Microfluidic Devices

Abstract

A microfluidic device is provided that can avoid the generation of air bubbles during well filling and ensure proper separation between filled wells. The microfluidic device includes an inlet opening (42), an outlet opening (43), a flow channel connecting the inlet opening and the outlet opening and defining a flow direction from the inlet opening through the flow channel (3) to the outlet opening, the flow channel including a first flow channel surface (31) and a second flow channel surface (41) opposite the first flow channel surface, and an array of wells provided in the first flow channel surface for fluid communication with the inlet opening and the outlet opening. Further, the first flow channel surface provides a first hydrophilicity, and at least a portion of the second flow channel surface provides a second hydrophilicity, wherein the first hydrophilicity is greater than the second hydrophilicity.

Description

[Technical Field]

[0001] Generally, the present invention relates to the technical field of microfluidic devices for diagnostic assays, where it is often an objective to be able to perform multiple different assays of one or more test samples on the same, usually disposable, microfluidic device. This allows for independent analysis of one or more test samples with multiple different reagents during a single analytical process, requiring only small amounts of test sample. More specifically, the present invention relates to a microfluidic device comprising an inlet opening, an outlet opening, and one or at least one flow channel, wherein the flow channel connects the inlet opening to the outlet opening and provides an array of wells within the flow channel in fluid communication with the inlet and outlet openings, the wells each intended as a reaction chamber for, for example, a chemical or biological reaction of at least one sample provided therein. In particular, the present invention relates to an improved microfluidic device that utilizes the volume of sample liquid as fully and efficiently as possible. [Background technology]

[0002] In the field of diagnostic assays, there is a general need to create diagnostic assays that are faster, cheaper, and simpler to perform while achieving the accuracy and efficiency of conventional laboratory processes. To this end, considerable efforts have been made to achieve miniaturization and integration of various assay operations, allowing for an increased number of parallel assays on a single device. However, reducing the reaction chamber volume to create such microfluidic structures presents several new problems, such as manufacturing limitations regarding the miniaturization of reaction chambers, cross-contamination between adjacent reaction chambers, air bubble entrapment in one or several reaction chambers, liquid evaporation, and an increased lack of accuracy and efficiency in metering sample liquid into miniaturized reaction chambers. In particular, microfluidic chips, also known as digital polymerase chain reaction (dPCR) chips, are known as such microfluidic devices, which provide microscale channels for accepting microliter- or nanoliter-scale samples in the form of streamable liquids. Generally, such dPCR chips feature inlet and outlet openings connected by flow channels, providing flow chambers containing multiple reaction sites in the form of small wells or arrays of microwells.

[0003] To perform a dPCR assay, known dPCR chips are first filled with an aqueous dPCR reaction mixture, typically consisting of a biological sample and a PCR master mix. This dPCR reaction mixture is introduced into the inlet opening, such as by a pipette, and typically passively flows into the array of wells of the chip by capillary forces until the capillary filling process is stopped. An immiscible separating or sealing liquid, such as silicone oil, is then forced through the inlet opening into the flow channel, first squeezing any remaining dPCR reaction mixture into any remaining empty wells and covering the filled wells, thereby fluidically isolating the individual wells from their surroundings and, in particular, from each other, to avoid any cross-contamination or contamination. After the initial filling and subsequent sealing processes are completed, the dPCR chip is typically subjected to thermal cycling, during which specific target nucleic acids are amplified through a series of repeated cycles over the course of a typical PCR run. The nucleic acids present in the dPCR reaction mixture are (a) denatured at a relatively high temperature, e.g., above 90°C, typically about 94°C-95°C, to separate double-stranded DNA. The reaction mixture is then cooled to an annealing temperature, e.g., about 52°C-56°C, at which short oligonucleotide primers bind to the single-stranded target nucleic acids for primer binding (annealing) with the separated DNA strands to provide templates. Then, (c) the primers are extended / extended using a polymerase enzyme at an extension temperature, e.g., about 72°C, to generate new DNA strands so that the original nucleic acid sequence is replicated. Typically, each well containing one or more targets produces a positive signal; after thermal cycling, the ratio of positive and negative signals allows accurate calculation of the initial target concentration in the sample, e.g., by luminescence assay measurement. Such techniques allow multiple assays to be performed simultaneously on a miniaturized scale.

[0004] To allow for the provision of sample liquids in such microfluidic devices without evaporation, US 6,143,496 A describes a microstructured fluidic device for analytical purposes, consisting of several layers in the form of a support and a cover attached to each other, with a flow-through channel provided between the support and the cover to provide multiple reaction sites, and a patterned additional layer provided between the support and the cover and attached to the support, where, according to one particular embodiment, the patterned layer may exhibit hydrophobic properties, and the cover surface facing the patterned layer may exhibit hydrophilic properties. Thus, US 6,143,496 A discloses a microfluidic consumable made of several different layers that need to be assembled in a complex manner and in a specific order. As further known prior art, US 6,027,695 A discloses another microstructured fluidic device comprising a plurality of adjacent microwells, where the walls of adjacent microwells intersect to form upwardly facing edges, and the microwells, for example in the form of hexagonal chambers, are arranged in a honeycomb configuration. Regarding the use of the microstructured fluidic device of US 6,027,695 A, the wells are filled by immersing the entire device in a solution containing beads that sink down over a long period of time and thus enter the wells so that at least one bead is provided in each well. The solution is then evaporated and the wells are fluidically separated from each other, which generally requires that the beads be denser than the liquid. However, this evaporation process to separate the wells is quite time-consuming, and providing a specific amount of solution to each well is a significant problem, as the variation between the contents of each well must be as small as possible.

