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EP3774044A1 - Procédé et dispositif microfluidique pour aliquoter un échantillon liquide au moyen d'un liquide de scellement, procédé de fabrication d'un dispositif microfluidique et système microfluidique - Google Patents

Procédé et dispositif microfluidique pour aliquoter un échantillon liquide au moyen d'un liquide de scellement, procédé de fabrication d'un dispositif microfluidique et système microfluidique

Info

Publication number
EP3774044A1
EP3774044A1 EP19714577.4A EP19714577A EP3774044A1 EP 3774044 A1 EP3774044 A1 EP 3774044A1 EP 19714577 A EP19714577 A EP 19714577A EP 3774044 A1 EP3774044 A1 EP 3774044A1
Authority
EP
European Patent Office
Prior art keywords
liquid
cavities
sample liquid
microfluidic device
sealing 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
EP19714577.4A
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 EP3774044A1 publication Critical patent/EP3774044A1/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
    • B01L3/502769—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
    • 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
    • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
    • 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
    • B01L2200/0684—Venting, avoiding backpressure, avoid gas bubbles
    • 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
    • B01L2300/16—Surface properties and coatings
    • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
    • 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 is based on a device or a method according to the preamble of the independent claims.
  • Microfluidic analysis systems so-called lab-on-a-chip systems, allow automated, reliable, compact and cost-effective processing of chemical or biological substances for medical diagnostics. By combining a variety of operations for a controlled system
  • Manipulation of fluids can realize complex microfluidic processes.
  • a fundamental operation is the aliquoting of a fluid, which forms the basis for highly multiplexed nucleic acid-based analysis methods, digital PCR applications or single-cell analyzes.
  • the literature has already presented a variety of mechanisms based approaches to aliquoting a fluid. A distinction can be made between droplet-based approaches and those based on the use of a microfluidic aliquoting structure with a plurality of
  • Compartments are based.
  • a monodisperse emulsion of droplets in a second, liquid, immiscible phase is produced and stabilized by the use of suitable surface-active substances, also called surfactants.
  • surfactants also called surfactants.
  • Reaction compartments generated fluidically which is a defined pre-storage of Reagents in the compartments can complicate.
  • the aliquoting takes place in a microfluidic structure, wherein the aliquots, ie the subsets, are generated in well-defined compartments.
  • target-specific reagents can be pre-stored in the individual compartments to enable highly multiplexed analyzes.
  • these approaches have the advantage that the aliquots are localized at defined positions, which allows a simpler evaluation.
  • Microfluidic chips in high throughput processes such as injection molding allow, but usually do not have sufficient gas permeability or elasticity.
  • the solution presented in US 8,895,295 B2 requires evacuation of the cavities.
  • PC polystyrene
  • PP polystyrene
  • PE polystyrene
  • COP polystyrene
  • COC polystyrene
  • PMMA polystyrene
  • some polymers in untreated form have a hydrophobic nature
  • microfluidic structures are required to achieve complete filling of the structures and prevent unwanted entrapment of air.
  • Cavity geometry depending on a contact angle or a wetting behavior of a sample liquid and a geometry of forming an interface on the sample liquid can be completely and reliably filled. This can be omitted evacuation of the cavities or an initial filling with a soluble in the sample liquid gas or using a gas-permeable substrate or a centrifugation perpendicular to a cavity plane. Furthermore, the approach presented here allows a subsequent overlay of the sample liquid with a
  • Microfluidic platform can be integrated and produced inexpensively.
  • a tempering of the sample liquid may be required, for example for carrying out a polymerase chain reaction, in short PCR.
  • gas solubility in liquids generally decreases.
  • the approach presented here optionally allows efficient removal of gas bubbles or on-chip degassing of liquids, so that not completely degassed liquids can be used.
  • Pre-storage of reagents in the cavities may be required.
  • the cavities which form, for example, an array structure
  • Carryover of upstream reagents is very important for the proper functionality of the cavities as it can lead to false positive or false negative results.
  • the approach presented here can significantly reduce such carry-over.
  • sample and sealing liquid are immiscible or only slightly miscible with each other.
  • Microfluidic device has a chamber that over specially shaped
  • Cavities features.
  • the shape of the cavities is designed so that a portion of the sample liquid remains in the cavities after the
  • Sealing liquid has been introduced into the chamber.
