EP4326438A1 - Procédé de détection en ligne automatisée d'au moins une substance cible biologique dans un liquide et analyseur en ligne - Google Patents

Procédé de détection en ligne automatisée d'au moins une substance cible biologique dans un liquide et analyseur en ligne

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Publication number
EP4326438A1
EP4326438A1 EP22722145.4A EP22722145A EP4326438A1 EP 4326438 A1 EP4326438 A1 EP 4326438A1 EP 22722145 A EP22722145 A EP 22722145A EP 4326438 A1 EP4326438 A1 EP 4326438A1
Authority
EP
European Patent Office
Prior art keywords
particles
reaction chamber
biomolecules
unit
liquid
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.)
Pending
Application number
EP22722145.4A
Other languages
German (de)
English (en)
Inventor
Timo Hillebrand
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.)
IST Innuscreen GmbH
Original Assignee
IST Innuscreen GmbH
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 IST Innuscreen GmbH filed Critical IST Innuscreen GmbH
Publication of EP4326438A1 publication Critical patent/EP4326438A1/fr
Pending legal-status Critical Current

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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
    • B01L3/502761—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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules
    • 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
    • B01L3/502715—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 interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/04—Exchange or ejection of cartridges, containers or reservoirs
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14—Process control and prevention of errors
    • B01L2200/143—Quality control, feedback systems
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/16—Reagents, handling or storing thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/046—Function or devices integrated in the closure
    • B01L2300/047—Additional chamber, reservoir
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0663—Whole sensors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics

Definitions

  • the invention relates to a method for automated online detection of at least one biological target substance in a liquid and an online analysis device that is designed to carry out the method automatically.
  • Target substances to be detected or monitored can be, for example, viruses, bacteria and plasmid-associated bacterial resistance genes.
  • Such biological target substances in liquids are detected using molecular genetic methods, preferably using amplification techniques such as PCR or real-time PCR.
  • samples of the liquid to be analyzed are taken and analyzed in specialized laboratories that have the necessary equipment systems.
  • Manually manageable or automatic sampling devices and sample collectors are known for automated sampling from processes, e.g.
  • the collected samples are usually transported to a laboratory for further analysis, where the extraction of the sample nucleic acids and the specific and possibly quantitative detection of the biological target substance is carried out manually or at least partially automatically.
  • online analysis devices which take a sample and carry out a quantitative determination of a mostly inorganic analyte in the sample in a fully automated manner.
  • Such an online analysis can be carried out continuously or discontinuously.
  • a sample collection device for flow-through sampling in bodies of water is known from US 2015/0224502 A1.
  • the device has several flow-through sample cartridges that are set up to receive water from the water to be monitored and, if necessary, to retain samples of components of the liquid or solids in a filter or adsorption medium, while the water received is returned from the cartridges to the body of water is routed back.
  • the samples can either be stored for later laboratory analysis or analyzed directly in the device.
  • biological substances adsorbed on the filter or adsorption medium can be released as lysate and made available to an analysis module for further analysis, e.g. by means of qPCR.
  • the object of the invention is to provide an improved method for automated online detection of a biological target substance in a liquid and an online analysis device suitable for carrying out the method.
  • the method and the device should enable an efficient concentration of the target substance during sampling and a low-loss forwarding of the concentrated target substance to a subsequent analysis unit.
  • the method according to the invention for the automated online detection of at least one biological target substance in a liquid, in particular water or waste water, using an online analysis device comprises the following steps:
  • the detection unit having a first microfluidic unit and a second microfluidic unit which can be fluidically connected to the first microfluidic unit; - Releasing and/or isolating nucleic acids from the biomolecules bound to the particles; - transporting an eluate comprising the released and/or isolated nucleic acids into the second microfluidic unit;
  • biomolecules are understood to mean bacteria, bacteriophages, viruses, subcellular particles and free nucleic acids or nucleic acid fragments.
  • the target nucleic acid to be amplified in the second microfluidic unit can either be the target substance to be detected or a nucleic acid of the target substance to be detected.
  • the first and the second microfluidic unit can be accommodated in two separate cartridges or in a common cartridge.
  • biomolecules By the biomolecules being bound to particles and transported with them into a first microfluidic unit in order to release and/or isolate nucleic acids for further analysis in the first microfluidic unit, a method in which biomolecules are bound to a filter medium are released by rinsing out the filter medium, a comparatively high proportion of the biomolecules obtained from the first volume of the liquid are concentrated in a small eluate volume.