[0005] In general, in the current technical field of diagnostic assay technology, and in particular in the field of dPCR performed by known microfluidic devices or chips, in order to overcome the above-mentioned problems of the known prior art, several technical requirements must be met, which are as follows: It has been found that not only the number of dPCR reaction wells / chambers but also their respective volumes must be maximal for a given area on a microfluidic device. However, the manufacturing process of such microfluidic devices, usually by injection molding, includes certain constraints regarding the maximum possible number of wells as well as their respective maximum volumes in the microfluidic device to be injection molded.

[0006] Furthermore, each well must reach a certain depth compared to its length and width, and certain aspect ratios of well length to flow channel height as well as minimum widths of any kind of rim between adjacent wells may be desirable, again constrained by restrictive manufacturing process conditions.

[0007] It may also be desirable for the wells to be filled with the dPCR reaction mixture in a passive manner, by capillary forces. However, in this regard, the miniaturization of microfluidic devices, and therefore the miniaturization of flow channels and wells, makes it difficult to fill any liquid, e.g., dPCR. Sufficient passive filling of the wells of a microfluidic device by capillary forces is difficult to achieve, as it creates problems where the reaction mixture cannot easily enter the wells or even enter the flow channel itself.

[0008] Furthermore, even if sufficient filling of the microfluidic device is achieved in some way, filling must be achieved without the generation of air bubbles and therefore without initial trapping of air bubbles in the wells. However, air bubble trapping in some or all wells and / or flow channels is still a severe problem resulting in undesirable dPCR analysis failure, since any air bubbles already trapped in a well will distort the detection signal that would otherwise be generated in that well, and furthermore, upon heating of the dPCR microfluidic device to the required maximum thermal cycling temperature of approximately 95° C., such bubbles will expand such that reliable separation of adjacent wells may no longer be guaranteed and the likelihood of undesirable cross-contamination becomes significantly increased.

[0009] As a further requirement for any microfluidic device, it is often desirable to fill wells to a certain maximum nominal volume. However, due to the separation of wells initially filled with dPCR reaction mixtures, during filling of the microfluidic device with immiscible sealing fluid, the ingressing sealing fluid often forms a meniscus that penetrates into each well. Thus, a significant portion of the dPCR reaction mixture filled in each well may be displaced by the sealing fluid again, thereby forcing the dPCR reaction mixture filled in the well out of the well. This significantly reduces the actual usable dPCR reaction mixture volume of the dPCR microfluidic device, which results in a decrease in the analytical performance of the device. Furthermore, due to the undesired displacement of the dPCR reaction mixture from the wells, the determination of the total amount of dPCR reaction mixture in the microfluidic device will be inaccurate, leading to inaccurate analytical results.

[0010] Furthermore, after filling the device with the dPCR reaction mixture and separating the wells with an immiscible fluid, the fluid separation of the wells must be maintained in a stable manner during the thermal cycling process, i.e., no leakage of the dPCR reaction mixture from one well to another must occur. However, complete fluid separation of adjacent wells is usually rarely achieved, and if not achieved, undesirable leakage between adjacent wells occurs. Therefore, dPCR products may migrate from one well to another and contaminate it, thereby generating false positive signals, which ultimately leads to false dPCR results. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] US 6,143,496 A [Patent Document 2] US 6,027,695 A Summary of the Invention [Problem to be solved by the invention]

[0012] The above list of requirements and problems for microfluidic devices is, of course, not exhaustive, but merely lists some of the current problems. In general, there is a need in the art to provide a microfluidic device that is capable of reliably and satisfactorily filling each single well of an array of wells, and in this regard, in particular for a microfluidic device that is capable of avoiding the creation of air bubbles during filling and ensuring adequate separation between filled wells. [Means for solving the problem]

[0013] The present invention addresses the above-mentioned needs and provides an improved microfluidic device for thermal cycling of reaction mixtures, which overcomes all of the above-mentioned problems and meets the enumerated requirements.

[0014] According to a first aspect of the present invention, there is provided a microfluidic device for thermal cycling of a reaction mixture, the microfluidic device comprising an inlet opening for a fluid inlet, an outlet opening for a fluid outlet, and a flow channel connecting the inlet opening and the outlet opening and serving as a channel for fluid flow from the inlet to the outlet, wherein the flow direction is defined by the structural arrangement from the inlet opening through the flow channel to the outlet opening. The microfluidic device as a whole may be disposable and may be made of a transparent material, such as cyclic olefin copolymer COC or cyclic olefin polymer COP, that typically provides a contact angle of about 80° to 90°, making the material transparent and suitable for visual analysis of dPCR results. Furthermore, the flow channel, or particularly the internal volume of the flow channel, comprises a first flow channel surface and a second flow channel surface opposite the first flow channel surface, and an array of wells or microwells is provided in the first flow channel surface such that fluid communication is established between the array of wells and the inlet and outlet openings. Regarding specific characteristics of the flow channels, the first flow channel surface, and preferably particularly the well-covered region of the first flow channel surface, exhibits / includes a first hydrophilicity, and at least a portion of the second flow channel surface, preferably the portion of the second flow channel surface directly opposite the well-covered region, exhibits / includes a second hydrophilicity, where the first hydrophilicity, i.e., the respective surface characteristics of the first flow channel surface, is greater than the second hydrophilicity, i.e., the respective surface characteristics of the second flow channel surface opposite the first flow channel surface. Here, for example, the first hydrophilicity, i.e., the hydrophilicity of the first flow channel surface, exhibits a surface contact angle in the range of about 30° to 50°, e.g., 40°, but may also be <30°, and the second hydrophilicity, i.e., the hydrophilicity of the second flow channel surface, exhibits a surface contact angle in the range of about 80° to 90°, resulting in the first flow channel surface of the device of the present invention being more hydrophilic than the second flow channel surface.This particular setup, with a defined relationship between a first flow channel surface provided with an array of wells and an opposing second flow channel surface disposed on the opposite side of the array of wells, achieves a hydrophilic relationship within the flow channel, which results in improved filling performance of the microfluidic device. See also below for further details.

