Fluidic module, and method for creating spatially separated liquid partitions

Abstract

The invention relates to a fluidic module and to a method for creating spatially separated liquid partitions in an array of depressions formed in a surface. The method comprises providing the fluidic module and rotating the fluidic module. The fluidic module has a fluid chamber which comprises the surface in which the array of depressions is formed. The fluidic module is rotated about a centre of rotation in order to apply a centrifugal force to a liquid, as a result of which the liquid is introduced into the fluid chamber, and as a result of which at least one portion of the liquid is transported from radially outward to radially inward over the array of depressions. The method also comprises removing the liquid from areas of the surface outside the depressions in order to create the spatially separated liquid partitions of the liquid in the depressions.

Description

[0001] Fluidics module and method for generating spatially separated fluid partitions

[0002] Description

[0003] The present invention relates to fluidic modules and methods for generating spatially separated fluid partitions that enable controlled transport of fluid across an array of wells against centrifugal force. In particular, the invention relates to such devices and methods suitable for handling fluids in a centrifugal microfluidic system.

[0004] Digital assays, such as digital PCR (dPCR), are essential technologies for the absolute and reference-free quantification of nucleic acids or other molecules or particles in unknown samples [Witters_2014]. Another typical application for digital reactions is the labeling (barcoding) of individual analytes such as nucleic acids or cells in order to be able to infer the initial analytes in a later process step [Kivioja _2012]. The implementation of a digital assay typically requires the partitioning of a reaction fluid into a large number of sub-volumes. Depending on the application, the number of partitions (typically several 10,000 to several million partitions) and the volume of an individual partition (typically in the picoliter to nanoliter range) vary significantly. A combination of partitions of different sizes can also be intended for the implementation of digital assays, e.g.to expand the dynamic range of an assay [Schulz_2021]. However, the same challenge applies to most digital assays: The precision and reproducibility of the generated partitions in terms of number, volume, and stability determine the performance of the digital assay. Another performance parameter is the lowest possible dead volume, i.e., the portion of the reaction fluid that is lost in the system and not available for analysis in the partitions. The dead volume results from the delta between the input volume of the reaction fluid and the analyzed volume.

[0005] ^Dead ^Input ^Analysis

[0006] A high dead volume has two negative effects:

[0007] • It reduces the proportion of sample that is analyzed as part of the reaction fluid. For rare analytes, such as certain mutations in the analysis of cell-free DNA, a high dead volume can result in the target analyte not being located in the analyzed partitions but in the unanalyzed dead volume, thus resulting in a false negative or at least an insufficient signal being measured.

[0008] • In addition, a high dead volume means an unnecessarily large use of sometimes very expensive reagents, e.g. polymerases in a PCR mix or antibodies in an ELISA mix.

[0009] A widely used method is the generation of partitions using emulsions, i.e., droplet microfluidics [Teh_2008]. There are also already studies based on centrifugal microfluidics [Schuler_2015]. Several commercialized systems for digital assays are also based on droplet microfluidics [Bio-Rad_2023; Stilla_2023]. Droplet-based systems are often characterized by low dead volumes, but have several technical disadvantages:

[0010] • Instability of the droplets towards coalescence, e.g. under temperature application during a PCR thermocycling protocol.

[0011] • Instability of the droplets under mechanical stress (shear forces), e.g. due to gas bubbles that arise as a result of an increase in temperature and move through the droplet accumulation.

[0012] • Compatibility problems of, for example, proteins or enzymes with the detergents required to stabilize the drops.

[0013] The alternative to droplet-based systems are geometrically defined systems, typically referred to as well arrays, microwells, or nanowells [Liao_2017]. Analogous to droplet-based systems, several commercialized systems already exist [QIAGEN_2023; Quanterix_2023; ThermoFisher_2023_1; Fluidigm_2023; JN-MEDSYS_2023; Roche_2023; illumina_2023]. By partitioning using geometrically defined wells (so-called wells) introduced into the fluidics module, the previously mentioned problems of droplet-based systems can be avoided:

[0014] • The use of detergents to stabilize against coalescence is usually no longer required, since the walls of the wells already provide sufficient mechanical stability of the partition.

[0015] • Shear forces caused by temperature-induced gas bubble formation affect the compartments to a lesser extent, since gas bubbles either do not come into contact with the wells at all or only marginally when transported through the oil.

[0016] In addition to solving droplet-associated problems, the following advantages also arise from geometric partitioning using recesses in the fluidics module:

[0017] • The option to pre-store reagents, e.g. primers for a digital PCR

[0018] • The greatly simplified optical evaluation of the partitions due to the defined geometric arrangement, e.g. in the fluorescence evaluation at the end of a digital PCR

[0019] There are already various systems that use geometric partitioning using recesses. However, the existing systems suffer from at least one of the following disadvantages:

[0020] • For many of the available systems, the chips (usually disposable products) are manufactured using photolithographic processes in glass or silicon. This results in high costs per chip during production [lllumina_2023; ThermoFisher_2023_2; JN-MEDSYS_2023, Podbiel_2021, Henley_2020].

[0021] • The wells are often filled via a network of inlet channels. The inlet channels define, among other things, the dead volume of the system and are therefore typically kept small in cross-section. This, in turn, causes high flow resistance, which limits the filling rate. Another problem with channel networks with small cross-sections is the undesirable adsorption of biomolecules on the channel walls, so that they are no longer available for analysis in the partitions. Furthermore, the inlet channels limit the achievable integration density of partitions [QIAGEN_2023; ThermoFisher_2023_1; Fluidigm_2023; US8277759B2; US9487822B2, EP3357575B1]

[0022] • The filling of the wells is achieved through targeted use of capillary forces. This requires local hydrophilization and / or hydrophobization of the fluidic module, which incurs additional costs in chip manufacturing [US10967370B2] [Roche_2023],

[0023] • The only known system that both dispenses with expensive chip materials such as glass or silicon and eliminates inlet channels by applying a vacuum to fill the wells, is the Quanterix Simoa system [Quanterix_2023]. However, this system has a very high dead volume. This is caused by a large gap (height: 500 pm) between the wells (height: 3.25 pm, diameter: 4.25 pm, center-to-center distance: 8 pm) and the opposite chamber side. The dead volume exceeds the analyzed volume of a sample by a factor of more than 100 [2012_Kan].

[0024] WO 2021 / 211754 A2 discloses devices and methods for immobilizing objects relative to examination sites using pressure or negative pressure. Centrifugal force is used to remove fluid from a chamber.

[0025] WO 2012 / 103447 A1 describes systems, devices, and / or methods relating to the introduction of a plurality of beads into test sites, the sealing of test sites, and the imaging of test sites. The objects are introduced into the test sites, for example, using a magnetic field generator that creates relative movement between the magnetic objects and the test sites. After objects have been introduced into the test sites of a first chamber, the device is rotated by one position to introduce further objects into the test sites of another chamber.

[0026] Furthermore, a device exists [2010_Rissin] in which a short piece of PVC tubing is attached to an etched end of a fiber bundle to create a reservoir for a particle solution, which is pipetted into this reservoir. The fiber bundle is then centrifuged at 1300 g for 10 minutes to force the particles into the etched wells.

[0027] In view of this, there is a need for a concept that enables a better compromise between improving precision and reproducibility in spatial separation of liquids and reducing dead volume. Furthermore, despite high precision and reproducibility, rapid and efficient separation of liquids with minimal loss of material to be analyzed, such as particles or molecules, should be achieved. Furthermore, a reduction in costs is desirable.

[0028] This is achieved by the subject matter of the independent claims of the present application. Further embodiments of the invention are defined by the subject matter of the dependent claims of the present application.

[0029] Description of the invention

[0030] The present invention is based on the partitioning of liquids using geometrically defined recesses introduced into the fluidics module. In this regard, the inventors discovered that centrifugal force enables controlled filling of a fluid chamber and the recesses arranged therein. This is based on the finding that centrifugal force can be used to create a defined liquid front within the fluid chamber, thus controlling the spread of the liquid within the fluid chamber when filling the fluid chamber from radially outside to radially inside, counter to the centrifugal force.A further advantage of filling via centrifugal force is that capillary forces within the fluid chamber become negligible. This allows for a reduction in chamber height and thus in dead volume, and also prevents or reduces unwanted air bubbles in the wells. This increases the proportion of liquid that ends up in the wells and can be analyzed. Thus, high precision and reproducibility are achieved in the spatial separation of liquids.

[0031] One embodiment relates to a method for generating spatially separated liquid partitions in an arrangement, e.g., an array or pattern, of depressions, e.g., wells, formed in a surface. The method comprises providing a fluidic module and rotating the fluidic module. The fluidic module comprises a fluid chamber having the surface in which the arrangement of depressions is formed. The fluidic module is rotated about a center of rotation to exert a centrifugal force on a liquid, by which the liquid is introduced into the fluid chamber and by which at least a portion of the liquid is transported from radially outside to radially inside across the arrangement of depressions. The method further comprises removing the liquid from regions of the surface outside the depressions to generate the spatially separated liquid partitions of the liquid in the depressions.

[0032] In embodiments, the liquid in the areas outside the recesses is removed from the fluid chamber via an outlet by rotating the fluidic module. The rotation during removal of the liquid can occur in a similar manner to the rotation during introduction of the liquid, i.e., at the same rotational frequency and / or by continuing the rotation during introduction of the liquid, e.g., without interruption of the rotation between introduction of the liquid and removal of the liquid. Alternatively, the fluidic module can be subjected to a rotational frequency that is higher than the rotational frequency when introduction of the liquid into the fluid chamber. The rotation of the fluidic module can cause the liquid not located in the recesses to be ejected from the fluid chamber.Since the centrifugal force acting on the small volumes of liquid remaining in the wells is smaller than the capillary forces acting on them, the liquid remains in the wells. This allows the spatially separated liquid partitions to be created very efficiently and reproducibly.

[0033] Optionally, after removal, a second liquid that is not homogeneously miscible with the liquid can be introduced into the fluid chamber by rotating the fluidics module in order to seal the wells with the liquid partitions contained therein. The rotation frequency when introducing the second liquid can correspond to the rotation frequency when introducing the liquid. By sealing the liquid partitions with the second liquid by rotating the fluidics module, the liquid partitions are efficiently separated from one another. This prevents two liquid partitions from becoming fluidically coupled to one another via any residual liquid that may remain on the surface. High precision can be achieved in the analysis of the liquid partitions because the sealing with the second liquid ensures precisely defined liquid volumes in the wells.

[0034] In embodiments, the entire fluid chamber is first emptied with the exception of the depressions, i.e. the liquid in the areas outside the depressions is completely removed from the fluid chamber via the outlet by rotating the fluidics module, before the second liquid is introduced. This achieves greater precision in the separation of the liquid partitions than if the liquid in the areas outside the depressions is removed from the fluid chamber via the outlet by introducing the second liquid. In embodiments, the fluid chamber forms a gap between the surface in which the arrangement of depressions is formed and a surface opposite this surface. A gap height of the gap is, for example, a maximum of 10 times a depth of the depressions and / or the gap height is a maximum of 200 pm, preferably a maximum of 100 pm. The gap height and the depth describe dimensions orExtensions perpendicular to the surface in which the depressions are arranged. The rotation of the fluidic module takes place at a speed such that the centrifugal force acting on the liquid volume is higher than the capillary force acting on the liquid in the gap. The speed is, for example, at least 30 Hz, preferably at least 40 Hz. Because the centrifugal force is higher than the capillary force, in contrast to capillary filling, in which the liquid would always move to where the highest capillary pressure prevails, a uniform liquid front can be created, through which a controlled filling of the depressions is possible even with a small gap height. The capillary forces, which could otherwise disrupt the defined filling process, for example by the inclusion of air bubbles in the area of ​​the depressions due to uneven transport of the liquid within the fluid chamber, ieduring transport via an arrangement of recesses, can be neglected. In addition, by avoiding trapped air, high precision and reproducibility are achieved in the creation of liquid partitions. In addition to the speed, the centrifugal force also depends, for example, on radial fill levels. The radial fill levels are defined, for example, by the liquid volume and the chamber and / or channel geometries. The radial fill levels are designed by the liquid volume and chamber and / or channel geometries, and the rotation of the fluidic module takes place at such a speed that the centrifugal force acting on the liquid volume is higher than the capillary force acting on the liquid in the gap.It is particularly advantageous if the centrifugal force acting on the liquid volume is higher by a factor of at least five, preferably at least ten, than a capillary force acting on the liquid in the gap.

