Microfluidic device for handling a fluid sample

Abstract

The invention relates to a microfluidic device (100, 200, 300) for handling a fluid sample by dividing it into a plurality of aliquots, with at least one microfluidic well (105) configured to receive the fluid sample. The at least one microfluidic well (105) comprising an inlet end (120), an outlet end (125) and a microfluidic circuit (110) with a plurality of microchambers (112). The inlet end and the outlet end are respectively coupled to the microfluidic circuit via at least one microfluidic channel (130, 130', 130'', 130'''). The plurality of microchambers is arranged such that each microchamber is at least indirectly via further microchambers fluidly coupled with the inlet end and the outlet end such that each microchamber is configured to receive fluid from the inlet end via the at least one microfluidic channel and to provide gas displaced by the fluid downstream to the outlet end. Furthermore, the at least one microfluidic channel comprises at least one fluidic delay structure (140, 240, 240', 340, 340', 440, 440', 540, 540', 640, 740, 840), which is configured to delay a fluid flow.

Description

[0001] E ITLE 066045 K t8

[0002] AV2025-565 17 December 2025 QIAGEN GmbH

[0003] QIAGEN Stral e 1

[0004] 40724 Hilden

[0005] GERMANY

[0006] Microfluidic device for handling a fluid sample

[0007] FIELD OF THE INVENTION

[0008] The invention relates to a microfluidic device for handling a fluid sample by dividing it into a plurality of aliquots, in particular for providing a digital polymerase chain reaction analysis. Furthermore, the invention relates to a system for providing a molecular analysis of a fluid sample, in particular for providing a digital polymerase chain reaction analysis, and to a corresponding method.

[0009] BACKGROUND OF THE INVENTION

[0010] Microfluidic devices are well known in the field of molecular analysis. Providing microchambers for aliquots in order to facilitate certain thermal reactions is particularly well known in the field of polymerase chain reaction (PCR) analysis. PCR methods usually rely on thermal cycling, which involves a thermal treatment of reactants permitting different temperature-dependent reactions.

[0011] Digital PCR describes a PCR analysis where a large number of separate aliquots is analyzed in order to detect after the thermal treatment, whether a molecule of target DNA was present in the respective aliquot or not. In view of the large number of aliquots, an original concentration of target DNA can be determined by using Poisson statistics. The use of digital PCR is particularly advantageous in the case of rare target DNA compared to a background of nontarget DNA. The concentration of such rare target DNA can be reliably determined if the number of aliquots used for the digital PCR is large enough.

[0012] US 2021 / 0379593 A1 describes a microfluidic device with a microfluidic circuit including an array of fluidly coupled microchambers. Each microchamber includes a reaction chamber and an associated vent chamber. The microfluidic circuit may be arranged such that a fluid sample introduced to the microfluidic device flows into the reaction chamber and gas present in the reaction chamber is vented from the microchamber through the vent chamber. US 6 637463 B1 relates to a multi-channel microfluidic system design with balanced fluid flow distribution. US 7 067 086 B2 states a microfluidic accumulating and proportioning component. US2014 / 0141438 A1 concerns devices and a method for positioning dried reagent in microfluidic devices.

[0013] It is an object of the invention to provide a microfluidic device with an improved filling characteristic, in particular with a homogeneous filling of the respective microfluidic circuit.

[0014] SUMMARY OF THE INVENTION

[0015] According to a first aspect of the invention, a microfluidic device for handling a fluid sample by dividing it into a plurality of aliquots is provided.

[0016] The microfluidic device is arranged and configured for handling a fluid sample by dividing it into a plurality of aliquots, in particular for providing a digital polymerase chain reaction analysis, with at least one microfluidic well configured to receive the fluid sample, the at least one microfluidic well comprising

[0017] - a microfluidic circuit with a plurality of microchambers, which provide a respective reaction space for an aliquot of the plurality of aliquots,

[0018] - an inlet end coupled to the microfluidic circuit via at least one microfluidic channel,

[0019] - an outlet end coupled to the microfluidic circuit via the at least one microfluidic channel, wherein the plurality of microchambers is arranged such that each microchamber is at least indirectly via further microchambers fluidly coupled with the inlet end and the outlet end such that each microchamber is configured to receive fluid from the inlet end via the at least one microfluidic channel and to provide gas displaced by the fluid downstream to the outlet end, and

[0020] wherein the at least one microfluidic channel comprises at least one fluidic delay structure, which is configured to delay a fluid flow.

[0021] The microfluidic device according to the first aspect of the invention provides an improved filling characteristic by avoiding an unnecessary large amount of fluid that is not provided in a respective reaction chamber. The invention is based on the finding that an uncontrolled filling edge during a filling process of the microfluidic device might lead to a microfluidic channel that is already completely filled with fluid while another microfluidic channel is still to be filled with fluid. In such a case, the fluid at the outlet end can prevent gas from the incompletely filled microfluidic channel to get out of the microfluidic circuit. This results in incompletely filled microchambers, the creation of bubbles and thus in a diminished quality of the aimed analysis.

[0022] The at least one fluidic delay structure provided according to this invention can support an even filling of the microfluidic device. The filling edge of a microfluidic channel that is filled rather fast might be delayed before reaching the outlet end or before reaching certainmicrochambers in order to allow more of these filling edges to reach the outlet end at approximately the same time.

[0023] The microfluidic device according to this first aspect of the invention is simple to build, since only the at least one fluidic delay structure has to be added to already available microdevices with a plurality of microchambers.

[0024] A further advantage is the variability of the provided solution. Different microfluidic circuits can be improved by providing at least one fluidic delay structure. A useful position of such a delay structure might be tested by simply analyzing the respective time-dependent filling edge of different variants of positions for the delay structure within the microfluidic circuit.

[0025] The at least one delay structure according to this invention allows an improved control of the fluid. In particular, the at least one delay structure can prevent the fluid from unintentionally entering regions of the microfluidic device that the fluid should not enter or should enter only at a later time.

[0026] The at least one fluidic delay structure can for example be configured to slow down the fluid flow and / or to inhibit the fluid flow until a predefined fluid pressure is reached.

[0027] According to the invention, the microchambers of the microfluidic circuit are coupled to other microchambers via a respective microfluidic channel. In one embodiment there is only one microfluidic channel that connects all microchambers of the respective microfluidic circuit with each other. In preferred embodiments, there are several microfluidic channels that form parallel flow paths from the inlet end to the outlet end and thereby connect a respective number of microchambers. In that sense, the respective microfluidic channel also comprises channel segments between microchambers.

[0028] In the following, embodiments of the microfluidic device according to the first aspect of the invention will be described.

[0029] In a preferred embodiment of the microfluidic device, the at least one fluidic delay structure is at least arranged between the inlet end and the plurality of microchambers. In this embodiment, the fluidic delay structure can prevent a pre-wetting of the microfluidic circuit or at least of the part of the circuit connected to the respective microfluidic channel comprising the delay structure, for example before a controlled pressure profile is applied to the fluid sample. Thereby a controlled filling of the microfluid device is advantageously supported. In a preferred variant of this embodiment, every microfluidic channel of the microfluidic circuit comprises a respective delay structure after the inlet end in order to prevent a pre-wetting of the respective microfluidic channel.In a further embodiment of the microfluidic device, the at least one fluidic delay structure is at least arranged between the plurality of microchambers and the outlet end. In this embodiment, the respective flow from the microfluidic channel out to the outlet end is controlled by the delay structure. This allows further microfluidic channels to vent the gas out of the respective microchambers without being hindered by fluid that already reached the outlet end. In a preferred variant of this embodiment, every microfluidic channel of the microfluidic circuit comprises a respective delay structure before the outlet end in order to prevent the outlet end from being impermeable for gas during the filling of the microchambers.

[0030] In a further embodiment of the microfluidic device, the microfluidic device comprises at least two microfluidic channels, wherein the at least one fluidic delay structure combines the at least two microfluidic channels to provide a combined fluid flow to the outlet end. In this embodiment, the flow through two different microfluidic channels is controlled by a single fluidic delay structure. This advantageously reduces the number of delay structures needed to provide a control of every microfluidic channel.

[0031] In a further embodiment of the microfluidic device, the at least one fluidic delay structure is at least provided between two microchambers of the microfluidic circuit. Such a position of the delay structure allows a controlled filling of certain regions of the microfluidic circuit with the sample fluid. For example, only after the predefined fluid pressure is reached, the region behind the fluidic delay structure can be filled with fluid. This leads to a very precise control of the filling edge. Irrespective of this, in a preferred variant, multiple fluidic delay structures are provided along a respective microfluidic channel. Thereby, the control of the flow of fluid can also be further improved.

