Abstract
A method for aliquoting a sample liquid using a sealing liquid in a microfluidic device includes combining the sample liquid and the sealing liquid, which have different wetting behaviors, to form a two-phase system separated by a boundary surface. The microfluidic device includes a chamber with at least one inlet channel for introducing the liquids and a plurality of cavities configured to be filled via the inlet channel. The inlet channel and the cavities have a geometry that is defined in dependence on the respective wetting behaviors of the sample liquid and the sealing liquid. The method first includes introducing the sample liquid to form a first meniscus configured by the defined geometry, e.g. concave, to fill the cavities. The method further includes introducing the sealing liquid to form a second meniscus configured by the existing, greater contact angle and the defined geometry, e.g. convex, to cover the filled cavities.
Claims
1. A method of aliquoting a sample liquid using a sealing liquid in a microfluidic device, the sample liquid and the sealing liquid having different wetting characteristics and being configured to be combined with one another to form a biphasic system composed of two phases separated from one another by an interface, the microfluidic device having a chamber with at least one inlet channel configured for introduction of the sample liquid and of the sealing liquid and with a multitude of cavities configured to be filled via the at least one inlet channel, the at least one inlet channel and the cavities having a geometry defined depending on the respective wetting characteristics of the sample liquid and of the sealing liquid, the method comprising: introducing a sample liquid, having a contact angle wetting characteristic, such that a meniscus of the sample liquid is suitably shaped by the defined geometry and the contact angle present in the sample liquid in order to fill the cavities with the sample liquid; and introducing a sealing liquid, having a contact angle wetting characteristic greater than the contact angle of the sample liquid, after the sample liquid has been introduced such that a meniscus of the sealing liquid, by virtue of the contact angle present in the sealing liquid, and the defined geometry, is suitably shaped in order to blanket the filled cavities with the sealing liquid.
2. The method as claimed in claim 1, further comprising introducing one or more of at least one reagent and at least one additive into the cavities prior to the introduction of the sample liquid.
3. The method as claimed in claim 2, wherein the one or more of the at least one reagent and the at least one additive is dried in the cavities in the introduction.
4. The method as claimed in claim 3, wherein introducing further includes: a first drying process in which the reagent is dried, and a second drying process in which the additive is dried, the second drying process following the first drying process.
5. The method as claimed in claim 1, further comprising: adjusting a temperature of the sample liquid to a reaction temperature; and one or more of putting the chamber in an oblique position and setting the chamber in a rotating motion.
6. The method as claimed in claim 1, further comprising heating a liquid-guiding section of the microfluidic device one or more of upstream and downstream of the cavities to a degassing temperature so as to degas one or more of the sample liquid and the sealing liquid.
7. The method as claimed in claim 1, wherein introducing the sealing liquid includes introducing the sealing liquid at a temperature at least as high as a temperature of a liquid present in the cavities.
8. A microfluidic device for aliquoting a sample liquid using a sealing liquid, the sample liquid and the sealing liquid having different wetting characteristics and being configured to be combined with one another to form a biphasic system composed of two phases separated from one another by an interface, the microfluidic device comprising: a chamber having (i) at least one inlet channel configured to introduce the sample liquid and the sealing liquid and (ii) a multitude of cavities configured to be filled via the at least one inlet channel, the at least one inlet channel and the cavities having a geometry defined depending on the respective wetting characteristics of the sample liquid and the sealing liquid, wherein the geometry of the at least one inlet channel and the cavities is defined using wetting information representative of the wetting characteristics of the sample liquid and the wetting characteristics of the sealing liquid, the chamber with the at least one inlet channel and the cavities configured in accordance with the defined geometry.
9. The microfluidic device as claimed in claim 8, wherein the cavities are rounded.
10. The microfluidic device as claimed in claim 8, wherein a respective width of the cavities is greater than a maximum extent of a meniscus of the sample liquid.
11. The microfluidic device as claimed in claim 8, wherein the cavities have one or more of at least partly hydrophilic surface characteristics, different geometries, and different volumes.
