Microfluidic device for handling a fluid sample

Abstract

The invention relates to a microfluidic device (100,200) for handling a fluid sample by dividing it into a plurality of aliquots, with at least one microfluidic well (102) configured to receive the fluid sample. The at least one microfluidic well comprising an inlet end (108), an outlet end (112) and a microfluidic circuit (104) with a plurality of microchambers (106). The inlet end and the outlet end are respectively coupled to the microfluidic circuit via at least one microfluidic channel (110,110',110'',110'''). The plurality of microchambers is arranged such that each microchamber is at least indirectly via one or more further microchambers fluidly coupled with the inlet end and the outlet end such that each microchamber is configured to receive sample fluid from the inlet end via the at least one microfluidic channel and to provide gas displaced by the sample fluid downstream to the outlet end. Furthermore, a protective layer (114,214) is applied to the at least one microfluidic well for sealing the inlet end and / or the outlet end of the microfluidic well, wherein the protective layer is at least partially permeable to gas.

Description

[0001] E ITLE 066047 K t8

[0002] AV2025-562 17 December 2025

[0003] QIAGEN GmbH

[0004] QIAGEN Stral e 1

[0005] 40724 Hilden

[0006] GERMANY

[0007] Microfluidic device for handling a fluid sample

[0008] FIELD OF THE INVENTION

[0009] 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 handling apparatus for handling a microfluidic device, to a system comprising such a handling apparatus, and to a method for handling a microfluidic device.

[0010] BACKGROUND OF THE INVENTION

[0011] 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.

[0012] 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.

[0013] 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.

[0014] The fluid sample is typically introduced into an inlet end of the microfluidic device fluidly coupled to the microfluidic circuit. In order to push the sample fluid from the inlet end into themicrofluidic circuit and thus into the microchambers of the microfluidic circuit, an elastic layer is usually applied over the inlet end after the fluid sample has been introduced into the inlet end. The elastic layer is then pushed into the inlet end, for example by means of a metal pin or the like, thereby compressing the air present between the elastic layer and the sample fluid. In this way, a filling pressure is applied to the sample fluid, causing the sample fluid to flow from the inlet end through the microfluidic circuit and thus into the microchambers. However, this procedure is rather laborious and also does not yet offer the desired reliability with respect to the filling of the microfluidic circuit and the microchambers with sample fluid. In addition, it has not yet been possible to achieve the desired degree of automation with the known procedure without negatively affecting the reliability of the process.

[0015] It is therefore an object of the invention to provide a microfluidic device, a handling apparatus, a system and a method which make it possible to fill the microfluidic circuit and its microchambers with sample fluid more reliably and with less effort.

[0016] SUMMARY OF THE INVENTION

[0017] 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.

[0018] 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

[0019] - a microfluidic circuit with a plurality of microchambers, which provide a respective reaction space for an aliquot of the plurality of aliquots,

[0020] - an inlet end coupled to the microfluidic circuit via at least one microfluidic channel,

[0021] - 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 one or more further microchambers fluidly coupled with the inlet end and the outlet end such that each microchamber is configured to receive sample fluid from the inlet end via the at least one microfluidic channel and to provide gas displaced by the sample fluid downstream to the outlet end,

[0022] wherein a protective layer, which is preferably an adhesive layer, is applied to the at least one microfluidic well for sealing the inlet end and / or the outlet end of the microfluidic well, and wherein the protective layer is at least partially permeable to gas.

[0023] By providing a protective layer sealing the inlet end and / or the outlet end which is partially permeable to gas, it is possible to feed gas via a gas flow through the protective layer into the inlet end and / or to suck gas via a gas flow out of the outlet end and thereby generate a filling pressure in the form of an overpressure at the inlet end and / or an underpressure at the outletend, as a result of which the sample fluid located in the inlet end flows from there through the microfluidic circuit and into its microchambers. The invention has recognized that by feeding gas into the inlet end and / or sucking gas out of the outlet end instead of pushing an elastic layer into the inlet end by means of physical pressuring means, such as moveable pistons, not only the filling pressure applied to the sample fluid can be controlled more precisely, thus allowing a more reliable filling of the microfluidic circuit and the microchambers with sample fluid, but also the effort can be reduced. This is because, the application of the protective layer to the microfluidic well can be less complex than that of an elastic layer which is pushed into the inlet end for the application of the filling pressure to the sample fluid since the forces acting on the protective layer during use are significantly lower than that acting on such an elastic layer during use. In contrast to the application of an elastic layer which is pushed into the inlet end for the pressurization of the fluid sample, the application of the protective layer can therefore also be automated without negatively affecting the reliability of the process. In addition, the protective layer can be less complex to manufacture than an elastic layer used in the prior art.

[0024] Furthermore, using constant gas flows can avoid pressure differences between different microfluidic wells which may occur due to mechanical or hydrodynamic reasons if an elastic layer is pushed into the inlet ends by means of pistons or other mechanical pressuring means. Thus, using the gas-permeable protective layer allows the use of pressurizing gas flows and thereby a particularly advantageous control over a parallel filling of the microchambers in all microfluidic wells within the microfluidic device. The parallel filling can support a homogenous development of a respective filling state in all microfluidic wells of the microfluidic device.

[0025] For the sake of simplicity, the protective layer can be at least permeable to gas at those regions that are essentially near the at least one inlet end and / or the at least one outlet end of the at least one microfluidic well. Then, the gas can flow through the protective layer directly into the at least one inlet end and / or out of the at least one outlet end directly through the protective layer. Preferably the whole protective layer is permeable to gas allowing a unique layer design for different arrangements of microfluidic wells and thus different locations of the at least one inlet end and / or the at least one outlet end on the microfluidic device.

[0026] The protective layer can be permeable to gas due to material characteristics of the layer material and / or due to at least one inlet valve and / or at least one outlet valve.

[0027] In a preferred embodiment, the protective layer is an adhesive layer. The adhesive layer can be self-adhesive or adhesive due to a certain processing step, such as a pressing or a chemical bonding or the like. Alternatively or additionally, the protective layer can be elastic. However, this does not need to be the case.

[0028] Preferably, the protective layer is not permeable for dust and fluids in order to provide a reliable protection against a contamination of the fluid sample. The protective layer allows a reliableprotection of the sample fluid against a leakage and against a respective contamination of the surrounding of the microfluidic device. Thereby, the cleanness of further devices and / or of a respective laboratory can be ensured.

[0029] It is also within the scope of the present invention to use multiple protective layers for the microfluidic device in order to avoid a contamination of the fluid sample. At least a number of these protective layers has to be at least partially permeable to gas. Preferably, just one protective layer is used for the microfluidic device in order to simplify the attachment of the layer to the microfluidic device.

[0030] It can be sufficient if only the at least one inlet end or the at least one outlet end is sealed by the protective layer. However, with respect to a simple protection against contamination it can be preferred if the at least one inlet end and the at least one outlet end of the at least one microfluidic well are sealed by the at least one protective layer.

[0031] The protective layer preferably comprises an elastomer which reseals itself after being punctured, for example with a needle. In this way, the protective layer can seal the inlet end and / or the outlet end reliably even if punctures, for example needle punctures, are necessary during the treatment of the fluid sample. For example, it is possible in this way to apply the protective layer to the at least one microfluidic well before the fluid sample is provided to the at least one inlet end through the protective layer by means of a needle and still ensure a reliable protection against contamination of the fluid sample by the protective layer after extraction of the needle. In this way, a very reliable protection against contamination can be achieved with little effort. Irrespective of this, it can be simple and expedient if the elastomer is provided in the form of a rubber coating.

[0032] The protective layer can also be attached to the at least one microfluidic well after the fluid sample has been brought into the at least one inlet end. Thereby, the filling of the inlet end is easily possible before the inlet end can be protected against contamination by the attached protective layer.

[0033] The application of the protective layer to the at least one microfluidic well might be performed manually by a user of the microfluidic device or by a certain apparatus that is configured to put the protective layer at the intended position over the at least one inlet end and / or the at least one outlet end.

[0034] It can be sufficient if the microfluidic device comprises only one microfluidic well. Then, the entire sample fluid of the fluid sample can be introduced into the inlet end of the microfluidic well. However, it can be preferred if the microfluidic device comprises a plurality of microfluidic wells, for example at least two, at least five or at least ten microfluidic wells. Then, the samplefluid of the fluid sample can be divided and sample fluid of the fluid sample can be introduced into each of the microfluidic wells.

[0035] The microfluidic device according to this first aspect of the invention is simple to build, since only the protective layer that is at least partially permeable to gas has to be added to already available microdevices with a plurality of microchambers.

[0036] A further advantage is the variability of the provided solution. Different microfluidic devices with different microfluidic circuits can be improved by simply providing the protective layer for the at least one inlet end and / or the at least one outlet end of the at least one microfluidic well.

[0037] According to the invention, the microchambers of the at least one microfluidic circuit are coupled to other microchambers of the microfluidic circuit via at least one microfluidic channel. In one embodiment, there is only one microfluidic channel that connects all microchambers of the 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 at least one microfluidic channel also comprises channel segments between microchambers.

[0038] In an embodiment of the microfluidic device according to the first aspect of the invention, the protective layer comprises at least one inlet valve for enabling at least one gas flow through the protective layer into the at least one inlet end and / or at least one outlet valve for enabling at least one gas flow through the protective layer out of the at least one outlet end. Such an inlet valve and / or outlet valve can for example be a Duckbill-valve. Using such an inlet valve and / or outlet valve can be advantageous due to a reduced gas resistance compared to a layer that is permeable to gas due to its material characteristics. In a preferred variant of this embodiment, the protective layer comprises a rubber coating. The rubber coating can advantageously improve a sealing of the inlet valve and / or outlet valve if the valve is closed.

[0039] In a preferred embodiment, the microfluidic device comprises a sealing layer that covers the at least one microfluidic circuit at least partially, preferably at least substantially. This can help to avoid a contamination of the sample fluid 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 of the at least one microfluidic circuit 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 causefluidic 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.

[0040] 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.

[0041] According to a second aspect of the invention, a handling apparatus for handling a microfluidic device, preferably of at least one of the aforementioned embodiments, is provided. The handling apparatus comprising

[0042] - a pressurization unit configured to apply a filling pressure to sample fluid within at least one microfluidic well of the microfluidic device such that the sample fluid flows through at least a part of a microfluidic circuit of the microfluidic well and into at least some of a plurality of microchambers of the microfluidic circuit as a result of the applied filling pressure, and - an electronic control unit configured to control the application of the filling pressure to the sample fluid within the at least one microfluidic well,

[0043] wherein the pressurization unit is configured to apply the filling pressure to the sample fluid within the at least one microfluidic well by feeding gas via at least one gas flow into and / or sucking gas via at least one gas flow out of the microfluidic device and the at least one microfluidic well.

[0044] Similar to the microfluidic device according to the first aspect, the handling apparatus according to the second aspect of the invention allows precise control of the filling pressure and thus reliable filling of the microfluidic circuit with the sample fluid with reduced effort.

[0045] The handling apparatus can for example be an analysis apparatus for providing a molecular analysis of the fluid sample, preferably for providing a digital polymerase chain reaction analysis of the fluid sample. In this way, a high degree of automation can be achieved. Then, the analysis apparatus can autonomously perform at least the steps from applying the fillingpressure to the sample fluid within the at least one microfluidic well to providing the molecular analysis, in particular the digital PCR analysis, of the fluid sample. Irrespective of this, the analysis apparatus can expediently comprise a robotic gripper unit configured to move the microfluidic device, a partitioning unit configured to fluidly separate the plurality of microchambers of the at least one microfluidic well from each other, preferably by deforming a sealing layer of the microfluidic device into channel segments fluidly coupling the microchambers, particularly preferably by applying a compressive force to the sealing layer, a thermal treatment unit configured to subject the fluid sample within the microfluidic device to a thermal treatment, preferably a thermal cycling, and / or optical means configured to acquire one or more images of the fluid sample within the microfluidic device during and / or after the thermal treatment. Alternatively or additionally, it can be expedient if the analysis apparatus comprises a, preferably at least substantially closed, handling room for receiving and handling the microfluidic device.

[0046] As an alternative to an analysis apparatus, the handling apparatus can be a robotic liquid handling apparatus. Robotic liquid handling apparatuses are typically not used for the application of a filling pressure to sample fluid within microfluidic wells to cause flow of the respective sample fluid through the microfluidic circuit of the microfluidic well. However, the invention has recognized that the modification of known robotic liquid handling apparatuses according to the invention can be easy to realize. Irrespective of this, the robotic handling apparatus can expediently comprise a plurality of pipettes and / or syringes and a robot unit configured to actuate and, preferably, move the plurality of pipettes and / or syringes. For example, the plurality of pipettes and / or syringes can be held on the robot unit.

[0047] Irrespective of the type of the handling apparatus, it can be useful if the pressurization unit is configured to apply the filling pressure to the sample fluid within the at least one microfluidic well such that the sample fluid flows through at least a predominant part of the microfluidic circuit and into at least a majority of the plurality of microchambers, preferably through at least substantially the entire microfluidic circuit and into at least substantially all of the plurality of microchambers, as a result of the applied filling pressure. In this way, reliable filling of the microchambers with sample fluid can be ensured. Alternatively or additionally, it can be preferred if the pressurization unit is configured to apply a filling pressure at least partially simultaneously to sample fluids within different microfluidic wells of the microfluidic device by feeding gas via a plurality of gas flows into and / or sucking gas via a plurality of gas flows out of the different microfluidic wells. This can be time saving.

