Method and apparatus for generating isolated liquid subvolumes

Abstract

Method for generating isolated liquid subvolumes in a plurality of micro-wells of a reservoir comprising a base, at least one sidewall and a single opening at least partially spanning the base. The plurality of micro-wells is arranged on the base and / or on the at least one sidewall. The method comprises introducing a sample liquid into the reservoir through the single opening; and introducing a sealing material into the reservoir through the single opening to seal subvolumes of the sample liquid in the plurality of micro-wells using a movement of the sealing material towards the base due to a body force applied to the sealing material.

Description

[0001] Method and apparatus for generating isolated liquid subvolumes

[0002] Description

[0003] Embodiments according to the invention relate to a method and an apparatus for generating isolated liquid subvolumes, e.g., like a high-density digital assay apparatus and a method for generating isolated liquid compartments.

[0004] The presented invention relates to so called “digital assays” for performing various types of biochemical analysis in molecular biology and microbiology [1999_Vogelstein] [2012_Kan], In such digital assays, the sample to be analyzed is mixed with liquid reagents and this assay mix is split into a large number of small volumes confined in individual compartments (typically thousands to millions) that are separated from each other. Following the paradigm of digital assays, the final result of the analysis can be determined by reading the either positive or negative signals in all of the individual compartments and determining the analytical result by using statistical methods. This approach allows for higher precision, sensitivity and the possibility to perform reference free absolute quantification [2017_Zhang] [2023_Kuo], In particular, to perform such an assay, apparatus and methods are needed to separate the assay mix into individual compartments. According to the state of the art, compartments consisting of liquid micro droplets in oil [2008_Teh], or physical micro-well arrays [1998_J ackman], have been used to generate physically separated liquid micro volumes as compartments for the assay reaction. The presented invention relates to a novel apparatus and method for producing isolated, closed liquid compartments from a bulk liquid volume by enclosing the liquid compartments physically into micro-wells that are sealed by a sealing material that can be either a non-miscible liquid or a solid material. The apparatus and the method of the presented invention are particularly suitable for generating isolated compartments for use in digital assays. The invention provides various advantages compared to the state-of-the art in terms of 1. simpler instrumentation (and therefore less costly and more robust) to produce the compartments and to run the assays, as well as 2. enabling a higher degree of integration (i.e. more compartments per unit area e.g. per well) and parallelization (i.e. more different samples per unit area e.g. per assay plate) for higher sensitivity and increased throughput, as well as 3. providing the opportunity to easily recover the sample liquid 60 from the micro compartments into a bulk liquid solution for subsequent analysis (e.g. by NGS methods).

[0005] In general, two strategies for generation of liquid compartments for digital assays are described in the state of the art:

[0006] II24EM02-2024323325.DOCX mh • Micro droplets take advantage of non-miscibility between a first liquid (e.g. a PCR mastermix) and a second liquid (e.g. a mineral oil) [2008_Teh], The liquid-liquid interface might be stabilized by adding surfactants. However, micro droplets are prone to merge, in particular during thermocycling [2021_Schlenker], Thus, identification of robust chemistry is challenging, time-consuming and might interfere with the desired reaction inside the micro droplets.

[0007] • Arrays of micro-wells circumvent this issue by significantly reducing the liquid-liquid interface [1998_Jackman] [2012_Kan], However, sealing of at least one open surface of the micro-wells is still required in order to realize further process steps, such as thermocycling without significant loss of assay liquid by evaporation.

[0008] Two kind of well array technologies can be distinguished:

[0009] • Arrays of micro-wells with fluidic channels between individual wells [US20190217298A1] [2014_Zhu], Here, a major limitation is the fluidic resistance caused by such a network of thousands of channels, causing long process times or requires high system pressure for filling. Additionally, the risk of losing target biomolecules is increased as the network of channels causes a high surface-to-volume ratio.

[0010] • Consequently, arrays of micro-wells without fluidic channels between individual wells [1998_Jackman] [2012_Kan] are desirable, as they significantly reduce flow resistance during filling and, due to overall less surface area, reduce the risk of biomolecule adsorption.

[0011] Filling of micro-well arrays without fluidic channels between individual wells is typically solved by a suitable contact angle between the sample liquid and the micro-well surface (wetting behavior, if required using surface treatment for hydrophilization of hydrophobization or additives in the sample liquid for increased or decreased contact angle) and / or by optimizing the micro-well geometry, e.g. using tilted wall angles or hexagonal well shapes [EP3714977A1],

[0012] Subsequent sealing of liquid filled micro-wells without fluidic channels between individual wells can be based on:

[0013] • A solid material used as sealing layer, for example using an adhesive tape or a flexible material such as silicone or PDMS. However, such systems typically require additional instruments for defined and continuous application of the sealing layer [US10385383B2] [2012_Men],

[0014] I I24EM02-2024323325. DOCX mh • A liquid sealing material, for example a non-miscible liquid such as mineral oil. However, systems in the state-of-the-art typically require either an additional instrument in order to press or suck the non-miscible liquid inside the system [2012_Kan] or a geometry of microfluidic channels and chambers in order to guide the flow of the non-miscible liquid inside the system [EP3714977A1] [US20220305493A1], Avoiding additional instruments is always desirable in order to reduce cost. Avoiding microfluidic channels and chambers in the system is desirable because they can cause long process time (due to flow resistance), loss of target biomolecules (due to increased probability of biomolecule adsorption at surfaces), and increased fabrication cost (due to high requirements on fabrication tolerances).

