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WO2014060998A1 - Composant microfluidique intégré pour l'enrichissement et l'extraction de composants cellulaires biologiques - Google Patents

Composant microfluidique intégré pour l'enrichissement et l'extraction de composants cellulaires biologiques Download PDF

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Publication number
WO2014060998A1
WO2014060998A1 PCT/IB2013/059450 IB2013059450W WO2014060998A1 WO 2014060998 A1 WO2014060998 A1 WO 2014060998A1 IB 2013059450 W IB2013059450 W IB 2013059450W WO 2014060998 A1 WO2014060998 A1 WO 2014060998A1
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WO
WIPO (PCT)
Prior art keywords
chamber
microfluidic component
integrated microfluidic
component according
separation element
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Application number
PCT/IB2013/059450
Other languages
German (de)
English (en)
Inventor
Gregory DAME
Sydney HAKENBERG
Hendrik HUBBE
Gerald Urban
Original Assignee
Albert-Ludwigs-Universität Freiburg
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Application filed by Albert-Ludwigs-Universität Freiburg filed Critical Albert-Ludwigs-Universität Freiburg
Publication of WO2014060998A1 publication Critical patent/WO2014060998A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44791—Microapparatus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/08—Preparation using an enricher
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column
    • G01N30/6095—Micromachined or nanomachined, e.g. micro- or nanosize
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0631—Purification arrangements, e.g. solid phase extraction [SPE]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0636—Integrated biosensor, microarrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0421—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic electrophoretic flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8827—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving nucleic acids

Definitions

  • 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.
  • a microfluidic component 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.
  • 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.
  • RNA ribonucleic acid
  • RNA is a small, strong Therefore, it is eluted in a relatively pure form by an electrophoresis gel
  • 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 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).
  • a functional coating for example NH 2 -COOH or OH-edge groups.
  • an actuation is always necessary and the magnetic carriers must be added in each case.
  • 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.
  • 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.
  • an integrated microfluidic component 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.
  • 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 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.
  • a pressure difference can also provide for the transport in the case of an alternative separating element.
  • 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.
  • the separating element has a liquid for performing a liquid-liquid separation.
  • 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.
  • a system for enriching cell particles (but also non-cell particles such as nanoparticles, toxins or ⁇ ) 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.
  • this flows through the Abrei- cationshunt of the chip.
  • 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.
  • 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.
  • this nonspecific accumulation can also be replaced or supplemented by a specific enrichment by functionalizing the accumulation surface with specifically interacting components.
  • different gel combinations for the accumulation of different particles can be used.
  • 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 Miniaturizier ashamed and cost efficiency can be increased.
  • the integrated microfluidic component according to the invention with regard to the extraction of nucleic acids by electroporation.
  • 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.
  • 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.
  • it is possible, for example, ⁇ extract from blood plasma, and in particular advantageously directly in flow mode.
  • 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,
  • 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.
  • FIG. 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 Elektroporations Marins; 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
  • FIG. 1 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.
  • a miniaturized PCR (polymerase chain reaction) analysis system 100 for biological samples, for example bacteria.
  • tmRNA transfer messenger ribonucleic acid
  • a bacterial RNA which has both messenger and transfer properties, can be advantageously used.
  • 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.
  • a biological sample 104 is introduced via an inflow 108 into a depletion chamber 105.
  • the cells to be identified accumulate in the depletion chamber 105, while the depleted biological sample leaves the system through the effluent 11.
  • 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 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.
  • cell particles are disrupted in the depletion chamber 105 by corresponding activation of electrodes, for example by lysis or electroporation.
  • the extracts within the "Lab” can be carried out with a lysis cell. and purification step are combined.
  • the cell particles 104 are accumulated in the depletion chamber 105 at an interface to the separation element 101.
  • the cell particles in the same chamber 105 are directly lysed.
  • 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 Dortmund, Freiburg
  • 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
  • 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 Aufalismssch Ritt carried a transport of the to be detected or to be obtained analytes through the separating element
  • 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.
  • 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).
  • the module 1 14 combines an enrichment module with an extraction chip, for example for cell particles, proteins or nucleic acids.
  • 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.
  • 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.
  • 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.
  • direct depletion in the flow can also be achieved with the aid of free-flow electrophoresis in the DC mode consequences.
  • smaller charged particles from a sample such as free ⁇ enriched in a plasma matrix at the interface to the extraction section and transported through the extraction section 101 into the collection chamber 106.
  • 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.
  • a gel permeation matrix may be provided in the extraction section 101, so that the collection chamber 106 then initially becomes a waste chamber.
  • the separation section usually consists of a hydrogel, for example polyacrylamide, but can be coated with separation matrices.
  • 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.
  • 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.
  • the control of the electrodes is selectively changed.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the analyte molecules can be purified without delay.
  • 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.
  • 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.
  • 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.
  • FIGS. 18 to 20 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.
  • the outer electrode 1 18c is used for both electroporation and electrophoresis.
  • 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.
  • FIGS. 18 to 20 can also be used for the cell transformation described above, in which substances are introduced into the cells.
  • FIGS. 18 to 20 can also be combined with the exemplary embodiment shown in FIG.
  • the analyte extraction module 14 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.
  • this nonspecific accumulation can also be replaced or supplemented by a specific one by positioning specifically interacting components on the accumulation surface.
  • various gel combinations may also be provided for the accumulation of different particles.
  • 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.
  • 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 ⁇ or toxins from blood plasma, advantageously directly in flow mode.
  • 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.
  • 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.
  • the cells deposit in a mostly multilayered layer during the entire sample flow over continuously.
  • 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
  • the same electrode configuration that was used for the enrichment also serves as the separation electrode pair.
  • Umpolungsvor bland 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne un composant microfluidique, utilisable notamment pour identifier des bactéries sur la base de leur information génétique dans le domaine du diagnostic moléculaire. Un composant microfluidique intégré comprend au moins une chambre d'appauvrissement (105) possédant au moins une entrée (108) servant à introduire un fluide à traiter et au moins une sortie (110) servant à évacuer le fluide appauvri, l'entrée (108) et la sortie (110) étant disposées de telle façon qu'un courant de fluide peut s'écouler entre l'entrée (108) et la sortie (110); au moins un élément séparateur (101) qui limite par au moins une surface la chambre d'appauvrissement (105); au moins une chambre de collecte (106) servant à recevoir les constituants du fluide à traiter enrichis et purifiés par l'élément séparateur (101), la chambre de collecte (106) étant reliée par le biais de l'élément séparateur (101) à la chambre d'appauvrissement (105) de façon à permettre un transport entre la chambre d'appauvrissement (105) et la chambre de collecte (106) à travers l'élément séparateur (101).
PCT/IB2013/059450 2012-10-19 2013-10-18 Composant microfluidique intégré pour l'enrichissement et l'extraction de composants cellulaires biologiques WO2014060998A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DEDE102012219156.9 2012-10-19
DE201210219156 DE102012219156A1 (de) 2012-10-19 2012-10-19 Integriertes mikrofluidisches bauteil zur anreicherung und extraktion biologischer zellbestandteile

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