[0048] The electronic control unit can for example comprise distributed components in the manner of a distributed system. Alternatively, the control unit can be a single control device. Irrespective of this, that the control unit is configured to control the application of the filling pressure to the sample fluid within the at least one microfluidic well can in particular be understood to mean or comprise that the control unit is configured to effect that the application of the filling pressureto the sample fluid within the at least one microfluidic well is carried out by means of or by the pressurization unit.

[0049] In the following, particularly preferred embodiments of the handling apparatus according to the second aspect of the invention will be described.

[0050] In a preferred embodiment, the pressurization unit comprises at least one compressor unit for generating the at least one gas flow. A compressor unit can be particularly space-saving. This can be particularly useful if the handling apparatus is an analysis apparatus for providing a molecular analysis of the fluid sample since such apparatuses are typically compact in design and provide only limited space. Irrespective of a compressor unit, the pressurization unit can be fluidly coupled to a gas reservoir of the handling apparatus. The gas reservoir can then provide the gas to be fed into the at least one microfluidic well. Providing a separate gas reservoir allows for the use of a suitable gas, such as for example clean air, in order to avoid a contamination of the fluid sample by ambient air. If the pressurization unit comprises a compressor unit, it is expedient if the compressor unit is fluidly connected to the gas reservoir.

[0051] In an alternative embodiment, the pressurization unit comprises at least one pipette and / or syringe for generating the at least one gas flow. This can be particularly cost-efficient. In addition, the at least one pipette and / or syringe can then also be used to introduce the fluid sample into the at least one microfluidic well. In this way, the amount of equipment required can be reduced. Against this background it can be preferred if the control unit is configured to effect that the fluid sample is introduced into the at least one microfluidic well by means of and / or by the at least one pipette and / or syringe. The use of a pipette and / or syringe for generating the gas flow can be particularly preferred if the handling apparatus is a liquid handling robot as such apparatuses typically comprise pipettes and / or syringes anyway. Irrespective of this, the at least one pipette and / or syringe can be a piston-driven pipette and / or syringe. This can be useful with respect to a precise and reliable pressurization of the sample fluid. Particularly preferably, the at least one pipette and / or syringe is a piston-driven air displacement pipette and / or syringe. In this way, contamination of the fluid sample by the piston of the pipette and / or syringe can be avoided when introducing the fluid sample into the microfluidic well. Irrespective of the type of the at least one pipette and / or syringe, it can be time-saving if the pressurization unit comprises a plurality of, for example at least two, at least four or at least five, pipettes and / or syringes for generating simultaneously a plurality of gas flows.

[0052] In a preferred embodiment, the pressurization unit comprises at least one gas guiding channel for guiding the at least one gas flow into and / or out of the at least one microfluidic well. This can contribute to a precise and reliable pressure application to the sample fluid within the at least one microfluidic well. The pressurization unit can also comprise a plurality of, for example at least two, at least four or at least five, gas guiding channels for guiding a plurality of gasflows at least partially simultaneously into and / or out of different microfluidic wells of the microfluidic device. This can be time saving. Then, the control unit can be configured to control the plurality of gas flows independently of each other. This allows for precise filling of the microfluidic circuit of each of the microfluidic wells with sample fluid. Irrespective of this, the pressurization unit can comprise one compressor unit or one pipette and / or syringe for generating the plurality of gas flows. This can be cost-efficient. However, with respect to a precise control of the different gas flows independently of each other, it can be advantageous if the pressurization unit comprises a plurality of compressor units or a plurality of pipettes and / or syringes each for generating a respective one of the plurality of gas flows. Irrespective of this, the at least one gas guiding channel can for example be formed at least partially, for example at least substantially, by the at least one pipette and / or syringe.

[0053] In a preferred embodiment, the pressurization unit comprises at least one sealing element for contacting the microfluidic device and sealing at least one flow path of the at least one gas flow in the contact area where the sealing element contacts the microfluidic device. This can contribute to a reliable and precise application of the filling pressure to the sample fluid. Then, it can be simple and expedient if the at least one sealing element forms at least one, in particular circumferential, section of the at least one gas guiding channel. Alternatively or additionally, the at least one sealing element can be made of a soft and / or elastic sealing material at least in a sealing section for contacting the microfluidic device. Then, the sealing material can be compressed when the sealing element is pressed against the microfluidic device. In this way, reliable sealing of the at least one flow path of the at least one gas flow can be achieved easily and cost-efficiently. For the same reason, it can be particularly preferred if the at least one sealing section of the at least one sealing element extends circumferentially around the at least one gas guiding channel of the pressurization unit, preferably around at least one opening of the at least one gas guiding channel. In some cases, it is conceivable that the at least one sealing element is made entirely of a soft and / or elastic sealing material. However, this does not have to be the case. Irrespective of whether the sealing element is made at least partially of a soft and / or elastic sealing material or not, the at least one sealing element can be at least partially, preferably at least substantially, injection molded. This can be simple and cost-efficient. If the pressurization unit comprises a plurality of gas guiding channels, it can alternatively or additionally be simple and expedient if at least one sealing element is provided per gas guiding channel.

[0054] If the pressurization unit comprises the at least one pipette and / or syringe, the at least one sealing element can be detachably connected to the at least one pipette and / or syringe. Then, the sealing element can be connected to the pipette and / or syringe for the pressure application to the sample fluid and disconnected from the pipette and / or syringe if other steps are to be performed by means of the pipette and / or syringe, for example the introduction of the fluid sample into the microfluidic device. In this way it is possible to perform various tasks reliably with the handling apparatus, with little expenditure on equipment. In addition, a detachableconnection between the sealing element and the pipette and / or syringe allows for existing handling apparatuses, for example liquid handling robots, to be easily modified according to the invention. Irrespective of this it can be simple and expedient if the at least one sealing element is detachably connected to at least one free end of the at least one pipette and / or syringe. For the same reason, it can alternatively or additionally be preferred if the at least one sealing element is detachably connected to the at least one pipette and / or syringe such that at least one section of the at least one gas guiding channel formed by the at least one sealing element is, preferably tightly, coupled at least one section of the at least one gas guiding channel formed by the at least one pipette and / or syringe. Irrespective of the design of the detachable connection between the sealing element and the pipette and / or syringe, the handling apparatus can comprise a storage unit for holding the at least one sealing element, preferably a plurality of sealing elements, available in a non-use state. If the at least one sealing element is not in use, the at least one sealing element can then be stored in the storage unit.

[0055] In a preferred embodiment, the handling apparatus comprises at least one gas pressure sensor configured to detect gas pressure information characteristic of a gas pressure. This can be useful for control of the handling apparatus. Then, the control unit can be configured to control the at least one gas flow provided by the pressurization unit based on the detected gas pressure information, preferably by controlling the compressor unit or the at least one pipette and / or syringe based on the detected gas pressure information. This allows for a very precise control of the filling pressure applied to the sample fluid in a simple way. Against this background, it can be particularly preferred if the control unit is configured to control a flow rate of the at least one gas flow based on the detected gas pressure information. Then, the control unit can for example effect a change in the flow rate if the detected gas pressure information differs from a predetermined set point or set point range, which can depend on, for example, the type of the microfluidic device used. Alternatively or additionally, the control unit can be configured to control the at least one gas flow provided by the pressurization unit based on the detected gas pressure information and based on a time information characteristic of the time the filling pressure has been applied to the sample fluid within the at least one microfluidic well. Then, the control unit can be configured to keep the filling pressure applied to the sample fluid within the at least one microfluidic well at least substantially constant for a predetermined period of time, preferably until the at least one microfluidic circuit is at least substantially filled with sample fluid. Preferably, the control unit can then be configured to stop the application of the filling pressure to the sample fluid within the at least one microfluidic well when the predetermined period of time has lapsed.

[0056] It can be simple and at the same time expedient if the detected gas pressure information is characteristic of a gas pressure in the at least one gas guiding channel. If the pressurization unit comprises the at least one pipette and / or syringe, for the same reason, the detected gas pressure information can be characteristic of a gas pressure in the at least one gas guiding channel in the at least one pipette and / or syringe. Alternatively or additionally, the at least onegas pressure sensor can be arranged at least partially in the at least one gas guiding channel and / or the at least one pipette and / or syringe. If the pressurization unit comprises a plurality of gas guiding channels for guiding a plurality of gas flows at least partially simultaneously into and / or out of different microfluidic wells of the microfluidic device, it can alternatively or additionally be preferred if at least one gas pressure sensor is provided per gas guiding channel. Then, the control unit can be configured to control each of the plurality of gas flows based on the gas pressure information detected by the respective gas pressure sensor. Irrespective of this, gas pressure information characteristic of a gas pressure can be, for example, the gas pressure itself. This may be preferred, but need not be the case. For example, gas pressure information characteristic of a gas pressure can also be a value proportional to the gas pressure.

[0057] In a preferred embodiment, the handling apparatus is configured to analyze a surface of the microfluidic device with optical means. The optical means can comprise a camera, such as a high-resolution camera or a further appropriate optical sensor. By analyzing the surface of the microfluidic device, a present filling state of the at least one microfluidic well might be detected. Preferably, the analyzed surface of the microfluidic device allows an optical detection of a filling state of the microchambers forming reaction spaces for the sample fluid. The optical means can track the filling flow front of all microfluidic wells at the same time in real time.

[0058] In a preferred variant of the aforementioned embodiment, the control unit is configured to control the at least one gas flow provided by the pressurization unit, for example by the compressor unit, 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. Preferably, the control unit is configured to control the at least one gas flow, for example by controlling the compressor unit, based on a received sensor signal provided by the optical means. This variant allows a precise control of the gas flow und thus of the filling pressure inside the microfluidic circuit based on the present filling state of the microfluidic well. In a particularly advantageous example of this variant, the control unit is configured to control the at least one gas flow provided by the pressurization unit, for example by the compressor unit, based on a current filling edge of the sample fluid within the at least one microfluidic circuit. The filling edge is preferably indicated by the present filling state that is provided for further analysis by the optical means. The current filling edge is an important characteristic of the present filling state since it includes the information which microchambers still have to be filled and which are already filled. Therefore, the current filling edge is a good measure to decide whether more pressure has to be applied to the sample fluid via the provided gas flow or not. Furthermore, knowing the current filling edge can help to decide if the filling is finished or not.

[0059] In a preferred embodiment, the handling apparatus comprises a layer application unit configured to apply a protective layer to the at least one microfluidic well for sealing at leastone inlet end and / or at least one outlet end of the at least one microfluidic well. In this way, contamination and leakage of the fluid sample can be prevented during further handling of the microfluidic device. Then, the control unit is preferably configured to control the application of the protective layer to the at least one microfluidic well. This can in particular be understood to mean or comprise that the control unit is configured to effect that the application of the protective layer to the at least one microfluidic well is carried out by means of or by the layer application unit. In this way, the application of the protective layer can be automated. In contrast to the application of an elastic layer which is pushed into the microfluidic well in order to apply the filling pressure to the sample fluid, the application of the protective layer can be automated without compromising the reliability of the process. For example, the control unit can be configured to control the application of the protective layer to the at least one microfluidic well before controlling the application of the filling pressure to the sample fluid within the microfluidic well. In this way, contamination and leakage of the sample fluid can be prevented also during the application of the filling pressure. Then, it can be preferred if the protective layer is at least partially permeable to gas such that the gas can be fed into and / or sucked out of the at least one microfluidic well through the protective layer. This can be useful with regard to a simple design of the microfluidic device. Alternatively, the control unit can be configured to control the application of the protective layer to the at least one microfluidic well after controlling the application of the filling pressure to the sample fluid within the microfluidic well. This can be particularly simple and can be preferred if contamination and leakage of the sample fluid is not critical. For the sake of simplicity and for cost reasons, it can then be preferred if the protective layer is not permeable to gas. Irrespective of this, it can be expedient if the control unit is configured to control the application of the protective layer after controlling a fluidic separation of the plurality of microchambers of the at least one microfluidic circuit from each other.

[0060] According to a further aspect of the invention, a system, for example for providing a molecular analysis of a fluid sample, in particular for providing a digital polymerase chain reaction analysis of a fluid sample, is provided. The system comprising

[0061] - a microfluidic device for handling a fluid sample by dividing it into a plurality of aliquots 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, and 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 one or more further microchambers fluidly coupled with the inlet end and the outlet end such that each microchamber is configured to receive sample fluid from the inlet end via the at least one microfluidic channel and to provide gas displaced by the sample fluid downstream to the outlet end, and- a handling apparatus of at least one of the aforementioned embodiments.

[0062] Since the system comprises a handling apparatus according to the second aspect of the invention, the advantages described for the handling apparatus according to the second aspect of the invention do also apply to the system.

[0063] The microfluidic device of the system a can be a microfluidic device according to the first aspect of the invention. Then, the pressurization unit can be configured to apply the filling pressure to the sample fluid within the at least one microfluidic well by feeding the gas through the protective layer into and / or sucking the gas through the protective layer out of the at least one microfluidic well. However, the microfluidic device of the system does not necessarily need to be a microfluidic device according to the first aspect of the invention. The microfluidic device of the system thus does not necessarily need to comprise a protective layer applied to the at least one microfluidic well for sealing the inlet end and / or the outlet end of the at least one microfluidic well, which is at least partially permeable to gas.

