WO2014060998A1 - Integrated microfluidic component for enriching and extracting biological cell components - Google Patents

Abstract

The invention relates to a microfluidic component, such as those which can be used in particular in the identification of bacteria on the basis of genetic information of the bacteria in the field of molecular diagnostics. An integrated microfluidic component comprises at least one depletion chamber (105) with at least one inlet (108) for introducing a fluid to be processed and at least one outlet (110) for discharging the depleted fluid, said inlet (108) and outlet (110) being arranged such that the fluid can flow between the inlet (108) and the outlet (110); at least one separating element (101), at least one surface of which delimits the depletion chamber (105); and at least one collection chamber (106) for receiving the enriched components of the fluid to be processed, said components having been purified by the separating element (101), wherein the collection chamber (106) is connected to the depletion chamber (105) via the separating element (101) such that a transportation between the depletion chamber (105) and the collection chamber (106) can be carried out through the separating element (101).

Description

 INTEGRATED MICROFLUIDIC COMPONENT FOR ENRICHMENT AND

 EXTRACTION OF BIOLOGICAL CELL COMPONENTS

The present invention relates to a microfluidic component, as it can be used in particular in the identification of bacteria on the basis of their genetic information in the field of molecular diagnostics. For fast, low-cost, near-patient laboratory diagnostics (also referred to as point-of-care testing, POCT), pre-treatment of samples prior to actual analysis is an essential factor. For a fully automated overall diagnostic system, this pretreatment must also be automated as far as possible, miniaturized and integrated with the detection method. The so-called "lab on a chip" technology (laboratory on a chip) represents a suitable platform for such automated analysis systems.

Such a lab on a chip with an automatic extraction of RNA (ribonucleic acid) from bacterial cells, for example, in the thesis Paul Vulto: "A Lab on a chip for automated RNA extraction from bacteria", Faculty of Applied Sciences of the Albert-Ludwigs-Universität Freiburg, Freiburg 2008. The chip disclosed in this dissertation uses the Joule thermal effect for thermoelectric lysis of the cells, so that the RNA is not degraded by RNAse enzymes, the lysis step is followed directly by an electrophoretic purification step RNA is a small, strong Therefore, it is eluted in a relatively pure form by an electrophoresis gel After the elution, various detection methods, such as a real-time PCR (real-time polymerase chain reaction), can be used to detect the RNA molecules.

The combined lysis and purification mechanism can be used to perform fully automatic sample preparation, thus meeting the requirements for POCT systems in terms of time, cost, and accuracy.

The basic principle of such a microfluidic component for purifying analyte molecules is correspondingly also disclosed in German Patent DE 10 2006 050 871 B4.

However, it has been shown that with the aid of this known arrangement, the required sensitivities often can not be achieved, and thus the required low detection limits are not met. Basically, it is useful in bioanalytics to reduce the detection limits for a measurement method by preceded by additional enrichment steps, so that the sample solution to be examined before the actual measurement contains a higher proportion of the material to be analyzed. For example, the article Puchberger-Enengl et al. "Microfluidic Concentration of Bacteria by On-Chip Electrophoresis", Biomicrofluidics 5 (4) December 201 1 An electrophoretic enrichment device based on an elongated channel flanked by two walls of gel behind which are specially coated electrodes Enrichment method no possibility to analyze analyte molecules, such as from cells in direct connection with this enrichment step.

The patent US 8,097,140 B2 discloses another method for the enrichment of species to be analyzed, wherein the enrichment is shown as a possible sample preparation or as the actual analysis. However, this known arrangement does not provide any sample solution transport transversely to the gel surface, so that the enrichment is limited by the passage through the gel.

The published patent application DE 101 49 803 A1 relates to a method for non-specific accumulation of bacterial cells by means of cationic polymers and magnetic carriers. These magnetic carriers have a functional coating (for example NH ₂ -COOH or OH-edge groups). However, in this known arrangement, an actuation is always necessary and the magnetic carriers must be added in each case. Furthermore, the bacterial polymer solution has to be pipetted up and down for thorough mixing. This contradicts the desired principle of a fully automatic flow analysis.