  • a retention of the sample liquid in the cavities can be ensured by the different wetting behavior of the sample and sealing liquid as well as the shape of the two-phase interface which forms between the two liquids and the substrate surface.
  • the cavities have a hydrophilic surface finish, so that a filling of the cavities supported by capillary forces takes place. This also allows a filling of cavities, which is a larger
  • the volume of the aliquots can be determined by the structure geometry and the
  • the method is particularly suitable for small cavities with volumes of less than 10 pL, as due to the large surface-to-volume ratio, the
  • Two-phase interface can be well stabilized by the occurring surface energies. This makes it possible to find a suitable process window of the flow rate, which leads only to a small volume variation of the aliquots.
  • Optional reagent pre-storage in the wells allows independent reactions to be performed in the individual aliquots. This makes it possible, for example, to carry out highly multiplexed applications which allow a sample to be examined with regard to a large number of different targets.
  • a suitable additive or embedding the upstream reagents in an additive Carryover of the upstream reagents during filling and sealing can be sufficiently prevented.
  • the thermal stability of the structure can be ensured by an efficient removal of gas bubbles, for example when carrying out a polymerase chain reaction, without completely degassed liquids being required for this purpose. In particular, it can thus be prevented that the formation of gas bubbles
  • Sealing liquid is affected or the sample liquid evaporated from the cavities in gas bubbles and thereby lost from the cavities.
  • a suitable design of the geometry of the microfluidic structures allows them to be completely filled with the sample liquid or to provide a more general microfluidic functionality, which is based on the forming capillary surface or interface on the introduced fluid or between several introduced fluids.
  • an analytical description of the capillary interfaces forming in microfluidic structures is possible at most in individual cases, and the calculation of general capillary interfaces in arbitrary microfluidic geometries by means of numerical methods can be very computationally expensive.
  • Calculation method for the efficient calculation of capillary interfaces described in order to design microfluidic structures with regard to a given microfluidic functionality suitable makes it possible to determine a suitable value range of the parameters by specifying existing contact angles and a class of test structures with suitable parameterization, in order to achieve a desired microfluidic functionality, such as complete filling and defined overlaying with a second fluid.
  • microfluidic functionalities include a complete filling of the cavities or a controlled partial displacement of fluids from the Cavities.
  • the calculation method may be used to properly design a microfluidic cavity array structure such that aliquoting of a fluid to a plurality of cavities may be achieved.
  • the central step of the calculation procedure is based on a geometric description of the progressive
  • Fluid meniscus by circle segments of different curvature which include a fixed angle with the limiting structure. From the model, conditions can be derived from the geometry of the structure, which ensure the desired microfluidic functionality, such as a complete filling up to a certain predetermined contact angle.
  • conditions can be derived that can be fulfilled after complete parameterization of the test structures of a subarea of the parameter space. After the identification of this subarea, the subarea can be used as a starting point in order to design a microfluidic structure appropriately under possible additional given boundary conditions.
  • the calculation method is particularly suitable for the design of structures with surfaces that are not wetted by the fluid, ie, where there is a large contact angle.
  • a suitable geometry of the microfluidic structure can be found without requiring a chemical surface modification of the substrate, ie an adaptation of the wetting behavior.
  • substrates with less suitable surface properties can also be used because they may still provide a given microfluidic functionality by appropriate design of the microfluidic structure.
  • the approach presented here now provides a method for aliquoting a sample liquid using a sealing fluid in a microfluidic device, wherein the sample fluid and the
  • Sealing liquid have different wetting behavior and can be combined with each other or combined into a two-phase system of two separated by an interface phases, the microfluidic device having a chamber with at least one inlet channel for introducing the sample liquid and the sealing liquid and a plurality of the inlet channel having fillable cavities, wherein the inlet channel and the cavities have a defined depending on a respective wetting behavior of the sample liquid and the sealing liquid geometry, wherein the
  • Procedure includes the following steps:
  • Sample liquid suitable, for example, concave is formed to the
  • the meniscus of the sealing liquid by the defined geometry and the present contact angle of the sealing liquid, which in particular exceeds the contact angle of the sample liquid is suitably shaped, for example, convexly formed to the filled cavities with the sealing liquid to overlay.
  • Aliquoting can be understood as a division or portioning of a total sample into several subsets, also called aliquots or aliquots.
  • a sample liquid for example, a body fluid, a PCR master mix or a cell suspension can be understood.