12. The microfluidic device as claimed in claim 8, further comprising: a deaeration chamber fluidically coupled to the chamber and configured to deaerate the microfluidic device; and a temperature controller configured to heat the deaeration chamber and to degas one or more of the sample liquid and the sealing liquid.
13. A microfluidic system, comprising: a microfluidic device configured to aliquot a sample liquid using a sealing liquid, the sample liquid and the sealing liquid having different wetting characteristics and being configured to be combined with one another to form a biphasic system composed of two phases separated from one another by an interface, the microfluidic device including a chamber that has (i) at least one inlet channel configured to introduce the sample liquid and the sealing liquid and (ii) a multitude of cavities configured to be filled via the at least one inlet channel, the at least one inlet channel and the cavities having a geometry defined depending on the respective wetting characteristics of the sample liquid and the sealing liquid, wherein the geometry of the at least one inlet channel and the cavities is defined using wetting information representative of the wetting characteristics of the sample liquid and the wetting characteristics of the sealing liquid, the chamber with the at least one inlet channel and the cavities configured in accordance with the defined geometry; a pump unit configured to pump liquids through the chamber of the microfluidic device; and a controller configured to actuate the pump unit.
14. The method of claim 1, wherein: the defined geometry of the inlet channel and cavities and the contact angle present in the sample liquid produce a concave meniscus within the cavities; and the defined geometry of the inlet channel and cavities and the contact angle present in the sealing liquid produce a convex meniscus within the cavities.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) Working examples of the disclosure are shown in the drawings and elucidated in detail in the description that follows. The figures show:
(2) FIGS. 1a-c schematic diagrams of a microfluidic device in one working example;
(3) FIGS. 2a-c schematic diagrams of a microfluidic device from FIG. 1 during a layering process;
(4) FIG. 3 a schematic diagram of a microfluidic device in one working example in top view;
(5) FIGS. 4a-c schematic diagrams of a microfluidic device from FIG. 1 with incorporated reagents;
(6) FIGS. 5a-c schematic diagrams of a microfluidic device from FIG. 1 during a degassing process;
(7) FIG. 6 a schematic diagram of a deaeration chamber in one working example;
(8) FIG. 7 a schematic diagram of parameters for two-dimensional geometric description of a phase interface in a microfluidic device in one working example;
(9) FIG. 8 a schematic cross-sectional diagram of a cavity in one working example;
(10) FIG. 9 a schematic diagram of a maximum extent of a meniscus in a cavity in one working example;
(11) FIG. 10 schematic diagrams of a cavity and a chamber in one working example during a filling process;
(12) FIG. 11 schematic diagrams of a cavity and a chamber with unsuitable geometry during a filling process;
(13) FIG. 12 schematic diagrams of a propagation of a biphasic interface during a layering process in a cavity in one working example;
(14) FIG. 13 schematic diagrams of a propagation of a biphasic interface during a layering process in a cavity in one working example;
(15) FIG. 14 schematic diagrams of a chamber in one working example during a filling process in top view;
(16) FIG. 15 schematic diagrams of a chamber from FIG. 14 during a layering process in top view;
(17) FIG. 16 a flow diagram of a method of aliquoting in one working example;
(18) FIG. 17 a flow diagram of a method of producing a microfluidic device in one working example; and
(19) FIG. 18 a schematic diagram of a microfluidic system in one working example.
DETAILED DESCRIPTION
(20) In the description of advantageous working examples of the present disclosure that follows, similar or identical reference signs are used for the elements having a similar effect that are shown in the different figures, dispensing with repeated description of these elements.
(21) FIGS. 1a to 1c show schematic diagrams of a microfluidic device 1 in one working example. The device comprises a chamber 100 having at least one inlet channel 101 and at least one outlet channel 102 for introduction and discharge of liquids, and a multitude of cavities 105 fillable via the inlet channel 101. A cross section of the chamber 100 is shaped with a geometry defined by the respective wetting characteristics of the liquids introduced. FIGS. 1a and 1b show the propagation of a sample liquid 10 on introduction into the chamber 100. It can be seen how the cavities 105 are filled completely owing to the concave meniscus of the sample liquid 10 which is curved counter to a flow direction. Subsequently, the cavities 105 filled with the sample liquid 10 are layered with a sealing liquid 20, as shown in FIGS. 2a to 2c.