[0035] In embodiments, when the liquid is introduced into the fluid chamber, a liquid flow into the fluid chamber is limited. This is based on the knowledge that when the fluidics module begins to rotate, e.g. when starting up a centrifuge that has the fluidics module, the rotational frequency increases slowly and a desired rotational frequency is not achieved instantly. In order for the fluidics module to rotate about the center of rotation at a rotational frequency with which a centrifugal force is exerted on the liquid, by means of which centrifugal force at least part of the liquid is transported from radially outside to radially inside via the arrangement of recesses, it is advantageous to limit the liquid flow into the fluid chamber. By limiting the flow, the desired rotational frequency is reached before the liquid reaches the recesses.In this way, a predetermined rotational frequency for transporting the liquid over the arrangement of depressions can be achieved before the liquid reaches the arrangement. This makes it possible to achieve controlled transport of the liquid over the arrangement of depressions because, for example, a correspondingly high rotational frequency prevails during transport. This ensures precise and reproducible filling of the depressions. During the introduction of the liquid, i.e. the filling process, the rotational speed does not have to be kept constant at a predetermined speed. However, it is advantageous if the rotational speed is already at a certain level, i.e. at least reaches the predetermined speed, e.g. the 30 to 40 Hz mentioned above.

[0036] In embodiments, limiting the flow of liquid into the fluid chamber prevents the liquid in the fluid chamber from reaching the arrangement of recesses before the centrifugal force acting on the liquid volume is higher than the capillary force acting on the liquid in the gap. It is particularly advantageous if the centrifugal force acting on the liquid volume is at least five or ten times higher than the capillary force acting on the liquid in the gap. The high centrifugal force, compared to the capillary force, improves filling precision because the liquid is transported from radially outside to radially inside with a controlled liquid front over the arrangement of recesses, thus reducing the likelihood of air bubbles becoming trapped in the liquid during this transport.

[0037] The fluid flow can be limited in various ways. The inventors consider the use of an inlet resistance channel, a pneumatic backpressure in the fluid chamber, or a pneumatic negative pressure in the inlet chamber to be particularly advantageous.

[0038] In embodiments, the fluidic module has an inlet resistance channel that opens into the fluid chamber to limit the flow of fluid into the fluid chamber. The cross-section and length of the inlet resistance channel are designed, for example, such that a predetermined rotational frequency is achieved when the fluidic module rotates before the fluid reaches the recesses in the fluid chamber. The inlet resistance channel has, for example, a small cross-section, e.g., a diameter or a height and / or width of a few tens of pm, preferably with a maximum of 100 pm, 50 pm, 30 pm, 20 pm, or 10 pm. The small cross-section causes a flow resistance that limits the filling rate.The inventors recognized that despite the disadvantages of a channel with a small cross-section, such as the undesirable adsorption of molecules on the channel walls, an inlet resistance channel with a small cross-section is advantageous in this case, as it allows for controlled filling of the recesses. Furthermore, the undesirable adsorption of molecules on the channel walls is minimized because no connecting channels are required between the recesses and the inlet resistance channel can be kept short. In contrast to channel networks with a small channel cross-section, in this case, despite the inlet resistance channel, a large number of recesses can be filled with the liquid very quickly, since the liquid is transported over a large area across the arrangement of recesses and resistances in inlet channels of individual recesses are eliminated.This achieves a good compromise between minimizing the problems caused by adsorption of biomolecules on channel walls and a fast and precise generation of liquid partitions.

[0039] In embodiments, a pneumatic backpressure is generated in the fluid chamber to limit the flow of fluid into the fluid chamber. This, for example, only transports the fluid toward the fluid chamber when the centrifugal force acting on the fluid exceeds the pneumatic backpressure. The pneumatic backpressure can be adjusted so that the fluid only reaches the recesses within the fluid chamber when a predetermined rotational frequency, or a predetermined centrifugal force acting on the fluid, is reached during rotation of the fluidic module.Limiting the fluid flow by means of pneumatic backpressure not only has the advantage that, upon reaching the predetermined rotational frequency, the fluid is transported from radially outside to radially inside with a controlled fluid front across the arrangement of depressions, but also that adsorption of biomolecules on channel walls can be minimized or avoided, since an inlet channel that opens into the fluid chamber and has a small cross-section can be dispensed with.

[0040] In exemplary embodiments, the pneumatic backpressure is achieved by a flow-limited venting of the fluid chamber. For example, when the fluid is introduced into the fluid chamber, the flow-limited venting creates an overpressure therein, which limits the flow of fluid into the fluid chamber. For example, via the flow-limited venting, less air escapes from the fluid chamber per unit of time than fluid is transported toward the fluid chamber. The flow-limited venting is designed, for example, such that the fluid only reaches the recesses within the fluid chamber when a predetermined rotational frequency, or a predetermined centrifugal force acting on the fluid, is reached upon rotation of the fluidic module.

[0041] In embodiments, a pneumatic vacuum is generated in the inlet chamber to limit the flow of fluid into the fluid chamber. This, for example, delays the fluid transport toward the fluid chamber. The pneumatic vacuum can be adjusted so that the fluid only reaches the recesses within the fluid chamber when a predetermined rotational frequency, or a predetermined centrifugal force acting on the fluid, is reached upon rotation of the fluidic module.Limiting the fluid flow by means of pneumatic vacuum not only has the advantage that by reaching the predetermined rotational frequency the fluid is transported from radially outside to radially inside with a controlled fluid front over the arrangement of depressions, but also that adsorption of biomolecules on channel walls can be minimized, since a small cross-section of the inlet channel leading into the fluid chamber can be dispensed with.

[0042] In one embodiment, the pneumatic negative pressure is achieved by flow-limited venting of the inlet chamber. By means of flow-limited venting, for example, when the liquid is introduced into the fluid chamber or when the liquid is transported from the inlet chamber to the fluid chamber, a negative pressure is created in the inlet chamber that limits the flow of liquid into the fluid chamber. Via flow-limited venting, for example, less air is introduced into the inlet chamber per unit of time than liquid is transported from the inlet chamber towards the fluid chamber. The flow-limited venting is designed, for example, such that the liquid only reaches the recesses within the fluid chamber when a predetermined rotational frequency, or a predetermined centrifugal force acting on the liquid, is reached when the fluidic module rotates.

[0043] In embodiments, a volume-controlled switch is used which is fluidically coupled to an outlet of the fluid chamber. The volume-controlled switch enables emptying of the fluid chamber, i.e., the removal of liquid from areas of the surface outside the depressions, only after the array of depressions has been completely overflowed by the liquid or the fluid chamber has been completely filled. The volume-controlled switch can, for example, be designed as an inverted siphon, the apex of which lies radially further inward than the radially inner end of the array of depressions. The volume-controlled switch can, for example, be designed in the form of a siphon which is fluidically coupled to the fluid chamber at a radially outermost point thereof. The siphon runs, for example, parallel to the fluid chamber, from the outlet, i.e.from the radially outermost point of the fluid chamber, at least as far as a radially innermost point of the arrangement of recesses radially inward, before the siphon changes direction and runs radially outward. As a result, a radial fill level of the siphon reflects a radial fill level of the chamber and only allows emptying of the fluid chamber after the arrangement of recesses has been completely overflowed by the liquid, since a radially innermost point, e.g. an apex, of the siphon is only reached when the arrangement of recesses has been completely overflowed by the liquid. The volume-controlled switch can very efficiently ensure that all recesses in the arrangement are filled.At the same time, the volume-controlled switch can be designed in such a way that only a very small dead volume is created, which allows saving valuable samples or expensive reagents, for example by using a small cross-section of the siphon.

[0044] In embodiments, an outlet resistance channel fluidically coupled to the outlet of the fluid chamber is used as an alternative to the volume-controlled switch. The outlet resistance channel prevents emptying of the fluid chamber, i.e., the removal of liquid from areas of the surface outside the recesses, until the arrangement of recesses has been completely overflowed by the liquid or until the fluid chamber is completely filled. The outlet resistance channel limits the outflow volume flow of the liquid from the chamber through the outlet. By means of the outlet resistance channel, the liquid volume flows out of the fluid chamber much more slowly than it is introduced into the fluid chamber.The flow resistance of the resistance channel at the outlet should be significantly higher than the flow resistance at the inlet (the flow resistance at the inlet can be defined, for example, by an inlet resistance channel or by a flow-limiting vent of the fluid chamber, see the above explanations) to prevent the fluid chamber from being emptied by centrifugal force directly via the resistance channel at the outlet. A cross-section and / or a length of the outlet resistance channel is, for example, designed such that the outlet resistance channel prevents the fluid chamber from being emptied until the arrangement of recesses has been completely overflowed by the liquid or the fluid chamber has been completely filled. The dimensioning of the outlet resistance channel and / or the inlet channel can be determined, for example, based on a calculation of the hydrodynamic resistances of the inlet channel and outlet channel.The outlet resistance channel ensures very efficient filling of all wells of the array. At the same time, dead volume is minimized, as the outlet resistance channel is preferably designed with a small cross-section to provide the necessary flow resistance. This allows for the saving of valuable samples or costly reagents.

[0045] One embodiment relates to a fluidics module for generating spatially separated fluid partitions using one of the methods described herein. The fluidics module has a fluid chamber, inlet structures, and outlet structures. The fluid chamber has the surface in which the arrangement of depressions is formed. The inlet structures are designed to introduce fluid into the fluid chamber by means of centrifugal force upon rotation of the fluidics module in order to transport the fluid from radially outside to radially inside over the arrangement of depressions. The outlet structures are designed to remove the same fluid, after flowing over the surface in which the arrangement of depressions is formed, from regions of the surface outside the depressions in order to generate the spatially separated fluid partitions of the fluid in the depressions.

[0046] The outlet structures and / or inlet structures are designed, for example, such that when the fluidic module rotates, more liquid is introduced into the fluid chamber by centrifugal force than is removed from the fluid chamber during introduction. This enables efficient filling of the fluid chamber from radially outside to radially inside. This can be achieved, for example, by designing the outlet structures and inlet structures such that a flow resistance at the outlet of the fluid chamber is higher than a flow resistance at the inlet of the fluid chamber. Alternatively, this can be achieved by the outlet structures having a siphon which is fluidically coupled to an outlet of the fluid chamber and is designed to be filled together with the fluid chamber from radially outside to radially inside and whose apex is arranged radially further inward than a radially innermost point of the arrangement of recesses.Preferably, the siphon has a cross-section perpendicular to the filling direction that is smaller than a cross-section of the fluid chamber perpendicular to the filling direction in order to minimize dead volume. The filling direction runs from radially outside to radially inside, i.e., in the direction of a center of rotation.

[0047] In embodiments, the fluid chamber forms a gap, e.g. a narrow hollow space or a narrow cavity, between the surface in which the arrangement of depressions is formed and a surface opposite this surface. The surface in which the arrangement of depressions is formed and the surface opposite this surface face the hollow space of the fluid chamber, i.e. the gap, or delimit the gap, e.g. on two opposite sides. A gap height of the gap is, e.g. a maximum of 10 times the depth of the depressions and / or the gap height is a maximum of 200 μm, preferably a maximum of 100 μm. The gap height corresponds, e.g. to an extension of the gap perpendicular to the surface in which the arrangement of depressions is formed. The gap height corresponds, e.g. to a distance between the surface in which the arrangement of depressions is formed and the surface opposite this surface.The depth of the depressions corresponds, for example, to an extension of the depressions perpendicular to the surface in which the arrangement of depressions is formed. The inventors recognized that by filling a fluid chamber from radially outside to radially inside by rotating the fluidics module, centrifugal forces can be used to fill the fluid chamber, compared to which capillary forces become negligible. This allows controlled and efficient filling of the fluid chamber even in fluid chambers with a low chamber height, i.e. with a low gap height. The special fluidics module enables a gap height that is a maximum of 10 times the depth of the depressions and / or a maximum of 200 μm, preferably a maximum of 100 μm, thereby minimizing dead volume. According to one embodiment, the gap height of the gap has a minimum height of 10 μm. This ensures, for example, that when using typical rotation frequencies (e.g.At frequencies of up to 100 Hz, the centrifugal force exceeds the capillary force by 5 to 10 times. Furthermore, this minimum height allows for controlled production of the fluid chamber, for example, if a plastic material rather than glass or silicon is used to manufacture the fluidics module. If the fluidics module is made of glass or silicon, a lower minimum height could potentially be achieved.