From the patent DE 10 2006 050 871 B4 an integrated microfluidic device for purifying analyte molecules is known in which cells are split by a thermoelectric see AC lysis and the released analytes are separated by gel electrophoresis. For example, as lysis and purification take place in the same integrated microfluidic system, in the detection of RNA, the time in which the susceptible RNA-degrading RNase molecule is exposed to molecules can be minimized, thus preventing RNA degradation , However, this document does not give any specific teaching as to how additional enrichment could be realized in this integrated concept. In particular, in conjunction with the arrangements shown in Figures 41 to 44 of the patent DE 10 2006 050 output, which is used for both depleted sample solution as well as for the enriched analyte molecules.

Generally, there are various approaches to perform an electrical lysis. In this case, special arrangements and forms of electrodes are necessary in order to ensure destruction of the membrane, for example of bacterial cells, by means of a sufficiently strong field. In the article Lee Sang-Wook et al.: "A micro cell lysis device", Sensors and Actuators 73 (1999), pages 74 to 79, for example sawtooth interlocking PTFE (polytetrafluoroethylene) coated electrodes are shown with which as low as possible A similar system was later presented by another group, in particular the low required voltage of only 8 V at a frequency of 10 kHz for a microfluidic component with potential for point-of-care application is of great importance (see, for example, Lu Hang et al.: "A microfluidic electroporation device for cell lysis", Lab on a Chip, 2005, 5, pages 23 to 29). Another method of electronic lysis is known in the article Cheng Jing et al.: "Preparation and hybridization analysis of DNA / RNA from E coli on microfabricated bioelectronic chips" Nature Biotechnology, Volume 16, June 1998, pages 541 to 546. Here discloses a checkerboard distribution of 25 platinum electrodes on silicon that perform both separation of E. coli and blood by dielectrophoresis and subsequent lysis.From the article Liu RH et al., Self-Contained, Fully Integrated Biochip for Sample Preparation, Polymerase Chain Reaction Amplification, and DNA Microarray Detection, Analytical Chemistry, Volume 76, no. 7, April 1, 2004, pages 1824 to 1831, a fully integrated DNA analysis system is known. However, this known system uses numerous actuated elements such as valves, electrochemical and thermo-pneumatic pumps, piezoelectric actuators, control of immunomagnetic particles, etc. Furthermore, the fact that enriched with the aid of immunomagnetic particles means that only a highly selective enrichment can be performed.

The object underlying the present invention is therefore to provide a system for rapid, cost-effective and efficient analysis and processing of biological material, which is achieved by the advantageous combination of enrichment, in particular unspecific enrichment, lysis and purification fully automatable and of a degree of integration that allows mobility outside of a laboratory. This object is solved by the subject matter of the independent patent claims. Advantageous developments of the present invention are the subject of the dependent claims.

The present invention is based on the idea of microfluidic scale on a chip enrichment of cells, virus particles, spores or other analytically relevant particles (in the following is simplified spoken of cell particles) in the simplest possible way with a lysis and purification step combine.

In particular, an integrated microfluidic component according to the present invention comprises at least one depletion chamber with at least one inlet for introducing a fluid to be processed and at least one outlet for discharging a depleted fluid. The inlet and the outlet are arranged so that a fluid flow can flow between the inlet and the outlet. Furthermore, at least one separating element is provided which delimits the depletion chamber with at least one surface. A collection chamber is provided for receiving the enriched and purified by the separating element components of the fluid to be processed. The collection chamber is connected via the separation element with the depletion chamber, that a transport between the depletion chamber and the collection chamber can be carried through the separation element.

On the one hand, such an integrated solution with at least one separate outlet for the separated biological sample has the advantage that considerably larger sample quantities can be processed in a significantly shorter time than if the entire quantity of liquid had to pass through the separating element. Furthermore, the components to be detected no longer leave the protected inner area of the component, so that, in particular, no damage is possible by decomposing the enzymes, for example RNAse. It is also possible to parallelize the processes more intensively, since, for example, the separation and lysis can be carried out continuously and subsequent processes are operated independently thereof. A particular advantage is that with the approach, a direct processing while avoiding dead volumes can be performed. The separation element can comprise, for example, a gel filtration section, and the enrichment at a surface of this gel filtration section delimiting the reduction chamber can be carried out by means of an electrophoretic separation. The membrane charges on the surface of the cell particles are used by ing electric potential gradient between the enrichment surface and, for example, the collection chamber are applied.