[0015] Typically, upon filling of wells provided within a first flow channel surface, the initially filled fluid advances more rapidly through a second flow channel surface that does not include any wells than through a first flow channel surface that includes an array of wells. Thus, during initial filling, fluid advance through the flow channel advances more rapidly through the second flow channel surface than through the first flow channel surface, resulting in the possibility that, during well filling, the fluid may entrap gas within the wells being filled, resulting in undesirable trapped air bubbles. With the microfluidic device of the present invention, the above-mentioned different advancement rates of fluid can be avoided by modifying the inner surface of the well region of the microfluidic device at the first flow channel surface in a manner that is more hydrophilic than the hydrophilicity of the flow channel surface opposite the well region, thereby achieving a hydrophilic relationship between opposing flow channel surfaces such that the head or front of fluid volume advancement within the flow channel of the microfluidic device assumes a substantially upright position / vertical orientation. In other words, the contact area between the second flow channel surface of the flow channel and the front of the initial fill fluid entering the inlet opening and proceeding through the flow channel to the outlet opening flows through the flow channel at a faster rate than the contact area between the first flow channel surface and the front of the initial fill fluid; i.e., the flow velocity of the initial fill fluid stream on the surface of the well is faster than the velocity of the initial fill fluid through the inner surface of the flow channel located opposite the well area. As a result, filling of the well with the initial fill fluid occurs more quickly than general filling of the flow channel itself, thereby preventing air bubbles from being trapped below the fill fluid in the well when the fill fluid rises above the air bubbles in the well. Therefore, progress of the fluid through the flow channel is substantially equal on both flow channel surface sides, allowing the well to be fully filled with fluid and avoiding air bubble trapping.In terms of performing digital PCR in the microfluidic devices of the present invention, this is particularly important for the initial filling of wells with an aqueous dPCR reaction mixture, also referred to as dPCR or PCR master mix, e.g., LightCycler480® Master Mix, because the initial filling is performed passively and various capillary forces are important, while active filling pressure is applied during the separation process that fluidically separates the filled wells from each other. Thus, providing a side of the flow channel containing the array of wells with higher hydrophilicity than the other side improves the filling performance of the microfluidic device and thus avoids bubble trapping by increasing the affinity of the side of the flow channel containing the array of wells for the aqueous dPCR reaction mixture. In other words, to achieve bubble-free filling according to the present invention, it is preferable to construct the microfluidic device so that the side of the flow channel not covered by the wells has less affinity for the dPCR reaction mixture than the well area.

[0016] According to certain embodiments of the present invention, the first hydrophilicity and / or the second hydrophilicity are provided either by the material properties of the microfluidic device, by a surface treatment of the first flow channel surface and / or the second flow channel surface, for example, by a plasma hydrophilization treatment, or by a hydrophilic coating, for example, a SiO2 coating, provided on the first flow channel surface and / or the second flow channel surface. As an example for comparison purposes, during the course of experiments performed by the inventors of the present invention, the well region of a microfluidic device was coated with a SiO2 coating and compared to a microfluidic device without any hydrophilization treatment. A LightCycler 480® master mix containing 100 nM fluorescein was then loaded into the microfluidic device through the inlet opening and into the flow channels, and subsequently a silicon fluid, for example, PMX silicon fluid 200 The wells were sealed by pumping 50 cs of sealing or separation fluid into the flow channel. Experimental results showed that passive filling was either incomplete or incomplete in the untreated microfluidic device, whereas successful passive filling and little or no bubble entrapment were detectable in the hydrophilized microfluidic device.

[0017] Furthermore, the shape of the wells may be an important factor when considering avoiding bubble entrapment. It has been observed by the present inventors that a circular well shape aids in the entrapment of normally circular bubbles, since such circular bubbles may actually completely contact the edges and seal off the entire circular well. On the other hand, a well shape that provides a minimized contact area between a trapped bubble and the inner wall of the well may further avoid bubble entrapment, since this reduced contact between the bubble and the inner wall of the well aids in the removal of the bubble from the well. Thus, according to a further specific aspect of the present invention, at least a portion of the array of wells may present a hexagonal well shape at the first flow channel surface, and all of the wells may present a hexagonal well shape at the first flow channel surface. "Well shape" in this sense refers to the shape of the well as seen from a top view of the first flow channel surface. Selecting a hexagonal well shape not only provides the benefit of an optimized well shape to reduce the inclusion of air bubbles during the filling process, but also maximizes the amount of wells and their respective internal volumes, for example, by arranging hexagonal wells in a honeycomb structure, which provides improved space utilization in the grid. Here, as an example of typical dimensions for hexagonal wells in a microfluidic device for dPCR, the hexagonal well shape may exhibit width x length x depth dimensions ranging from approximately 25 μm x 50 μm x 25 μm to approximately 150 μm x 300 μm x 200 μm. Thus, each well may have a well length in the flow direction ranging from 50 μm to 300 μm, a well width perpendicular to the well length ranging from 25 μm to 150 μm, and / or a well depth ranging from 25 μm to 200 μm. Furthermore, in this regard, it may be preferable if the wells have an elongated hexagonal shape, i.e., an elongated or elongated hexagonal well shape in the flow direction determined by the flow channel from the inlet opening to the outlet opening, which may further reduce contact between air bubbles and the inner well walls and may also enlarge the internal volume of each well.

