DE102012219156A1 - INTEGRATED MICROFLUIDIC COMPONENT FOR ENRICHMENT AND EXTRACTION OF BIOLOGICAL CELL COMPONENTS - Google Patents

Abstract

The present invention relates to a microfluidic component, as can be used in particular in the identification of bacteria on the basis of their genetic information in the field of molecular diagnostics. Such a microfluidic component comprises at least one depletion chamber (105) with an inlet (108) for introducing a fluid to be processed and with an outlet (110) for discharging the depleted fluid, the inlet (108) and the outlet (110) being arranged in this way are that a fluid flow can flow between inlet (108) and outlet (110). Furthermore, at least one separation element (101) is provided, which delimits the depletion chamber (105) with at least one surface. At least one collecting chamber (106) receives the enriched components of the fluid to be processed, which are cleaned up by the separating element (101), the collecting chamber (106) being connected to the depletion chamber (105) via the separating element (101) in such a way that a transport between the depletion chamber (105) and the collecting chamber (106) can take place through the separating element (101).

Description

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 dissertation Paul Vulto: "A Lab on a Chip for automated RNA extraction from bacteria", Faculty of Applied Sciences of the University of Freiburg, Freiburg 2008 , described. The chip disclosed in this dissertation uses the Joule heat 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, highly negatively charged cell substance and is therefore eluted in relatively pure form by an electrophoresis gel. After elution, various detection methods, such. As a real-time PCR (real-time polymerase chain reaction), are 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 in the German patent specification DE 10 2006 050 871 B4 disclosed.

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 shows Puchberger-Enengl et al. "Microfluidic concentration of bacteria by on-chip electrophoresis", Biomicrofluidics 5 (4) December 2011 , an electrophoretic enrichment device based on an elongated channel flanked by two gel walls, behind which are specially lapped electrodes. However, this enrichment method does not allow for analysis of analyte molecules such as cells in direct association with this enrichment step.

The patent US 8,097,140 B2 discloses another method for enrichment of species to be analyzed, wherein the enrichment is presented 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 publication 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 (eg, NH ₂ -COOH or OH-edge groups). However, in this known arrangement always an actuation is necessary and it must be added to the magnetic carrier 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 is an integrated microfluidic device for purifying analyte molecules is known in which cells are split by a thermoelectric 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 given in the 41 to 44 the patent DE 10 2006 050 871 B4 are disclosed, it is assumed that the sample chamber always has only the only output that is suitable for both depleted sample solution as well as for the enriched analyte molecules is used.

Generally, there are various approaches to perform an electrical lysis. In this case, special arrangements and forms of electrodes are necessary to ensure destruction of the membrane, for example of bacterial cells, by a sufficiently strong field. In the article Lee Sang-Wook et al .: "A microcell lysis device", Sensors and Actuators 73 (1999), pages 74 to 79 For example, sawtooth-like intermeshing electrodes coated with PTFE (polytetrafluoroethylene) are shown, with which the smallest possible electrode spacing and thus a high field strength is achieved.

A similar system was later presented by another group, and in particular the low required voltage of only 8 V at a frequency of 10 kHz is of great importance for a microfluidic component with potential for point-of-care application (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 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 , known. Here a checkerboard distribution of 25 platinum electrodes on silicon is disclosed, which perform both the separation of E. coli and blood by means of dielectrophoresis, as well as a 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 the rapid, cost-effective and efficient analysis and processing of biological material, which can be fully automated by the advantageous combination of enrichment, in particular non-specific enrichment, lysis and purification 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) to unite in the simplest possible way with a lysis and purification step.

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.

Such an integrated solution with at least one separate outlet for the depleted biological sample on the one hand has the advantage that much larger sample quantities can be processed in a much shorter time than if the entire amount of liquid had to pass through the separating element. Furthermore, the components to be detected no longer leave the protected inner region of the component, so that, in particular, no damage by decomposition of the enzymes, such as, for example, RNAse, is possible. 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 corresponding electric potential difference between the Enrichment surface and, for example, the collection chamber are created.

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 separation 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 lysed 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 μRNA) from a flowing sample into a microfluidic system for lysing the cell particles and for extracting nucleic acids from the lysate is integrated. To enrich the sample, this flows through the depletion chamber 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 is thus a compact accumulation of bacteria on a surface, according to a first embodiment, for example on a hydrogel.