  • the method according to the invention not only brings about an efficient concentration of the target substance to be determined, but also makes it possible to select a comparatively small first volume of liquid to be removed from the process, even with a low concentration of the target substance in the liquid to be examined.
  • the method is therefore universally suitable for monitoring liquids in a large number of different industrial processes, regardless of whether the first volume transported into the process unit has to be discarded after enrichment and, if necessary, disposed of, or whether it can be returned to the process.
  • the sample feed line can be fluidically connected to a sample receiver.
  • the sample template can contain a supply of the liquid to be examined. It can be connected to a sampling device which is set up to take liquid from a body of water, a liquid-carrying line of a supply network, or a process container, e.g. Alternatively, the sampling device can also be connected directly to the sample feed line of the online be connected to the analyzer. The transport of liquid into the process unit of the online analysis device can be automatically controlled by the control electronics of the analysis device.
  • the first volume of liquid can be drained from the reaction chamber and a second volume of liquid can be introduced into the reaction chamber.
  • Biomolecules contained in the second volume of the liquid can be concentrated in the reaction chamber by binding to the particles remaining in the reaction chamber.
  • the particles can be magnetic or paramagnetic particles to which the biomolecules bind, in particular non-selectively.
  • the concentration of biomolecules contained in the liquid can include introducing one or more reaction components into the reaction chamber of the process unit.
  • the reaction components can, for example, comprise an alginate solution and a salt of a di- or polyvalent cation or an acid into the reaction chamber, so that an alginate gel-biomolecule complex is formed on the particles.
  • the particles can be added to the reaction chamber at the same time as the reaction components, or can also be placed in the latter before or after the first volume of liquid has been transported into the reaction chamber.
  • the particles are first moved by means of the magnet (for example in the direction of an opening through which the particles can be removed) and then by means of the needle, the magnetic needle or the pipetting device in the said area be transferred to the reaction chamber.
  • the reverse order can be provided, so that the particles are first transferred by means of the needle, the magnetic needle or the pipetting device and are then moved by means of the magnet.
  • the magnet can be a permanent magnet or an electromagnet.
  • the magnetic part of the magnetic needle can also be designed as a permanent magnet or as an electromagnet.
  • the particles can be flushed into the first microfluidic unit by introducing a small partial volume of the liquid contained in the reaction chamber.
  • the transport of the liquid and the particles can be brought about by the effect of gravity or a hydrostatic pressure of the liquid in the reaction chamber by arranging the area in which the fluid line opens into the reaction chamber somewhat lower than the bottom of the reaction chamber.
  • the liquid remaining in the reaction chamber can be drained out of the reaction chamber via an outlet.
  • the majority of the liquid can first be drained from the reaction chamber while the particles are still retained in the reaction chamber by means of the magnet. The particles can then be flushed into the detection unit or into the first microfluidic unit with a remainder of the liquid.
  • Releasing and/or isolating nucleic acids from the biomolecules bound to the particles in the first microfluidic unit can include at least the following steps:
  • the same particles are advantageously used not only for extracting and enriching the biomolecules from the liquid to be monitored, but also for binding and purifying the released nucleic acids.
  • the method can include detaching the nucleic acids from the particles by elution and transporting an eluate comprising the nucleic acids into the second microfluidic unit for subsequent amplification.
  • the release and/or isolation of nucleic acids from the biomolecules bound to the particles in the first microfluidic unit can include the following steps:
  • a buffer solution for example in a phosphate-buffered saline solution, in water or a Tris buffer, which optionally contains a complexing reagent for complexing of divalent or polyvalent cations bound to the particles with the biomolecules, and
  • the thermal release can include dispersing the particles in the buffered solution, optionally containing the complexing agent, and incubation.
  • the alginate gel structure present on the particle surfaces is thereby dissolved and the biomolecules bound therein are dissolved.
  • This variant of the method is suitable, for example, for applications in which the target substance to be determined is a nucleic acid that is already freely present in the liquid to be analyzed. In this alternative embodiment of the method, a classic lysis can therefore be omitted.
  • the nucleic acids bound to the particles in an alginate complex can only be dissolved by adding the buffer solution to the particles, optionally with further addition of the complexing agent, eg a chelating agent such as EDTA. It is also possible to use the method when the target substance is a bacterium or a virus as a biomolecule. In this case, the biomolecules bound to the particles can be thermally destroyed and the nucleic acid contained can be released.
  • the complexing agent eg a chelating agent such as EDTA.
  • the amplification carried out in the second microfluidic unit can be carried out, for example, using conventional PCR-based methods.