[0032] In a preferred embodiment of the microfluidic device, the at least one fluidic delay structure comprises at least one delay channel section which is longer than the geometrical distance between a fluid inlet and a fluid outlet of the delay channel section. Then, the fluid preferably has to cover a relatively long distance to get through the fluidic delay structure compared to the extent of the delay structure. In this way, with a compact design of the delay structure, a significant pressure loss can be created in the fluid so that the fluid flow is slowed down considerably. This allows a precise control of the fluid. Against this background, it is particularly preferred if the delay channel section is at least 1.5 times, preferably at least 2 times, in particular at least 3 times, particularly preferably at least 5 times, longer than the geometrical distance between the fluid inlet and the fluid outlet of the delay channel section. Irrespective of the ratio of the length of the delay channel section to the geometrical distance of its fluid inlet and outlet, the delay channel section can have a depth of at most 35 pm, preferably at most 30 pm, in particular at most 25 pm. A small depth can be advantageous with regard to a large pressure drop and thus to a sharp slowdown of the fluid flow in the delay structure. For manufacturing reasons, it can alternatively or additionally be useful if the delay channel section has a depth of at least 5 pm, preferably at least 10 pm, in particular at least 15 pm. A depth ofapproximately 20 pm can be particularly preferred in this context. Irrespective of the depth, it can be preferred for the same reasons if the delay channel section has a width of at most 20 pm, preferably at most 15 pm, in particular at most 12 pm, and / or at least 2 pm, preferably at least 5 pm, in particular at least 8 pm. A delay channel section having such dimensions can for example be manufactured by means of injection molding. Irrespective of this and of a specific depth and / or width, it can be simple and functional if the depth and / or the width of the delay channel section is at least substantially constant.

[0033] In a preferred variant of the aforementioned embodiment, the delay channel section has a meander-like form. A meander-like form can be particularly preferred with regard to compactness and a large pressure drop, and is also simple to manufacture. The meander-like channel section can comprise a plurality of changes of direction, preferably a plurality of reversals of direction. For example, the meander-like channel section can comprise at least 3, preferably at least 4, in particular at least 5, changes of direction, preferably reversals of direction. Irrespective of the specific number, the changes of direction, in particular the reversals of direction, can be provided by curved segments of the meander-like channel section. This can be preferred from a hydraulic point of view. As an alternative or in addition, the meander-like channel section can comprise a plurality of, for example at least 3, preferably at least 4, in particular at least 5, at least substantially straight segments. The straight segments can then be fluidly connected to each other by the curved segments. Irrespective of this, the straight segments can be arranged at least substantially parallel to each other and / or have at least substantially the same length. This can lead to a particular compact design of the meander-like channel section. For the same reason, it can alternatively or additionally be preferred if the meander-like channel section comprises at most 20, preferably at most 15, in particular at most 10, straight segments and / or changes of direction, in particular reversals of direction.

[0034] In an embodiment of the microfluidic device, the at least one fluidic delay structure comprises at least one fluidic stop structure, which is configured to inhibit a fluid flow until a predefined fluid pressure is reached. Such a microfluidic stop structure also allows a precise control of the fluid. The fluid pressure might be controlled in a way that allows to reach the predetermined fluid pressure only when the respective fluid can pass the stop structure without blocking the vented gas of another microfluidic channel. Irrespective of this, the at least one fluidic stop structure can preferably comprise one or more of the following structures: a capillary stop structure, a bifurcational stop structure, a hydrophobic stop structure. Typical designs for such fluidic stop structures are presented in the description of the drawings. These structures combine the advantage that their commercial use and construction is well known since such structures are extensively used for lab-on-a-chip-applications and the like.

[0035] In a preferred variant of the aforementioned embodiment, the at least one fluidic stop structure, in particular the at least one capillary stop structure, comprises at least one suddenenlargement of the cross section of the microfluidic channel. This allows simple manufacture of a fluidic stop structure, in particular of a capillary stop structure, and reliable stopping of the fluid flow. By means of a sudden enlargement of the cross section, hydraulic, in particular capillary, forces can be generated which counteract the fluid flow through the microfluidic channel. At the sudden enlargement of the cross section, the fluid can then be stopped until a predetermined pressure is reached, due to which the fluid flows into the channel section of the microfluidic channel having the greater cross section. A sudden enlargement of the cross section of the microfluidic channel means in particular an enlargement in the intended direction of flow of the fluid in the microfluidic channel. Irrespective of this, it can be sufficient if the depth or the width of the at least one microfluidic channel is abruptly increased at the at least one sudden enlargement of the cross section. However, with respect to a reliable stopping of the fluid flow, it is preferred if the depth and the width of the at least one microfluidic channel is abruptly increased at the at least one sudden enlargement of the cross section. With respect to a simple manufacture, it can alternatively or additionally be preferred if the at least one fluidic stop structure is at least substantially formed by the at least one sudden enlargement of the cross section. Then, the at least one fluidic stop structure does not have to comprise any components other than the at least one sudden enlargement of the cross section. Alternatively or additionally, the at least one sudden enlargement of the cross section can be formed at least substantially by at least one stop chamber having at least partially a greater depth and / or a greater width than adjacent channel segments of the microfluidic channel. The at least one stop chamber can be tapered in the direction of a fluid outlet of the stop chamber. Such a design of the at least one stop chamber can be expedient and space-saving. Alternatively or additionally, the at least one stop chamber can have an at least substantially triangular or an at least substantially rectangular cross section. This can be both simple and functional, wherein a triangular cross section can be preferred with regard to a space-saving design.

[0036] It can be expedient, if the at least one stop chamber has a depth of at least 100 pm, preferably at least 150 pm, in particular at least 175 pm, and / or of at most 300 pm, preferably at most 250 pm, in particular at most 225 pm. A depth of approximately 200 pm can be particularly expedient. Regardless of the depth of the stop chamber, it can also be expedient if at least one, preferably each, of the channel segments adjacent to the at least one stop chamber has a width of at least 2 pm, preferably at least 5 pm, in particular at least 8 pm, and / or of at most 20 pm, preferably at most 15 pm, in particular at most 12 pm. In this context, a width of approximately 10 pm can be particularly expedient. Alternatively or additionally to a respective width, at least one, preferably each, of the channel segments adjacent to the at least one stop chamber can expediently have depth of at least 5 pm, preferably at least 10 pm, in particular at least 15 pm, and / or of at most 40 pm, preferably at most 30 pm, in particular at most 25 pm, wherein a depth of approximately 20 pm can be particularly expedient.

[0037] In an embodiment, the at least on fluidic delay structure, preferably the at least one hydrophobic stop structure, comprises at least one hydrophilic channel section, in which thesurface of the microfluidic channel is hydrophilic, and at least one hydrophobic channel section, in which the surface of the microfluidic channel is hydrophobic. A change from a hydrophilic to a hydrophobic surface can slow down or even stop the fluid flow. Therefore, it can be preferred if the hydrophobic channel section is arranged adjacent to and, when viewed in the intended direction of flow of the fluid, behind the hydrophilic channel section. In order to increase the effect, the fluidic delay structure can also comprise a plurality of, for example at least two or at least three, hydrophilic and hydrophobic channel sections, which can be provided alternately one after the other. The different properties of the hydrophilic and hydrophobic surface sections can for example be provided by structuring and / or coating the at least one hydrophobic surface section and / or the at least one hydrophilic surface section and / or different materials of the at least one hydrophobic surface section and the at least one hydrophilic surface section. Irrespective of this, the at least one hydrophilic channel section and the at least one hydrophobic channels section can be easily combined with other kinds of fluidic delay structures, for example with a sudden enlargement of the cross section of the microfluidic channel and / or a delay channel section as described above.

[0038] In a further embodiment, the microfluidic device comprises at least two microfluidic channels, which fluidly couple a basically equal number of microchambers between inlet end and outlet end, wherein the microfluidic channels provide similar delay structure positions of a respective fluidic delay structure. A similar delay structure position is a position at a similar point along the microfluidic channel. The use of similar positions ensures a similar flow characteristic for the at least two microfluidic channels. Using a basically equal number of microchambers allows a comparable flow duration for the respective fluid flow between inlet end and outlet end. This supports a fast, homogeneous and controlled filling of the microchambers with the fluid.

[0039] In a variant of the aforementioned embodiment, the fluidic delay structures arranged at similar delay structure positions are at least substantially identical. This can be even more preferred with respect to a similar flow characteristic for the at least two microfluidic channels. For example, it can be ensured in this way that meander-like channel sections arranged at similar delay structure positions create at least substantially the same pressure drop.