Advantageously, this method has an extremely low selectivity, so that also unknown and mixed cells can 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 constructed 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. Detachment of the concentrated layer from the breaker surface is possible for example by a voltage reversal. In principle, with the present inventive integrated microfluidic component all in the DE 10 2006 050 871 B4 addressed applications, in particular the extraction of messenger RNA. Other applications that use only parts of the possible processing, but can also be envisaged with the help of the arrangement according to the invention in an advantageous manner. Thus, it is possible, for example, to extract μRNA from blood plasma, specifically in an advantageous manner directly in flow mode.

• 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, 2011, Volume 11, No. 9, 1561–1700 .

In order to define the meniscus of a phase boundary when filling or emptying the microfluidic component according to the invention, phase guides can be used, as described, for example, in the laid-open European patent application EP 2 213 364 A1 or known 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, 2011, Volume 11, no. 9, 1561-1700 ,

The use of phaseguide structures makes it possible, in particular with 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 subject 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:

1 an overview of the overall concept of a microfluidic platform technology;

2 a plan view of an exemplary realization of a fully integrated microfluidic component according to a possible embodiment;

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

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

5 a schematic representation of the pulsed depletion in the arrangement of 3 ;

6 a schematic representation of a second embodiment of an integrated microfluidic component;

7 a schematic representation of the arrangement 6 in the presence of differently charged cell particles;

8th a schematic representation of the arrangement 3 accumulation of cells during flow;

9 a schematic representation of the arrangement 8th during the lysis step;

10 a schematic representation of the arrangement 8th and 9 during the separation of the lysed particles in the separation distance;

11 a schematic representation of an integrated microfluidic device with alternative electrode design;

12 the component off 11 during an electroporation step;

13 the component off 11 and 12 during separation on the separation element;

14 a schematic representation of another mode for the transformation of cells;

15 the arrangement off 14 in the next process step;

16 a representation of the transformed cells;

17 a schematic representation of the resuspension step of the transformed cells;

18 a schematic representation of an integrated microfluidic device with a second electrode pair for electroporation of cell particles on a second side;

19 a representation of the release of nucleic acids from enriched cell particles;

20 a schematic representation of the step of extraction and transfer of the nucleic acids into the collection chamber after the step of 19 ,

The present invention will be explained below in detail with reference to the drawings. One possible overall system in which the principles according to the invention are used is in 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 in the patent DE 10 2006 050 871 B1 described, that can inventive system 100 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 according to the invention 100 a biological sample 104 over a tributary 108 into a depletion chamber 105 initiated. As will be explained in more detail below, the cells to be identified accumulate in the depletion chamber 105 while the depleted biological sample through the drain 110 leave the system again. In particular, the biological cells to be detected accumulate at the interface to a separation element 101 comprising, in particular, a separation and extraction section. In a collection chamber 106 , which is also referred to as an elution chamber or detection chamber depending on their mode of operation, accumulate the enriched and purified components to be detected (for example tmRNA), which can subsequently be detected by on-chip PCR or isothermal amplification, for example via fluorescence detection.

The required amplification reagents are mixed with the purified and enriched sample components to be analyzed and after the appropriate amplification in the detection chamber 112 detected and quantified.

The present invention is directed to the in 1 focusing on components for enrichment, lysis, and purification that focuses on an analyte recovery module 114 form, which will be described below.

According to the invention are in the depletion chamber 105 Cell particles disrupted by appropriate control of electrodes, for example by lysis or electroporation. Without further processing steps, the extracts can be cleaned and processed within the "Lab on a Chip" system. As already mentioned, an enrichment of cells, virus particles or other analytically relevant particles, such as spores, generally referred to as cell particles, can be combined with a lysis and purification step with the aid of the inventive arrangement on a microfluidic scale.

As in 1 By utilizing induced membrane charges on the surface of a cell particle, an electrophoretic separation process through a gel layer is demonstrated 101 used through.

Here are first during a flow between inflow 108 and drain 110 the cell particles 104 in the depletion chamber 105 at an interface with the severing element 101 accumulated. In a subsequent step, the cell particles become in the same chamber 105 directly lysed. Subsequently, the analytes can be passed through, for example, a gel filtration section 101 be purified and eluted from the collection chamber or analyzed there directly.

That is, according to the invention, the accumulation of cell particles from a depletion chamber 105 flowing through sample combined with a microfluidic system for lysis of the cell particles and for the extraction of analyte molecules such as nucleic acids from the lysate.