Alternatively, however, a pressure difference can also provide for the transport in the case of an alternative separating element. In addition, a controllable or permanent magnetic field can be used in combination with magnetic beads on the cell particles in order to effect the required deflection of the cell particles.

The separating element may have pores of a size which is smaller than the diameter of the components to be enriched, such as bacterial cells. Before the actual purification, the bacterial cells can then be destroyed or opened to produce sufficiently small analyte elements.

Alternatively, however, it is also possible to provide a gel permeation chromatography section which has pores of a size which is greater than a diameter of the components to be enriched.

Depending on the application, it may also be provided that the separating element has a liquid for performing a liquid-liquid separation. In any case, the cell particles are accumulated on a wall of the separation element, for example the gel filtration section, in the depletion chamber and can be directly lyzed in the same chamber. Subsequently or during this time, the analyte particles obtained can be purified directly via the gel filtration section and eluted or analyzed directly.

According to the present invention, a system for enriching cell particles (but also non-cell particles such as nanoparticles, toxins or μΡιΝΑ) from a flowing sample in a microfluidic system for lysis of the cell particles and for the extraction of nucleic acids from the lysate is integrated. To enrich the sample, this flows through the Abrei- cationskammer of the chip. According to an advantageous embodiment, an electric field is generated in this depletion chamber via integrated electrodes. Due to their membrane charge, the cell particles are nonspecifically deflected from the direction of the flow and accumulate on the surface of the separation element, for example on a polyacrylamide gel. Thus, advantageously, a homogeneously distributed layer of bacteria can be produced over the entire length of the interface to the separating element. The result of the enrichment tion is thus a compact accumulation of bacteria on a surface, according to a first embodiment, for example on a hydrogel.

This method advantageously has an extremely low selectivity, so that unknown and mixed cells can also be enriched for a total analysis in a single step.

Alternatively, however, this nonspecific accumulation can also be replaced or supplemented by a specific enrichment by functionalizing the accumulation surface with specifically interacting components. Furthermore, different gel combinations for the accumulation of different particles can be used. In particular, several chambers, which may be separated by gels, can be cascaded with specific enrichment modules and own sampling and analysis devices.

The main advantage of the electrophoretic enrichment and separation is that no moving parts such as valves or centrifuge constructions are required and thus both the reliability as well as the Miniaturizierbarkeit and cost efficiency can be increased.

Particular advantages are provided by the integrated microfluidic component according to the invention with regard to the extraction of nucleic acids by electroporation. With such an arrangement, a very compact, relatively homogeneous bacterial accumulation is produced at the boundary to the separating element, for example a gel border. This achieves a lower electrode separation, which is particularly advantageous for this type of nucleic acid recovery (lysis in general) and electroporation. A detachment of the concentrated layer from the separating element surface is possible for example by a voltage reversal. In principle, all the applications mentioned in DE 10 2006 050 871 B4, in particular the extraction of messenger RNA, can be covered with the present inventive integrated microfluidic component. Other applications which use only parts of the possible processing, however, can also advantageously be envisaged with the aid of the arrangement according to the invention. Thus, it is possible, for example, μΡιΝΑ extract from blood plasma, and in particular advantageously directly in flow mode.

In order to define the meniscus of a phase boundary when filling or emptying the microfluidic component according to the invention, phaseguides can be used, as are known, for example, from the published European patent application EP 2 213 364 A1 or from the following articles:

Vulto, P. et al.: "A microfluidic appproach for high efficiency extraction of low molecular weight RNA" Lab on a Chip, 2010, Volume 10, No. 5, 610-616, Vulto, P. et al. Microfluidic Channel fabrication in dry film resist for production and prototyping of hybrid chips ", Lab on a Chip, 2005, Volume 5, no. 2, 158 - 162,

Vulto, P. et al: "Phaseguides: A paradigm shift in microfluidic priming and emptying", Lab on a Chip, 201 1, Volume 1 1, No. 9, 1561-1700.