[0040] In a further embodiment of the microfluidic device, the at least one microfluidic well is arranged such that the microchambers of the microfluidic well form respective sub-wells with at least one fluidic connection between two of these sub-wells and wherein a respective fluidic delay structure is arranged at the at least one fluidic connection between two sub-wells. Such a controllable connection between two sub-wells provides the possibility to fill just a predefined number of sub-wells and further sub-wells later. Thereby a better control of the filling process is possible.

[0041] In a preferred embodiment of the microfluidic device, the at least one microfluidic channel comprises at least two fluidic delay structures, for example at least two fluidic stop structures,between the inlet end and the outlet end. Two fluidic delay structures can combine different advantages of such fluidic delay structures. On the one hand they can prevent a pre-wetting of the microchambers before the start of the filling process, and on the other hand they can ensure that gas can vent out of the microfluidic circuit via the outlet end before the fluid reaches the outlet end. For this reason, for example, at least one of the fluidic delay structures can be arranged between the inlet end and the plurality of microchambers and at least one other of the fluidic delay structures can be arranged between the plurality of microchambers and the outlet end. Alternatively or additionally, at least two of the fluidic stop structures can be arranged in series one after another. Then, there is preferably no microchamber arranged between the fluidic stop structures arranged in series. In this way, a fluid flow can be stopped very reliably.

[0042] In another preferred variant of the aforementioned embodiment, the two fluidic stop structures between the inlet end and the outlet end are configured such that two different predefined fluid pressures are needed to enable a flow through the respective fluidic stop structure. Such different predefined fluid pressures enable a separate control of both stop structures by controlling the fluid pressure. After reaching a first pressure value, fluid passes the first fluidic stop structure, and after reaching a second pressure value, which is preferably larger than the first pressure value, fluid passes the second fluidic stop structure. In another variant, an even longer cascade of stop structures with further predefined pressure values is provided in a respective microfluidic channel. With a larger amount of stop structures a more precise control of the fluid flow becomes possible.

[0043] In a preferred embodiment, the microfluidic device comprises a sealing layer that covers the microfluidic circuit at least partially, preferably at least substantially. This can help to avoid a contamination of the fluid sample in the microfluidic circuit. The sealing layer can be a film, preferably a plastic film. This can be simple and functional. For the same reason, the sealing layer can alternatively or additionally be an adhesive sealing layer. Regardless of whether the sealing layer is adhesive or not, the sealing layer can be configured to fluidly separate the microchambers from each other. A sealing layer allows easy and reliable fluidic separation of the microchambers and thus of the aliquots contained therein. This applies even more if the sealing layer is configured to fluidly separate the microchambers by deformation, for example elastic deformation and / or plastic deformation, into channel segments of the at least one microfluidic channel that fluidly couple the microchambers. Then, the sealing layer can seal the channel segments and can thus cause fluidic separation of the microchambers. A deformation of the sealing layer into the channel segments can be achieved, for example, by heating the sealing layer. However, fluidic separation of the microchambers can be achieved particularly easily and reliably if the sealing layer is configured to deform into the channel segments upon application of a compressive force to the sealing layer. As a result of the applied compressive force, the sealing layer can then be pressed into the channel segments. The compressive force can for example be applied by means of a roll and / or a clamping plate.In principle, it is conceivable that the microchambers have the same depth and width as channel segments of the at least one microfluidic channel fluidly coupling the microchambers. In this case, the microfluidic device can comprise a forming layer, for example a forming plate, having one or more protrusions configured to press the sealing layer into the channel segments when the forming layer is pressed against the sealing layer. However, for the sake of simplicity, it is preferred if the microchambers have a greater depth and, preferably, a greater width than channel segments of the at least one microfluidic channel that fluidly couple the microchambers. Then, the microfluidic device does not have to comprise a respective forming layer, although this should not be excluded in principle.

[0044] In a preferred variant of the microfluidic device according to the first aspect of the invention, a plurality of delay structures is arranged in parallel and / or in series. In this variant, the plurality of delay structures can lead to a desired fluid flow through the microfluidic device. In an example of this variant, cascades of stop structures are formed within the microfluidic device, such as series-connected stop-structure systems, such as series-connected triangular systems of stop structures.

[0045] According to a second aspect of the invention, a system for providing a molecular analysis of a fluid sample, in particular for providing a digital polymerase chain reaction analysis, is provided. The system comprising

[0046] - the microfluidic device of at least one of the aforementioned embodiments,

[0047] - an analysis apparatus, configured to receive the microfluidic device and to analyze a surface of the microfluidic device with optical means,

[0048] wherein the analysis apparatus is further configured to provide a fluid pressure, in particular the predefined fluid pressure, to a fluid within the microfluidic device in order to enable a flow of the respective fluid through the at least one fluidic delay structure, in particular through the at least one fluidic stop structure.

[0049] The system according to the second aspect of the invention comprises the microfluidic device according to the first aspect and therefore all mentioned advantages of the first aspect of the invention.

[0050] The optical means are needed in order to analyze the result of the reactions within the microchambers. In a preferred embodiment, the analysis apparatus is configured to provide different predefined fluid pressures for different stop structures within the microfluidic circuit.

[0051] In the following, embodiments of the system according to the second aspect of the invention will be described.In a preferred embodiment of the system according to the second aspect of the invention, the fluid pressure, in particular the predefined fluid pressure, is provided by a piston, which is arranged to be pushed against an elastic layer that is sealing the respective inlet end of the at least one well of the microfluidic device. Such a piston can be easily controlled and is known to be rather robust and easy to build.

[0052] In a further embodiment, the system is further comprising a control unit configured to control the pressure provided to the fluid within the microfluidic device based on an analysis of a present filling state of the at least one microfluidic well, wherein this analysis is facilitated by the optical means. In this embodiment, the advantages of the provided fluidic delay structures lead to an easy and reliable control of the fluid flow and in particular of the filling edge of the current fluid flow. After the fluid flow has reached a certain area of the respective well, the pressure of the flow might be increased in order to provide a flow through a respective number of fluidic delay structures, in particular through a respective number of fluidic stop structures.

[0053] According to a third aspect of the invention, a method for providing a molecular analysis of a fluid sample, in particular for providing a digital polymerase chain reaction analysis, is provided. The method comprising the steps:

[0054] - providing an analysis apparatus and a microfluidic device with at least one microfluidic well configured to receive the fluid sample;

[0055] - providing the fluid sample to the at least one microfluidic well via a respective inlet end of this microfluidic well;

[0056] - insertion of the microfluidic device into the analysis apparatus; and

[0057] - applying a pressure profile on the fluid sample in order to push the fluid sample into a microfluidic circuit of the microfluidic device and through at least one fluidic delay structure provided within at least one microfluidic channel of the microfluidic device, wherein a flow of fluid is delayed by means of the at least one fluidic delay structure.

[0058] The method of the third aspect of the invention shares the advantages described in the context of the system according to the second aspect of the invention. In particular, the method allows a controlled filling of the at least one microfluidic well. The provided fluidic delay structure may prevent pre-wetting and / or a blocked venting of the microfluidic channel.

[0059] The steps of the method according to the third aspect are preferably performed in the presented order.

[0060] The pressure profile is formed by a sequence of applied pressures, preferably by a predefined sequence of applied pressures.

[0061] By means of the at least one fluidic delay structure, the flow of fluid can for example be slowed down and / or stopped until a predefined fluid pressure is reached.In a preferred embodiment of the method according to the third aspect of the invention, the applied pressure surpasses at least at a certain stage of the molecular analysis a predefined fluid pressure that is needed to enable a flow of fluid through at least one fluidic stop structure of the at least one fluidic delay structure. This allows a precise control of the fluid. The predefined fluid pressure is preferably applied at a predetermined time of the molecular analysis. Irrespective of this, it can be preferred for the sake of simplicity if the at least one fluidic delay structure is at least substantially formed by the at least one fluidic stop structure. Then, the at least one fluidic delay structure preferably does not comprise other components than the at least one fluidic stop structure.

[0062] In a further embodiment, the method is further comprising the steps

[0063] - analyzing a present filling state of a plurality of microchambers provided by the microfluidic well; and

[0064] - determining the pressure profile to be applied on the fluid sample based on the present filling state of the plurality of microchambers.

[0065] The detailed control of the pressure according to this embodiment allows a precise filling of the microchambers. Furthermore, it can be decided whether to fill all microchambers or not. The present filling state might be analyzed by optical means, such as a camera.