[0048] In embodiments, the inlet structures have an inlet chamber that is at least partially arranged radially further inward than an innermost section of the arrangement of recesses. Furthermore, the inlet structures have a fluid channel that connects the inlet chamber to the fluid chamber. This special arrangement of the inlet chamber enables filling of the fluid chamber from radially outside to radially inside, since upon rotation of the fluidic module, a liquid arranged in the inlet chamber is transported radially outward via the fluid channel into the fluid chamber to a radially outermost point of the fluid chamber and fills the fluid chamber within the fluid chamber from radially outside to radially inside.

[0049] If, for example, an inlet of the fluid chamber, to which the fluid channel is fluidically coupled, is located at a radially innermost point of the fluid chamber, then all of the liquid from the inlet chamber can be introduced into the fluid chamber from radially outside to radially inside. If the inlet is arranged radially further out than the radially innermost point of the fluid chamber, or even at the radially outermost point of the fluid chamber, then the fluid chamber is filled from radially outside to radially inside until a radial fill level within the fluid chamber corresponds to a radial fill level within the inlet structures, i.e. the fluid channel. However, because the inlet chamber is arranged at least partially radially further in than a radially innermost section of the arrangement of recesses, it is ensured that the arrangement of recesses is completely flooded with liquid.the fluid chamber is filled up to a radially innermost point of the arrangement by means of rotation. If the inlet is not located at the radially outermost point of the fluid chamber, the fluid chamber preferably has a bypass in the lateral region of the chamber with lower flow resistance. This prevents the fluid from initially being transported from radially inside to radially outside via the arrangement of recesses, since the fluid is transported from the inlet via the bypass, past the arrangement of recesses, directly to the radially outermost point of the fluid chamber and from there fills the fluid chamber from radially outside to radially inside.

[0050] In embodiments, the inlet structures comprise an inlet resistance channel configured to limit the flow of fluid into the fluid chamber. Alternatively, the fluid chamber comprises a flow-limited vent configured to generate a pneumatic backpressure in the fluid chamber upon introduction of the fluid into the fluid chamber in order to limit the flow of fluid into the fluid chamber. Alternatively, the inlet chamber comprises a flow-limited vent configured to generate a pneumatic negative pressure in the inlet chamber upon introduction of the fluid into the fluid chamber in order to limit the flow of fluid into the fluid chamber.Flow limitation allows a liquid, for example, to reach the array of recesses at the earliest when a predetermined rotational frequency is reached, thus ensuring controlled transport of the liquid across the array from radially outside to radially inside. This is based on the realization that when the fluidic module begins to rotate, e.g., when starting up a centrifuge containing the fluidic module, the rotational frequency increases with a delay, and the desired rotational frequency is not achieved instantly.

[0051] In embodiments, the outlet structures comprise a volume-controlled switch that allows the fluid chamber to be emptied only after the assembly has been completely overflown by the liquid. Alternatively, the outlet structures comprise an outlet resistance channel that prevents the fluid chamber from being emptied until the assembly has been completely overflown by the liquid. This can very efficiently ensure that all recesses of the assembly are filled.

[0052] Embodiments relate to a device for carrying out one of the methods described herein. The device comprises one of the fluidic modules described herein and a drive device for rotating the fluidic module. Brief description of the figures

[0053] Embodiments according to the present invention are explained in more detail below with reference to the accompanying figures. With regard to the schematic figures shown, it should be noted that the illustrated functional blocks are to be understood both as elements or features of the device according to the invention and as corresponding method steps of the method according to the invention, and corresponding method steps of the method according to the invention can also be derived therefrom. They show:

[0054] Fig. 1 is a schematic representation of a method for generating spatially separated fluid partitions and a fluidics module for use in the method;

[0055] Fig. 2 schematic representations of a fluid chamber and

[0056] Embodiments of a surface of the fluid chamber;

[0057] Fig. 3a-b schematic representations of depressions in a surface of a fluid chamber;

[0058] Fig. 4 schematic representations of a fluid chamber when it is filled with a liquid;

[0059] Fig. 5a) to 5e) schematic representation of a method for generating spatially separated liquid partitions in a fluid chamber;

[0060] Fig. 6 schematic representation of a process of a microfluidic

[0061] Filling and sealing of recesses in a fluidic module with volume-controlled switch;

[0062] Fig. 7 schematic representation of a process of a microfluidic

[0063] Filling and sealing of recesses in a fluidic module with outlet resistance channel; and

[0064] Fig. 8A and 8B are schematic representations of examples of inventive

[0065] Fluid handling devices.

[0066] Detailed description of the embodiments according to the figures

[0067] Before exemplary embodiments of the present invention are explained in more detail below with reference to the drawings, it is pointed out that identical, functionally equivalent or equivalent elements, objects and / or structures in the different figures are provided with the same or similar reference numerals, so that the description of these elements shown in different exemplary embodiments is interchangeable or can be applied to one another. A repeated description of elements provided with the same or similar reference numerals is typically omitted. In particular, identical or similar elements can each be provided with reference numerals that have the same number with a different or no lowercase letter or with a different or no subscript number.In the following description, many details are described to provide a more thorough explanation of examples of the disclosure. However, other examples may be implemented without these specific details. Features of the various described examples may be combined with each other unless features of a corresponding combination are mutually exclusive or such a combination is expressly excluded.

[0068] Before further explaining examples of the present disclosure, definitions of some terms used herein are provided.

[0069] The term "liquid" as used herein includes, in particular, liquids containing solid components, such as suspensions, biological samples, and reagents. A reaction liquid, for example, refers to a mixture of substances whose reaction enables a pre-analytical or analytical function, e.g., the direct detection of nucleic acids using a fluorescence reaction (digital PCR). A sealing liquid refers to a substance or mixture of substances whose properties enable the sealing of the wells, thus preventing an interface between the reaction liquid and air or other gases or gas mixtures.

[0070] A siphon or siphon channel is understood herein to be a microfluidic channel or section of a microfluidic channel in a fluidic module (a centrifugal microfluidic cartridge) in which the inlet and outlet of the channel (when used in a centrifugal microfluidic system or a centrifugal microfluidic platform) are at a greater distance from the center of rotation than an intermediate region of the channel. A siphon apex or apex of the siphon is the region of a siphon channel in a fluidic module with the minimum distance from the center of rotation. Since the apex of such a siphon is inverted in the centrifugal field compared to the arrangement of a typical siphon in the Earth's gravitational field, such a siphon could also be referred to as an inverted siphon in the centrifugal field.A fluidic module is understood herein to mean a module, for example a cartridge, that has microfluidic structures designed to enable liquid handling as described herein. A fluidic module is, for example, a self-contained unit consisting of a fluidic structure and a cover thereof. A centrifugal microfluidic fluidic module (cartridge) is understood to mean a corresponding module that can be subjected to rotation, for example in the form of a fluidic module that can be inserted into a rotating body or a rotating body. In examples of the present invention, the fluidic module is a centrifugal microfluidic fluidic module.

[0071] A fluidic structure or a microfluidic structure, for example, has structures or cavities that are open on one side, e.g. for channels and chambers of a fluidic module.

[0072] A cover, for example, has a film or a plate that is glued or welded to the fluidic structure.

[0073] When reference is made here to a fluid channel, this means a structure whose length dimension from a fluid inlet to a fluid outlet is greater, for example more than 5 times or more than 10 times greater, than the dimension or dimensions that define the flow cross-section. A fluid channel therefore has a flow resistance for flow through it from the fluid inlet to the fluid outlet. A channel or fluid channel refers, for example, to a volume in a fluidic module that is delimited by a cover and at least one part, e.g. a channel part, of the fluidic structure. As a rule, a fluid channel is smaller than 0.5 mm in at least two dimensions and considerably longer in the third dimension. A fluid channel serves, for example, as a fluidic connection between fluid chambers, i.e. a fluid channel fluidically couples, for example, at least two fluid chambers to one another.

[0074] A fluid chamber, however, is defined herein as a chamber with dimensions such that, when flowing through the chamber, a flow resistance occurs that is negligible compared to connected channels. This flow resistance can, for example, be 1 / 100 or 1 / 1000 of the flow resistance of the channel structure connected to the chamber with the smallest flow resistance. A chamber or fluid chamber refers, for example, to a volume in a fluidic module delimited by the cover and at least one part, e.g., a chamber part, of the fluidic structure. Typically, a fluid chamber is larger than 0.5 mm in at least two dimensions.

[0075] When the term "depression" is used herein, it refers, for example, to a geometrically defined structure that is generally smaller than 0.5 mm in all three dimensions. A depression may also be referred to herein as a well or partition. A depression in a surface of a fluid chamber wall refers, for example, to a recess in the surface or a locally limited depression in the surface.

[0076] An arrangement of depressions is understood herein to mean, for example, a plurality of depressions arranged in a specific formation on a surface. The formation can be uniform or irregular, or evenly or irregularly formed. The arrangement of depressions defines, for example, a locally limited area on a surface in which the depressions are arranged. Examples of the invention can be a planar arrangement of depressions, which corresponds, for example, to an arrangement of the depressions in a plane. The arrangement of depressions can also be referred to as a pattern of depressions or as an array of depressions.

[0077] An array can be understood here as a planar arrangement of depressions, typically in the form of a repetitive pattern, e.g. in the form of a matrix of rows and columns or a hexagonal arrangement of the depressions.

[0078] Examples of the invention can be applied in particular in the field of centrifugal microfluidics, which involves the processing of liquids in the picoliter to milliliter range. Accordingly, the fluidic structures can have suitable dimensions in the micrometer range for handling corresponding liquid volumes.

[0079] Partitioning is defined here as the division of a liquid, e.g. a reaction liquid, into sub-units (“partitions”). The purpose is to carry out digital reactions, whereby the partitions ideally contain one (digital “1”) or no (digital “0”) molecule or particle. In practice, partitions can also be occupied by more than one molecule or particle, but this effect can be compensated for using statistical methods. In order to prevent or at least drastically reduce any exchange between the partitions during a reaction, a sealing liquid, for example, fills the space between two or more partitions. A sealing liquid is understood to be a liquid that is not homogeneously miscible with a liquid divided into partitions, i.e. with the liquid partitions.

[0080] When the term radial is used herein, it means radial with respect to the center of rotation around which the fluidic module or the rotating body is rotatable. In the centrifugal field, a radial direction away from the center of rotation is radially decreasing, and a radial direction towards the center of rotation is radially increasing. A fluid channel whose beginning is closer to the center of rotation than its end is thus radially decreasing, while a fluid channel whose beginning is further from the center of rotation than its end is radially increasing. A channel with a radially increasing section therefore has directional components that increase radially or run radially inwards. It is clear that such a channel does not have to run exactly along a radial line, but can run at an angle to the radial line or be curved.Radially further out means further away from the center of rotation and radially further in means closer to the center of rotation.

[0081] A rotational frequency, also referred to herein as speed, is the number of revolutions N per unit of time t in the unit 1 / second (s -1 ) or 1 / minute (min -1 ) or Hz.

[0082] A dead volume is understood, for example, as a part of the liquid introduced into the system (e.g. a reaction liquid), i.e. a part of the liquid from which spatially separated liquid partitions are to be created, which does not end up in the partitions and is therefore not analyzed.

[0083] Examples of the present disclosure provide devices and methods for partitioning molecules or particles into arrays, i.e., liquids comprising molecules and / or particles. Examples are directed to devices and methods for generating spatially separated liquid partitions that are geometrically defined in location and size. The goal of the partitioning is to implement digital assays, e.g., digital PCR or digital immunoassays, but the examples disclosed herein are not limited to these. The core of the invention is a device with defined wells (partitions) in a surface that are filled with liquids by centrifugation. The wells are filled with a first liquid, e.g., an aqueous reaction mix, by flowing over them. Excess liquid is then removed from the wells by spinning.In a further optional centrifugation step, the open side of the wells is sealed with a second liquid, e.g. oil.

[0084] An embodiment of a method 100 according to the invention is shown in Fig. 1. The method is used to partition a liquid 102.

[0085] In the method 100, a fluidics module 200 is provided 110. Fig. 1 shows a top view of the schematic fluidics module 200. The fluidics module 200 includes a fluid chamber 210. The fluid chamber 210 has a surface that includes an array 220 of recesses 222. The method 100 creates spatially separated fluid partitions in the array 220 of recesses 222 formed in the surface.

