Microfluidic device for handling a fluid sample

Abstract

The invention relates to a microfluidic device (100,200,700) 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 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, a storage section (140,240,740) with a microfluidic storage circuit (142,242,342,742) is provided in order to store fluid that does not reach and / or flows beyond the microchambers of the microfluidic well during a filling process of the microfluidic device and can therefore not be provided at the intended reaction space, wherein a storage opening structure (144,344) is provided in the storage section.

Description

FIELD OF THE INVENTION

[0001] The invention relates to a microfluidic device for handling a fluid sample by dividing it into a plurality of aliquots. 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.BACKGROUND OF THE INVENTION

[0002] 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.

[0003] 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 non-target 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.

[0004] 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.

[0005] It is an object of the invention to provide a microfluidic device with an improved usability of the analyzed fluid sample, in particular with an improved further access to the fluid sample.SUMMARY OF THE INVENTION

[0006] 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.

[0007] The microfluidic device according to the first aspect of the invention has 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 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, wherein a storage section with a microfluidic storage circuit is provided in order to store fluid that does not reach and / or flows beyond the microchambers of the microfluidic well during a filling process of the microfluidic device and can therefore not be provided at the intended reaction space, and wherein a storage opening structure is provided in the storage section.

[0008] The microfluidic device according to the first aspect of the invention provides an improved access to the fluid sample after a filling of the microfluidic device. It furthermore enables an amplification of those parts of the fluid sample that did not form parts of the aliquots within the microfluidic chambers. The invention is based on the finding that a rather large amount of the fluid sample remains unused during the treatment of the microfluidic device. Often, parts of the fluid sample can even not be extracted out of the microfluidic device in view of the microscopic dimensions of the respective microfluidic circuit. The microfluidic device according to the first aspect of the invention solves this problem by providing the storage section and a related storage opening structure for the fluid that does not belong to the aliquots within the microchambers.

[0009] By providing the storage section, the stored fluid in the storage section might also be activated like the aliquots within the microchambers. Thus, further analysis after a thermal treatment might be possible with the stored fluid. This is particularly advantageous in view of the difficult access to the aliquots after the applied thermal treatment. These aliquots are within microstructures on a micrometer scale and therefore hard to remove from the microfluidic device in a controlled way. Thus, the provided storage section allows a further analysis of activated stored fluid with an easy access to the stored fluid via the provided storage opening structure.

[0010] The access to activated stored fluid is particularly advantageous in view of the small volume of fluid that is typically stored in a respective microchamber. Such small volumes of preferably less than 100 nanoliters typically do not allow a controlled fluid extraction out of a microchamber. The storage section according to the first aspect of the invention thus enables the access, especially the controlled access, to activated fluid. Against this background, it can be preferred if the volume of the microfluidic storage circuit is larger than the volume of at least one of the microchambers, and even more preferred if the volume of the microfluidic storage circuit is larger than the volume of each of the microchambers.

[0011] Furthermore, the storage section allows a precise control over the amount of stored fluid that is extracted after the thermal treatment. A scale, a respective stopping valve or other arrangements that are obvious for a skilled person within the field of microfluid dynamics might be provided in order to enable the extraction of a predefined amount of activated stored fluid after the thermal treatment via the storage opening structure. Alternatively or additionally, such arrangements might restrict the amount of sample fluid that is allowed to flow into the storage section.

[0012] The storage section can be provided such that fluid that does not reach the microchambers during the filling process and can therefore not be provided at the intended reaction space can be stored in the storage section. In this way, it is possible to take advantage of the fact that during the filling process, excess fluid usually remains upstream of the microchambers since all microchambers are aimed to be filled with the fluid sample. Typically, the division of the fluid sample will not be precise enough to avoid remaining fluid outside the microchambers. For the same reason, there is usually also excess fluid that passes the microchambers during the filling process. To take advantage of this, the storage section can alternatively or additionally be provided such that fluid that flows beyond the microchambers during the filling process and can therefore not be provided at the intended reaction space can be stored in the storage section. This can be particularly preferred since the amount of excess fluid passing the microchambers can be particularly difficult to control.

[0013] The microfluidic device according to this first aspect of the invention is simple to build, since only the storage section has to be added to already available microdevices with a plurality of microchambers.

[0014] A further advantage is the variability of the provided solution. Different microfluidic circuits can be improved by providing the storage section, for example at the downstream end of the respective microfluidic well.

[0015] 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.

[0016] The microfluidic storage circuit is fluidly coupled with the microfluidic circuit. Thereby, the fluid that passes the storage section can preferably flow directly into the microchambers and / or the fluid that passes the microchambers can preferably flow directly into the storage section. Irrespective of this, the microfluidic storage circuit can be fluidly coupled with the outlet end such that fluid can flow, preferably directly, from the microfluidic storage circuit into the outlet end. Thus, if too much fluid reaches the storage section, the amount of fluid that is too much can simply flow into the outlet end.

[0017] The storage opening structure is preferably arranged at an end, downstream end or upstream end, of the microfluidic storage circuit. At such a location the whole amount of fluid stored in the storage section can be extracted at once.

[0018] The microfluidic storage circuit can consist of a single channel, preferably a single microfluidic channel.

[0019] In the following, embodiments of the microfluidic device according to the first aspect of the invention will be described.

[0020] In a preferred embodiment of the microfluidic device according to the first aspect of the invention, the storage section is arranged between the inlet end and the microfluidic circuit. In this way, fluid not reaching the microchambers during the filling process can be easily stored in the storage section. Then the inlet end is in particular fluidly coupled to the microfluidic circuit via the microfluidic storage circuit. In addition, the storage section can be arranged geometrically between the inlet end and the microfluidic circuit. With respect to an easy storage of fluid flowing beyond the microchambers during the filling process, it can alternatively or additionally be preferred if the storage section is arranged between the microfluidic circuit and the outlet end. Then, the microfluidic circuit is in particular fluidly coupled to the outlet end via the microfluidic storage circuit. The position of the microfluidic storage circuit downstream, preferably right before, the outlet end allows storage of all the fluid that has passed the microchambers of a certain microfluidic channel during the filling process. Irrespective of this, the storage section can additionally be arranged geometrically between the microfluidic circuit and the outlet end.

