EP1807208A1 - Dispositif d'analyse de proteine et d'adn integree et automatisee dans une cartouche a usage unique, procede de production de cette cartouche et procede de fonctionnement de l'analyse de proteine et d'adn a l'aide de cette cartouche - Google Patents

Dispositif d'analyse de proteine et d'adn integree et automatisee dans une cartouche a usage unique, procede de production de cette cartouche et procede de fonctionnement de l'analyse de proteine et d'adn a l'aide de cette cartouche

Info

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
Application number
EP05801513A
Other languages
German (de)
English (en)
Other versions
EP1807208B1 (fr
Inventor
Heike Barlag
Siegfried Birkle
Walter Gumbrecht
Daniela KÜHN
Peter Paulicka
Manfred Stanzel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Siemens AG
Original Assignee
Siemens AG
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Siemens AG filed Critical Siemens AG
Publication of EP1807208A1 publication Critical patent/EP1807208A1/fr
Application granted granted Critical
Publication of EP1807208B1 publication Critical patent/EP1807208B1/fr
Ceased legal-status Critical Current
Anticipated expiration legal-status Critical

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
    • 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

L'invention concerne une analyse de protéine et d'ADN automatisée à l'aide d'une cartouche (carte) dotée d'un système de microcanaux et/ou de microcavités. Les microcanaux ou les microcavités ont des structures géométriques permettant la réception de réactifs secs. Aux fins de production industrielle, la cartouche est produite à partir d'un corps carte plat, par exemple, par une technique de moulage par injection. Les réactifs sont appliqués ponctuellement et séchés dans les canaux ouverts puis les canaux sont fermés par un film. Cela permet de doter une cartouche finie d'un échantillon pour essai et de commencer le procédé de mesure entièrement automatique par son introduction dans l'appareil d'évaluation.
EP05801513A 2004-10-15 2005-10-17 Dispositif d'analyse de proteine et d'adn integree et automatisee dans une cartouche a usage unique, procede de production de cette cartouche et procede de fonctionnement de l'analyse de proteine et d'adn a l'aide de cette cartouche Ceased EP1807208B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102004050576 2004-10-15
PCT/EP2005/055303 WO2006042838A1 (fr) 2004-10-15 2005-10-17 Dispositif d'analyse de proteine et d'adn integree et automatisee dans une cartouche a usage unique, procede de production de cette cartouche et procede de fonctionnement de l'analyse de proteine et d'adn a l'aide de cette cartouche

Publications (2)

Publication Number Publication Date
EP1807208A1 true EP1807208A1 (fr) 2007-07-18
EP1807208B1 EP1807208B1 (fr) 2013-03-20

Family

ID=35432485

Family Applications (2)

Application Number Title Priority Date Filing Date
EP05801513A Ceased EP1807208B1 (fr) 2004-10-15 2005-10-17 Dispositif d'analyse de proteine et d'adn integree et automatisee dans une cartouche a usage unique, procede de production de cette cartouche et procede de fonctionnement de l'analyse de proteine et d'adn a l'aide de cette cartouche
EP05803183.2A Ceased EP1796838B1 (fr) 2004-10-15 2005-10-17 Procede de realisation d'une mesure electrochimique sur un echantillon de mesure liquide dans une chambre de mesure accessible par des conduites

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP05803183.2A Ceased EP1796838B1 (fr) 2004-10-15 2005-10-17 Procede de realisation d'une mesure electrochimique sur un echantillon de mesure liquide dans une chambre de mesure accessible par des conduites

Country Status (5)

Country Link
US (2) US7851227B2 (fr)
EP (2) EP1807208B1 (fr)
JP (1) JP4546534B2 (fr)
CN (2) CN101039751B (fr)
WO (2) WO2006042734A1 (fr)

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CN101039750A (zh) 2007-09-19
EP1796838A1 (fr) 2007-06-20
WO2006042838A1 (fr) 2006-04-27
EP1807208B1 (fr) 2013-03-20
US7851227B2 (en) 2010-12-14
JP2008517259A (ja) 2008-05-22
US20090130658A1 (en) 2009-05-21
US20090136922A1 (en) 2009-05-28
WO2006042734A1 (fr) 2006-04-27
JP4546534B2 (ja) 2010-09-15
EP1796838B1 (fr) 2014-10-08
CN100534619C (zh) 2009-09-02
CN101039751A (zh) 2007-09-19
CN101039751B (zh) 2010-05-05

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