[0018] Regarding the specific arrangement of hexagonal wells within the first flow channel surface, certain embodiments of the present invention provide for orienting the vertices of each hexagonal well, also referred to as corners of the hexagonal shape of the well, toward the inlet opening in the flow direction, where the two vertices / corners of each hexagonal well, arranged relative to one another, are oriented parallel to the flow direction defined by the flow channel from the inlet opening to the outlet opening. In this regard, because the flow channels of the microfluidic devices of the present invention are filled with the dPCR reaction mixture from the inlet opening side, alignment of the vertices of each hexagonal well with the flow direction can significantly improve the filling performance of the microfluidic device. In other words, if one of the six corners of the hexagon of the well is pointing in the filling direction, i.e., toward the inlet opening, capillary suction from this corner is improved, which significantly facilitates the initial filling of the well with the dPCR reaction mixture. Therefore, the hexagonal shape of the wells is optimally selected in a manner that aids in filling of the flow channels by capillary forces, particularly by providing the corners of the hexagons in the flow direction, thereby facilitating fluid entry into each well and, in particular, preventing the entering fluid from simply flowing over the well without filling it. In particular, an elongated hexagonal well shape may be more advantageous than a regular hexagonal shape, since, when bubbles are trapped, as already mentioned above, it is possible not only to reduce the contact surface of the bubbles with the inner wall of the well, but also to achieve the effect of forcing large bubbles into an energetically unfavorable elongated shape and facilitating their release from the well, an effect that is not achieved when using circular or regular hexagonal well shapes.

[0019] According to a more specific embodiment of the present invention, the edge of each well in the first flow channel surface facing the inlet opening is a rounded edge. Here, the term "rounded edge" refers to the edge between the first flow channel surface and the inner wall of the well, which does not provide a sharp corner but presents a curved surface, i.e., a curved surface connecting the flow channel surface and each inner wall of the well. By providing each well with such a rounded edge, the filling characteristics of each well can be significantly improved, thereby improving the sufficient filling of each well with the dPCR reaction mixture. As an example of such a curved edge surface, each rounded well edge can be rounded with a radius of less than 10 μm (<10 μm). Alternatively or additionally, a rim can be provided between adjacent wells to fluidically separate them. In this context, a rim is understood to be a portion of the first flow channel surface that separates adjacent wells from one another, where such a rim may include a width or thickness greater than 10 μm (>10 μm) to achieve sufficient distance between adjacent wells to further improve fluidic separation between the adjacent wells after they are filled. Therefore, the specific shape of the rim between adjacent wells is implemented so that the fluid layer between the first flow channel surface and the separation fluid is sufficiently suppressed, thereby ensuring fluidic separation between adjacent wells after filling. Furthermore, the chemical composition of the dPCR reaction mixture may be modified in a manner that prevents fluidic bridging across the rim region.

[0020] According to a further specific embodiment of the microfluidic device of the present invention, the aspect ratio h / l between the height h of the flow channel and the length l of each well ranges from 0.3 to 0.7, e.g., approximately 0.5. This provides an optimal aspect ratio h / l for ensuring sufficient fluidic separation between adjacent filled wells, in addition to the characteristics already described in this regard. For example, the height h of the flow channel may range from 25 μm to 200 μm, and the length l of the well may range from 50 μm to 300 μm, where the aspect ratios defined above must be met within these ranges. For illustrative purposes, an aspect ratio h / l of less than 0.3 leads to filling of wells with too little fluid volume, while an aspect ratio h / l of approximately 1.0 also leads to wells that are sufficiently filled, but with the problem that adjacent wells are no longer sufficiently fluidically separated from each other. Generally, in this context, the length l of each well shall be understood as the longitudinal extension of the well parallel to the flow direction, and the height h of the flow channel shall be understood as the distance between the first and second flow channel surfaces of the flow channel of the microfluidic device. To achieve proper separation of the wells, the channel height h must be smaller than the well length l so that surface tension forces will force some of the initially filled dPCR reaction mixture out of each well. Therefore, the channel height h is modified within a predetermined aspect ratio to allow neat separation of the wells with only minimal displacement of the dPCR reaction mixture out of the wells.

[0021] According to another specific embodiment of the microfluidic device of the present invention, the microfluidic device consists of two parts that can be attached to each other, and the device is divided into two parts along its longitudinal axis. More specifically, a flow channel containing an array of wells is provided in one part of the device, providing a first flow channel surface, e.g., a support, and the other part constitutes a second flow channel surface and a cover part providing inlet and outlet openings, preferably a flat component in the form of a thin cover foil that covers the flow channel and provides inlets for introducing fluids into the flow channel and outlets for discharging fluids from the flow channel. Alternatively, the inlet and outlet openings may also be provided in the part of the device that provides the first flow channel surface, in which case the other part of the device simply constitutes the cover part, e.g., in the form of a thin cover foil. Such a microfluidic device may be used for digital PCR, dPCR, or biochemical assays of samples provided in the form of reaction mixtures to each well of the flow channel. Here, to help improve the fillability of the microfluidic device, a surfactant, e.g., TWEEN® 20, may be provided to the dPCR reaction mixture, i.e., the chemical composition of the aqueous dPCR reaction mixture was adjusted in a manner that facilitated the filling process, e.g., by the addition of a surfactant.

[0022] In other words, to provide a microfluidic device that can ensure reliable filling of each well, (a) a cover foil or plate with a lower affinity for the dPCR reaction mixture, (b) a particular well shape, well structure, and orientation, and (c) a particular aspect ratio of the flow channel height h to the well length l can allow liquid to fill the wells for a longer period than would otherwise be possible, slowing the initial fluid flow during filling and thereby significantly improving the subsequent separation of adjacent filled wells. In this regard, typically, after initially filling a portion of the flow channel and some of the wells, a sealing fluid that is immiscible with the dPCR reaction mixture is forced into the flow channel, and this sealing fluid forces the dPCR reaction mixture through the remainder of the flow channel and into the remaining wells that are also filled. Furthermore, the sealing fluid forces any dPCR reaction mixture that is not already in a well out of the flow channel and fluidically separates the filled wells from each other. As mentioned above, to achieve proper separation of the wells, the height of the flow channel must be approximately half the length of the wells, and to further improve the separation capability of the microfluidic device, the wells must be separated by a rim with a specific width. Furthermore, to minimize displacement of the dPCR reaction mixture out of the wells, the speed of the separation process, i.e., the force required to push the second fluid through the flow channel, may be increased. Generally, the hydrophilicity ratio of the flow channel's inner surface appears to have a more significant impact on improving the initial filling of the wells with the dPCR reaction mixture. While the aspect ratio of the flow channel's height to the well's length not only improves the initial filling of the wells with the dPCR reaction mixture, but also significantly improves the sealing or separation fluid and the separability of adjacent wells from each other due to the different dynamics of passive initial filling of the wells based on various "pulling forces" in terms of active pressure application (appliance) during separation. Therefore, using the microfluidic device presented herein, overall improvements in the initial filling characteristics, as well as the subsequent sealing process, can be achieved.

