EP4076751A1 - Unité réceptrice destinée à recevoir un fluide, procédé et appareil destinés à produire une unité réceptrice, procédé et appareil destinés à commander une unité réceptrice, et dispositif récepteur - Google Patents

Unité réceptrice destinée à recevoir un fluide, procédé et appareil destinés à produire une unité réceptrice, procédé et appareil destinés à commander une unité réceptrice, et dispositif récepteur

Info

Publication number
EP4076751A1
EP4076751A1 EP20841672.7A EP20841672A EP4076751A1 EP 4076751 A1 EP4076751 A1 EP 4076751A1 EP 20841672 A EP20841672 A EP 20841672A EP 4076751 A1 EP4076751 A1 EP 4076751A1
Authority
EP
European Patent Office
Prior art keywords
receiving
receiving unit
microcavities
microcavity
fluid
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
EP20841672.7A
Other languages
German (de)
English (en)
Inventor
Daniel Sebastian Podbiel
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.)
Robert Bosch GmbH
Original Assignee
Robert Bosch 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 Robert Bosch GmbH filed Critical Robert Bosch GmbH
Publication of EP4076751A1 publication Critical patent/EP4076751A1/fr
Pending legal-status Critical Current

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
    • 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
    • B01L3/502769—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 multiphase flow arrangements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
    • B01J2219/00317—Microwell devices, i.e. having large numbers of wells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0642—Filling fluids into wells by specific techniques
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0689—Sealing
    • 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
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0803—Disc shape
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0893—Geometry, shape and general structure having a very large number of wells, microfabricated wells
    • 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
    • B01L2300/16—Surface properties and coatings
    • 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
    • B01L2300/16—Surface properties and coatings
    • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic

Definitions

  • Receiving unit for receiving a fluid for receiving a fluid, method and device for
  • the invention is based on a receiving unit for receiving a fluid, a method and a device for producing a receiving unit, a method and a device for operating a receiving unit and a receiving device according to the preamble of the independent claims.
  • the present invention also relates to a computer program.
  • microfluidic analysis systems so-called lab-on-chips, which enable fully automated analysis of patient samples, are particularly suitable.
  • Complex tests often require several, mutually independent detection reactions to be carried out in order to address different targets in a sample to be examined.
  • the approach presented here enables a more reliable filling of microcavities compared to the state of the art in combination with a simple introduction and pre-storage of reagents dried in the microcavities as well as a risk of carry-over of reagents pre-stored in the microcavities during the controlled filling of the microcavities with a Fluid, for example a sample liquid, avoided.
  • a cross-talk of reactions taking place in the microcavities filled with a fluid is achieved by sealing with a second fluid, i. E. H. a suitable sealing liquid of the microcavities filled with sample liquid, for example in order to carry out different independent amplification reactions in the microcavities, for example for the detection of different DNA targets.
  • a receiving unit for receiving a fluid which has a receiving element with a receiving surface which has at least one microcavity which is arranged in the receiving element on the receiving surface and is shaped to receive the fluid and the one at least in one to the at least one Microcavity adjoining subregion of the receiving surface has a hydrophilic surface quality.
  • the receiving unit can be used, for example, in a receiving device which is designed to test samples, for example.
  • the fluid can be implemented, for example, as a liquid, such as a sample liquid.
  • the sample liquid can be, for example, an aqueous solution, for example obtained from a biological substance, for example of human origin, such as a body fluid, a smear, a secretion, sputum, a tissue sample or a device with attached sample material.
  • the sample liquid contains, for example, species of medical, clinical, diagnostic or therapeutic relevance such as bacteria, viruses, cells, circulating tumor cells, cell-free DNA, proteins or other biomarkers or, in particular, components from the objects mentioned.
  • the sample liquid is a master mix or components thereof, for example for carrying out at least one amplification reaction in the receiving element, for example for DNA detection at the molecular level such as an isothermal amplification reaction or a polymerase chain reaction.
  • the receiving element is formed in particular as a sample carrier, the receiving surface of which is made hydrophilic, for example, at least in a partial area adjoining the at least one microcavity.
  • the microcavity which is arranged in the receiving surface can also be referred to, for example, as a cavity or recess, in particular which is characterized as a cavity with a dimension in the sub-millimeter range.
  • the microcavity can accordingly have a cavity in order to be able to accommodate the fluid.
  • the microcavity can have a surface quality which is inert and has a high biocompatibility in order to carry out, for example, a molecular DNA detection reaction such as an isothermal amplification reaction or a polymerase chain reaction.
  • Capillary and surface forces are important for the functionality of the device, in particular for covering the aqueous phase present in the microcavities with a second immiscible phase. This functionality cannot be guaranteed for large macroscopic cavities.
  • the receiving unit has a plurality of further microcavities which can be arranged in the receiving element on the receiving surface and can be shaped in order to receive the fluid.
  • the microcavity and the plurality of further microcavities can be arranged in an arrangement area, in particular a square, circular, rectangular or oval area of the receiving surface, in particular at a predetermined distance from the edge of the receiving surface, in particular according to a hexagonal, square or rectangular scheme, where in particular between the microcavity and the plurality of further microcavities, the receiving surface has a hydrophilic surface quality.
  • the arrangement area of the microcavities that is particularly relevant for the functionality of the receiving unit and can lead to possible contamination of the surface or the microcavities there.
  • the microcavities By arranging the microcavities according to a hexagonal scheme, a particularly high surface density of the microcavities can be achieved with a constant distance between adjacent microcavities.
  • the cavities By arranging the cavities in a square or rectangular scheme, the cavities can be assigned particularly easily.
  • the receiving unit has further structures, in particular also adjacent to the receiving surface outside the arrangement area of the cavities, which are used to assign or reference the microcavities.
  • These are, for example, adjustment marks for the standardized introduction of reagents into the microcavities by means of an array spotting system, for example a piezo-based fine dispensing system or for the assignment of the cavities in an optical readout device, which, for example, is based on the detection reactions in the microcavities of the Recording unit detects outgoing fluorescence signals.
  • an array spotting system for example a piezo-based fine dispensing system
  • an optical readout device which, for example, is based on the detection reactions in the microcavities of the Recording unit detects outgoing fluorescence signals.
  • different reagents are introduced or held available in the different microcavities so that, for example, different detection reactions can be carried out in the microcavities.
  • the microcavity can have at least one side wall oriented essentially perpendicular to the receiving surface.
