, characterized in that prior to treatment with binding buffer, an additional Verdauschritt

is carried out.

5. The method according to any one of the preceding claims, characterized in that after the treatment with washing buffer, the filter (10; 20; 30; 50; 64; 710) is dried.

6. The method according to any one of the preceding claims, characterized in that prior to pumping the liquid with the biological cells via the filter, a pretreatment of the liquid is carried out, preferably in the liquid present human cells are selectively lysed.

7. The method as claimed in one of the preceding claims, characterized in that at least some of the method steps are carried out with the introduction of heat, in particular cell disruption and / or optionally performed digestion step and / or optional drying of the filter (10, 20, 30, 50, 64) 710).

8. The method according to any one of the preceding claims, characterized in that the pumping direction in one or more of the method steps one or more times is reversed.

9. The method according to any one of the preceding claims, characterized in that the method is carried out in a microfluidic device (700).

10. Apparatus for carrying out a purification of biological molecules, in particular of nucleic acids or proteins, with at least one pump (12; 22; 32; 52; 702) for pumping liquids and at least one device for fixing at least one filter (10; 20; 30; 50; 64; 710), characterized in that the device comprises a conduit system (11; 21; 31; 51; 701) for pumping liquids circularly over the filter.

Apparatus according to claim 10, characterized in that the means for fixing the filter (64; 712) is a filter chamber (60; 713; 813; 913), which preferably has a multilayer structure, the filter chamber (60; 713; 813, 913) for inserting, in particular, a filter membrane (64) or another filter material, in particular a particle bed.

12. The device according to claim 11, characterized in that the filter chamber (813, 913) has an extension (815, 915) for coupling ultrasound, wherein preferably the extension with a cover membrane

closed aperture (816; 916).

13. Device according to one of claims claim 10 to 12, characterized

characterized in that the device comprises at least one vented vessel (24; 34; 44; 704) for introducing fluids into the system.

14. Device according to one of claims 10 to 13, characterized in that the filter (10; 20; 30; 50; 64; 710) and / or the pump (12; 22; 32; 52; 702) and / or the optionally provided vessel (24; 34; 44; 704) and / or the conduit system (11; 21; 31; 51; 701) are heatable.

15. Device according to one of claims 10 to 14, characterized in that the device is designed as a microfluidic system (700).