(22) Shown by way of example in FIGS. 1a to 1c is a cross section through a section of a cavity array structure in a given substrate, for instance made of PC, PP, PE, COP, COC, PMMA, float glass, anodically bondable glass, photostructurable glass, silicon, metal or a combination of these materials and/or with modified surface properties, for instance with a surface having high biocompatibility. The sample liquid 10 forms a contact angle θ.sub.1 with the substrate that permits complete filling of the cavities 105 with the sample liquid 10.
(23) After the structure has been filled with the sample liquid 10, in a second step, the filled cavities 105 are layered with the sealing liquid having zero or only low miscibility with the sample liquid 10, such that a stable microfluidic interface is formed between the liquids. The properties of the sealing liquid are such that it has a contact angle θ.sub.2 with respect to the substrate surface of the filled cavity array structure which is greater than the contact angle θ.sub.1 to a sufficient degree that a portion of the sample liquid 10 remains in the cavities 105, as apparent from FIGS. 2a to 2c. The cavities 105 can be suitably designed, for instance, by a calculation method described hereinafter for geometric design of microfluidic structures. In this way, it is possible to achieve well-defined aliquoting of the sample liquid 10 in the cavities 105.
(24) In one working example, the cavities 105 have roundings 106, 108 on their flanks 107. By means of the rounding 108 adjoining a base 109 of the cavities 105, it is possible to prevent trapping of air in the cavities 105. This is especially relevant if the aim is for the cavities 105 to become filled with a non-wetting liquid having a large contact angle with respect to the substrate. Suitable dimensions of the roundings 106, 108 are likewise found, for example, in the calculation method just mentioned. By means of the rounding 106 adjoining the chamber 100, it is possible to prevent or at least significantly reduce unwanted pinning of the liquid meniscus, which would occur in the case of abrupt widening of the chamber 100. This pinning is disadvantageous for complete filling of the cavities 105 since it can lead to abrupt changes in the capillary pressure present and hence also to relatively large variations in the flow rate during the filling process. These variations can have an adverse effect on the filling characteristics.
(25) In a particularly advantageous working example, the cavities 105 have hydrophilic surface properties that permit capillary-assisted filling. Owing to the small contact angle θ.sub.1 that the sample liquid 10, generally an aqueous phase, forms with the substrate in this case, it is still possible to fill even cavities having a relatively high aspect ratio completely with an aqueous phase. This is advantageous in turn since a relatively small contact angle θ.sub.2 of the sealing liquid is then already sufficient for a portion of the sample liquid 10 not to be displaced from the cavities 105. This allows the use of various fluids as sealing liquid.
(26) FIGS. 2a to 2c show schematic diagrams of a microfluidic device 1 from FIG. 1 during a layering process with the sealing liquid 20. It can be seen that the sealing liquid 20, by contrast with the sample liquid 10, has a convex meniscus, i.e. curved in flow direction, defined by the contact angle θ.sub.2. As a result, an interface forms between the liquids superposed in the cavities 105, curved in the direction of a respective base of the cavities 105.
(27) FIG. 3 shows a schematic diagram of a microfluidic device in a working example in top view. What is shown is a microscope image of a cavity array structure that has been filled first with a dark-colored aqueous solution as sample solution and then with a colorless liquid, shown in a light color here, as sealing liquid, which is miscible with the aqueous phase only to a small degree, if at all, such that the dark-colored liquid remains in the cavities and hence aliquoting of the dark-colored liquid is achieved. Depending on the geometric shape of the cavities, the volume of the individual aliquots of the first liquids can be adjusted.
(28) The cavity array structure has, by way of example, two different cavity geometries that correspond to two different volumes of the aliquots. The color contrast present in the microscope image results from the different volumes of the aliquots. By suitable arrangement of the two different cavity shapes, by way of example, the pattern of a double-T anchor in top view was replicated.