[0086] The surface comprising the arrangement 220 of depressions 222 can be a planar surface or an inclined or curved surface. An inclined or curved surface has the advantage that gas bubbles that may arise in the fluid chamber 210, e.g., during the introduction 126 of a liquid 102 or during the heating of a liquid 102, can be removed in a targeted manner.

[0087] The arrangement 220 of depressions 222 shown schematically in Fig. 1 has depressions 222 with a round cross-section. The depressions 222 correspond, for example, to cylindrical and / or hemispherical and / or spherical segment-shaped and / or conical and / or truncated cone-shaped recesses. Alternatively or additionally, the arrangement 220 can have depressions 222 with a different geometrically defined structure, see, for example, Fig. 3 and the associated explanations. Furthermore, the depressions 222 shown in Fig. 1 are arranged unevenly. Alternatively or additionally, the surface of the fluid chamber 210 can also have a uniform arrangement of depressions, see, for example, Figs. 2 to 7 and the associated explanations. A further feature of the depressions 222 shown in Fig. 1 is their different size, i.e. their different volume or capacity.Alternatively, the arrangement 220 may also have only recesses 222 of the same size, see, for example, Figs. 2 to 7 and the associated explanations. The arrangement 220 of recesses 222 shown in Fig. 1 covers at least a portion of the surface of the fluid chamber 210 on which the arrangement 220 is arranged. Alternatively, the recesses 222 may also be arranged on the entire surface comprising the arrangement 220.

[0088] By rotating 120 the fluidic module 200 around a rotation center 122, a centrifugal force F z exerted on the liquid 102, through which the liquid 102 is introduced 126 into the fluid chamber 210, and through which at least a portion of the liquid 102 is transported from radially outside to radially inside, ie, in a radially ascending direction 124, via the arrangement 220 of recesses 222. The liquid 212 is introduced 126 into the fluid chamber 210 via an inlet 212 of the latter.

[0089] The center of rotation 122 is only shown schematically in Fig. 1 and can alternatively be located further away from the fluidic module 200 than shown.

[0090] Furthermore, Fig. 1 illustrates a counterclockwise rotation 120. Alternatively, it is also possible for the fluidic module 200 to be rotated 120 clockwise.

[0091] By rotating 120 the fluidic module 200, the liquid 102 is introduced 126, for example, from a radially further inward region of the fluidic module 200 into the fluid chamber 210. Radially further inward is understood in relation to the radially innermost position of the arrangement 220. The radially further inward region is, for example, at least partially arranged radially further inward than the arrangement 220 of recesses 222. The centrifugal force F acting on the liquid 102 zthus transports the liquid 102 from the radially inner region of the fluidic module 200 from radially inside to radially outside, ie in a radially descending direction, into the fluid chamber 210. The fluid chamber 210 itself, however, is filled from radially outside to radially inside. This is based on the fact that the centrifugal force F z, which is exerted on the liquid 102, the liquid 102 is initially transported within the fluid chamber 210 to its radially outermost point, and then a radial fill level of the fluid chamber 210 increases radially inward with increasing liquid 102 that is introduced 126 into the fluid chamber 210. The radially inner region of the fluidic module 200 is fluidly coupled to the inlet 212 of the fluid chamber 210 via a fluid channel, e.g., an inlet channel 232. The radially inner region can have a liquid inlet 234 and / or a plurality of further fluid chambers and / or one or more further fluid channels. In Fig. 1, the radially inner region has, by way of example, an inlet chamber 230 or reservoir chamber. In other words, the inlet chamber 230 is at least partially arranged radially further inward than a radially innermost section of the arrangement 220 of recesses 222.The inlet chamber 230 optionally has a liquid inlet 234, through which the liquid 102 can be introduced into the inlet chamber 230. The inlet chamber 230 is coupled / connected to the inlet 212 of the fluid chamber 210 via the inlet channel 232, i.e., a fluid channel. In Fig. 1, the fluidic module 200 thus has, by way of example, an inlet chamber 230 in which the liquid 102 is provided.

[0092] The fluidic module 200 has, for example, inlet structures 236 that are fluidically coupled to the inlet 212 of the fluid chamber 210. The inlet structures 236 include, for example, the radially inner region of the fluidic module 200 and the fluid channel via which the radially inner region is fluidically coupled to the inlet 212 of the fluid chamber 210. The inlet structures 236 in Fig. 1 include, for example, the inlet chamber 230 and the inlet channel 232.

[0093] In Fig. 1, the inlet 212 of the fluid chamber 210 is arranged, by way of example, at a radially outermost point of the fluid chamber 210. This enables the arrangement 220 of recesses 222 to be overflowed by the liquid 102 only from radially outside to radially inside during the introduction 126 of the liquid 102. If, however, the inlet 212 is arranged radially further inward than the radially outermost point of the fluid chamber 210, the arrangement 220 of recesses 222 could be overflowed at least partially by the liquid 102 from radially inside to radially outside, since the centrifugal force F acting on the liquid 102 during the rotation 120 z The liquid 102 is initially transported radially outward from the inlet 212 to the radially outermost point of the fluid chamber 210. Thus, the inlet 212 at the radially outermost point of the fluid chamber 210 improves controlled filling of the fluid chamber 210.

[0094] Alternatively, the inlet 212 could also be arranged radially further inward than the radially outermost point of the fluid chamber 210. In such a case, however, it would be advantageous if the fluid chamber 210 also had a bypass with low flow resistance laterally of the arrangement 220, i.e., leading past the arrangement, in order to prevent the arrangement 220 from being overflowed from the radial inside to the radial outside.

[0095] If the inlet 212 of the fluid chamber 210 is located at a radially innermost point of the fluid chamber 210, the entire liquid 102, e.g. from the inlet chamber 230, can be introduced into the fluid chamber 210. If, however, the inlet 212 is arranged radially further outward than the radially innermost point of the fluid chamber 210, or even at the radially outermost point of the fluid chamber 210, the fluid chamber 210 is filled from radially outside to radially inside until a radial fill level within the fluid chamber 210 corresponds to a radial fill level within the inlet structures, ie the inlet channel 232. In this regard, the liquid volume of the liquid 102 and the chamber geometry of the fluid chamber 210 should be designed such that athe filling, the radial filling level achieved in the fluid chamber 210 is arranged radially further inward than a radially innermost point of the arrangement 220 of recesses 222 in order to ensure complete filling of all recesses 222.

[0096] The method 100 further comprises removing 130 the liquid 102 from areas of the surface outside the recesses 222, i.e., excess liquid 102, in order to create the spatially separated liquid partitions of the liquid 102 in the recesses 222. This means that the liquid 102 not located in the recesses 222 is removed 130. The bottom right of Fig. 1 schematically shows that, after the removal 130 of excess liquid 102, the fluid chamber 210 contains the liquid 102 only in the recesses 222. The liquid 102 is removed 130 from the fluid chamber 210 through an outlet 214 of the fluid chamber.

[0097] The removal 130 of the excess liquid 102 from the fluid chamber 210 can, for example, be carried out by rotating 132 the fluidics module 200 about the center of rotation 122. This allows the excess liquid 102 to be ejected from the fluid chamber 210. The rotation 120 for introducing 126 the liquid 102 into the fluid chamber 210 and the rotation 132 for removing 130 the excess liquid 102 from the fluid chamber 210 do not differ, for example. Alternatively, the rotation 120 for introducing 126 the liquid 102 and the rotation 132 for removing 130 the excess liquid 102 could differ in a speed or rotational frequency and / or direction of rotation with which the fluidics module 200 is subjected in each case. The excess liquid 102 could, for example, B. by rotating 132 at a higher speed than the rotating 120 when introducing 126 the liquid 102, removed 130.By removing the excess fluid by rotating the fluidic module 200, e.g., at a constant or even increased rotational speed, a backflow of the fluid 102 into the inlet structures 236 is avoided. The rotation 120 for introducing 126 and the rotation 132 for removing 130 occur, for example, in the same direction of rotation, e.g., clockwise or counterclockwise.

[0098] The fluidics module 200 has, for example, outlet structures 240 that are fluidically coupled to the outlet 214 of the fluid chamber 210. The outlet structures 240 are designed to remove 130 the liquid 102 from the areas of the surface outside the depressions 222 after it has flowed over the surface in which the arrangement 220 of depressions 222 is formed. The outlet structures 240 are designed to prevent premature emptying of the fluid chamber 210, i.e., before all depressions 222 of the arrangement 220 have been filled with the liquid 102. This special design of the outlet structures 240 further supports the filling of the fluid chamber from radially outside to radially inside, since the outlet structures 240 are designed to remove 130 less liquid 102 from the fluid chamber 210 during the introduction 126 of the liquid into the fluid chamber 210 than is introduced 126 into the fluid chamber 210 per unit of time.This can be achieved, for example, in that the outlet structures 240 have a volume-controlled switch 242, a flow-limiting unit, or a pressure-controlled switch, which is designed to prevent premature emptying of the fluid chamber 210. In Fig. 1, for example, a volume-controlled switch 242 is implemented in the form of an inverted siphon, whose apex 243 is arranged radially further inward than a radially innermost point of the arrangement 220 of recesses 222. The volume-controlled switch 242 is explained in detail in connection with Fig. 6. Alternatively, the outlet structures 240 can, for example, have a flow-limiting unit, such as an outlet resistance channel, which is designed to limit a liquid flow out of the fluid chamber 210. A flow-limiting unit is explained in detail in connection with Fig. 7.Alternatively, the outlet structures 240 may, for example, comprise a pressure-controlled switch, such as a pressure-dependent valve, which is configured to remove 130 the liquid 102 from the fluid chamber 210 only when a predetermined pressure exerted thereon by the liquid 102 is exceeded. Upon removal 130, or shearing off, of the excess liquid 102, the liquid 102 remains in the recesses 222 and is only removed 130 from the surrounding areas. The liquid 102 remains in the recesses 222 because the centrifugal force F acting on the small liquid volumes arranged in the recesses 222. z is lower than the capillary forces acting on them, ie in each of the depressions 222 a capillary force caused by this holds the respective liquid volume within the respective depression 222.

[0099] The subject of the invention is a device, see the fluidics module 200, and a method 100 for generating spatially separated and geometrically defined partitions in terms of location and size, which:

[0100] • Can be cost-effectively integrated into a polymer chip

[0101] • No channel network is required for filling, ie all depressions 222 are overflowed when the liquid 102 is introduced 126 into the fluid chamber 210 and it is not necessary to fill each depression 222 individually by means of a channel

[0102] • No hydrophilization or hydrophobization required for filling.

[0103] • Drastically reduce the dead volume compared to the state of the art.

[0104] Fig. 2 schematically shows a concept for a fluid chamber 210. One of the fluidic modules 200 described herein may include a fluid chamber 210 having features as explained in connection with Fig. 2.

[0105] Fig. 2 shows, on the left, a cross-section or a side view of the fluid chamber 210, and in the center, a top view of the fluid chamber 210 or a detailed view of the surface 216 of the fluid chamber 210, which has the arrangement 220 of recesses. An alternative design for the surface 216 is shown on the right.

[0106] On the far left, it can be seen that the fluid chamber 210 can have fluidic connections 213. Furthermore, the fluid chamber 210 has a surface 216 in which the arrangement 220 of depressions 222 is formed. Fig. 2 shows, on the far left, in combination with the detailed view in the middle, an exemplary embodiment in which the depressions 222 are completely distributed in the surface 216, i.e., the arrangement 220 of depressions 222 completely occupies the surface 216. Alternatively, as shown by way of example on the far right, the surface 216 of the fluid chamber 210 could have two or more arrangements, see 220i and 2202, of depressions 222. Fig. 2 shows in the middle and right exemplary designs of the arrangement 220 of depressions 222 as locally limited areas of the depressions (see right) and depressions 222 completely covering a surface 216 of a fluid chamber 210 as a global arrangement (see the middle).In other words, the fluid chamber 210 may have a global arrangement 220 of recesses, as shown in the center, or local arrangements 220i and 2202 of recesses, as seen on the right.

[0107] According to one embodiment, a fluidics module 200 described herein can consist of at least two parts. A first part, e.g., a fluidics part 211a, has, for example, a fluidics structure composed of fluid chambers and / or fluid channels for implementing microfluidic functions. A second part, e.g., a cover part 211b, is designed to cover the fluidics structure. At least one of the two parts or both parts have flatly arranged recesses 222 in one region of the surface 216 (see 220), or in several regions of the surface 216 (see arrangements 220i and 2202). The recesses 222 are shown in Fig. 2 on the left as an example in the cover part 211b, but can also be integrated into the fluidics part 211a or into both parts.