  • the sealing liquid may be, for example Mineral, paraffin or silicone oil, a silicone prepolymer or a fluorinated oil such as Fomblin, Fluorinert FC-40 / FC-70.
  • a wetting behavior can be understood to mean a behavior of liquids on contact with a solid surface. Depending on the type of liquid and depending on the material and nature of the solid surface, the liquid can wet the solid surface more or less.
  • the wetting behavior is characterized by a contact angle, also called edge or wetting angle.
  • An angle of contact can be understood as meaning an amount of liquid for the
  • Amount of liquid and solid surface depends on the
  • a cavity can be understood to mean a depression in a substrate.
  • the cavities may be arranged in an array structure with multiple columns or rows. The cavities can over the
  • Inlet channel fluidly connected to each other.
  • the cavities when introducing a liquid on the
  • Inlet channel to be filled simultaneously or successively with the liquid.
  • cavities, each belonging to one row, can be filled simultaneously, while cavities, each belonging to one column, can be filled one after the other.
  • a defined geometry for example, a defined height, a defined width, a defined length, a defined volume, a defined radius of curvature or another geometric parameter of the
  • Inlet channel and in particular the cavities are understood.
  • Geometry may be defined in particular according to a calculation method described in more detail below depending on the respective wetting behavior of the liquids to be introduced and on the respective material of the inlet channel and the cavities.
  • a meniscus the curvature of a surface of a liquid can be understood, the curvature being due to an interaction between the liquid and a surface of an adjacent wall.
  • a concave meniscus can be understood to mean an inwardly curved surface of the liquid.
  • a convex meniscus can be understood to mean an outwardly curved surface of the liquid.
  • Air bubbles are avoided when the sample liquid flows into the cavities.
  • the forming, for example, convexly shaped meniscus of the sealing liquid makes it possible for the interface, which forms in the cavities between the sample and sealing liquid, to be curved in the direction of a respective bottom of the cavities. Thus, it can be prevented that in the
  • Cavities located subsets of the sample liquid are largely displaced by the inflowing sealing liquid. Furthermore, an escape of the sample liquid from the cavities can be effectively prevented.
  • At least one reagent and / or additive may be introduced into the cavities prior to introduction of the
  • Sample liquid are introduced. Under a reagent can
  • a primer or a probe such as
  • An additive may include an auxiliary or additive such as, for example, polyethylene glycol, xanthan, trehalose, agarose, gelatin,
  • Detection reactions in different subsets of the sample liquid can be carried out targeted and reproducible.
  • the reagent and / or the additive can be dried in the cavities. This allows a long-term stable storage of the reagent or of the additive. Also, by doing so a carryover of the reagent or the additive when filling the cavities are avoided.
  • the reagent in the step of introduction in a first drying step, can be dried in and, in a second drying step following the first drying step, the additive can be dried. This can cause the carryover of the
  • Reagent be reduced to a minimum.
  • the method may include a step of tempering the
  • a reaction temperature can be understood as meaning a predetermined temperature at which certain reactions take place in the sample liquid, for example a polymerase chain reaction or a detection reaction for detecting specific molecules in the sample liquid. This can be used to ensure that gas bubbles that are generated during heating of the sample liquid.
  • Sample liquid can arise, rise quickly and be removed from the cavities.
  • a liquid-conducting portion of the microfluidic device upstream and / or downstream of the cavities can be brought to a degassing temperature for degassing the sample liquid and / or the sealing liquid.
  • a liquid-carrying section can be understood as meaning a section of the device which is fluidically coupled to the cavities, for example in the form of a further chamber or a channel.
  • the liquid-carrying section may comprise a temperature-controlled ventilation chamber.
  • the sealing liquid is introduced at a temperature which is at least as high as a temperature of one located in the cavities Liquid is. As a result, evaporation of the sample liquid in the cavities can be avoided.
  • the approach presented here also provides a microfluidic device for aliquoting a sample fluid using a
  • said microfluidic device comprising: a chamber having at least one inlet channel for introducing the
  • Sample liquid and the sealing liquid and a plurality of fillable via the inlet channel cavities, wherein the inlet channel and the cavities have a defined depending on a respective wetting behavior of the sample liquid and the sealing liquid geometry.
  • the microfluidic device can be realized, for example, as a lab-on-a-chip unit from a suitable substrate such as PC, PP, PE, COP, COC or PMMA. As a result, the device is inexpensive and high
  • the cavities may be rounded.