(29) FIGS. 4a to 4c show schematic diagrams of a microfluidic device 1 from FIG. 1 with reagents 30, 31 incorporated in the cavities 105. These are, for instance, primers and probes which, after performance of a (quantitative) polymerase chain reaction, allow the conclusion of the presence of target-specific DNA base sequences in the sample liquid 10. This geometric multiplexing allows analysis of the sample liquid 10, depending on the number of cavities, for the presence of a multitude of different target molecules. For example, it is also possible in this way to pre-store DNA template molecules in order to perform a multitude of defined standard amplification reactions as references. By comparison of fluorescence signals of the amplification reactions in the aliquots of the sample liquid 10 with signals of standard amplification reactions, it is possible to conclude the starting amounts of the targets in the sample liquid 10.
(30) In a further working example, the reagents 30, 31 are incorporated in an additive 40 that prevents unwanted going-into-solution and entraining of the pre-stored reagents 30, 31 during the filling of the cavities 105 with the sample liquid 10 before layering of the aliquots with the sealing liquid 20. The reagents 30, 31 in the additive 40 are incorporated, for instance, by defined spotting and drying of an aqueous solution of the reagents 30, 31 and the additive 40.
(31) In a further working example, in a first step, the reagents 30, 31 are dried in the cavities 105 and then, in a second step which is executed after the first step, the additive 40 is spotted and dried. Such successive drying enables a significant reduction of the entrainment of the reagents 30, 31.
(32) In one working example, the second step is executed repeatedly in succession. In this way, it is possible to achieve particularly stable incorporation of the reagents 30, 31 in the additive 40.
(33) By addition of a suitable additive and the choice of a suitable process regime, it is possible to prevent unwanted entrainment. More particularly, the time between filling and sealing should not be too long. For example, an additive of sparing or zero water solubility that brings about release of the pre-stored reagents within the periods characteristic of the filling process only at elevated temperature is used.
(34) FIGS. 5a to 5c show schematic diagrams of a microfluidic device 1 from FIG. 1 during a degassing process. The temperature of the sample liquid 10 is adjusted here to a reaction temperature T.sub.2, which is above an ambient temperature T.sub.1 of the device 1 here. By suitable control of the temperature of the sample liquid 10, it is possible, for example, to perform multiple independent polymerase chain reactions in the aliquots of the sample liquid 10. Since the gas solubility of liquids is temperature-dependent and usually decreases with rising temperature, it is generally necessary in the case of use of incompletely degassed liquids to remove gas bubbles 50 that precipitate out from the aliquots of the sample liquid 10 in a suitable manner, for instance in order to prevent unwanted evaporation of the sample liquid 10 into the gas bubbles 50, which can lead to a loss of sample liquid 10 from the cavities 105.
(35) For avoidance of gas bubbles 50, for example, the entire structure of the device 1 or at least the chamber 100 is in a tilted alignment relative to the direction of action of a gravitational force 60, as shown in FIG. 5b. For instance, a resulting buoyancy force 61 acting on the gas bubbles 50 and in particular a force component at right angles to the plane of the cavities 105 may be utilized in order to remove the gas bubbles 50 that form from the region of the cavities 105.
(36) In a further working example, the device 1 is additionally or alternatively set in a rotating motion, such that the buoyancy force 61 resulting from a centrifugal force 62 makes it possible to conduct the gas bubbles 50 away. This is shown in FIG. 5c.
(37) It is particularly advantageous when the sealing liquid has a low viscosity, such that precipitating gas bubbles have a low fluidic resistance and high mobility in the liquid, in order to be able to be efficiently removed.
(38) Optionally, the device 1 has a bubble formation unit designed to bring about condensation of precipitating gases at well-defined sites. In this way, it is possible to prevent bubble formation in the region of the cavities 105.
(39) FIG. 6 shows a schematic diagram of a deaeration chamber 202 in one working example. The deaeration chamber 202 is fluidically connected to the chamber in which the cavities are present, also called cavity array chamber, and comprises a deaeration channel 201 coupled to a surrounding atmosphere. By means of a heat source 70, the deaeration chamber 202 is heatable to a degassing temperature T.sub.3 which is especially greater than or equal to the reaction temperature T.sub.2. In this way, degassing of liquids, especially of the sealing liquid 20, in the deaeration chamber 202 is achieved, such that unwanted bubble formation in the cavity array chamber is avoided.