[0021] Irrespective of the arrangement of the storage section, it can be preferred if the storage opening structure is provided between the microfluidic circuit and the microfluidic storage circuit. Such a position of the storage opening structure ensures the presence of fluid at the storage opening structure. While it is hard to predict, how much of the volume of the microfluidic storage circuit is filled with fluid, it is clear that fluid is present at least between the microfluidic circuit and the microfluidic storage circuit.

[0022] In a further embodiment, the storage opening structure is configured to stay closed during the filling process of the plurality of microchambers and to allow a retrieval of a storage sample of the stored fluid by an external device which pierces the storage opening structure. The storage opening structure of this embodiment avoids a contamination of the fluid sample prior to the retrieval of the storage sample. Using an external device, such as a syringe or the like, to pierce the storage opening structure is a secure way to avoid any contamination of the storage sample during the retrieval. Furthermore, after the storage sample is in the syringe, it can be easily used for further steps of analysis.

[0023] In a preferred variant of the aforementioned embodiment, the microfluidic device comprises a cover layer that is arranged to hold the storage opening structure closed during the filling process. Such a cover layer is particularly suitable for avoiding a contamination of the fluid. In a preferred example of this embodiment, the cover layer also holds the inlet end and the outlet end of the microfluidic device closed during the filling process, after the fluid sample has been inserted into the inlet end. Irrespective of this, the cover layer can be an adhesive cover layer. An adhesive cover layer allows easy and at the same time reliable closure of the storage opening structure. Alternatively or additionally, the cover layer can be a film, preferably a plastic film. This can be simple and functional.

[0024] In a preferred embodiment, the microfluidic storage circuit comprises a meander-like structure. Such a meander-like structure can lead to small dimensions of the storage section, compared to an elongated structure of the microfluidic storage circuit. In addition, a meander-like structure, in particular ribs between adjacent sections of the meander-like structure, can provide good support for a layer resting on the storage section, for example the cover layer and / or a sealing layer. In a preferred variant of this embodiment, the microfluidic storage circuit consists of a single channel, especially a single microfluidic channel. This can lead to a robust structure that is easy to manufacture.

[0025] The meander-like structure has preferably a spiralled configuration. In a preferred variant, the spiralled configuration comprises a plurality of circuit sections wherein these circuit sections respectively comprise a linear channel and the circuit sections of the storage circuit are arranged such that the linear channels are at least partially parallel to each other. Preferably, the linear circuit sections of said spiralled configuration are at least partially identical to each other. In a related variant of this embodiment, the circuit sections show essentially identical geometric measurements. Preferably, two successive circuit sections of the spiralled configuration are connected to each other via a respective turning point that redirects respective fluid from a first linear channel of a first circuit section to a second linear channel of a second circuit section. Preferably, the storage circuit comprises between 3 and 12 turning points, especially between 5 and 10 turning points, in particular at least 6 turning points. In a preferred variant, adjacent circuit sections show a similar length of the respective linear channel. In a preferred variant, all linear channels of the microfluidic storage circuit have a similar length.

[0026] As an alternative or in addition to the meander-like structure, the microfluidic storage circuit can comprise a receiving space for the fluid to be stored. Then, a plurality of spacers, for example at least 5, 10, 15 or 20, can be arranged in the receiving space. Such a structure can for example be preferred if, in addition to thermal cycling, a continuous imaging of a fluorescence signal (RT-PCR) is to be performed. The spacers can then support a layer resting on the storage section, for example the cover layer and / or the sealing layer. In this way, it is possible to easily and reliably keep the layer out of the receiving space. Irrespective of this, the spacers can be simply and expediently designed as pillars. Alternatively or additionally, the receiving space can for example be defined by a basin-like structure of the storage section.

[0027] In a preferred embodiment, a depth of the microfluidic storage circuit is essentially equal to a depth of the microchambers. Similar depths of microfluid storage circuit and microchambers of the respective microfluidic well ensure a similar thermal treatment during the activation of the sample fluid.

[0028] In a preferred embodiment, the cross section of the microfluidic storage circuit is essentially equal to the cross section of the at least one microfluidic channel. Similar cross sections enable a reduced fluidic resistance compared to different cross sections.

[0029] In a preferred embodiment, the microfluidic storage circuit is configured to store at least 5 µL, preferably at least 10 µL of the fluid sample. This amount of fluid sample is usually enough for a future processing, e.g. sequencing, of unusual and / or particularly interesting stored fluid. Therefore, the microfluidic storage circuit of this embodiment is very advantageous for the use of the microfluidic device for a polymerase chain reaction analysis, such as a digital polymerase chain reaction analysis, since it allows a further sequencing if enough sample fluid has been inside the microfluidic storage circuit.

[0030] With respect to a sufficiently large amount of fluid sample for a future sequencing, it can alternatively or additionally be preferred if the volume of the microfluidic storage circuit is at least 50 times the volume of at least one of the microchambers. For the same reason it can be even more preferred if the volume of the microfluidic storage circuit is at least 200 times, preferably at least 500 times, the volume of at least one of the microchambers. Alternatively or additionally, it can also be particularly preferred in this context if the volume of the microfluidic storage circuit is larger than the volume of each of the microchambers by a respective factor.

[0031] In an embodiment of the microfluidic device according to the first aspect of the invention, the at least one microfluidic channel comprises at least one fluidic delay structure, which is configured to delay a fluid flow, in particular in the direction of the microfluidic storage section. The fluidic delay structure of this embodiment allows a control of the filling of the storage section with fluid. In a variant of this embodiment, the at least one fluidic delay structure can comprise at least one fluidic stop structure, which is configured to inhibit a fluid flow until a predefined fluid pressure is reached. A fluidic stop structure allows particularly good control of the filling of the storage section. Only after reaching the predefined fluid pressure, the fluid can pass the fluidic stop structure and reach those parts of the microfluidic circuit and / or of the microfluidic storage circuit that are behind the stop structure in a downstream direction. Possible designs of such stop structures are well-known in the art. Typically, the hydrodynamic characteristics of such a stop structure are based on capillary forces that act due to different cross sections, on hydrophobic characteristics of a chosen material and / or surface, and / or on a turbulent flow guidance that decelerates a fluid flow.