2 shows a possible actual realization of a combined analyte recovery module 114 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 dissertation of Paul Vulto "A Lab on a Chip for Automated RNA Extraction from Bacteria", Faculty of Applied Sciences, Albert-Ludwigs-University Freiburg, July 2008 , to be discribed. In contrast to the known systems, however, the analyte production module according to the invention has 114 a depletion chamber 105 that have at least one inlet 108 and at least one outlet 110 for the sample flow possesses. Furthermore, the depletion chamber is adjacent 105 so to a separating element 101 (which is formed here by a gel path) that can accumulate at this interface cell particles. After enrichment, lysis of the cell particles can be performed (this procedure is described with reference to 9 explained in detail below) and in a subsequent separation and purification step, the analytes to be detected or recovered are transported by the separation element 101 through into the collection chamber 106 into it.

To facilitate the filling and emptying of the liquids, as already mentioned, phase guide elements 116 be provided. In the case of the separating element 101 serve the phaseguides 116 to facilitate filling with the gel during manufacture.

Both the enrichment, as well as the transport through the separating element 101 through and also an optionally provided lysis or electroporation of the cell particles 104 is done with the help of planar, integrated in the microsystem electrodes 118 controlled. It is an essential idea of the present invention is that by the change between an inactive and an active state and by applying different Voltages different functions can be met.

A first embodiment and a first mode of operation of the analyte recovery module 114 according to the present invention will be described below with reference to 3 and 4 explained. The analyte recovery module 114 has a depletion chamber 105 with an inlet 108 and a process 110 wherein a wall of the depletion chamber 105 is formed by a separation path, for example a gel matrix. The exit side of the gel matrix is adjacent to the collection chamber 106 , According to the invention, the analyte extraction module has 114 According to a first embodiment, three electrodes 118a to 118c of which are the electrodes filled in black 103 are active and the dotted electrode is inactive (reference numeral 102 ).

As in 3 shown, unites the module 114 an enrichment module with an extraction chip, for example for cell particles, proteins or nucleic acids. On the same chip is first, as in 3 shown during the flow at the separating element 101 an enrichment of the cell particles to be detected 104 carried out. It is to the electrodes 118a and 118c applied a voltage that is suitable to the cell particles 104 towards the interface of the gel matrix 101 to accelerate and hold on 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 the propagation of living cells, in which the flow between inflow 108 and drain 110 is stopped, an elution over the expiration 110 he follows. Usually, however, in a next step, the pervious particles to be analyzed are passed through the gel matrix 101 extracted through.

As an alternative to this one-stage extraction can also, as in 4 shown to follow a direct depletion in the flow using a free-flow electrophoresis in the DC mode. During the flow, smaller charged particles from a sample, such as free μRNA, are enriched in a plasma matrix at the interface to the extraction section and through the extraction pathway 101 through into the collection chamber 106 transported.

As in 5 Alternatively, the flow can be stopped and then a pulsed depletion into the collection chamber 106 into it. 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, a gel permeation matrix may be present in the extraction section 101 be provided so that the collection chamber 106 then first to 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 in the 6 and 7 shown. According to this embodiment, the analyte recovery module has 114 an example, centrally arranged depletion chamber 105 (which thus acts as a collection chamber), which on two sides to a respective separation element 101 . 101b borders.

By appropriate control of the electrodes 118a and 118c can be bioparticles 104 be accumulated with opposite polarities at the two gel fronts during the flow. Finally, they can be extracted directly or it can, as in 7 shown differently charged smaller particles in the free flow electrophoresis in DC mode in two directions. According to this embodiment, then two collection chambers 106a and 106b intended.

8th again shows the module assembly 3 to 5 and it will be explained below, the operation of the module in the event that an 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 118a and 118c initially charged with a DC potential, so that the charged cells at the interface to the separating element 101 attach. Then the flow is stopped and it gets between the electrodes 118b and 118c an alternating voltage is applied, which leads to the lysis of the enriched cells. That is, the inactive during the enrichment electrode 118b changes its status and becomes an active electrode 103 while the electrode 118a now an inactive electrode 102 is.

Subsequently, as soon as the lysis is completed, the control of the electrodes is again changed so that now in the separation distance 101 the various components of the lysate, such as sugars, proteins and nucleic acids, can be separated by electrophoresis. The purified nucleic acids can then be either detected or eluted in the collection chamber.