(40) In one working example, the sealing liquid 20 in the deaeration chamber 202 is degassed before being introduced into the cavity array chamber.
(41) In a further working example, the sealing liquid 20 is brought to a temperature greater than or equal to the temperature of the sample liquid present in the cavities. In this way, it is possible to prevent evaporation of the sample liquid and condensation on the top side of the structure.
(42) Illustrative dimensions of the device 1 are listed hereinafter: thickness of the polymer substrates: 0.1 mm to 10 mm, preferably 1 mm to 3 mm; channel cross sections: 10×10 μm.sup.2 to 3×3 mm.sup.2, preferably 100×100 μm.sup.2 to 1×1 mm.sup.2; dimensions of the chamber: 1×1×0.3 mm.sup.3 to 100×100×10 mm.sup.3, preferably 3×3×1 mm.sup.3 to 30×30×3 mm.sup.3; lateral dimensions of the overall system: 10×10 mm.sup.2 to 200×200 mm.sup.2, preferably 30×30 mm.sup.2 to 100×100 mm.sup.2; number of cavities for (multiplex) digital PCR: 100-1 000 000, preferably 1000-100 000; volume of cavities for (multiplex) digital PCR: 1 p1 to 1 μl, preferably 10 pl to 100 μl; number of cavities for multiplex (quantitative) PCR: 2-1000, preferably 10-100; volume of cavities for multiplex (quantitative) PCR: 10 pl to 10 μl, preferably 100 pl to 1 μl.
(43) There follows a description of a calculation method for design of the geometry of the chamber and the cavities of the microfluidic device described above.
(44) This involves first fixing a class of test structures which is defined by a set of parameters. The characteristics of this class may be such that the test structures present meet existing boundary conditions with regard to the geometry.
(45) In the next step, the microfluidic functionality of the test structures is evaluated by calculation by modeling of the biphasic interface described hereinafter. In the course of this evaluation, adjustment or extension of the parameter space may become necessary, for instance if no entity from the class of the test structures provides the desired microfluidic functionality. According to the model-based (iterative) interpretation of the structure, in the last step of the method, the functionality is evaluated experimentally.
(46) On the basis of the experimental result, it may be the case that a further adjustment or extension of the parameter space that describes the structure geometry is required. This may be the case, for instance, when the real surface properties or the dynamics of the microfluidic filling process lead to contact angles outside the range of tolerance which is limited by an angle e as described in detail hereinafter. Conversely, by means of additional microfluidic elements such as throttles etc., it is possible to control the dynamics of the filling process such that the real (dynamic) contact angle is within the given tolerance range.
(47) FIG. 7 shows a schematic diagram 700 of parameters for two-dimensional geometric description of a phase interface in a microfluidic device in one working example. The central step of the method is the two-dimensional geometric description of the phase interface between two fluids that are insoluble or barely soluble in one another, for instance water and air or water and oil, in a boundary structure as the third, solid phase, for instance composed of a polymer such as PC, PP, PE, COP, COC or PMMA, by a circle segment, under the boundary conditions that the tangents of the circle segment and the boundary structure each form a given angle e with one another at the two three-phase points A, B. The modeling of the phase interface by circle segments may be motivated by the surface tension that exists at the phase interface. The capillary pressure that corresponds thereto leads to a constant curvature of the two-dimensional interface (cf. Young-Laplace equation). The simplifying description of the biphasic interface by circle segments is particularly advantageous since it firstly permits efficient analytical calculation of cross sections of capillary interfaces and secondly, for geometries with virtually fixed main planes of curvature, provides a very good approximation to the cross sections of the exact three-dimensional interface that exist within the main planes of curvature (cf. FIGS. 10a to 10i and FIGS. 11a to 11g). The specification of an angle θ formed by the tangents to the biphasic meniscus and the boundary structure at the three-phase points A, B can be motivated by the formation of a contact angle that results from the interfacial energies or surface tensions. The specified angle θ thus defines the boundary of a tolerance range within which the real contact angle must lie so that the desired microfluidic functionality is provided. The real contact angle present during the filling process may be subject to certain (small) fluctuations that may be caused, for instance, by dynamic effects without any resultant restriction of the applicability of the method.