The use of phaseguide structures makes it possible, in particular for very small dimensions of the individual chambers, to carry out a complete, complete and bubble-free filling or a complete emptying.

With reference to the advantageous embodiments shown in the accompanying drawings, the present invention will be explained in more detail below. Similar or corresponding details of the article according to the invention are provided with the same reference numerals. Furthermore, individual features or combinations of features from the embodiments shown and described can be considered as stand alone, inventive or inventive solutions. Show it:

Figure 1 is an overview of the overall concept of a microfluidic platform technology; FIG. 2 shows a plan view of an exemplary realization of a completely integrated microfluidic component according to a possible embodiment;

FIG. 3 a schematic representation of a microfluidic component with an accumulation of cells on a gel front according to a first embodiment;

Figure 4 is a schematic representation of the arrangement of Figure 3 during direct depletion with free flow electrophoresis;

Figure 5 is a schematic representation of the pulsed depletion in the arrangement of Figure 3; a schematic representation of a second embodiment of an integrated microfluidic component; a schematic representation of the arrangement of Figure 6 in the presence of differently charged cell particles; a schematic representation of the arrangement of Figure 3 with accumulation of cells during the flow; a schematic representation of the arrangement of Figure 8 during the lysis step; a schematic representation of the arrangement of Figure 8 and 9 during the separation of the lysed particles in the separation distance; a schematic representation of an integrated microfluidic device with alternative electrode design; the component of Figure 1 1 during a Elektroporationsschritts; the component of Figure 1 1 and 12 during the separation of the separating element; a schematic representation of another mode for the transformation of cells; the arrangement of Figure 14 in the next step; a representation of the transformed cells; a schematic representation of the resuspension step of the transformed cells; a schematic representation of an integrated microfluidic device with a second electrode pair for electroporation of cell particles on a second side; a representation of the release of nucleic acids from enriched cell particles; FIG. 20 shows a schematic representation of the step of extraction and transfer of the nucleic acids into the collecting chamber after the step of FIG. 19.

The present invention will be explained below in detail with reference to the drawings. A possible overall system in which the principles according to the invention are used is shown in FIG. 1 using the example of a miniaturized PCR (polymerase chain reaction) analysis system 100 for biological samples, for example bacteria. For the detection and especially for the genomic identification of bacteria, tmRNA (transfer messenger ribonucleic acid), a bacterial RNA which has both messenger and transfer properties, can be advantageously used. As described in the patent DE 10 2006 050 871 B1, the system 100 according to the invention can be used for example by means of an integrated on-chip PCR for the identification of bacteria by means of the detection of tmRNA.

In particular, in the overall system 100 according to the invention, a biological sample 104 is introduced via an inflow 108 into a depletion chamber 105. As will be explained in more detail below, the cells to be identified accumulate in the depletion chamber 105, while the depleted biological sample leaves the system through the effluent 11. In particular, the biological cells to be detected accumulate at the interface with a separation element 101, which in particular comprises a separation and extraction section. The enriched and purified components to be detected (for example, tmRNA) are collected in a collection chamber 106, which is also referred to as an elution chamber or detection chamber, depending on their mode of operation, which are subsequently detected by means of on-chip PCR or isothermal amplification, for example via fluorescence detection can.

The required amplification reagents are mixed with the purified and enriched sample components to be analyzed and detected and quantified after the corresponding amplification in the detection chamber 12.

The present invention focuses on the enrichment, lysis and purification components detailed in Figure 1 which form an analyte recovery module 14, which will be described below. According to the invention, cell particles are disrupted in the depletion chamber 105 by corresponding activation of electrodes, for example by lysis or electroporation. Without further processing steps, the extracts within the "Lab As already mentioned, with the aid of the arrangement according to the invention on the microfluidic scale on-chip, an enrichment of cells, virus particles or other analytically relevant particles such as spores, generally referred to as cell particles, can be carried out with a lysis cell. and purification step are combined.

As shown in Figure 1, by utilizing induced membrane charges on the surface of a cell particle, an electrophoretic separation method through a gel layer 101 is utilized.