[0108] Figs. 3a and 3b show exemplary geometries and sections of arrangements 220 of depressions 222 in plan view. The three arrangements 220 shown in Fig. 3a and the arrangement 220 shown in Fig. 3b represent arrays of depressions 222, ie, the depressions 222 are arranged in rows and lines in the surface 216 of a fluid chamber.

[0109] An arrangement 220 of wells can, for example, only have wells of the same size, see right and left in Fig. 3a and Fig. 3b, or wells of different sizes, see center in Fig. 3a. The central arrangement 220 of wells 222 in Fig. 3a has, by way of example, wells 222 with two different sizes. The use of arrangements 220 with wells 222 of different sizes enables, for example, an efficient analysis of different analyte concentrations in the liquid (so-called dynamic range). The combination of more than two different sizes of wells can be useful for certain applications. Likewise, the combination of different geometries can be advantageous if, for example, a different geometry proves advantageous for filling small wells than for filling large wells. The wells 222 of the arrangement 220 shown in Fig.The arrangements 220 shown in Figure 3a are pyramid-shaped, i.e., the depressions 222 form pyramid-shaped recesses in the surface 216. Four-sided pyramid-shaped depressions 222 are shown as examples on the left and center, and six-sided pyramid-shaped depressions 222 are shown as examples on the right. Apart from the geometries shown, other geometries for the depressions 222 are also feasible, e.g., truncated pyramids, cylinders, hemispheres, spherical segments, semi-ellipsoids, ellipsoid segments, cones, truncated cones, cuboids, or prisms.

[0110] Fig. 3b shows an example embodiment of a surface 216 with an arrangement 220 of recesses 222 in the form of a truncated cone.

[0111] As illustrated in Fig. 4, the core of the invention is the minimization of the dead volume in the device, i.e. in the fluidic module 200 described herein, by means of a gap 218 or a chamber height that is as small as possible between a surface 216 in which the arrangement 220 of recesses 222 is arranged and a surface opposite this surface 216. The gap is formed, for example, between the fluidic part 211a and the cover part 211b. The fluid chamber 210 of a fluidic module 200 described herein has, for example, a gap 218, as described in connection with Fig. 4. In Fig. 4, a completely filled fluid chamber 210 is shown schematically on the left and the fluid chamber 210 during a filling process on the right.

[0112] When the fluid chamber 210 is completely filled, the portion of the liquid 102 located in the recesses 222 of the fluid chamber 210 defines an analysis volume Vanalysis, and the remaining portion of the liquid 102 defines the dead volume V-rot. The chamber height or the gap 218 largely defines the ratio of dead volume to analysis volume. A cross-section of the inlet and outlet channels or the inverted siphon can also influence the dead volume and thus the ratio of dead volume to analysis volume. The smaller the chamber height, the smaller the dead volume. In the methods 100 and fluidic modules 200 described herein, a gap height of the gap 218 can be less than or equal to 200 μm, preferably less than or equal to 100 μm. The gap height can be a maximum of 10 times the depth of the recesses 222.The dead volume can be minimized by filling the recesses 222 in a defined manner from radially outside to radially inside, as shown on the right in Fig. 4, by means of high centrifugal force (Fzen). The center of rotation 122 is shown schematically as a reference for the direction of filling. By using correspondingly high centrifugal forces (Fzen), capillary forces (Fxa) are reduced. P) are negligible, which could otherwise disrupt the filling process, for example by the inclusion of air bubbles in the area of ​​the recesses 222. The capillary forces increase with decreasing chamber height and can result in uncontrolled filling of the partitions. Due to high centrifugal forces, the capillary forces become relatively negligible and a defined filling of the chamber occurs from radially outside to radially inside. Radial fill levels of the fluid chamber 210 are designed by a liquid volume of the liquid 102 to be introduced and a chamber geometry of the fluid chamber 210 in such a way, and the rotation 120 of the fluidic module 200 takes place at such a speed that the centrifugal force acting on the liquid volume, or on the first liquid 102, is higher than the capillary force acting on the liquid 102 in the gap 218.In contrast to pressure-driven systems (negative and / or positive pressure) as are common in the prior art, the artificial gravitational field created by centrifugation is used here, whereby capillary forces become negligible. In the invention described here, a defined liquid front 103 is thus created along the same hydrostatic pressure (i.e., along the same radius in the centrifugal microfluidic system), which fills the array chamber, i.e., the fluid chamber 210, from radially outside to radially inside. This allows the realization of a much smaller gap 218 than in the prior art and thus the minimization of dead volume. While the chamber height in the prior art is 500 pm [Kan_2012], the invention described here allows a chamber height of less than 100 pm.Depending on the design of the wells 222, the dead volume can thus be reduced from several hundred times the volume of the input volume to a maximum of 1 to 100 times. Depending on the design, a ratio of less than 1:1 between dead volume and analysis volume could be achieved.

[0113] Fig. 5 shows an example of a method 100 for partitioning a liquid, which comprises, for example, molecules or particles, into planar arrangements 220 of depressions 22 in four steps. Below each step, an enlargement of a depression 222 of a fluid chamber 210, or an array chamber, is shown. Methods 100 described herein for generating spatially separated liquid partitions may have features as described in connection with Fig. 5.

[0114] Fig. 5a schematically shows a fluidics module 200 for use in the method 100. The fluidics module 200 has, by way of example, an inlet chamber 230, an inlet resistance channel 232, and a fluid chamber 210 with a surface having the arrangement 220 of recesses 222. Figures 5b to 5e show side views of the fluid chamber 210 together with an enlargement of a recess 222, respectively. Fig. 5b shows a side view of the fluid chamber 210 before filling with a first liquid 102, Fig. 5c shows a side view of the fluid chamber 210 after filling the recesses 222 with the first liquid 102, Fig. 5d shows a side view of the fluid chamber 210 after spinning off excess liquid 102 by centrifugation, and Fig. 5e shows a side view of the fluid chamber 210 after sealing the recesses 222 filled with the first liquid 102 by means of a second liquid 104.

[0115] The core of the method 100 is a precise and reproducible filling of the recesses 222 with a first liquid 102, as well as the separation of the liquid partitions 102p in the recesses 222 from one another by a seal in the form of a second liquid 104. The method 100 comprises one or more of the following individual steps:

[0116] • Advantageously, the condition Fzen » F Kapby centrifuging or rotating the fluidics module 200. It is particularly advantageous if these conditions are achieved before a first liquid 102 reaches the recesses 222, so that the capillary forces in the region of the recesses 222, caused by the dead volume-minimizing gap 218, are significantly exceeded (see Figure 5b). In other words, a centrifugal pressure is built up in the first liquid 102 before it reaches the fluid chamber 210 with the recesses 222. This can be achieved, on the one hand, by a very fast start-up of a centrifuge having the fluidic module 200, or by a delaying fluidic element or a flow-imitating element, for example a resistance channel 232 (see Fig. 5a), a pneumatic back pressure in the fluid chamber, a pneumatic negative pressure in the inlet chamber or a volume-based siphon located in front of the fluid chamber 210 as a switch.• The depressions 222 are filled by the first liquid 102, e.g. an aqueous reaction mix, flowing over them from radially outside to radially inside, i.e. in a radially ascending direction 124, see Figure 5c). The channel, i.e. the inlet resistance channel 232, between the inlet chamber 230 and the fluid chamber 210 can either be connected to the radially outer end of the fluid chamber 210, as shown in Fig. 5a, or laterally, i.e. radially further inward than the radially outermost point of the fluid chamber 210, or at the radially innermost point of the fluid chamber 210, insofar as the geometry of the fluid chamber 210 ensures that it is filled from radially outside to radially inside (e.g. through a bypass in the lateral region of the fluid chamber 210 with lower flow resistance, if the inlet 212 of the fluid chamber 210 is not located at the radially outermost point of the fluid chamber 210).

[0117] • In a further step, any excess liquid is removed, e.g., by centrifugation from the wells 222. This step is also achieved by centrifugation of the fluidic module 200 and provides precisely defined liquid volumes, ie, liquid partitions 102 p , in the recesses 222 (see Figure 5d). Capillary forces within the recesses 222 are, for example, greater than a centrifugal force acting on the liquid 102 during the centrifugation, whereby the liquid 102 is only removed from areas outside the recesses. Thus, following the centrifugation, the fluid chamber 210 has liquid partitions 102 p within the recesses 222 and the rest of the fluid chamber 210 is filled with a gas 105, such as air.

[0118] • In a further step, for example, an open side of the recesses 222 is sealed with a second liquid 104, e.g., oil. The second liquid 104 must not mix homogeneously with the first liquid 102 (Figure 5e). The second liquid 104, like the first liquid 104, is introduced into the fluid chamber 210 in a radially ascending direction 124, for example.

[0119] Figures 6 and 7 show embodiments of a method 100 for generating spatially separated liquid partitions 102 pTop views of a fluidic module 200 used in the respective method 100 are shown at the top, and side views of a fluid chamber 210 of the fluidic module 200 used in the respective method 100 are shown below, or cross-sections of an exemplary fluid chamber 210 with recesses 222. Figures 6 and 7 show an exemplary process for the microfluidic filling and sealing of recesses 222. For example, a first liquid 102 is used to fill the recesses 222, and a second liquid 104 is used to seal the recesses 222.

[0120] Figures 6 and 7 show, by way of example, a fluidics module 200 having a fluid chamber 210, inlet structures 236, and outlet structures 240. As can be seen in the detailed view in the figures below, the fluid chamber 210 has a surface 216 in which a plurality of recesses 222 are arranged. The arrangement of the recesses 222 is designated by reference numeral 220.

[0121] The method 100 includes providing 110 the fluidic module 200. The fluidic module 200 is provided 110, for example, together with a first liquid 102. The inlet structures 236 include, for example, an inlet chamber 230 and an inlet channel 232. The inlet chamber 230 is fluidically coupled or connected to an inlet 212 of the fluid chamber 210 via the inlet channel 232. The first liquid 102 is provided, for example, in the inlet chamber 230.

[0122] During the method 100, the fluidics module 200 is at least temporarily rotated about a center of rotation 122 in order to introduce 126 one or more liquids into the fluid chamber 210 via the inlet structures 236 and optionally to remove 130 the same from the fluid chamber 210 via the outlet structures 240. The method 100 comprises, for example, rotating 120 the fluidics module 200 to introduce 126 the first liquid 102 into the fluid chamber 210.

[0123] The fluid chamber 210 has, for example, an inlet 212 and an outlet 214 at opposite radially outer ends or corners. Thus, the first liquid 102 can be introduced 126 into the fluid chamber 210 from the radial outside, as well as removed 130 therefrom radially outwards. To completely remove the first liquid 102 from the fluid chamber 210, it can be advantageous if the outlet 214 is connected to the radially outermost point of the fluid chamber 210, e.g., by a radial gradient of the lower chamber side of the fluid chamber 210, i.e., by a radial gradient of the surface 216 in which the recesses 222 are arranged.The positioning of inlet 212 and outlet 214 radially outward has the advantage that a liquid can be introduced 126 into the fluid chamber 210 and can also be removed 130 therefrom in a controlled manner, and no liquid flows back into the inlet chamber 230 during removal 130 from the fluid chamber 210. Since outlet 214 is arranged radially outward, the first liquid can be ejected from the fluid chamber 210 by rotating 132 the fluidics module 200. The rotating 132 for removing 130 the first liquid 102 can take place at the same rotational frequency as the rotating 120 for introducing 126 the first liquid 102, or at a lower or higher rotational frequency. During removal 130 of the first liquid 102, the first liquid 102 is removed from areas of the surface 216 outside the recesses 222.Such removal may also be referred to as emptying the fluid chamber 210, since only the fluid partitions 102. p remain in the recesses 222 in the fluid chamber.

