EP1807208A1 - 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 cartridgeInfo
- Publication number
- EP1807208A1 EP1807208A1 EP05801513A EP05801513A EP1807208A1 EP 1807208 A1 EP1807208 A1 EP 1807208A1 EP 05801513 A EP05801513 A EP 05801513A EP 05801513 A EP05801513 A EP 05801513A EP 1807208 A1 EP1807208 A1 EP 1807208A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- arrangement according
- cartridge
- dna
- reagents
- pcr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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
-
- 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
- the invention relates to an arrangement for integrated and automated DNA or protein analysis in a single-use cartridge.
- Cartridge is a flat card in credit card format.
- the invention contemplates the production of such a cartridge.
- the invention also relates to an operating method for DNA or protein analysis using such a cartridge.
- a PCR polymerase chain reaction
- amplification selective DNA amplification
- the object of the invention is the realization of a low-cost, easy-to-handle, complete DNA or protein analysis process in a miniaturized cartridge.
- the following improvements compared to the laboratory method should be realized: - A complete integration of all substances (possibly except
- the cartridge used is small and inexpensive to produce.
- the invention is based, in particular, on WO 02/072262 A1 and the further prior art mentioned therein.
- an analysis device with dry-stored in fluid channels, stable at room temperature reagents is described, which are brought into solution by supplying water shortly before their intended use.
- the invention is furthermore based on the unpublished DE 10 2004 021780 A1 and the unpublished DE 10 2004 021822 A1.
- the subject of the invention is such a disposable cartridge with a system of microchannels and / or microcavities for a given process flow after sample taking, the cartridge having structures for receiving the dry reagents and these structures means for performing both the cell digestion on the one hand and the PCR on the other hand, but also the electrochemical detection are associated.
- the channels have different, problem-adapted structures. Spe ⁇ essential the digestion channel advantageously has stepped cross sections for optimum wetting of the dry reagent, while the PCR chamber and the Elisa reagent channels have potty-shaped wells.
- the inventive system of the geometric structures ren in microchannels or microcavities for receiving dry reagents to provide suitable conditions for DNA analysis of a hand and the other hand, protein analysis ⁇ .
- the following features and measures are essential:
- the changes introduced in the microchannel or the microcavity reagents are dryable substances with vernachläs ⁇ sigbarem vapor pressure.
- the substances which are stable at room temperature retain their properties for cell digestion and / or PCR and / or detection.
- mixtures may be formed with excipients thin films of the materials and the mixtures may thin Pa ⁇ refine layers be watertight covered.
- the reagents and auxiliaries are already introduced into depressions of the cartridge channels 4 as dry substances in the production of the cartridge.
- the reagents are not washed away by a so-called water flow, but are retained when the entire channel is filled. Only after filling the channel, the reagent spots dissolve via diffusion processes and there is a homogeneous reagent solution.
- the recesses are located at predefined intervals along the reagent channel.
- the distances can be equidistant or be arranged particularly advantageous in variable spacing patterns.
- the depressions can advantageously be filled with variable amounts of dry reagent. Through the combination of different dry reagent quantities and well spacing patterns, the desired concentration profiles of the final reagent solutions can be adjusted.
- the magnetic beads are dispensed as a suspension in the lysis channel. Evaporation of the solvent is observed to cause the beads to enter the
- the lysis reagents and the magnetic beads may be contained together in a single dry matrix.
- the card also contains the reagents for an ELISA assay. Two reagents are described in detail for the ELISA assay required, that the first agent Re ⁇ a label enzyme, and as the second reagent is an enzyme substrate.
- a detection module for the electrical detection of the hybridization processes is arranged in the cartridge.
- the detection module advantageously consists of an el-metal / plastic composite or a semiconductor-processed Silicon chip with precious metal electrodes.
- electrochemical, magnetic or piezoelectric measuring methods are suitable for electrical detection.
- an input port for a whole blood sample before ⁇ handen is in particular an input port for a whole blood sample before ⁇ handen.
- means are for supplying water vorhan ⁇ to, for example, inflow ports for connection to an ex ternal ⁇ water supply or an integrated water reservoir.
- dry buffer substances have a defined ionic strength after the supply of water.
- means for mixing a whole blood sample with water or a buffer solution are advantageously present.
- means for flowing through the lysis / bead / reagent coated microchannel or microcavity with blood or blood / water or blood / buffer mixtures are present.
- a magnetic field spe ⁇ for the invention in the cartridge in use essential for DNA analysis carried out PCR are still With ⁇ tel for generating for fixing the DNA / magnetic bead complex in the PCR cavity there.
- the PCR cavity must be able to be suitably closed and means for thermocyclization must be present.
- FIG. 2 shows the plan view of a cell disruption channel
- FIG. 3 shows the cross section through the cell disruption channel
- FIG. 5 FIG.
- FIG. 5 the top view of the PCR chamber in FIG. 1, FIGS. 6 and 7 the cross section through the PCR chamber according to FIG. 5,
- Figure 8 is a plan view of an ELISA reagent channel in Fi gur ⁇ 1, 9, 10 in accordance with the cross section of the ELISA Reagenzkanals
- FIG. 8 and FIGS. 11 to 23 show the plan view of the cartridge according to FIG. 1 in various process states during an automated evaluation.
- a cartridge 100 for an ELISA ( "enzyme-linked immunosorbent assay") - test with an elevation of the existing therein microchannel or Darge cavities system ⁇ is, the clarity, the associated function ⁇ designations are also referred to ,
- the cartridge 100 consists in detail of a plastic body 101 with there introduced fluidic structures, the one of
- FIGS. 2 to 10. 1 shows a sample port 102 with adjoining metering section 105, via which defined liquid samples for, in particular, nucleic acid analysis, for example for the analysis of white blood cells from whole blood, can be obtained in order to answer human-genomic questions.
- a channel region 110 for the cell disruption of the sample and furthermore, especially for a DNA analysis, a region 120 for a PCR (Polymerase Chain Reaction) for selective DNA amplification (amplification) to increase the concentration of the DNA to be detected so far that it can be detected in a third station.
- the actual PCR chamber can be closed by valves 122, 122 '. The detection of the thus prepared sample, in particular according to the ELISA method, then takes place in the region 130.
- From Figure 1 may also be water ports 103-103 '''he ⁇ clearly.
- water can be introduced into the Cartridge 100 as a transport and solvent.
- the cartridge 100 has an input port 102 for a whole blood sample.
- means for supplying water are present.
- microchannels or microcavities 101-131 are filled in the Nor mally ⁇ with dry buffer substances which ensure a de ⁇ finêt ionic strength to the water supply.
- dry buffer substances which ensure a de ⁇ finiert ionic strength to the water supply.
- For the blood analysis are means for mixing whole blood samples and water or the buffer solution and / or means for flowing through a microsphere coated with a lysis bead reagent or the microcavity with blood or with blood-water or blood-buffer mixture available.
- In the channel system are wide areas 106, 107, 108, 109 is provided as re ⁇ servoirs for receiving waste ( "waste").
- reference numerals 101 again identify the cartridge base body.
- the body includes a spe ⁇ essential for cell disruption ( "lysis") a shaped in a special way flow passage 111 with edges formed by the sides ⁇ step-shaped recesses 112 is used to receive me of dry reagents.
- the recesses 112 in this case have a plurality of steps with step heights of 10 to 500 .mu.m and ha ⁇ ben an extension of approximately 1 mm and a depth of about microns 100th
- reagents of this kind which in particular also contain magnetic beads for binding the released DNA, are distributed uniformly between the steps 112 via the flow channel 111.
- Magnetic beads have DNA- and protein-binding properties, provided that they have been pretreated accordingly. They may interfere with DNA-binding properties. optionally also be coated with antibodies.
- FIGS. 5 to 7 show the structure of a PCR chamber 120 in the cartridge main body 101 with flow channel 111.