[0066] In a further embodiment, the method comprises the step of fluidly separating a plurality of microchambers of the microfluidic circuit from each other, in particular after the microchambers have been at least partially filled with the fluid sample. This prevents the exchange of fluid between the microchambers. In principle, the application of the pressure on the fluid sample can be stopped before the microchambers are fluidly separated from each other. However, with respect to a controlled filling of the microchambers with the fluid sample, it can be useful to maintain the pressure on the fluid sample at least partially until the microchambers are fluidly separated from each other. Irrespective of this, the fluidic separation of the microchambers can be achieved easily and reliably by deforming a sealing layer into channel segments of the at least one microfluidic channel that fluidly couple the microchambers. The sealing layer can cover the microfluidic circuit at least partially, preferably at least substantially. Alternatively or additionally, it can be particularly preferred with respect to an easy and reliable fluidic separation of the microchambers if the deformation of the sealing layer into the channel segments is achieved by applying a compressive force to the sealing layer, preferably in a direction at least partially, in particular at least substantially, perpendicular to the sealing layer. Irrespective of the direction, the compressive force can for example be applied to the sealing layer by means of a roll, a clamping plate, and / or a gas pressure, wherein the gas can be air for the sake of simplicity. The gas pressure can then be applied to the sealing layer from outside.It shall be understood that the microfluidic device of the first aspect of the invention, the system for providing a molecular analysis of a fluid sample of the second aspect of the invention, and the method for providing a molecular analysis of a fluid sample of the third aspect of the invention have similar or identical embodiments.

[0067] These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter.

[0068] BRIEF DESCRIPTION OF THE DRAWINGS

[0069] In the following drawings:

[0070] Fig. 1 shows a first embodiment of a microfluidic device according to a first aspect of the invention;

[0071] Fig. 2 shows a second embodiment of the microfluidic device according to the first aspect of the invention;

[0072] Fig. 3 shows a third embodiment of the microfluidic device according to the first aspect of the invention;

[0073] Figs. 4a, 4b, 5a, 5b, 6, 7, 8a, 8b show seven different embodiments of fluidic delay structures for the microfluidic device according to the first aspect of the invention; Figs. 9a-d show details of the first embodiment of the microfluidic device shown in Fig. 1;

[0074] Fig. 10 shows a first embodiment of a system according to a second aspect of the invention;

[0075] Fig. 11 shows a second embodiment of a system according to the second aspect of the invention;

[0076] Fig. 12 shows a flow diagram of an embodiment of a method according to a third aspect of the invention.

[0077] DETAILED DESCRIPTION OF EMBODIMENTS

[0078] Fig. 1 shows an embodiment of a microfluidic device 100 according to a first aspect of the invention. The microfluidic device 100 is configured to receive a fluid sample and to divide it into a plurality of aliquots, in particular for providing a digital polymerase chain reaction analysis. One microfluidic well 105 of the microfluidic device 100 is shown in Fig. 1. In a preferred variant of this embodiment, the microfluidic device 100 comprises at least one further microfluidic well.

[0079] The microfluidic well 105 comprises a microfluidic circuit 110 with a plurality of microchambers 112, which provide a respective reaction space for an aliquot of the plurality of aliquots. The microfluidic well 105 further comprises an inlet end 120 coupled to the microfluidic circuit 110via at least one microfluidic channel 130, 130’, 130”, 130”’ and an outlet end 125 coupled to the microfluidic circuit 110 via the at least one microfluidic channel 130, 130’, 130”, 130’”.

[0080] The plurality of microchambers 112 is arranged such that each microchamber 112 is at least indirectly via further microchambers 112 fluidly coupled with the inlet end 120 and with the outlet end 125. In the present embodiment, four microfluidic channels 130, 130’, 130”, 130’” are arranged parallel to each other and each connect five microchambers 112 in a row. The connections between two respective microchambers 112 are formed by the respective microfluidic channel 130, 130’, 130”, 130’”. As a consequence of this microfluidic structure each microchamber 112 is configured to receive fluid from the inlet end 120 via the at least one microfluidic channel 130, 130’, 130”, 130’” and to provide gas displaced by the fluid downstream to the outlet end 125. Possible structures of such microchambers 112 are known in the art and are therefore not described in detail in the following. As a non-limiting example, US 2021 / 0379593 A1 describes an embodiment of such microchambers.

[0081] The embodiment according to the invention further comprises at least one microfluidic channel 130, 130’, 130”, 130’” with at least one fluidic delay structure 140, which is configured to delay a fluid flow. The fluidic delay structure 140 enables a control of the flow of the fluid sample into the microfluidic circuit 110 and of gas displaced by the fluid out of the microfluidic circuit 110. Possible embodiments of such fluidic delay structures are shown in Figs. 4, 5, 6, 7 and 8. The fluidic delay structure 140 can for example comprise a fluidic stop structure, which is configured to inhibit a fluid flow until a predefined fluid pressure is reached. Typically, the predefined fluid pressure to reach is defined by the physical structure and / or the material of the fluidic stop structure. These characteristics lead to capillary forces and / or other hydraulic forces that act as force against the fluid pressure provided during the filling of the inlet end 120.

[0082] In the embodiment shown in Fig. 1, each of the microfluidic channels 130, 130’, 130”, 130’” comprises a respective fluidic delay structure 140 between the last microchamber 112 and the outlet end 125. In a preferred variant of this embodiment, all fluidic delay structures 140 comprise a similar structure. Alternatively, different fluidic delay structures are used in a further variant of this embodiment.

[0083] Using the fluidic delay structures 140 at the shown positions within the microfluidic circuit 110 allows that all the microchambers 112 are filled with the fluid sample before fluid of the fluid sample can reach regions where it would prevent gas from unfilled or only partially filled microchambers 112 to stream out of the outlet end 125. In case the fluidic delay structures 140 comprise fluidic stop structures configured to inhibit a fluid flow until a predefined fluid pressure is reached, the microchambers 112 can be filled with a pressure below the predefined fluid pressure leading to completely filled microfluidic channels 130, 130’, 130”, 130’”. The predefined pressure might not be reached during the filling process, or it is reached at the endof the filling process ensuring that all channels are vented before the outlet end 125 is filled with fluid.

[0084] The microfluidic well 105 shown in Fig. 1 is just one of at least four microfluidic wells of the microfluidic device 100, which are not depicted in Fig. 1 for clarity reasons. In a preferred embodiment, the microfluidic device comprises at least 10 microfluidic wells, preferably at least 20 microfluidic wells. In a further preferred embodiment, each microfluidic well of the microfluidic device comprises at least 50 microchambers, in particular at least 200 microchambers. The different microfluidic wells do not share a fluidic connection. Therefore, each inlet end 120 has to be filled with the fluid sample separately.

[0085] The microfluidic device 100 according to the first aspect of the invention is particularly advantageous for handling a fluid sample within the scope of a polymerase chain reaction analysis, especially within the scope of a digital polymerase chain reaction (dPCR) analysis. This is the case since a large number of aliquots has to be applied to a thermal treatment for a reasonable interpretation of the final analysis. The provided microfluidic device 100 allows a particularly homogenous filling of the microchambers in order to assure a large number of aliquots for the dPCR analysis.

[0086] Fig. 2 shows a second embodiment of the microfluidic device 200 according to the first aspect of the invention.

[0087] The microfluidic device 200 differs from the microfluidic device 100 shown in Fig. 1 in view of the respective position of the fluidic delay structures 240, 240’. There are four fluidic delay structures 240 which are respectively located at each microfluidic channel 130, 130’, 130”, 130’” between the inlet end 120 and the first microchamber 112. This location can prevent a pre-wetting of the microchambers 112, if there is no pressure applied to the fluid sample in the inlet end 120.

[0088] A further difference towards the microfluidic device 100 is the second fluidic delay structure 240’ arranged at a further location downstream compared to the first fluidic delay structure 240. The second fluidic delay structure 240’ respectively combines at least two microfluidic channels 130, 130’, 130”, 130’” to provide a combined fluid flow to the outlet end 125. Thus, two microfluidic channels 130, 130’, 130”, 130’” share the last microchambers 112, respectively. It should be understood that a microfluidic channel is every direct connection between inlet end 120 and outlet end 125, even if two channels share a number of microchambers 112 like in the depicted embodiment.

[0089] Furthermore, the two fluidic delay structures 240, 240’ in each microfluidic channel 130, 130’, 130”, 130’” can for example be formed by different fluidic stop structures 240, 240’ such that two different predefined fluid pressures are needed to enable a flow through the respectivefluidic stop structure 240, 240’. For the first fluidic stop structure 240 a lower pressure level may be needed in order to press fluid through the fluidic stop structure 240 compared to the pressure level of the second fluidic stop structure 240’. Then, the last microchambers 112 of each microfluidic channel 130, 130’, 130”, 130’” are filled with fluid only after a pressure level is reached that goes beyond the pressure level needed to fill the first microchambers 112 of each microfluidic channel 130, 130’, 130”, 130’”.