  • all of the side walls of the microcavity can also be oriented essentially perpendicular to the receiving surface. This makes it possible, for example, to manufacture the receiving element in a particularly simple manner.
  • the substantially perpendicular side wall (s) can, for example, have an angle of 80 ° to 100 ° with the receiving surface.
  • the Microcavity and / or using an additive which has been introduced into the microcavity - a carryover or discharge of reagents stored in the microcavity, for example, during filling can be reduced to less than 10% of the amount held in the microcavity, for example.
  • DNA target-specific primers and / or probes can be stored upstream in the at least one microcavity in order to carry out at least one specific detection reaction therein.
  • a microcavity contains at least one upstream reagent and / or additive.
  • a “reagent” can be understood to mean a substance which is used to carry out a specific reaction in the microcavity.
  • An “additive”, on the other hand, can be understood to mean a substance that is generally present in several cavities and enables the microcavity to be filled and / or less carryover of upstream reagent. The “additive” is therefore particularly decisive for the fluidic functionality, while the “reagent” is particularly decisive for the precise detection reaction.
  • the at least one upstream reagent can cause a predetermined desired reaction with the fluid, i. H. in particular a sample liquid and in particular certain components of the sample liquid, so-called targets, so that the sample liquid can be examined for the presence of certain features.
  • the receiving unit has several microcavities in which at least two different detection reactions for detecting at least two different targets can be carried out.
  • highly complex molecular diagnostic tests which address a large number of different targets with a large number of different detection reactions, can be carried out in the recording unit.
  • detection reactions with a reduced multiplex performance can be used in order to carry out verifications in the single plex format in the individual fluid partitions in the microcavities (geometric multiplexing).
  • isothermal DNA detection reactions independent of one another can be carried out in the microcavities, which on the one hand have a high reaction rate and on the other hand only a low multiplex compatibility (for example due to undesired interactions between primers and / or probes).
  • a receiving unit with a plurality of cavities can be used in a particularly advantageous manner in order to carry out rapid DNA highly multiplex tests therein using isothermal detection reactions in the single plex format.
  • there is a multiplex pre-amplification in particular by means of polymerase chain reactions, in order to increase the sensitivity of the sample analysis.
  • the detection time for multiplex pre-amplification and the singleplex detection of several DNA targets in the recording unit is less than 60 minutes, the detection time for the singleplex detection of several DNA targets in the recording unit is less than 30 minutes.
  • the receiving unit by means of the receiving unit according to the invention, a very simple and quick examination of the sample liquid for a large number of different targets is possible in a single device, in particular also using detection reactions with a limited multiplex capability.
  • the use of the recording unit also makes it possible to easily adapt multiplex tests, i. E. H.
  • the addition of a detection reaction to a multiplex test is possible, since the detection reactions in the microcavities of the receiving unit take place independently of one another and accordingly no significant interactions between the different primers and probes used in the multiple microcavities can occur.
  • the receiving surface can be designed at least partially as a silicon nitride layer and / or silicon oxide layer and / or as a silane layer, for example as a polyethylene glycol-silane layer.
  • the hydrophilicity of the receiving surface allows the fluid to penetrate into the at least one microcavity can be made possible or significantly improved, such a type of receiving surface being able to be produced with technically simple, inexpensive and well-engineered processes.
  • the receiving element can be used in combination with a microfluidic device in order to enable a fully automated introduction of the fluid into the at least one microcavity of the receiving unit.
  • the receiving element and / or the receiving unit can also be formed from a silicon substrate.
  • the silicon substrate can be implemented as a silicon wafer, for example.
  • material costs can be reduced during production, for example, since substrates of this type are already used in semiconductor technology and production methods of semiconductor technology can therefore be used for the production of the approach presented here.
  • several recording units can be manufactured in parallel by processing a silicon wafer.
  • predetermined breaking points can be introduced into the silicon substrate simultaneously with the etching of the microcavities.
  • the receiving unit can have a further plurality of microcavities, which are located in the receiving element on the
  • the receiving surface can be arranged and shaped to receive the fluid, the further plurality of microcavities in a further arrangement area, in particular a square, circular, rectangular or oval area of the receiving surface, in particular at a predetermined distance from the edge of the receiving surface, in particular after can be arranged in a hexagonal, square or rectangular scheme.
  • a spacing area in which no microcavities are provided can be arranged between the arrangement area and the further arrangement area.
  • several groups of microcavities can be used to carry out a multiplex test, for example if the individual microcavities are provided with different reagents held therein. In this way, for example, a plurality of tests can advantageously be carried out at the same time by, for example, further reagents being upstream in the further plurality of microcavities.
  • the spacing area can have a width which, for example, can correspond to at least twice the minimum spacing between adjacent microcavities of the arrangement area or the further arrangement area.
  • the arrangement areas can thereby be distinguished from one another in a clearly recognizable manner, so that an evaluation of the individual groups of microcavities is facilitated.
  • the spacing area facilitates handling of the chips after the receiving unit has been singulated.
  • the microcavities or groups of microcavities have different dimensions and / or different volumes.
  • different volumes of sample liquid in the individual microcavities ie reaction compartments
  • statistically different numbers of target units for example DNA copies
  • smaller reaction compartments can be used for detection reactions with a high sensitivity
  • larger reaction compartments can be used for detection reactions with a low sensitivity in order to reliably detect different targets in the sample liquid using specific detection reactions with different detection characteristics.
  • a larger range of quantification can be achieved in this way when using a digital detection methodology.
  • the receiving surface can have an optically recognizable feature which can have a predefined position relative to the arrangement of the at least one microcavity, in particular wherein the optically recognizable feature can have a predetermined quality with regard to its size and / or optical properties.
  • a receiving device which has a receiving unit in one of the variants presented above, a housing for receiving the receiving unit, a chamber for introducing at least one fluid, for example a sample liquid, into at least one microcavity of the receiving unit and optionally for the subsequent introduction of a second fluid, i . H. a sealing liquid, which is not or only slightly miscible with the sample liquid and allows a layering / sealing of the sample liquid enclosed in the microcavities of the receiving device, as well as at least one channel which is designed to supply the sample liquid to the microcavities of the receiving unit and then the microcavities to be covered with the sealing liquid and / or to allow ventilation and / or to remove excess sample and sealing liquid.
  • a sealing liquid which is not or only slightly miscible with the sample liquid and allows a layering / sealing of the sample liquid enclosed in the microcavities of the receiving device, as well as at least one channel which is designed to supply the sample liquid to the microcavities of the receiving unit and then the microcavities
  • the housing can be shaped, for example, to protect the receiving unit and the sample liquid from environmental influences and / or, conversely, to prevent contamination of the environment by the sample liquid.