[0032] In a further variant of the aforementioned embodiment, the at least one fluidic delay structure comprises alternatively or additionally to the at least one fluidic stop structure 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. 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. The at least one delay channel section can for example have 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.

[0033] In a further variant of the aforementioned embodiment, at least one further delay structure is provided within the microfluidic circuit. Thereby, the fluidic delay structure might enable a homogenous filling of the microchambers. Delay structures can lead to a filling that prevents gas from the microchambers to be trapped in the circuit. A trapping of gas might occur, if a microfluidic channel is completely filled with fluid that flows into the storing section while a further microfluidic channel is not completely filled so that gas that cannot vent through the filled storing section is trapped within this channel. Therefore, in the present embodiment, a trapping of gas might be avoided.

[0034] In a preferred variant of the aforementioned embodiment, the at least one fluidic delay structure, in particular the at least one fluidic stop structure, is arranged between the plurality of microchambers and the microfluidic storage circuit. This allows particularly good control of the filling of the storage section with fluid. In the case of the at least one fluidic stop structure, preferably, the storage section is not filled with fluid and the sample fluid rests in the microfluidic circuit, as long as the predefined fluid pressure is not reached. But after reaching the fluid pressure during the filling process, the fluidic stop structure opens for the fluid and, preferably, the storage section gets filled with fluid that has passed the microfluidic circuit.

[0035] In a further embodiment of the microfluidic device, a cross section of the microfluidic storage circuit is larger than a cross section of the at least one microfluidic channel such that capillary forces do not cause a flow of fluid into the storage section. In this embodiment, the physical structure of the respective circuit can ensure that fluid is not brought into the storage section by capillary forces without any applied pressure. Thereby, it can be ensured that fluid is not accidentally within the storage section. It can be furthermore ensured that fluid is not pressed out of a microchamber by capillary forces of the storage section.

[0036] 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. For simplicity, the sealing layer and the cover layer can be the same layer. However, this does not necessarily have to be the case. Irrespective of this, 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.

[0037] 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.

[0038] The inlet end can be fluidly coupled to the microchambers, via a, in particular common, filling channel. The filling channel can for example be part of the at least one microfluidic channel. However, this does not have to be the case. Irrespective of this, it can be preferred for technical reasons if the filling channel has at least substantially the same depth as one or more, preferably at least substantially all, of the microchambers. This can contribute to a simple manufacture of the microfluidic device. As an alternative or in addition, the filling channel can have a depth of at least 20 µm, preferably at least 40 µm, in particular at least 50 µm. This can be useful for good and reliable filling of the at least one microfluidic channel and thus of the microchambers. With respect to an economic use of the fluid sample and a flat design of the microfluidic device, it can alternatively or additionally be preferred if the filling channel has a depth of at most 800 µm, preferably at most 600 µm, particularly at most 500 µm. Irrespective of its depth, the filling channel can have a width of at least 50 µm, preferably at least 75 µm, in particular at least 100 µm. This can also be useful for good and reliable filling of the microchambers. Alternatively or additionally, the filling channel can have a width of at most 300 µm, preferably at most 250 µm, in particular at most 200 µm. This can not only be advantageous with respect to an economic use of the fluid sample but can also ensure that the fluorescence signal of the fluid cycled in the filling channel does not outshine the signal of the fluid in the microchambers and thus contribute to a reliable analysis.

[0039] The microchambers can have a depth of at least 20 µm, preferably at least 40 µm, in particular at least 50 µm. This helps to ensure that the microchambers can accommodate a sufficient volume of the fluid sample. As an alternative or in addition, the microchambers can have a depth of at most 800 µm, preferably at most 600 µm, particularly at most 500 µm. This can contribute to a compact design of the microfluidic device and an economic use of the fluid sample. For the same reasons, the microchambers can alternatively or additionally have a width of at least 25 µm, preferably at least 40 µm, in particular at least 50 µm, and / or of at most 130 µm, preferably at most 105 µm, particularly at most 85 µm. The depth and the volume of the microchambers depend in particular on the purpose of the analysis to be performed with the microfluidic device. Depending on the type of application, many small microchambers or a few large microchambers or a combination thereof can be expedient.

[0040] The at least one microfluidic channel can have a depth of at least 5 µm, preferably at least 10 µm, in particular at least 15 µm. This can not only be useful with respect to a reliable filling of the microchambers but also with respect to the fluidic separation of the microchambers. This is because if the sealing layer is deformed into the microfluidic channel by applying a compressive force to the sealing layer, a channel depth that is too small leads to excessive variations in back pressure as the production tolerance of the microfluidic channel is typically at least + / - 2 µm. As an alternative or in addition, the at least one microfluidic channel can have a depth of at most 80 µm, preferably at most 60 µm, in particular at most 50 µm. This allows the microfluidic channel to be reliably sealed in a simple manner. Irrespective of its depth, the at least one microfluidic channel can have a width of at least 5 µm, preferably at least 10 µm, and / or of at most 120 µm, preferably at most 100 µm, particularly at most 80 µm. In this way, a good filling behavior can be ensured for different kinds of fluids. Irrespective of this, the at least one microfluidic channel can have the respective depth and / or width preferably at least in channel segments fluidly coupling the microchambers.

[0041] The microchambers can be fluidly coupled to the microfluidic storage circuit via a, in particular common, collecting channel. The collecting channel can for example be part of the at least one microfluidic channel. However, this does not have to be the case. Irrespective of this, the collecting channel can have a depth of at least 55 µm, preferably at least 85 µm, in particular at least 110 µm. This can be useful to provide a sufficient collection volume. As an alternative or in addition, the collecting channel can have a depth of at most 330 µm, preferably at most 275 µm, in particular at most 220 µm. This can contribute to a flat design of the microfluidic device. Irrespective of its depth, it can be preferred with respect to a sufficient collection volume if the collecting channel has a width of at least 50 µm, preferably at least 75 µm, in particular at least 100 µm. Alternatively or additionally, the collecting channel can have a width of at most 300 µm, preferably at most 250 µm, particularly at most 200 µm.