In this case, first during a throughflow between inflow 108 and outflow 10, the cell particles 104 are accumulated in the depletion chamber 105 at an interface to the separation element 101. In a subsequent step, the cell particles in the same chamber 105 are directly lysed. Subsequently, the analytes can be purified through, for example, a gel filtration section 101 and eluted from the collection chamber or analyzed there directly. That is, according to the invention, the enrichment of cell particles from a sample flowing through the depletion chamber 105 is combined with a microfluidic system for lysing the cell particles and for extracting analyte molecules such as nucleic acids from the lysate.

FIG. 2 shows a possible actual realization of a combined analyte recovery module 14 according to the present invention. The production of such a microfluidic chip can be carried out according to the micromechanical principles described, for example, in the already mentioned Paul Vulto dissertation "A Lab on a Chip for Automated RNA Extraction from Bacteria", Faculty of Applied Sciences of the Albert-Ludwigs-University Freiburg, Freiburg However, in contrast to the known systems, the analyte production module 1 14 according to the invention has a depletion chamber 105 which has at least one inlet 108 and at least one outlet 10 for the sample flow, and the depletion chamber 105 also adjoins a separation element 101 (which is formed here by a gel line), that cell particles can accumulate at this interface After lysing, lysis of the cell particles can be carried out (this process will be explained in detail with reference to Figure 9) and in a subsequent separation and separation Aufreinigungssch Ritt carried a transport of the to be detected or to be obtained analytes through the separating element 101 into the collection chamber 106 inside. In order to facilitate the filling and emptying of liquids, as already mentioned, phase guide elements 1 16 may be provided. In the case of the separating element 101, the Phaseguides 1 16 serve to facilitate the filling with the gel during manufacture.

Both the enrichment, as well as the transport through the separating element 101 and also an optionally provided lysis or electroporation of the cell particles 104 is controlled by means of planar, integrated in the microsystem electrodes 1 18. In this case, an essential idea of the present invention is that different functions can be fulfilled by changing between an inactive and an active state and by applying different voltages. A first embodiment and a first mode of operation of the analyte recovery module 14 according to the present invention will be explained below with reference to FIGS. 3 and 4. The analyte recovery module 14 has a depletion chamber 105 with an inlet 108 and a drain 110, wherein a wall of the depletion chamber 105 is formed by a separation line, for example a gel matrix. According to a first embodiment, the analyte extraction module 14 according to the invention has three electrodes 11a to 18c, of which the electrodes 103 filled in black are in each case active and the dotted electrode is inactive (reference numeral FIG 102).

As shown in FIG. 3, the module 1 14 combines an enrichment module with an extraction chip, for example for cell particles, proteins or nucleic acids. First, as shown in FIG. 3, an enrichment of the cell particles 104 to be detected is carried out on one and the same chip during the flow through the separation element 101. In this case, a voltage is applied to the electrodes 1 18a and 1 18c which is suitable for accelerating the cell particles 104 in the direction of the interface of the gel matrix 101 and holding them there. According to a first mode of operation of the arrangement according to the invention, it can be provided that the gel matrix 101 serves only as a holding surface for the cell particles to be examined, and after an optionally provided sufficient incubation for propagating living cells, in which the flow between inflow 108 and outflow 1 10 stopped is, an elution on the process 1 10 takes place. Normally, however, in a next step, the pervious particles to be analyzed are extracted through the gel matrix 101.

As an alternative to this one-stage extraction, as shown in FIG. 4, direct depletion in the flow can also be achieved with the aid of free-flow electrophoresis in the DC mode consequences. During the flow, smaller charged particles from a sample, such as free μΡιΝΑ enriched in a plasma matrix at the interface to the extraction section and transported through the extraction section 101 into the collection chamber 106. As shown in FIG. 5, however, alternatively the flow can also be stopped and then a pulsed depletion into the collecting chamber 106 can take place. This can also be done without electrical current flow. On the other hand, if one wishes to analyze the larger components, for example proteins, and separate off smaller components thereof, then a gel permeation matrix may be provided in the extraction section 101, so that the collection chamber 106 then initially becomes a waste chamber. Otherwise, the separation section usually consists of a hydrogel, for example polyacrylamide, but can be coated with separation matrices.