  • the channel can, for example, be realized in the form of a tube or hose and, for example, have an almost rectangular cross section.
  • the receiving device can for example be manufactured inexpensively from a polymer material such as polycarbonate (PC), polypropylene (PP), polyethylene (PE), cycloolefin copolymer (COP, COC), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS) or thermoplastic elastomers (TPE) such as polyurethane (TPU) or styrene block copolymer (TPS) or a combination of polymer materials and are manufactured by high-throughput processes such as injection molding, thermoforming, stamping and / or using of joining technologies such as laser transmission welding.
  • a polymer material such as polycarbonate (PC), polypropylene (PP), polyethylene (PE), cycloolefin copolymer (COP, COC), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS) or thermoplastic elastomers (TPE) such as polyurethane (TPU) or styrene block copo
  • the receiving device can have a pump device which can be designed to pump the at least one fluid, for example the sample liquid and / or the sealing liquid, through the channel.
  • a pump device which can be designed to pump the at least one fluid, for example the sample liquid and / or the sealing liquid, through the channel.
  • the pumping device can, for example, be controlled by a processing unit via a pneumatic interface.
  • the pumping device is based in particular on an elastic membrane which is integrated into the receiving device and can be deflected into recesses within the receiving device by applying an overpressure or underpressure, whereby a controlled displacement of the sample or sealing liquid can be achieved.
  • microfluidic elements such as pump chambers and valves can be implemented.
  • a controlled transport of the sample and sealing liquid can be achieved through a suitable sequential actuation of several elements of the pump device, in particular using peristaltic pump mechanisms.
  • the receiving device has in particular at least one opening for entering the sample and optionally a further opening which serves as a vent.
  • the receiving device has further recesses for pre-storing liquid or solid reagents and a microfluidic network which is used to process the reagents within the receiving device.
  • a method for producing a receiving unit is presented in one of the variants presented above, the method comprising a step of providing and a step of introducing.
  • the receiving surface of the receiving element is made available.
  • the step of introducing the at least one microcavity into the Introduced receiving surface for receiving the fluid in order to produce the receiving unit.
  • a photoresist layer / photoresist can also be applied and / or a lithography substep can be provided in the step of introducing in a partial step, as well as structuring using reactive ion deep etching (Bosch process) to introduce the microcavities (and / or or other optically detectable features).
  • the photoresist can, for example, be spun on and exposed in the lithography step before excess material can be removed.
  • the receiving element can be treated, for example, in such a way that, for example, excess photoresist can be removed.
  • the receiving surface and / or the microcavities can also be coated in an optional step in order to produce a hydrophilic surface quality of the receiving surface and / or microcavities.
  • a surface quality of the receiving surface and / or the microcavities can optionally be changed in such a way that it becomes hydrophilic, for example by using it as a silicon nitride surface or as a silicon oxide surface and / or as a silane layer, for example is formed as a polyethylene glycol-silane layer.
  • reagents are introduced into the microcavity (s) of the receiving unit.
  • the method can include a step of dividing, in which, for example, the receiving element can be divided. The dividing can be achieved in particular by introducing predetermined breaking points in the receiving surface of the receiving element, which is advantageously carried out together with the introduction of the microcavities, and then mechanically breaking them.
  • a method for operating a receiving unit having a step of filling and sealing, a step of performing and a step of evaluation includes.
  • the filling and sealing step at least one microcavity is first filled with a sample liquid and then covered with a sealing liquid as the second fluid, so that a partition of the sample liquid is present as a fluidic reaction compartment in the at least one microcavity.
  • the sealing liquid is, for example, a liquid with a low solubility in water in order to prevent undesired mixing with the sample liquid and / or a low viscosity, high mobility, ie good removal of, for example, a thermal processing of the device To achieve gas bubbles and / or a low thermal conductivity in order to keep the parasitic heat losses occurring during temperature control as low as possible and / or a low heat capacity in order to keep the thermal mass to be processed - for example when carrying out a polymerase chain reaction - as small as possible and / or with contained surfactants in order to stabilize the interface with the sample liquid.
  • the sealing liquid is, for example, a fluorinated hydrocarbon.
  • the carrying out step at least one reaction is carried out in the at least one microcavity in order to obtain a reaction result.
  • the receiving element and in particular the reaction compartment present in the at least one microcavity has, in particular, a predetermined temperature which enables the reaction, for example an isothermal amplification reaction, to proceed.
  • thermal cycling of the receiving device takes place in the step of carrying out, for example in order to carry out a polymerase chain reaction in the at least one reaction compartment.
  • a fluorescence signal emanating from the at least one reaction compartment is also recorded, which allows conclusions to be drawn about the progress of a reaction.
  • the reaction result is evaluated.
  • the step of evaluating takes place on the basis of the fluorescence signal that was recorded in the step of performing. Takes place in an advantageous manner an evaluation of the reaction result already parallel to the implementation of the at least one reaction on the basis of the fluorescence signal curve and the implementation of the reaction is stopped as soon as the reaction result can be determined with sufficient accuracy.
  • An exemplary embodiment of the method presented here is particularly favorable, with mutually independent, in particular different, detection reactions being carried out in the microcavities.
  • a step of multiplex pre-amplification of the sample material and a subsequent detection of targets in the single plex array format can be carried out in a variant of the recording unit presented here.
  • Variants of the methods presented here can be implemented, for example, in software and / or hardware or in a mixed form of software and hardware, for example in a control device.
  • the approach presented here also creates a device which is designed to carry out, control or implement the steps of a variant of one of the methods presented here in corresponding devices.
  • the object on which the invention is based can also be achieved quickly and efficiently by means of this embodiment variant of the invention in the form of a device.
  • the device can have at least one processing unit for processing signals or data, at least one storage unit for storing signals or data, at least one interface to a sensor or an actuator for reading in sensor signals from the sensor or for outputting data or control signals to the Have actuator and / or at least one communication interface for reading in or outputting data, which are embedded in a communication protocol.
  • the computing unit can be, for example, a signal processor, a microcontroller or the like, wherein the storage unit can be a flash memory, an EEPROM or a magnetic storage unit.
  • the communication interface can be designed to read in or output data wirelessly and / or wired, with one communication interface, the wired Data can be fed in or output, this data can for example be fed in electrically or optically from a corresponding data transmission line or can be output in a corresponding data transmission line.