  • a respective outer edge of the cavities can be formed with a suitable rounding.
  • an inner edge at the respective bottom of the cavities may be rounded in a suitable manner.
  • a respective width of the cavities is greater than a maximum extent of a meniscus of the sample liquid.
  • a maximum extent for example, a maximum width can be understood which the meniscus can assume when it flows into a cavity.
  • the geometry may be defined by the following conditions:
  • d height of a side wall of the cavities.
  • the geometry can be defined with relatively little computational effort.
  • the cavities may have an at least partially hydrophilic surface finish, and / or have divergent geometries and / or different volumes. Due to the hydrophilic surface quality, a better fillability of the cavities with aqueous media can be achieved. As a result, in particular the filling of cavities with a larger aspect ratio of cavity depth to cavity width becomes possible. Furthermore, different reaction volumes can be provided by deviating cavity geometries.
  • the microfluidic device according to a further embodiment, a venting chamber fluidly coupled to the chamber for venting the microfluidic device and a tempering device for heating the Have venting chamber and for degassing the sample liquid and / or the sealing liquid.
  • This embodiment enables a particularly efficient, precisely controllable degassing of introduced liquids outside the cavities.
  • the approach presented here also provides a method for producing a microfluidic device according to one of the preceding embodiments, the method comprising the following steps:
  • the device in the step of forming, may be made of a polymer in a suitable additive manufacturing process such as 3-D printing or stereolithography, a subtractive manufacturing process such as ultrashort pulse laser ablation or micro-milling, or a high-throughput process such as injection molding or thermoforming.
  • a suitable additive manufacturing process such as 3-D printing or stereolithography
  • a subtractive manufacturing process such as ultrashort pulse laser ablation or micro-milling
  • a high-throughput process such as injection molding or thermoforming.
  • microfluidic device according to one of the preceding
  • Embodiments a pumping device for pumping liquids through the chamber of the microfluidic device; and a control device for controlling the pumping device.
  • a control device can be understood as meaning an electrical device which processes sensor signals and, as a function thereof, controls and / or
  • the control unit may have an interface, which may be formed in hardware and / or software.
  • the interfaces may be part of a so-called system ASIC, which performs various functions of the system
  • Control unit includes.
  • the interfaces are their own integrated circuits or at least partially consist of discrete components.
  • the interfaces may be software modules that are present, for example, on a microcontroller in addition to other software modules.
  • FIGS. 1a-c show schematic representations of a microfluidic device according to an embodiment
  • Fig. 2a-c are schematic representations of a microfluidic device
  • FIG. 3 is a schematic representation of a microfluidic device according to an embodiment in plan view
  • Fig. 4a-c are schematic representations of a microfluidic device
  • Fig. 5a-c are schematic representations of a microfluidic device
  • FIG. 6 is a schematic representation of a venting chamber according to an embodiment
  • FIG. 8 is a schematic cross-sectional view of a cavity according to one embodiment
  • Fig. 10 are schematic representations of a cavity and a chamber
  • FIG. 11 shows schematic representations of a cavity and a chamber with unsuitable geometry during a filling process
  • Fig. 12 are schematic representations of a propagation of a
  • Fig. 13 are schematic representations of a propagation of a
  • Fig. 14 are schematic representations of a chamber according to a
  • FIG. 15 shows schematic representations of a chamber from FIG. 14 during a superposing process in plan view
  • 16 is a flowchart of a method of aliquoting according to an embodiment
  • 17 is a flowchart of a method for manufacturing a
  • microfluidic device according to an embodiment
  • Fig. 18 is a schematic representation of a microfluidic system according to an embodiment.
  • Figures la to lc show schematic representations of a microfluidic device 1 according to an embodiment.
  • the device 1 comprises a chamber 100 with at least one inlet channel 101 and at least one outlet channel 102 for introducing or discharging liquids and a plurality of cavities 105 that can be filled via the inlet channel 101
  • Cross-section of the chamber 100 is with one of a respective
  • the cavities 105 filled with the sample liquid 10 are covered with a sealing liquid 20, as shown in FIGS. 2a to 2c.
