US20090130658A1 - Arrangement for integrated and automated dna or protein analysis in a single-use cartridge, method for producing such a cartridge and operating method for dna or protein analysis using such a cartridge - Google Patents
Arrangement for integrated and automated dna or protein analysis in a single-use cartridge, method for producing such a cartridge and operating method for dna or protein analysis using such a cartridge Download PDFInfo
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- US20090130658A1 US20090130658A1 US11/665,380 US66538005A US2009130658A1 US 20090130658 A1 US20090130658 A1 US 20090130658A1 US 66538005 A US66538005 A US 66538005A US 2009130658 A1 US2009130658 A1 US 2009130658A1
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- 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
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- 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
- B01L3/502707—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 the manufacture of the container or its components
-
- 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
- B01L3/502723—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 venting arrangements
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/11—Automated chemical analysis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
- Y10T436/2575—Volumetric liquid transfer
Definitions
- Embodiments of the invention generally relate to an arrangement for integrated and automated DNA or protein analysis in a single-use cartridge.
- a flat card in check card format is described as a cartridge.
- embodiments of the invention generally relate to the production of such a cartridge.
- embodiments of the invention also generally relate to an operating method for DNA or protein analysis using such a cartridge.
- a PCR Polymerase Chain Reaction
- amplification selective DNA multiplication
- thermocycler a PCR device which is suitable for quantitative PCR
- electro-phoresis apparatus a hybridization stage
- optical reader so-called Eppendorf tubes
- pipetting devices a refrigerated container for reagents
- At least one embodiment of the invention is directed to the implementation of an inexpensive, simply manageable, complete DNA or protein analysis process in a miniaturized cartridge.
- the following improvements compared to the laboratory method should be implemented:
- At least one embodiment of the invention is in particular based on WO 02/072262 A1 and the further state of the art mentioned there. Therein is described an analytical device with reagents stable at room temperature, dry-stored in fluid channels, which are brought into solution through the introduction of water shortly before their use as intended. At least one embodiment of the invention is further based on the unpublished DE 10 2004 021780 A1 and the unpublished DE 10 2004 021822 A1. In addition, the specific use of electrically readable detection modules is also known.
- an object of at least one embodiment of the invention is such a single-use cartridge with a system of microchannels and/or microcavities for a predefined process sequence after sample uptake, wherein the cartridge has structures to accommodate the dry reagents and devices for the implementation both of the cell disintegration on the one hand and of the PCR on the other, but also of the electrochemical detection, are allocated to these structures.
- the channels therein have different, problem-adapted structures.
- the disintegration channel advantageously has stepped cross-sections for optimal wetting with the dry reagent, while the PCR chamber and the Elisa reagent channels have pot-shaped depressions.
- the reagents and additives are already introduced as dry substances into depressions of the cartridge channels during production.
- the depressions are located at predefined intervals along the reagent channel.
- the intervals can be equidistant or particularly advantageously can be arranged in variable spacing patterns.
- the depressions can advantageously be filled with variable quantities of dry reagent. Through the combination of different amounts of dry reagent and spacing patterns of the depressions, the desired concentration profiles of the finished reagent solutions can be established.
- the magnetic beads are dispensed into the lysis channel as a suspension. On evaporation of the solvent, it is observed that the beads are drawn back into the edge region of the lysis channel and an even distribution does not result from this. Through stepped structuring of the lysis channel cross section, the magnetic beads distribute themselves across the steps and an even distribution is achieved.
- substrates with DNA-binding properties in particular the DNA-binding magnetic beads
- the lysis reagents and the magnetic beads can be contained together in a single dry matrix.
- the reagents for an ELISA assay are also present in the card.
- two reagents are needed, i.e. a labeling enzyme as the first reagent and an enzyme substrate as the second reagent.
- a detection module for the electrical detection of the hybridization processes is arranged in the cartridge.
- the detection module advantageously consists of a noble metal/plastic composite or a semiconductor-processed silicon chip with noble metal electrodes.
- Especially suitable for electric detection here are electrochemical, magnetic or piezoelectric measurement procedures.
- an input port for a whole blood sample is present in the cartridge according to at least one embodiment of the invention.
- device(s) for the addition of water are present, for example inlet ports for connection to an external water supply or an integrated water reservoir.
- dry buffer substances have defined ionic strength after the addition of water.
- device(s) for mixing of a whole blood sample with water or a buffer solution are advantageously present.
- device(s) for passing blood or blood/water mixtures or blood/buffer mixtures through the micro-channel or microcavity coated with lysis/bead/reagent are present.
- PCR for the PCR to be performed in the cartridge according to at least one embodiment of the invention during use specifically for DNA analysis, device(s) for generation of a magnetic field for immobilization of the DNA/magnetic bead complex in a PCR cavity are present.
- the PCR cavity must be capable of being suitably sealed and device(s) for thermocycling must be present.
- the cartridge it is essential that device(s) for storage of used sample material and used reagents be present, which constitute waste reservoirs.
- the device(s) must be suitable for the germproof, cell-free and particle-free venting of at least one waste reservoir.
- device(s) for the immobilization of the cartridge must be present.