[0042] For technical reasons, it can be preferred if the microfluidic storage circuit has at least substantially the same depth as one or more, preferably at least substantially all, of the microchambers. This can contribute to a simple manufacture of the microfluidic device. Irrespective of this, the microfluidic storage circuit can have a depth of at least 20 µm, preferably at least 40 µm, in particular at least 50 µm. This allows the storage circuit to store a sufficient amount of the fluid sample. Alternatively or additionally, the microfluidic storage circuit can have a depth of at most 800 µm, preferably at most 600 µm, in particular at most 500 µm. This can contribute to a flat design of the microfluidic device. Irrespective of its depth, a width of the cross section of the microfluidic storage circuit, in particular of the meander-like structure, can be at least 35 µm, preferably at least 55 µm, in particular at least 65 µm. This can also help to provide a sufficient storage volume. As an alternative or in addition, a width of the cross section of the microfluidic storage circuit, in particular of the meander-like structure, can be at most 300 µm, preferably at most 250 µm, in particular at most 200 µm. This can not only help to provide good support for a layer, for example the cover layer, resting on the storage section, but can also ensure that the fluorescence signal of the fluid cycled in the microfluidic storage circuit does not outshine the signal of the fluid in the microchambers and thus contribute to a reliable analysis.

[0043] 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 the microfluidic device of at least one of the aforementioned embodiments, an analysis apparatus configured to receive the microfluidic device and to amplify the plurality of aliquots within the plurality of microchambers by providing a predefined thermal treatment, and wherein the microfluidic storage circuit is arranged such that the fluid stored in the storage section is also amplified by the analysis apparatus.

[0044] 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.

[0045] The position of the storage section and of the microchambers at the microfluidic device is preferable a position at a surface of the microfluidic device so that the amplification of the stored fluid and of the aliquots is similar to each other. Details concerning the amplification process are well known in the art and therefore not described in the following.

[0046] The analysis apparatus can be configured to, preferably optically, analyze the fluid stored in the storage section during the amplification. In this way, the result of the molecular analysis can be further improved.

[0047] In the following, embodiments of the system according to the second aspect of the invention will be described.

[0048] In a preferred embodiment, the analysis apparatus is further configured to provide the predefined fluid pressure to a fluid within the microfluidic device in order to enable a flow of the respective fluid through the microfluidic circuit and at least partly through the microfluidic storage circuit. In order to provide the fluid pressure, the analysis apparatus comprises in a variant of this embodiment a number of pistons that are arranged to be pressed inside the inlet end after it has been filled with the fluid sample. In an alternative variant, the analysis apparatus provides a fluid flow, preferably a gas flow, in order to provide the fluid pressure. In this alternative variant, the fluid flow, preferably the gas flow, is pressed inside the inlet end after it has been filled with the fluid sample. Preferably, a membrane is provided in order to prevent a contact of surrounding laboratory and / or device with the fluid sample. The membrane is preferably a semi-permeable membrane covering the inlet end. Then, the gas flow can be applied to the fluid within the microfluidic device through the membrane. At the same time, the membrane can effectively prevent the release of the fluid and / or of aerosols from the microfluidic device, in particular during the amplification of the fluid. If a fluidic stop structure is provided in the microfluidic device, the pressure applied to the fluid within the inlet end preferably exceeds at least at a certain moment of the filling process the predefined pressure threshold of the respective fluidic stop structure. In a preferred variant, the analysis apparatus is configured to provide different predefined fluid pressures for different stop structures within the microfluidic circuit.

[0049] In a preferred embodiment, the system further comprises optical means, which are arranged and configured to enable an analysis of a present filling state of the at least one microfluidic well. The optical means are furthermore advantageous 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.

[0050] In a further embodiment, the optical means are connected to a control unit of the analysis apparatus and the control unit is configured to control a filling of the microfluidic circuit as a function of data provided by the optical means. In an advantageous variant of this embodiment, the pressure provided by the analysis apparatus to provide a fluid flow of the sample fluid depends on the data provided by the optical means.

[0051] As an alternative or in addition to the optical means for the analysis of the present filling state of the microfluidic well, the system can comprise optical means for providing a fluorescence signal. The fluorescence signal can then represent one or more pictures of the plurality of aliquots and / or of the fluid stored in the storage section, wherein at least one picture can show one or more of the plurality of aliquots and / or the fluid stored in the storage section in a fluorescent state. Irrespective of this, the optical means for providing the fluorescence signal and the optical means for enabling the analysis of the present filling state can simply be the same. However, this does not necessarily have to be the case. Alternatively or additionally, the optical means for providing the fluorescence signal can be connected to a control unit, preferably the control unit mentioned above. The control unit can then be configured to control, for example stop, the amplification of the plurality of aliquots and / or the fluid stored in the storage section based on the fluorescence signal.

[0052] 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 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 filling a microfluidic circuit of the microfluidic device at least partially, preferably at least substantially, with fluid of the fluid sample, wherein fluid that does not reach and / or flows beyond microchambers of the microfluidic well during that filling process is stored in a microfluidic storage circuit of the microfluidic device.

[0053] 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 storing of surplus fluid of the fluid sample in the microfluidic storage circuit. The pressure is preferably applied at a predetermined time of the molecular analysis.

[0054] The steps of the method according to the third aspect are preferably performed in the presented order.

[0055] In the following, embodiments of the method according to the third aspect of the invention will be described.

[0056] In a preferred embodiment, a pressure is applied to the fluid sample in order to push the fluid sample into the microfluidic circuit, wherein fluid that does not reach and / or flows beyond the microchambers during the application of the pressure is stored in the microfluidic storage circuit. In this way, the microfluidic circuit can be easily and reliably filled with the fluid sample. The pressure can for example be applied to the fluid sample by means of one or more pistons. The at least one piston can then be pushed into the at least one inlet end after the fluid sample has been filled into the at least one inlet end. In order to prevent a contamination of the fluid sample and a release of the fluid sample from the microfluidic device, the at least one inlet end can be covered with an elastic layer after the fluid sample has been filled into the at least one inlet end. Then, the at least one piston can be pushed into the elastic layer, thereby deforming the elastic layer into the inlet end and applying the pressure to the fluid sample. As an alternative to the application of the pressure by means of the at least one piston, the pressure can also be applied to the fluid sample by means of a, in particular controlled, gas flow. The gas flow can then be applied to the fluid sample via the at least one inlet end. In order to prevent a contamination of the fluid sample and a release of the fluid sample from the microfluidic device, the at least one inlet end can then be covered with a, preferably semi-permeable, membrane after the fluid sample has been filled into the at least one inlet end. Then, the gas flow can be applied to the fluid sample through the membrane. Irrespective of this, for the sake of simplicity, the gas of the gas flow can be air.