[0090] Fig. 3 shows a third embodiment of the microfluidic device 300 according to the first aspect of the invention.

[0091] The microfluidic device 300 differs from the microfluidic device 200 of Fig. 2 by its structure of the microfluidic circuit 110 and the positions of the fluidic delay structures 340, 340’.

[0092] The microfluidic device 300 with the depicted microfluidic well 105 comprises a number of subwells 307. In the given embodiment, four sub-wells 307 are arranged to fluidly connect the inlet end 120 with the outlet end 125. Each sub-well 307 is fluidly connected to a further sub-well 307 via a fluidic connection, wherein a respective fluidic delay structure 340 is arranged at the fluidic connection between two sub-wells 307. The fluidic connection forms a part of the respective microfluidic channel that connects inlet end 120 and outlet end 125 according to the first aspect of the invention. Each sub-well 307 comprises a respective microfluidic sub-circuit, wherein all sub-circuits form together the microfluidic circuit 110. The detailed structure of a respective sub-well 307 is not shown in Fig. 3 due to clarity reasons.

[0093] The fluidic delay structures 340 which are arranged at the fluidic connection between two subwells 307 are the same as the fluidic delay structures 340’ which are arranged between the microfluidic circuit 110 and the outlet end 125. Possible structures of such fluidic delay structures are shown in Figs. 4a, 4b, 5a, 5b, 6, 7, 8a and 8b.

[0094] The microfluidic device 300 allows a controlled filling of certain regions of the microfluidic circuit 110. The fluidic delay structures 340, 340’ can for example comprise fluidic stop structures configured to inhibit a fluid flow until a predefined fluid pressure is reached such that the microfluidic device 300 allows a pressure dependent filling of certain regions of the microfluidic circuit 110. In a not shown embodiment, at least three different fluidic delay structures are used in the microfluidic circuit in order to allow a controlled, for example pressure dependent, filling of the microchambers.

[0095] Figs. 4a, 4b, 5a, 5b, 6 show five different embodiments of fluidic delay structures 440, 440’, 540, 540’, 640 in the form of fluidic stop structures for the microfluidic device according to the first aspect of the invention. The intended direction of the fluid flow is shown by a respective arrow.The fluidic stop structure 440 shown in Fig. 4a is a capillary stop structure. The capillary stop structure 440 comprises two regions of reduced thickness 441 , 442 while all other parts of the depicted structure share an essentially similar larger thickness. Between the two regions of reduced thickness 441, 442 is a capillary stop 443, which holds fluid that is pressed through the first region of reduced thickness 441 after for example a thermal heating. Capillary forces that act between the microfluidic channel with larger thickness and the regions of reduced thickness 441, 442 lead to the defined level of fluid pressure that has to be reached in order to pass this fluidic stop structure 440.

[0096] Fig. 4b shows a capillary stop structure 440’ similar to the capillary stop structure 440 of Fig.

[0097] 4a. It has a region of reduced thickness 44 T while other parts of the depicted structure share an essentially similar thickness. Capillary forces that act between the microfluidic channel with larger thickness und the regions of reduced thickness 44 T lead to the defined level of fluid pressure that has to be reached in order to pass this fluidic stop structure 440’.

[0098] Other capillary stop structures are well known in the art and might also be used in an embodiment of the microfluidic device according to the first aspect of the invention. Such capillary stop structures usually combine at least one region of reduced thickness with a microfluidic channel with larger thickness on both sides of the stop structure. Capillary stop structures might also be combined with other stop structures like a bifurcational stop structure and / or a hydrophobic stop structure, as shown in the following.

[0099] The fluidic stop structure 540 shown in Fig. 5a is a bifurcational stop structure. The bifurcational stop structure 540 comprises six loops 544 which respectively divide an incoming fluid flow into two streams, wherein one stream is directed into a direction opposite to the direction of the other stream. Therefore, the loops 544 of this embodiment lead to a deceleration of the incoming flow, wherein the degree of deceleration depends on the number of loops used for the bifurcational stop structure. As a result, the bifurcational stop structure can inhibit the filling of microchambers behind that structure unless the predetermined filling pressure is reached.

[0100] Fig. 5b shows a bifurcational stop structure 540’ similar to the bifurcational stop structure 540 of Fig. 5a. The bifurcational stop structure 540’ comprises four loops 544’ which respectively divide an incoming fluid flow into two streams. One of these streams is in line with an incoming flow direction and afterwards directed into a direction opposite to the incoming flow at the end of the respective loop 544’. Therefore, the loops 544’ of this embodiment lead to a deceleration of the incoming flow, wherein the degree of deceleration depends on the number of loops used for the bifurcational stop structure. As a result, the bifurcational stop structure can inhibit the filling of microchambers behind that structure unless the predetermined filling pressure is reached.Other bifurcational stop structures might also be used in an embodiment of the microfluidic device according to the first aspect of the invention. Such stop structures might also comprise bifurcational elements that divide an incoming fluid flow in more than two streams, like three streams, as it is obvious to someone skilled in the art of microfluid dynamics. Such bifurcational stop structure usually combine a stream between two bifurcational elements, as for example two loops, with a further stream that is directed away from a next bifurcational element, as shown in Figs. 5a and 5b. Bifurcational stop structures might also be combined with other stop structures like a capillary stop structure and / or a hydrophobic stop structure.

[0101] The fluidic stop structure 640 shown in Fig. 6 is a hydrophobic stop structure. The hydrophobic stop structure 640 comprises a hydrophobic channel section 645 having a hydrophobic surface that is configured to stop a fluid flow due to its hydrophobic characteristics. In the shown embodiment, the fluid to cross the fluidic stop structure first crosses a hydrophilic channel section 646 having a hydrophilic surface and is afterwards stopped by the hydrophobic channel section 645. After a certain pressure of filling is reached, the hydrophobic characteristics can no longer prevent the fluid from flowing through that stop structure 640. Other hydrophobic stop structures might also be used in an embodiment of the microfluidic device according to the first aspect of the invention.

[0102] Fig. 7 shows an embodiment of a fluidic delay structure 740 for the microfluidic device according to the first aspect of the invention in a top view. The intended direction of the fluid flow is shown by an arrow.

[0103] The fluidic delay structure 740 is at least substantially formed by a delay channel section 747 which is at least 10 times longer than the geometrical distance between a fluid inlet 748 and a fluid outlet 749 of the delay channel section 747. The delay channel section 747 has a meander-like form and comprises a plurality of curved segments 751 providing a plurality of reversals of direction and a plurality of straight segments 752 fluidly coupling the curved segments 751. In the depicted embodiment, the delay channel section 747 has an at least substantially constant width wD of approximately 10 pm and an at least substantially constant depth of approximately 20 pm.

[0104] Figs. 8a and 8b show an embodiment of two fluidic delay structures 840 in the form of two fluidic stop structures 840 for the microfluidic device according to the first aspect of the invention in a top view (Fig. 8a) and in a sectional view (Fig. 8b) along the section plane VII Ib-VI 11 b shown in Fig. 8a. The intended direction of the fluid flow is shown by an arrow.

[0105] The fluidic stop structures 840 are each at least substantially formed by a sudden enlargement 853 of the cross section of a microfluidic channel 830, which can, for example, be one of the microfluidic channels 130, 130’, 130”, 130”’ shown e.g. in Fig. 1. At the sudden enlargements 853 of the cross section, the depth and the width of the microfluidic channel 830 is abruptlyincreased. The sudden enlargements 853 of the cross section are each at least substantially formed by a stop chamber 854, namely at a respective fluid inlet 855 of the respective stop chamber 854. In the depicted embodiment, the stop chambers 854 have a triangular cross section and taper in the direction of a respective fluid outlet 856 of the respective stop chamber 854. The stop chambers 854 have a greater depth and partially a greater width than adjacent channel segments of the microfluidic channel 830. In the shown embodiment, the channel segments of the microfluidic channel 830 adjacent to the stop chambers 854 have a width wC of approximately 10 pm and a depth dC of approximately 20 pm and the stop chambers 854 have a depth dS of approximately 200 pm.

[0106] Other fluidic delay structures different from those shown in Figs. 4a, 4b, 5a, 5b, 6, 7, 8a and 8b can also be used to provide the microfluidic device according to the first aspect of the invention.

[0107] Figs. 9a-d show details of the microfluidic device 100 shown in Fig. 1 in a sectional view along a section plane along the microfluidic channel 130 in the region of two of the microchambers 112 (Figs. 9a, 9c) and in a sectional view along a section plane perpendicular to the microfluidic channel 130 in a region between the two microchambers 112 (Figs. 9b, 9d). As far as shown in Figs. 9a-d, the microfluidic device 200 shown in Fig. 2 and / or the microfluidic device 300 shown in Fig. 3 can have at least substantially the same structure.