An advantageous development of this concept is shown in FIGS. 6 and 7. According to this embodiment, the analyte extraction module 1 14 has an example, centrally arranged depletion chamber 105 (which thus acts as a collection chamber), which borders on two sides of a respective separation element 101 a, 101 b.

By appropriate activation of the electrodes 18a and 18c, bioparticles 104 with opposite polarities can be accumulated on the two gel fronts during the flow. Finally, they can be extracted directly or, as shown in FIG. 7, differently charged smaller particles can be guided in a bidirectional manner with free-flow electrophoresis in the DC mode in two directions. According to this embodiment, two collection chambers 106a and 106b are then provided.

FIG. 8 again shows the module arrangement from FIGS. 3 to 5, and the mode of operation of the module will be explained below for the case in which additional lysis of the cell particles 104 is carried out.

According to the invention, the control of the electrodes is selectively changed. In particular, the electrodes 1 18a and 1 18c are first supplied with a DC potential, so that the charged cells are deposited at the interface to the separation element 101. Subsequently, the flow is stopped and an alternating voltage is applied between the electrodes 1 18b and 1 18c, which leads to the lysis of the enriched cells. That is, the inactive electrode 1 18b changes during the enrichment Status and becomes an active electrode 103, while the electrode 1 18a is now an inactive electrode 102.

Subsequently, as soon as the lysis has been completed, the activation of the electrodes is again changed, so that now in the separation path 101, the various constituents of the lysate, such as sugars, proteins and nucleic acids, can be separated electrophoretically. The purified nucleic acids can then be either detected or eluted in the collection chamber.

For some applications, it is better not to lyse the cells completely, but to open only by means of electroporation. For electroporation of the cell particles very high field strengths and thus preferably very small electrode distances are required. In order to enable these small distances, as shown in FIG. 11, a homogeneous bacterial film distributed over a defined distance is first concentrated at the interface to the separation section 101. In contrast to the previous embodiments, according to the present embodiment, an electrode pair 1 18b, 1 18d is placed in the immediate vicinity of the enrichment surface and remains inactive during the enrichment step (reference numeral 102).

As shown in FIG. 12, after enrichment with DC voltage between the electrodes 1 18a and 1 18c, the activation of the electrodes is changed so that a sufficiently large DC voltage is applied between the electroporation electrodes 1 18b and 1 18d. In addition to the beam-shaped electrode geometries shown here, other geometries can be used in an advantageous manner, such as a sawtooth structure, which generates the required extremely high field strengths in the tips by bundling the field lines.

When the cells have been sufficiently lysed or have delivered a sufficient portion of their contents to the lysate through the generated pores, the analyte molecules can be purified without delay. For this purpose, as shown in FIG. 13, an electric field is once again generated with the aid of the electrodes 1 18a and 1 18c which, according to the known principle of gel electrophoresis, removes the analyte molecules from the lysate and leads them into the collecting chamber 106. However, the analyte production module 14 according to the present invention can also be used conversely to subject cells to electroporation and then to introduce nucleic acids or nanoparticles into the cells for transforming these cells. smuggle. As shown in Figure 14, after the cellular particles are accumulated on the gel matrix, for example, a nucleic acid-containing fluid is added and prepared by an electrode for electroporation of the cell particles. FIG. 15 shows the process of electropuration in which the nucleic acids were incorporated into the cell. Subsequently, the electrodes 1 18b and 1 18c are inactivated again, the polarity of the voltage applied between the electrodes 18a and 18c is reversed in comparison with the enrichment, and the transformed cells 104 can be resuspended and drained through the process.

The principle of electroporation and subsequent analysis can also be extended in two directions according to another embodiment. This is illustrated in FIGS. 18 to 20. According to this embodiment, the cell particles are first enriched in two gel matrices 101 during the flow, wherein additional electrodes 1 18d, 1 18e are provided for the electroporation so that a pair of small electrode spacing pair of electrodes is formed on each enrichment surface. As shown in Figure 19, the outer electrode 1 18c is used for both electroporation and electrophoresis. Again, the nucleic acids are dissolved out by electroporation and then conveyed into the collection chamber 106 by activating the electrodes 1 18a and 18c accordingly.