[0057] As an alternative or in addition to the application of a pressure to the fluid sample, the microfluidic circuit and / or the microfluidic storage circuit can be filled with the fluid sample at least partially, for example at least substantially, due to capillary forces. Then, an application of pressure to the fluid sample can be dispensed with. This can be advantageous with respect to the technical effort, will, however, typically be less reliable than a filling by means of an application of a pressure to the fluid sample.

[0058] In a further preferred embodiment, the method comprises the step of fluidly separating the microchambers 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 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 and / or a clamping plate.

[0059] In a further embodiment, the method further comprises the amplification of the fluid within the microchambers and within the microfluidic storage circuit by applying a thermal treatment to that fluid, preferably after the fluidic separation of the microchambers. Details of such an amplification depend on the applied analysis and are well-known in the art. In particular, the thermal treatment for the digital polymerase chain reaction analysis is well-known. During the amplification, the fluid within the microfluidic storage circuit can be, preferably optically, analyzed. In this way, the result of the molecular analysis can be further improved.

[0060] In a further embodiment of the method according to the third aspect of the invention, the method comprises, preferably as a last step, the retrieval of the fluid stored in the microfluidic storage circuit. This additional step allows a further analysis of the retrieved fluid. The retrieval of the fluid stored in the microfluidic storage circuit is preferably performed after the amplification of the fluid within the microchambers and within the microfluidic storage circuit. Irrespective of this, the fluid stored in the microfluidic storage circuit can be easily and expediently retrieved through a storage opening structure of the microfluidic device. For the same reason, it can be even more preferred if the fluid stored in the microfluidic storage circuit is retrieved by means of a syringe through the storage opening structure.

[0061] Preferably, a plurality of storage sections for a respective plurality of microfluidic wells is provided by the microfluidic device and a further step of the method comprises a selection of the storage sections whose stored fluid appears to be interesting for a further analysis after the amplification. This selection is preferable based on results of an analysis provided by the analysis apparatus after the amplification and / or during the amplification of the fluid.

[0062] 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.

[0063] These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter.BRIEF DESCRIPTION OF THE DRAWINGS

[0064] In the following drawings: Fig. 1 shows a first embodiment of a microfluidic device according to a first aspect of the invention; Fig. 2 shows a second embodiment of the microfluidic device according to the first aspect of the invention; Fig. 3 shows a cross-section of an embodiment of storage opening structure of the microfluidic device according to the first aspect of the invention; Figs. 4A-D show details of the first embodiment of the microfluidic device shown in Fig. 1; Fig. 5 shows an embodiment of a system according to a second aspect of the invention; Fig. 6 shows a flow diagram of an embodiment of a method according to a third aspect of the invention; Fig. 7 shows a third embodiment of the microfluidic device according to the first aspect of the invention. DETAILED DESCRIPTION OF EMBODIMENTS

[0065] 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. 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.

[0066] 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 110 via 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'".

[0067] 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.

[0068] In the shown embodiment, the microfluidic well 105 comprises a filling channel 132 and a collecting channel 134. Via the filling channel 132, the inlet end 120 is fluidly coupled to the microfluidic channels 130, 130', 130", 130'". And via the collecting channel 134, the microfluidic channels 130, 130', 130", 130‴ are fluidly coupled to the outlet end 125.

[0069] The microfluidic device 100 according to the first aspect of the invention further comprises a storage section 140 with a microfluidic storage circuit 142 that is arranged between the microfluidic circuit 110 and the outlet end 125 in order to store fluid that flows beyond the microchambers 112 of the microfluidic well 105 during a filling process of the microfluidic device 100. The stored fluid can therefore not be provided at the intended reaction space. In the shown embodiment, every microfluidic well 105 comprises a separate storage section 140. For reasons of clarity just one microfluidic well is depicted in Fig. 1.

[0070] As an alternative or in addition to the storage section 140 arranged between the microfluidic circuit 110 and the outlet end 125, the microfluidic device 100 could comprise a storage section with a microfluidic storage circuit that is arranged between the inlet end 120 and the microfluidic circuit 110 in order to store fluid that does not reach the microchambers 112 during the filling process of the microfluidic device 100 and can therefore not be provided at the intended reaction space. The microfluidic storage circuit of this storage section could for example be designed as a large distribution channel.

[0071] The microfluidic well 105 shown in Fig. 1 is just one of at least four microfluidic wells of the microfluidic device 100. 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 and provides a respective storage section 140. In a not shown embodiment, there is at least one microfluidic well that does not provide a respective storage section. Providing certain microfluidic wells without the storage section might be necessary due to structural reasons and / or to simplify a manufacturing of the microfluidic device.

[0072] In the embodiment shown in Fig. 1, a storage opening structure 144 is provided in the storage section 140 to allow a retrieval of the stored fluid. The storage opening structure 144 is preferably closed by a layer, a cap or the like. A possible structure of this storage opening structure 144 is shown in Fig. 3. The providing of the storage section allows an easy access to amplified fluid that is free from contamination. Thereby, a further analysis, especially a specific analysis, of certain parts of the fluid sample is enabled.

[0073] In a not shown embodiment, every microfluidic channel of the microfluidic circuit is fluidly connected to a separate storage section with a separate microfluidic storage circuit. Such separate storage section might be connected to separate outlet ends or to a combined outlet end of the respective microfluidic well.

[0074] The microfluidic storage circuit 142 of the shown embodiment has a larger cross section than the microfluidic channels 130, 130', 130", 130''', respectively. Thereby, capillary forces that might press fluid at least partly into the microfluidic channels 130, 130', 130", 130''', for example during an unintended pre-wetting of the microfluid well 105, cannot cause a flow of fluid into the storage section 140.

[0075] 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 further analysis, such as for example a sequencing of stored fluid. If the fluid of a certain microfluidic well 105 appears to be of special interest, the stored fluid of the respective storage section 140 can be retrieved to provide further analysis.