[0064] In a preferred embodiment of the system, the handling apparatus is configured to analyze a surface of the microfluidic device with optical means, wherein the plurality of microchambers is arranged at a bottom of the microfluidic device and the optical means are arranged and configured to analyze a bottom surface of the microfluidic device. In this variant, the microchambers are arranged and configured such that they can be analyzed by analyzing the optical characteristics of the bottom surface of the microfluidic device. Details of this embodiment of the system are already known for digital PCR arrangements, which per se requires an analysis of the result of the applied thermal cycling for each aliquot. An analysis with optical means provides an easy and fast way for such an analysis of a large number of aliquots. The optical means, such as a camera, and optionally illumination means, such as LEDs, can be arranged at the bottom. This allows to monitor the filling process of all wells per video and provide feedback to the control unit.

[0065] In a further embodiment of the system, the microfluidic device comprises at least two microfluidic wells with a respective inlet end and a respective outlet end, wherein the control unit is configured to control the gas flow provided at each inlet end and / or at each outlet end separately based on the filling state of the respective microfluidic well. In this embodiment, separate gas flows might be provided by the pressurization unit allowing a particularly detailed control of the pressure applied to the respective sample fluid by means of the gas flow. According to this embodiment, the pressurization unit preferably comprises a respective gas guiding channel for each inlet end and / or for each outlet end in order to control the gas flows separately. In a further variant of this embodiment, the microfluidic wells of the microfluidic device are separated in certain groups and the gas flow to each group for applying a pressure to the respective sample fluid is controlled separately.According to a third aspect of the invention, a method for handling a microfluidic device, preferably of at least one of the aforementioned embodiments, preferably performed at least partially by a handling apparatus of at least one of the aforementioned embodiments, is provided. The method comprising the steps

[0066] - introducing a fluid sample into the microfluidic device and at least one microfluidic well of the microfluidic device via at least one inlet end of the at least one microfluidic well; and

[0067] - applying a filling pressure to the sample fluid within the at least one microfluidic well by means of a pressurization unit such that the sample fluid flows through at least a part of a microfluidic circuit of the microfluidic well and into at least some of a plurality of microchambers of the microfluidic circuit as a result of the applied filling pressure, wherein the filling pressure is applied to the sample fluid within the at least one microfluidic well by feeding gas, preferably air, via at least one gas flow into and / or sucking gas, preferably air, via at least one gas flow out of the microfluidic device and the at least one microfluidic well.

[0068] The method of the third aspect of the invention shares the advantages described in the context of the handling apparatus according to the second aspect of the invention. In particular, the method allows reliable filling of the at least one microfluidic circuit with reduced effort.

[0069] Preferably, the method is a method for providing a molecular analysis of a fluid sample, in particular for providing a digital polymerase chain reaction analysis of a fluid sample. However, this does not necessarily need to be the case.

[0070] The method can be performed at least partially by a handling apparatus of at least one of the aforementioned embodiments. In particular, one or more steps of the method can be performed by the handling apparatus. Preferably, the method is performed at least predominantly, in particular at least substantially, by the handling apparatus. Irrespective of whether the method is at least partially performed by a handling apparatus or not, the method can expediently comprise the following steps:

[0071] - providing a handling apparatus, for example an analysis apparatus, and the microfluidic device configured to receive the fluid sample; and

[0072] - inserting the microfluidic device into the handling apparatus.

[0073] The fluid sample can expediently comprise different components. In particular the fluid sample can comprise a material to be analyzed and one or more reagents. Irrespective of this, for the sake of simplicity, the gas fed into and / or the gas sucked out of the at least one microfluidic well can be air.

[0074] Preferably, the filling pressure can be applied to the sample fluid within the at least one microfluidic well such that the sample fluid flows through at least a predominant part of the microfluidic circuit, in particular at least substantially the entire microfluidic circuit, and / or intoat least a majority of the plurality of microchambers, in particular at least substantially all of the plurality of microchambers, as a result of the applied filling pressure. However, although it should not be excluded, it is not absolutely necessary that the entire microfluidic circuit and / or all of the plurality of microchambers are filled with sample fluid as a result of the applied filling pressure. Rather, it can be preferred with regard to an economic use of the sample fluid, if some of the plurality of microchambers of the at least one microfluidic well are free from sample fluid after the application of the filling pressure to the sample fluid within the microfluidic well.

[0075] For the sake of simplicity, it can be preferred if the gas is fed into the at least one microfluidic well via the at least one inlet end. Then, the at least one gas flow can simply be directed to the sample fluid within the at least one microfluidic well via the inlet end of the microfluidic well. It can be particularly preferred if the fluid sample is introduced into the at least one microfluidic well via at least one inlet opening of the at least one inlet end and the gas is fed into the at least one microfluidic well via the at least one inlet opening. This can be useful with respect to a simple design of the microfluidic device.

[0076] In the following, particularly preferred embodiments of the method according to the third aspect of the invention will be described.

[0077] In a preferred embodiment, the at least one gas flow is generated by means of at least one pipette and / or syringe of the pressurization unit. This can be simple and cost-efficient. In addition, the at least one pipette and / or syringe can then be used to introduce the fluid sample into the at least one microfluidic well. In this way, the amount of equipment required can be reduced. Irrespective of this, it can be preferred with respect to a precise and reliable pressurization of the sample fluid if the at least one pipette and / or syringe is a piston-driven pipette and / or syringe. With respect to preventing contamination of the fluid sample during introduction into the microfluidic well, it can be further preferred if the at least one pipette and / or syringe is a piston-driven air displacement pipette and / or syringe. Irrespective of the type of the pipette and / or syringe it can be preferred if a predetermined volume of gas, preferably a predetermined volume of air, is sucked into the at least one pipette and / or syringe before the application of the filling pressure to the sample fluid. If the at least one pipette and / or syringe contains a predetermined volume of gas when starting the application of the filling pressure to the sample fluid, this can facilitate precise control of the applied pressure.

[0078] In a preferred embodiment, the method is further comprising the step

[0079] - pressing at least one sealing element of the pressurization unit against the microfluidic device during the application of the filling pressure to the sample fluid within the at least one microfluidic well.

[0080] In this way, at least one flow path of the at least one gas flow can be sealed in the contact area where the pressurization unit contacts the microfluidic device. This can contribute to reliable and accurate application of the filling pressure to the sample fluid. Against this background itcan be particularly preferred if at least one soft and / or elastic sealing section of the at least one sealing element is pressed against the microfluidic device during the application of the filling pressure to the sample fluid within the at least one microfluidic well. Then, the at least one sealing section can be compressed when being pressed against the microfluidic device. In this way, reliable sealing can be achieved easily and cost-efficiently. For the same reason, it can alternatively or additionally be preferred if the at least one sealing element, in particular the at least one soft and / or elastic sealing section of the at least one sealing element contacts the microfluidic device circumferentially around at least opening of the at least one microfluidic well when the at least one sealing element is pressed against the microfluidic device.

[0081] In a preferred variant of the aforementioned embodiment, the method is further comprising one or both of the following steps

[0082] - detachably connecting the at least one sealing element to the at least one pipette and / or syringe before applying the filling pressure to the sample fluid within the at least one microfluidic well, and / or

[0083] - disconnecting the at least one sealing element from the at least one pipette and / or syringe after applying the filling pressure to the sample fluid within the at least one microfluidic well.

[0084] In this way, the pipette and / or syringe can also be used to perform tasks other than the application of the filling pressure to the sample fluid in a reliable manner, for example the introduction of the fluid sample into the microfluidic device. If the at least one sealing element is not connected to the at least one pipette and / or syringe, for example, at least one other piece of equipment can be connected to the at least one pipette and / or syringe, for example an injection needle in order to introduce the fluid sample into the microfluidic device. Irrespective of this, it can be simple and expedient if the at least one sealing element is connected to and / or disconnected from the at least one sealing element at a storage unit for holding the at least one sealing element available in a non-use state. After the at least one sealing element has been connected to the at least one pipette and / or syringe, the at least one pipette and / or syringe can then be moved together with the at least one sealing element to the microfluidic device in order to apply the filling pressure to the sample fluid within the at least one microfluidic well. During the movement from the storage unit to the microfluidic device, the predetermined volume of gas can then be sucked into the at least one pipette and / or syringe. This can be time saving.

[0085] In a preferred embodiment, the method is further comprising the steps

[0086] - detecting, preferably by means of at least one gas pressure sensor, gas pressure information characteristic of a gas pressure, and

[0087] - controlling, preferably by means of a control unit, the at least one gas flow based on the gas pressure information.This allows for a very precise control of the filling pressure applied to the sample fluid and thus of the filling of the microfluidic circuit in a simple manner. Against this background, it can be particularly preferred if a flow rate of the at least one gas flow is controlled based on the gas pressure information. Alternatively or additionally, the at least one gas flow can be controlled based on the gas pressure information and based on a time information characteristic of the time the filling pressure has been applied to the sample fluid within the at least one microfluidic well. Then, the filling pressure applied to the sample fluid within the at least one microfluidic well can be kept at least substantially constant for a predetermined period of time, preferably until the at least one microfluidic circuit is at least substantially filled with sample fluid. After the predetermined period of time has lapsed, the application of the filling pressure to the sample fluid within the at least one microfluidic well can then be stopped.

[0088] If the at least one gas flow is generated by means of a compressor or the at least one pipette and / or syringe, it can alternatively or additionally be simple and expedient if the at least one gas flow, in particular a flow rate of the at least one gas flow, is controlled based on the gas pressure information by controlling the compressor unit or the at least one pipette and / or syringe based on the gas pressure information. Irrespective of this, it can be preferred for the same reason if the gas pressure information is characteristic of a gas pressure in at least one gas guiding channel of the pressurization unit guiding the at least one gas flow into and / or out of the at least one microfluidic well, preferably in the at least one pipette and / or syringe. If a plurality of gas flows are guided by means of a plurality of gas guiding channels into and / or out of a plurality of microfluidic wells of the microfluidic device, it can alternatively or additionally be preferred if gas pressure information characteristic of a gas pressure in each of the gas guiding channels is detected and each of the plurality of gas flows is controlled based on the respective gas pressure information.

[0089] In a preferred embodiment, the method according to the third aspect of the invention is further comprising the steps

[0090] - analyzing a present filling state of the plurality of microchambers of the at least one microfluidic well; and

[0091] - determining the filling pressure to be applied to the sample fluid within the at least one microfluidic well based on the present filling state of the plurality of microchambers of the microfluidic well.

[0092] In this embodiment, the knowledge about the current filling state is advantageously used to control the filling pressure applied to sample fluid by means of the gas flow. Thereby, the gas flow can be stopped if all microchambers or a desired number of microchambers of the microfluidic well is filled with sample fluid. It can also be detected if the gas flow has to be increased in order to overcome any capillary forces or the like that may interfere with the desired flow of the sample fluid through the microfluidic device.In another embodiment, the method according to the third aspect of the invention is further comprising the step

[0093] - applying a protective layer to the at least one microfluidic well for sealing the inlet end of the microfluidic well and / or an outlet end of the microfluidic well fluidly coupled to the inlet end.

[0094] This can contribute to reliable protection of the fluid sample from contamination and to reliable protection against leakage of the fluid sample and thus contamination of the environment of the microfluidic device. The protective layer can for example be applied to the at least one microfluidic well after the filling pressure has been applied to the sample fluid within the at least one microfluidic well. This can be easy and sufficient if contamination of the fluid sample and the environment of the microfluidic device is not particularly critical. Then, it can be preferred for cost reasons if the protective layer is not permeable to gas. However, if contamination of the fluid sample and / or the environment of the microfluidic device is critical, it can be preferred if the protective layer is applied to the at least one microfluidic well before the filling pressure is applied to the sample fluid within the at least one microfluidic well. Then, it can be useful with regard to a simple design of the microfluidic device if the protective layer is at least partially permeable to gas and the filling pressure is applied to the sample fluid within the at least one microfluidic well by feeding the gas through the protective layer into and / or sucking the gas through the protective layer out of the microfluidic well. A permeable protective layer allows the gas to flow through its layer-material and thereby a reliable way to ensure a controlled pressure level while avoiding any contamination of the fluid sample. If the protective layer is at least partially permeable to gas, it can be useful to apply, preferably glue, a further protective layer to the at least partially gas-permeable protective layer after the filling pressure has been applied to the sample fluid within the at least one microfluidic well. The further protective layer can then be at least substantially impermeable to gas. In this way, leakage of aerosols and / or nucleic acids can be avoided during a subsequent thermal treatment of the fluid sample.

[0095] With respect to reliable prevention of contamination, it can be particularly preferred if the protective layer is applied to the at least one microfluidic well before the fluid sample is introduced into the at least one microfluidic well. Then, the fluid sample can be introduced into the at least one microfluidic well through the protective layer. This can be particularly reliable with respect to the prevention of contamination. In order to introduce the fluid sample through the protective layer into the at least one microfluidic well, the protective layer can, for example, be punctured with at least one, preferably metallic, needle and the fluid sample can be introduced into the at least one microfluidic well by means of the at least one needle puncturing the protective layer. Then, it can be useful if the protective layer comprises an elastomer which reseals after being punctured with the at least one needle. Such elastomers are known from medicine, for example, where they are used in ampules. Alternatively or additionally, the protective layer can comprise at least one valve, for example a duckbill valve, for introducing the fluid sample through the protective layer into the at least one microfluidic well. Then, the fluid sample can be introduced into the at least one microfluidic well through the valve.Irrespective of whether the protective layer is applied to the microfluidic well before or after providing the fluid sample to the microfluidic device, it can be preferred if the inlet end and the outlet end of the at least one microfluidic well are sealed by means of the protective layer. In this way, contamination can be very reliably prevented.