[0023] As used herein, and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless clearly indicated otherwise. Similarly, the words "comprise," "contain," and "encompass" are inclusive rather than exclusive; i.e., they shall be interpreted as meaning "including, but not limited to." Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The terms "plurality," "multiple," or "amount" refer to two or more, i.e., 2 or >2, and an integer multiple; the term "single" or "single" refers to one, i.e., =1. Additionally, the term "at least one" is understood to mean one or more, i.e., 1 or >1, also an integer multiple. Thus, words using singular or plural numerical values ​​also include the plural and singular numerical values, respectively. Furthermore, the words "herein," "above," "above," and "below," and words of similar import, when used herein, refer to the specification as a whole and not to any particular portions of the specification.

[0024] Furthermore, specific terms are used for convenience and are not intended to limit the invention. The terms "right," "left," "up," "down," "below," and "up" refer to directions in the figures. These terms include the terms explicitly mentioned as well as variations and terms of similar meaning. Spatially relative terms, such as "below," "bottom," "lower," "upper," "superior," "near," "distal," etc., may also be used to describe the relationship of one element or feature to another, as illustrated in the figures. These spatially relative terms are intended to encompass different positions and orientations of the device in use or operation in addition to the position and orientation shown in the figures. For example, if a device in the figures were inverted, an element described as "below" or "below" another element or feature would then be "above" or "above" the other element or feature. Thus, the exemplary term "below" can encompass both upper and lower positions and orientations. The device may be otherwise oriented (rotated 90 degrees or at other orientations), and the spatially relative descriptors used herein will be interpreted accordingly.

[0025] In the illustrations and descriptions of the various aspects and exemplary embodiments, it should be understood that, to avoid repetition, many features are common to many aspects and embodiments. The description of particular embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise forms disclosed. Specific embodiments of the disclosure, and examples thereof, are described herein for illustrative purposes; however, as those skilled in the relevant art will recognize, various equivalent modifications may be within the scope of the disclosure, as defined by the appended claims. Specific elements of any of the foregoing embodiments may be combined with or substituted for elements in other embodiments. Furthermore, although advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily need to exhibit such advantages in order to fall within the scope of the present disclosure as defined by the accompanying claims. The omission of an aspect from a description or figure does not indicate that the aspect is missing from the embodiment(s) that incorporate it. Instead, the aspect may have been omitted for clarity and to avoid redundant description. In this context, the following applies to the remainder of this description: for clarity of the figures, if a figure contains a reference sign that is not explained in the directly relevant part of the description, reference is made to the preceding or following description section. Furthermore, for clarity, if a section of a figure does not provide a reference sign for every feature of a part, reference is made to another section of the same figure. In two or more figures, like numerals indicate the same or similar elements.

[0026] The following examples are intended to illustrate various specific embodiments of the present invention. As such, the specific modifications discussed hereinafter are not to be considered limitations on the scope of the present invention. It will be apparent to those skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the present invention, and therefore, it is to be understood that such equivalent embodiments are encompassed herein. Further aspects and advantages of the present invention will become apparent from the following description of specific embodiments illustrated in the drawings. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 is a schematic exploded view of a microfluidic device according to an embodiment of the present invention. [Figure 2] 2a-d are schematic illustrations of the progression of aqueous fluids through flow channels, including, for comparison purposes, one exemplary well of the microfluidic device of FIG. 1 shown in cross section with similar hydrophilic flow channel interior surfaces. [Figure 3]3a-d are schematic illustrations of fluid progression through a flow channel, including one exemplary well of the microfluidic device of FIG. 1 shown in cross-section, with the inner surfaces of the flow channels having different hydrophilicities, in accordance with the present invention. [Figure 4] 4a-c are schematic diagrams of an array of hexagonal wells of a microfluidic device shown in top view with different elongation levels of the hexagonal wells for size comparison. [Figure 5] 5a-c are schematic diagrams of one of the hexagonal wells of FIGS. 4a-c shown in cross section along lines AA, BB, and CC as shown in FIGS. 4a-c for aspect ratio comparison. [Figure 6] 6a-d are schematic illustrations of the progression of a separating or sealing fluid through a flow channel, including the wells of FIGS. 4a and 5a shown in cross section, in accordance with the present invention. [Figure 7] 7a-d are schematic illustrations of fluid progression through a flow channel, including the exemplary well of FIGS. 4b and 5b shown in cross-section. [Figure 8] 8a-d are schematic illustrations of fluid progression through a flow channel, including the exemplary wells of FIGS. 4c and 5c shown in cross-section. [Figure 9] 9a and b are schematic diagrams of different hexagonal well dimensions shown in top view with trapped air bubbles for comparison. DETAILED DESCRIPTION OF THE INVENTION

[0028] 1 shows a schematic diagram of a microfluidic device 1 according to a particular embodiment of the invention in an exploded perspective view. The microfluidic device 1 basically comprises two parts, a support 2 and a cover 4 in the form of a plate or foil, which parts 2, 4 can be attached to each other. On one surface of the support 2, a flow channel 3 is provided, which provides a first flow channel surface 31 in which an array of hexagonal wells / microwells 32 is introduced in the exemplary honeycomb structure type; the area in which the array of wells 32 is located is also referred to as the flow chamber of the microfluidic device 1. Here, for illustrative purposes, only a few empty wells 32 in the flow chamber are shown.