[0076] Fig. 2 shows a second embodiment of the microfluidic device 200 according to the first aspect of the invention.

[0077] The microfluidic device 200 differs from the microfluidic device 100 shown in Fig. 1 in view of the structure of the storage section 240. The microfluidic storage circuit 242 comprises a meander-like structure. Such a structure allows a large volume of stored fluid allocated at a little space of the microfluidic device 200 while a small cross section of the microfluidic storage circuit 242 avoids gas bubbles or the like during the retrieval of stored fluid. In a not shown embodiment, such a meander-like structure can also have channels with a larger cross section than the microfluidic channels of the microfluidic circuit in order to avoid an unintended flow of fluid into the storage section due to capillary forces.

[0078] The meander-like structure of the embodiment shown in Fig. 2 has a spiralled configuration. The spiralled configuration comprises a plurality of circuit sections wherein these circuit sections respectively comprise a linear channel and the circuit sections of the storage circuit 242 are arranged such that the linear channels are at least partially parallel to each other. Two successive circuit sections of the spiralled configuration are connected to each other via a respective turning point that redirects respective fluid from a first linear channel of a first circuit section to a second linear channel of a second circuit section. The depicted storage circuit 242 comprises 7 turning points. All linear channels between respective turning points show a similar length in the depicted embodiment. In a not shown embodiment, the meander-like structure has another number of turning points, preferably between 3 and 12 turning points, especially between 5 and 10 turning points, in particular at least 6 turning points. It is obvious for a person skilled in the art that other structures of the storage section, such as a circular structure, a loop-shaped structure, a coiled structure, an angular structure or the like, can also be used for a microfluidic device according to the first aspect of the invention. In that sense, the depicted storage sections form non-limiting examples of the storage section according to the first aspect of the invention.

[0079] In the depicted embodiment, a depth of the microfluidic storage circuit 242 is essentially equal to a depth of the microchambers 112. Similar depths of microfluid storage circuit 242 and microchambers 112 of the respective microfluidic well ensure a similar thermal treatment during the activation of the sample fluid.

[0080] The microfluidic storage circuit 242 is configured to store at least 5 µL, preferably at least 10 µL of the fluid sample. This amount of stored fluid is usually enough for further analysis purposes. For example, this amount of stored fluid usually allows a further sequencing analysis.

[0081] Furthermore, the microfluidic channels 130, 130', 130", 130‴ of the microfluidic circuit 110 comprise at least one fluidic delay structure 250 that is in the depicted embodiment a fluidic stop structure 250, which is configured to inhibit a fluid flow until a predefined fluid pressure is reached. In the shown embodiment, the respective fluidic stop structure 250 is arranged between the plurality of microchambers 112 and the microfluidic storage circuit 242. The fluidic stop structure 250 allows at the provided position a control of the flow of fluid into the storage section 240. Only after the predefined fluid pressure is reached, the fluid can flow into the microfluidic storage circuit 242. Thereby, an unintended filling of the storage section 240 is avoided. In a not shown embodiment, further fluidic delay structures, preferably fluidic stop structures, are arranged within the microfluidic circuit 110 and / or in the microfluidic storage circuit 242. Further fluidic delay structures, in particular fluidic stop structures, may advantageously allow a detailed control of a filling process for the microfluidic device. Thereby, an unintended filling of areas of the circuit and / or a trapping of gas might be avoided. Possible designs for fluidic stop structures are well known in the art and therefore not described in the following. In general, stop structures provide a flow resistance due to capillary forces and / or due to flow turbulences.

[0082] Fig. 3 shows a cross-section of an embodiment of a storage opening structure 344 of the microfluidic device according to the first aspect of the invention.

[0083] The storage opening structure 344 is configured to stay closed during the filling process of the plurality of microchambers and to allow a retrieval of a storage sample of the stored fluid 360 by an external device 370 which pierces the storage opening structure 344. The external device 370 is in the depicted embodiment a syringe.

[0084] In the shown embodiment, the storage opening structure 344 stays closed due to a cover layer 365 arranged at the storage opening structure 344. In the shown embodiment, the cover layer 365 itself has multiple layers. The cover layer 365 comprises a glue layer 366 configured to attach the cover layer 365 at the microfluidic storage circuit 342. Furthermore, the cover layer 365 comprises a carrier layer 367 which carries in this embodiment the pressure sensitive adhesive (PSA). Using a PSA carrier allows the simply piercing of the carrier with the external device 370, in particular with the syringe.

[0085] Preferably, the cover layer 365 is arranged to hold the storage opening structure 344 closed during the filling process. Thereby, a contamination of the stored fluid can be reliably avoided.

[0086] Figs. 4A-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. 4A, 4C) and in a sectional view along a section perpendicular to the microfluidic channel 130 in a region between the two microchambers 112 (Figs. 4B, 4D). As far as shown in Figs. 4A-D, the microfluidic device 200 shown in Fig. 2 can have at least substantially the same structure.

[0087] The microfluidic device 100 comprises an adhesive sealing layer 480, for example in the form of an adhesive plastic film, that is glued to the microfluidic well 105 and covers the microchambers 112. In the depicted embodiment, the sealing layer 480 and the cover layer 365 (cf. Fig. 3) are arranged on opposite sides of the microfluidic device 100 and are different layers. However, in an alternative embodiment, the sealing layer 480 and the cover layer 365 could be arranged on the same side of the microfluidic device 100 and could be the same layer.

[0088] In Figs. 4A, 4B, the sealing layer 480 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 480 in the direction of the microfluidic well 105.

[0089] In Figs. 4C, 4D, a compressive force F has been applied to the sealing layer 480 in a direction at least substantially perpendicular to the sealing layer 480, 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 480 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 480. In this way, the microchambers 112 are fluidly separated from each other. The sealing layer 480 is also deformed into the microchambers 112. However, due to the greater depth of the microchambers 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.

[0090] Fig. 5 shows an embodiment of a system 500 according to a second aspect of the invention.

[0091] The system 500 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 500 comprises the microfluidic device 100 according to at least one of the preceding embodiments and an analysis apparatus 590.

[0092] As an example, the microfluidic device 100 comprises eight 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 590 by using a holding structure 592 of the analysis apparatus 590.