[0096] In a further embodiment, the method according to the third aspect of the invention is further comprising the step

[0097] - applying a water-immiscible layer, e.g. a mineral oil layer, to the sample fluid within the at least one microfluidic well, preferably before applying the protective layer.

[0098] A water-immiscible layer can also contribute to reliable protection against contamination of the fluid sample. The water immiscible layer, such as a mineral oil layer, can just be applied to seal the fluid sample reliably but must not be pressed into a microchamber of the microfluidic circuit. Irrespective of this, it can be expedient if the water immiscible layer is applied to the sample fluid before the filling pressure is applied to the sample fluid by means of the gas flow. For the same reason, the water immiscible layer can alternatively or additionally be applied to the sample fluid before the protective layer is applied to the at least one microfluidic well. Then, the water-immiscible layer can be applied to the sample fluid before the filling pressure is applied to the sample fluid and the protective layer can be applied to the at least one microfluidic well after the filling pressure has been applied to the sample fluid. In this way, contamination of the fluid sample can be avoided very reliably in a simple manner without the protective layer having to be permeable to gas. However, with respect to prevention of contamination not only of the fluid sample but also of the environment of the microfluidic device, it can be even more preferred if both the water immiscible layer is applied to the sample fluid and the protective layer is applied to the at least one microfluidic well before the filling pressure is applied to the sample fluid.

[0099] For providing the water-immiscible liquid, hydrophobic liquids may be used that have a lighter density than water. Suitable examples include hydrocarbons, hydrocarbon mixtures. According to one embodiment a water-immiscible alkane is used for providing the layer. The use of mineral oil is particularly preferred to apply the water-immiscible layer to the sample fluid. Using such water-immiscible layer is advantageous since such a layer does typically not react with the fluid sample that is usually being used in arrangement according to the present invention.

[0100] In another embodiment, the method is further comprising the step

[0101] - fluidly separating the plurality of microchambers of the at least one microfluidic circuit from each other after applying the filling pressure to the sample fluid within the microfluidic well. In this way, exchange of sample fluid between the microchambers can be prevented after the microchambers have been filled at least partially with sample fluid. In principle, the application of the filling pressure to the sample fluid can be stopped before the microchambers are fluidlyseparated from each other. However, with respect to a controlled filling of the microchambers with sample fluid, it can be useful to maintain the filling pressure on the sample fluid at least partially until the microchambers are fluidly separated from each other. Alternatively or additionally, the step of fluidly separating the plurality of microchambers of the at least one microfluidic circuit from each other can preferably be performed before the protective layer is applied to the at least one microfluidic well and / or before a thermal treatment or the like is started. Regardless of when it is carried out, the fluidic separation of the plurality of microchambers of the at least one microfluidic circuit can be achieved easily and reliably by deforming a sealing layer into channel segments of at least one microfluidic channel that fluidly couples the plurality of microchambers. The sealing layer can cover the at least one 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 plurality of microchambers of the at least one microfluidic circuit 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. Further details about the fluidic separation of the microchambers by means of a sealing layer are for example described in US 2021 / 0379593 A1.

[0102] In a preferred variant of the aforementioned embodiment, a duration of the application of the filling pressure to the sample fluid within the at least one microfluidic well, a pressure level of the filling pressure applied to the sample fluid within the at least one microfluidic well, and / or a pressure profile with which the filling pressure is applied to the sample fluid within the at least one microfluidic well depends on an amount of sample fluid that is usually displaced by the fluidic separation of the plurality of microchambers of a microfluidic well of the microfluidic device. If a large amount of sample fluid is usually displaced by the fluidic separation of the microchambers for a specific microfluidic device, the handling apparatus does not have to provide the at least one gas flow until all of the plurality of microchambers of the at least one microfluidic device are filled since the last microchambers might get filled by the displaced amount of sample fluid during the fluidic separation. This can be considered by the handling apparatus automatically. Therefore, in this variant, less sample fluid is discharged out of the microfluidic circuit unused during the fluidic separation of the microchambers. In this way, the so-called dead volume, i.e. the part of the volume of the fluid sample that is provided to the microfluidic well, but not analyzed during a subsequent molecular analysis, can be minimized. The pressure profile of the filling pressure is preferably a profile over time. For example, the pressure profile can be a stepped profile. Then, the filling pressure applied to the sample fluid within the at least one microfluidic well can be increased stepwise over time. This can be useful, for instance, to open different pressure valves at different times and, in this way, control the flow of the sample fluid. Irrespective of the pressure profile, the amount of sample fluid that is usually displaced might depend on the used design of the microfluidic device. Therefore, theduration of the application of the filling pressure, the pressure level of the filling pressure, and / or the pressure profile of the filling pressure preferably further depends on an indicated model type of the microfluidic device. Irrespective of this, the amount of sample fluid that is usually displaced by the fluidic separation of the plurality of microchambers of a microfluidic well of the microfluidic device can for example be an empirical value, which can, for example, be stored in a memory of the handling apparatus.

[0103] In a further embodiment of the method according to the third aspect of the invention, a number of finally not filled microchambers of the microfluidic device is detected and a subsequent molecular analysis is provided as a function of the detected number of not filled microchambers. Taking not filled microchambers into account improves an accuracy of the statistics that are finally used to interpret the result of the reactions trigged within the microchambers. Therefore, using optical means to detect the filling state and / or the filling edge can be used to decide when to stop the filling process as well as to improve the accuracy of the applied analytical and / or statistical methods. Against this background, it can alternatively or additionally also be useful to detect a number of microchambers that are filled with sample fluid in another way than by the application of the filling pressure to the sample fluid within the at least one microfluidic well. Then, a subsequent molecular analysis can also be provided as a function of the detected number of microchambers filled in another way than by the application of the filling pressure to the sample fluid. The microchambers that are filled other than by the application of the filling pressure to the sample fluid can for example be filled with sample fluid as a consequence of the fluidic separation of the plurality of microchambers of the at least one microfluidic well.

[0104] With respect to the accuracy of the applied analytical and / or statistical methods, it can alternatively or additionally also be preferred if a degree of filling of at least one microchamber of the microfluidic device with sample fluid is detected and a subsequent molecular analysis is provided as a function of the at least one detected degree of filling. For the said reason, it is particularly useful if not only a degree of filling of one microchamber, but a respective degree of filling of a plurality of microchambers, in particular of at least substantially all microchambers, of the microfluidic device are detected. Then, the subsequent molecular analysis can expediently be provided as a function of the detected degrees of filling. Irrespective of this, a degree of filling of a microchamber means in particular the relation of the volume of the microchamber filled with sample fluid to the total volume of the microchamber. Alternatively or additionally to the detection of a degree of filling, a filling speed of at least one microchamber of the microfluidic device can be detected. Then, a subsequent molecular analysis can be provided as a function of the at least one detected filling speed. This can also contribute to a high accuracy of the applied analytical and / or statistical methods. Against this background, it is even more preferred if not only a filling speed of one microchamber, but a respective filling speed of a plurality of microchambers, in particular of at least substantially all microchambers, of the microfluidic device are detected. Then, a subsequent molecular analysis can expedientlybe provided as a function of the detected filling speeds. Irrespective of this, a filling speed of a microchamber means in particular a speed at which the microchamber is filled with sample fluid. Against the same background, it can be useful if a filling duration of at least one microchamber of the microfluidic device, preferably a respective filling duration of a plurality of microchambers, in particular of at least substantially all microchambers of the microfluidic device, is detected and a subsequent molecular analysis is provided as a function of the at least one detected filling duration.

[0105] It shall be understood that the microfluidic device of the first aspect of the invention, the handling apparatus of the second aspect of the invention, the system of the further aspect of the invention, and the method of the third aspect of the invention can have similar or identical embodiments. Thus, the disclosure of a feature for one of the microfluidic device of the first aspect, the handling apparatus of the second aspect, the system of the further aspect, and the method of the third aspect of the invention shall also be understood as disclosure of that feature and / or a corresponding feature for the other three of the microfluidic device, the handling apparatus, the system and the method.

[0106] These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter.

[0107] BRIEF DESCRIPTION OF THE DRAWINGS

[0108] In the following drawings:

[0109] Fig. 1 shows schematically a first embodiment of a microfluidic device according to a first aspect of the invention;

[0110] Fig. 2 shows schematically a second embodiment of the microfluidic device according to the first aspect of the invention;

[0111] Fig. 3A-D show schematically details of the first embodiment of the microfluidic device shown in Fig. 1;

[0112] Fig. 4 shows schematically a first embodiment of a handling apparatus according to a second aspect of the invention;

[0113] Fig. 5 shows schematically a second embodiment of the handling apparatus according to the second aspect of the invention;

[0114] Figs. 6A, 6B show schematically a third embodiment of the handling apparatus according to the second aspect of the invention;

[0115] Fig. 7 shows schematically a fourth embodiment of the handling apparatus according to the second aspect of the invention;

[0116] Figs. 8A-C show schematically details of the fourth embodiment of the handling apparatus and of a microfluidic device shown in Fig. 7; and

[0117] Fig. 9 shows a flow diagram of an embodiment of a method according to a third aspect of the invention.DETAILED DESCRIPTION OF EMBODIMENTS

[0118] 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 102 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.

[0119] The microfluidic well 102 comprises a microfluidic circuit 104 with a plurality of microchambers 106, which provide a respective reaction space for an aliquot of the plurality of aliquots. The microfluidic well 102 further comprises an inlet end 108 coupled to the microfluidic circuit 104 via at least one microfluidic channel 110, 110’, 110”, 110”’ and an outlet end 112 coupled to the microfluidic circuit 104 via the at least one microfluidic channel 110, 110’, 110”, 110’”.

[0120] The plurality of microchambers 106 is arranged such that each microchamber 106 is at least indirectly via one or more further microchambers 106 fluidly coupled with the inlet end 108 and with the outlet end 112. In the present embodiment, four microfluidic channels 110, 110’, 110”, 110’” are arranged parallel to each other and each connect five microchambers 106 in a row. The connections between two respective microchambers 106 are formed by the respective microfluidic channel 110, 110’, 110”, 110’”. As a consequence of this microfluidic structure each microchamber 106 is configured to receive sample fluid from the inlet end 108 via the at least one microfluidic channel 110, 110’, 110”, 110’” and to provide gas displaced by the sample fluid downstream to the outlet end 112. Possible structures of such microchambers 106 are known in the art and are therefore not described in detail in the following. As a nonlimiting example, US 2021 / 0379593 A1 describes an embodiment of such microchambers.

[0121] Furthermore, the microfluidic device 100 comprises a protective layer 114 that is applied to the at least one microfluidic well 102 for sealing the inlet end 108 and the outlet end 112. The protective layer 114 in this embodiment is an adhesive layer. The protective layer can be self-adhesive or adhesive due to a certain processing step, such as a pressing or a chemical bonding or the like. In not shown embodiments, the protective layer is not adhesive but attached to the microfluidic device via further processing steps. In the shown embodiment, the protective layer 114 is attached to the depicted surface of the microfluidic device 100 so that the perspective of Fig. 1 shows right though the protective layer 114, which is for reasons of clarity transparent in Fig. 1. The protective layer 114 is at least partially permeable to gas. In the embodiment shown in Fig. 1, the whole protective layer 114 is permeable to gas. It is attached to the microfluidic device 100 after the sample fluid has been put into the inlet end 108. Preferably, the protective layer 114 is not permeable for dust and fluids. Thereby, a contamination of the sample fluid can be reliably avoided.The microfluidic well 102 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 108 has to be filled with sample fluid of the fluid sample separately.

[0122] 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 controlled filling of the microchambers in order to assure a large number of aliquots for the dPCR analysis.

[0123] Fig. 2 shows a second embodiment of the microfluidic device 200 according to the first aspect of the invention.

[0124] The microfluidic device 200 comprises eight microfluidic wells 102 that are at least similar to the microfluidic well shown in Fig. 1. The respective microfluidic circuits are not shown in view of the protective layer 214 that protects the fluid sample against contamination. Furthermore, the protective layer 214 protects the surrounding of the microfluidic device 200 against a contamination with the sample fluid. The positions of respective microfluidic wells are shown by dashed lines.

[0125] In contrast to the protective layer 114 of Fig. 1, the protective layer 214 comprises inlet valves 216 for the inlet ends of the microfluidic wells 102 and outlet valves 218 for the outlet ends of the microfluidic wells 102. The valves 216, 218 of this embodiment are designed as Duckbillvalves. Other one-way valves are also possible as inlet valves and / or as outlet valves. Providing valves for the inlet end and the outlet end can be advantageous not only if gas is to be fed into the inlet end and sucked out of the outlet end, but also if gas is only to be fed into the inlet end or only to be sucked out of the outlet end in order to avoid any problems with positive pressure at the outlet end or negative pressure at the inlet end.

[0126] The protective layer 214 of this embodiment is made of a material that is not permeable to gas since the layer itself is permeable to gas due to the provided valves 216, 218.

[0127] Fig. 3A-D show details of the microfluidic device 100 shown in Fig. 1 in a sectional view along a section plane along the microfluidic channel 110 in the region of two of the microchambers 106 (Figs. 3A, 3C) and in a sectional view along a section plane perpendicular to the microfluidic channel 110 in a region between the two microchambers 106 (Figs. 3B, 3D). Asfar as shown in Figs. 3A-D, the microfluidic device 200 shown in Fig. 2 can have at least substantially the same structure.