- FIG. 1 shows a cartridge with an overview of individual microchannel/microcavity systems with the relevant function designations
- FIG. 2 shows a top view of a cell disintegration channel
- FIG. 3 shows the cross section through the cell disintegration channel according to FIG. 5 .
- FIG. 4 shows two alternatives for the throughflow channel cross section, shown enlarged
- FIG. 5 shows the top view of the PCR chamber in FIG. 1 .
- FIGS. 6 and 7 show the cross section through the PCR chamber according to FIG. 5 .
- FIG. 8 shows the top view of an ELISA reagent channel in FIG. 1 .
- FIGS. 9 and 10 show the cross section of the ELISA reagent channel according to FIG. 8 and
- FIGS. 11 to 23 show the top view of the cartridge according to FIG. 1 in various process states during an automated evaluation.
- FIGS. 1 to 10 are described together, and FIG. 11 to are described together.
- FIG. 1 shows a cartridge 100 for an ELISA (“Enzyme Linked Immuno Sorbent ASSAY”) test with a front view of the micro-channel or microcavity system present therein, wherein for clarity the relevant function designations are also shown.
- the cartridge 100 consists of a plastic base 101 with fluidic structures incorporated therein, which are covered by a plastic film. The structures are further described below on the basis of FIGS. 2 to 10 .
- This is connected to a channel area 110 for the cell disintegration of the sample and moreover specifically for a DNA analysis an area 120 for a PCR (Polymerase Chain Reaction) for selective DNA multiplication (amplification) in order to increase the concentration of the DNA to be detected sufficiently for it to be detectable in a third stage.
- the actual PCR chamber is sealable by valves 122 and 122 ′. The detection of the samples thus prepared, in particular according to the ELISA method, then takes place in the area 130 .
- water ports 103 to 103 ′′′ are also visible in FIG. 1 .
- water ports 103 to 103 ′′′ are also visible in FIG. 1 .
- venting ports 104 to 104 ′′′ are present.
- the cartridge 100 has in particular an input port 102 for a whole blood sample.
- at least one device for the introduction of water is present.
- An inlet port for connection to an external water source can be present, or the feed port can be connected to an integrated water reservoir.
- the microchannels or microcavities 101 to 131 are filled with dry buffer substances which ensure a defined ionic strength after the introduction of water.
- at least one device for mixing of whole blood samples and water or the buffer solution and/or at least one device for passing a flow of blood or blood-water or blood buffer mixture through a micro-channel coated with a lysis bead reagent or the microcavity are present.
- wide regions 106 , 107 , 108 and 109 are provided as reservoirs to accommodate waste. Apart from this, a region with channels 131 or 131 ′ to accommodate different ELISA reagents is present.
- reference symbols 101 again indicate the cartridge base.
- the base contains a throughflow channel 111 shaped in a particular way especially for cell disintegration (“lysis”) with step-shaped depressions 112 formed by the side edges to accommodate reagents.
- the depressions 112 have several steps with step heights from 10 to 500 ⁇ m and have an extent of ca. 1 mm and a depth of about 100 ⁇ m.
- FIGS. 5 to 7 show the structure of a PCR chamber 120 in the cartridge base 101 with a flow channel 111 .
- the valve arrangement for closure of the PCR chamber during use as intended is not shown here. It is essential that circular cylindrical depressions 124 and 124 ′ are present in the PCR chamber 120 to accommodate specific reagents 127 and 127 ′, which are needed in the implementation of the PCR.
- FIG. 7 it is also shown that a dry, storable PCR reagent 127 or 127 ′, storable at room temperature, is firstly covered with a paraffin wax layer 128 or 128 ′.
- FIGS. 8 to 10 show the design and the structure of the ELISA reagent channels 131 and 131 ′ of FIG. 1 .
- Dish-shaped depressions 132 to 132 6′ respectively are present, which are suitable to accommodate pre-dispensed and pre-apportioned quantities of reagents for the ELISA process according to FIG. 9 .
- This has already been described in detail in WO 02/072262 A1, mentioned at the outset as state of the art, to which in the present connection reference is also expressly made (“Incorporation by Reference”).
- the circular cylindrical depressions 132 to 132 6′ are shown filled with dry reagents 133 to 133 6′ .
- a first reagent embodies a labeling enzyme and a second reagent an enzyme substrate, such as is known to be needed in the hybridization of the sample, also prepared by a PCR if necessary, with specific capture probes.
- a detection zone 130 shown only schematically, different sensors for detection of biochemical reactions can be located in a module of a noble metal/plastic composite. Especially during electrochemical measurements with semiconductor-processed chips, i.e. in particular silicon-based sensors, the signals can be detected electrically and immediately further processed. Apart from the electrochemical measurement methods, magnetic and/or piezo-electric measurement methods with corresponding sensors are also possible.
- FIGS. 11 to 23 the cartridge 100 according to FIG. 1 is shown in top view, the zone of the cartridge 100 active in the analytical process being marked in each: for this the cartridge 100 is inserted into an analytical device, which is not shown in detail in the diagrams and is not an object of the present patent application.
- the evaluation is now illustrated on the basis of eleven concrete component process steps a) to m), after the cartridge has been inserted into an evaluation device with at least one device for accommodating the cartridge, and the evaluation device with the cartridge immobilized therein has been activated.