[0108] The microfluidic device 100 comprises an adhesive sealing layer 960, for example in the form of an adhesive plastic film, which is glued to the microfluidic well 105 and covers the microchambers 112.

[0109] In Figs. 9a, 9b, the sealing layer 960 is arranged at least substantially outside the microfluidic channel 130, thus allowing fluid flow through the microfluidic channel 130. In this way, fluid of the fluid sample can flow from one microchamber 112 to another microchamber 112 via the microfluidic channel 130 during a filling process of the microfluidic device 100. In order to fluidly separate the microchambers 112 from each other after the microchambers 112 have been at least partially filled with the fluid sample, a compressive force F can be applied to the sealing layer 960 in the direction of the microfluidic well 105.

[0110] In Figs. 9c, 9d, a compressive force F has been applied to the sealing layer 960 in a direction at least substantially perpendicular to the sealing layer 960, for example by means of a roll and / or a clamping plate. As a result of the application of the compressive force F, the sealing layer 960 is deformed into channel segments of the microfluidic channel 130 that fluidly couple the microchambers 112 such that the channel segments are sealed by the sealing layer 960. In this way, the microchambers 112 are fluidly separated from each other. The sealing layer 960 is also deformed into the microchambers 112. However, due to the greater depth of themicrochambers 112 compared to the depth of the channel segments of the microfluidic channel 130, a volume for the fluid sample remains in each microchamber 112.

[0111] Fig. 10 shows a first embodiment of a system 1000 according to a second aspect of the invention.

[0112] The system 1000 is arranged and configured for providing a molecular analysis of a fluid sample, in particular for providing a digital polymerase chain reaction analysis. The system 1000 comprises the microfluidic device 100 according to at least one of the preceding embodiments and an analysis apparatus 1070.

[0113] As an example, the microfluidic device 100 comprises four microfluidic wells 105 as depicted in Fig. 1. The microfluidic device 100 is shown in an inserted state, where it is inserted into the analysis apparatus 1070 by using a holding structure 1072 of the analysis apparatus 1070.

[0114] The analysis apparatus 1070 is configured to receive the microfluidic device 100 via the holding structure 1072 and to analyze a surface of the microfluidic device 100 with optical means 1074. The optical means 1074 are in the shown embodiment formed by a camera.

[0115] Furthermore, the analysis apparatus 1070 comprises pressuring means 1076 that are formed by a plate with a series of pistons 1077, which are arranged to be pressed inside the respective inlet end in order to provide a fluid pressure on the inserted sample fluid. Preferably, the applied fluid pressure enables a flow of fluid through the fluidic delay structures of the microfluidic device 100. In the shown embodiment, an elastic layer 1009 is provided at the microfluidic device 100 in order to prevent a contamination of the sample fluid in the analysis apparatus 1070. The layer 1009 is elastic in order to enable the pistons 1077 of the pressuring means 1076 to press into the inlet end through the layer 1009 without damaging that layer 1009. In a preferred variant of this embodiment, the elastic layer is also adhesive. Thereby a reliable sealing of the inlet end is provided. In a not shown embodiment, no layer is used in order to seal the inlet end. In such an embodiment, other precautionary measures might ensure that the fluid sample is not contaminated.

[0116] Preferably, the sample fluid is brought into the inlet end of the microfluidic device 100 prior to the insertion of this microfluidic device 100 into the analysis apparatus 1070. After the fluid sample is brought into the respective inlet ends, the elastic layer 1009 is brought onto the microfluidic device 100 in order to seal the inlet end and afterwards the microfluidic device 100 is brought into the holding structure 1072, formed for example as sliding rails.

[0117] In order to fluidly separate the microchambers from each other, channel segments of the microfluidic channel that are arranged between microchambers are closed, before a thermal treatment or the like starts. The channel segments are closed by applying a compressive forceto the sealing layer as described above with regard to Figs. 9a-d. Further details about the closing of the microfluidic channel are for example described in US 2021 / 0379593 A1.

[0118] After the sample fluid is pressed inside the microchambers, the analysis apparatus 1070 is further configured to apply a certain predefined analysis of the fluid sample in the different microchambers, in particular to apply a predefined thermal treatment of the sample fluid.

[0119] Fig. 11 shows a second embodiment of a system 1100 according to the second aspect of the invention.

[0120] The system 1100 differs from the system 1000 shown in Fig. 10 in view of a control unit 1178 of the analysis apparatus 1170. The control unit 1178 is configured to control the pressure provided to the fluid within the microfluidic device based on an analysis of a present filling state of the at least one microfluidic well, wherein this analysis is facilitated by the optical means 1074. If the optical means 1074 detect that there are still unfilled microchambers, the control unit 1178 may provide a control signal to the pressuring means in order to increase the pressure on the fluid sample in the inlet end and thereby fill the respective microchambers.

[0121] Since the pressure is tuned depending on the filling state, the filling process of the microchambers can be controlled very detailed by the control unit 1178 of the depicted embodiment.

[0122] Fig. 12 shows a flow diagram of an embodiment of a method 1200 according to a third aspect of the invention.

[0123] The method 1200 is configured for providing a molecular analysis of a fluid sample, in particular for providing a digital polymerase chain reaction analysis. The method 1200 comprising steps as given in the following.

[0124] A first step 1210 is the providing of an analysis apparatus and of a microfluidic device with at least one microfluidic well configured to receive the fluid sample.

[0125] A second step 1220 is a providing of the fluid sample to the at least one microfluidic well via a respective inlet end of this microfluidic well.

[0126] A third step 1230 is an insertion of the microfluidic device into the analysis apparatus.

[0127] A fourth step 1240 is an applying of a pressure profile on the fluid sample in order to push the fluid sample into a microfluidic circuit of the microfluidic device and through at least one fluidic delay structure provided within at least one microfluidic channel of the microfluidic device, wherein a flow of fluid is delayed by means of the at least one fluidic delay structure.The steps 1210, 1220, 1230, 1240 of the method 1200 according to the third aspect of the invention are preferably performed in the given order.

[0128] In a preferred variant of the embodiment shown in Fig. 12, the applied pressure surpasses at least at a certain stage of the molecular analysis a predefined fluid pressure that is needed to enable a flow of fluid through at least one fluidic stop structure of the at least one fluidic delay structure.

[0129] Usually, after the method 1200, the molecular analysis is provided by further steps of the particular analysis method.

[0130] In a preferred variant of the embodiment shown in Fig. 12, the method further comprises the steps

[0131] - analyzing a present filling state of a plurality of microchambers provided by the microfluidic well; and

[0132] - determining the pressure profile to be applied on the fluid sample based on the present filling state of the plurality of microchambers.

[0133] These further steps are preferably provided between the insertion of the microfluidic device and the applying of the pressure.

[0134] In a further variant of the embodiment shown in Fig. 12, the method further comprises the step - fluidly separating a plurality of microchambers of the microfluidic circuit from each other, preferably by deforming a sealing layer into channel segments of the at least one microfluidic channel fluidly coupling the microchambers, in particular by applying a compressive force to the sealing layer.

[0135] This further step is preferably provided after the application of the pressure on the fluid sample. Then, it can be further preferred if the pressure on the fluid sample is at least partially maintained until the microchambers are fluidly separated from each other.

[0136] The following numbered items further describe the present invention, solve its object, and form part of the disclosure of the present document.

[0137] Item 1 : A microfluidic device for handling a fluid sample by dividing it into a plurality of aliquots, in particular for providing a digital polymerase chain reaction analysis, with at least one microfluidic well configured to receive the fluid sample, the at least one microfluidic well comprising a microfluidic circuit with a plurality of microchambers, which provide a respective reaction space for an aliquot of the plurality of aliquots, an inlet end coupled to the microfluidic circuit via at least one microfluidic channel, an outlet end coupled to the microfluidic circuit via the at least one microfluidic channel, wherein the plurality of microchambers is arranged suchthat each microchamber is at least indirectly via further microchambers fluidly coupled with the inlet end and the outlet end such that each microchamber is configured to receive fluid from the inlet end via the at least one microfluidic channel and to provide gas displaced by the fluid downstream to the outlet end, and wherein the at least one microfluidic channel comprises at least one fluidic delay structure, which is configured to delay a fluid flow.

[0138] Item 2: The microfluidic device of item 1, wherein the at least one fluidic delay structure is at least arranged between the inlet end and the plurality of microchambers.