[0128] The microfluidic device 100 comprises an adhesive sealing layer 320, for example in the form of an adhesive plastic film, that is glued to the microfluidic well 102 and covers the microchambers 106. In the depicted and thus preferred embodiment, the sealing layer 320 and the protective layer 114 (not shown in Figs. 3A-D) are arranged on opposite sides of the microfluidic device 100. However, in an alternative embodiment, the sealing layer 320 and the protective layer 114 could be arranged on the same side of the microfluidic device 100.

[0129] In Figs. 3A, 3B, the sealing layer 320 is arranged at least substantially outside the microfluidic channel 110, thus allowing sample fluid to flow through the microfluidic channel 110. In this way, sample fluid can flow from one microchamber 106 to another microchamber 106 via the microfluidic channel 110 during a filling process of the microfluidic device 100. In order to fluidly separate the microchambers 106 from each other after the microchambers 106 have been at least partially filled with sample fluid, a compressive force F can be applied to the sealing layer 320 in the direction of the microfluidic well 102.

[0130] In Figs. 3C, 3D, a compressive force F has been applied to the sealing layer 320 in a direction at least substantially perpendicular to the sealing layer 320, 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 320 is deformed into channel segments of the microfluidic channel 110 that fluidly couple the microchambers 106 such that the channel segments are sealed by the sealing layer 320. In this way, the microchambers 106 are fluidly separated from each other. The sealing layer 320 is also deformed into the microchambers 106. However, due to the greater depth of the microchambers 106 compared to the depth of the channel segments of the microfluidic channel 110, a volume of the fluid sample remains in each microchamber 106.

[0131] Fig. 4 shows a first embodiment of a handling apparatus 400 according to a second further aspect of the invention and a microfluidic device 100. Together, the handling apparatus 400 and the microfluidic device 100 form a system 450 according to a further aspect of the invention.

[0132] The shown handling apparatus 400 is an analysis apparatus 400 for providing a molecular analysis of a fluid sample, in particular for providing a digital polymerase chain reaction analysis. In the shown embodiment, the microfluidic device 100 is a microfluidic device according to at least one of the preceding embodiments. However, this does not necessarily need to be the case. In particular, the microfluidic device 100 does not necessarily need to comprise a protective layer permeable to gas.As an example, the microfluidic device 100 comprises eight microfluidic wells 102 as depicted in Fig. 1. The microfluidic device 100 is shown in an inserted state, where it is inserted into the analysis apparatus 400 by using a holding structure 422 of the analysis apparatus 400. The holding structure 422 of the shown embodiment comprises sliding rails. The analysis apparatus 400 is configured to receive the microfluidic device 100 via the holding structure 422.

[0133] The analysis apparatus 400 further comprises a gas reservoir 424 and a fluidly connected pressurization unit 426 for feeding gas from the gas reservoir 424 via a controlled gas flow 428 through the protective layer 114 of the microfluidic device 100 into the microfluidic device 100 and into the inlet ends 108 (not shown in Fig. 4) of the microfluidic wells 102. The pressurization unit 426 comprises a compressor unit 430 for generating the gas flow 428 and a gas guiding structure 432 forming a gas guiding channel 434 for guiding the gas flow 428 from the compressor unit 430 to the microfluidic device 100 and through the protective layer 114 of the microfluidic device 100 into the inlet ends 108 of the microfluidic wells 102. In this way, a filling pressure can be applied to sample fluid of a fluid sample present within the microfluidic wells 102 such that the sample fluid flows through at least a part of the microfluidic circuit 104 (not shown in Fig. 4) of the respective microfluidic well 102 and into at least some of the plurality of microchambers 106 (not shown in Fig. 4) of the microfluidic circuit 104. As an alternative or in addition to feeding gas into the inlet ends 108 of the microfluidic wells 102, the pressurization unit 426 could also be configured to suck gas out of the outlet ends 112 (not shown in Fig. 4) of the microfluidic wells 102 in order to cause a flow of the sample fluid through a part of the microfluidic circuit 104 of the respective microfluidic well 102.

[0134] The analysis apparatus 400 further comprises an electronic control unit 436. The control unit 436 is configured to control the application of the filling pressure to the sample fluid within the microfluidic wells 102 of the microfluidic device 100. For example, the control unit 436 can be configured to effect that the end of the gas guiding structure 432 assigned to the microfluidic device 100 is moved to the microfluidic device 100 before the application of the filling pressure to the sample fluid, that the pressure application is started and stopped, and that the end of the gas guiding structure 432 assigned to the microfluidic device 100 is moved away from the microfluidic device 100 after the pressure application. In addition, the control unit 436 is configured to control the flow rate of the gas flow 428 provided by the pressurization unit 426 to the microfluidic device 100 by controlling the compressor unit 430.

[0135] The analysis apparatus 400 is preferably further configured to amplify the plurality of aliquots within the plurality of microchambers 106 of the microfluidic device 100 by providing a predefined thermal treatment after the filling process of the microchambers 106 according to the present invention is finished.

[0136] Preferably, the sample fluid is brought into the inlet ends of the microfluidic device 100 prior to a sealing of the inlet ends and the outlet ends with the protective layer 114 and prior to theinsertion of this microfluidic device 100 into the analysis apparatus 400. However, this does not necessarily need to be the case.

[0137] In order to fluidly separate the plurality microchambers 106 of the microfluidic wells 102 from each other, channel segments of the microfluidic channels 110, 110’, 110”, 110”’ (not shown in Fig. 4) that are arranged between microchambers 106 are closed, before a thermal treatment or the like starts. The channel segments 110, 110’, 110”, 110’” are closed by applying a compressive force to the sealing layer as described above with regard to Figs. 3A-D. Further details about the closing of the microfluidic channels 110, 110’, 110”, 110’” are shown in Fig. 5 below and are for example described in US 2021 / 0379593 A1.

[0138] Fig. 5 shows a second embodiment of the handling apparatus 500 according to the second aspect of the invention and a microfluidic device 100, which together form a second embodiment of the system 550 according to the further aspect of the invention. Like the handling apparatus 400 in Fig. 4, the shown handling apparatus 500 is an analysis apparatus 500 for providing a molecular analysis, in particular a digital polymerase chain reaction analysis, of a fluid sample.

[0139] The analysis apparatus 500 differs from the analysis apparatus 400 shown in Fig. 4 by the design of the gas guiding structure 532 of the pressurization unit 526. At the end assigned to the compressor unit 430, the gas guiding structure 532 forms a gas guiding channel 538 for guiding the gas flow 540 generated by the compressor 430 unit away from the compressor unit 430. In the direction of the end of the gas guiding structure 532 assigned to the microfluidic device 100, the gas guiding channel 538 splits up into a plurality of gas guiding channels 534 such that also the gas flow 540 generated by the compressor unit 430 splits up into a plurality of gas flows 528 which can be guided by the plurality of gas guiding channels 534 exactly towards and into the inlet ends 108 (not shown in Fig. 5) of the microfluidic wells 102 (not shown in Fig. 5) under the gas-permeable protective layer 114 of the microfluidic device 100. In an alternative embodiment, it could be provided that the microfluidic device 100 does not comprise the gas-permeable protective layer 114 such that the plurality of gas guiding channels 534 can guide the plurality of gas flows 528 directly into the inlet ends 108 of the microfluidic wells 102. A, for example gas-impermeable, protective layer sealing the inlet ends 108 and the outlet ends 112 of the microfluidic wells 102 could then be applied to the microfluidic wells 102 after the application of the filling pressure to the sample fluid within the microfluidic wells 102.

[0140] The analysis apparatus 500 further comprises optical means 542, such as for example a camera, to analyze a surface of the microfluidic device 100. In the present embodiment, the optical means 542 are arranged to analyze a bottom surface of the microfluidic device 100. This is advantageous since the microchambers 106 of the microfluidic wells 102 are arranged at a bottom of the microfluidic device 100 in the present embodiment. Therefore, analyzing thebottom surface enables the optical means 542 to detect a present filling state of the microfluidic circuits.

[0141] The electronic control unit 536 is configured to receive a respective sensor signal from the optical means 542 and to control the compressor unit 430 and thus the gas flow 540 as well as the plurality of gas flows 528 based on the sensor signal and therefore based on the present filling state. Preferably, the analysis apparatus 500 is also configured to detect a current filling edge during the filling process and to control the compressor 430 accordingly. In addition, the control unit 536 could be configured to control the plurality of gas flows 528 guided into the inlet ends 108 of the microfluidic wells 102 independently from each other based on the detected filling state of the respective microfluidic well 102, for example by means of valves or the like provided in the plurality of gas guiding channels 534.

[0142] The analysis apparatus 500 furthermore comprises a roll 544 to apply a compressive force on the bottom surface of the microfluidic device 100 and thus to the sealing layer of the microfluidic device 100, which is not shown in Fig. 5. The compressive force aims to close channel segments of the microfluidic channels 110, 110’, 110”, 110”’ (not shown in Fig. 5) within the microfluidic circuits 104 in order to fluidly separate the plurality of microchambers 106 of each of the microfluidic wells 102 from each other. The embodiment shown in Fig. 5 provides pressuring rails 546 at the bottom surface of the microfluidic device 100, which are arranged to allow a controlled rolling of the roll 544 along the sealing layer of the microfluidic device 100 in order to apply the external compressive force. After the microchambers 106 are fluidly separated from each other, a thermal treatment or the like can start. Further details about the closing of the microfluidic channels 110, 110’, 110”, 110’” are for example described in US 2021 / 0379593 A1.

[0143] The optical means 542 of this embodiment may also detect for each of the microfluidic wells 102 an amount of sample fluid that is displaced by the fluidic separation of the plurality of microchambers 106. Thereby, a future filling pressure application might be shortened since the last unfilled microchambers 106 might be filled by the sample fluid displaced by the roll 544 during the application of the compressive force to the sealing layer.

[0144] Figs. 6A, 6B show a third embodiment of the handling apparatus 500’ according to the second aspect of the invention and a microfluidic device 100, which together form a third embodiment of the system 550’ according to the further aspect of the invention. Like the handling apparatuses 400, 500 in Fig. 4 and 5, the shown handling apparatus 500’ is an analysis apparatus 500’ for providing a molecular analysis of a fluid sample, in particular a digital polymerase chain reaction analysis.

[0145] Fig. 6A shows a part of the analysis apparatus 500’ with the same gas guiding structure 532 as the analysis apparatus 500 shown in Fig. 5. The gas guiding channels 534 are directedexactly towards the respective inlet end 108 (not shown in Fig. 6A) under the protective layer 114 of the microfluidic device 100. The protective layer 114 forms an elastic seal that allows air to pass the protective layer 114 without an amplification of the sample fluid provided in the microfluidic device 100.

[0146] Furthermore, optical means 542’ are arranged to analyze a bottom surface of the microfluidic device 100 while the microfluidic device 100 is in the depicted inserted state. The optical means 542’ are formed by a camera. A possible picture provided by these optical means 542’ is shown in Fig. 6B. In the shown embodiment, the roll 544 is at the side during the application of the filling pressure to the sample fluid within the microfluidic wells 102 to allow monitoring of the filling state. The life camera 542’ can track the filling state of all microfluidic wells 102 at the same time and may stop the application of the filling pressure.

[0147] The picture in Fig. 6B shows a detected filling state 548 with a certain filling edge 552. The depicted microfluidic well 102 is filled from left to right. The microchambers 106 cannot be recognized due to their small size. The depicted filling state 548 is reached by applying the filling pressure to the sample fluid within the microfluidic well 102 without activating the roll 544 (not shown in Fig. 6B). After the depicted filling state 548 is reached, the roll 544 might apply a compressive force on the bottom surface of the microfluidic device 100, for example by rolling from left to right over the bottom surface. The compressive force aims to close channel segments of the microfluidic channels 110, 110’, 110”, 110’” within the microfluidic circuit 104 in order to fluidly separate the microchambers 106 from each other. Thereby, a dead volume of sample fluid not being in any microchamber 106 is avoided and further empty microchambers 106 within an empty region 554 behind the filling edge 552 might be filled with displaced sample fluid.

[0148] In a not shown embodiment of the system, the optical means are arranged to detect another surface of the microfluidic device, and the microfluidic device allows a detection of a present filling state from the detected other surface, for example by detecting a filling state of at least one microfluidic channel of the microfluidic device.

[0149] Fig. 7 shows a fourth embodiment of a handling apparatus 700 according to the second aspect of the invention and a microfluidic device 756, which together form a fourth embodiment of a system 750 according to the further aspect of the invention.

[0150] The shown handling apparatus 700 is a robotic liquid handling apparatus 700. The microfluidic device 756 is similar to the microfluidic device 100 shown in Fig. 1 but does not comprise a gas-permeable protective layer 114 at its upper side. For the rest, the design of the microfluidic device 756 can be the same as that of the microfluidic device 100 shown in Fig. 1.

[0151] The robotic liquid handling apparatus 700 comprises a pressurization unit 726. The pressurization unit 726 comprises a plurality of pipettes 758 and / or syringes 758 in the form ofpiston-driven air displacement pipettes 758 and / or syringes 758. In addition, the pressurization unit 726 comprises a plurality of sealing elements 760, wherein some of the sealing elements 760 are detachably connected to the free ends of the pipettes 758 and / or syringes 758. Each of the sealing elements 760 comprises a soft and / or elastic sealing section 762 and a connecting section 764 for connecting the sealing element 760 to one of the pipettes 758 and / or syringes 758. The plurality of pipettes 758 and / or syringes 758 are held on a robot unit 766 of the robotic liquid handling apparatus 700. The robot unit 766 is configured to actuate and move the pipettes 758 and / or syringes 758, for example in all three spatial directions.