- the following component steps are involved:
- the signals arising can be read electrically and evaluated using a processor in accordance with a preset program.
- the cartridge described in detail with channels and cavities in FIG. 1 is produced from a polymeric material, such as for example polycarbonate, for example by injection molding technology.
- a polymeric material such as for example polycarbonate
- the card base 101 with structures open upwards is first produced, and the reagents are spotted into the initially open channels or cavities and then dried.
- the detection module is introduced in a suitable manner, in particular glued, into the cartridge.
- the channels and the cavities are fitted for example with an elastic film as the upper covering and is thus sealed for use as directed.
- the specific measurement method is illustrated on the basis of FIGS. 11 to 23 for one specific case of DNA analysis of a sample of whole blood.
- the use of the cartridge described is envisaged for DNA analysis on the one hand and/or protein analysis on the other, wherein, as already mentioned above, an appropriate reading device and corresponding evaluation algorithm are utilized.
- an appropriate reading device and corresponding evaluation algorithm are utilized.
- operating methods follow from this, which for the first time render the cartridge described on the basis of the examples suitable in practice for decentralized use in the context of a medical “Point of Care” application.
- electrochemical measurements firstly the rinsing of the detection chamber with an antibody solution bearing an enzyme label (ELISA reagent 1) takes place. Then the rinsing of the detection chamber with enzyme substrate (ELISA reagent 2) takes place.
- the electrochemical measurements are performed in a manner in itself known at predefinable, different temperatures and variable flow rates of the enzyme-substrate solution.
Abstract
A cartridge (card) having a system of microchannels and/or microcavities is used for automated DNA or protein analysis. In at least one embodiment, the microchannels or microcavities include geometrical structures for receiving dry reagents. For the purpose of industrial production, the cartridge is produced from a flat card support, e.g., by injection moulding. The reagents are spotted into the open channels, dried and then the channels are sealed by way of a film. A finished cartridge can thus be provided with a test sample and the fully automated measuring sequence can be initiated by inserting said cartridge into a read-out device.
Description
- This application is the national phase under 35 U.S.C. § 371 of PCT International Application No. PCT/EP2005/055303 which has an International filing date of Oct. 17, 2005, which designated the United States of America and which claims priority on German Patent Application number 10 2004 050 576.4 filed Oct. 15, 2004, the entire contents of which are hereby incorporated herein by reference.
- Embodiments of the invention generally relate to an arrangement for integrated and automated DNA or protein analysis in a single-use cartridge. Herein, a flat card in check card format is described as a cartridge. In addition, embodiments of the invention generally relate to the production of such a cartridge. Finally, embodiments of the invention also generally relate to an operating method for DNA or protein analysis using such a cartridge.
- For nucleic acid analysis, for example for the analysis of white blood cells from whole blood with the aim of answering human genome questions, firstly, in a first stage as the sample preparation step, the cells must be broken up and then the DNA thus liberated must be isolated. In a second stage, a PCR (Polymerase Chain Reaction) for selective DNA multiplication (amplification) is performed in order to increase the concentration of the DNA to be detected sufficiently for it to be detectable in a third stage.
- In the laboratory, the latter component processes are carried out separately according to the known state of the art. The three stages mentioned above each include several working steps and are carried out independently of one another with different equipment. The individual work steps are largely performed manually.
- The implementation of these steps is dependent on the presence of laboratory equipment—such as a cell disintegration apparatus, a PCR device (so-called thermocycler), possibly a PCR device which is suitable for quantitative PCR, an electro-phoresis apparatus, a hybridization stage, an optical reader, so-called Eppendorf tubes, several pipetting devices and a refrigerated container for reagents, and must be carried out by trained personnel with observation of safety procedures with regard to infection risk, waste disposal or the like. In particular, several volumetric, i.e. accurate, dispensings (pipettings) of reagent solutions must be performed. Such work steps are time-consuming and cost-intensive.
- From the state of the art, devices for biochemical analysis are known which according to WO 02/073153 A1 make use in particular of silicon-based measurement modules, which can be integrated into a chip card. Moreover, according to WO 02/072262 A1, the reagents used for the analysis are already integrated into the analysis module in dry-stored form.
- At least one embodiment of the invention is directed to the implementation of an inexpensive, simply manageable, complete DNA or protein analysis process in a miniaturized cartridge. In particular in at least one embodiment, the following improvements compared to the laboratory method should be implemented:
-
- complete integration of all substances (possibly except for water) in a closed, single-use cartridge;
- preparation of the reagents in a form stable on storage at room temperature;
- automatic implementation of all processes in the cartridge;
- no manual working steps, apart from injection of the sample to be analyzed, e.g. blood;
- no direct contact with substances hazardous to health (blood and reagent wastes remain in the cartridge);
- cartridge geometry allows efficient and rapid thermocycling;
- all detection processes should operate electrically and be easy to read;
- the cartridge used is small and inexpensive to produce.
- At least one embodiment of the invention is in particular based on WO 02/072262 A1 and the further state of the art mentioned there. Therein is described an analytical device with reagents stable at room temperature, dry-stored in fluid channels, which are brought into solution through the introduction of water shortly before their use as intended. At least one embodiment of the invention is further based on the unpublished DE 10 2004 021780 A1 and the unpublished DE 10 2004 021822 A1. In addition, the specific use of electrically readable detection modules is also known.