  • the qualitative or quantitative determination of the target substance can also be carried out in a conventional manner by using a sensor to record at least one measured value of a measured variable at a specific point in time or at a plurality of points in time, which corresponds to the number present at the respective point in time due to the amplification generated copies of a target nucleic acid. From the measured value obtained or the course of measured values as a function of time, the presence of the target nucleic acid and thus the biological target substance in the liquid to be monitored can be qualitatively detected, or a quantitative value, e.g. a concentration, of the biological target substance in the liquid can be determined and used by the control electronics as a measurement result spend.
  • a quantitative value e.g. a concentration
  • control electronics of the online analysis device advantageously carry out all the method steps described here in an automated manner.
  • it can, for example, effect liquid transport by actuating controllable valves and/or pumps or a relative movement of the magnet with respect to the reaction chamber by a drive moving the magnet and/or the reaction chamber.
  • the invention also includes an online analysis device for detecting at least one biological target substance in a liquid, in particular according to the method described above.
  • the online analysis device comprises at least the following components: control electronics, a processing unit and a detection unit, with the processing unit having at least one reaction chamber and a sample feed line opening into the reaction chamber, and with the processing unit being set up to measure a predetermined volume of the liquid in the reaction chamber absorb and concentrate biomolecules contained in the liquid by binding to particles; and wherein the detection unit comprises: a first microfluidic unit which can be fluidically connected to the reaction chamber and is set up to release and/or isolate nucleic acids from the biomolecules bound to the particles; a second microfluidic unit which is connected to the first microfluidic unit and is set up to receive an eluate comprising the released and/or isolated nucleic acids and to amplify a target nucleic acid; and a sensor, in particular an optical sensor, which is set up to generate a measurement signal dependent on the progress of an amplification carried out in
  • the analysis device can also have a sample pump, with the control electronics being set up to control the sample pump to transport a predetermined volume of the sample into the reaction chamber.
  • the sample feed line can be fluidically connectable to a sample receiver or directly to a body of water or a line in a process plant, a fermenter or another process container.
  • the analysis device can comprise at least one reaction component reservoir, which contains at least one reaction component for concentrating the biomolecules in the reaction chamber.
  • the analyzer can, for example, hold an alginate solution, a solution containing a salt of a di- or polyvalent cation, or alternatively a weak acid as a reaction component in one or more separate reservoirs.
  • the reaction component reservoir can be a container that can be fluidically connected to the reaction chamber via a reaction component supply line that can be selectively blocked or released, for example with a valve.
  • the particles can be placed in the reaction chamber or can also be added as reaction components from a reaction component reservoir to the liquid transported into the reaction chamber to enrich the biomolecules.
  • the particles can be magnetic and/or paramagnetic particles.
  • the control electronics of the analysis device can advantageously be set up to meter a predeterminable amount of the at least one reaction component or all reaction components into the reaction chamber.
  • one or more pumps can be provided for transporting the at least one reaction component, which controls the control electronics.
  • the control electronics can control the already mentioned valve.
  • the analysis device can have means for moving, in particular stirring, the liquid contained in the reaction chamber.
  • the means can include, for example, a stirrer, a feed line for an inert gas into the reaction chamber and a discharge line for the inert gas, which are arranged such that the inert gas flows through the liquid contained in the reaction chamber, or have a drive for moving the reaction chamber.
  • the analysis device can further comprise a magnet, which can be oriented relative to the reaction chamber by means of the control electronics in such a way that magnetic or paramagnetic particles contained in the reaction chamber are transported by magnetic force into an area of the reaction chamber in which a fluid line opens into the reaction chamber, which contains the reaction chamber fluidly connected to the first microfluidic unit.
  • the analysis device can have a switchable electromagnet, which is arranged with respect to the reaction chamber in such a way that magnetic or paramagnetic particles contained in the reaction chamber are transported by magnetic force into an area of the reaction chamber in which a fluid line opens into the reaction chamber fluidically connects the reaction chamber to the first microfluidic device.
  • the control electronics can be designed to switch the electromagnet.
  • the first and the second microfluidic unit can be arranged together in one, in particular replaceable, cartridge.
  • the first microfluidic unit can be arranged in a first cartridge and the second microfluidic unit can be arranged in a second cartridge that is different from the first cartridge, the first and second cartridges being fluidically connectable to one another in order to transport liquid from the first cartridge into the second .
  • the first and the second cartridge can be designed to be exchangeable.
  • the first microfluidic unit can be set up to receive the particles with biomolecules bound thereto and to add one or more lysis reagents to release nucleic acid of the adsorbed biomolecules, then to bind the released nucleic acids to the particles and to absorb the particles with the nucleic acids bound thereto. or to wash several times with a washing solution.