  • a device can be understood to mean an electrical device that processes sensor signals and outputs control and / or data signals as a function thereof.
  • the device can have an interface which can be designed in terms of hardware and / or software.
  • the interfaces can, for example, be part of a so-called system ASIC which contains a wide variety of functions of the device.
  • the interfaces are separate, integrated circuits or at least partially consist of discrete components.
  • the interfaces can be software modules that are present, for example, on a microcontroller alongside other software modules.
  • the device controls a method for operating a recording unit.
  • the device can, for example, access sensor signals such as a read-in signal, which represents information that has been read, and / or a control signal in order to control the steps of one of the methods.
  • the control takes place via actuators such as a read-in unit, an evaluation unit and / or a supply unit.
  • a computer program product or computer program with program code which can be stored on a machine-readable carrier or storage medium such as a semiconductor memory, a hard disk memory or an optical memory, and for carrying out, implementing and / or controlling the steps of the method according to one of the embodiments described above is also advantageous is used, especially when the program product or program is executed on a computer or device.
  • FIG. 1 shows a schematic side illustration of a receiving device according to an exemplary embodiment
  • 2A shows a schematic side illustration of a receiving unit according to an exemplary embodiment
  • FIG. 2B shows a schematic illustration in plan view of a receiving unit according to an exemplary embodiment
  • FIG. 3 shows a schematic side view of a receiving unit according to an exemplary embodiment
  • FIG. 5 shows a flow chart of a method for producing a recording unit according to an exemplary embodiment
  • FIG. 6A shows a perspective illustration of a receiving unit according to an exemplary embodiment
  • FIG. 6B shows a perspective illustration of a receiving unit according to a further exemplary embodiment
  • FIG. 7 shows an illustration to explain the procedure for determining a reaction result of a polymerase chain reaction obtained in a receiving unit according to an exemplary embodiment
  • FIG. 8 shows a representation of a reaction result obtained in a receiving unit according to an exemplary embodiment after a carry-over test
  • 9 shows an illustration to explain the procedure for determining a reaction result of a multiplex test obtained in a recording unit according to an exemplary embodiment
  • FIG. 10 shows a flow chart of a method for operating a recording unit according to an exemplary embodiment
  • FIG. 11 shows a block diagram of a device according to an exemplary embodiment.
  • the receiving device 100 is designed to introduce a fluid into a receiving unit 105 and / or around the receiving unit 105 on the receiving surface 130 at least in partial areas thereof and in particular in the area of the cavities and in particular after the fluid has been introduced into the receiving unit 105 with another fluid to coat the so-called sealing liquid.
  • the receiving device 100 has the receiving unit 105 for receiving the fluid, a housing 110 for receiving the receiving unit 105, a chamber 115 which is designed to introduce the fluid into the receiving unit 105, and at least one channel 120 which is designed to to supply the fluid to the receiving unit 105 and / or to discharge it from the receiving unit 105 and / or to enable the chamber 115 and the microcavities 135, 150 to be vented.
  • the receiving device 100 has a pump device which is designed to pump the fluid and possibly the sealing liquid through the at least one channel 120.
  • the receiving unit 105 has a receiving element 125 with a receiving surface 130 with a hydrophilic surface quality and at least one microcavity 135 which is arranged in the receiving element 125 on the receiving surface 130 and is shaped to receive the fluid.
  • the receiving element 125 is formed from a silicon substrate, for example.
  • the receiving surface 130 is designed, for example, at least partially as a silicon nitride layer, silicon oxide layer and / or as a silane layer, for example as a polyethylene glycol-silane layer, which, for example, facilitates penetration of the fluid into the microcavity 135.
  • the microcavity 135 has side walls 140 which are oriented essentially perpendicularly to the receiving surface 130 and which, for example, are oriented at an angle 145 between 80 ° and 100 ° to the receiving surface 130.
  • the microcavity 135 is shaped to be almost cylindrical.
  • the receiving unit 105 according to this exemplary embodiment has a plurality of further microcavities 150, which are arranged in the receiving element 125 on the receiving surface 130 and are shaped in order to receive the fluid.
  • the microcavity 135 and the plurality of further microcavities 150 are arranged in an arrangement area not shown here, in particular a square, circular, rectangular or oval area of the receiving surface, in particular at a predetermined distance from the edge of the receiving surface, in particular after a hexagonal, square or rectangular scheme, wherein in particular between the microcavity 135 and the plurality of further microcavities 150, the receiving surface 130 has a hydrophilic surface quality.
  • the receiving unit 105 according to this exemplary embodiment has an optically recognizable feature 155 which has a position defined relative to the arrangement of the at least one microcavity 130. This means that the optically recognizable feature 155 according to this exemplary embodiment has a predetermined quality with regard to size and optical properties.
  • a microcavity array chip that is to say the receiving unit 105, is used for aliquoting a fluid, which is also referred to as sample liquid, ie filling the microcavities 135, 150 in the receiving unit 105 with the sample liquid by wetting the receiving surface 130 with the sample liquid and successive wetting of the Receiving surface 130 with a sealing liquid, the sample liquid remaining at least partially in the microcavities 135, 150 of the receiving unit, the implementation of mutually independent reactions in the aliquots, ie the partitions of sample liquid present in the microcavities 135, 150 after an aliquoting of the sample liquid, which in each case can contain specific reagents stored upstream in the microcavities 135, 150, and a method for producing the receiving unit 105 is presented.
  • microcavities 135, 150 a device for distributing a sample liquid to a large number of compartments, which are also referred to as microcavities 135, 150, as well as carrying out a large number of mutually independent reactions in the microcavities 135, 150 the implementation of the reactions, for example in an automated manner, in a microfluidic system which, according to this exemplary embodiment, is designated as receiving device 100.
  • the approach described here also creates a solution which, according to this exemplary embodiment, allows easy introduction and pre-storage of, for example, dried reagents in the microcavities 135, 150 by means of the receiving unit 105, a carryover of the upstream reagents during the controlled distribution of the fluid to the Microcavities 135, 150 are sufficiently reduced, has only very little cross-talk of reactions between the different microcavities 135, 150, enables (automatable) microfluidic aliquoting of the fluid in the microcavities 135, 150, can be produced cost-effectively and / or can be integrated into a receiving device 100 so that fully automated microfluidic processing is achieved.
  • the receiving device 100 has in particular the chamber 115 with advantageously predetermined dimensions 160, which is provided for introducing the fluid into the microcavities 135, 150 and / or for sealing the microcavities 135, 150 with a second fluid that is immiscible with the fluid .