For some applications, it is better not to completely lyse the cells, but rather only with the help of an electroporation to open. For electroporation of the cell particles very high field strengths and thus preferably very small electrode distances are required. To enable these small distances, as in 11 shown, first at the interface to the separation line 101 a homogeneous and distributed over a defined distance bacterial film concentrated. In contrast to the previous embodiments, according to the present embodiment, a pair of electrodes 118b . 118d placed in the immediate vicinity of the enrichment surface and remains inactive during the enrichment step (reference numeral 102 ).

As in 12 is shown after accumulation at DC voltage between the electrodes 118a and 118c the driving of the electrodes changed so that between the electroporation electrodes 118b and 118d a sufficiently large DC voltage is applied. 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 in 13 shown, again with the help of the electrodes 118a and 118c generates an electric field which, according to the known principle of gel electrophoresis, removes the analyte molecules from the lysate and into the collection chamber 106 leads.

The analyte recovery module 114 However, in accordance with the present invention, it is also possible, conversely, to subject cells to electroporation and then to introduce nucleic acids or nanoparticles for transforming these cells into the cells. As in 14 For example, after the cell particles are accumulated on the gel matrix, a nucleic acid-containing fluid is added and prepared by an electrode for electroporation of the cell particles. 15 shows the process of electroporation, in which the nucleic acids were incorporated into the cell.

Subsequently, the electrodes 118b and 118c again inactivates the polarity of the between the electrodes 118a and 118c applied voltage is reversed compared to the accumulation and the transformed cells 104 can through the process 110 be resuspended and removed.

The principle of electroporation and subsequent analysis can also be extended in two directions according to another embodiment. This is in the 18 to 20 illustrated. According to this embodiment, the cell particles are first attached to two gel matrices 101 enriched during the flow, wherein for electroporation additional electrodes 118d . 118e are provided, so that at each enrichment surface a Porationselektrodenpaar is formed with a small electrode spacing. As in 19 shown is the outer electrode 118c used both for electroporation and for electrophoresis. Again, the nucleic acids are eluted by electroporation and then by appropriate activation of the electrodes 118a and 118c in the collection chamber 106 promoted.

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

The double electrode arrangement of 18 to 20 Of course you can also use the in 7 combined embodiment shown.

In summary, the analyte extraction module has 114 according to the present invention, 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, however, 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 several chambers, which may be separated by gels, each with specific enrichment modules and a separate sampling or analysis part may be provided.

Advantageously, the electrophoretic approach requires no moving parts, such as valves or centrifuges. This offers special advantages inventive system with respect 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 μRNA 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 after the enrichment or even during the enrichment itself.

The lysis may be as described, for example, in the international published application WO 2008/049638 A1 describe using the Joule heat generated by applied AC voltage or as mentioned by electroporation of the cell walls.

• 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 der TUHH: „Mechanischer Aufschluss von eukaryontischen Zellen“, herunterzuladen von http://www.spe.tuharburg.de/de/forschung/biopartikel.html

so genannte Nanomesseranordnungen bekannt, die in dem mikrofluidischen Bauteil gemäß der vorliegenden Erfindung integriert werden können. 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 .
• Institute for Bioprocess and Biosystems Technology of the TUHH: "Mechanical digestion of eukaryotic cells", downloadable from http://www.spe.tuharburg.de/de/forschung/biopartikel.html

So-called nano-knife arrangements are known, which can be integrated in the microfluidic component 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. 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 making the enrichment, especially a nonspecific enrichment, completely automatable with lysis and purification and with a degree of integration which also allows mobility outside the laboratory.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• DE 102006050871 B4 [0004, 0009, 0009, 0028]
• US8097140 B2 [0007]
• DE 10149803 A1 [0008]
• EP 2213364 A1 [0029]
• DE 102006050871 B1 [0052]
• WO 2008/049638 A1 [0084]

Cited non-patent literature

• Paul Vulto: "A Lab on a Chip for Automated RNA Extraction from Bacteria", Faculty of Applied Sciences, Albert-Ludwigs-University Freiburg, Freiburg 2008 [0002]
• Puchberger-Enengl et al. "Microfluidic concentration of bacteria by on-chip electrophoresis", Biomicrofluidics 5 (4) December 2011 [0006]
• Lee Sang-Wook et al .: "A micro cell lysis device", Sensors and Actuators 73 (1999), pages 74 to 79 [0010]
• Lu Hang et al .: "A microfluidic electroporation device for cell lysis", Lab on a Chip, 2005, 5, pages 23 to 29 [0011]
• 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 [0011]
• 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 [0012]
• 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 [0029]
• 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 [0029]
• Vulto, P. et al: "Phaseguides: A paradigm shift in microfluidic priming and emptying", Lab on a Chip, 2011, Volume 11, no. 9, 1561-1700 [0029]
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Claims (18)