  • the control electronics can be set up to control a transport of the particles and optionally the reagents through the first microfluidic unit.
  • the first microfluidic unit can also be set up to detach the nucleic acids from the particles by elution and to transport the eluate into the second microfluidic unit via a fluid line connecting the first microfluidic unit to the second microfluidic unit.
  • the control electronics can be set up to control the elution and the transport of the eluate.
  • the first microfluidic unit can be set up to receive the particles with biomolecules bound to them and the biomolecules bound to the particles or the nucleic acids contained in these biomolecules in a buffer solution, for example in a phosphate-buffered saline solution (PBS buffer), water or a Tris buffer, which optionally contains a complexing agent, for example a chelating agent such as EDTA, thermally release, the control electronics being set up to control the thermal release of the biomolecules and the buffer solution with the biomolecules or nucleic acids dissolved therein in the second microfluidic unit to be transported for subsequent amplification.
  • PBS buffer phosphate-buffered saline solution
  • Tris buffer which optionally contains a complexing agent, for example a chelating agent such as EDTA
  • thermally release the control electronics being set up to control the thermal release of the biomolecules and the buffer solution with the biomolecules or nucleic acids dissolved therein in the second microfluidic unit to be transported for
  • the first microfluidic unit can contain reaction components required for the release and/or isolation of the nucleic acid in solid form, for example in the form of pellets obtained by lyophilization, or in reagent chambers closed by microvalves or in reagent packs closed by foils, which are automated by the action of force or heat, in particular by means of the control electronics, can be opened.
  • Reagents for the amplification can be stored in a corresponding manner in the second microfluidic unit. It can also contain one or more detection chambers, in which the amplification of a target nucleic acid or else several different target nucleic acids can be carried out in parallel.
  • the second microfluidic unit can include a temperature control device for the detection chambers.
  • the temperature control device can, for example, have thermoelectric elements that can be controlled by the control electronics.
  • the control electronics can have an electronic data processing device with at least one processor and a memory in which one or more operating programs are stored that can be executed by the processor in order to automatically control the analysis device and to determine qualitative or quantitative analysis results from the measurement signals of the sensor .
  • the operating programs can be designed in such a way that the control electronics control the analysis device to carry out the method described above.
  • the control electronics can also have a user interface, e.g., a touch screen or other display in combination with an input keyboard.
  • the control electronics can be set up to be connected wirelessly by radio or via a data line to another data processing device, e.g. a computer, a process controller, a display or operating device, in particular a portable one, for communication.
  • the analyzer can thus perform on-line determinations of biological target substances in a liquid without requiring manual sampling or preparation or transport of samples to a laboratory.
  • the analysis device can carry out sampling and detection cycles fully automatically, which can be event-controlled by the electronic control system or carried out at predetermined time intervals, for example.
  • FIG. 2 shows an exemplary embodiment of a process unit of the online system shown in FIG.
  • the analysis device 1 shows an exemplary embodiment of an online analysis device 1 for detecting a biological target substance in a liquid according to the invention.
  • the analysis device 1 has a housing 2 in which all the components of the device are accommodated. These components include in detail a process unit 3, a detection unit 4 and control electronics 5.
  • the process unit 3 forms the core of the invention, since it is designed to enrich biological target molecules in a volume of a liquid to be analyzed and for detection and, if necessary, preparatory steps such as Extraction and / or isolation of nucleic acids to the detection unit 4 pass.
  • the process unit 3 is fluidically connected via a sample feed line 6 to a sample receiver 7 or to a process container, e.g. a pipeline or a reactor or a fermenter, in which the liquid to be analyzed is contained.
  • a pump 9 which can be controlled by the electronic control unit 5 according to an operating program stored in the electronic control unit 5 and executed by it, is used to transport and dose the liquid from the sample receiver 7 into a reaction chamber 8 contained in the process unit 3.
  • the reaction chamber 8 can also be fluidically connected to reaction component reservoirs 10, 11.
  • the reaction component reservoirs 10, 11 contain reagents which serve to enrich the biomolecules contained in the liquid.
  • each reaction component reservoir 10, 11 is fluidically connected to the reaction chamber in the process unit 3 via a fluid line.
  • a pump 12 , 13 is used to transport the reaction components through the fluid lines. It is also possible for the reaction components to be integrated directly into the process unit 3 .
  • the transport of the reaction components can, of course, be effected by other means known to those skilled in the art, for example pneumatically or by utilizing hydrostatic pressure.