[0124] Since both the inlet 212 and the outlet 214 are arranged radially outward, the outlet structures 240 are designed, for example, to prevent the introduced liquid from directly escaping from the fluid chamber 210. The outlet structures 240 are designed to allow less of the first liquid 102 to escape from the fluid chamber 210 during the introduction 126 of the first liquid 102 than is introduced into it. The outlet structures 240 thus enable the first liquid 102 to completely flow over the arrangement 220 of recesses 222 within the fluid chamber 210 from radially outward to radially inward. In Fig. 6, this is achieved by the outlet structures 240 having a siphon 242, and in Fig. 7, this is achieved by the outlet structures 240 having an outlet resistance channel 244. The siphon 242 or alternatively the outlet resistance channel 244 is fluidically coupled to the outlet 214 of the fluid chamber 210.

[0125] The siphon 242 shown in Fig. 6 can be regarded as a volume-controlled switch, since a vertex 243 of the siphon 242 is arranged radially further inward than a radially innermost point of the arrangement 220 of recesses 222. As a result, the fluid chamber 210 can be filled to a maximum radial fill level upon introduction 126 of the first liquid 102, which lies at the same radial height as the vertex 243 of the siphon 242. The maximum radial fill level cannot be exceeded, since additionally introduced liquid would lead to exceeding the vertex 243 of the siphon 244, and thus the liquid is removed from the fluid chamber 210 via the siphon 242.In order to realize a complete overflow of all depressions 222, the siphon 242 should be designed such that the maximum radial fill level is arranged radially further inward than a position of a radially innermost depression 222 of the arrangement 220 of depressions 222. The siphon 242 is designed, for example, to enable the removal 130 of the first liquid 102 from regions of the surface 216 outside the depressions 222 only after the arrangement 220 of depressions 222 has been completely overflowed by the first liquid 102.

[0126] The outlet resistance channel 244 shown in Fig. 7 is designed to limit the outflow of the first liquid 102 from the fluid chamber 210. The outlet resistance channel has, for example, a smaller diameter than the inlet channel 232. As a result, more of the first liquid 102 can be introduced into the fluid chamber 210 via the inlet channel 232 than is simultaneously removed from it via the outlet resistance channel 244. The cross-section of the outlet resistance channel 244 is, for example, B. dimensioned such that when the first liquid 102 is introduced 126, the fluid chamber 210 is filled until at least the complete arrangement 220 of depressions 222 has been overflowed by the first liquid 102, ie until a radial filling level of the fluid chamber 210 is arranged radially further inward than a radially innermost depression 222 of the arrangement 220 of depressions 222. The outlet resistance channel 244 is e.g.designed to enable the removal 130 of the first liquid 102 from areas of the surface 216 outside the depressions 222 only after the arrangement 220 of depressions 222 has been completely overflowed by the first liquid 102.

[0127] Since both the inlet 212 and the outlet 214 are arranged radially outward, and for controlled filling of the fluid chamber 210, introduction of the first liquid 102 from radially outside to radially inside is necessary, it is further advantageous if the inlet structures 136 have a vent 238 for the fluid chamber 210. The fluid chamber 210 has, for example, an opening via which the fluid chamber 210 is connected to the vent 238. The opening is arranged, for example, at a radially innermost point of the fluid chamber 210 or radially further inward than a radially innermost point of the arrangement 220 of recesses 222.

[0128] Efficiency and control during the introduction 126 of the first liquid 102 into the fluid chamber 210 can be increased if the inlet structures 236 are configured to delay the entry of the first liquid 102 into the fluid chamber 210 or to delay the first liquid 102 reaching the arrangement 220 of recesses 222 (see Fig. 6 and Fig. 7). This ensures that the fluidic module 200 rotates 120 at a predetermined rotational frequency about the center of rotation 122 when the first liquid 102 flows over the arrangement 220 of recesses 222 from radially outside to radially inside.This enables a controlled liquid front during the flow over the arrangement 220 of depressions 222 and reduces the formation of air bubbles, since, for example, the delay can ensure that a centrifugal force acts on the first liquid 102 that is greater than a capillary force acting on the first liquid 102 due to a low chamber height 218 of the fluid chamber 210. In other words, the inlet structures 236 are designed to limit the liquid flow into the fluid chamber 210 in order to prevent the first liquid 102 in the fluid chamber 210 from reaching the arrangement 220 of depressions 222 before the centrifugal force acting on the first liquid 102 is greater than the capillary force acting on the first liquid 102 in the gap 218. Such a delay or limitation can be realized by the inlet channel 232, for example. B. is implemented as a resistance channel or the vent 238 e.g.is implemented as a flow-imitating vent or a vent 231 of the inlet chamber 230 is implemented, for example, as a flow-imitating vent.

[0129] If the inlet channel 232 is implemented as a resistance channel, i.e., as an inlet resistance channel, it limits a flow of the first liquid 102 from the inlet chamber 230 into the fluid chamber 210, i.e., a liquid flow into the fluid chamber 210 is limited. A cross-section and a length of the inlet channel 232 are dimensioned, for example, such that the first liquid 102 only reaches the arrangement 220 of the recesses 222 within the fluid chamber 210 when a predetermined rotational frequency is reached during the rotation 120 for introducing the first liquid 102 into the fluid chamber 210. In a preferred embodiment, inlet structures 236, which have an inlet resistance channel, are combined with outlet structures 240, which have a volume-controlled switch, see the siphon 242 in Fig. 6. This enables a very efficient introduction 126 and removal 130 of the first liquid 102.Alternatively, inlet structures 236 having an inlet resistance channel could also be combined with outlet structures 240 having an outlet resistance channel 244, see Fig. 7. However, it should be noted that the outlet resistance channel 244 should be designed to limit the liquid flow more strongly than the inlet resistance channel.

[0130] If the vent 238 of the fluid chamber 210 is designed as a flow-imitating vent, it limits a flow of the first liquid 102 from the inlet chamber 230 into the fluid chamber 210 by the vent 238 generating an overpressure in the fluid chamber 210 during the introduction 126 of the first liquid 102, i.e., generating a pneumatic counterpressure in the fluid chamber 210. The vent 238, e.g., a cross-section of a vent channel, is designed / dimensioned such that the first liquid 102 only reaches the arrangement 220 of the recesses 222 within the fluid chamber 210 when a predetermined rotational frequency is reached during the rotation 120 for introducing the first liquid 102 into the fluid chamber 210. When inlet structures 236 having a flow-imitating vent are combined with outlet structures 240 having a volume-controlled switch, see the siphon 242 in Fig.6, the flow-imitating vent should be designed such that an overpressure generated thereby in the fluid chamber 210 does not lead to a premature overflow of the siphon 242. The siphon 242 and the flow-limiting vent should be coordinated with one another such that the introduction 126 of the first liquid 102 into the fluid chamber 210 is delayed, but the arrangement 220 of recesses 222 is nevertheless completely overflowed by the first liquid 102. Alternatively, inlet structures 236 having a flow-limiting vent could also be combined with outlet structures 240 having an outlet resistance channel 244, see Fig. 7. In this case, it should be noted that the outlet resistance channel 244 should be designed to limit the liquid flow more strongly than the overpressure generated by the flow-imitating vent in the fluid chamber 210.In particular, the outlet resistance channel 244 and the flow-imitating vent should be coordinated with one another in such a way that the introduction 126 of the first liquid 102 into the fluid chamber 210 is delayed, but the arrangement 220 of recesses 222 is nevertheless completely overflowed by the first liquid 102.

[0131] If the vent 231 of the inlet chamber 230 is designed as a flow-imitating vent, it limits a flow of the first liquid 102 from the inlet chamber 230 into the fluid chamber 210 by the vent 231 generating a negative pressure in the inlet chamber 230 during the introduction 126 of the first liquid 102, i.e., generating a pneumatic negative pressure in the inlet chamber 230. The vent 231, e.g., a cross-section of a vent channel, is designed / dimensioned, for example, such that the first liquid 102 only reaches the arrangement 220 of the recesses 222 within the fluid chamber 210 when a predetermined rotational frequency is reached during the rotation 120 for introducing 126 the first liquid 102 into the fluid chamber 210. When inlet structures 236 having a flow-imitating vent are combined with outlet structures 240 having a volume-controlled switch, see the siphon 242 in Fig.6, the flow-imitating vent should be designed such that a negative pressure generated thereby in the inlet chamber 230 does not lead to a premature exceeding of the siphon 242. The siphon 242 and the flow-limiting vent should be coordinated with one another such that the introduction 126 of the first liquid 102 into the fluid chamber 210 is delayed, but the arrangement 220 of recesses 222 is nevertheless completely overflowed by the first liquid 102. Alternatively, inlet structures 236 having a flow-limiting vent could also be combined with outlet structures 240 having an outlet resistance channel 244, see Fig. 7. In this case, it should be noted that the outlet resistance channel 244 should be designed to limit the liquid flow more strongly than the negative pressure generated by the flow-imitating vent in the fluid chamber 210.In particular, the outlet resistance channel 244 and the flow-imitating vent should be coordinated with one another in such a way that the introduction 126 of the first liquid 102 into the fluid chamber 210 is delayed, but the arrangement 220 of recesses 222 is nevertheless completely overflowed by the first liquid 102.

[0132] During the removal 130 of the first liquid 102, it remains in the depressions 222 and is only removed from areas of the surface 216 outside the depressions 222 in order to form the spatially separated liquid partitions 102 pof the first liquid 102 in the recesses 222 (see Fig. 6 and Fig. 7). As already mentioned, the removal 130 of the first liquid 102 can be carried out by rotating 132, i.e. by rotation, the fluidics module 200 around the center of rotation 122, so that the first liquid 102 is ejected from the fluid chamber 210. The first liquid 102 is removed from the fluid chamber 210 via the outlet 214. This is based on the fact that the siphon 242 in Fig. 6 runs radially outwards after its apex 243 and the alternative outlet resistance channel 244 in Fig. 7 also runs radially outwards from the fluid chamber 210 and thus the centrifugal force acting on the first liquid 102 transports the first liquid 102 radially outwards from the fluid chamber 210. In the case of the siphon 242 in Fig. 6, for example, during the introduction 126 of the first liquid 102 at a time the apex

[0133] 243 of the siphon 242 is exceeded, as a result of which the first liquid 102 is transported radially outward within the siphon 242 and thus removed from the fluid chamber 210. The first liquid 102 is removed during the removal 130, for example, via the siphon 242 in Fig. 6 or via the outlet resistance channel

[0134] 244 in Fig. 7 into a protrusion chamber 246 of the outlet structures 240. The protrusion chamber 246 of the fluidic module 200 is arranged radially further outward than the fluid chamber 210.

[0135] Optionally, the method 100 may further comprise providing 140 a second liquid 104. This is provided, for example, in the inlet chamber 230. The second liquid is not homogeneously miscible with the first liquid and serves, for example, as a sealing liquid.

[0136] After the first liquid 102 has been removed 130 from the fluid chamber 210, the cartridge, i.e., the fluidic module 200, could be stopped to introduce the second liquid 104 into the inlet chamber. Alternatively, the second liquid 104 could also be added while rotating using a further automated process. A structure of the supernatant chamber 246, i.e., the collecting chamber, which is located radially on the very outside, for example, can be designed so as not to transport the first liquid 102 back into the inlet structure 236 or fluid chamber 210 at the moment of stopping. The subsequent rotation 120 for introducing the second liquid 104 can help ensure that the first liquid 102 remains in the supernatant chamber 246. This reduces contamination of the inlet structure 236.

[0137] In one embodiment, the second liquid 104 is introduced 142 into the emptied fluid chamber 210 in the method 100 by rotating 120 the fluidic module 200 about the rotation axis 122 in order to fill the liquid partitions 102 p to seal in the recesses 222. In other words, after the removal 130 of the first liquid 102, a second liquid 104, which is not homogeneously miscible with the first liquid 102, is introduced into the fluid chamber 210 by rotating 120 the fluidic module 200.

[0138] As already mentioned, a first embodiment is directed to a combination of an inlet resistance channel with a fill level-coupled siphon 242, see Fig. 6. An array chamber, i.e. the fluid chamber 210, is completely filled from the inlet chamber 230, or until at least the arrangement 220 of depressions 222 has been at least completely overflowed. A flow resistance between the inlet chamber 230 and the array chamber ensures that the filling of the depressions 222 is delayed after the centrifuge has started up and therefore only occurs at a sufficiently high rotation frequency (i.e. centrifugal force). The fill level is controlled via the siphon 242, which allows the fluid chamber 210 to be emptied into a third chamber (overflow chamber 246) only after the array chamber has been completely filled or only after at least the arrangement 220 of depressions 222 has been at least completely overflowed.The array chamber can then be filled with a second liquid 104 to seal the recesses 222. In a similar embodiment, the third chamber can be expanded by an additional, radially inner chamber, since penetrating second liquid 104 can displace the first liquid 102 from the supernatant chamber 246 due to density differences.