Of course, the electrode arrangement of FIGS. 18 to 20 can also be used for the cell transformation described above, in which substances are introduced into the cells.

Of course, the double electrode arrangement of FIGS. 18 to 20 can also be combined with the exemplary embodiment shown in FIG.

In summary, the analyte extraction module 14 according to the present invention has a depletion chamber in which an electric field is generated via integrated electrodes. Due to their membrane charge, the cell particles are nonspecifically deflected from the direction of the flow between inflow and outflow and accumulate on the surface of a separation element, for example on a polyacrylamide gel. This produces a homogeneously distributed layer of bacteria along the entire length of the gel interface. The result of this enrichment is a compact accumulation of bacteria on a defined surface, such as a hydrogel. As mentioned, this enrichment has a low selectivity, so that even unknown and mixed cells can be enriched for a total analysis in a single step. Of course, this nonspecific accumulation can also be replaced or supplemented by a specific one by positioning specifically interacting components on the accumulation surface. In addition, various gel combinations may also be provided for the accumulation of different particles. In particular, a cascading, not shown in the figures, of a plurality of chambers, which may be separated by gels, may be provided, each with specific enrichment modules and a separate sampling or analysis part.

Advantageously, the electrophoretic approach requires no moving parts, such as valves or centrifuges. The system according to the invention offers particular advantages with regard to the extraction of nucleic acids by electroporation. With this arrangement, a very compact, relatively homogeneous bacteria accumulation is produced at the gel border or other surface. This allows a low, for this type of nucleic acid recovery and in particular the electroporation advantageous electrode spacing. A detachment of the concentrated layer from the gel boundary by a voltage reversal is possible. Alternatively, only a part of the possible processing can be carried out, for. B. for extracting μΡιΝΑ or toxins from blood plasma, advantageously directly in flow mode. In its basic embodiment, the system has at least one chamber, which is delimited at least by a gel surface, whose pores are smaller than the cell diameter, and has at least one inlet and outlet channel. The channels form a flow-through system through which it is possible to enrich even samples of multiples of the chamber volume. For enrichment, an electric field is generated via the electrodes, so that cells / particles are accelerated out of the sample by their charge in the direction of the gel boundary. There, the cells deposit in a mostly multilayered layer during the entire sample flow over continuously. By providing additional gel boundaries, cells of reversed polarity can also be enriched and, if necessary, a separation of these two basic cell types can be achieved. The enriched cells are lysed according to the invention directly following the enrichment or even during the enrichment itself. The lysis can be carried out, as described for example in the international published application WO 2008/049638 A1, using the Joule heat generated by applied alternating voltage or as mentioned by electroporation of the cell walls. Furthermore, other suitable methods for lysing the cell particles according to the present invention may be used. So z. B. from the publications

Dino Di Carlo, Ki-Hun Jeong and Luke P. Lee: "Reagentless mechanical cell lysis by nanoscale barbs in microchannels for sample preparation", Lab Chip, 2003, 3, 287-291, Matthias Wurm and An-Ping Zeng: "Mechanical Disruption of mammalian cells in a microfluidic system and its numerical analysis based on computational fluid dynamics ", Lab Chip, 2012, 12, 1071,

Institut für Bioprozess- und Biosystemtechnik of the TUHH: "Mechanical digestion of eukaryotic cells", download from http://www.spe.tu-harburg.de/de/forschung/biopartikel.html so-called nanometer device arrangements known in the microfluidic component can be integrated according to the present invention.

Subsequently, according to the invention, a purification of the lysed components, a subsequent detection of the biomolecules or else an elution or other further processing of the extracts takes place. In this case, preferably the same electrode configuration that was used for the enrichment also serves as the separation electrode pair.

Umpolungsvorgänge the electrode potentials can also be used in an advantageous manner to loosen the enriched cell particle layer in order to avoid clumping or clogging. Of course, however, at least one corresponding pump, in particular a micropump, and at least one valve may be provided in order to build up a suitable pressure gradient.