(48) FIG. 7 shows the detailed geometric construction of the two-dimensional phase interface in a boundary structure which is planar on one side. The biphasic interface is constructed by a circle segment with center M and radius of curvature r in a channel cross section described by the channel width y and the opening angle −α. Among others, the following coordinates and relationships are shown: M=(M.sub.x|M.sub.y), A=(x|f(x)), B=(x+d|0), Y=|f(x)−0|, s=y/cos(−α/2) r=s/2/cos(α/2−θ+π) α=arctan(f′(x))
(49) By exploiting the trigonometric relationships that exist between the parameters involved, it is possible to conclude the radius of curvature as a function of the angles α, θ and the local channel width y:
(50)
(51) The calculation method described hereinafter is then employed in order to design a microfluidic cavity array structure in such a way that the cavities are completely filled when a liquid wets the inlet and outlet channel present above the cavities. In order to assure the applicability of the method, the dimensions of the microfluidic structure and the flow rate should be chosen such that the shape of the biphasic interface is stabilized by the surface tension and kinetic effects have only a limited influence on the process. It can thus be ensured that the dynamic (wetting) contact angle is within the range of tolerance and does not exceed the angle θ which is used for the design of the structure.
(52) FIG. 8 shows a schematic cross-sectional diagram of a cavity 105 in a working example. For parametrization of a class of test structures, a two-dimensional channel cross section to be suitably designed is considered with an upper straight boundary and a lower boundary of arbitrary shape. In addition, a two-dimensional channel cross section is considered, which, at least in part, is shaped with mirror symmetry with respect to an axis of symmetry that lies at right angles to the upper straight boundary, in such a way that the cavity 105 is formed. A class of test structures of relevance in respect of this problem may be defined by the following five parameters: s as the minimum channel width (without shaping of the cavity), r.sub.1 as the rounding radius of the top side of the cavity, d as the height of the side flank of the cavity, r.sub.2 as the rounding radius of the bottom side of the cavity and w as the inner width of the base of the cavity.
(53) FIG. 8 shows, by way of example, the test structure that results for the choice of parameters s=r.sub.1=d=r.sub.2=w/3. Likewise shown is the model-based construction of the biphasic meniscus for various positions of the meniscus and θ=120°.
(54) A crucial factor for the complete wetting of the cavity 105 by a liquid seems to be the condition that the liquid does not come into contact with both flanks of the cavity 105 before the medium initially present in the cavity 105, for instance air, has been displaced from the entire volume that adjoins the base of the cavity 105. The satisfaction of this condition can be decided on the basis of the maximum meniscus tilt that occurs, i.e. a maximum distance t between the three-phase point B and a point A′ at the upper boundary, where A′ is given by the orthogonal projection of the three-phase point A on the axis defined by the upper, straight boundary of the structure (cf. FIG. 7). The meniscus tilt t thus defined can be determined in the geometry under consideration as t=y tan(−α/2) (see FIG. 7) and becomes (for r.sub.2<s+r.sub.1+d) a maximum at a critical point C that marks the lower conclusion of the (left-hand) vertical (|α|=90°) flank of the cavity 105.
(55) FIG. 9 shows a schematic diagram of a maximum extent of a meniscus in a cavity 105 in one working example. FIG. 9 delineates the maximum extent of the meniscus that exists (for r.sub.2<s+r.sub.1+d) at the critical point C. The range of relevance for possibly incomplete filling of the cavity 105 is the range of 90°<θ≤180°, i.e.
(56)
With regard to the cavity geometry defined above (cf. FIG. 8), the two following sufficient conditions are found for complete filling of the cavity 105:
2r.sub.2+w>c+r (I)
r.sub.2>f=c+r−α (regions I and II in FIG. 9) (II)
(57) With the geometric relationships c=r sin(θ−π/2), r=a/√{square root over (2)}/sin(θ−π/4) and with a=s+r.sub.1+d, the result for θ>90° is the following conditions for complete filling of the cavity 105 under the above criterion:
(58)
(59) The conditions restrict the space of the geometry parameters to a region in which the structure is filled completely for a maximum angle θ. The aspect ratios
(60)
can therefore be regarded as characteristic parameters of a cavity geometry with regard to complete filling.