[0029] From the perspective of bubble trapping, well shape is an important factor. As already described further above, a circular well shape favors trapping of bubbles that are typically circular, because such circular bubbles can actually fill the entire circular well with complete surface edge contact. Therefore, noncircular wells are preferred because such well shapes can minimize the contact area between the trapped bubbles and the inner wall of the well. Here, elongated hexagonal well shapes are more suitable than regular hexagonal shapes. Experiments conducted by the inventors of the present invention have led to the results that, in circular wells, approximately 40% of the wells contain trapped bubbles, and approximately >80% of small regular hexagonal wells contain trapped bubbles, while in larger hexagonal wells, only >1% contain trapped bubbles, and in elongated hexagonal wells, such as well 32 of microfluidic device 1, a significantly lower number, less than 0.01%, contain trapped bubbles. As an illustrative example, Figure 9a shows a regular hexagonal shape of well 32' in which a bubble 6 is trapped and the bubble 6 can still achieve six contact points 61 with the well interior wall, while the elongated hexagonal shape of well 32 of the microfluidic device 1 of the present invention reduces the number of potential bubble contact points 61 to two. See Figure 9b. Thus, the elongated hexagonal shape of well 32 may reduce the surface contact of bubble 6 with the interior wall of well 32 when a bubble is trapped. Also, in such an elongated well shape, larger bubbles are forced into an energetically unfavorable elongated shape, and thus, expedites the release of bubble 6 from well 32.

[0030] 1 , the surface of cover 4, disposed opposite the mentioned surface of support 2, provides second flow channel surface 41 opposite first flow channel surface 31. Generally, when attached to one another, support 2 and cover plate 4 provide microfluidic device 1 in such a way that a continuous duct is established, starting from inlet opening 42 in cover 4, continuing in flow channel 3 bounded by first flow channel surface 31 and second flow channel surface 41, and finally terminating at outlet opening 43, which also defines the flow direction in microfluidic device 1 from inlet opening 42 to outlet opening 43, i.e., parallel to the longitudinal axis of flow channel 3 in support 2. In the microfluidic device 1, the wells 32 are oriented in the flow direction, which means that the longitudinal axis of the elongated hexagonal shape of the wells 32 is arranged parallel to the flow direction of the microfluidic device 1, i.e., the apex 321 of each hexagonal well 32 is oriented in the flow direction toward the inlet opening 42, which significantly improves the filling performance of the microfluidic device 1 because the capillary suction force from the well apex 321 promotes filling of the wells 32.

[0031] As an example of dimensions, the microfluidic device, i.e., its two portions 2, 4, may exhibit an overall length of about 75 mm and an overall width of about 25 mm, the width of the flow channel 3 being about 6 mm, and the length of the region of the flow channel 3 covering the wells 32 being about 47 mm. Here, the number of hexagonal elongated wells 32 may be greater than 16,000, where each well 32 comprises a length of about 60 μm, a width of about 30 μm, and a depth of about 60 μm, and the rim 33 between adjacent wells comprises a width of more than 10 μm. Furthermore, the height of the flow channel 3 is 30 μm, resulting in a preferred aspect ratio of 0.5 of the flow channel height h to the well length l to ensure sufficient fluidic separation between adjacent wells 32 after filling with an initial fluid, e.g., a dPCR reaction mixture 5, and fluidic separation by the sealing fluid 7.

[0032] Regarding the effect of providing different hydrophilicities of first flow channel surface 31 and second flow channel surface 41 in the present invention, FIGS. 2a-2d illustrate the flow of an initial dPCR reaction mixture through flow channel 3, including one exemplary well 32, of microfluidic device 1, as described above. 2A-2D, the dPCR reaction mixture 5 proceeds through the flow channel 3 during filling, proceeding with a front 51 more rapidly on the second flow channel surface 41' than on the first flow channel surface 31'. As a result, during filling of the well 32, the dPCR reaction mixture 5 entering the well 32 traps pre-existing gas, e.g., air, within the well 32. This means that air bubbles in the form of air bubbles 6 are trapped by the dPCR reaction mixture 5 in the well 32, precisely at the well bottom and in contact with the sidewalls of the well 32.

[0033] 2a-2d, FIGS. 3a-3d show a microfluidic device 1 that is structurally essentially identical to the microfluidic device 1 shown in FIGS. 2a-2d, with the significant difference being that, in accordance with the present invention, first flow channel surface 31 and second flow channel surface 41 exhibit different hydrophilicities, with first flow channel surface 31 of microfluidic device 1 being coated with a SiO2 coating. This results in first flow channel surface 31 providing a first hydrophilicity with a surface contact angle in the range of approximately 30°-50°, and at least a portion of second flow channel surface 41 providing a second hydrophilicity with a surface contact angle in the range of approximately 80°-90°, depending on the material properties, with the first hydrophilicity being greater or more pronounced than the second hydrophilicity. As can be seen in Figures 3a-3d, when observing the progression of the initial dPCR reaction mixture 5 through flow channel 3, front 51 of dPCR reaction mixture 5 progresses through flow channel 3 in a substantially upward manner compared to Figures 2a-2d. Thus, the contact area of ​​front 51 with first flow channel surface 31 and second flow channel surface 41 progressing through flow channel 3 toward outlet opening 43 flows at a faster rate on first flow channel surface 31 than the rate at which liquid follows second flow channel surface 41 through flow channel 3, resulting in more rapid filling of well 32 than filling of flow channel 3. See particularly Figures 3b and 3c. This avoids trapping of air bubbles as the progress of fluid through the flow channel 3 is substantially equal on both flow channel surfaces 31, 41, resulting in the wells 32 being completely filled with the dPCR reaction mixture 5 without trapping air bubbles and leading to improved filling performance of the microfluidic device 1.