Thus, the system according to the invention allows a rapid cost-effective and efficient analysis of biological material by the enrichment, especially a nonspecific enrichment, with lysis and purification completely automated and with a level of integration that allows mobility outside the laboratory.

Claims

Integrated microfluidic component comprising: at least one depletion chamber (105) with at least one inlet (108) for introducing a fluid to be processed and with at least one outlet (1 10) for discharging the depleted fluid, the inlet (108) and the outlet (1 10) are arranged so that a fluid stream can flow between the inlet (108) and the outlet (1 10); at least one separation element (101) which delimits the depletion chamber (105) with at least one surface; at least one collection chamber (106) for receiving the enriched components of the fluid to be processed that have been purified by the separation element (101), the collection chamber (106) being connected to the depletion chamber (105) via the separation element (101) in such a way that transport between the depletion chamber (105) and the collecting chamber (106) can take place through the separation element (101). Integrated microfluidic component according to claim 1, wherein the separation element (101) has a gel filtration section with pores of a size that is smaller than a diameter of the components to be enriched. Integrated microfluidic device according to claim 1 or 2, wherein the separation element (101) has a gel permeation chromatography section with pores of a size that is larger than a diameter of the components to be enriched. Integrated microfluidic component according to one of the preceding claims, wherein the separation element (101) has a liquid for carrying out a liquid-liquid separation. Integrated microfluidic component according to one of the preceding claims, further comprising: at least a first and a second electrode for applying a first voltage which generates an electric field through which electrically charged or polarizable components contained in the fluid flow as the fluid flows through the separation. cherungskammer (105) can be accelerated towards the surface of the separation element (101). Integrated microfluidic component according to one of the preceding claims, wherein a controllable or permanent magnetic field and / or a pressure gradient is provided to accelerate cell particles as the fluid flows through the depletion chamber (105) towards the surface of the separation element (101). 7. Integrated microfluidic component according to one of the preceding claims, wherein the at least one separation element (101) is provided with at least one component which specifically interacts with a component of the fluid to be enriched. 8. Integrated microfluidic component according to one of the preceding claims, wherein the depletion chamber (105) has at least one phase guide structure for guiding the meniscus of a phase boundary when filling or emptying the microfluidic component (100). 9. Integrated microfluidic component according to one of the preceding claims, wherein a flow direction of the fluid stream between the inlet (108) and the outlet (1 10) of the depletion chamber (105) is transverse to a transport direction between the depletion chamber (105) and the collection chamber ( 106) runs. 10. Integrated microfluidic component according to one of the preceding claims, wherein a plurality of depletion chambers (105) are arranged cascaded one behind the other, each of which is separated from one another by specifically enriching gels. 1 1 . Integrated microfluidic component according to one of the preceding claims, wherein at least one separation element (101) and, connected thereto, one collecting chamber (106) are arranged on two opposite sides of the depletion chamber (105), and wherein a first and a second electrode are each in a collecting chamber (106) are arranged, so that components of the fluid to be enriched with different electrical polarity are enriched on different separation elements (101). 12. Integrated microfluidic component according to one of the preceding claims, further comprising: at least pair of electrodes are generated to generate an electric field that leads to the lysis or electroporation of the enriched components. 13. Integrated microfluidic component according to claim 12, wherein the at least one electrode is arranged at the interface between the depletion chamber (105) and the separation element (101). 14. Integrated microfluidic component according to one of the preceding claims, wherein a detection unit for detecting the purified components is further arranged on the collecting chamber (106). 15. Integrated microfluidic component according to one of the preceding claims, further comprising at least one reagent chamber for feeding further reagents into the collection chamber (106). 16. Integrated microfluidic component according to one of the preceding claims, which is designed as a micromechanically produced one-piece, essentially flat chip. 17. Use of the integrated microfluidic component (100) according to one of the preceding claims for enriching cells and/or cell particles, wherein the purified components obtained in the at least one collection chamber (106) comprise nucleic acid molecules. 18. Use according to claim 17, wherein an incubation step is further carried out in the microfluidic component (100) in order to multiply the cells.