[0093] The analysis apparatus 590 is configured to receive the microfluidic device 100 and to amplify the plurality of aliquots within the plurality of microchambers by providing a predefined thermal treatment. For that reason, the analysis apparatus 590 comprises a heating unit 594 that is controlled by a control unit 595 of the analysis apparatus 590. The microfluidic storage circuit is arranged such that the fluid stored in the storage section is also amplified by the analysis apparatus 590.

[0094] In a not shown embodiment, the analysis apparatus is configured to receive the microfluidic device via the holding structure and to analyze a surface of the microfluidic device with optical means. The optical means are preferably formed by a camera. The optical means can for example enable an analysis of a present filling state of the microfluidic wells 105. Then, the control unit 595 can control the filling of the microfluidic device 100 based on data provided by the optical means. Alternatively or additionally, the optical means can provide a fluorescence signal representing one or more pictures of the plurality of aliquots and / or the fluid stored in the storage section, wherein in at least one picture at least one of the aliquots and / or the fluid stored in the storage section is in a fluorescent state. Then, the control unit 595 can control, for example stop, the amplification based on the fluorescence signal.

[0095] Furthermore, the analysis apparatus 590 comprises pressuring means 596 that are formed by a plate with a series of pistons 598, which are arranged to be pressed inside the respective inlet end in order to provide a fluid pressure of the inserted sample fluid. Preferably, the applied fluid pressure enables a flow of fluid through the microfluidic circuit of the microfluidic device 100. In the shown embodiment, an elastic layer 599 is provided at the microfluidic device 100 in order to prevent a contamination of the sample fluid in the analysis apparatus 590. The layer 599 is elastic in order to enable the pistons 598 of the pressuring means 596 to press into the inlet end through the layer 599 without damaging that layer 599. 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.

[0096] As an alternative or in addition to the plate with the pistons 598, the analysis apparatus 590 could also comprise means for providing a gas flow into the respective inlet end in order to apply a pressure to the sample fluid within the microfluidic device 100 and cause a flow of fluid through the microfluidic circuit of the microfluidic device 100. Then, instead of the elastic layer 599, a semi-permeable membrane could be provided such that the gas flow can be applied through the membrane to the fluid within the microfluidic device 100 and, at the same time, the fluid cannot escape from the microfluidic device 100.

[0097] The elastic layer 599 is preferably configured to allow a piercing of that layer by an external device, such as a syringe, in order to retrieve sample fluid through the elastic layer 599 and the storage opening structure. In that way, the elastic layer 599 may form an additional layer compared to the storage opening structure. Alternatively, the elastic layer may form the only closure for the storage opening structure according to the first aspect of the invention.

[0098] 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 590. After the fluid sample is brought into the respective inlet ends, the elastic layer 599 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 592, formed for example as sliding rails.

[0099] 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 force to the sealing layer as described above with regard to Figs. 4A-D. Further details about the closing of the microfluidic channel are for example described in US 2021 / 0379593 A1. In order to provide a reliable structural support against the external pressure it can be advantageous to provide the microfluidic storage circuit with a small width of the respective channels in a direction perpendicular to the external pressure. Thereby the structure of the microfluidic device can be particularly robust.

[0100] After the sample fluid is pressed inside the microchambers, the analysis apparatus 590 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 via the heating unit 594. The control unit 595 is configured to control the heating unit 594 and the pressuring means 596 meaning that the filling process can be completed before the heating via the heating unit 594 starts. In the shown embodiment, the retrieval of sample fluid can be done manually with an external device after the microfluidic device 100 is removed from the analysis apparatus 590. In a not shown embodiment, the analysis apparatus is configured to retrieve a fluid sample out of the respective storage opening structure.

[0101] Fig. 6 shows a flow diagram of an embodiment of a method 600 according to a third aspect of the invention.

[0102] The method 600 is configured for providing a molecular analysis of a fluid sample, in particular for providing a digital polymerase chain reaction analysis. The method 600 comprising steps as given in the following.

[0103] A first step 610 comprises a providing of an analysis apparatus and of a microfluidic device with at least one microfluidic well configured to receive the fluid sample.

[0104] A second step 620 comprises a providing of the fluid sample to the at least one microfluidic well via a respective inlet end of this microfluidic well.

[0105] A further step 630 comprises an insertion of the microfluidic device into the analysis apparatus.

[0106] A last step 640 comprises a filling of a microfluidic circuit of the microfluidic device at least partially, preferably at least substantially, with fluid of the fluid sample, wherein fluid that does not reach and / or flows beyond microchambers of the microfluidic well during that filling process is stored in a microfluidic storage circuit of the microfluidic device. In order to fill the microfluidic circuit at least partially with the fluid, preferably a pressure is applied to the fluid sample, for example by means of at least one piston or a gas flow, thereby pushing the fluid sample into the microfluidic circuit.

[0107] The steps 610, 620, 630, 640 of the method 600 according to the third aspect of the invention are preferably performed in the given order.

[0108] Usually, after the method 600, the molecular analysis is provided by further steps of the particular analysis method.

[0109] In an embodiment, the method comprises a fluidic separation of the microchambers from each other, preferably by deforming a sealing layer into channel segments of at least one microfluidic channel fluidly coupling the microchambers, in particular by applying a compressive force to the sealing layer. This 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.

[0110] The analysis method preferably comprises an amplification of the fluid within the microchambers and within the microfluidic storage circuit by applying a thermal treatment to that fluid. This step is preferably provided after the fluidic separation of the microchambers.

[0111] In a preferred embodiment, the method comprises an additional step, preferably provided at the end of the method, namely a retrieval of the fluid stored in the microfluidic storage circuit, preferably through a storage opening structure of the microfluidic device. This retrieved fluid might also be further analyzed according to a particular analysis method.

[0112] Fig. 7 shows a third embodiment of the microfluidic device 700 according to the first aspect of the invention.