- In contrast to this, an object of at least one embodiment of the invention is such a single-use cartridge with a system of microchannels and/or microcavities for a predefined process sequence after sample uptake, wherein the cartridge has structures to accommodate the dry reagents and devices for the implementation both of the cell disintegration on the one hand and of the PCR on the other, but also of the electrochemical detection, are allocated to these structures. In particular, the channels therein have different, problem-adapted structures. Specifically, the disintegration channel advantageously has stepped cross-sections for optimal wetting with the dry reagent, while the PCR chamber and the Elisa reagent channels have pot-shaped depressions.
- It can thus be achieved that the introduction and preparation of the sample, the DNA amplification and the actual detection of the DNA is possible in the course of one process.
- By way of the system of geometric structures in the micro-channels or microcavities to accommodate dry reagents according to at least one embodiment of the invention, suitable conditions are obtained for DNA analysis on the one hand and protein analysis on the other. In at least one embodiment, the following features and measures are essential:
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- the reagents introduced into the microchannel or into the microcavity are dryable substances with negligible vapor pressure. As the substances are stable at room temperature, their properties for cell disintegration and/or PCR and/or detection are retained. In addition, mixtures of the substances with additives can form thin films, and the mixtures can be water-tightly covered with thin layers of paraffin wax.
- In at least one embodiment of the invention, the reagents and additives are already introduced as dry substances into depressions of the cartridge channels during production. The following advantages result from this:
-
- simple and precise application of the reagents during the production of the cartridge;
- protection of the reagents during the filling of the reagent channels, i.e. the reagents are not washed away during a so-called water flow, but are retained during the filling of the whole channel. Only after the filling of the channel do the reagent spots dissolve by diffusion processes and a homogeneous solution is produced.
- In a further example embodiment, the depressions are located at predefined intervals along the reagent channel. Here, the intervals can be equidistant or particularly advantageously can be arranged in variable spacing patterns.
- The depressions can advantageously be filled with variable quantities of dry reagent. Through the combination of different amounts of dry reagent and spacing patterns of the depressions, the desired concentration profiles of the finished reagent solutions can be established.
- For certain functions, such as for example cell disintegration in the presence of magnetic beads and lysis reagents, an even distribution of the insoluble components, i.e. the beads, in the dry reagent is necessary. For this, the magnetic beads are dispensed into the lysis channel as a suspension. On evaporation of the solvent, it is observed that the beads are drawn back into the edge region of the lysis channel and an even distribution does not result from this. Through stepped structuring of the lysis channel cross section, the magnetic beads distribute themselves across the steps and an even distribution is achieved.
- In order that a cell disintegration, a PCR and a so-called DNA/protein ELISA test can equally be performed with the cartridge according to the invention, it is advantageous that substrates with DNA-binding properties, in particular the DNA-binding magnetic beads, be present in the microchannels or microcavities. Here, the lysis reagents and the magnetic beads can be contained together in a single dry matrix. Further, the reagents for an ELISA assay are also present in the card. In particular, for the ELISA assay two reagents are needed, i.e. a labeling enzyme as the first reagent and an enzyme substrate as the second reagent.
- In particular, a detection module for the electrical detection of the hybridization processes is arranged in the cartridge. The detection module advantageously consists of a noble metal/plastic composite or a semiconductor-processed silicon chip with noble metal electrodes. Especially suitable for electric detection here are electrochemical, magnetic or piezoelectric measurement procedures.
- For the application of at least one embodiment of the invention, in particular an input port for a whole blood sample is present in the cartridge according to at least one embodiment of the invention. Moreover, device(s) for the addition of water are present, for example inlet ports for connection to an external water supply or an integrated water reservoir. In the microchannels or microcavities, dry buffer substances have defined ionic strength after the addition of water.
- In the application of at least one embodiment of the invention for the analysis of white blood cells from whole blood, device(s) for mixing of a whole blood sample with water or a buffer solution are advantageously present. At the same time, device(s) for passing blood or blood/water mixtures or blood/buffer mixtures through the micro-channel or microcavity coated with lysis/bead/reagent are present.
- Further, for the PCR to be performed in the cartridge according to at least one embodiment of the invention during use specifically for DNA analysis, device(s) for generation of a magnetic field for immobilization of the DNA/magnetic bead complex in a PCR cavity are present. For this purpose, the PCR cavity must be capable of being suitably sealed and device(s) for thermocycling must be present.
- Finally, in the cartridge according to at least one embodiment of the invention, it is essential that device(s) for storage of used sample material and used reagents be present, which constitute waste reservoirs. At the same time, the device(s) must be suitable for the germproof, cell-free and particle-free venting of at least one waste reservoir. Finally, for the reading of the cartridge in a reader device, which is not an object of at least one embodiment of the invention, device(s) for the immobilization of the cartridge must be present.