Integrated microfluidic component comprising: 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, wherein the inlet ( 108 ) and the outlet ( 110 ) are arranged so that a fluid flow between the inlet ( 108 ) and the outlet ( 110 ) can flow; at least one separating element ( 101 ) having at least one surface the depletion chamber ( 105 ) limited; at least one collecting chamber ( 106 ) for receiving the enriched and by the separating element ( 101 ) purified components of the fluid to be processed, the collection chamber ( 106 ) over the separating element ( 101 ) so with the depletion chamber ( 105 ), that a transport between the depletion chamber ( 105 ) and the collection chamber ( 106 ) by the separating element ( 101 ) can be done through. Integrated microfluidic component according to claim 1, wherein the separating element ( 101 ) has a gel filtration path having pores of a size smaller than a diameter of the components to be enriched. Integrated microfluidic component according to claim 1 or 2, wherein the separating element ( 101 ) has a gel permeation chromatographic stretch having pores of a size greater than a diameter of the constituents to be enriched. Integrated microfluidic component according to one of the preceding claims, wherein the separating element ( 101 ) has a liquid for performing a liquid-liquid separation. An integrated microfluidic device according to any one of the preceding claims, further comprising: at least first and second electrodes for applying a first voltage generating an electric field through the electrically charged or polarizable components contained in the fluid as the fluid passes through the depletion chamber (16). 105 ) in the direction of the surface of the separating element ( 101 ) are accelerated. Integrated microfluidic component according to one of the preceding claims, wherein a controllable or permanent magnetic field and / or a pressure gradient is provided in order to remove cell particles as the fluid flows through the depletion chamber (FIG. 105 ) in the direction of the surface of the separating element ( 101 ) to accelerate. Integrated microfluidic component according to one of the preceding claims, wherein the at least one separating element ( 101 ) is provided with at least one component which interacts specifically with a component of the fluid to be enriched. Integrated microfluidic component according to one of the preceding claims, wherein the depletion chamber ( 105 ) at least one phase guide structure for guiding the meniscus of a phase boundary during filling or emptying of the microfluidic component ( 100 ) having. Integrated microfluidic component according to one of the preceding claims, wherein a flow direction of the fluid flow between the inlet ( 108 ) and the outlet ( 110 ) of the depletion chamber ( 105 ) transversely to a transport direction between the depletion chamber ( 105 ) and the collection chamber ( 106 ) runs. Integrated microfluidic component according to one of the preceding claims, wherein a plurality of depletion chambers ( 105 ) are arranged in cascade one behind the other, each separated by specific enriching gels. Integrated microfluidic component according to one of the preceding claims, wherein on two opposite sides of the depletion chamber ( 105 ) at least one separating element ( 101 ) and, associated therewith, one collecting chamber each ( 106 ) are arranged, and wherein a first and a second electrode in each case in a collecting chamber ( 106 ) are arranged so that to be enriched components of the fluid with different electrical polarity at each different separation elements ( 101 ) are enriched. An integrated microfluidic device according to any one of the preceding claims, further comprising: generates at least electrode pair for generating an electric field, which leads to the lysis or electroporation of the enriched components. The integrated microfluidic device of claim 12, wherein the at least one electrode is located at the interface between the depletion chamber (16). 105 ) and the separating element ( 101 ) is arranged. Integrated microfluidic component according to one of the preceding claims, wherein at the collecting chamber ( 106 ) is further arranged a detection unit for detecting the purified components. An integrated microfluidic device according to any one of the preceding claims, further comprising at least one reagent chamber for introducing further reagents into the collection chamber ( 106 ). Integrated microfluidic component according to one of the preceding claims, which is designed as a micromechanically produced one-piece, substantially planar chip. Use of the integrated microfluidic component ( 100 ) according to one of the preceding claims for enriching cells and / or cell particles, wherein the purified, in the at least one collecting chamber ( 106 ) include nucleic acid molecules. Use according to claim 17, wherein in the microfluidic component ( 100 ), an incubation step is further performed to proliferate the cells.

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