- the valve arrangement for completing the PCR chamber in the case of appropriate use is not shown here. It is essential that in the PCR chamber 120 round cylindrical recesses 124, 124 'for receiving specific reagents 127, 127 ', which are needed in carrying out the PCR, are present. Specifically, it is shown in FIG. 7 that a dry-storable, room temperature-stable PCR reagent 127, 127 'is initially covered by a paraffin layer 128, 128'.
- FIGS. 8 to 10 show the structure and the structure of the ELISA reagent channels 131 and 131 'from FIG. 1.
- well-shaped depressions 132 to 132 6' are present which are suitable for receiving pre-dosed and pre-ported quantities Reagents for the ELISA process according to FIG. 9 are suitable.
- This is described in detail in WO 02/072262 A1, which has already been cited at the outset as state of the art, to which reference is likewise expressly made in the present context (“Incorporation by Reference").
- the circular cylindrical recesses 132-132 6 'with dry reagents 133-133 6' filled dar ⁇ provided.
- a first reagent realizes a labeling enzyme and a second reagent an enzyme substrate, as is known to be required in the hybridization of the sample optionally prepared by a PCR with specific capture probes.
- area 130 of the detection may be in a module of a precious metal / plastic composite are different sensors for Erfas ⁇ solution of biochemical reactions.
- semiconductor-processed chips ie in particular silicon-based sensors, the signals electrically detected and processed immediately.
- electrochemical, magnetic and / or piezoelectric measuring methods with related sensors are also possible.
- the cartridge 100 according to FIG. 1 is shown in plan view, with the area of the cartridge 100 active during the analysis process being marked.
- the cartridge 100 is transformed into an analyzer, which is not shown in detail in the figures and not Jacobs ⁇ tand the present patent application, is inserted.
- the enzyme-substrate solution flows via the Detektionsmo ⁇ module 130 for enzymatic-electrochemical detection of hybridization in the waste area 109 (Waste 4).
- the resulting signals can be read out electrically and processor-based evaluated according to a predetermined program.
- the cartridge described in detail with channels and cavities with reference to FIG. 1 is made of a polymer material, such as polycarbonate, for example by injection molding.
- the card base body 101 is produced with structures that are open at the top, and the reagents are spotted into the initially open channels or cavities and then dried.
- the detection module is incorporated in a suitable manner in the cartridge, insbesonde ⁇ re glued.
- the channels and the cavities are provided , for example, with an elastic film as the top cover and are therefore closed for the intended use.
- the detection chamber is first flushed with an antibody solution carrying an enzyme label (ELISA reagent 1). Then, the rinsing of the detection chamber with enzyme substrate (ELISA reagent 2). The electrochemical measurements are carried out in a manner known per se at predeterminable, different temperatures and changeable flow rates of the enzyme-substrate solution.
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Clinical Laboratory Science (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Dispersion Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Automatic Analysis And Handling Materials Therefor (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
Abstract
Description
Beschreibungdescription
Anordnung zur integrierten und automatisierten DNA- oder Pro¬ tein-Analyse in einer einmal verwendbaren Cartridge, Herstel- lungsverfahren für eine solche Cartridge und Betriebsverfah¬ ren der DNA- oder Protein-Analyse unter Verwendung einer sol¬ chen CartridgeArrangement for integrated and automated DNA or protein analysis in a single-use cartridge, production method for such a cartridge and method of operating the DNA or protein analysis using such a cartridge
Die Erfindung bezieht sich auf eine Anordnung zur integrier- ten und automatisierten DNA- oder Protein-Analyse in einer einmal verwendbaren Cartridge. Als Cartridge wird dabei eine flache Karte im Scheckkartenformat bezeichnet. Daneben be¬ zieht sich die Erfindung auf die Herstellung einer solchen Cartridge. Schließlich bezieht sich die Erfindung auch auf ein Betriebsverfahren für eine DNA- oder Protein-Analyse un¬ ter Verwendung einer solchen Cartridge.The invention relates to an arrangement for integrated and automated DNA or protein analysis in a single-use cartridge. Cartridge is a flat card in credit card format. In addition, be ¬ the invention contemplates the production of such a cartridge. Finally, the invention also relates to an operating method for DNA or protein analysis using such a cartridge.
Zur Nukleinsäureanalytik, beispielsweise zur Analytik von weißen Blutzellen aus Vollblut, zwecks Beantwortung von hu- mangenomischen Fragestellungen müssen zunächst in einer ers¬ ten Station als Probenvorbereitungsschritt die Zellen auf¬ gebrochen und anschließend die dabei freigesetzte DNA iso¬ liert werden. In einer zweiten Station erfolgt eine PCR (Po¬ lymerase Chain Reaction = Polymerase-Ketten-Reaktion) zur se- lektiven DNA-Vervielfältigung (Amplifikation) , um die Kon¬ zentration der nachzuweisenden DNA soweit zu erhöhen, dass diese in einer dritten Station nachgewiesen werden kann.For nucleic acid analysis, for example for the analysis of white blood cells from whole blood, for the purpose of answering hu mangenomischen questions the cells ¬ must be broken first in a ers¬ th station as a sample preparation step and then the DNA iso ¬ be profiled liberated. In a second station, a PCR (polymerase chain reaction) for selective DNA amplification (amplification) is carried out in order to increase the concentration of the DNA to be detected to such an extent that it is detected in a third station can be.
Im Labor werden letztere Teilprozesse separat nach bekanntem Stand der Technik durchgeführt. Die vorstehend erläuterten drei Stationen beinhalten jeweils mehrere Arbeitsschritte und werden voneinander unabhängig mit unterschiedlichen Geräten durchgeführt. Die einzelnen Arbeitsschritte erfolgen weitge¬ hend manuell.In the laboratory, the latter sub-processes are carried out separately according to the known state of the art. The three stations explained above each involve several work steps and are performed independently of each other with different devices. Carried out the individual steps weitge ¬ starting manually.
Die Realisierung dieser Schritte ist vom Vorhandensein von Laborgeräten - wie einer Zellaufschluss-Apparatur, einem PCR- Gerät (sog. Thermocycler) , evtl. einem PCR-Gerät, das für quantitative PCR geeignet ist, einer Elektrophorese-Appara¬ tur, einer Hybridisierungsstation, einem optischen Reader, sog. Eppendorf-Tubes, mehreren Pipettier-Geräten sowie einem Kühlbehälter für Reagenzien - abhängig und muss von geschul- tem Personal unter Einhaltung von Sicherheitsvorschriften be¬ züglich Infektionsgefahr, Abfallentsorgung od. dgl. durchge¬ führt werden. Insbesondere müssen mehrere volumetrische, d.h. genaue, Dosierungen (Pipettieren) von Reagenzlösungen durch¬ geführt werden. Solche Arbeitsschritte sind zeitaufwendig und kostenintensiv.The realization of these steps is the presence of laboratory equipment - such as a cell disruption apparatus, a PCR device (so-called thermocycler), possibly a PCR device, which is for Quantitative PCR is suitable for an electrophoresis Appara ¬ ture, a hybridization station, an optical reader, so-called Eppendorf tubes, multiple pipetting equipment including cooling containers for reagents -. dependent and by trained personnel in compliance with safety regulations must be¬ züglich Danger of infection, waste disposal od. Like. Durchge leads. In particular, several volumetric, ie precise, dosages (pipetting) of reagent solutions must be carried out. Such steps are time consuming and costly.