  • the microfluidic chamber 115 has at least one channel 120, which is also referred to as a supply and / or discharge channel and which is provided for a controlled supply and discharge of the fluid or fluids to the receiving unit 105.
  • it further comprises a channel system (not shown here) and / or a pumping device (not shown) in order to enable fully automated microfluidic processing of the receiving unit 105.
  • the receiving unit 105 has the receiving surface 130, which is also referred to as a planar surface and which has an arrangement of microcavities 135, 150 made on the receiving element 125 formed from a substrate material.
  • the receiving surface 130 in particular in the immediate vicinity of the microcavities 135, 150, has hydrophilic wetting properties.
  • the microcavities 135, 150 are characterized in particular by almost vertical side walls 140, with the receiving surface 130 on the microcavities 135, 150, or openings thereof, at an almost 90 ° angle 145 to the side walls 140 of the microcavities 135 , 150 includes.
  • microcavities 135, 150 there is optionally in particular at least one upstream substance, which is also referred to as a reagent or additive.
  • the microcavities 135, 150 optionally have an almost cylindrical shape, which allows the receiving unit 105 to be manufactured in a particularly simple manner.
  • the arrangement of the microcavities 135, 150 follows in particular a square, hexagonal or rectangular scheme in order to optionally enable standardized introduction of reagents into the microcavities, in particular using an array spotting system, in particular a piezo-based fine dispensing system.
  • the receiving surface 130 only optionally has the optically recognizable feature 155 which, for example, has a defined position relative to the arrangement of microcavities (20) and has a suitable quality in terms of size and optical properties.
  • the feature 155 can thereby be detected in particular by an optically sensitive device such as a camera of an array spotting system and for a defined, fully automated introduction of reagents into the arrangement Can be used from micro cavities 135, 150.
  • the feature 155 is used to determine the relative position of the microcavities 135,
  • 150 can be used, in particular in the event that an optical evaluation of the reactions carried out in the microcavities 135, 150 takes place.
  • a receiving unit 105 with a combination of a hydrophilically designed receiving surface 130 with which the fluid comes into contact at least in partial areas for filling the microcavities 135, 150, at least partially vertical side walls 140 of the microcavities 135, 150, which in particular counteract a carryover of reagents stored upstream in microcavities 135, 150, upstream reagents which allow different, specific detection reactions to be carried out in microcavities 135, 150 and / or at least one upstream additive, such as a substance that ensures wetting and complete Filling of the microcavities 135, 150 ensures that no air is trapped in the microcavities 135, 150, and / or leads to a reduction in the carryover of the above-mentioned reagents upstream in the microcavities 135, 150 and a use of cavities 135, 150 with a solid bottom. This means that there are no through holes, so that a preliminary storage of reagents and / or at least one additive in the cavities 135, 150
  • the approach presented here ensures, in addition to a microfluidic functionality with regard to the filling and / or sealing of the reaction compartments, low cross-talk of reactions carried out in the compartments, that is, microcavities 135, 150.
  • the approach presented here describes wetting properties of the receiving surface 130, for example made of silicon nitride, silicon oxide or a hydrophilic silane layer, in particular a polyethylene glycol silane layer, the microcavities 135 (for example with polyethylene glycol as a dried additive and primers and probes for a molecular DNA Detection reaction as a dried reagent and / or a silicon oxide layer, silicon nitride layer or a silane layer as hydrophilic surface) and for example a flow cell made of polymer, for example made of polycarbonate.
  • the receiving surface 130 for example made of silicon nitride, silicon oxide or a hydrophilic silane layer, in particular a polyethylene glycol silane layer
  • the microcavities 135 for example with polyethylene glycol as a dried additive and primers and probes for a molecular DNA Detection reaction as a dried reagent and / or a silicon oxide layer, silicon nitride layer or a silane layer as hydro
  • a component manufactured in an alternative method not described here can also be used to provide the functionalities mentioned here, but in this case the receiving unit 105 is somewhat more complex to manufacture with, for example, two lithography steps than that described here Receiving unit 105 produced approach.
  • FIG. 2A shows a schematic side view of a receiving unit 105 according to an exemplary embodiment.
  • the receiving unit 105 shown here can correspond or be similar to the receiving unit 105 described in FIG. 1.
  • the receiving unit 105 is only shown enlarged so that at least one upstream reagent 200 according to this exemplary embodiment can be seen in the microcavity 135.
  • the receiving unit 105 according to this exemplary embodiment has at least one upstream reagent.
  • a center point of the microcavities 135, 150 has the same distance 205 to an adjacent microcavity 135, 150 as a center point of the optically recognizable element 155 has to the center point of the respectively adjacent microcavity 135,
  • the receiving unit 105 is described, which enables the distribution of the fluid to the microcavities 135, 150 and the implementation of a large number of mutually independent reactions in the microcavities 135, 150, with dried reagents 200 being stored in the microcavities 135, 150. Furthermore, a method for producing the receiving unit 105, which is described in one of the following figures, is presented.
  • the receiving unit 105 allows a reliable introduction of reagents 200 into the microcavities 135, 150, reduces the carryover of the reagents 200 upstream in the microcavities 135, 150 during a distribution of the fluid to the microcavities 135, 150 sufficiently, for example to ⁇ 10%, If the recording device 105 shows only a very small ( ⁇ 1%) cross-talk of reactions between the various microcavities 135, 150 after the microcavities have been sealed with a suitable sealing liquid, it enables automated microfluidic aliquoting of the fluid in the microcavities 135, 150 and is shown in FIG a microfluidic system, like the receiving device 100, can be integrated.
  • the receiving unit 105 thus has the microcavities 135, 150, which are used to form microfluidic compartments.
  • the microcavities 135, 150 have almost vertical side walls, in particular at an interface with a side of the receiving unit 105 that comes into contact with the fluid, and in particular have upstream reagents 200 and a limited aspect ratio, for example to prevent undesired inclusion of air in the microcavities 135, 150 during the filling of the microcavities 135, 150 with the fluid and to enable a complete filling of the microcavities 135, 150 with the fluid.
  • the receiving surface of the receiving unit 105 which comes into contact with the fluid and via which the microcavities 135, 150 are filled, has, according to this exemplary embodiment, a hydrophilic surface quality, in particular in the immediate vicinity of the microcavities 135, 150, around the penetration of the fluid in the microcavities 135, 150 to enable.