  • the process unit 3 can optionally have a temperature control unit (not shown in FIG. 1 ), which is used to set a specific temperature in the reaction chamber 8 .
  • the temperature control unit can include resistance heating, cooling and/or thermoelectric elements for selective heating or cooling. It can be connected to the control electronics 5 , the control electronics 5 being set up to control or regulate the temperature in the reaction chamber 8 .
  • the process unit 3 can also have a device 14 for moving or stirring a liquid or liquid mixture contained in the reaction chamber 8 .
  • the device 14 comprises a drive for moving, for example shaking, the reaction chamber 8 .
  • the device 14 in particular the drive, can be controlled by the control electronics 5 .
  • the reaction chamber 8 can be fluidically connected to the detection unit 4 and to a liquid outlet 17 via the fluid line 15 optionally by means of a valve 16 that can be actuated by the control unit 5 .
  • the detection unit 4 has an analysis cartridge in which two microfluidic units fluidically connected to one another via the fluid line 18, namely a first microfluidic unit 19 and a second microfluidic unit 20, are integrated.
  • the microfluidic units can also be accommodated in separate cartridges.
  • the analysis cartridge can be replaced.
  • the first microfluidic unit 19 is set up to receive a sample containing the concentrated biomolecules from the reaction chamber and to prepare it for subsequent amplification and detection in the second microfluidic unit 20 . This can be done by means of extraction and isolation processes known per se.
  • the first microfluidic unit 19 includes reagents and optionally a temperature control unit for incubating reaction mixtures produced in the microfluidic unit 19 .
  • the detection unit comprises suitable means known to those skilled in the art, which can be controlled by the control electronics 5 .
  • the second microfluidic unit 20 includes means for amplification, i.e.
  • the detection unit has a sensor 21 which can, for example, carry out fluorescence measurements in the second microfluidic unit 20 and can output measurement signals to the control electronics 5 .
  • the control electronics 5 are set up to determine a qualitative or quantitative analysis result based on the measurement signals.
  • the control electronics 5 can be a central processing unit, e.g. a CPU with a processor and memory and operating programs stored therein. It can also be divided among several computing units within the analysis device, e.g. the process unit 3 and the detection unit 4 can each have their own on-site electronics, which are connected to higher-level electronics for communication, with the higher-level electronics and the on-site electronics together Control electronics 5 form.
  • a central processing unit e.g. a CPU with a processor and memory and operating programs stored therein. It can also be divided among several computing units within the analysis device, e.g. the process unit 3 and the detection unit 4 can each have their own on-site electronics, which are connected to higher-level electronics for communication, with the higher-level electronics and the on-site electronics together Control electronics 5 form.
  • FIG. 2 shows the process unit 4 of the online analysis device 1 in detail.
  • the structure of the process unit 4 shown in FIG. 2 is only one possible exemplary embodiment. A person skilled in the art can find numerous variants without departing from the spirit of the invention.
  • the process unit 4 has a reaction chamber 8 with various inlets and outlets.
  • the supply lines 22, 23, 24 are connected to storage containers for reagents, which serve as reaction component reservoirs 10, 11.
  • the opening 25 is connected to the sample feed line 6 .
  • a specific volume of the liquid to be analyzed is fed into the reaction chamber 8 via it.
  • a further opening 26 is connected to the fluid line 15 which connects the reaction chamber 8 to a sample outlet and to the detection unit 4 .
  • valves are arranged in the supply lines, which can be actuated by the control electronics.
  • the process unit 3 in the present exemplary embodiment includes a magnet 27 that is movably arranged on the housing of the reaction chamber 8.
  • the process steps according to the invention running in the process unit 3 are as follows.
  • the control electronics 5 controls the pump 9 to transport a first volume of the liquid from the sample template 7 via the sample feed line 6 and the opening 25 into the reaction chamber 8.
  • the Liquid can be, for example, water from a body of water, a water network or from a water treatment process.
  • reaction components are added to the liquid in the reaction chamber 8 via the feed lines 22 , 23 and 24 . This can take place before, during or after the liquid is introduced into the reaction chamber 8 via the sample feed line 6 .
  • magnetic or paramagnetic particles, an alginate solution and a calcium chloride solution are added as reaction components.
  • reaction components make it possible to enrich the biomolecules present in the liquid and to prepare them for a nucleic acid extraction.
  • the method is basically described in DE 10 015215 894 A1. If particles, calcium chloride and alginate are present, it is therefore possible to bind biomolecules, e.g. viruses, bacteria or also free nucleic acids to the particles in the form of an alginate complex. This makes it possible to reduce a large-volume water sample to a much smaller sample volume in such a way that the biomolecules from the first volume of the liquid then accumulate on the particles.