Vom Stand der Technik sind Einrichtungen zur biochemischen Analyse bekannt, die entsprechend der WO 02/073153 Al Verwen¬ dung von insbesondere siliziumbasierten Messmodulen machen, welche in eine Chipkarte integriert werden können. Dabei wer¬ den entsprechend der WO 02/072262 Al bereits die zur Analyse verwendeten Reagenzien in trockengelagerter Form in das Ana¬ lysemodul integriert .Devices are known for biochemical analysis of the prior art, which accordingly make dung WO 02/073153 Al USAGE ¬ of particularly silicon-based measurement modules, which can be integrated in a chip card. Here, whoever the WO 02/072262 Al already lysis module integrated in accordance with ¬ the reagents used for analysis in dry-stored form into the Ana¬.
Davon ausgehend ist Aufgabe der Erfindung die Realisierung eines preisgünstigen, einfach handhabbaren, kompletten DNA- oder Protein-Analyse-Vorganges in einer miniaturisierten Cartridge. Insbesondere sollen folgende Verbesserungen im Vergleich zur Labormethode realisiert werden: - Eine vollständige Integration aller Stoffe (evtl. außerOn this basis, the object of the invention is the realization of a low-cost, easy-to-handle, complete DNA or protein analysis process in a miniaturized cartridge. In particular, the following improvements compared to the laboratory method should be realized: - A complete integration of all substances (possibly except
Wasser) in einer geschlossenen einmal verwendbaren Cartrid¬ ge;Water) in a closed single-use Cartrid¬ ge;
- eine Bevorratung der Reagenzien in einer bei Raumtemperatur lagerstabilen Form; - eine automatische Durchführung aller Prozesse in der Cart¬ ridge;a storage of the reagents in a storage-stable form at room temperature; - ridge automatic execution of all processes in the Cart ¬;
- keine manuellen Arbeitsschritte, außer Injektion der zu analysierenden Probe, z.B. Blut;no manual operations, except injection of the sample to be analyzed, e.g. Blood;
- Kein direkter Kontakt mit gesundheitsgefährdenden Stoffen (Blut und Reagenzien-Abfall verbleiben in der Cartridge) ;- No direct contact with hazardous substances (blood and reagent waste remain in the cartridge);
- Cartridge-Geometrie erlaubt eine effiziente und schnelle Thermozyklisierung;- Cartridge geometry allows efficient and fast thermocycling;
- alle Detektionsvorgänge sollen elektrisch ablaufen und ein¬ fach auszulesen sein;- All detection processes should be electrically and be read out ein¬ easy;
- die verwendete Cartridge ist klein und kostengünstig herzu- stellen.- The cartridge used is small and inexpensive to produce.
Die Aufgabe ist erfindungsgemäß durch eine Anordnung mit ei¬ ner Cartridge gemäß Patentanspruch 1 gelöst. Die Herstellung einer solchen Cartridge ist Gegenstand des Patentanspruches 34. Betriebsverfahren mit solchen Cartridges sind Gegenstand der nebengeordneten Patentansprüche 38 und 42. Dabei ist An¬ spruch 38 auf die DNA-Analyse und Anspruch 42 auf die Prote¬ in-Analyse gerichtet. Weiterbildungen der Anordnung, des Her¬ stellungsverfahrens und der Betriebsweise bei der Analyse sind in den jeweiligen Unteransprüchen angegeben.The object is achieved by an arrangement with egg ¬ ner cartridge according to claim 1. The preparation of such cartridge is the subject of claim 34. Operating methods with such cartridges are the subject of independent claims 38 and 42. In this case, at ¬ is demanding directed to the Prote in ¬ analysis 38 on DNA analysis and claim 42nd Further developments of the arrangement, of the production method and of the mode of operation during the analysis are specified in the respective subclaims.
Die Erfindung geht insbesondere von der WO 02/072262 Al und dem dort genannten weiteren Stand der Technik aus. Dort wird eine Analyseeinrichtung mit in Fluidkanälen trockengelager- ten, bei Raumtemperatur stabilen Reagenzien beschrieben, die durch Zuführen von Wasser kurz vor ihrer bestimmungsgemäßen Verwendung in Lösung gebracht werden. Die Erfindung geht wei¬ terhin von der nicht vorveröffentlichten DE 10 2004 021780 Al und der nicht vorveröffentlichten DE 10 2004 021822 Al aus. Daneben ist es auch bekannt, speziell elektrisch auslesbare Detektionsmodule zu verwenden.The invention is based, in particular, on WO 02/072262 A1 and the further prior art mentioned therein. There, an analysis device with dry-stored in fluid channels, stable at room temperature reagents is described, which are brought into solution by supplying water shortly before their intended use. The invention is furthermore based on the unpublished DE 10 2004 021780 A1 and the unpublished DE 10 2004 021822 A1. In addition, it is also known to use specially electrically readable detection modules.
Demgegenüber ist Gegenstand der Erfindung eine solche einmal verwendbare Cartridge mit einem System aus Mikrokanälen und/oder Mikrokavitäten für einen vorgegebenen Prozessablauf nach der Probenaufnahme, wobei die Cartridge Strukturen zur Aufnahme der Trockenreagenzien aufweist und diesen Strukturen Mittel zur Durchführung sowohl des Zellaufschlusses einer¬ seits und der PCR andererseits, aber auch der elektrochemi- sehen Detektion zugeordnet sind. Insbesondere haben dabei die Kanäle unterschiedliche, problemangepasste Strukturen. Spe¬ ziell der Aufschlusskanal hat vorteilhafterweise gestufte Querschnitte zur optimalen Benetzung mit dem Trockenreagenz, während die PCR-Kammer und die Elisa-Reagenzkanäle töpfchen- förmige Vertiefungen aufweisen.In contrast, the subject of the invention is such a disposable cartridge with a system of microchannels and / or microcavities for a given process flow after sample taking, the cartridge having structures for receiving the dry reagents and these structures means for performing both the cell digestion on the one hand and the PCR on the other hand, but also the electrochemical detection are associated. In particular, the channels have different, problem-adapted structures. Spe ¬ essential the digestion channel advantageously has stepped cross sections for optimum wetting of the dry reagent, while the PCR chamber and the Elisa reagent channels have potty-shaped wells.
Es kann somit erreicht werden, dass in einem Verfahrensab- lauf die Zuführung und Aufbereitung der Probe, die DNA-It can thus be achieved that, in a process sequence, the supply and preparation of the sample, the DNA
Amplifikation und die eigentliche Detektion der DNA möglich ist .Amplification and the actual detection of the DNA is possible.
Durch das erfindungsgemäße System der geometrischen Struktu- ren in den Mikrokanälen bzw. Mikrokavitäten zur Aufnahme von Trockenreagenzien ergeben sich geeignete Randbedingungen für die DNA-Analyse einerseits und die Protein-Analyse anderer¬ seits. Folgende Merkmale und Maßnahmen sind wesentlich:The inventive system of the geometric structures ren in microchannels or microcavities for receiving dry reagents to provide suitable conditions for DNA analysis of a hand and the other hand, protein analysis ¬. The following features and measures are essential:
- Die im Mikrokanal bzw. in der Mikrokavität eingebrachten Reagenzien sind trockenbare Substanzen mit vernachläs¬ sigbarem Dampfdruck. Durch die bei Raumtemperatur stabi¬ len Substanzen bleiben deren Eigenschaften für Zellauf- schluss und/oder PCR und/oder Detektion erhalten. Dabei können Gemische aus den Substanzen mit Hilfsstoffen Dünnfilme bilden und können die Gemische mit dünnen Pa¬ raffinschichten wasserdicht abgedeckt sein.- The changes introduced in the microchannel or the microcavity reagents are dryable substances with vernachläs ¬ sigbarem vapor pressure. The substances which are stable at room temperature retain their properties for cell digestion and / or PCR and / or detection. Here, mixtures may be formed with excipients thin films of the materials and the mixtures may thin Pa ¬ refine layers be watertight covered.