[0139] Item 3: The microfluidic device of item 1 or 2, wherein the at least one fluidic delay structure is at least arranged between the plurality of microchambers and the outlet end.

[0140] Item 4: The microfluidic device of at least one of the preceding items, comprising at least two microfluidic channels, wherein the at least one fluidic delay structure combines the at least two microfluidic channels to provide a combined fluid flow to the outlet end.

[0141] Item 5: The microfluidic device of at least one of the preceding items, wherein the at least one fluidic delay structure is at least provided between two microchambers of the microfluidic circuit.

[0142] Item 6: The microfluidic device of at least one of the preceding items, wherein the at least one fluidic delay structure comprises at least one delay channel section, which is, preferably at least 1.5 times, in particular at least 2 times, particularly preferably at least 3 times, longer than the geometrical distance between a fluid inlet and a fluid outlet of the delay channel section, and wherein, preferably, the delay channel section has a meander-like form.

[0143] Item 7: The microfluidic device of at least one of the preceding items, wherein the at least one fluidic delay structure comprises at least one fluidic stop structure configured to inhibit a fluid flow until a predefined fluid pressure is reached and wherein, preferably, the at least one fluidic stop structure comprises one or more of the following structures: a capillary stop structure, a bifurcational stop structure, a hydrophobic stop structure.

[0144] Item 8: The microfluidic device of item 7, wherein the at least one fluidic stop structure comprises at least one sudden enlargement of the cross section of the microfluidic channel, and wherein, preferably, the at least one sudden enlargement of the cross section is at least substantially formed by at least one stop chamber having at least partially a greater depth and / or a greater width than adjacent channel segments of the microfluidic channel, and, preferably, an at least substantially triangular or rectangular cross section.

[0145] Item 9: The microfluidic device of at least one of the preceding items, wherein the at least one fluidic delay structure, in particular the at least one hydrophobic stop structure, comprises atleast one hydrophilic channel section having a hydrophilic surface and at least one hydrophobic channel section having a hydrophobic surface.

[0146] Item 10: The microfluidic device of at least one of the preceding items, comprising at least two microfluidic channels, which fluidly couple a basically equal number of microchambers between inlet end and outlet end, wherein the at least two microfluidic channels provide similar delay structure positions of a respective fluidic delay structure.

[0147] Item 11: The microfluidic device of at least one of the preceding items, wherein the at least one microfluidic well is arranged such that the microchambers of the microfluidic well form respective sub-wells with at least one fluidic connection between two of these sub-wells and wherein a respective fluidic delay structure is arranged at the at least one fluidic connection between two sub-wells.

[0148] Item 12: The microfluidic device of at least one of the preceding items, wherein at least one microfluidic channel comprises at least two fluidic delay structures, preferably at least two fluidic stop structures, between the inlet end and the outlet end, and wherein, preferably, the two fluidic stop structures between the inlet end and the outlet end are configured such that two different predefined fluid pressures are needed to enable a flow through the respective fluidic stop structure.

[0149] Item 13: The microfluidic device of at least one of the preceding items, wherein the microfluidic device comprises a sealing layer at least partially covering the microfluidic circuit and configured to fluidly separate the microchambers from each other, preferably by deformation into channel segments of the at least one microfluidic channel fluidly coupling the microchambers, in particular upon application of a compressive force to the sealing layer.

[0150] Item 14: A system for providing a molecular analysis of a fluid sample, in particular for providing a digital polymerase chain reaction analysis, comprising the microfluidic device of at least one of the preceding items, an analysis apparatus, configured to receive the microfluidic device and to analyze a surface of the microfluidic device with optical means, wherein the analysis apparatus is further configured to provide a fluid pressure, in particular the predefined fluid pressure, to a fluid within the microfluidic device in order to enable a flow of the respective fluid through the at least one fluidic delay structure, in particular through the at least one fluidic stop structure.

[0151] Item 15: The system according to item 14, wherein the fluid pressure, in particular the predefined fluid pressure, is provided by a piston, which is arranged to be pushed against an elastic layer that is sealing the respective inlet end of the at least one microfluidic well of the microfluidic device.Item 16: The system according to item 14 or 15, further comprising a control unit configured to control the pressure provided to the fluid within the microfluidic device based on an analysis of a present filling state of the at least one microfluidic well, wherein this analysis is facilitated by the optical means.

[0152] Item 17: A method for providing a molecular analysis of a fluid sample, in particular for providing a digital polymerase chain reaction analysis, comprising the steps providing an analysis apparatus and a microfluidic device with at least one microfluidic well configured to receive the fluid sample; providing the fluid sample to the at least one microfluidic well via a respective inlet end of this microfluidic well; insertion of the microfluidic device into the analysis apparatus; and applying a pressure profile on the fluid sample in order to push the fluid sample into a microfluidic circuit of the microfluidic device and through at least one fluidic delay structure provided within at least one microfluidic channel of the microfluidic device, wherein a flow of fluid is delayed by means of the at least one fluidic delay structure.

[0153] Item 18: The method of item 17, wherein the applied pressure surpasses at least at a certain stage of the molecular analysis a predefined fluid pressure that is needed to enable a flow of fluid through at least one fluidic stop structure of the at least one fluidic delay structure.

[0154] Item 19: The method of item 17 or 18, further comprising the steps analyzing a present filling state of a plurality of microchambers provided by the microfluidic well; and determining the pressure profile to be applied on the fluid sample based on the present filling state of the plurality of microchambers.

[0155] Item 20: The method of at least one of the preceding items, further comprising the step fluidly separating a plurality of microchambers of the microfluidic circuit from each other, preferably by deforming a sealing layer into channel segments of the at least one microfluidic channel fluidly coupling the microchambers, in particular by applying a compressive force to the sealing layer.LIST OF REFERENCE SIGNS

[0156] 100, 200, 300 microfluidic device

[0157] 105 microfluidic well

[0158] 110 microfluidic circuit

[0159] 112 microchamber

[0160] 120 inlet end

[0161] 125 outlet end

[0162] 130, 130’, 130”, 130’” microfluidic channel

[0163] 830

[0164] 140, 240, 240’, 340, 340’, fluidic delay structure

[0165] 440, 440’, 540, 540’, 640,

[0166] 740, 840

[0167] 307 sub-well

[0168] 441, 442, 44T, 442 region of reduced thickness 443 capillary stop

[0169] 544, 544’ loop

[0170] 645 hydrophobic area

[0171] 646 hydrophilic area

[0172] 747 delay channel section

[0173] 748 fluid inlet

[0174] 749 fluid outlet

[0175] 751 curved segment

[0176] 752 straight segment

[0177] 853 sudden enlargement

[0178] 854 stop chamber

[0179] 855 fluid inlet

[0180] 856 fluid outlet

[0181] 960 sealing layer

[0182] 1000, 1100 system

[0183] 1009 elastic layer

[0184] 1070, 1170 analysis apparatus

[0185] 1072 holding structure

[0186] 1074 optical means

[0187] 1076 pressuring means

[0188] 1077 piston

[0189] 1178 control unit

[0190] 1200 method

[0191] 1210, 1220, 1230, 1240 steps of the methoddC depth of a channel segment of a microfluidic channel dS depth of a stop chamber

[0192] F compressive force

[0193] wC width of a channel segment of a microfluidic channel wD width of a delay channel section

Claims

CLAIMS1. A microfluidic device (100, 200, 300) for handling a fluid sample by dividing it into a plurality of aliquots, in particular for providing a digital polymerase chain reaction analysis, with at least one microfluidic well (105) configured to receive the fluid sample, the at least one microfluidic well (105) comprising- a microfluidic circuit (110) with a plurality of microchambers (112), which provide a respective reaction space for an aliquot of the plurality of aliquots,- an inlet end (120) coupled to the microfluidic circuit (110) via at least one microfluidic channel (130, 130’, 130”, 130’”),- an outlet end (125) coupled to the microfluidic circuit (110) via the at least one microfluidic channel (130, 130’, 130”, 130’”),wherein the plurality of microchambers (112) is arranged such that each microchamber (112) is at least indirectly via further microchambers (112) fluidly coupled with the inlet end (120) and the outlet end (125), wherein parallel flow paths are formed from the inlet end (120) to the outlet end (125) to thereby connect a respective number of microchambers (112) with the respective microfluidic channel (130, 130’, 130”, 130’”) also comprising channel segments between microchambers, such that each microchamber (112) is configured to receive fluid from the inlet end (120) via the at least one microfluidic channel (130, 130’, 130” ,130’”) and to provide gas displaced by the fluid downstream to the outlet end (125), andwherein the at least one microfluidic channel (130, 130’, 130”, 130’”) comprises at least one fluidic delay structure (140, 240, 240’, 340, 340’, 440, 440’, 540, 540’, 640, 740, 840), which is configured to delay a fluid flow by being configured to slow down the fluid flow and / or to inhibit the fluid flow until a predefined fluid pressure is reached.