  • a further advantage results from the fact that it is possible with the approach according to the invention to bind bacteria, viruses and also free nucleic acids to one and the same type of particle in a completely unselective manner.
  • no modified particles ie for example particles coupled to an antibody or other ligands, are required for the enrichment of the target substance.
  • methods that use particles modified with antibodies or other ligands for the enrichment of the target substance a large number of different particles are required for the parallel determination of several target substances in a sample. Such a method is significantly more complex and so expensive that its use for a continuous monitoring of biological parameters in process industry applications would not be profitable.
  • the target substance to be detected is a bacterium. Bacteria adsorb non-specifically to small particles. Although this adsorption is not as efficient as the binding in the presence of alginate and calcium ions, the bacteria contained in the liquid can be enriched on the particles in this way, while the free nucleic acids and viruses remain in the liquid and therefore not in the subsequent extraction and analysis steps.
  • biomolecules to the particles, they can be dispersed in the liquid, e.g. by means of a stirring device that can be actuated by the control electronics 5 or by means of a drive that causes the processing unit 3 to move (not shown in FIG. 2).
  • the particles with the bound biomolecules are subsequently sedimented and concentrated by means of the movable magnet 27 .
  • the magnet 27 then moves the particles to the opening 26 on the processing unit 4. This opening 26 is arranged somewhat recessed. The particles are moved into this depression by means of the magnet 27 and placed there.
  • the reaction chamber 8 can be moved with respect to the magnet.
  • the magnet can be a switchable electromagnet, which is arranged in the area of the opening 26 and is switched on by the control electronics 5 in order to attract the particles into the area of the opening 26 by means of its magnetic field.
  • a needle which can also be designed magnetically
  • a pipetting device or a combination of magnet, needle and/or pipetting device is used for the movement or the transfer of the particles.
  • the opening 26 is fluidly connected to the fluid line 15 which can be connected via the valve 16 to the detection unit 4 on the one hand and to the liquid outlet 17 on the other hand.
  • the particles together with a small volume of the liquid contained in the reaction chamber are flushed through the fluidic line 15 into the first microfluidic unit 19 via the fluidic line 15 by means of the control electronics 5 .
  • a further pump, which is controlled by the control electronics 5, can be used for this purpose.
  • the valve 16 is also actuated by the control electronics 5 . Automated nucleic acid extraction and/or isolation takes place in the first microfluidic unit 19, and automated amplification and detection by means of PCR or real-time PCR takes place in the second microfluidic unit 20 connected downstream of the first microfluidic unit 19.
  • Microfluidic units suitable for the method described here for automated nucleic acid extraction, amplification and detection are basically known in the prior art.
  • a centrifugal platform such as that described in WO 2013/045631 A1 and EP 2 621 632, for example, is advantageous for the present application A1 is described, but it is also possible to use other microfluidic platforms or lab-on-chip systems that transport liquids and reagents by means of capillary forces,
  • the nucleic acids are extracted and/or isolated in the first microfluidic unit 19 using known reagents and methods, as already described in the introduction. If the biological target substance is not already freely present in the liquid taken from the sample, the biomolecules bound to the particles are lysed. The free nucleic acids are bound to the particles again. The same particles are therefore used to enrich the biomolecules in the original sample as well as for the subsequent isolation of the released nucleic acids. The particles with the bound nucleic acids are then washed in a known manner and the nucleic acids are finally detached from the particles. The eluate containing the dissolved nucleic acids is transported into the second microfluidic unit 20, again controlled by the control electronics 5.
  • the second microfluidic unit 20 specific volumes of the eluate are transported into one or more detection chambers and amplification reagents are added. An amplification then takes place in the detection chambers, the progress of which is monitored by means of fluorescence measurements with the sensor 21 .
  • the sensor 21 outputs measurement signals to the control electronics 5 . From the measurement signals, this determines either a qualitative value that indicates whether the specific biological target substance is contained in the liquid taken from the sample holder 7 .
  • the control electronics 5 can also determine a quantitative value from the measurement signals, which represents a concentration of the biological target substance in the liquid.
  • different target nucleic acids can be amplified in a number of detection chambers and a number of different biological target substances in the liquid can thus be determined qualitatively or quantitatively in parallel.
  • the liquid remaining in the reaction chamber 8 is discharged from the process unit 3 through the liquid outlet 17 .
  • the process unit 8 can be sufficiently rinsed with a rinsing medium, in particular with liquid sucked in from the sample holder 7, and the process can now be repeated for a further measurement.