Bei der Erfindung werden die Reagenzien und Hilfsstoffe be¬ reits bei der Herstellung der Cartridge als Trockensubstanzen in Vertiefungen der Cartridgekanäle4 eingebracht. Daraus er¬ geben sich folgende Vorteile:In the invention, the reagents and auxiliaries are already introduced into depressions of the cartridge channels 4 as dry substances in the production of the cartridge. This gives the following advantages:
- einfache und präzise Applikation der Reagenzien bei der Herstellung der Cartridge- easy and precise application of the reagents in the production of the cartridge
- Schutz der Reagenzien beim Befüllen der Reagenzkanäle, d.h. die Reagenzien werden durch einen sog. Wasserflow nicht weggespült, sondern bleiben beim Füllen des gesam¬ ten Kanals erhalten. Erst nach dem Befüllen des Kanals lösen sich die Reagenz-Spots über Diffusionsvorgänge auf und es entsteht eine homogene Reagenzlösung.Protection of the reagents during filling of the reagent channels, i. The reagents are not washed away by a so-called water flow, but are retained when the entire channel is filled. Only after filling the channel, the reagent spots dissolve via diffusion processes and there is a homogeneous reagent solution.
In einer weiteren besonderen Ausführungsform sind die Vertie¬ fungen in vordefinierten Abständen entlang des Reagenzkanals lokalisiert. Dabei können die Abstände äquidistant sein oder besonders vorteilhaft in variablen Abstandsmustern angeordnet sein.In a further particular embodiment, the recesses are located at predefined intervals along the reagent channel. The distances can be equidistant or be arranged particularly advantageous in variable spacing patterns.
Die Vertiefungen können vorteilhafterweise mit variablen Tro- ckenreagenzmengen befüllt sein. Durch die Kombination von verschiedenen Trockenreagenzmengen und Abstandsmustern der Vertiefungen können die gewünschten Konzentrationsprofile der fertigen Reagenzlösungen eingestellt werden.The depressions can advantageously be filled with variable amounts of dry reagent. Through the combination of different dry reagent quantities and well spacing patterns, the desired concentration profiles of the final reagent solutions can be adjusted.
Für bestimmte Funktionen wie z.B. ZellaufSchluss in Gegenwart von Magnet-Beads und Lyse-Reagenzien ist eine gleichmäßige Verteilung der unlöslichen Komponenten, d.h. der Beads, im Trockenreagenz erforderlich. Dazu werden die Magnet-Beads als Suspension in den Lyse-Kanal dispensiert. Beim Verdampfen des Lösungsmittels wird beobachtet, dass sich die Beads in dieFor certain functions, such as Cell disruption in the presence of magnetic beads and lysis reagents is a uniform distribution of the insoluble components, i. of the beads, in dry reagent required. For this purpose, the magnetic beads are dispensed as a suspension in the lysis channel. Evaporation of the solvent is observed to cause the beads to enter the
Randbereiche des Lyse-Kanals zurückziehen und eine gleichmä¬ ßige Verteilung dadurch nicht gegeben ist. Durch stufenartige Strukturierung des Lyse-Kanal-Querschnittes verteilen sich die Magnet-Beads über die Stufen und eine gleichmäßige Ver- teilung wird erreicht.Retract edge regions of the lysis channel and a gleichmä ¬ lar distribution is thus not given. By stepwise structuring of the lysis channel cross section, the magnetic beads are distributed over the steps and a uniform distribution is achieved.
Damit mit der erfindungsgemäßen Cartridge gleichermaßen ein ZellaufSchluss, eine PCR und ein so genannter DNA-/Protein- ELISA-Test durchgeführt werden kann, ist es vorteilhaft, dass in den Mikrokanälen bzw. Mikrokavitäten Substrate mit DNA- bindenden Eigenschaften, insbesondere die DNA-bindenden Mag¬ net-Beads, vorhanden sind. Dabei können die Lyse-Reagenzien und die Magnet-Beads zusammen in einer einzigen Trockenmatrix enthalten sein. Weiterhin sind in der Karte auch die Reagen- zien für einen ELISA-Assay vorhanden. Im Einzelnen werden für das ELISA-Assay zwei Reagenzien benötigt, d.h. als erstes Re¬ agenz ein Label-Enzym und als zweites Reagenz ein Enzym-Substrat.In order to be able to carry out a cell disruption, a PCR and a so-called DNA / protein ELISA test in the same way with the cartridge according to the invention, it is advantageous that in the microchannels or microcavities substrates with DNA-binding properties, in particular the DNA-binding Magnetic beads are present. The lysis reagents and the magnetic beads may be contained together in a single dry matrix. Furthermore, the card also contains the reagents for an ELISA assay. Two reagents are described in detail for the ELISA assay required, that the first agent Re ¬ a label enzyme, and as the second reagent is an enzyme substrate.
Insbesondere ist in der Cartridge ein Detektionsmodul zur e- lektrischen Detektion der Hybridisierungsvorgänge angeordnet. Das Detektionsmodul besteht vorteilhafterweise aus einem E- delmetall/Plastik-Verbund oder einem halbleiterprozessierten Siliziumchip mit Edelmetallelektroden. Für eine elektrische Detektion geeignet sind dabei insbesondere elektrochemische, magnetische oder piezoelektrische Messverfahren.In particular, a detection module for the electrical detection of the hybridization processes is arranged in the cartridge. The detection module advantageously consists of an el-metal / plastic composite or a semiconductor-processed Silicon chip with precious metal electrodes. In particular, electrochemical, magnetic or piezoelectric measuring methods are suitable for electrical detection.
Zur Anwendung der Erfindung der erfindungsgemäßen Cartridge ist insbesondere ein Eingabeport für eine Vollblutprobe vor¬ handen. Weiterhin sind Mittel zur Zufuhr von Wasser vorhan¬ den, beispielsweise Zufluss-Ports zum Anschluss an eine ex¬ terne Wasserzufuhr oder ein integriertes Wasserreservoir. In den Mikrokanälen bzw. Mikrokavitäten haben trockene Puffer¬ substanzen definierte Ionenstärke nach der Wasserzufuhr.To apply the invention of the cartridge according to the invention is in particular an input port for a whole blood sample before ¬ handen. Furthermore, means are for supplying water vorhan¬ to, for example, inflow ports for connection to an ex ternal ¬ water supply or an integrated water reservoir. In the microchannels or microcavities, dry buffer substances have a defined ionic strength after the supply of water.
Bei Anwendung der Erfindung zur Analytik von weißen Blutzel¬ len aus Vollblut sind vorteilhafterweise Mittel zum Mischen einer Vollblutprobe mit Wasser bzw. einer Pufferlösung vor¬ handen. Dabei sind Mittel zum Durchströmen des mit Lyse- /Bead/Reagenz beschichteten Mikrokanals bzw. Mikrokavität mit Blut bzw. Blut/Wasser- oder Blut/Puffer-Gemische vorhanden.When applying the invention for the analysis of white blood cells from whole blood, means for mixing a whole blood sample with water or a buffer solution are advantageously present. In this case, means for flowing through the lysis / bead / reagent coated microchannel or microcavity with blood or blood / water or blood / buffer mixtures are present.
Für die in der erfindungsgemäßen Cartridge bei Anwendung spe¬ ziell zur DNA-Analyse durchzuführende PCR sind weiterhin Mit¬ tel zum Generieren eines Magnetfeldes zum Fixieren des DNA- /Magnetbead-Komplexes in einer PCR-Kavität vorhanden. Für diesen Zweck muss die PCR-Kavität geeignet verschlossen wer- den können und müssen Mittel zur Thermozyklisierung vorhanden sein.A magnetic field spe¬ for the invention in the cartridge in use essential for DNA analysis carried out PCR are still With ¬ tel for generating for fixing the DNA / magnetic bead complex in the PCR cavity there. For this purpose, the PCR cavity must be able to be suitably closed and means for thermocyclization must be present.