  • FIGS. 1 a to 1 c Shown by way of example in FIGS. 1 a to 1 c is a cross section through a section of a cavity array structure in a given substrate, such as PC, PP, PE, COP, COC, PMMA, float glass, anodically bondable glass, photoimageable glass, silicon, Metal or a combination of these materials and / or with a modified surface texture, such as a surface having a high biocompatibility.
  • the sample liquid 10 encloses a contact angle qi with the substrate, which permits a complete filling of the cavities 105 with the sample liquid 10.
  • Sample liquid 10 is miscible, so that forms a stable microfluidic interface between the liquids.
  • the sealing liquid is such that it has a contact angle 0 2 to the substrate surface of the filled cavity array structure sufficiently larger than that
  • Contact angle 0i is such that a portion of the sample liquid 10 remains in the cavities 105, as can be seen in FIGS. 2a to 2c.
  • a suitable design of the shape of the cavities 105 for example, according to a calculation method for geometrical design described below Microfluidic structures take place. In this way, a well-defined aliquoting of the sample liquid 10 in the cavities 105 can be achieved.
  • the cavities 105 have rounded portions 106, 108 on their flanks 107.
  • the rounding 108 adjoining a bottom 109 of the cavities 105 inclusion of air in the cavities 105 can be avoided. This is particularly relevant for the case where complete filling of the cavities 105 with a non-wetting liquid having a large contact angle to the substrate is desired.
  • the suitable dimensioning of the fillets 106, 108 also takes place, for example, in the aforementioned calculation method.
  • undesirable pinning of the liquid meniscus may be prevented or at least significantly reduced, which would occur upon abrupt expansion of chamber 100.
  • This pinning is disadvantageous for a complete filling of the cavities 105, since it can lead to abrupt changes in the capillary pressure present and thus also to larger fluctuations in the flow rate during the filling process. These fluctuations can adversely affect the filling behavior.
  • the cavities 105 have a hydrophilic surface finish which allows capillary-assisted filling. Due to the small contact angle qi, the
  • Sample liquid 10 usually an aqueous phase, in this case includes with the substrate, can also be cavities with a larger
  • FIGS. 2 a to 2 c show schematic representations of a microfluidic device 1 from FIG. 1 during a superposing process with the sealing liquid 20. It can be seen that the
  • FIG. 3 shows a schematic illustration of a microfluidic device 1 according to an embodiment in plan view. Shown is a
  • the cavity array structure has, for example, two different ones
  • FIGS. 4a to 4c show schematic representations of a microfluidic device 1 from FIG. 1 with reagents 30, 31 stored in the cavities 105. These are, for example, primers and probes which are present after carrying out a (quantitative) polymerase chain reaction Close the target of specific DNA base sequences in the sample liquid 10 back. As a result of this geometric multiplexing, the sample liquid 10 can be examined for the presence of a multiplicity of different target molecules, depending on the number of cavities. For example, DNA template molecules can also be pre-stored in this way in order to carry out a multiplicity of defined standard amplification reactions as references.
  • the reagents 30, 31 are incorporated in an additive 40, which prevents unwanted in-going and carry over the upstream reagents 30, 31 during the filling of the cavities 105 with the sample liquid 10, before overlaying the aliquots with the sealing liquid 20 takes place.
  • the incorporation of the reagents 30, 31 in the additive 40 takes place, for example, by defined spotting and drying of an aqueous solution of the reagents 30, 31 and the additive 40.
  • the reagents 30, 31 are dried in the cavities 105 and then in a second step, which is carried out after the first step, a spotting and drying of the additive 40.
  • a successive drying is a Significant reduction of the carryover of the reagents 30, 31 possible.
  • the second step is repeated several times
  • the time between filling and sealing should not be too long.
  • a poorly or not water-soluble additive is used, which causes a release of the upstream reagents within the characteristic for the filling process periods only at elevated temperature.
  • FIGS. 5a to 5c show schematic representations of a microfluidic device 1 from FIG. 1 during a degassing process.
  • a temperature control of the sample liquid 10 to a reaction temperature T 2 which is here above an ambient temperature Ti of the device 1.
  • suitable temperature control of the sample liquid 10 for example several independent polymerase Ketenre forceen in the aliquots of the sample liquid 10 are performed. Since the gas solubility of
  • Liquids is temperature dependent and usually decreases with increasing temperature, it is generally necessary when using non-degassed liquids to dissipate leaking gas bubbles 50 in a suitable manner from the aliquots of the sample liquid 10, such as to prevent unwanted evaporation of the sample liquid 10 in the gas bubbles 50 , which can lead to a loss of sample liquid 10 from the cavities 105.