[0152] The robotic liquid handling apparatus 700 further comprises a workdeck 768. On the workdeck 768, a holding structure 722 is provided for holding the microfluidic device 756. In addition, a storage unit 770, for example in the form of a rack or the like, is provided on the workdeck 768 for holding the sealing elements 760 available when they are not in use. The storage unit 770 comprises a plurality of receptacles 772 for the sealing elements 760. Further, a layer application unit 774 is provided on the workdeck 768 for applying a protective layer to the upper side of the microfluidic device 756 and thus to a plurality of microfluidic wells 702 of the microfluidic device 756 in order to seal inlet ends and outlets ends of the microfluidic wells 702. The layer application unit 774 comprises a transport device 776, for example in the form of a movable slide, on which the microfluidic device 756 can be placed and by means of which the microfluidic device 756 can be moved into the layer application unit 774 for the application of the protective layer. Such layer application units are known in the prior art.

[0153] Further, the robotic liquid handling apparatus 700 comprises an electronic control unit 736. The control unit 736 is configured to control the robot unit 766 and the layer application unit 774. For example, the control unit 736 can comprise distributed components in the manner of a distributed system.

[0154] By means of the pipettes 758 and / or syringes 758 a fluid sample can be introduced into the inlet ends of the microfluidic wells 702 of the microfluidic device 756. For this purpose, the sealing elements 760 can be disconnected from the pipettes 758 and / or syringes 758 and stored in the storage unit 770. Then, for example, pipette tips can be connected to the pipettes 758 and / or syringes 758 in order to pipette the fluid sample into the inlet ends of the microfluidic wells 702.

[0155] After the fluid sample has been introduced into the microfluidic device 756 and the pipette tips have been disconnected from the pipettes 758 and / or syringes 758, one of the sealing elements 760 can be connected to each of the pipettes 758 and / or syringes 758 at the storage unit 770. Thereafter, the robot unit 766 can move the pipettes 758 and / or syringes 758 together with the connected sealing elements 760 from the storage unit 770 to the microfluidic device 756. During this movement, a predetermined volume of gas, in particular a predetermined volume of air, can be sucked into each of the pipettes 758 and / or syringes 758 by actuatingthe pistons 778 of the pipettes 758 and / or syringes 758 and pulling them away from the sealing element 760 connected to the respective pipette 758 and / or syringe 758. Then, the robot unit 766 can press the sealing elements 760 connected to the pipettes 758 and / or syringes 758 with their respective sealing section 762 against the upper side of the microfluidic device 756 in the region of the inlet ends of the microfluidic wells 702. Thereafter, the pistons 778 of the pipettes 758 and / or syringes 758 can be pushed in the direction of the sealing element 760 connected to the respective pipette 758 and / or syringe 758, thereby feeding gas via a plurality of gas flows into the inlet ends of the microfluidic wells 702. In this way, a filling pressure can be applied to the sample fluid within each of the microfluidic wells 702 as a result of which the sample fluid flows through at least a predominant part of a microfluidic circuit of the respective microfluidic well 702 and into at least a majority of a plurality of microchambers of the microfluidic circuit.

[0156] After the filling pressure has been applied to the sample fluid within the microfluidic wells 702, the robot unit 766 can move the microfluidic device 756 from the holding structure 722 onto the transport device 776 of the layer application unit 774, for example by means of a not shown gripper device. The transport device 776 can then move the microfluidic device 756 into the layer application unit 774, where a protective layer, for example in the form of a gas-impermeable protective layer, can be applied to the upper side of the microfluidic device 756 and thus to the microfluidic wells 702 in order to seal the inlet ends and outlet ends of the microfluidic wells 702 by the layer application unit 774.

[0157] The control unit 736 is configured to control the introduction of the fluid sample into the inlet ends of the microfluidic wells 702, the application of the filling pressure to the sample fluid within the microfluidic wells 702 and the application of the protective layer to the microfluidic wells 702 by controlling the robot unit 766 and the layer application unit 774.

[0158] After the protective layer has been applied to the microfluidic device 756, the microfluidic device 756 can be transferred, for example manually, from the robotic liquid handling apparatus 700 into an analysis apparatus, for example similar to one of them shown in Figs.

[0159] 4-6, for further handling of the microfluidic device 756, in particular for fluidic separation of the microchambers of the microfluidic wells 702 and thermal treatment of the fluid sample.

[0160] Fig. 8A shows a detail of the robotic liquid handling apparatus 700 and the microfluidic device 756 shown in Fig. 7 in the region of the free end of one of the pipettes 758 and / or syringes 758 of the robotic liquid handling apparatus 700 and of one of the microfluidic wells 702 of the microfluidic device 756.

[0161] At its free end, the pipette 758 and / or syringe 758 comprises a common tip adapter 880 not shown in detail. Such tip adapters are known in the prior art. To the tip adapter 880, a common pipette tip 882 is detachably connected. Such pipette tips are also known in the prior art.By means of the pipette 758 and / or syringe 758 and the connected pipette tip 882, the sample fluid 884 taken up with the pipette tip 882 is pipetted into the inlet end 808 of the microfluidic well 702 via an inlet opening 886 of the inlet end 808. The piston 778 of the pipette 758 and / or syringe 758 is pushed in the direction of the connected pipette tip 882, thereby pushing the gas present between the piston 778 and the sample fluid 884 within the pipette tip 882 in the direction of the free end of the pipette tip 882 and thus the sample fluid 884 out of the pipette tip 882 into the inlet end 808 of the microfluidic well 702. At least partially simultaneously, sample fluid 884 of the fluid sample is also introduced into the other microfluidic wells 702 of the microfluidic device 756 by means of the other pipettes 758 and / or syringes 758 and connected pipette tips 882.

[0162] Fig. 8B shows a detail of the robotic liquid handling apparatus 700 and the microfluidic device 756 shown in Fig. 7 similar to the detail shown in Fig. 8A.

[0163] Instead of the pipette tip 882, one of the sealing elements 760 is connected to the tip adapter 880 of the pipette 758 and / or syringe 758 with its connecting section 764. The pipette 758 and / or syringe 758 and the sealing element 760 together form a gas guiding channel 834 for guiding a gas flow 828 along a flow path 888 into the inlet end 808 of the microfluidic well 702. The section of the gas guiding channel 834 formed by the pipette 758 and / or syringe 758 and the section of the gas guiding channel 834 formed by the sealing element 760 are tightly connected by the tip adapter 880 of the pipette 758 and / or syringe 758 and the connecting section 764 of the sealing element 760. The sealing section 762 of the sealing element 760 extends circumferentially around the gas guiding channel 834, namely around an outlet opening 890 of the gas guiding channel 834.

[0164] The robot unit 766 (not shown in Fig. 8B) presses the sealing element 760 with its soft and / or elastic sealing section 762 against the upper side of the microfluidic device 756 such that the sealing section 762 contacts the microfluidic device 756 circumferentially around the inlet opening 886 of the inlet end 808 and the sealing section 762 is slightly compressed. In this way, the flow path 888 is sealed in the contact area where the sealing element 760 contacts the microfluidic device 756.

[0165] Simultaneously, the robot unit 766 (not shown in Fig. 8B) actuates the pipette 758 and / or syringe 758 and pushes the piston 778 in the direction of the sealing element 760, thereby feeding the gas in the gas guiding channel 834 via a gas flow 828 partially into the inlet end 808 of the microfluidic well 702. In this way, the gas pressure in the inlet end 808 is increased and thus a filling pressure is applied to the sample fluid 884 within the inlet end 808 as a result of which the sample fluid 884 flows through at least substantially the entire microfluidic circuit 804 of the microfluidic well 702 and into at least substantially all of the microchambers 806 of the microfluidic circuit 804. If some of the microchambers 806 are not filled with sample fluid884 as a result of the application of the filling pressure, these microchambers 806 can be filled during a subsequent fluidic separation of the microchambers 806 in which the sealing layer 820 of the microfluidic device 756 is deformed into the channel segments of the microfluidic channels 810 (only one of which is shown in Fig. 8B) between the microchambers 806 (cf. explanations on Figs. 3A-D).

[0166] During the application of the filling pressure to the fluid sample 888, a gas pressure sensor 892 in the pipette 758 and / or syringe 758 detects gas pressure information characteristic of a gas pressure inside the gas guiding channel 834 in the pipette 758 and / or syringe 758 and transmits the detected gas pressure information to the control unit 736 (not shown in Fig. 8B). Based on the detected gas pressure information, the control unit 736 controls the movement of the piston 778 of the pipette 758 and / or syringe 758 and thus the flow rate of the gas flow 828 into the inlet end 808 of the microfluidic well 702.

[0167] At least partially simultaneously to the application of the filling pressure to the sample fluid 884 within the shown microfluidic well 702, a filling pressure is also applied to the sample fluid 884 within the other microfluidic wells 702 of the microfluidic device 756 by means of the other pipettes 758 and / or syringes 758 and connected sealing elements 760. In the course of this, the control unit 736 controls the plurality of gas flows 828 provided to the different microfluidic wells 702 independently from each other based on the gas pressure information detected by the respective gas pressure sensor 892 in the respective pipette 758 and / or syringe 758.

[0168] In an alternative or supplemental embodiment not shown, gas could also be sucked via a gas flow out of the outlet end 812 of the microfluidic well 702 by means of a pipette 758 and / or syringe 758 and a sealing element 760 adapted to the shape of the outlet end 812 in order to reduce the gas pressure in the outlet end 812 and apply in this way a filling pressure to the sample fluid 884 within the inlet end 808 as a result of which the sample fluid 884 flows through the microfluidic circuit 804 and into the microchambers 806.

[0169] Fig. 8C shows a detail of the microfluidic device 756 shown in Fig. 7 similar to the detail shown in Figs. 8A-B.

[0170] In Fig. 8C, at least substantially the entire sample fluid 884 has been pushed from the inlet end 808 into the microfluidic circuit 804 and the microchambers 806 of the microfluidic circuit 804. Some microchambers 806 close to the outlet end 812 are still free from sample fluid 884 and can be filled with sample fluid 884 during a subsequent fluidic separation of the microchambers 806 in which the sealing layer 820 is deformed into the microfluidic circuit 804. In addition, a, for example gas-impermeable, protective layer 814 has been applied to the upper side of the microfluidic device 756 which seals the inlet ends 808 and the outlet ends 812 of the microfluidic wells 702.Fig. 9 shows a flow diagram of an embodiment of a method 900 according to a third aspect of the invention.

[0171] The method 900 is a method for handling a microfluidic device, preferably for providing a molecular analysis of a fluid sample, in particular for providing a digital polymerase chain reaction analysis of a fluid sample.

[0172] A, for example first, step 910 of the method 900 is a step of introducing a fluid sample into the microfluidic device and at least one microfluidic well of the microfluidic device via a at least one inlet end of the at least one microfluidic well.

[0173] A further, for example second, step 920 of the method 900 is a step of applying a filling pressure to the sample fluid within the at least one microfluidic well by feeding gas via at least one gas flow into and / or sucking gas via at least one gas flow out of the microfluidic device and the at least one microfluidic well. As a result of the applied filling pressure, the sample fluid within the at least one microfluidic well flows through at least a predominant part of a microfluidic circuit of the microfluidic well and into at least the majority of a plurality of microchambers of the microfluidic circuit.

[0174] A further step 930 of the method 900 is a step of fluidly separating the plurality of microchambers of the at least one microfluidic circuit from each other, preferably by deforming a sealing layer into channel segments of at least one microfluidic channel fluidly coupling the microchambers.

[0175] A further step 940 of the method 900 is a step of applying a protective layer to the at least one microfluidic well for sealing the inlet end of the microfluidic well and / or an outlet end of the microfluidic well fluidly coupled to the inlet end. The step 940 of applying the protective layer to the at least one microfluidic well can for example be performed before the step 910 of introducing the fluid sample into the at least one microfluidic well. Then, the fluid sample can be introduced into the at least one microfluidic well through the protective layer, for example by means of one or more injection needles. In addition, the protective layer can then be at least partially permeable to gas such that the filling pressure can be applied to the sample fluid within the at least one microfluidic well by feeding and / or sucking the gas through the protective layer into and / or out of the at least one microfluidic well. As an alternative, the step 940 of applying the protective layer to the at least one microfluidic well can for example be performed after the step 910 of introducing the fluid sample into the at least one microfluidic well and before the step 920 of applying the filling pressure to the sample fluid within the at least one microfluidic well. Then, the fluid sample is not introduced into the at least one microfluidic well through the protective layer, but the filling pressure can be applied to the sample fluid within the at least one microfluidic well by feeding and / or sucking the gas through the protective layer into and / or out of the at least one microfluidic well if the protective layer is permeable to gas. According toa further alternative, the step 940 of applying the protective layer to the at least one microfluidic well can be performed after the step 920 of applying the filling pressure to the sample fluid within the at least one microfluidic well. Then, also the filling pressure can be applied to the sample fluid within the at least one microfluidic well without feeding and / or sucking the gas through the protective layer.

[0176] In a variant of the method 900, the steps 910 and 940 of introducing the fluid sample into the at least one microfluidic well and applying the protective layer to the at least one microfluidic well are performed manually by an operator. Thereafter, the microfluidic device is be inserted by the operator into an analysis apparatus, for example into an analysis apparatus as that shown in Fig. 4, 5 or 6. The steps 920 and 930 of applying the filling pressure to the sample fluid within the at least one microfluidic well and separating the plurality of microchambers of the at least one microfluidic circuit from each other are then performed autonomously by the analysis apparatus.