Schließlich ist es bei der erfindungsgemäßen Cartridge we¬ sentlich, dass Mittel zum Lagern von verbrauchtem Probenmate- rial und verbrauchten Reagenzien vorhanden sind, die Abfall- Reservoirs bilden. Dabei müssen die Mittel zur keimdichten, zell- bzw. partikelfreien Entlüftung von mindestens einem Ab¬ fallreservoir geeignet sein. Zum Auslesen der Cartridge in einem Auslesegerät, das nicht Gegenstand der Erfindung ist, müssen schließlich Mittel zur Fixierung der Cartridge vorhan¬ den sein.Finally, in the case of the cartridge according to the invention, it is essential that there are means for storing spent sample material and spent reagents which form waste reservoirs. In this case, the means for germ-proof, cell-free or particle-free venting of at least one waste reservoir must be suitable. Finally, to read out the cartridge in a read-out device, which is not the subject of the invention, means for fixing the cartridge must be present.
Weitere Einzelheiten und Vorteile der Erfindung ergeben sich aus der nachfolgenden Figurenbeschreibung von Ausführungsbei¬ spielen anhand der Zeichnung in Verbindung mit den Patentan¬ sprüchen. Es zeigen in jeweils schematischer DarstellungFurther details and advantages of the invention will become apparent from the following description of the figures Ausführungsbei¬ play with reference to the drawing in conjunction with the patent claims. They show in a schematic representation
Figur 1 eine Cartridge mit einer Übersicht über einzelne1 shows a cartridge with an overview of individual
Mikrokanal-/ Kavitäten-Systeme mit den zugehörigen Funktionsbezeichnungen,Microchannel / cavity systems with the associated function designations,
Figur 2 die Draufsicht auf einen Zeilaufschlusskanal, Figur 3 den Querschnitt durch den Zeilaufschlusskanal ge- maß Figur 5,FIG. 2 shows the plan view of a cell disruption channel, FIG. 3 shows the cross section through the cell disruption channel, FIG. 5, FIG.
Figur 4 zwei Alternativen des Durchflusskanalquerschnittes in vergrößerter Darstellung,4 shows two alternatives of the flow channel cross-section in an enlarged view,
Figur 5 die Draufsicht auf die PCR-Kammer in Figur 1, Figuren 6 und 7 den Querschnitt durch die PCR-Kammer gemäß Figur 5,5 the top view of the PCR chamber in FIG. 1, FIGS. 6 and 7 the cross section through the PCR chamber according to FIG. 5,
Figur 8 die Draufsicht auf einen ELISA-Reagenzkanal in Fi¬ gur 1, Figuren 9, 10 den Querschnitt des ELISA-Reagenzkanals gemäßFigure 8 is a plan view of an ELISA reagent channel in Fi gur ¬ 1, 9, 10 in accordance with the cross section of the ELISA Reagenzkanals
Figur 8 sowie die Figuren 11 bis 23 die Draufsicht auf die Cartridge gemäß Fi¬ gur 1 in verschiedenen Verfahrenszuständen während einer automatisierten Auswertung.FIG. 8 and FIGS. 11 to 23 show the plan view of the cartridge according to FIG. 1 in various process states during an automated evaluation.
In den Figuren haben gleiche bzw. gleichwirkende Elemente gleiche Bezugszeichen. Insbesondere die Figuren 1 bis 10 ei¬ nerseits und die Figuren 11 bis 23 andererseits werden ge¬ meinsam beschrieben.In the figures, identical or equivalent elements have the same reference numerals. In particular, Figures 1 to 10 ei ¬ hand, and Figures 11 to 23 on the other hand ge ¬ be described jointly.
In Figur 1 ist eine Cartridge 100 für einen ELISA("Enzyme Linked Immuno Sorbent ASSAY")- Test mit einem Aufriss des darin vorhandene Mikrokanal- bzw. Kavitäten-System darge¬ stellt, wobei der Übersicht halber die zugehörigen Funktions¬ bezeichnungen zusätzlich bezeichnet sind. Die Cartridge 100 besteht im Einzelnen aus einem Kunststoff-Grundkörper 101 mit dort eingebrachten Fluidik-Strukturen, die von einerIn Figure 1, a cartridge 100 for an ELISA ( "enzyme-linked immunosorbent assay") - test with an elevation of the existing therein microchannel or Darge cavities system ¬ is, the clarity, the associated function ¬ designations are also referred to , The cartridge 100 consists in detail of a plastic body 101 with there introduced fluidic structures, the one of
Kunstofffolie abgedeckt sind. Die Strukturen werden anhand der Figuren 2 bis 10 weiter unten beschrieben. Aus der Draufsicht gemäß Figur 1 ist ein Probenport 102 mit daran anschließender Dosierstrecke 105, über den definiert flüssige Proben zur insbesondere Nukleinsäureanalytik, bei¬ spielsweise zur Analytik von weißen Blutzellen aus Vollblut, zwecks Beantwortung von humangenomischen Fragestellungen ein¬ gebracht werden können, ersichtlich. Es schließt sich ein Ka¬ nalbereich 110 für den ZellaufSchluss der Probe und weiterhin speziell für eine der DNA-Analyse ein Bereich 120 für eine PCR(Polymerase Chain Reaction = Polymerase-Ketten-Reaktion) zur selektiven DNA-Vervielfältigung (Amplifikation) an, um die Konzentration der nachzuweisenden DNA soweit zu erhöhen, dass diese in einer dritten Station nachgewiesen werden kann. Die eigentliche PCR-Kammer ist durch Ventile 122, 122' ver¬ schließbar. Die Detektion der so aufbereiteten Probe, insbe- sondere gemäß dem ELISA-Verfahren, erfolgt dann im Bereich 130.Plastic film are covered. The structures will be described below with reference to FIGS. 2 to 10. 1 shows a sample port 102 with adjoining metering section 105, via which defined liquid samples for, in particular, nucleic acid analysis, for example for the analysis of white blood cells from whole blood, can be obtained in order to answer human-genomic questions. This is followed by a channel region 110 for the cell disruption of the sample, and furthermore, especially for a DNA analysis, a region 120 for a PCR (Polymerase Chain Reaction) for selective DNA amplification (amplification) to increase the concentration of the DNA to be detected so far that it can be detected in a third station. The actual PCR chamber can be closed by valves 122, 122 '. The detection of the thus prepared sample, in particular according to the ELISA method, then takes place in the region 130.
Aus Figur 1 sind weiterhin Wasserports 103 bis 103''' er¬ sichtlich. Darüber ist bei der Aufbereitung einer Probe Was- ser als Transport- und Lösungsmittel in die Cartrridge 100 einbringbar. Weiterhin sind Entlüftungsports 104 bis 104''' vorhanden.From Figure 1 may also be water ports 103-103 '''he ¬ clearly. When preparing a sample, water can be introduced into the Cartridge 100 as a transport and solvent. Furthermore, vent ports 104 to 104 '''are present.
Wie erwähnt hat die Cartridge 100 insbesondere einen Eingabe- Port 102 für eine Vollblutprobe. Dazu sind Mittel zur Zufuhr von Wasser vorhanden. Es kann ein Zuflussport zum Anschluss an eine externe Wasserzufuhr vorhanden sein oder der Zufluss¬ port kann an ein integriertes Wasser-Reservoir angeschlossen werden.In particular, as mentioned, the cartridge 100 has an input port 102 for a whole blood sample. For this purpose, means for supplying water are present. There may be an inflow port for connection to an external water supply or the inflow port may be connected to an integrated water reservoir.