- Further details and advantages of embodiments of the invention follow from the following descriptions of the diagrams of practical examples on the basis of the drawings in combination with the patent claims. In diagrammatic form, respectively:
-
FIG. 1 shows a cartridge with an overview of individual microchannel/microcavity systems with the relevant function designations, -
FIG. 2 shows a top view of a cell disintegration channel, -
FIG. 3 shows the cross section through the cell disintegration channel according toFIG. 5 , -
FIG. 4 shows two alternatives for the throughflow channel cross section, shown enlarged, -
FIG. 5 shows the top view of the PCR chamber inFIG. 1 , -
FIGS. 6 and 7 show the cross section through the PCR chamber according toFIG. 5 , -
FIG. 8 shows the top view of an ELISA reagent channel inFIG. 1 , -
FIGS. 9 and 10 show the cross section of the ELISA reagent channel according toFIG. 8 and -
FIGS. 11 to 23 show the top view of the cartridge according toFIG. 1 in various process states during an automated evaluation. - In the figures, the same or similarly operating components have the same reference symbols. In particular,
FIGS. 1 to 10 are described together, andFIG. 11 to are described together. -
FIG. 1 shows acartridge 100 for an ELISA (“Enzyme Linked Immuno Sorbent ASSAY”) test with a front view of the micro-channel or microcavity system present therein, wherein for clarity the relevant function designations are also shown. In detail, thecartridge 100 consists of aplastic base 101 with fluidic structures incorporated therein, which are covered by a plastic film. The structures are further described below on the basis ofFIGS. 2 to 10 . - In the top view according to
FIG. 1 , there can be seen asample port 102 with adispensing section 105 connected thereto, through which liquid samples in particular for nucleic acid analysis, for example for the analysis of white blood cells from whole blood, for answering human genome questions can be introduced in a defined manner. This is connected to achannel area 110 for the cell disintegration of the sample and moreover specifically for a DNA analysis anarea 120 for a PCR (Polymerase Chain Reaction) for selective DNA multiplication (amplification) in order to increase the concentration of the DNA to be detected sufficiently for it to be detectable in a third stage. The actual PCR chamber is sealable byvalves area 130. - Also visible in
FIG. 1 arewater ports 103 to 103′″. Through these, water can be introduced into thecartridge 100 as a transport agent and solvent during the preparation of a sample. Further, ventingports 104 to 104′″ are present. - As mentioned, the
cartridge 100 has in particular aninput port 102 for a whole blood sample. In addition, at least one device for the introduction of water is present. An inlet port for connection to an external water source can be present, or the feed port can be connected to an integrated water reservoir. - In the normal case, the microchannels or
microcavities 101 to 131 are filled with dry buffer substances which ensure a defined ionic strength after the introduction of water. For blood analysis, at least one device for mixing of whole blood samples and water or the buffer solution and/or at least one device for passing a flow of blood or blood-water or blood buffer mixture through a micro-channel coated with a lysis bead reagent or the microcavity are present. - In the channel system,
wide regions channels - In each of
FIGS. 2 to 4 ,reference symbols 101 again indicate the cartridge base. The base contains athroughflow channel 111 shaped in a particular way especially for cell disintegration (“lysis”) with step-shapeddepressions 112 formed by the side edges to accommodate reagents. Here thedepressions 112 have several steps with step heights from 10 to 500 μm and have an extent of ca. 1 mm and a depth of about 100 μm. - Specifically in the presentation according to
FIG. 4 a, there arises the alternative option, with a throughflow channel with no additional depressions, of providing for the accommodation of the lysis reagents only in theregion 113 of the edges of thethroughflow channel 111. On the other hand, inFIG. 4 b, such reagents, which in particular also contain magnetic beads for the binding of the DNA liberated, are evenly distributed between thesteps 112 across thethroughflow channel 111. Magnetic beads have DNA- and protein-binding properties, if they have been appropriately pretreated. They can be coated with DNA-binding properties and if necessary also with antibodies. For the introduction of dry substances as a matrix with lysis reagent and magnetic beads, reference is in particular made to the applicant's prior DE 10 2004 021780 A1 and prior DE 10 2004 021822. -
FIGS. 5 to 7 show the structure of aPCR chamber 120 in thecartridge base 101 with aflow channel 111. The valve arrangement for closure of the PCR chamber during use as intended is not shown here. It is essential that circularcylindrical depressions PCR chamber 120 to accommodatespecific reagents FIG. 7 , it is also shown that a dry,storable PCR reagent paraffin wax layer - The correct implementation of the PCR with valve-controlled thermocycling within a cartridge is described in detail in the applicant's parallel applications DE 10 2004 050576.4 and DE 10 2004 050510.1 with the same application priority, to which in the present connection reference is expressly made (“Incorporation by Reference”). In particular, the use of magnetic beads for DNA binding and concentration of the magnetic beads with the DNA in the
PCR chamber 120 by way of controllable magnetic fields is described therein, concerning which no more detailed description will be given here. -
FIGS. 8 to 10 show the design and the structure of theELISA reagent channels FIG. 1 . Dish-shapeddepressions 132 to 132 6′ respectively are present, which are suitable to accommodate pre-dispensed and pre-apportioned quantities of reagents for the ELISA process according toFIG. 9 . This has already been described in detail in WO 02/072262 A1, mentioned at the outset as state of the art, to which in the present connection reference is also expressly made (“Incorporation by Reference”). InFIG. 10 , the circularcylindrical depressions 132 to 132 6′ are shown filled withdry reagents 133 to 133 6′. Here a first reagent embodies a labeling enzyme and a second reagent an enzyme substrate, such as is known to be needed in the hybridization of the sample, also prepared by a PCR if necessary, with specific capture probes. In thedetection zone 130, shown only schematically, different sensors for detection of biochemical reactions can be located in a module of a noble metal/plastic composite. Especially during electrochemical measurements with semiconductor-processed chips, i.e. in particular silicon-based sensors, the signals can be detected electrically and immediately further processed. Apart from the electrochemical measurement methods, magnetic and/or piezo-electric measurement methods with corresponding sensors are also possible. - In each of
FIGS. 11 to 23 , thecartridge 100 according toFIG. 1 is shown in top view, the zone of thecartridge 100 active in the analytical process being marked in each: for this thecartridge 100 is inserted into an analytical device, which is not shown in detail in the diagrams and is not an object of the present patent application. - The evaluation is now illustrated on the basis of eleven concrete component process steps a) to m), after the cartridge has been inserted into an evaluation device with at least one device for accommodating the cartridge, and the evaluation device with the cartridge immobilized therein has been activated. In detail, with reference to
FIG. 1 , the following component steps are involved: - a) Ca. 10 μl of blood are introduced as the measurement sample. 1 μl is automatically dispensed via the dispensing
capillary 105. - b) The excess blood is washed into the cavity 106 (waste 1).