  • the entire structure of the device 1 or at least the chamber 100 is tilted to the direction of action of a gravitational force 60, as shown in Fig. 5b.
  • a gravitational force 60 as shown in Fig. 5b.
  • a force component perpendicular to the plane of the cavities 105 can be used to guide away the forming gas bubbles 50 from the region of the cavities 105.
  • the device 1 is additionally or alternatively offset in a rotational movement, so that the resulting from a centrifugal force 62 buoyancy force 61 a Wegnd the
  • the sealing liquid 20 has a low viscosity, so that leaking gas bubbles have a low fluid resistance and a high mobility in the liquid in order to be able to be discharged efficiently.
  • the apparatus 1 comprises a bubbler which is adapted to effect condensation of precipitating gases at well-defined locations. In this way, a formation of bubbles in the region of the cavities 105 can be prevented.
  • venting chamber 202 shows a schematic representation of a venting chamber 202 according to an exemplary embodiment.
  • the venting chamber 202 is fluidic with the chamber in which the cavities are located, also cavities array chamber and comprises a venting channel 201 coupled to a surrounding atmosphere.
  • a heat source 70 is the
  • Venting chamber 202 heated to a degassing temperature T 3 , which is in particular greater than or equal to the reaction temperature T 2 . In this way, a degassing of the liquids, in particular the
  • the sealing liquid 20 in the venting chamber 202 is degassed before being introduced into the cavity array chamber.
  • the sealing liquid 20 is brought to a temperature which is greater than or equal to the temperature of the sample liquid located in the cavities. In this way, the sample liquid can be prevented from evaporating and condensing at the top of the structure.
  • Thickness of the polymer substrates 0.1 mm to 10 mm, preferably 1 mm to 3 mm;
  • Number of wells for (multiplexed) digital PCR 100-1,000,000, preferably
  • test structures are defined, defined by a set of parameters. This class can be designed so that the test structures contained meet already given boundary conditions to the geometry.
  • Modeling the two-phase interface In the context of this evaluation, an adaptation or extension of the parameter space may become necessary, for example in the event that no entity from the class of the test structures provides the desired microfluidic functionality. After the model-based (iterative) design of the structure, an experimental evaluation of the functionality takes place in the last step of the procedure.
  • FIG. 7 shows a schematic representation 700 of parameters for
  • the central step of the method consists in the two-dimensional geometric description of the phase interface between two not or hardly into each other soluble fluids, such as water and air or water and oil, in a limiting structure as a third, solid phase, such as a polymer such as PC, PP, PE, COP, COC or PMMA, through a circular segment under the
  • Two-phase meniscus and the limiting structure at the three-phase points A, B with each other, can be motivated by the formation of a contact angle, which from the interfacial energies or
  • the predetermined angle Q thus defines the limit of a tolerance range within which the real contact angle may lie, so that the desired microfluidic functionality is provided.
  • the actual contact angle during the filling process may be subject to certain (small) fluctuations, which may be caused by dynamic effects, for example, without limiting the applicability of the method.
  • Fig. 7 the detailed geometric construction of the two-dimensional phase interface in a limiting semi-planar structure is shown.
  • the construction of the two-phase interface is carried out by a circular segment with center M and radius of curvature r in a channel cross-section, which is described by the channel width y and the opening angle - a.
  • the following coordinates and relationships are shown:
  • the radius of curvature can be deduced as a function of the angles a, Q and the local channel width y:
  • the dimensions of the microfluidic structure and the flow velocity should be chosen such that the shape of the
  • FIG. 8 shows a schematic cross-sectional illustration of a cavity 105 according to one exemplary embodiment.
  • Test structures will be a suitably interpreted two-dimensional
  • Channel cross-section viewed with an upper, straight boundary and a lower, arbitrarily shaped boundary. Furthermore, a two-dimensional channel cross-section is considered, which is formed at least in sections mirror-symmetrically to an axis of symmetry perpendicular to the upper, straight
  • Limitation lies in the way that the cavity 105 is formed.
  • One for This problem relevant class of test structures can be defined by the following five parameters: s as a minimum channel width (without forming the cavity),
  • Liquid the circumstance appears that the liquid does not touch both flanks of the cavity 105 before the medium initially present in the cavity 105, such as air, has been displaced from the entire volume which adjoins the bottom of the cavity 105.