Die Mikrokanäle bzw. Mikrokavitäten 101 bis 131 sind im Nor¬ malfall mit trockenen Puffersubstanzen gefüllt, die eine de¬ finierte Ionenstärke nach Wasserzufuhr gewährleisten. Für die Blutanalyse sind Mittel zum Mischen von Vollblutproben und Wasser bzw. der Pufferlösung und/oder Mittel zum Durchströmen eines mit einem Lyse-Bead-Reagenz beschichteten Mikrokanals bzw. der Mikrokavität mit Blut oder mit Blut-Wasser- bzw. Blut-Puffer-Gemisch vorhanden. Im Kanalsystem sind weite Bereiche 106, 107, 108, 109 als Re¬ servoirs für die Aufnahme von Abfall ("Waste") vorgesehen. Daneben ist ein Bereich mit Kanälen 131 bzw. 131' für die Aufnahme unterschiedlicher ELISA-Reagenzien vorhanden.The microchannels or microcavities 101-131 are filled in the Nor mally ¬ with dry buffer substances which ensure a de ¬ finierte ionic strength to the water supply. For the blood analysis are means for mixing whole blood samples and water or the buffer solution and / or means for flowing through a microsphere coated with a lysis bead reagent or the microcavity with blood or with blood-water or blood-buffer mixture available. In the channel system are wide areas 106, 107, 108, 109 is provided as re ¬ servoirs for receiving waste ( "waste"). In addition, there is an area with channels 131 and 131 'for receiving different ELISA reagents.
In den Figuren 2 bis 4 kennzeichnen jeweils Bezugszeichen 101 wieder den Cartridge-Grundkörper. Der Körper beinhaltet spe¬ ziell für einen ZellaufSchluss ("Lyse") einen in besonderer Weise ausgeformten Durchflusskanal 111 mit durch die Seiten¬ kanten gebildeten stufenförmigen Vertiefungen 112 zur Aufnah¬ me von Trockenreagenzien. Die Vertiefungen 112 weisen dabei mehrere Stufen mit Stufenhöhen von 10 bis 500 μm auf und ha¬ ben eine Ausdehnung von ca. 1 mm und eine Tiefe von etwa 100 μm.In FIGS. 2 to 4, reference numerals 101 again identify the cartridge base body. The body includes a spe¬ essential for cell disruption ( "lysis") a shaped in a special way flow passage 111 with edges formed by the sides ¬ step-shaped recesses 112 is used to receive me of dry reagents. The recesses 112 in this case have a plurality of steps with step heights of 10 to 500 .mu.m and ha ¬ ben an extension of approximately 1 mm and a depth of about microns 100th
Speziell in der Darstellung gemäß der Figur 4a ergibt sich die alternative Möglichkeit, bei einem Durchflusskanal ohne Zusatzvertiefungen die Aufnahme der Lyse-Reagenzien nur im Bereich 113 der Kanten des Durchflusskanals 111 vorzusehen. In Figur 4b sind dagegen derartige Reagenzien, die insbeson¬ dere auch Magnet-Beads zur Bindung der freigesetzen DNA ent¬ halten, gleichmäßig zwischen den Stufen 112 über den Durch¬ flusskanal 111 verteilt. Magnet-Beads haben DNA- und Protein- bindende Eigenschaften, sofern sie entsprechend vorbehandelt sind. Sie können mit DNA-bindenden Eigenschaften u. gegebenenfalls auch mit Antikörpern beschichtet sein. Zum Einbringen von Trockensubstanzen als Matrix mit Lyse-Reagenz und Magnet-Beads wird insbesondere auf die ältere DE 10 2004 021780 Al und die ältere DE 10 2004 021822 der Anmelderin verwiesen.Especially in the illustration according to FIG. 4a, the alternative possibility arises of providing the lysis reagents only in the area 113 of the edges of the flow channel 111 in the case of a flow channel without additional depressions. On the other hand, in FIG. 4b, reagents of this kind, which in particular also contain magnetic beads for binding the released DNA, are distributed uniformly between the steps 112 via the flow channel 111. Magnetic beads have DNA- and protein-binding properties, provided that they have been pretreated accordingly. They may interfere with DNA-binding properties. optionally also be coated with antibodies. For introducing dry substances as matrix with lysis reagent and magnetic beads, reference is made in particular to the older DE 10 2004 021780 A1 and the earlier DE 10 2004 021822 of the applicant.
In den Figuren 5 bis 7 ergibt sich die Struktur einer PCR- Kammer 120 im Cartridge-Grundkörper 101 mit Strömungskanal 111. Die Ventilanordnung zum Abschluss der PCR-Kammer bei be¬ stimmungsgemäßer Anwendung ist hier nicht dargestellt. We¬ sentlich ist, dass in der PCR-Kammer 120 rundzylindrische Vertiefungen 124, 124' zur Aufnahme spezifischer Reagenzien 127, 127', die bei der Durchführung der PCR benötigt werden, vorhanden sind. Speziell in Figur 7 ist dazu dargestellt, dass ein trocken lagerbares, bei Raumtemperatur stabiles PCR- Reagenz 127, 127' zunächst von einer Paraffinschicht 128, 128' abgedeckt ist.FIGS. 5 to 7 show the structure of a PCR chamber 120 in the cartridge main body 101 with flow channel 111. The valve arrangement for completing the PCR chamber in the case of appropriate use is not shown here. It is essential that in the PCR chamber 120 round cylindrical recesses 124, 124 'for receiving specific reagents 127, 127 ', which are needed in carrying out the PCR, are present. Specifically, it is shown in FIG. 7 that a dry-storable, room temperature-stable PCR reagent 127, 127 'is initially covered by a paraffin layer 128, 128'.
Die sachgerechte Durchführung der PCR mit ventilgesteuerter Thermozyklisierung innerhalb einer Cartridge wird im Einzel¬ nen in den parallelen Anmeldungen DE 10 2004 050576.4 und DE 10 2004 050510.1 Anmelderin mit gleicher Anmeldepriorität be¬ schrieben, auf die im vorliegenden Zusammenhang ausdrücklich verwiesen wird ("Incorporation by Reference") . Insbesondere wird darin die Verwendung von Magnet-Beads zur DNA-Bindung und die Konzentration der Magnet-Beads mit der DNA in der PCR-Kammer 120 durch steuerbare Magnetfelder beschrieben, worauf hier nicht mehr im Einzelnen eingegangen wird.The proper implementation of PCR with valve-controlled thermocycling in a cartridge is nen in individual ¬ in the parallel applications DE 10 2004 050576.4 and DE 10 2004 050510.1 applicant with the same application priority be ¬ wrote, is expressly referred to in the present context ( "Incorporation by Reference "). In particular, it describes the use of magnetic beads for DNA binding and the concentration of the magnetic beads with the DNA in the PCR chamber 120 by means of controllable magnetic fields, which will not be discussed in detail here.