[0034] For comparison, Figures 4a-4c show different types of well arrays provided within first flow channel surface 31. Here, Figure 4a shows a honeycomb structure of wells 32 having an elongated hexagonal shape, shown in top view, with left-hand well apex 321 oriented toward inlet opening 42 and hexagonally shaped well edges 322; Figure 4b shows a honeycomb structure of wells 32' having a regular or regular hexagonal shape, shown in top view; and Figure 4c shows a honeycomb structure of wells 32" having a highly elongated hexagonal shape, shown in top view. The left side of each figure shows at least a portion of the honeycomb well structure, and the right side of each figure provides an enlarged detail view, where, in particular, each representative well shape is shown in top view. Figures 5a-5c show a honeycomb structure of wells 32' having a regular or regular hexagonal shape, shown in top view. 5c show each of the wells 32, 32', 32" of Figures 4a-c in cross-section along lines AA, BB and CC of Figures 4a-c, where Figure 5a shows the elongated hexagonal well 32 of Figure 4a shown in cross-section along line AA in an enlarged detail of Figure 4a, Figure 5b shows the regular hexagonal well 32' of Figure 4b shown in cross-section along line BB in an enlarged detail of Figure 4b, and Figure 5c shows the highly elongated hexagonal well 32" of Figure 4c shown in cross-section along line CC in an enlarged detail of Figure 4c. In all of Figures 5a-5c, the height h of the flow channel 3 remains the same, while the lengths of the wells 32, 32', 32" are different. In particular, the well length l of well 32 as shown in FIG. 5a satisfies an aspect ratio h / l of 0.5, which provides an optimal aspect ratio h / l for ensuring sufficient fluid separation between adjacent filled wells, while the well length l' of well 32' satisfies an aspect ratio h / l' of 1.0, and the well length l" of well 32" satisfies an aspect ratio h / l" of 0.25.

[0035] The progression of the sealing process by sealing fluid 7 of the filled elongated well 32 of FIGS. 4a and 5a is shown in FIGS. 6a to 6d, the progression of the sealing process by sealing fluid 7 of the filled regular hexagonal well 32′ of FIGS. 4b and 5b is shown in FIGS. 7a to 7d, and the progression of the sealing process by sealing fluid 7 of the filled highly elongated well 32″ of FIGS. 4c and 5c is shown in FIGS. 8a to 8d. In FIGS. 6a to 6d, it can be inferred that the sealing fluid 7 enters the flow channel 3 from the side of the inlet opening 42 and proceeds towards the outlet opening 43. As soon as the sealing fluid 7 reaches the elongated well 32 where the aspect ratio of 0.5 is fulfilled, i.e., the well length l is twice the height h of the flow channel, the sealing fluid 7 is drawn into the well 32 by capillary forces, i.e., surface tension, and by contact angle conditions with respect to the walls of the well 32. 6b. As the sealing fluid 7 is pushed further through the flow channel 3, it closes off the well 32, and a substantial portion of the dPCR reaction mixture 5 remains at the bottom of the well 32, as shown in FIG. 6c, while the dPCR reaction mixture 5 within the flow channel 3 is pushed further toward the outlet opening 42 until the flow channel 3 is completely filled with the sealing fluid 7, except for a sufficient amount of the dPCR reaction mixture 5 that is trapped at the bottom of the well 32, as shown in FIG. 6d. Thus, the elongated well 32 is fully filled with the dPCR reaction mixture 5, and adjacent elongated wells 32 are safely fluidically separated from each other by the sealing fluid 7.

[0036] For illustrative comparison purposes, Figures 7a-7d show a similar sealing process, except that the regular hexagonal well 32' meets an aspect ratio of 1.0, i.e., the height h of the flow channel 3 and the length l' of the well are identical. Here, it can be assumed that the sealing fluid 7 again enters the flow channel 3 from the side of the inlet opening 42 and proceeds toward the outlet opening 43. As soon as the sealing fluid 7 reaches the regular hexagonal well 32', it is forced into the well 32' by capillary forces, i.e., surface tension, and by contact angle conditions with the walls of the well 32', and as seen in Figure 7b, it pushes a small amount of the dPCR reaction mixture 5 out of the well 32', clearly less than the amount pushed out of the well 32' in Figure 6b. As sealing fluid 7 is pushed further through flow channel 3, it closes off well 32', and a significant portion of dPCR reaction mixture 5 remains in well 32', as shown in Figure 7c, while dPCR reaction mixture 5 within flow channel 3 is pushed further toward outlet opening 42 until flow channel 3 is completely filled with sealing fluid 7, except that a large amount of dPCR reaction mixture 5 is trapped in well 32', as shown in Figure 7d. Thus, well 32' is largely filled with dPCR reaction mixture 5.

[0037] Again, for illustrative comparison purposes, FIGS. 8a-8d show a similar sealing process, except that the highly elongated hexagonal well 32″ meets an aspect ratio of 0.25, i.e., the height h of the flow channel 3 is one-quarter of the well length l″. Here, the sealing fluid 7 can again be assumed to enter the flow channel 3 from the side of the inlet opening 42 and proceed toward the outlet opening 43. As soon as the sealing fluid 7 reaches the highly elongated hexagonal well 32″, it is forced into the well 32″ by capillary forces, i.e., surface tension, and by contact angle conditions with respect to the walls of the well 32″, and forms a large meniscus within the well 32″, which begins to push the dPCR reaction mixture 5 out of the well 32″, as seen in FIG. 8b. As the sealing fluid 7 is pushed further through the flow channel 3, the meniscus of the sealing fluid 7 almost completely fills the well 32″, and only a very small portion of the dPCR reaction mixture 5 remains at the bottom outer edge of the well 32″, as seen in FIG. 8c. As the sealing fluid 7 proceeds towards the outlet opening 43, the sealing fluid 7 forms a large meniscus within the well 32″, which begins to push the dPCR reaction mixture 5 out of the well 32″, as seen in FIG. 8b. A small portion of the dPCR reaction mixture 5 remains in the well 32" until the well 32" is closed and the flow channel 3 is completely filled with the sealing fluid 7, as shown in FIG. 8d. Thus, as can be inferred from FIG. 8d, the well 32" is almost completely filled with the sealing fluid 7, while only a small amount of the dPCR reaction mixture 5 remains in the well 32". This reduction in the amount of dPCR reaction mixture that can actually be used in the dPCR microfluidic device not only significantly deteriorates the analytical performance of the device, but also makes it impossible to clearly determine the actual total amount of dPCR reaction mixture remaining in the microfluidic device, which would make any analytical results inaccurate and unusable.