[0139] As also already mentioned above, a second embodiment is directed to a resistance channel at the outlet 214, i.e. to an outlet resistance channel 244, see Fig. 7. A fluid chamber 210 (array chamber) with a locally limited array, i.e. with a locally limited arrangement 220 of recesses 222, is completely filled from the inlet chamber 230, or until at least the arrangement 220 of recesses 222 has been at least completely overflowed. The fill level is controlled via the resistance of the outlet channel, i.e. via the outlet resistance channel 244, in order to enable a temporary complete filling of the fluid chamber 210, or a complete filling of all recesses 222 of the arrangement 220 of recesses 222. For this purpose, the first liquid 102 should flow into the array chamber significantly faster than it can flow out through the outlet 214.Nevertheless, as previously described, a high centrifugal force should already be present when the first liquid 102 flows into the array chamber. This can be achieved, for example, by a very fast-starting centrifuge, by an inlet resistance channel, by a volume-coupled siphon after the inlet chamber 230, i.e., between the inlet chamber 230 and the fluid chamber 210, or by a flow-imitating vent 238. The array chamber is then filled, for example, with a second liquid 104 to seal the depressions 222. In a similar embodiment, the supernatant chamber 246 can be expanded by a further, radially inner chamber, since the penetrating second liquid 104 can displace the first liquid 102 from the supernatant chamber 246 due to density differences. Further embodiments are based on the previously mentioned flow-limited venting of the fluid chamber 210, i.e.,The vent 238 is implemented as a flow-limited vent. The fluid chamber 210 (array chamber) with a locally limited array, i.e., with a locally limited arrangement 220 of recesses 222, is completely filled from the inlet chamber 230, or until at least the arrangement 220 of recesses 222 has been completely overflowed. The fill level is controlled via the flow-limited resistance of the array chamber vent 238 in order to enable a temporary complete filling of the fluid chamber 210, or a complete filling of all recesses 222 of the arrangement 220 of recesses 222. For this purpose, the siphon 242, see Fig. 6, should be designed such that, on the one hand, an overpressure of air is established when filling the array chamber, but this does not lead to a premature overpressure of the siphon 242. For a combination of flow-limited venting and resistance channel at the outlet 214, i.e. the outlet resistance channel 244, see Fig.7, the resistance of the outlet resistance channel 244 or the ratio of the resistance of the outlet resistance channel 244 to a resistance caused by the flow-limited venting at the inlet 212 should be selected such that, on the one hand, an air overpressure is created in the array chamber during filling (due to the flow-limited venting), and on the other hand, the array chamber is temporarily filled in order to fill all recesses 222 with the first liquid 102 (through the resistance channel at the outlet 214).

[0140] The present invention achieves the general advantages of geometrically defined systems over droplet-based systems, as already explained in the introduction to the present application. The following points relate to the advantages of the invention over existing geometrically defined systems in the prior art:

[0141] • The invention enables particularly low costs per microfluidic module because the device can be realized entirely in plastic, i.e., the fluidic module 200 can comprise plastic material. In contrast to typical prior art systems, expensive materials such as glass or silicon in the chip can be omitted.

[0142] • The filling of a large number of wells 222 can be accomplished very quickly because it is performed over a large area, i.e., without individual inlet channels. This allows a virtually unlimited number of wells 222 to be filled in an acceptable time, since resistance in the inlet channels is eliminated. Problems caused by the adsorption of biomolecules on the channel walls are also minimized.

[0143] • The method 100 enables a high spatial integration density of the recesses 222 since, in contrast to the prior art, no connecting channels are required between the recesses 222.

[0144] • By using centrifugal force to fill the recesses 222, no hydrophilization or hydrophobization is required. This saves manufacturing costs and enables the use of fluids with different contact angle properties.

[0145] • A key advantage over the prior art is the significantly reduced dead volume. Centrifugal forces overcome the disruptive capillary forces, allowing a defined filling of the wells 222 despite the small gap 218 in the array chamber, i.e., the fluid chamber 210. A reduced dead volume is essential in many applications, e.g., to save valuable samples or costly reagents.

[0146] The device presented here, i.e., the fluidics module 200, and the method 100 allow the microfluidic filling of planar arrangements 220 of wells 222 with a particularly low dead volume. A person skilled in the art would either use droplet-based systems for this purpose (see above for disadvantages) or accept the high dead volume of well-based systems.

[0147] It is also surprising that in the described method 100 for filling and sealing the recesses 222, no instabilities of the partitions 102 p observed. Under centrifugation, one would expect the first liquid 102, which preferably has a lower density, to rise in the vicinity of the second liquid 104, which preferably has a higher density.

[0148] The following describes additional embodiments and aspects that may be used alone or in combination with the features and functions described herein.

[0149] One embodiment relates to a device, ie, a fluidics module 200, which has geometrically defined recesses 222 for separating particles and / or molecules. The recesses 222 can be filled using centrifugal forces. To achieve high centrifugal forces when a liquid, e.g., the first liquid 102, enters the fluid chamber 210, the fluidics module 200 can

[0150] • have a resistance channel, ie the inlet resistance channel, or

[0151] • be designed to exert a pneumatic counterpressure on the liquid to be introduced, e.g. via the flow-limited venting of the fluid chamber, or

[0152] • be designed to exert a pneumatic pressure on the liquid to be introduced, e.g. via the flow-limited venting of the inlet chamber, or

[0153] • have a volume-controlled switch, e.g. the Siphon 242, or

[0154] • have a very fast accelerating centrifuge.

[0155] One embodiment relates to a device, ie a fluidic module 200, which has the geometrically defined recesses 222 for separating particles and / or molecules in a fluidic part and / or in a cover part of the fluidic module 200.

[0156] An initial creation of the recesses 222 is carried out, for example, by means of photolithography, interference lithography, laser lithography, micro-milling, laser machining, electric discharge machining, chemical processes such as etching processes, roll processes such as roll-to-roll, roll-to-foil, roll-to-plate or additive processes such as 2D or 3D printing processes.

[0157] Optionally, the device, ie the fluidic module 200, is made entirely of plastic.

[0158] According to one embodiment, the recesses 222 of the arrangement 220 of recesses 222 are limited to a defined area of ​​the fluidic module 200, i.e., to a limited region on the surface 216 of the fluid chamber 210 of the fluidic module 200. Alternatively, the arrangement 220 of recesses 222 can extend over the entire surface 216, i.e., the recesses 222 are completely distributed over a single surface 216, i.e., a surface 216 of an inner wall of the fluid chamber 210. The recesses 222 can, for example, be round, rectangular, pyramidal, cylindrical, hemispherical, spherical segment-shaped, conical, truncated cone-shaped, truncated pyramid-shaped, semi-ellipsoidal, ellipsoidal segment-shaped, prism-shaped, and / or cuboid-shaped.

[0159] In one embodiment, recesses 222 of different sizes or volumes and / or different geometries are combined.

[0160] A further embodiment relates to a method 100 in which the filling of the depressions 222 with a first liquid 102, ie the introduction 126 of the first liquid 102, and a sealing of these with a second liquid 104, ie the introduction 142 of the second liquid 104, takes place by a surface flow over the arrangement 220 of depressions 222.

[0161] The first liquid 102 comprises, for example, a reaction mix for detecting nucleic acids or a reaction mix for detecting proteins and / or metabolites.

[0162] The second liquid 104 is, for example, a mineral oil or a synthetic oil.

[0163] Optionally, the fluidic module 200 is subjected to a defined temperature to carry out a reaction (isothermal assay). The fluidic module 200 can, for example, have a heater or cooler configured to heat the liquid partitions 102. pin the recesses 222 of the fluid chamber 210 to the predetermined temperature. Optionally, the fluidic module 200 is subjected to a temperature profile to carry out the reaction (thermo-cycling assay).

[0164] The reaction in the partitions 102 p can be observed in real time. Optionally, the reaction is displayed in partitions 102 p evaluated at one endpoint.

[0165] Optionally, the method 100 comprises removing products of the reaction from the partitions 102 p on.

[0166] The liquid, e.g., the first liquid 102, which is in the liquid partitions 102 p may comprise, for example, molecules such as nucleic acids, proteins and / or metabolites. Additionally or alternatively, the liquid, e.g., the first liquid 102, which is divided into the liquid partitions 102 pdivided into particles such as cells, extracellular vesicles and / or beads.

[0167] Referring now to Figures 8A and 8B, examples of fluid handling devices in the form of centrifugal microfluidic systems according to examples of the invention will be described, respectively, using or including a fluidic module 200 as described herein. In other words, the fluidic module 200 in the systems in Figures 8A and 8B may be any of the fluidic modules described herein.

[0168] Fig. 8A shows a fluid handling device with a fluidics module 200 in the form of a rotating body 1110, which has a substrate 1112 and a cover 1114. The substrate 1112 and the cover 1114 can be circular in plan view, with a central opening through which the rotating body 1110 can be attached to a rotating part 1118 of a drive device 1120 via a conventional fastening device 1116. The rotating part 1118 is rotatably mounted on a stationary part 1122 of the drive device 1120. The drive device 1120 can be, for example, a conventional centrifuge, which can have an adjustable rotation speed, or a CD or DVD drive. A control device 1124 may be provided which is designed to control the drive device 1120 in order to subject the rotation body 1110 to a rotation or rotations at different rotational frequencies.The control device 1124 can, as will be apparent to those skilled in the art, be implemented, for example, by an appropriately programmed computing device or a user-specific integrated circuit. The control device 1124 can further be configured to control the drive device 1120 in response to manual inputs from a user in order to effect the required rotations of the rotating body. In any case, the control device 1124 can be configured to control the drive device 1120 in order to impart the required rotation to the rotating body in order to implement examples of the invention as described herein. A conventional centrifuge with only one direction of rotation can be used as the drive device 1120.

[0169] The rotating body 1110 includes the fluidic structures that form the fluidic modules 200 as described herein. The fluidic structures may be formed by cavities and channels in the cover 1114, the substrate 1112, or in the substrate 1112 and the cover 1114. The substrate is also referred to herein as the fluidic part, and the cover is also referred to herein as the cover part. In examples, fluidic structures may be depicted in the substrate 1112, while fill openings and vent openings are formed in the cover 1114. In examples, the structured substrate (including fill openings and vent openings) is arranged on top, and the cover is arranged on the bottom.

[0170] In an alternative example shown in Fig. 8B, fluidic modules 200 are inserted into a rotor 1130 and, together with the rotor 1130, form the rotating body 1110. The fluidic modules 200 can each have a substrate and a cover, in which corresponding fluidic structures can be formed. The rotating body 1110 formed by the rotor 1130 and the fluidic modules 200 can, in turn, be subjected to rotation by the drive device 1120, which is controlled by the control device 1124.

[0171] In Figures 8A and 8B, the center of rotation around which the fluidic module or the rotation body can rotate is again denoted by R.

[0172] In examples of the invention, the fluidic module or the rotating body comprising the fluidic structures can be formed from any suitable material, for example a plastic such as PMMA (polymethyl methacrylate), PC (polycarbonate), PVC (polyvinyl chloride) or PDMS (polydimethylsiloxane), glass or the like. The rotating body 1110 can be considered a centrifugal microfluidic platform. In preferred examples, the fluidic module or the rotating body can be formed from a thermoplastic such as PP (polypropylene), PC, COP (cyclic olefin polymer), COC (cyclo olefin copolymer) or PS (polystyrene).

[0173] In examples, the drive device 1120 and any of the fluidic modules 200 described herein form an example of a fluid handling device according to the invention. In examples, the drive device 1120 is configured, for example, controlled by the controller 1124, to, in a first phase, rotate the fluidic module 200 at a rotational frequency to exert a centrifugal force on a liquid 102, by which the liquid 102 is introduced into the fluid chamber 210 of the fluidic module 200 and by which at least a portion of the liquid 102 is transported from radially outside to radially inside via the array 220 of recesses 222 within the fluid chamber 210, and to, in a second phase, continue to rotate the fluidic module 200 at the rotational frequency or increase the rotational frequency to remove the liquid 102 from areas of the surface 216 outside the recesses 222.