Aus den Figuren 8 bis 10 ergibt sich der Aufbau und die Struktur der ELISA-Reagenz-Kanäle 131 und 131' aus Figur 1. Es sind jeweils näpfchenförmige Vertiefungen 132 bis 1326' vorhanden, die zur Aufnahme von vordosierten und vorportio¬ nierten Mengen an Reagenzien für den ELISA-Prozess entspre¬ chend Figur 9 geeignet sind. Dies ist im Einzelnen in der eingangs bereits als Stand der Technik genannten WO 02/072262 Al beschrieben, auf die im vorliegenden Zusammenhang eben¬ falls ausdrücklich verwiesen wird ("Incorporation by Referen¬ ce") . In Figur 10 sind die kreiszylindrischen Vertiefungen 132 bis 1326' mit Trockenreagenzien 133 bis 1336' befüllt dar¬ gestellt. Dabei realisiert ein erstes Reagenz ein Markie- rungsenzym und ein zweites Reagenz ein Enzymsubstrat, wie es bekanntermaßen bei der Hybridisierung der gegebenenfalls durch eine PCR aufbereiteten Probe mit spezifischen Fänger¬ sonden benötigt wird. Im nur schematisch angedeuteten Bereich 130 der Detektion können sich in einem Modul aus einem Edel- metall-/Plastik-Verbund unterschiedliche Sensoren zur Erfas¬ sung biochemischer Reaktionen befinden. Speziell bei elektro¬ chemischen Messungen mit halbleiterprozessierten Chips, d.h. insbesondere siliziumbasierten Sensoren, können die Signale elektrisch erfasst und unmittelbar weiterverarbeitet werden. Neben elektrochemischen sind auch magnetische und/oder piezo¬ elektrische Messmethoden mit diesbezüglichen Sensoren mög¬ lich.FIGS. 8 to 10 show the structure and the structure of the ELISA reagent channels 131 and 131 'from FIG. 1. In each case, well-shaped depressions 132 to 132 6' are present which are suitable for receiving pre-dosed and pre-ported quantities Reagents for the ELISA process according to FIG. 9 are suitable. This is described in detail in WO 02/072262 A1, which has already been cited at the outset as state of the art, to which reference is likewise expressly made in the present context ("Incorporation by Reference"). In Figure 10, the circular cylindrical recesses 132-132 6 'with dry reagents 133-133 6' filled dar¬ provided. In this case, a first reagent realizes a labeling enzyme and a second reagent an enzyme substrate, as is known to be required in the hybridization of the sample optionally prepared by a PCR with specific capture probes. In only schematically indicated area 130 of the detection may be in a module of a precious metal / plastic composite are different sensors for Erfas ¬ solution of biochemical reactions. Especially in elektro¬ chemical measurements with semiconductor-processed chips, ie in particular silicon-based sensors, the signals electrically detected and processed immediately. In addition to electrochemical, magnetic and / or piezoelectric measuring methods with related sensors are also possible.
In den Figuren 11 bis 23 ist jeweils die Cartridge 100 gemäß Figur 1 in der Draufsicht dargestellt, wobei der jeweils beim Analysevorgang aktive Bereich der Cartridge 100 markiert ist: Dazu wird die Cartridge 100 in ein Analysegerät, das in den Figuren nicht im Einzelnen dargestellt ist und nicht Gegens¬ tand vorliegender Patentanmeldung ist, eingeschoben.In FIGS. 11 to 23, the cartridge 100 according to FIG. 1 is shown in plan view, with the area of the cartridge 100 active during the analysis process being marked. For this purpose, the cartridge 100 is transformed into an analyzer, which is not shown in detail in the figures and not Gegens ¬ tand the present patent application, is inserted.
Die Auswertung wird nachfolgend anhand elf konkreter Verfah¬ rens-Teilschritte a) bis m) verdeutlicht, nachdem die Cart- ridge in ein Auswertegerät mit Mitteln zur Aufnahme der Cart¬ ridge eingeschoben und das Auswertegerät bei darin fixierter Cartridge aktiviert ist. Unter Bezugnahme auf Figurl ergeben sich im Einzelnen folgende Teilschritte:The evaluation is illustrated below with reference to eleven specific procedural ¬ RENS steps a) to m) after the cartridge is inserted into an evaluation unit with means for receiving the Cart ¬ ridge and the evaluation device is activated at a fixed therein cartridge. With reference to FIG. 1, the following sub-steps result in detail:
a) Es wird ca. 10 μl Blut als Messprobe eingefüllt. Über die Dosierkapillare 105 wird 1 μl automatisch dosiert. b) Das überschüssige Blut wird in den Leerraum 106 (Waste 1) gewaschen. c) Anschließend werden 1 μl Blutprobe mit Wasser verdünnt und in den Zeilaufschlusskanal 110 transferiert. Dort finden der ZellaufSchluss (Lyse) der Blutzellen sowie das Binden der freigesetzten DNA an die Magnet-Beads statt. d) Anschließend werden die Magnet-Beads in die PCR-Kammer transferiert und dort gesammelt. Es findet ein Wasch¬ vorgang statt, wobei die Waschlösung im Leerraum 107a) About 10 μl of blood is added as a test sample. 1 μl is dosed automatically via the dosing capillary 105. b) The excess blood is washed into the void 106 (Waste 1). c) Subsequently, 1 μl of blood sample is diluted with water and transferred to the cell digestion channel 110. There, cell lysis of the blood cells and binding of the liberated DNA to the magnetic beads take place. d) Subsequently, the magnetic beads are transferred to the PCR chamber and collected there. There is a washing ¬ process takes place, the washing solution in the void 107th
(Waste 2) gesammelt wird. e) Nunmehr ist der Waschvorgang abgeschlossen. f) Anschließend werden die PCR-Kammerventile 122, 122' ge- schlössen und die PCR durchgeführt. g) Während der PCR wird gleichermaßen der ELISA-Reagenz- kanal 131, der das Enzymsubstrat enthält, mit Wasser gefüllt. h) Gleichermaßen wird während der PCR der ELISA-Reagenz kanal 131', der das Label-Enzym enthält, mit Wasser ge¬ füllt. i) Nach der PCR werden die PCR-Kammerventile 122, 122' ge- öffnet und das PCR-Produkt wird über das Detektionsmo- dul 130 (in den Waste 3, Kanal 108) geleitet, wo die Hybridisierung mit den spezifischen Fängersonden er¬ folgt. j) Der Enzym-Substrat-Kanal wird in den Abfallkanal 108 (Waste 3) entlüftet. k) Der Label-Enzym-Kanal wird in den Abfallkanal 108(Waste 2) is collected. e) Now the washing process is completed. f) Subsequently, the PCR chamber valves 122, 122 'are closed and the PCR is carried out. g) Similarly, the ELISA reagent channel 131, which contains the enzyme substrate, is filled with water during the PCR. h) Similarly, during the PCR of the ELISA reagent channel 131 ', which contains the label enzyme, ge filled with water ¬ . i) After the PCR, the PCR chamber valves 122, 122 'are opened and the PCR product is passed via the detection module 130 (into the waste 3, channel 108), where the hybridization with the specific capture probes er¬ follows , j) The enzyme-substrate channel is vented into the waste channel 108 (waste 3). k) The label enzyme channel is in the waste channel 108th
(Waste 3) entlüftet. 1) Die Label-Enzym-Lösung fließt über das Detektionsmodul(Waste 3) vented. 1) The label enzyme solution flows over the detection module
130 zum Labein in den Abfallbereich 109 (Waste 4) . m) Die Enzym-Substrat-Lösung fließt über das Detektionsmo¬ dul 130 zur enzymatisch-elektrochemischen Detektion der Hybridisierung in den Abfallbereich 109 (Waste 4) .130 to Labein in the waste area 109 (Waste 4). m) The enzyme-substrate solution flows via the Detektionsmo ¬ module 130 for enzymatic-electrochemical detection of hybridization in the waste area 109 (Waste 4).
Damit ist das Analyseverfahren abgeschlossen. Insbesondere bei einer elektrochemischen Detektion können die anfallenden Signale elektrisch ausgelesen und prozessorgestützt gemäß vorgegebenem Programm ausgewertet werden.This concludes the analysis process. In particular, in an electrochemical detection, the resulting signals can be read out electrically and processor-based evaluated according to a predetermined program.
Die anhand der Figur 1 im Einzelnen mit Kanälen und Kavitäten beschriebene Cartridge wird aus einem Polymermaterial, wie z.B. Polycarbonat, beispielsweise in Spritzgusstechnik, her¬ gestellt. Dabei wird zunächst der Karten-Grundkörper 101 mit nach oben offenen Strukturen hergestellt und es werden die Reagenzien in die zunächst offenen Kanäle bzw. Kavitäten ge- spottet und anschließend getrocknet. Das Detektionsmodul wird in geeigneter Weise in die Cartridge eingebracht, insbesonde¬ re eingeklebt. Zum Abschluss werden die Kanäle und die Kavi¬ täten z.B. mit einer elastischen Folie als obere Abdeckung versehen und ist damit für den bestimmungsgemäßen Gebrauch abgeschlossen.The cartridge described in detail with channels and cavities with reference to FIG. 1 is made of a polymer material, such as polycarbonate, for example by injection molding. Initially, the card base body 101 is produced with structures that are open at the top, and the reagents are spotted into the initially open channels or cavities and then dried. The detection module is incorporated in a suitable manner in the cartridge, insbesonde ¬ re glued. Finally, the channels and the cavities are provided , for example, with an elastic film as the top cover and are therefore closed for the intended use.