- c) Next, 1 μl of blood sample is diluted with water and transferred to the
cell disintegration channel 110. There the cell disintegration (lysis) of the blood cells and the binding of the liberated DNA to the magnetic beads take place. - d) Next, the magnetic beads are transferred to the PCR chamber and collected there. A washing process takes place, the wash solution being collected in the cavity 107 (waste 2).
- e) The washing process is now concluded.
- f) Next, the
PCR chamber valves - g) During the PCR, the
ELISA reagent channel 131 which contains the enzyme substrate is simultaneously filled with water. - h) Simultaneously during the PCR reaction, the
ELISA reagent channel 131′ which contains the label enzyme is filled with water. - i) After the PCR, the
PCR chamber valves detection module 130 where the hybridization with the specific capture probes takes place (into waste 3, channel 108). - j) The enzyme substrate channel is vented into the waste channel 108 (waste 3).
- k) The label enzyme is vented into the waste channel 108 (waste 3).
- l) The label enzyme solution flows via the
detection module 130 for the labeling into the waste zone 109 (waste 4). - m) The enzyme substrate solution flows via the
detection module 130 for the enzymatic-electrochemical detection of the hybridization into the waste zone 109 (waste 4). - Thereby the analytical process is concluded. In particular in the case of electrochemical detection, the signals arising can be read electrically and evaluated using a processor in accordance with a preset program.
- The cartridge described in detail with channels and cavities in
FIG. 1 is produced from a polymeric material, such as for example polycarbonate, for example by injection molding technology. During this, thecard base 101 with structures open upwards is first produced, and the reagents are spotted into the initially open channels or cavities and then dried. The detection module is introduced in a suitable manner, in particular glued, into the cartridge. In conclusion, the channels and the cavities are fitted for example with an elastic film as the upper covering and is thus sealed for use as directed. - It is also possible to apply certain special fittings, for example as sealing materials and/or venting materials onto the
open card base 101 before the closing and finishing of the cartridge on the cover side. - The specific measurement method is illustrated on the basis of
FIGS. 11 to 23 for one specific case of DNA analysis of a sample of whole blood. In general, the use of the cartridge described is envisaged for DNA analysis on the one hand and/or protein analysis on the other, wherein, as already mentioned above, an appropriate reading device and corresponding evaluation algorithm are utilized. Defined operating methods follow from this, which for the first time render the cartridge described on the basis of the examples suitable in practice for decentralized use in the context of a medical “Point of Care” application. - In conclusion, specifically for DNA analysis, the integrated operating method for the cartridge described in detail above is once again summarized as a combination or in the sequence of the individual component steps:
-
- introduction of the sample into the cartridge
- insertion of the cartridge into the reading device
- starting of the completely automatic assay
- sample dispensing via dispensing section
- washing of the dispensing section
- dilution of the sample and introduction into lysis channel
- residence in the lysis channel
- collection of the DNA-bead complex by bead collectors in the PCR chamber
- washing of the DNA-bead complex with water
- closure of the PCR chamber
- implementation of the PCR
- during the PCR: filling of both ELISA reagent channels with water
- opening of the PCR chamber
- transport of the PCR product into detection chamber
- hybridization in the detection chamber
- venting of both ELISA reagent channels
- filling and rinsing of the detection chamber with ELISA reagent 1
- filling and rinsing of the detection chamber with ELISA reagent 2
- implementation of the electrochemical measurements.
- In the electrochemical measurements, firstly the rinsing of the detection chamber with an antibody solution bearing an enzyme label (ELISA reagent 1) takes place. Then the rinsing of the detection chamber with enzyme substrate (ELISA reagent 2) takes place. The electrochemical measurements are performed in a manner in itself known at predefinable, different temperatures and variable flow rates of the enzyme-substrate solution.