[0177] In an alternative variant of the method 900, the steps 910, 920 and 940 of introducing the fluid sample into the at least one microfluidic well, applying the filling pressure to the sample fluid within the at least one microfluidic well and applying the protective layer to the at least one microfluidic well are performed autonomously by a robotic liquid handling apparatus, for example a robotic liquid handling apparatus as that shown in Figs. 7 and 8A-B. Thereafter, the microfluidic device is transferred, for example manually by an operator, to an analysis apparatus, for example an analysis apparatus similar to that shown in Fig. 4, 5 or 6. The step 930 of fluidly separating the plurality of microchambers of the at least one microfluidic circuit is then performed autonomously by the analysis apparatus.

[0178] Usually, after the step 930, a molecular analysis of the fluid sample is provided by further steps of the method 900.

[0179] In a preferred variant of the method 900, the following two steps are provided after and / or during step 920 of applying the filling pressure to the sample fluid within the at least one microfluidic well:

[0180] - analyzing a present filling state of a plurality of the plurality of microchambers of the at least one microfluidic well; and

[0181] - determining the filling pressure to be applied to the sample fluid within the at least one microfluidic well based on the present filling state of the plurality of microchambers of the microfluidic well.

[0182] In this embodiment, a detailed control of the filling of the microchambers by means of the at least one gas flow is possible.

[0183] In an alternative or supplemental variant of the method 900, the following two steps are provided during step 920 of applying the filling pressure to the sample fluid within the at least one microfluidic well:- detecting gas pressure information characteristic of a gas pressure in at least one gas guiding channel guiding the at least one gas flow into and / or out of the at least one microfluidic well; and

[0184] - controlling the at least one gas flow based on the detected gas pressure information.

[0185] In this way, the filling pressure applied to the fluid sample and thus the filling of the microchambers can be controlled very precisely in a simple manner.

[0186] In a further variant of the method 900, the following step is provided during step 920 of applying the filling pressure to the sample fluid within the at least one microfluidic well:

[0187] - pressing at least one sealing element against the microfluidic device.

[0188] This can contribute to reliable and accurate application of the filling pressure to the sample fluid.

[0189] In a further variant of the method 900, the following step is provided after step 920 of applying the filling pressure to the sample fluid within the at least one microfluidic well:

[0190] - applying, preferably glueing, a further protective layer to the at least partially gas-permeable protective layer, wherein the further protective layer is at least substantially impermeable to gas.

[0191] In this way, leakage of aerosols can be avoided during a subsequent thermal treatment.

[0192] In a further variant of the method 900, the following step is provided after step 910 of introducing the fluid sample into the at least one microfluidic well and prior to step 940 of applying the protective layer to the at least one microfluidic well:

[0193] - applying a water-immiscible layer, such as a mineral oil layer, to the sample fluid within the at least one microfluidic well.

[0194] Using such a water-immiscible layer, such as a mineral oil layer, may help to prevent a contamination of the fluid sample. Suitable water-immiscible liquids are described elsewhere herein and it is referred to the corresponding disclosure. In addition, a water-immiscible layer, for example an oil layer, can also avoid leakage of aerosols of the sample fluid. Therefore, it is not necessary to apply a further protective layer, which is impermeable to gas, to the gas-permeable protective layer if a water-immiscible layer is applied to the sample fluid.

[0195] In an advantageous variant of the method 900, a duration of the application of the filling pressure to the sample fluid within the at least one microfluidic well, a pressure level of the filling pressure applied to the sample fluid within the at least one microfluidic well, and / or a pressure profile with which the filling pressure is applied to the sample fluid within the at least one microfluidic well depends on an amount of sample fluid that is usually displaced by the fluidic separation of the plurality of microchambers of a microfluidic well of the microfluidic device. If a large amount of sample fluid is displaced by the fluidic separation, the last microchambers might not be filled by the filling pressure applied to the sample fluid, but as aresult of the fluidic separation of the microchambers. Thereby, the method leads to a more efficient filling of the microchambers.

[0196] If there is a number of finally not filled microchambers of the microfluidic device, this number might be detected. Likewise, if there is a number of microchambers of the microfluidic device that are not filled by the application of the filling pressure to the sample fluid within the at least one microfluidic well, but in another way, for example as a consequence of the fluidic separation of the plurality of microchambers of the at least one microfluidic well, this number can be detected. Alternatively or additionally, one or more degrees of filling and / or filling speeds of one or more microchambers of the microfluidic device can be detected. A subsequent molecular analysis can then preferably be provided as a function of the detected number of not filled microchambers, the detected number of microchambers filled other than by the application of the filling pressure to the sample fluid, the one or more detected degrees of filling and / or the one or more detected filling speeds. By taking one or more of detected values into account, final statistics to interpret the result of the thermal treatment of the fluid sample can become more precise. Preferably, the number of not filled microchambers, the number of microchambers filled other than by the application of filling pressure to the sample fluid, the at least on degree of filling and / or the at least one filling speed can be detected by the optical means of the handling apparatus.

[0197] The following numbered items further describe the present invention, solve its object, and form part of the disclosure of the present document.

[0198] Item 1 : A microfluidic device for handling a fluid sample by dividing it into a plurality of aliquots, 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, and 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 one or more further microchambers fluidly coupled with the inlet end and the outlet end such that each microchamber is configured to receive sample fluid from the inlet end via the at least one microfluidic channel and to provide gas displaced by the sample fluid downstream to the outlet end, wherein a protective layer is applied to the at least one microfluidic well for sealing the inlet end and / or the outlet end of the microfluidic well, and wherein the protective layer is at least partially permeable to gas.

[0199] Item 2: The microfluidic device of item 1, wherein the protective layer comprises at least one inlet valve for enabling at least one gas flow through the protective layer into the at least oneinlet end and / or at least one outlet valve for enabling at least one gas flow through the protective layer out of the at least one outlet end.

[0200] Item 3: The microfluidic device of item 1 or 2, wherein the microfluidic device comprises a sealing layer at least partially covering the at least one microfluidic circuit and configured to fluidly separate the microchambers of the microfluidic circuit 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.

[0201] Item 4: A handling apparatus for handling a microfluidic device, preferably of one of items 1 to 3, comprising a pressurization unit configured to apply a filling pressure to sample fluid within at least one microfluidic well of the microfluidic device such that the sample fluid flows through at least a part of a microfluidic circuit of the microfluidic well and into at least some of a plurality of microchambers of the microfluidic circuit as a result of the applied filling pressure, and an electronic control unit configured to control the application of the filling pressure to the sample fluid within the at least one microfluidic well, wherein the pressurization unit is configured to apply the filling pressure to the sample fluid within the at least one microfluidic well by feeding gas via at least one gas flow into and / or sucking gas via at least one gas flow out of the microfluidic device and the at least one microfluidic well.

[0202] Item 5: The handling apparatus of item 4, wherein the pressurization unit comprises at least one compressor unit for generating the at least one gas flow and / or wherein the pressurization unit, in particular the compressor unit, is fluidly coupled to a gas reservoir for providing the gas to be fed into the at least one microfluidic well.

[0203] Item 6: The handling apparatus of item 4, wherein the pressurization unit comprises at least one, preferably piston-driven, in particular air displacement, pipette and / or syringe for generating the at least one gas flow.

[0204] Item 7: The handling apparatus of one of items 4 to 6, wherein the pressurization unit comprises at least one gas guiding channel for guiding the at least one gas flow into and / or out of the at least one microfluidic well, preferably a plurality of gas guiding channels for guiding a plurality of gas flows at least partially simultaneously into and / or out of different microfluidic wells of the microfluidic device, and wherein, further preferably, the control unit is configured to control the plurality of gas flows independently of each other.

[0205] Item 8: The handling apparatus of one of items 4 to 7, wherein the pressurization unit comprises at least one sealing element for contacting the microfluidic device and sealing at least one flow path of the at least one gas flow in the contact area, and wherein, preferably, the at least one sealing element is made of a soft and / or elastic sealing material at least in a sealing section for contacting the microfluidic device and / or the at least one sealing element isdetachably connected to the at least one pipette and / or syringe, preferably to at least one free end of the at least one pipette and / or syringe.

[0206] Item 9: The handling apparatus of one of items 4 to 8, wherein the handling apparatus comprises at least one gas pressure sensor configured to detect gas pressure information characteristic of a gas pressure, preferably in the at least one gas guiding channel, in particular in the at least one pipette and / or syringe, and wherein, preferably, the control unit is configured to control the at least one gas flow provided by the pressurization unit based on the detected gas pressure information.

[0207] Item 10: The handling apparatus of one of items 4 to 9, wherein the handling apparatus is configured to analyze a surface of the microfluidic device with optical means, wherein, preferably, the control unit is configured to control the at least one gas flow provided by the pressurization unit based on an analysis of a present filling state of the at least one microfluidic well, wherein the analysis of the present filling state is facilitated by the optical means, and wherein, further preferably, the control unit is configured to control the at least one gas flow provided by the pressurization unit based on a current filling edge of the sample fluid within the at least one microfluidic circuit.

[0208] Item 11: The handling apparatus of one of items 4 to 10, wherein the handling apparatus comprises a layer application unit configured to apply a protective layer to the at least one microfluidic well for sealing at least one inlet end and / or at least one outlet end of the at least one microfluidic well, and wherein, preferably, the control unit is configured to control the application of the protective layer to the at least one microfluidic well.

[0209] Item 12: A method for handling a microfluidic device, preferably of one of items 1 to 3, preferably performed at least partially by a handling apparatus of one of items 4 to 11, comprising the steps introducing a fluid sample into the microfluidic device and at least one microfluidic well of the microfluidic device via at least one inlet end of the at least one microfluidic well; and applying a filling pressure to the sample fluid within the at least one microfluidic well by means of a pressurization unit such that the sample fluid flows through at least a part of a microfluidic circuit of the microfluidic well and into at least some of a plurality of microchambers of the microfluidic circuit as a result of the applied filling pressure, wherein the filling pressure is applied to the sample fluid within the at least one microfluidic well by feeding gas via at least one gas flow into and / or sucking gas via at least one gas flow out of the microfluidic device and the at least one microfluidic well.

[0210] Item 13: The method of item 12, wherein the at least one gas flow is generated by means of at least one, preferably piston-driven, in particular air displacement, pipette and / or syringe of the pressurization unit and wherein, preferably, the fluid sample is introduced into the at least one microfluidic well by means of the at least one pipette and / or syringe.Item 14: The method of item 12 or 13, further comprising the step pressing at least one sealing element of the pressurization unit, preferably at least one soft and / or elastic sealing section of the at least one sealing element, against the microfluidic device during the application of the filling pressure to the sample fluid within the at least one microfluidic well, wherein, preferably, the at least one sealing element, in particular the at least one sealing section of the at least one sealing element, pressed against the microfluidic device contacts the microfluidic device circumferentially around at least one opening of the at least one microfluidic well.

[0211] Item 15: The method of one of items 12 to 14, further comprising the steps detecting gas pressure information characteristic of a gas pressure, preferably in at least one gas guiding channel of the pressurization unit guiding the at least one gas flow into and / or out of the at least one microfluidic well; and controlling the at least one gas flow based on the detected gas pressure information.

[0212] Item 16: The method of one of items 12 to 15, further comprising the steps analyzing a present filling state of the plurality of microchambers of the at least one microfluidic well; and determining the filling pressure to be applied to the sample fluid within the at least one microfluidic well based on the present filling state of the plurality of microchambers of the microfluidic well.

[0213] Item 17: The method of one of items 12 to 16, further comprising the step applying a protective layer to the at least one microfluidic well for sealing the inlet end of the microfluidic well and / or an outlet end of the microfluidic well fluidly coupled to the inlet end, wherein, for example, the protective layer is at least partially permeable to gas and the filling pressure is applied to the sample fluid within the at least one microfluidic well by feeding the gas through the protective layer into and / or sucking the gas through the protective layer out of the microfluidic well.

[0214] Item 18: The method of at least one of items 12 to 17, further comprising the step fluidly separating the plurality of microchambers of the at least one microfluidic circuit from each other after applying the filling pressure to the sample fluid within the microfluidic well, preferably by deforming a sealing layer into channel segments of at least one microfluidic channel fluidly coupling the plurality of microchambers, in particular by applying a compressive force to the sealing layer.

[0215] Item 19: The method of item 18, wherein a duration of the application of the filling pressure to the sample fluid within the at least one microfluidic well, a pressure level of the filling pressure applied to the sample fluid within the at least one microfluidic well, and / or a pressure profile with which the filling pressure is applied to the sample fluid within the at least one microfluidic well depends on an amount of sample fluid that is usually displaced by the fluidic separation of the plurality of microchambers of a microfluidic well of the microfluidic device.Item 20: The method of at least one of items 12 to 19, wherein a number of finally not filled microchambers of the microfluidic device, a number of microchambers of the microfluidic device filled other than by the application of the filling pressure to the sample fluid within the at least one microfluidic well, in particular by the fluidic separation of the plurality of microchambers of the at least one microfluidic well, and / or a degree of filling, a filling speed and / or a filling duration of at least one microchamber of the microfluidic device is detected and a subsequent molecular analysis is provided as a function of the detected number of not filled microchambers, the detected number of microchambers filled other than by the application of the filling pressure to the sample fluid , the at least one detected degree of filling, the at least one detected filling speed, and / or the at least one detected filling duration.