Es ist auch möglich, dass auf den offenen Kartengrundkörper 101 vor dem abdeckseitigen Abschluss und Fertigstellung der Cartridge bestimme Sondereinrichtungen, beispielsweise als Dichtungsmaterialien und/oder Entlüftungsmaterialien, aufge¬ bracht werden.It is also possible that on the open card body 101 before the cover side completion and completion of the Cartridge certain special equipment, for example, as sealing materials and / or ventilation materials, aufge¬ be introduced.
Das konkrete Messverfahren wurde anhand der Figuren 11 bis 23 für einen spezifischen Fall der DNA-Analyse einer Probe aus Vollblut erläutert. Allgemein ist vorgesehen, die beschriebe¬ ne Cartridge für die DNA-Analyse einerseits und/oder die Pro¬ teinanalyse andererseits einzusetzen, wobei - wie vorstehend bereits erwähnt - ein entsprechendes Auslesegerät und zugehö¬ rige Auswertealgorithmen zu Hilfe genommen werden. Damit er¬ geben sich definierte Betriebsverfahren, welche die anhand der Beispiele beschriebene Cartridge erst praxistauglich zum dezentralen Einsatz im Rahmen einer medizinischen "Point of Care"-Anwendung machen.The concrete measuring method was explained with reference to Figs. 11 to 23 for a specific case of DNA analysis of a sample of whole blood. Generally is provided which ne-described ¬ Cartridge for DNA analysis of a hand and / or the Pro ¬ protein analysis on the other hand use, wherein - as already mentioned above - an appropriate reader and zugehö ¬ membered evaluation algorithms can be used as an aid. This results in defined operating methods which make the cartridge described in the examples only practical for decentralized use in the context of a medical "point of care" application.
Abschließend wird speziell für die DNA-Analyse nochmals das integrierte Betriebsverfahren der oben im Einzelnen beschrie¬ benen Cartridge als Kombination bzw. in der Abfolge der einzelnen Teilschritte zusammengefasst :Finally, especially for the DNA analysis, the integrated operating method of the cartridge described above in detail is summarized again as a combination or in the sequence of the individual substeps:
- Aufgeben der Probe in Cartridge- Place the sample in cartridge
- Einschieben der Cartridge in Auslesegerät- Insert the cartridge in the reader
- Starten des vollautomatischen Assays - Proben-Dosierung über Dosierstrecke- Start of the fully automatic assay - Sample dosing via dosing line
- Waschen der Dosierstrecke- Washing the dosing line
- Verdünnen der Probe und Einbringen in Lyse-Kanal- Dilute the sample and place in lysis channel
- Verweilen im Lyse-Kanal- Linger in the lysis channel
- Sammeln des DNA-Bead-Komplexes durch Beadsammler in der PCR-Kammer- Collection of the DNA bead complex by bead collectors in the PCR chamber
- Waschen des DNA-Bead-Komplexes mit Wasser- Washing the DNA bead complex with water
- Verschließen der PCR-Kammer- Close the PCR chamber
- Durchführung der PCR- Performing the PCR
- Während der PCR: Füllen der beiden ELISA-Reagenzkanäle mit Wasser- During the PCR: Fill the two ELISA reagent channels with water
- Öffnen der PCR-Kammer- opening the PCR chamber
- Transport des PCR-Produkts in Detektionskammer- Transport of the PCR product in detection chamber
- Hybridisierung in der Detektionskammer - Entlüften der beiden ELISA-Reagenzkanäle- Hybridization in the detection chamber - Vent the two ELISA reagent channels
- Füllen und Spülen der Detektionskammer mit ELISA Rea¬ genz 1Filling and rinsing the detection chamber with ELISA reagent 1
- Füllen und Spülen der Detektionskammer mit ELISA- Reagenz 2- Fill and rinse the detection chamber with ELISA reagent 2
- Durchführung der elektrochemischen Messungen.- Carrying out the electrochemical measurements.
Bei den elektrochemischen Messungen erfolgt zunächst das Durchspülen der Detektionskammer mit einer ein Enzymlabel tragenden Antikörperlösung (ELISA-Reagenz 1) . Dann erfolgt das Durchspülen der Detektionskammer mit Enzymsubstrat (ELISA-Reagenz 2) . Die elektrochemischen Messungen werden in an sich bekannter Weise bei vorgebbaren, unterschiedlichen Temperaturen und änderbarer Fließgeschwindigkeiten der Enzym- substrat-Lösung durchgeführt.In electrochemical measurements, the detection chamber is first flushed with an antibody solution carrying an enzyme label (ELISA reagent 1). Then, the rinsing of the detection chamber with enzyme substrate (ELISA reagent 2). The electrochemical measurements are carried out in a manner known per se at predeterminable, different temperatures and changeable flow rates of the enzyme-substrate solution.
Für die Protein-Analyse werden entsprechende Vorgehensweisen angewandt, wobei in diesem Fall die PCR nicht zum Tragen kommt . For protein analysis, appropriate procedures are used, in which case the PCR does not apply.
Claims
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE102004050576 | 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 (2)
| Publication Number | Publication Date |
|---|---|
| EP1807208A1 true EP1807208A1 (en) | 2007-07-18 |
| EP1807208B1 EP1807208B1 (en) | 2013-03-20 |
Family
ID=35432485
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP05801513A Ceased EP1807208B1 (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 |
| EP05803183.2A Ceased EP1796838B1 (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 |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP05803183.2A Ceased EP1796838B1 (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 |
Country Status (5)
| Country | Link |
|---|---|
| US (2) | US7851227B2 (en) |
| EP (2) | EP1807208B1 (en) |
| JP (1) | JP4546534B2 (en) |
| CN (2) | CN100534619C (en) |
| WO (2) | WO2006042838A1 (en) |
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- 2005-10-17 US US11/665,331 patent/US7851227B2/en active Active
- 2005-10-17 JP JP2007536193A patent/JP4546534B2/en not_active Expired - Fee Related
- 2005-10-17 EP EP05801513A patent/EP1807208B1/en not_active Ceased
- 2005-10-17 CN CNB2005800352122A patent/CN100534619C/en not_active Expired - Fee Related
- 2005-10-17 US US11/665,380 patent/US20090130658A1/en not_active Abandoned
- 2005-10-17 WO PCT/EP2005/011156 patent/WO2006042734A1/en active Application Filing
- 2005-10-17 CN CN2005800352245A patent/CN101039751B/en not_active Expired - Fee Related
- 2005-10-17 EP EP05803183.2A patent/EP1796838B1/en not_active Ceased
Non-Patent Citations (1)
| Title |
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| See references of WO2006042838A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| JP4546534B2 (en) | 2010-09-15 |
| CN100534619C (en) | 2009-09-02 |
| CN101039750A (en) | 2007-09-19 |
| US7851227B2 (en) | 2010-12-14 |
| EP1807208B1 (en) | 2013-03-20 |
| US20090130658A1 (en) | 2009-05-21 |
| WO2006042838A1 (en) | 2006-04-27 |
| EP1796838B1 (en) | 2014-10-08 |
| WO2006042734A1 (en) | 2006-04-27 |
| CN101039751B (en) | 2010-05-05 |
| EP1796838A1 (en) | 2007-06-20 |
| JP2008517259A (en) | 2008-05-22 |
| US20090136922A1 (en) | 2009-05-28 |
| CN101039751A (en) | 2007-09-19 |
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