  • the microfluidic units 19, 20 in the detection unit can be replaced by new microfluidic units 19, 20 for further measurement.
  • the liquid in an alternative embodiment of the method, which is particularly advantageous if the biological target substance is present in the liquid in a very low concentration, it is possible for the liquid to be discharged from the process unit 3 after the biomolecules contained in the liquid have been enriched by binding to the particles , but the particles are not yet transferred to the detection unit 4 for the subsequent nucleic acid extraction, but remain in the process unit 3 .
  • second volume of liquid from the sample template 7 in the Reaction chamber 8 are initiated.
  • the particles are resuspended in the second volume of liquid and the alginate solution and calcium chloride are again added. This starts a cumulative process that allows a larger volume of liquid to be used and, if necessary, also to increase sensitivity. Such runs can be repeated several times.
  • Example detection of bacteria and free DNA in a water sample
  • a sample volume of 50 ml of the water was transferred via the opening 26 into an exemplary process unit 3 .
  • a volume of 100 ⁇ l of an alginate solution was added to the sample via the opening 24 from a reservoir.
  • the process unit 3 was briefly swiveled for the purpose of mixing.
  • a volume of 100 ⁇ l of a calcium chloride solution from a reservoir was then added to the sample via the opening 23 and the process unit was pivoted again.
  • 50 ml of paramagnetic particles (MAG Suspension; AJ Innuscreen GmbH) from a reservoir were added to the sample via a further opening 22 and the process unit 3 was swiveled again. After an incubation time of 20 min, the particles were collected with the movable magnet 27 and transported to the depression at the outlet opening 26 and fixed there.
  • MAG Suspension AJ Innuscreen GmbH
  • the reaction chamber 8 was subsequently emptied via the outlet opening 26 .
  • the collected particles were removed from the process unit via the outlet opening 26 together with the remainder of the water sample (approx. 200 ml) remaining in the reaction chamber.
  • the particles are transported from this position into the detection unit 4 for extraction and further detection. Here the extraction of the nucleic acid and subsequent specific target detection takes place.
  • the entire process unit 3 is subsequently rinsed with water via the inlet opening 25 and outlet opening 26 and can subsequently be filled with a new sample for the next measurement.
  • the extraction and detection was carried out manually in order to demonstrate that it is possible using the method according to the invention to convert a large sample volume into a small sample volume as an online process and thus enable detection of biological target substances.
  • the extraction was carried out as follows. A lysis buffer (Lysis Solution RL; AJ Innuscreen GmbH) and proteinase K were added to the derived particles.
  • a binding buffer (Binding Solution SBS; AJ Innuscreen GmbH) was added. This binding buffer in combination with the lysis buffer causes the DNA released from the bacteria and the free control fragment DNA to bind to the particles.
  • the batch was mixed briefly and incubated for 2 minutes. The magnetic particles were then separated using a magnet. The supernatant was removed and the magnetic particles were washed with 1 ml of a washing buffer (Washing Solution HS; AJ Innuscreen GmbH). After the magnetic particles had been separated again, the supernatant was completely removed and the magnetic particles were washed twice more with 80% ethanol.
  • the bound DNA was eluted by adding 100 ⁇ l of RNAse-free water and incubating at 60° C. for 5 minutes. After separating the magnetic particles, the supernatant was removed and used in a real-time PCR device for the subsequent detection of the salmonella DNA and the control fragment DNA. Real Time PCR results are shown in the table below.
  • results show that it is possible to bind both bacteria and free DNA to particles from a volume of 50 ml of water, remove the initial water sample and concentrate the particles to a volume of 200 ⁇ l.
  • the process took place in the process unit according to the invention.
  • the particles were then used to extract the nucleic acid and finally the bacteria and free DNA were detected using real-time PCR.
  • the analysis device and method according to the invention thus solves the task in an ideal way by linking available device systems and enrichment technologies and enables online liquid analysis of biological targets to be carried out, which is particularly advantageous for monitoring water or waste water in networks, sewage treatment plants or treatment plants as well as bodies of water appears.
  • the method according to the invention makes it possible for the first time to implement molecular genetic monitoring of biological targets in a practical and economical manner without manual intervention, starting with a large-volume sample up to detection.