  • the receiving unit 105 can be part of a receiving device, as described in FIG. 1, in a particularly advantageous manner, in order, for example, to enable fully automated microfluidic processing and, if necessary, to carry out reactions in the microcavities 135, 150.
  • this ensures reliable filling through the hydrophilic surface properties of the receiving surface of the receiving unit 105 adjoining the microcavities 135, 150, pre-storage of reagents 200 and / or an additive in the microcavities 135, 150 and a suitable aspect ratio of the microcavities 135 , 150 with the appropriately procured fluid.
  • the receiving device that is used to process the receiving unit 105 can be manufactured inexpensively from a polymer or a combination of polymer materials.
  • the functionality provided by the receiving unit 105 is implemented in compact lab-on-chip systems which can be used in molecular laboratory diagnostics.
  • FIG. 2B shows a schematic illustration of the top view of a receiving unit 105 according to an exemplary embodiment.
  • the receiving unit 105 has microcavities 135, 150 which are arranged in a circular arrangement area 600 according to a hexagonal scheme. Furthermore, the outer boundary (marked by the dotted-dashed line) of the arrangement area 600 of the microcavities 135, 150 has a predetermined, minimum distance from the edge of the receiving surface of the receiving unit 105.
  • This edge area can be used in particular to enable simple handling of the receiving unit 105 with an automatic placement machine (pick-and-place robot) and thus, for example, to enable simple manufacture, for example of a receiving device 100 described above.
  • the receiving unit 105 in this exemplary embodiment has optically recognizable features 155, or reference markings referred to differently, which can be used, for example, for a clear assignment and / or symbolic designation of the microcavities 135, 150 and / or, for example, for determining the position of the receiving unit 105 in processing devices can be used with optical detection systems, for example to determine the position in a fine dispensing system for the automated introduction of reagents into the microcavities 135, 150 and / or, for example, to determine the position in a processing device, which can be used by means of an optical system in particular for the detection of fluorescence signals and which, for example can detect the fluorescence signal course of, for example, biochemical reactions in the microcavities 135, 150.
  • FIG. 3 shows a schematic side view of a receiving unit 105 according to an exemplary embodiment.
  • the receiving unit 105 shown here can correspond or be similar to the receiving unit 105 described in FIG. 1 or 2.
  • the illustration enlarged according to this exemplary embodiment is only different so that the optically recognizable element is not shown.
  • FIG. 4 shows a schematic representation of different stages of intermediate products of a possible manufacturing process 400 of a receiving unit 105 according to an exemplary embodiment.
  • the receiving unit 105 can correspond or be similar to the receiving unit 105 described in one of FIGS. 1 to 3 and can therefore also be used in a receiving device as described in FIG. 1.
  • a receiving element 125 made of silicon which is also referred to as a silicon wafer, serves as the substrate material.
  • the hydrophilic surface quality is produced on the substrate material on the receiving surface 130.
  • this is in particular a silicon nitride surface which, for example, by means of a method for the deposition of silicon oxide, silicon nitride and polysilicon, as well as metals, which is also known as low-pressure chemical vapor deposition (LPCVD) vapor deposition) is referred to, can be generated on the silicon substrate.
  • LPCVD low-pressure chemical vapor deposition
  • a layer system of, for example, 50 nm S1O2 and 140 nm S13N4 around a low-stress Si 3 / V 4 layer with a good connection to the silicon substrate is particularly suitable to manufacture.
  • silicon nitride is suitable as a surface coating, since it has, in particular, hydrophilic wetting properties.
  • HMDS hexamethyldisilazane
  • a photoresist 405 is applied, which is also referred to as photoresist and which serves as a mask for etching the microcavities in the silicon substrate.
  • the resist is developed.
  • the S13N4 and S1O2 are removed from the exposed areas 420 by means of CF4 dry etching 415, for example.
  • the microcavities 135, 150 are introduced into the silicon substrate.
  • the reactive ion deep etching 425 is advantageously optimized in terms of process technology for the production of microstructures with almost vertical side walls.
  • the remaining photoresist 405 is removed by treatment in, for example, an oxygen plasma 430.
  • One or more reagents 200 are introduced into the microcavities 135 according to this exemplary embodiment, for example, by means of a piezo-based fine dispensing system or an array spotting system.
  • a production process 400 can take place at wafer level, which enables a particularly cost-effective and parallelized production of the receiving unit 105.
  • the receiving units 105 produced in parallel can be singulated, for example, by sawing, breaking or some other, for example laser-based singling method, such as so-called Mahoh dicing, in particular after the reagents 200 have been introduced into the microcavities 135.
  • FIG. 5 shows a flow chart of a method 500 for producing a recording unit according to an exemplary embodiment.
  • the method 500 shown here can include eight sub-steps 502 according to the production process 400 described in FIG. 4 and produce a receiving unit as it was described in one of FIGS. 1 to 3.
  • the method 500 comprises a step 505 of providing the receiving area and a step 510 of introducing the at least one Microcavity in the receiving surface for receiving the fluid in order to produce the receiving unit.
  • steps 505, 510 and / or partial steps 502 of method 500 can be omitted in an advantageous embodiment and / or carried out in a different order.
  • FIG. 6A shows a perspective illustration of a semifinished product in the production of receiving units 105 according to an exemplary embodiment.
  • the receiving unit 105 shown here can correspond or be similar to the receiving unit 105 described in one of FIGS. 1 to 3.
  • the plurality of further microcavities 150 are formed in order to receive the fluid.
  • the microcavity 135 and the plurality of further microcavities 150 are arranged in an almost square arrangement area 600 in such a way that they follow a square scheme.
  • the receiving surface 130 has a hydrophilic surface quality, in particular between the micro-cavity 135 and the plurality of further micro-cavities 150.
  • the receiving unit 105 in addition to the microcavity 135 and the plurality of further microcavities 150, has a further plurality 605 of microcavities which are shaped to receive the fluid.
  • the further plurality 605 of microcavities is arranged in a further arrangement area 610 in such a way that they form a square, rectangular or hexagonal shape, in particular with a spacing area 615 in which no microcavities are arranged between the arrangement area 600 and the further arrangement area 610 135, 150, 605 are provided.
  • the spacing area 615 has a width that corresponds, for example, to at least twice the minimum spacing between adjacent microcavities of the arrangement area 600 or the further arrangement area 610.
  • a representation of a processed silicon wafer with microcavities 135, 150, 605 is reproduced, for example after carrying out the method described in FIG. 5 for producing a receiving unit 105.