2. The microfluidic device (200) of claim 1 , wherein the at least one fluidic delay structure (240) is at least arranged between the inlet end (120) and the plurality of microchambers (112).

3. The microfluidic device (100, 300) of claim 1 or 2, wherein the at least one fluidic delay structure (140, 340’) is at least arranged between the plurality of microchambers (112) and the outlet end (125).

4. The microfluidic device (200) of at least one of the preceding claims, comprising at least two microfluidic channels (130, 130’, 130”, 130’”), wherein the at least one fluidic delay structure (240’) combines the at least two microfluidic channels (130, 130’, 130”, 130’”) to provide a combined fluid flow to the outlet end (125).

5. The microfluidic device (200, 300) of at least one of the preceding claims, wherein the at least one fluidic delay structure (240’, 340) is at least provided between two microchambers (112) of the microfluidic circuit (110).

6. The microfluidic device (100, 200, 300) of at least one of the preceding claims, wherein the at least one fluidic delay structure (140, 240, 240’, 340, 340’, 740) comprises at least one delay channel section (747), which is, preferably at least 1.5 times, in particular at least 2 times, particularly preferably at least 3 times, longer than the geometrical distance between a fluid inlet (748) and a fluid outlet (749) of the delay channel section (747), and wherein, preferably, the delay channel section (747) has a meander-like form.

7. The microfluidic device (100, 200, 300) of at least one of the preceding claims, wherein the at least one fluidic delay structure (140, 240, 240’, 340, 340’, 440, 440’, 540, 540’, 640, 840) comprises at least one fluidic stop structure (140, 240, 240’, 340, 340’, 440, 440’, 540, 540’, 640, 840) configured to inhibit a fluid flow until a predefined fluid pressure is reached and wherein, preferably, the at least one fluidic stop structure (140, 240, 240’, 340, 340’, 440, 440’, 540, 540’, 640, 840) comprises one or more of the following structures: a capillary stop structure (440, 440’, 840), a bifurcational stop structure (540, 540’), a hydrophobic stop structure (640).

8. The microfluidic device (100, 200, 300) of claim 7, wherein the at least one fluidic stop structure (140, 240, 240’, 340, 340’, 840) comprises at least one sudden enlargement (853) of the cross section of the microfluidic channel (130, 130’, 130”, 130’”, 830), and wherein, preferably, the at least one sudden enlargement (853) of the cross section is at least substantially formed by at least one stop chamber (854) having at least partially a greater depth (dS) and / or a greater width than adjacent channel segments of the microfluidic channel (130, 130’, 130”, 130’”, 830), and, preferably, an at least substantially triangular or rectangular cross section.

9. The microfluidic device (100, 200, 300) of at least one of the preceding claims, wherein the at least one fluidic delay structure (140, 240, 240’, 340, 340’, 440, 440’, 540, 540’, 640, 740, 840), in particular the at least one hydrophobic stop structure (640), comprises at least one hydrophilic channel section (646) having a hydrophilic surface and at least one hydrophobic channel section (645) having a hydrophobic surface.

10. The microfluidic device (100, 200, 300) of at least one of the preceding claims, comprising at least two microfluidic channels (130, 130’, 130”, 130’”), which fluidly couple a basically equal number of microchambers (112) between inlet end (120) and outlet end (125), wherein the at least two microfluidic channels (130, 130’, 130”, 130’”)provide similar delay structure positions of a respective fluidic delay structure (140, 240, 240’, 340, 340’).

11. The microfluidic device (300) of at least one of the preceding claims, wherein the at least one microfluidic well (105) is arranged such that the microchambers (112) of the microfluidic well (105) form respective sub-wells (307) with at least one fluidic connection between two of these sub-wells (307) and wherein a respective fluidic delay structure (340) is arranged at the at least one fluidic connection between two sub-wells (307).

12. The microfluidic device (200, 300) of at least one of the preceding claims, wherein at least one microfluidic channel (130, 130’, 130”, 130’”) comprises at least two fluidic delay structures (240, 240', 340, 340’), preferably at least two fluidic stop structures (240, 240’, 340, 340’), between the inlet end (120) and the outlet end (125), and wherein, preferably, the two fluidic stop structures (240, 240’, 340, 340’) between the inlet end (120) and the outlet end (125) are configured such that two different predefined fluid pressures are needed to enable a flow through the respective fluidic stop structure (240, 240’, 340, 340’).

13. The microfluidic device (100, 200, 300) of at least one of the preceding claims, wherein the microfluidic device (100, 200, 300) comprises a sealing layer (960) at least partially covering the microfluidic circuit (110) and configured to fluidly separate the microchambers (112) from each other, preferably by deformation into channel segments of the at least one microfluidic channel (130, 130’, 130”, 130’”) fluidly coupling the microchambers (112), in particular upon application of a compressive force (F) to the sealing layer (960).

14. A system (1000, 1100) for providing a molecular analysis of a fluid sample, in particular for providing a digital polymerase chain reaction analysis, comprising- the microfluidic device (100, 200, 300) of at least one of the preceding claims, - an analysis apparatus (1070, 1170), configured to receive the microfluidic device (100, 200, 300) and to analyze a surface of the microfluidic device (100, 200, 300) with optical means (1074),wherein the analysis apparatus (1070, 1170) is further configured to provide a fluid pressure, in particular the predefined fluid pressure, to a fluid within the microfluidic device (100, 200, 300) in order to enable a flow of the respective fluid through the at least one fluidic delay structure (140, 240, 240’, 340, 340’, 440, 440’, 540, 540’, 640, 740, 840), in particular through the at least one fluidic stop structure (140, 240, 240’, 340, 340’, 440, 440’, 540, 540’, 640, 840).

15. The system (1000, 1100) according to claim 14, wherein the fluid pressure, in particular the predefined fluid pressure, is provided by a piston (1077), which is arranged to be pushed against an elastic layer (1009) that is sealing the respective inlet end (120) of the at least one microfluidic well (105) of the microfluidic device (100, 200, 300).

16. The system (1100) according to claim 14 or 15, further comprising a control unit (1178) configured to control the pressure provided to the fluid within the microfluidic device (100, 200, 300) based on an analysis of a present filling state of the at least one microfluidic well (105), wherein this analysis is facilitated by the optical means (1074).

17. A method (1200) for providing a molecular analysis of a fluid sample, in particular for providing a digital polymerase chain reaction analysis, comprising the steps- providing an analysis apparatus (1070, 1170) and a microfluidic device (100, 200, 300) with at least one microfluidic well (105) configured to receive the fluid sample; - providing the fluid sample to the at least one microfluidic well (105) via a respective inlet end (120) of this microfluidic well (105);- insertion of the microfluidic device (100, 200, 300) into the analysis apparatus (1070, 1170); and- applying a pressure profile on the fluid sample in order to push the fluid sample into a microfluidic circuit (110) of the microfluidic device (100, 200, 300) and through at least one fluidic delay structure (140, 240, 240’, 340, 340’, 440, 440’, 540, 540’, 640, 740, 840) provided within at least one microfluidic channel (130, 130’, 130”, 130’”) of the microfluidic device (100, 200, 300), wherein a flow of fluid is delayed by means of the at least one fluidic delay structure (140, 240, 240’, 340, 340’, 440, 440’, 540, 540’, 640, 740, 840) by being configured to slow down the fluid flow and / or to inhibit the fluid flow until a predefined fluid pressure is reached.

18. The method of claim 17, wherein the applied pressure surpasses at least at a certain stage of the molecular analysis a predefined fluid pressure that is needed to enable a flow of fluid through at least one fluidic stop structure (140, 240, 240’, 340, 340’, 440, 440’, 540, 540’, 640, 840) of the at least one fluidic delay structure (140, 240, 240’, 340, 340’, 440, 440’, 540, 540’, 640, 840).

19. The method of claim 17 or 18, further comprising the steps- analyzing a present filling state of a plurality of microchambers (112) provided by the microfluidic well (105); and- determining the pressure profile to be applied on the fluid sample based on the present filling state of the plurality of microchambers (112).

20. The method of at least one of the preceding claims, further comprising the step- 31 -- fluidly separating a plurality of microchambers (112) of the microfluidic circuit (110) from each other, preferably by deforming a sealing layer (960) into channel segments of the at least one microfluidic channel (130, 130’, 130”, 130”’) fluidly coupling the microchambers (112), in particular by applying a compressive force (F) to the sealing layer (960).

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