- For the protein analysis, corresponding procedures are used, but in this case the PCR is not used.
- Example embodiments being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.
Claims (48)
1. An arrangement for the integrated and automated DNA or protein analysis of a measurement sample in a single-use cartridge filled with dried reagents, the arrangement comprising:
a system of at least one of microchannels and microcavities for microfluidic process technology, present in the cartridge,
the at least one of microchannels and microcavities including predefined geometric structures to accommodate reagents, wherein
the reagents are stored ready in a storage-stable form at defined sites in the at least one of microchannels and microcavities of the cartridge; and
means for making the dry-stored reagents available for the relevant component process in suitable form.
2. The arrangement as claimed in claim 1 , wherein the structures include depressions to accommodate the dry, storage-stable reagents.
3. The arrangement as claimed in claim 2 , wherein the depressions include at least one step with step heights of 10 to 500 μm.
4. The arrangement as claimed in claim 2 , wherein the depressions have a length of ca. 1 mm and a depth of about 100 μm.
5. The arrangement as claimed in claim 3 , wherein the depressions are cylindrical, and wherein the length represents the diameter.
6. The arrangement as claimed in claim 1 , wherein the reagents introduced into the at least one of micro-channels and microcavities include the following properties:
they are dryable substances with negligible vapor pressure, which are stable at room temperature, so that the properties remain unchanged for one cell disintegration or one PCR or for the detection of biochemical quantities.
7. The arrangement as claimed in claim 1 , wherein that mixtures of the particular substance with additives form thin films which adhere to the walls.
8. The arrangement as claimed in claim 7 , wherein the substances or mixtures introduced into parts of the at least one of micro-channels and microcavities are watertightly covered with thin paraffin wax layers.
9. The arrangement as claimed in claim 1 , wherein the substances introduced into parts of the at least one of microchannels and microcavities include at least one of -DNA- and protein-binding properties.
10. The arrangement as claimed in claim 1 , wherein the substances introduced into parts of the at least one of microchannels and microcavities are magnetic beads with specific binding properties.
11. The arrangement as claimed in claim 10 , wherein the magnetic beads are coated with antibodies.
12. The arrangement as claimed in claim 10 , wherein the magnetic beads are coated with DNA-binding substances.
13. The arrangement as claimed in claim 1 , wherein lysis reagents and magnetic beads are simultaneously present, and wherein the lysis reagents and the magnetic beads are contained in a single dry matrix.
14. The arrangement as claimed in claim 1 , wherein a so-called DNA ELISA assay or protein ELISA assay is performable, and wherein a label enzyme and an enzyme substrate are present as reagents for the ELISA assay.
15. The arrangement as claimed in claim 1 , wherein a detection module for the electrical detection of the hybridization processes is present.
16. The arrangement as claimed in claim 15 , wherein the detection module consists of a noble metal/plastic composite.
17. The arrangement as claimed in claim 15 , wherein the detection module consists of a semiconductor-processed silicon chip with noble metal electrodes.
18. The arrangement as claimed in claim 15 , wherein at least one of electrochemical, magnetic and piezoelectric measurement methods are used by the module for the electrical detection.
19. The arrangement as claimed in claim 1 , wherein the cartridge includes an input port for a whole blood sample.
20. The arrangement as claimed in claim 1 , further comprising means for the introduction of water.
21. The arrangement as claimed in claim 20 , further comprising an inlet port for connection to an external water source.
22. The arrangement as claimed in claim 21 , wherein the inlet port is connected to an integrated water reservoir.
23. The arrangement as claimed in claim 1 , wherein the at least one of microchannels and microcavities are filled with dry buffer substances of defined ionic strength after addition of water.
24. The arrangement as claimed in claim 1 , further comprising means for the mixing of whole blood samples and water or the buffer solution.
25. The arrangement as claimed in claim 1 , further comprising means for passing at least one of blood, blood-water and blood-buffer mixture through the at least one of the microchannel and microcavity coated with lysis bead reagent.
26. The arrangement as claimed claim 1 , further comprising means for generating a magnetic field for the purpose of immobilizing at least one of the DNA/magnetic bead and protein/magnetic bead complex.
27. The arrangement as claimed in claim 1 , further comprising means for generating a magnetic field for the purpose of immobilizing the DNA/magnetic bead complex in a PCR cavity.
28. The arrangement as claimed in claim 21 , claim 1 , further comprising means for closure of the PCR cavity.
29. The arrangement as claimed in claim 1 , further comprising means for the thermocycling of the sample are present.
30. The arrangement as claimed in claim 1 , further comprising, in the cartridge, means for the storage of at least one of used sample material and used reagent.
31. The arrangement as claimed in claim 30 , wherein the means for the storage of at least one of used sample material and used reagents constitute waste reservoirs.
32. The arrangement as claimed in claim 1 , further comprising means for at least one of the germproof, particle- and cell-free venting of the waste reservoirs.
33. The arrangement as claimed in claim 1 , further comprising means for the immobilization of the cartridge in a reading device.