  • FIG. 6B shows a perspective illustration of a silicon substrate with predetermined breaking points introduced to form a plurality of receiving units before the substrate is separated.
  • the receiving units each have a microcavity and a plurality of further microcavities which are arranged in an (almost) circular arrangement area according to a hexagonal scheme. Furthermore, the receiving units each have optically recognizable features, for example for introducing reagents into the microcavities by means of a fine dispensing system and / or for determining the position of the receiving unit in a detection device and / or for clearly naming the microcavities.
  • FIG. 7 shows a representation to explain the procedure for determining a reaction result 700 of a polymerase chain reaction obtained in a receiving unit 105 according to an exemplary embodiment.
  • a reaction result 700 can be obtained in a receiving unit 105, as it was described in one of the FIGS. 1 to 3 presented above.
  • PCR master mix which contained a target gene with a concentration of 10 initial copies per microcavity (25 nl), was used, for example, as the sample liquid, which is also referred to as fluid.
  • the PCR master mix also contained a target-specific TaqMan fluorescence probe (Cy3), which indicates an amplification of the target gene.
  • FIG. 7a schematically illustrates a fluorescence microscope image of the fluid distributed over the microcavities of the receiving unit 105, which can also be referred to as a device, which was made during temperature cycling to carry out the polymerase chain reactions.
  • the microcavities with fluid in which a significant amount of the PCR product has already been generated appear, for example through the cleavage of the fluorescence probe, light.
  • the microcavities without a significant amount of the PCR product appear dark according to this embodiment.
  • FIG. 7b shows a signal profile belonging to the microcavity “F3”, which has a sigmoidal increase which can be attributed to the running of a polymerase chain reaction in this microcavity.
  • 7c shows normalized sigmoidal fitted amplification curves of the individual microcavities combined in a graph.
  • 89 of the 96 microcavities show an increase in the fluorescence signal with a mean ci value, that is, the PCR cycle at the turning point of the sigmoid signal increase, of 31.53 with a standard deviation of 0.81 temperature cycles.
  • 4 microcavities show no significant increase in the fluorescence signal during 50 temperature cycles.
  • the remaining 3 microcavities show an increase in the fluorescence signal at ci values> 45 temperature cycles. 7d illustrates this with the aid of a histogram of the ci values.
  • 7e illustrates this on the basis of a map with a spatial distribution of the ci values in a suitable false color representation.
  • 7c, d, e it becomes clear that in a large part of the microcavities (92.71%) an amplification takes place in a ci value range between 30 and 34 temperature cycles.
  • the fluctuation in the measured ci values can partly be traced back to the statistical fluctuation of the number of copies initially present in the microcavities.
  • a fluctuation between about 2 and 16 initial copies per microcavity, corresponding to the 4 PCR cycles mentioned above, can be assumed.
  • the number of negative cavities cannot be attributed solely to the statistical fluctuation of the number of copies in the cavities based on the binomial distribution. This is where she plays
  • Amplification characteristics of the detection reaction in particular the sensitivity, the limit-of-detection, play a decisive role.
  • the microcavities with a negative signal course can be attributed to the fact that with a low number of copies initially present in a microcavity, there is not always one Amplification occurs.
  • the sensitivity of the selected detection reaction is too low for this.
  • a statistical detection limit for the reaction used here was determined with a so-called limit-of-detection of around 2.5 initial copies per microcavity.
  • the microcavities with a negative signal profile also indicate, according to this exemplary embodiment, that there is no significant copy number generated by means of a PCR in these even after the amplification reaction has progressed in the adjacent microcavities.
  • microcavities can be used as an indicator of cross-talk between adjacent reaction compartments.
  • the 3 microcavities in which a delayed PCR takes place are potentially relevant for this.
  • the delay in the sigmoidal increase by more than 10 PCR cycles can namely not be attributed to the initial statistical fluctuation of the copy numbers according to this exemplary embodiment. Rather, these are possibly delayed-positive or false-positive amplification reactions that may have been initiated by the cross-talk of amplification reactions in neighboring microcavities.
  • the receiving unit 105 is suitable for carrying out (geometrically) multiplexed amplification reactions without significant cross-talk between adjacent reaction compartments, which are also referred to as microcavities.
  • FIG. 8 shows a schematic illustration of a reaction result 800 obtained in a receiving unit 105 according to an exemplary embodiment after a carry-over test.
  • a reaction result 800 can be obtained in a recording unit 105, as it was described in one of the FIGS. 1 to 3 presented above.
  • a carryover of reagents stored upstream in the microcavities is investigated, which may occur during what is known as microfluidic processing of the receiving unit 105, that is, when the microcavities are filled with a fluid in a controlled manner and the microcavities are subsequently sealed with a second fluid .
  • copies of a target gene for example an ABL gene, were introduced into (almost) every second microcavity in the form of a checkerboard pattern by means of a fine dispensing system / array spotting system and, for example, in dried form together with polyethylene glycol (PEG) upstream as an additive (see Fig. 8a).
  • PEG polyethylene glycol
  • FIG. 8b shows a schematic illustration of a fluorescence microscope image which was taken during temperature cycling. The image was taken after a significant increase in the fluorescence signal could already be seen in some microcavities, which indicates the generation of a PCR product.
  • a stronger fluorescence signal can be observed than in the microcavities without template DNA in front (no filling in Fig. 8a) . This consequently indicates a selective amplification and thus only a slight carryover of upstream reagents during the microfluidic processing.
  • FIG. 8c also shows a spatial distribution of the ci values.
  • a reliable amplification with ci values between 26.8 and 28.8 temperature cycles can be observed in the microcavities, in which 100 copies of template DNA were stored in each case. In the other microcavities, however, mostly no amplification can be observed within 50 temperature cycles. Delayed amplification with a delay of more than 4 temperature cycles only takes place in 8 microcavities.
  • the recording unit is therefore suitable
  • target-specific reagents such as primers and probes
  • FIG. 9 shows an illustration to explain the procedure for determining a reaction result 900 of a multiplex test, which is obtained in a recording unit 105 according to an exemplary embodiment.
  • a reaction result 900 can be obtained in a recording unit 105, as it was described in one of the FIGS. 1 to 3 presented above.
  • the reaction result 900 is shown, which emerged from a multiplex test with upstream primers and probes.
  • target-specific primers and probes were stored in front of the receiving unit 105 in 12 microcavities each, for example in dried form together with polyethylene glycol as an additive, which address the two target genes “ABL” and “el3a2”.