[0113] The microfluidic device 700 differs from the microfluidic device 100 shown in Fig. 1 in view of the structure of the storage section 740. The microfluidic storage circuit 742 comprises a receiving space 701 for the fluid to be stored in the microfluidic storage circuit 742. In the receiving space 701, a plurality of spacers 703 in the form of cylindrical pillars 703 are arranged. The spacers 703 support the cover layer, which covers in the depicted embodiment the entire microfluidic storage circuit 742 including the storage opening structure 144, so that the cover layer does not hang into the receiving space 701.LIST OF REFERENCE SIGNS

[0114] 100, 200, 700microfluidic device 105microfluidic well 110microfluidic circuit 112microchamber 120inlet end 125outlet end 130, 130', 130", 130‴microfluidic channel 132filling channel 134collecting channel 140, 240, 740storage section 142, 242, 342, 742microfluidic storage circuit 144, 344storage opening structure 250fluidic delay structure, fluidic stop structure 360stored fluid 365cover layer 366glue layer 367carrier layer 370external device 480sealing layer 500system 590analysis apparatus 592holding structure 594heating unit 595control unit 596pressuring means 598piston 599elastic layer 600method 610, 620, 630, 640steps of the method 701receiving space 703spacer Fcompressive force

Claims

1. A microfluidic device (100,200,700) 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 - 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) 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), wherein a storage section (140,240,740) with a microfluidic storage circuit (142,242,342,742) is provided in order to store fluid that does not reach and / or flows beyond the microchambers (112) of the microfluidic well (105) during a filling process of the microfluidic device (100,200,700) and can therefore not be provided at the intended reaction space, and wherein a storage opening structure (144,344) is provided in the storage section (140,240,740).

2. The microfluidic device (100,200,700) of claim 1, wherein the storage section (140,240,740) is arranged between the inlet end (120) and the microfluidic circuit (110) and / or between the microfluidic circuit (110) and the outlet end (125) and / or wherein the storage opening structure (144,344) is provided between the microfluidic circuit (110) and the microfluidic storage circuit (142,242,342,742).

3. The microfluidic device (100,200,700) of claim 1 or 2, wherein the storage opening structure (144,344) is configured to stay closed during the filling process of the plurality of microchambers (112) and to allow a retrieval of a storage sample of the stored fluid (360) by an external device (370) which pierces the storage opening structure (144,344).

4. The microfluidic device (100,200,700) of claim 3, wherein the microfluidic device (100,200,700) comprises a, preferably adhesive, cover layer (365) that is arranged to hold the storage opening structure (144,344) closed during the filling process.

5. The microfluidic device (200,700) of at least one of the preceding claims, wherein the microfluidic storage circuit (242) comprises a meander-like structure and / or wherein the microfluidic storage circuit (242) comprises a receiving space (701) for the fluid to be stored in which a plurality of spacers (703), preferably in the form of pillars (703), are arranged.

6. The microfluidic device (100,200,700) of at least one of the preceding claims, wherein the microfluidic storage circuit (142,242,342,742) is configured to store at least 5 µL, preferably at least 10 µL of the fluid sample, and / or wherein the volume of the microfluidic storage circuit (142,242,342,742) is at least 50 times, preferably at least 200 times, in particular at least 500 times, the volume of at least one of the microchambers (112), preferably the volume of each of the microchambers (112).

7. The microfluidic device (200) of at least one of the preceding claims, wherein the at least one microfluidic channel (130,130',130",130‴) comprises at least one fluidic delay structure (250) configured to delay a fluid flow, and wherein, preferably, the at least one fluidic delay structure (250) comprises at least one fluidic stop structure (250) configured to inhibit a fluid flow until a predefined fluid pressure is reached.

8. The microfluidic device (200) of claim 7, wherein the at least one fluidic delay structure (250), in particular the at least one fluidic stop structure (250), is arranged between the plurality of microchambers (112) and the microfluidic storage circuit (242).

9. The microfluidic device (100,200,700) of at least one of the preceding claims, wherein a cross section of the microfluidic storage circuit (142,242,342,742) is larger than a cross section of the at least one microfluidic channel (130,130',130",130‴) such that capillary forces do not cause a flow of fluid into the storage section (140,240,740).

10. The microfluidic device (100,200,700) of at least one of the preceding claims, wherein the microfluidic device (100,200,700) comprises a sealing layer (480) 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 (480).

11. A system (500) for providing a molecular analysis of a fluid sample, in particular for providing a digital polymerase chain reaction analysis, comprising - the microfluidic device (100,200,700) of at least one of the preceding claims, - an analysis apparatus (590) configured to receive the microfluidic device (100,200,700) and to amplify the plurality of aliquots within the plurality of microchambers (112) by providing a predefined thermal treatment, and wherein the microfluidic storage circuit (142,242,342,742) is arranged such that the fluid stored in the storage section (140,240,740) is also amplified by the analysis apparatus (590).

12. The system (500) of claim 11, wherein the analysis apparatus (590) is further configured to provide the predefined fluid pressure to a fluid within the microfluidic device (100,200,700) in order to enable a flow of the respective fluid through the microfluidic circuit (110) and at least partly through the microfluidic storage circuit (142,242,342,742).

13. A method (600) 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 (590) and a microfluidic device (100,200,700) 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 (100,200,700) into the analysis apparatus (590); and - filling a microfluidic circuit (110) of the microfluidic device (100,200,700) at least partially, preferably at least substantially, with fluid of the fluid sample, wherein fluid that does not reach and / or flows beyond microchambers (112) of the microfluidic well (105) during that filling process is stored in a microfluidic storage circuit (142,242,342,742) of the microfluidic device (100,200,700).

14. The method (600) of claim 13, wherein a pressure is applied to the fluid sample, preferably by means of at least one piston (598) or by means of a gas flow, in order to push the fluid sample into the microfluidic circuit (110), wherein fluid that does not reach and / or flows beyond the microchambers (112) during the application of the pressure is stored in the microfluidic storage circuit (142,242,342,742).

15. The method (600) of claim 13 or 14, further comprising one or more of the following steps - fluidly separating the microchambers (112) from each other, preferably by deforming a sealing layer (480) into channel segments of 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 (480), and / or - amplifying the fluid within the microchambers (112) and within the microfluidic storage circuit (142,242,342,742) by applying a thermal treatment to that fluid.

16. The method (600) of at least one of the preceding claims, further comprising the, preferably final, step - retrieval of the fluid (360) stored in the microfluidic storage circuit (142,242,342,742), preferably through a storage opening structure (144,344) of the microfluidic device (100,200,700).

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