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Abstract

L'invention concerne un procédé de détection en ligne automatisée d'au moins une substance cible biologique dans un liquide, en particulier de l'eau ou des eaux usées, au moyen d'un appareil d'analyse en ligne, comprenant les étapes suivantes : - transport d'un premier volume défini du liquide par une conduite d'amenée d'échantillon dans une chambre réactionelle d'une unité de traitement de l'appareil d'analyse en ligne ; - concentration de biomolécules contenues dans le premier volume du liquide par liaison à une pluralité de particules dans la chambre réactionnelle ; - transport des particules avec les biomolécules liées à ces dernières dans une unité de détection de l'appareil d'analyse en ligne, l'unité de détection présentant une première unité microfluidique et une deuxième unité microfluidique pouvant être reliée fluidiquement à la première unité microfluidique ; - libération et/ou isolement d'acides nucléiques à partir des biomolécules liées aux particules ; - transport d'un éluat contenant les acides nucléiques libérés et/ou isolés jusque dans la deuxième unité microfluidique ; - amplification d'un acide nucléique cible dans la seconde unité microfluidique ; - détection d'un signal de mesure qui représente une grandeur de mesure qui dépend de la progression de l'amplification et/ou d'un certain nombre de copies de l'acide nucléique cible dans la deuxième unité microfluidique, et - détermination qualitative et/ou quantitative de la substance cible dans l'échantillon à l'aide du signal de mesure détecté par une électronique de commande de l'appareil d'analyse. L'invention concerne en outre un appareil d'analyse en ligne pour la mise en oeuvre automatisée du procédé.
EP22722145.4A 2021-04-19 2022-04-08 Procédé de détection en ligne automatisée d'au moins une substance cible biologique dans un liquide et analyseur en ligne Pending EP4326438A1 (fr)

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DE102021109852.1A DE102021109852A1 (de) 2021-04-19 2021-04-19 Verfahren zur automatisierten Online-Detektion mindestens einer biologischen Zielsubstanz in einer Flüssigkeit und Online-Analysegerät
PCT/EP2022/059368 WO2022223314A1 (fr) 2021-04-19 2022-04-08 Procédé de détection en ligne automatisée d'au moins une substance cible biologique dans un liquide et analyseur en ligne

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DE102022113316A1 (de) 2022-05-25 2023-11-30 Ist Innuscreen Gmbh Verfahren zur automatisierten online-Detektion mindestens einer biologischen Zielsubstanz in einer Probeflüssigkeit mittels eines Online-Analysegeräts
DE102022119712A1 (de) * 2022-08-05 2024-02-08 Endress+Hauser BioSense GmbH Verfahren zum Einbringen einer aufzukonzentrierenden biologischen Probe mit biologischem Material in eine zentrifugal-mikrofluidische Kartusche

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EP2363205A3 (fr) * 2006-01-11 2014-06-04 Raindance Technologies, Inc. Dispositifs Microfluidiques Et Leurs Procédés D'utilisation Dans La Formation Et Le Contrôle De Nanoréacteurs
DK2001990T3 (en) 2006-03-24 2016-10-03 Handylab Inc Integrated microfluidic sample processing system and method for its use
EP2072133A1 (fr) * 2007-12-20 2009-06-24 Koninklijke Philips Electronics N.V. Dispositif à plusieurs compartiments doté de particules magnétiques
EP2271919A1 (fr) * 2008-04-16 2011-01-12 Cynvenio Biosystems, Inc. Système de séparation magnétique à modules de prétraitement et de post-traitement
US10787701B2 (en) * 2010-04-05 2020-09-29 Prognosys Biosciences, Inc. Spatially encoded biological assays
DE102010041621B4 (de) 2010-09-29 2016-11-03 Hahn-Schickard-Gesellschaft für angewandte Forschung e.V. Verfahren zum Transport magnetischer Partikel
DE102011083920B4 (de) 2011-09-30 2018-07-19 Albert-Ludwigs-Universität Freiburg Verfahren und vorrichtung zum erzeugen von fluidisch voneinander separierten teilvolumina einer flüssigkeit
EP2878375A1 (fr) 2013-11-29 2015-06-03 Genewave Cartouche microfluidique pour diagnostic moléculaire, station d'accueil utilisant une telle cartouche microfluidique et procédé pour analyser un échantillon biologique
US20150224502A1 (en) 2014-02-12 2015-08-13 Monterey Bay Aquarium Research Institute Flow-through cartridge-based system for collecting and processing samples from water
DE102015215894B4 (de) 2015-03-26 2017-11-30 Aj Innuscreen Gmbh Verfahren zur Anreicherung von Biomolekülen und zur Entfernung der Biomoleküle aus einer biologischen Probe
KR101971636B1 (ko) * 2017-07-24 2019-04-24 한국과학기술원 전자동 유전자 판별 통합칩

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