  • the probes had fluorophores corresponding to “Cy3” and “Cy5”, as outlined in FIG. 9a.
  • FIGS. 9b, c schematically illustrate two fluorescence recordings that were made before and after thermocycling.
  • the representations shown are each composed of two individual fluorescence microscopic recordings made with the fluorophore Cy3 shown in the horizontal pattern and the filter sets corresponding to the fluorophore Cy5 shown in the vertical pattern.
  • the recordings no significant carryover of reagents stored in the microcavities or cross-talk of reactions that take place in neighboring microcavities can be seen. Only the microcavities with upstream primers and probes show a clear fluorescence signal. Accordingly, according to this exemplary embodiment, the recordings confirm the previous experiments described in FIG.
  • 9d shows the sigmoidal signal curve of the microcavity “G4”, which indicates a positive detection of the ABL template DNA in the sample liquid by means of the polymerase chain reaction.
  • 9e shows a map of a spatial distribution of the ci values.
  • an amplification with ci values in the range between 27.3 and 29.6 can be observed.
  • 9f shows an associated graph with the normalized amplification curves of the twelve microcavities.
  • the measurement underlines the excellent suitability of the recording unit 105 for performing geometrically highly multiplexed sample analyzes by means of molecular diagnostic amplification methods.
  • FIG. 10 shows a flow chart of a method 1000 for operating a recording unit according to an exemplary embodiment.
  • the method 1000 can be used, for example, for a recording device as described in FIG. 1.
  • the method 1000 comprises a step 1005 of filling and sealing the at least one microcavity with a fluid or a second (sealing) fluid which, for example, is immiscible or only slightly miscible with the fluid, a step 1010 of performing at least one possible reaction in the at least one microcavity in order to obtain a reaction result, and a step 1015 of evaluating the reaction result.
  • the microcavities of the receiving unit are filled with the fluid.
  • the microcavities that have previously been filled with the fluid are then sealed with a second (sealing) fluid which is not or only very slightly miscible with the fluid.
  • the second fluid which is also referred to as the sealing liquid, is a fluorinated hydrocarbon.
  • mutually independent reactions are carried out, in particular amplification reactions, such as polymerase chain reactions or isothermal amplification reactions, for example for the detection of at least one target gene in the microcavities of the receiving unit.
  • suitable reaction conditions for this purpose are established by external action, for example heat input or heat dissipation.
  • steps 1005, 1010, 1015 take place automatically in a processing unit which is provided for processing the receiving device.
  • steps of the method 1000 can be omitted in an advantageous embodiment and / or carried out in a different order.
  • FIG. 11 shows a block diagram of a device 1100 according to an exemplary embodiment.
  • the device 1100 is designed to carry out or control one of the methods described in FIG. 5 or 10.
  • the device 1100 is designed to read in an input signal 1105, for example by means of a read-in unit 1110, and to provide a control signal 1115 by means of a supply unit 1120.
  • the device optionally has an evaluation unit 1125 which is designed to evaluate information represented by the read-in signal 1105.
  • an exemplary embodiment comprises an “and / or” link between a first feature and a second feature, this is to be read in such a way that the exemplary embodiment according to one embodiment has both the first feature and the second feature and, according to a further embodiment, either only the has the first feature or only the second feature.
  • nl 1 nl to 100 nl, preferably 5 nl to 40 nl
  • Target-specific primers and probes template DNA
  • PEG polyethylene glycol
  • Master mix for an amplification reaction such as a PCR or an isothermal amplification method or components thereof, in particular master mix without primers and / or probes which are present in the microcavities (135, 150)
  • Second fluid Fluorinated hydrocarbons such as 3M Fluorinert FC-40, Fluorinert FC-70, or Novec 7500
  • the chamber (115) having suitable dimensions such as 7mm x 7mm x 1mm (volume ⁇ 50mI):

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Abstract

L'invention porte sur une unité réceptrice (105) destinée à recevoir un fluide. L'unité réceptrice (105) a un élément récepteur (125) pourvu d'une face réceptrice (130) et d'au moins une microcavité (135) qui est agencée et formée dans l'élément récepteur (125) sur la face réceptrice (130) afin de recevoir le fluide. La face réceptrice (130) a en outre une caractéristique de surface hydrophile dans au moins une sous-zone joignant la ou les microcavités (135).
EP20841672.7A 2019-12-18 2020-12-17 Unité réceptrice destinée à recevoir un fluide, procédé et appareil destinés à produire une unité réceptrice, procédé et appareil destinés à commander une unité réceptrice, et dispositif récepteur Pending EP4076751A1 (fr)

Applications Claiming Priority (2)

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DE102019220017.6A DE102019220017A1 (de) 2019-12-18 2019-12-18 Aufnahmeeinheit zum Aufnehmen eines Fluids, Verfahren und Vorrichtung zum Herstellen einer Aufnahmeeinheit, Verfahren und Vorrichtung zum Betreiben einer Aufnahmeeinheit und Aufnahmeeinrichtung
PCT/EP2020/086753 WO2021122980A1 (fr) 2019-12-18 2020-12-17 Unité réceptrice destinée à recevoir un fluide, procédé et appareil destinés à produire une unité réceptrice, procédé et appareil destinés à commander une unité réceptrice, et dispositif récepteur

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US (1) US20230017412A1 (fr)
EP (1) EP4076751A1 (fr)
JP (1) JP2023507599A (fr)
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CN (1) CN114786815A (fr)
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DE102021200588A1 (de) 2021-01-22 2022-07-28 Robert Bosch Gesellschaft mit beschränkter Haftung Verfahren und Steuergerät zum Herstellen eines Trägerelements zum Aufnehmen einer Probenflüssigkeit, Trägerelement und Analysevorrichtung mit Trägerelement
DE102022202862A1 (de) * 2022-03-24 2023-09-28 Robert Bosch Gesellschaft mit beschränkter Haftung Mikrofluidisches Aufnahmeelement, mikrofluidische Vorrichtung mit Aufnahmeelement, Verfahren zum Herstellen eines mikrofluidischen Aufnahmeelements und Verfahren zum Verwenden eines mikrofluidischen Aufnahmeelements
DE102022209416B3 (de) 2022-09-09 2023-12-21 Robert Bosch Gesellschaft mit beschränkter Haftung Mikrofluidische Vorrichtung

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DE102019220017A1 (de) 2021-06-24
US20230017412A1 (en) 2023-01-19

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