(61) FIG. 10 shows schematic diagrams of a cavity 105 and a chamber 100 in one working example during a filling process. What are shown by way of example are measurement results for applicability of the calculation method. For the measurements, various microfluidic test structures have been manufactured in a polycarbonate substrate. Plate a shows biphasic interfaces calculated by the method that result for the specific cavity geometry with the parameter selection s=400 μm, r.sub.1=r.sub.2=200 μm, d=0, w=300 μm and an angle θ=110°. Plates b to i show schematics of eight microscope images that were taken during a filling process. The scale bar in plate b corresponds to 200 μm. The microscope images of the microfluidic biphasic interface that forms have a good agreement with the calculated shapes that result from the performance of the method. Plate j shows a schematic of the top view of the chamber 100, here in the form of a cavity array comprising, by way of example, 55 circular cavities 105 in a hexagonal arrangement that have a cross-sectional geometry that satisfies the same aspect ratios as the microfluidic shape shown on the left-hand side of plates a to i. Plates k to n show schematics of four microscope images that were made during the filling of the cavity array structure. The scale bar in plate k corresponds to 500 μm. The field of view of the images in plates k to n is marked by a frame in plate j. The images show complete homogeneous filling of the cavities 105.
(62) FIG. 11 shows schematic diagrams of a cavity 105 and a chamber 100 with unsuitable geometry during a filling process. The results shown are obtained for an unsuitable cavity geometry. The corresponding parameters are, by way of example: θ=110°, s=d=200 μm, r.sub.1=r.sub.2=w=100 μm. The microscope images of the biphasic interface in plates b to d that were taken during the filling process do show good agreement with the shapes calculated, but complete filling (given existence of a sufficiently large contact angle) does not take place with this cavity geometry since the meniscus spans both flanks of the cavity shape before the air present in the cavity 105 has been displaced completely from the cavity 105. This results in unwanted trapping of air in the cavity 105, which prevents complete filling. Nor is it possible to ensure complete filling of the cavities 105 for the array structure derived from the cavity geometry, as shown by the microscope images in plates i to l. The scale bars correspond to 200 μm in plate b and 500 μm in plates g and i.
(63) In order to evaluate filling characteristics in the course of the calculation method, it is also possible to employ the sufficient conditions derived above, as shown hereinafter for the geometries considered in FIGS. 10 and 11. For an assumed maximum permissible contact angle θ=110°, it follows that there is a contact angle parameter) g(110°)=1.047. For the cavity geometry shown in FIG. 10 with 3s=4w=6r.sub.1=6r.sub.2 and d=0, it follows that AR.sub.1=1.167>1.047=g(110°) and AR.sub.2=0.333>0.047=g(110°)−1, i.e. both conditions are satisfied, which indicates complete filling. By contrast, for the cavity geometry shown in FIG. 11 with s=d=2r.sub.1=2r.sub.2=2w, AR.sub.1=0.6<1.047=)g(110°) and AR.sub.2=0.2>0.047=g(110°)−1, and so complete filling cannot be assured here.
(64) FIG. 12 shows schematic diagrams of a propagation of a biphasic interface during a layering process in a cavity 105 in one working example. In addition to filling of microfluidic structures, the calculation method can also be applied to interfaces that form between two mutually immiscible liquids. FIG. 12 shows a schematic of four microscope images that show the propagation of a biphasic interface between water (dark-colored here) and mineral oil through a microfluidic cavity geometry. For this purpose, the cavity 105 was first filled completely with the mineral oil and then the dark-colored water was forced into the inlet channel. The experimental result again shows good agreement with the propagation of the biphasic interface construed geometrically via the calculation method. It is obvious that there is incomplete filling of the cavity 105 with the aqueous phase. This observation is consistent with the sufficient conditions derived above for complete filling, which are not met. With g(150°)=1.366 and s=r.sub.1=2d=2r.sub.2, w=0, it follows that AR.sub.1=0.4<1.366, AR.sub.2=0.2<0.366, such that the mineral oil cannot be displaced completely from the cavity 105 by the dark-colored water.