[0038] While the present invention has been described with reference to specific embodiments, it is to be understood that this description is for illustrative purposes only, and it is therefore intended that the invention be limited only by the scope of the appended claims. [Explanation of symbols]

[0039] 1. Microfluidic Device 2 Support 3 Flow Channel 31 first flow channel surface 31' first flow channel surface 32 Elongated hexagonal microwells 32' Regular hexagonal microwell 32" extra or extra elongated hexagonal microwells 321 Microwell Apex 322 Micro Well Edge 33 Rims between microwells 4 Cover 41 Second flow channel surface 41' Second flow channel surface 42 Inlet opening 43 Exit opening 5. dPCR Reaction Mixture 51 Front of dPCR reaction mixture 6. Air bubbles 7 Separation / Sealing Fluids

Claims

1. A microfluidic device (1) for thermal cycling a reaction mixture (5): Inlet opening (42); outlet opening (43); a flow channel (3) connecting said inlet opening (42) and said outlet opening (43) and defining a flow direction from said inlet opening (42) through said flow channel (3) towards said outlet opening (43), said flow channel (3) comprising a first flow channel surface (31) and a second flow channel surface (41) opposite said first flow channel surface (31); an array of wells (32; 32'; 32") provided in said first flow channel surface (31) for fluid communication with said inlet opening (42) and said outlet opening (43); Including, the first flow channel surface (31) provides a first hydrophilicity with a surface contact angle in the range of 30° to 50°, and at least a portion of the second flow channel surface (41) provides a second hydrophilicity with a surface contact angle in the range of 80° to 90°; the first hydrophilicity is greater than the second hydrophilicity; at least a portion of the array of wells (32; 32'; 32") exhibit hexagonal well shapes in the first flow channel surface (31); and Each hexagonal well (32; 32'; 32") comprises an elongated hexagonal shape elongated in the direction of flow; The microfluidic device (1).

2. The microfluidic device (1) of claim 1, wherein said first and / or second hydrophilicity is provided by material properties, by a surface treatment, or by a hydrophilic coating.

3. The surface treatment is a plasma hydrophilization treatment, and the hydrophilic coating is SiO ２ The microfluidic device (1) of claim 2, which is a coating.

4. The microfluidic device (1) according to any one of claims 1 to 3, wherein the array of all wells (32; 32'; 32") presents a hexagonal well shape in the first flow channel surface (31).

5. 5. The microfluidic device (1) of claim 4, wherein the vertex (321) of each hexagonal well (32) is oriented in the flow direction towards the inlet opening (42).

6. 6. The microfluidic device (1) of claim 4 or 5, wherein two vertices of each oppositely disposed hexagonal well (32) are oriented parallel to the flow direction.

7. 7. The microfluidic device (1) of any one of claims 1 to 6, wherein each well (32; 32'; 32") comprises a well length in the direction of flow ranging from 50 μm to 300 μm, and / or a well width perpendicular to the well length ranging from 25 μm to 150 μm, and / or a well depth ranging from 25 μm to 200 μm.

8. 8. The microfluidic device (1) of any one of claims 1 to 7, wherein the edge (322) of each well (32) on the first flow channel surface (31) facing the inlet opening (42) is a rounded edge.

9. The microfluidic device (1) of claim 8, wherein the rounded well edges (322) are rounded with a radius of <10 μm.

10. 10. The microfluidic device (1) of any one of claims 1 to 9, wherein rims (33) are provided between adjacent wells (32) for fluidic separation of the adjacent wells (32), each rim (33) comprising a width of > 10 μm.

11. The microfluidic device (1) of any one of claims 1 to 10, wherein the aspect ratio between the height of the flow channel (3) and the length of each well (32) ranges between 0.3 and 0.

7.

12. The microfluidic device (1) of any one of claims 1 to 11, wherein the height of the flow channel (3) is in the range of 25 μm to 200 μm.

13. the microfluidic device (1) consists of two parts (2, 4) that are attachable to each other, or The microfluidic device (1) consists of two parts (2, 4) that can be attached to each other and is divided into two parts (2, 4) along its longitudinal axis. A microfluidic device (1) according to any one of claims 1 to 12.

14. 14. A microfluidic device (1) according to claim 13, wherein a flow channel (3) comprising an array of wells (32) and inlet openings (42) and outlet openings (43) is provided in one part (2) of the microfluidic device (1) providing a first flow channel surface (31), and the other part (4) of the microfluidic device (1) constitutes a cover part providing a second flow channel surface (41), or wherein the other part (4) of the microfluidic device (1) constitutes a cover part providing the second flow channel surface (41) provided in the form of a cover plate or cover foil.

15. The microfluidic device (1) according to any one of claims 1 to 14, wherein the microfluidic device (1) is used for digital PCR or biochemical assays of samples provided in the form of reaction mixtures (5) in each of the wells (32) by the flow channels (3).

16. the microfluidic device (1) is a consumable product, or The microfluidic device (1) is a consumable product and is made of a transparent material, a cyclic olefin copolymer COC, or a cyclic olefin polymer COP; A microfluidic device (1) according to any one of claims 1 to 15.

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