  • the presence of this circumstance can be decided from the maximum occurring meniscus tilt, i. H. of a maximum distance t between the three-phase point B and a point A 'on the upper boundary, where A' is given by the orthogonal projection of the
  • 90 °) flank of the cavity 105.
  • 9 shows a schematic representation of a maximum extent of a meniscus in a cavity 105 according to one exemplary embodiment.
  • 9 outlines the maximum meniscus extent that exists (for r 2 ⁇ s + r + d) at the critical point C.
  • the conditions limit the space of geometry parameters to an area in which a complete filling of the structure for a maximum angle Q success s t.
  • the aspect ratios AR 2T ⁇ 2 + W
  • FIG. 10 shows schematic representations of a cavity 105 and a chamber 100 according to an embodiment during a filling process.
  • the panels b to i schematically show eight microscopic images taken during a
  • the scaling bar in panel b corresponds to 200 pm.
  • the micrographs of the microfluidic two-phase interface are in good agreement with the calculated shapes that result from performing the method.
  • the panel j shows a schematic sketch of the plan view of the chamber 100, here in the form of a cavity array, which comprises by way of example 55 hexagonal circular cavities 105 which have a cross-sectional geometry which satisfies the same aspect ratios as the microfluidic formation shown on the left side on the panels a to i.
  • the panels k to n show schematically four micrographs taken during the
  • the scaling bar in panel k corresponds to 500 pm.
  • the field of view of the images in the panels k to n is marked in panel j by a frame.
  • the photographs show a complete homogeneous filling of the cavities 105.
  • FIG. 11 shows schematic representations of a cavity 105 and a chamber 100 of inappropriate geometry during a filling process. Shown are results that result in an unsuitable cavity geometry.
  • this cavity geometry does not fully fill (when the contact angle is sufficiently large) since the meniscus is both Flanks spans the Kavticianenausformung before the air in the cavity 105 has been completely displaced from the cavity 105. This leads to an undesirable inclusion of air in the cavity 105, which prevents complete filling.
  • complete filling of the cavities 105 can not be ensured, as shown by the microscopic images in panels i to I.
  • the scaling bars correspond to 200 pm in panel b and 500 pm in panels g and i.
  • Fig. 12 shows schematic representations of a propagation of a
  • Two-phase interface during a lamination process in a cavity 105 according to an embodiment.
  • the calculation method can also
  • FIG. 12 shows schematically four
  • FIG. 13 shows schematic representations of a propagation of a
  • Two-phase interface during a lamination process in a cavity 105 Shown is an example of an application of the computational method to the design of a cavity that allows aliquoting a fluid by overlaying it with a second fluid that is not miscible with the first fluid.
  • the cavity 105 for example, with a PCR master mix as
  • FIG. 13 shows schematically four micrographs taken during an overlay with oil as
  • the contact angle of the oil, which sets in displacing the PCR master mix, is sufficiently large, so that a part of the PCR master mix in the cavity formation of the
  • microfluidic channel remains and is covered by the oil.
  • the part of the PCR master mix which remains in the cavity formation after being overlaid, ie the enclosed volume, can be adjusted both by the geometry of the cavity and by the contact angle that forms between the two fluids.
  • derived criterion (I) indicates incomplete displacement of the first fluid, resulting in the desired overlay of the first fluid.
  • FIG. 14 shows schematic representations of a chamber 100 according to an exemplary embodiment during a filling process in plan view.
  • FIG. 15 shows schematic representations of a chamber 100 from FIG. 14 during a superposing process in plan view.
  • Figures 14 and 15 show schematically an experimental result for aliquoting a fluid in an array of 55 cavities with a volume of 25 nl each.
  • the cross-sectional geometry of the cavities 105 is designed such that initially a complete filling of the cavities 105 with a PCR master mix is achieved, as shown in FIG. 14, and subsequently a
  • Coating of the cavities takes place by means of mineral oil, as shown in Fig. 15.
  • FIG. 16 shows a flow chart of an aliquoting method 1600 according to an embodiment.
  • the method 1600 can be carried out, for example, by means of a microfluidic device, as described above with reference to FIGS. 1 to 15.
  • a first step 1610 the sample liquid 10 is introduced into the chamber 100.