34. A method for the production of a cartridge comprising:
making from polymer, a cartridge base with at least one of channels and cavities;
spotting reagents into open channels and drying the reagents there; and
closing the at least one of channels and cavities with a film.
35. A production method as claimed in claim 34 , wherein the card base is produced by injection molding technology.
36. The production method as claimed in claim 34 , wherein special materials are applied onto the card body.
37. The production method as claimed in claim 34 , wherein, before the sealing of the card body, a detection module with measurement devices is introduced.
38. An operating method for DNA analysis in an arrangement as claimed in claim 1 , the method comprising:
introducing the sample into the cartridge;
inserting the cartridge into the reading device; and
starting a fully automatic assay.
39. The operating method as claimed in claim 38 , comprising the following steps during operation of the fully automatic assay:
sample dispensing via a dispensing section;
washing the dispensing section;
diluting the measurement sample and introducing it into the lysis channel;
having a cell disintegration take place by residence in the lysis channel;
carrying the DNA-bead complex formed into the PCR chamber by a liquid flow and holding it in the PCR chamber via a bead collector;
washing of the DNA-bead complex with water;
closing the PCR chamber is closed;
performing the PCR;
transporting, after completion of the PCR, the PCR product into the detection chamber;
having hybridization processes, with specific capture probes, take place in the detection chamber;
flushing the detection chamber with labeling enzyme;
flushing the detection chamber with enzyme substrate;
performing the electrochemical measurement; and
performing the electrochemical measurements at various temperatures and various flow rates of the enzyme-substrate solution.
40. The operating method as claimed in claim 39 , wherein, during the PCR, the ELISA reagent channels are filled with water.
41. The operating method as claimed in claim 39 , wherein, after the hybridization, both ELISA channels are vented, that next the detection chamber is firstly flushed gas bubble-free with the first ELISA reagent and then flushed gas bubble-free with the second ELISA reagent, and that the electrochemical measurement is then performed.
42. The operating method for protein analysis in an arrangement as claimed in claim 1 , the method comprising:
introducing the sample into the cartridge;
inserting the cartridge into the reading device; and
starting the fully automatic assay.
43. The operating method as claimed in claim 42 , with the following steps during operation of the fully automatic assay:
sample dispensing via a dispensing section;
washing the dispensing section;
diluting the measurement sample and transporting it into the detection chamber by a liquid flow;
having binding processes between the proteins of the measurement sample and specific capture antibodies or capture proteins take place in the detection chamber;
flushing the detection chamber with an antibody solution bearing an enzyme label (ELISA reagent 1);
flushing the detection chamber with enzyme substrate (ELISA reagent 2); and
performing the electrochemical measurements.
44. The operating method as claimed in claim 43 , wherein, after the hybridization, both ELISA channels are vented, that next the detection chamber is firstly flushed gas bubble-free with the first ELISA reagent and then flushed gas bubble-free with the second ELISA reagent, and that the electrochemical measurement is then performed.
45. The arrangement as claimed in claim 1 , wherein the dry-stored reagents are made available for the relevant component process as a liquid reagent.
46. The arrangement as claimed in claim 20 , wherein the means for the introduction of water includes an inlet port.
47. The arrangement as claimed in claim 1 , further comprising an inlet port for the introduction of water.
48. The production method as claimed in claim 36 , wherein special materials include at least one of sealing membranes and venting membranes.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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DE102004050576 | 2004-10-15 | ||
DE102004050576.4 | 2004-10-15 | ||
PCT/EP2005/055303 WO2006042838A1 (en) | 2004-10-15 | 2005-10-17 | Arrangement for integrated and automated dna or protein analysis in a single-use cartridge, method for producing such a cartridge and operating method for dna or protein analysis using such a cartridge |
Publications (1)
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US20090130658A1 true US20090130658A1 (en) | 2009-05-21 |
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US11/665,331 Active 2027-01-16 US7851227B2 (en) | 2004-10-15 | 2005-10-17 | Method for carrying out an electrochemical measurement on a liquid measuring sample in a measuring chamber that can be accessed by lines, and corresponding arrangement |
US11/665,380 Abandoned US20090130658A1 (en) | 2004-10-15 | 2005-10-17 | Arrangement for integrated and automated dna or protein analysis in a single-use cartridge, method for producing such a cartridge and operating method for dna or protein analysis using such a cartridge |
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EP (2) | EP1796838B1 (en) |
JP (1) | JP4546534B2 (en) |
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WO (2) | WO2006042734A1 (en) |
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Also Published As
Publication number | Publication date |
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JP4546534B2 (en) | 2010-09-15 |
CN101039751A (en) | 2007-09-19 |
JP2008517259A (en) | 2008-05-22 |
CN101039751B (en) | 2010-05-05 |
US20090136922A1 (en) | 2009-05-28 |
EP1807208B1 (en) | 2013-03-20 |
WO2006042838A1 (en) | 2006-04-27 |
CN101039750A (en) | 2007-09-19 |
EP1807208A1 (en) | 2007-07-18 |
US7851227B2 (en) | 2010-12-14 |
EP1796838B1 (en) | 2014-10-08 |
EP1796838A1 (en) | 2007-06-20 |
CN100534619C (en) | 2009-09-02 |
WO2006042734A1 (en) | 2006-04-27 |
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