[0174] In examples, the drive device 1120 is configured to perform a method 100 as described above with reference to Figures 1 to 7. Examples of the present invention provide corresponding methods 100 for effectively creating spatially separated fluid partitions. Such methods 100 may be performed using fluidic modules and fluid handling devices as described herein.

[0175] Although features of the invention have been described in each case with reference to device features or method features, it is obvious to those skilled in the art that corresponding features can also be part of a method or device. Thus, the device can be configured to perform corresponding method steps, and the respective functionality of the device can represent corresponding method steps.

[0176] In the foregoing Detailed Description, various features have been grouped together in examples in order to streamline the disclosure. This manner of disclosure should not be interpreted as intending that the claimed examples include more features than are expressly recited in each claim. Rather, as the following claims reflect, the subject matter may lie in fewer than all of the features of a single disclosed example. Accordingly, the following claims are hereby incorporated into the Detailed Description, with each claim being capable of standing as its own separate example.While each claim may stand as its own separate example, it should be noted that although dependent claims in the claims refer to a specific combination with one or more other claims, other examples also include a combination of dependent claims with the subject matter of any other dependent claim or a combination of any feature with other dependent or independent claims. Such combinations are intended to be encompassed unless it is stated that a specific combination is not intended. Furthermore, it is intended to encompass a combination of features of a claim with any other independent claim, even if that claim is not directly dependent on the independent claim.

[0177] The examples described above are merely illustrative of the principles of the present disclosure. It is understood that modifications and variations of the arrangements and details described will be apparent to those skilled in the art. Therefore, it is intended that the disclosure be limited only by the appended claims and not by the specific details set forth for the purpose of describing and explaining the examples.

[0178] References

[0179] [Witters_2014] Witters et al., Digital biology and chemistry, Lab Chip, 2014, 14, 3225-3232

[0180] [Kivioja _2012] Kivioja et al., Counting absolute numbers of molecules using unique molecular identifiers, Nature Methods volume 9, pages 72-74 (2012)

[0181] [Schulz_2021] Schulz et al., High Dynamic Range Digital Assay Enabled by DualVolume Centrifugal Step Emulsification, Anal. Chem. 2021, 93, 5, 2854-2860

[0182] [Schuler_2015] Schuler et al., Centrifugal step emulsification applied for absolute quantification of nucleic acids by digital droplet RPA, Lab Chip, 2015, 15, 2759-2766

[0183] [Teh_2008] Teh et al., Droplet microfluidics, Lab Chip, 2008, 8, 198-220

[0184] [Bio-Rad_2023] Bio-Rad QX200, htps: / / www.bio-rad.com / de-de / life- science / digital-pcr / qx200-droplet-digital-pcr-system, Website aufgerufen am 01.03.2023

[0185] [Stilla_2023] Stilla Naica, htps: / / www.stillatechnoloQies.com / multiplex- pcr / digital-pcr / , Website aufgerufen am 01 .03.2023

[0186] [Liao_2017] Liao et al., Digital PCR: Endless Frontier of ‘Divide and Conquer, Micromachines 2017, 8, 231

[0187] [QIAGEN_2023] QIAGEN QIAcuity, https: / / www.qiaqen.com / de- de / applications / digital-pcr / products, Website aufgerufen am 01.03.2023

[0188] [Quanterix_2023] Quanterix Simoa, htps: / / www.quanterix.com / simoa-technoloqv / ,

[0189] Website accessed on March 1, 2023 [ThermoFisher_2023._1] ThermoFisher QuantStudio Absolute Q, https: / / www.thermofisher.com / de / de / home / life- science / pcr / digital-pcr / quantstudio-absolute-q- system.html, Website accessed on March 1, 2023

[0190] [ThermoFisher_2023. _1] ThermoFisher QuantStudio 3D Digital PCR System, https: / / www.thermofisher.com / de / de / home / life-science / pcr / digital-pcr / quantstudio-3d-digital-pcr-system-emea.html, website accessed on March 1st, 2023

[0191] [Fluidigm_2023] Fluidigm Digital Array IFC for Digital PCR, https: / / store.standardbio.com / ccrz _ ProductDetails?sku=BMK-M-

[0192] 48,770&cclcl=en_US, website accessed on 01.03.2023

[0193] [JN-MEDSYS_2023] JN MEDSYS Clarity Digital PCR, https: / / www.jnmedsys.com / clarity-digital-pcr / , website accessed on March 1, 2023

[0194] [Roche_2023] Roche Digital LightCycler, https: / / diagnostics.roche.com / global / en / products / systems / digital- lightcycler-system-sys-362.html, Website aufgerufen am 01.03.2023

[0195] [lllumina_2023] Illumina Microarray Technology, https: / / www.illumina.com / science / technology / microarray.html, Website aufgerufen am 01.03.2023

[0196] [Podbiel_2021] Podbiel et al., Fusing MEMS technology with lab-on-chip, Microsystems & Nanoengineering (2020) 6:82

[0197] [Henley_2020] Henley et al., Spatially isolated reactions in a complex array: using magnetic beads to purify and quantify nucleic acids with digital and quantitative realtime PCR in thousands of parallel microwells, Lab Chip, 2020, 20, 1771-1779

[0198] [US8277759B2] University of Utah, Patent US 8,277,759 B2, MICROFLUIDIC FLOW CELL

[0199] [US9487822B2] Fluidigm, Patent US 9,487,822 B2, METHOD AND APPARATUS FOR DETERMINING COPY NUMBER VARATION USING DIGITAL PCR

[0200] [EP3357575B1] Roche, Patent EP 3 357 575 B1 , SEALABLE MICROFLUIDIC CHIP AND METHOD FOR THERMOCYCLING

[0201] [US10967370B2] Roche, Patent US 10,967,370 B2, MICROFLUIDIC DEVICE AND METHOD FOR MANUFACTURING THE SAME [2012_Kan] Kan et al., Isolation and detection of single molecules on paramagnetic beads using sequential fluid flows in microfabricated polymer array assemblies, Lab Chip, 2012, 12, 977

[0202] [WO2021211754A2] QUANTERIX CORP, Patent WO 2021 / 211754 A2, METHODS AND SYSTEMS RELATED TO HIGHLY SENSITIVE ASSAYS AND DELIVERING CAPTURE OBJECTS

[0203] [WO2012103447A1] QUANTERIX CORP, Patent WO 2012 / 103447 A1 , SYSTEMS, DEVICES, AND METHODS FOR ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES

[0204] [2010_Rissin] Rissin et al., Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations, nature biotechnology, 2010, 28, 595-599

Claims

Claims 1. A method (100) for generating spatially separated liquid partitions (102p) in an array (220) of depressions (222) formed in a surface (216), comprising the following features: Providing (110) a fluidic module (200); Rotating (120) the fluidic module (200), which has a fluid chamber (210) having the surface (216) in which the arrangement (220) of recesses (222) is formed, about a center of rotation (122) to exert a centrifugal force on a liquid (102), by which the liquid (102) is introduced (126) into the fluid chamber (210), and by which at least a portion of the liquid (102) is transported from radially outside to radially inside via the arrangement (220) of recesses (222); and Removing (130) the liquid (102) from areas of the surface (216) outside the depressions (222) in order to form the spatially separated liquid partitions (102 p) of the liquid (102) in the recesses (222).

2. Method (100) according to claim 1, wherein the liquid (102) in the areas outside the depressions (222) is removed (130) from the fluid chamber (210) by rotating (132) the fluidic module (200) via an outlet (214), wherein after the removal (130) a second liquid (104) which is not homogeneously miscible with the liquid (102) is introduced (142) into the fluid chamber (210) by rotating (120) the fluidic module (200) in order to fill the depressions (222) with the liquid partitions (102) contained therein. p ) to seal.

3. Method (100) according to claim 1 or 2, wherein the fluid chamber (210) forms a gap (218) between the surface (216) in which the arrangement (220) of depressions (222) is formed and a surface opposite this surface (216), wherein the rotation (120) of the fluidic module (200) takes place at a speed such that the centrifugal force acting on the liquid volume is higher than the capillary force acting on the liquid (102) in the gap (218).

4. The method (100) according to any one of claims 1 to 3, wherein a flow of liquid into the fluid chamber (210) is limited when the liquid (102) is introduced (126) into the fluid chamber (210).

5. The method (100) of claim 4 when appended to claim 3, wherein limiting the flow of liquid into the fluid chamber (210) prevents the liquid (102) in the fluid chamber (210) from reaching the array (220) of recesses (222) before the centrifugal force acting on the volume of liquid is higher than the capillary force acting on the liquid (102) in the gap (218).

6. The method (100) of claim 4 or 5, which uses an inlet resistance channel (232) in the fluidic module (200) opening into the fluid chamber (210) to limit the flow of fluid into the fluid chamber (210).

7. The method (100) of claim 4 or 5, wherein a pneumatic back pressure is generated in the fluid chamber (210) to limit the flow of fluid into the fluid chamber (210).

8. The method (100) of claim 7, wherein the pneumatic back pressure is effected by a flow-limited venting (238) of the fluid chamber (210).

9. The method (100) of claim 4 or 5, wherein a pneumatic vacuum is generated in the inlet chamber (230) to limit the flow of fluid into the fluid chamber (210).

10. The method (100) according to claim 9, wherein the pneumatic depression is effected by a flow-limited venting (231) of the inlet chamber (230).

11. Method (100) according to one of claims 1 to 10, in which a volume-controlled switch (242) which is fluidically coupled to one or the outlet (214) of the fluid chamber (210) is used, which enables emptying (130) of the fluid chamber (210) only after the arrangement (220) has been completely overflowed by the liquid (102).

12. The method (100) according to any one of claims 1 to 10, wherein an outlet resistance channel (244) fluidically coupled to one or the outlet (214) of the fluid chamber (210) is used, which prevents emptying (130) of the fluid chamber (210) until the arrangement (220) has been completely overflowed by the liquid (102).

13. A fluidics module (200) for generating spatially separated fluid partitions (102p) using a method (100) according to claim 1, comprising: the fluid chamber (210) having the surface (216) in which the array (220) of recesses (222) is formed; Inlet structures (236) designed to introduce (126) liquid (102) into the fluid chamber (210) by means of centrifugal force upon rotation (120) of the fluidic module (200) in order to transport the liquid (102) from radially outside to radially inside via the arrangement (220) of recesses (222); and Outlet structures (240) which are designed to remove the same liquid (102) from regions of the surface (216) outside the depressions (222) after flowing over the surface (216) in which the arrangement (220) of depressions (222) is formed, in order to separate the spatially separated liquid partitions (102 p ) of the liquid (102) in the recesses (222).

14. Fluidics module (200) according to claim 13, wherein the fluid chamber (210) forms a gap (218) between the surface (216) in which the arrangement (220) of depressions (222) is formed and a surface opposite this surface (216), wherein a gap height of the gap (218) is at most 10 times the depth of the depressions (222) and / or the gap height is at most 200 pm, preferably at most 100 pm.

15. Fluidic module (200) according to claim 13 or 14, wherein the inlet structures (236) comprise an inlet chamber (230) which is arranged at least partially radially further inward than an innermost portion of the arrangement (220) of Recesses (222), and a fluid channel (232) connecting the inlet chamber to the fluid chamber (210).

16. Fluidics module (200) according to one of claims 13 to 15, wherein the inlet structures (236) have an inlet resistance channel (232) which is designed to limit the liquid flow into the fluid chamber (210), or wherein the fluid chamber (210) has a flow-limited vent (238) which is designed to generate a pneumatic counterpressure in the fluid chamber (210) upon introduction (126) of the liquid (102) into the fluid chamber (210) in order to limit the liquid flow into the fluid chamber (210), or wherein the inlet chamber (230) has a flow-limited vent (231) which is designed to generate a pneumatic negative pressure in the inlet chamber (230) upon introduction (126) of the liquid (102) into the fluid chamber (210) in order to limit the liquid flow in to limit the fluid chamber (210).

17. Fluidics module (200) according to one of claims 13 to 16, wherein the outlet structures (240) comprise a volume-controlled switch (242) which only enables emptying of the fluid chamber (210) after the arrangement (220) has been completely overflowed by the liquid (102), or an outlet resistance channel (244) which prevents emptying of the fluid chamber (210) until the arrangement (220) has been completely overflowed by the liquid (102).

18. Apparatus for carrying out a method (100) according to one of claims 1 to 12, comprising: a fluidic module (200) according to one of claims 13 to 17; a drive device (1120) for rotating the fluidic module (200).

Images