  • the contact angle q of the sample liquid 10 defined geometry of the chamber 100 more precisely the
  • the meniscus of the sample liquid 10 is suitably shaped, for example, concave or convex, while the liquid 10 flows into the cavities 105. It can thereby be achieved that the cavities 105 are completely filled with the sample liquid 10.
  • the Sealing liquid 20 introduced into the chamber 100.
  • the meniscus of the sealing liquid 20 is shaped differently, for example convexly, by the larger contact angle 0 2 > 0 1 present here and the defined geometry of the chamber 100. It is thereby achieved that subsets of the sample liquid 10 are enclosed in the cavities 105 of the sealing liquid 20.
  • step 17 shows a flow chart of a method 1700 for producing a microfluidic device according to an exemplary embodiment, for example the device described above with reference to FIGS. 1 to 15.
  • wetting information is read in which represents the respective wetting behavior of the sample and sealing liquid, for example its contact angle depending on a material of the chamber of the device.
  • a geometry suitable for the complete filling and sealing of the cavities is defined using the wetting information.
  • the geometry can be selected from a plurality of predetermined, already calculated geometries that are assigned to each different wetting behavior.
  • the chamber is set according to the defined geometry in a suitable
  • Manufacturing process such as an additive or subtractive or a high-throughput process, molded.
  • FIG. 18 shows a schematic representation of a microfluidic system 1800 according to one exemplary embodiment.
  • the system 1800 includes the
  • Device 1 a fluidically coupled to the device 1 pumping device 1802 for pumping the sample and sealing liquid through the chamber of the device 1 and a control unit 1804 for driving the
  • the microfluidic system 1800 thus enables in particular a fully automated aliquoting of the sample liquid by means of the device 1.
  • an exemplary embodiment includes a "and / or" link between a first feature and a second feature, this is to be read such that the Embodiment according to an embodiment, both the first feature and the second feature and according to another embodiment, either only the first feature or only the second feature.

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Abstract

L'invention concerne un procédé d'aliquotage d'un échantillon liquide (10) au moyen d'un liquide de scellement (20) dans un dispositif microfluidique (1). L'échantillon liquide (10) et le liquide de scellement (20) présentent différents comportements au mouillage et peuvent être combinés de manière à former un système à deux phases composé de deux phases séparées l'une de l'autre par une interface. Le dispositif microfluidique (1) comprend une chambre (100) comportant au moins un canal d'entrée (101) pour introduire l'échantillon liquide (10) et le liquide de scellement (20) et une pluralité de cavités (105) pouvant être remplies par l'intermédiaire du canal d'entrée (101), ledit canal d'entrée (101) et les cavités (105) présentant une géométrie définie indépendamment d'un comportement au mouillage respectivement de l'échantillon liquide (10) et du liquide de scellement (20). Selon le procédé, dans un premier temps, l'échantillon liquide (10) est introduit. Un ménisque de l'échantillon liquide (10) se prête, de par la géométrie définie, par ex. concave, à remplir les cavités (105) avec l'échantillon liquide (10). Le liquide de scellement (20) est ensuite introduit, dans une autre étape. Un ménisque du liquide de scellement (20) se prête, de par l'angle de contact présent plus important et la géométrie définie, par ex. convexe, à recouvrir de liquide de scellement (20) les cavités (105) remplies.
EP19714577.4A 2018-03-27 2019-03-25 Procédé et dispositif microfluidique pour aliquoter un échantillon liquide au moyen d'un liquide de scellement, procédé de fabrication d'un dispositif microfluidique et système microfluidique Pending EP3774044A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102018204624.7A DE102018204624A1 (de) 2018-03-27 2018-03-27 Verfahren und mikrofluidische Vorrichtung zur Aliquotierung einer Probenflüssigkeit unter Verwendung einer Versiegelungsflüssigkeit, Verfahren zum Herstellen einer mikrofluidischen Vorrichtung und mikrofluidisches System
PCT/EP2019/057376 WO2019185508A1 (fr) 2018-03-27 2019-03-25 Procédé et dispositif microfluidique pour aliquoter un échantillon liquide au moyen d'un liquide de scellement, procédé de fabrication d'un dispositif microfluidique et système microfluidique

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CN111886075B (zh) 2021-11-30
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US20200406262A1 (en) 2020-12-31
US11565261B2 (en) 2023-01-31
CN111886075A (zh) 2020-11-03

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