[0216] LIST OF REFERENCE SIGNS

[0217] 100, 200 microfluidic device

[0218] 102, 702 microfluidic well

[0219] 104, 804 microfluidic circuit

[0220] 106, 806 microchamber

[0221] 108, 808 inlet end

[0222] 110, 110’, 110”, 110’”, 810 microfluidic channel

[0223] 112, 812 outlet end

[0224] 114, 214, 814 protective layer

[0225] 216 inlet valve

[0226] 218 outlet valve

[0227] 320, 820 sealing layer

[0228] 400, 500, 500’, 700 handling apparatus

[0229] 422, 722 holding structure

[0230] 424 gas reservoir

[0231] 426, 526, 726 pressurization unit

[0232] 428, 528, 828 gas flow

[0233] 430 compressor unit

[0234] 432, 532 gas guiding structure

[0235] 434, 534, 834 gas guiding channel

[0236] 436, 536, 736 control unit

[0237] 538 gas guiding channel

[0238] 540 gas flow

[0239] 542, 542’ optical means

[0240] 544 roll

[0241] 546 pressuring rail

[0242] 548 filling state450, 550, 550’, 750 system

[0243] 552 filling edge

[0244] 554 empty region

[0245] 756 microfluidic device 758 pipette and / or syringe 760 sealing element 762 sealing section

[0246] 764 connecting section 766 robot unit

[0247] 768 workdeck

[0248] 770 storage unit

[0249] 772 receptacle

[0250] 774 layer application unit 776 transport device 778 piston

[0251] 880 tip adapter

[0252] 882 pipette tip

[0253] 884 sample fluid

[0254] 886 inlet opening

[0255] 888 flow path

[0256] 890 outlet opening

[0257] 892 gas pressure sensor 900 method

[0258] 910, 920, 930, 940 steps of the method

[0259] F compressive force

Claims

-43 -CLAIMS1. A microfluidic device (100,200) for handling a fluid sample by dividing it into a plurality of aliquots, with at least one microfluidic well (102) configured to receive the fluid sample, the at least one microfluidic well (102) comprising- a microfluidic circuit (104) with a plurality of microchambers (106), which provide a respective reaction space for an aliquot of the plurality of aliquots,- an inlet end (108) coupled to the microfluidic circuit (104) via at least one microfluidic channel (110, 110’, 110”, 110”’),- an outlet end (112) coupled to the microfluidic circuit (104) via the at least one microfluidic channel (110, 110’, 110”, 110’”),wherein the plurality of microchambers (106) is arranged such that each microchamber (106) is at least indirectly via one or more further microchambers (106) fluidly coupled with the inlet end (108) and the outlet end (112) such that each microchamber (106) is configured to receive sample fluid from the inlet end (108) via the at least one microfluidic channel (110,110’, 110”, 110’”) and to provide gas displaced by the sample fluid downstream to the outlet end (112),wherein a protective layer (114,214) is applied to the at least one microfluidic well (102) for sealing the inlet end (108) of the microfluidic well (102), andwherein the protective layer (114,214) is at least partially permeable to gas.

2. The microfluidic device (200) of claim 1, wherein the protective layer (114, 214) is applied to the at least one microfluidic well (102) for sealing also the outlet end (112) of the microfluidic well (102).

3. The microfluidic device (200) of claim 1 or 2, wherein the protective layer (214) comprises at least one inlet valve (216) for enabling at least one gas flow (428,528) through the protective layer (214) into the at least one inlet end (108) and / or at least one outlet valve (218) for enabling at least one gas flow (428,528) through the protective layer (214) out of the at least one outlet end (112) .

4. The microfluidic device (100,200) of any of claims 1 to 3, wherein the microfluidic device (100,200) comprises a sealing layer (320) at least partially covering the at least one microfluidic circuit (104) and configured to fluidly separate the microchambers (106) of the microfluidic circuit (104) from each other, preferably by deformation into channel segments of the at least one microfluidic channel (110, 110’, 110”, 110’”) fluidly coupling the microchambers (106), in particular upon application of a compressive force (F) to the sealing layer (320).- 44 -5. A handling apparatus (400, 500, 500’, 700) for handling a microfluidic device (100,200,756) of one of claims 1 to 4, comprising- a pressurization unit (426,526,726) configured to apply a filling pressure to sample fluid (884) within at least one microfluidic well (102,702) of the microfluidic device (100,200,756) such that the sample fluid (884) flows through at least a part of a microfluidic circuit (104,804) of the microfluidic well (102,702) and into at least some of a plurality of microchambers (106,806) of the microfluidic circuit (104,804) as a result of the applied filling pressure, and- an electronic control unit (436,536,736) configured to control the application of the filling pressure to the sample fluid (884) within the at least one microfluidic well (102,702),wherein the pressurization unit (426,526,726) is configured to apply the filling pressure to the sample fluid (884) within the at least one microfluidic well (102,702) by feeding gas via at least one gas flow (428,528,540,828) into and / or sucking gas via at least one gas flow out of the microfluidic device (100,200,756) and the at least one microfluidic well (102,702).

6. The handling apparatus (400,500,500’) of claim 5, wherein the pressurization unit (426,526) comprises at least one compressor unit (430) for generating the at least one gas flow (428,528,540) and / or wherein the pressurization unit (426,526), in particular the compressor unit (430), is fluidly coupled to a gas reservoir (424) for providing the gas to be fed into the at least one microfluidic well (102,702).

7. The handling apparatus (700) of claim 5, wherein the pressurization unit (726) comprises at least one, preferably piston-driven, in particular air displacement, pipette (758) and / or syringe (758) for generating the at least one gas flow (828).

8. The handling apparatus (400, 500, 500’, 700) of one of claims 5 to 7, wherein the pressurization unit (426,526,726) comprises at least one gas guiding channel (434,534,538,834) for guiding the at least one gas flow (428,528,540,828) into and / or out of the at least one microfluidic well (102,702), preferably a plurality of gas guiding channels (534,538,834) for guiding a plurality of gas flows (528, 828) at least partially simultaneously into and / or out of different microfluidic wells (102,702) of the microfluidic device (100,200,756), and wherein, further preferably, the control unit (536,736) is configured to control the plurality of gas flows (528, 828) independently of each other.

9. The handling apparatus (400, 500, 500’, 700) of one of claims 5 to 8, wherein the pressurization unit (426,526,726) comprises at least one sealing element (760) for contacting the microfluidic device (100,200,756) and sealing at least one flow path (888) of the at least one gas flow (428,528,540,828) in the contact area, and wherein, preferably, the at least one sealing element (760) is made of a soft and / or elastic sealing-45 -material at least in a sealing section (762) for contacting the microfluidic device (100,200,756) and / or the at least one sealing element (760) is detachably connected to the at least one pipette (758) and / or syringe (758), preferably to at least one free end of the at least one pipette (758) and / or syringe (758).

10. The handling apparatus (400, 500, 500’, 700) of one of claims 5 to 9, wherein the handling apparatus (400, 500, 500’, 700) comprises at least one gas pressure sensor (892) configured to detect gas pressure information characteristic of a gas pressure, preferably in the at least one gas guiding channel (434,534,538,834), in particular in the at least one pipette (758) and / or syringe (758), and wherein, preferably, the control unit (436,536,736) is configured to control the at least one gas flow (428,528,540,828) provided by the pressurization unit (426,526,726) based on the detected gas pressure information.

11. The handling apparatus (400, 500, 500’, 700) of one of claims 5 to 10, wherein the handling apparatus (400, 500, 500’, 700) is configured to analyze a surface of the microfluidic device (100,200,756) with optical means (542,542’), wherein, preferably, the control unit (436,536,736) is configured to control the at least one gas flow (428,528,540,828) provided by the pressurization unit (426,526,726) based on an analysis of a present filling state (548) of the at least one microfluidic well (102,702), wherein the analysis of the present filling state (548) is facilitated by the optical means (542,542’), and wherein, further preferably, the control unit (436,536,736) is configured to control the at least one gas flow (428,528,540,828) provided by the pressurization unit (426,526,726) based on a current filling edge (552) of the sample fluid (884) within the at least one microfluidic circuit (104,804).

12. The handling apparatus (400, 500, 500’, 700) of one of claims 5 to 11, wherein the handling apparatus (400, 500, 500’, 700) comprises a layer application unit (774) configured to apply a protective layer (114,214,814) to the at least one microfluidic well (102,702) for sealing at least one inlet end (108,808) and / or at least one outlet end (112,812) of the at least one microfluidic well (102,702), and wherein, preferably, the control unit (436,536,736) is configured to control the application of the protective layer (114,214,814) to the at least one microfluidic well (102,702).

13. A method (900) for handling a microfluidic device (100,200,756) of one of claims 1 to 4, preferably performed at least partially by a handling apparatus (400, 500, 500’, 700) of one of claims 5 to 12, comprising the steps- introducing a fluid sample into the microfluidic device (100,200,756) and at least one microfluidic well (102,702) of the microfluidic device (100,200,756) via at least one inlet end (108,808) of the at least one microfluidic well (102,702); and- applying a filling pressure to the sample fluid (884) within the at least one microfluidic well (102,702) by means of a pressurization unit (426,526,726) such that the sample fluid (884) flows through at least a part of a microfluidic circuit (104,804) of the microfluidic well (102,702) and into at least some of a plurality of microchambers (106,806) of the microfluidic circuit (104,804) as a result of the applied filling pressure, wherein the filling pressure is applied to the sample fluid (884) within the at least one microfluidic well (102,702) by feeding gas via at least one gas flow (428,528,540,828) into and / or sucking gas via at least one gas flow out of the microfluidic device (100,200,756) and the at least one microfluidic well (102,702).

14. The method (900) of claim 13, wherein the at least one gas flow (828) is generated by means of at least one, preferably piston-driven, in particular air displacement, pipette (758) and / or syringe (758) of the pressurization unit (726) and wherein, preferably, the fluid sample is introduced into the at least one microfluidic well (102,702) by means of the at least one pipette (758) and / or syringe (758).

15. The method (900) of claim 13 or 14, further comprising the step- pressing at least one sealing element (760) of the pressurization unit (426,526,726), preferably at least one soft and / or elastic sealing section (762) of the at least one sealing element (760), against the microfluidic device (100,200,756) during the application of the filling pressure to the sample fluid (884) within the at least one microfluidic well (102,702), wherein, preferably, the at least one sealing element (760), in particular the at least one sealing section (762) of the at least one sealing element (760), pressed against the microfluidic device (100,200,756) contacts the microfluidic device (100,200,756) circumferentially around at least one opening (886) of the at least one microfluidic well (102,702).

16. The method (900) of one of claims 13 to 15, further comprising the steps- detecting gas pressure information characteristic of a gas pressure, preferably in at least one gas guiding channel (434,534,538,834) of the pressurization unit (426,526,726) guiding the at least one gas flow (428,528,540,828) into and / or out of the at least one microfluidic well (102,702); and- controlling the at least one gas flow (428,528,540,828) based on the detected gas pressure information.

17. The method (900) of one of claims 13 to 16, further comprising the steps- analyzing a present filling state (548) of the plurality of microchambers (106,806) of the at least one microfluidic well (102,702); and- determining the filling pressure to be applied to the sample fluid (884) within the at least one microfluidic well (102,702) based on the present filling state (548) of the plurality of microchambers (106,806) of the microfluidic well (102,702).

18. The method (900) of one of claims 13 to 17, further comprising the step - applying a protective layer (114,214,814) to the at least one microfluidic well (102,702) for sealing the inlet end (108,808) of the microfluidic well (102,702) and / or an outlet end (112,812) of the microfluidic well (102,702) fluidly coupled to the inlet end (108,808), wherein, for example, the protective layer (114,214,814) is at least partially permeable to gas and the filling pressure is applied to the sample fluid (884) within the at least one microfluidic well (102,702) by feeding the gas through the protective layer (114,214,814) into and / or sucking the gas through the protective layer (114,214,814) out of the microfluidic well (102,702).

19. The method (900) of at least one of claims 13 to 18, further comprising the step- fluidly separating the plurality of microchambers (106,806) of the at least one microfluidic circuit (104,804) from each other after applying the filling pressure to the sample fluid (884) within the microfluidic well (102,702), preferably by deforming a sealing layer (320,820) into channel segments of at least one microfluidic channel (110, 110’, 110”, 110’”, 810) fluidly coupling the plurality of microchambers (106,806), in particular by applying a compressive force (F) to the sealing layer (320,820).

20. The method (900) of claim 19, wherein a duration of the application of the filling pressure to the sample fluid (884) within the at least one microfluidic well (102,702), a pressure level of the filling pressure applied to the sample fluid (884) within the at least one microfluidic well (102,702), and / or a pressure profile with which the filling pressure is applied to the sample fluid (884) within the at least one microfluidic well (102,702) depends on an amount of sample fluid (884) that is usually displaced by the fluidic separation of the plurality of microchambers (106,806) of a microfluidic well (102,702) of the microfluidic device (100,200,756).

21. The method (900) of at least one of claims 13 to 20, wherein a number of finally not filled microchambers (106,806) of the microfluidic device (100,200,756), a number of microchambers (106,806) of the microfluidic device (100,200,756) filled other than by the application of the filling pressure to the sample fluid (884) within the at least one microfluidic well (102,702), in particular by the fluidic separation of the plurality of microchambers (106,806) of the at least one microfluidic well (102,702), and / or a degree of filling, a filling speed and / or a filling duration of at least one microchamber (106,806) of the microfluidic device (100,200,756) is detected and a subsequent molecular analysis is provided as a function of the detected number of not filled microchambers (106,806), the detected number of microchambers (106,806) filled other than by the application of the filling pressure to the sample fluid (884) , the at least one detected degree of filling, the at least one detected filling speed, and / or the at least one detected filling duration.

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