(65) FIG. 13 shows schematic diagrams of propagation of a biphasic interface during a layering process in a cavity 105 in one working example. What is shown is an example of an application of the calculation method with regard to the design of a cavity that permits the aliquoting of a fluid by blanketing with a second fluid immiscible with the first fluid. In a first step, the cavity 105 is filled, for example, with a PCR master mix as sample liquid 10. FIG. 13 shows a schematic of four microscope images that have been taken during layering with oil as sealing liquid 20. The contact angle of the oil which is established in the displacement of the PCR master mix is sufficiently large that a portion of the PCR master mix remains in the cavity shape of the microfluidic channel and is layered with the oil. The portion of the PCR master mix that remains in the cavity shape after layering, i.e. the volume enclosed, can be adjusted both via the geometry of the cavity and via the contact angle which is established between the two fluids. With g(130°)=1.166 and s=r.sub.1=r.sub.2, d=w=0, it follows
(66)
such that the criterion (I) derived indicates incomplete displacement of the first fluid, which leads to the desired layering of the first fluid.
(67) FIG. 14 shows schematic diagrams of a chamber 100 in a working example during a filling process in top view.
(68) FIG. 15 shows schematic diagrams of a chamber 100 from FIG. 14 during a layering process in top view.
(69) FIGS. 14 and 15 show, in schematic form, an experimental result for aliquoting of a fluid in an array of 55 cavities each with a volume of 25 nl. The cross-sectional geometry of the cavities 105 is designed such that complete filling of the cavities 105 with a PCR master mix is first achieved, as shown in FIG. 14, and then layering of the cavities by means of mineral oil, as shown in FIG. 15.
(70) FIG. 16 shows a flow diagram of a method 1600 of aliquoting in one working example. The method 1600 may be executed, for example, by means of a microfluidic device as described above for FIGS. 1 to 15. In a first step 1610, the sample liquid 10 is introduced into the chamber 100. The geometry of the chamber 100 defined depending on the wetting characteristics, especially the contact angle θ.sub.1 of the sample liquid 10, more specifically of the inlet channel and especially of the cavities 105, achieve the effect that the meniscus of the sample liquid 10 is suitably shaped, for example in concave or convex form, while the liquid 10 flows into the cavities 105. This can achieve the effect that the cavities 105 are completely filled with the sample liquid 10. Subsequently, in a further step 1620, the sealing liquid 20 is introduced into the chamber 100. By contrast with the sample liquid 10, the meniscus of the sealing liquid 20 is shaped differently, for example in convex form, by virtue of the greater contact angle θ.sub.2>θ.sub.1 that exists here and the defined geometry of the chamber 100. This achieves the effect that portions of the sample liquid 10 are enclosed by the sealing liquid 20 in the cavities 105.
(71) FIG. 17 shows a flow diagram of a method 1700 of producing a microfluidic device in one working example, for instance the device described above with reference to FIGS. 1 to 15. In a step 1710, wetting information representative of the respective wetting characteristics of the sample liquid and sealing liquid, for instance the contact angles thereof depending on a material of the chamber of the device, is read in. In a further step 1720, using the wetting information, a geometry suitable for complete filling and sealing of the cavities is defined. For example, the geometry here may be selected from a multitude of defined, already calculated geometries that are each assigned to different wetting characteristics. The geometries have been calculated, for example, using the above-described calculation method. In a step 1730, the chamber is shaped in accordance with the defined geometry in a suitable manufacturing method, for instance an additive or subtractive or high-throughput method.
(72) FIG. 18 shows a schematic diagram of a microfluidic system 1800 in one working example. The system 1800 comprises the device 1, a pump unit 1802 fluidically coupled to the device 1 for pumping of the sample liquid and sealing liquid through the chamber of the device 1, and a controller 1804 for actuating the pump unit 1802. The microfluidic system 1800 therefore especially enables fully automated aliquoting of the sample liquid by means of the device 1.
(73) If a working example comprises an “and/or” linkage between a first feature and a second feature, this should be read such that the working example in one embodiment has both the first feature and the second feature, and in a further embodiment has either only the first feature or only the second feature.