[0015] In summary, according to the state-of-the-art, there are no satisfactory solutions known, to fill and seal liquid filled micro-wells by a layer of non-miscible liquid, without the use of either fluidic channels between individual wells or an additional instrument or the use of microfluidic channels or more than one of these characteristics.

[0016] Prior art US20220305493A1 describes a microfluidic device for processing and aliquoting a sample liquid. However, this prior art requires a pump for processing the liquids and a network of microfluidic channels to realize sample partitioning and sealing of micro-wells.

[0017] Prior art EP3357575B1 describes a favorable relation of densities between sample liquid, displacement liquid and sealing liquid (density sample liquid > density sealing liquid). Same as US20220305493A1, the invention EP3357575B1 describes a system using microfluidic channels in order to realize functionality.

[0018] Prior art EP3714977A1 describes a method for filling and sealing of micro-wells employing different liquid contact angles at different walls inside a microfluidic chip, in order to assure robust filling and sealing of micro-wells. Such differences are typically achieved by surface treatments, such as plasma treatment or SiC>2 coating. Such treatments cause extra cost during production and, thus, are not favorable. Additionally, same as US20220305493A1 and EP3357575B1, the invention EP3714977A1 requires a microfluidic channel in order to realize filling and sealing of micro-wells.

[0019] Prior art US10213761 B2 describes use of a higher density liquid in order to displace parts of a coating liquid from the surface of a micro-well array. However, the purpose of this invention is to realize a coating process for micro-well arrays, and not a method for filling and sealing of well arrays with a sample liquid, which is not possible with this prior-art method.

[0020] I I24EM02-2024323325. DOCX mh Prior art US10767226B2 describes the use of the interaction of surface tension and capillary forces between a first sample liquid and a second sealing liquid in order to fill and seal microwells. However, same as US20220305493A1 and other prior art, the system described in US10767226B2 requires microfluidic channels in order to realize its function.

[0021] Therefore, it is desired to provide a concept for producing isolated, closed liquid compartments from a bulk liquid volume which makes a better compromise between simplifying instrumentation, enabling a high degree of integration and parallelization for high sensitivity and high throughput, as well as providing the opportunity to easily recover the assay liquid from the micro compartments into a bulk liquid solution for subsequent analysis.

[0022] This is achieved by the subject matter of the independent claims of the present application.

[0023] Further embodiments according to the invention are defined by the subject matter of the dependent claims of the present application.

[0024] In summary, none of the available prior art discloses an apparatus and method for isolating liquids into micro compartments without the use of any micro channels and by exploiting a body force to the sealing material, as proposed by the present invention.

[0025] Summary of the Invention

[0026] In accordance with an aspect of the present invention, the inventors of the present application realized that one problem encountered when trying to generate isolated liquid subvolumes stems from the fact that the usage of fluidic channels causes long process times or requires high system pressure and increases the risk of losing sample material. According to this aspect of the present application, this difficulty is overcome by introducing a sample liquid and a sealing liquid into a reservoir with micro-wells through an opening of the reservoir, e.g., without using channels for directing the liquids towards the opening, without using channels for directing the sample liquid over a surface area in which the micro-wells are arranged, and without using channels for directing the sample liquid towards a further reaction chamber or an outlet. The inventors found, that it is possible to generate well isolated liquid subvolumes using a body force and that the usage of the body force makes channels for controlling a flow of liquids obsolete. This is based on the idea that a sealing material may move through the bulk layer of sample liquid to a base of the reservoir by applying the body force to the sealing material and that the sealing material thus seals subvolumes of the sample liquid in the microwells. In particular, it was noted that due to the small dimensions of the micro-wells, surface

[0027] I I24EM02-2024323325. DOCX mh tension forces are higher compared to the body forces typically applied for moving the sealing material towards the base, which prevents a displacement of the sample liquid out of the microwells, even if the sealing material is a liquid. Thus, the sealing material efficiently displaces the bulk sample liquid outside the micro-wells by a movement induced by the body force and separates a sample liquid subvolume within a micro-well from sample liquid subvolumes within neighboring micro-wells. This approach simplifies significantly the instrumentation, since channels are not necessary and the body force can be applied to all micro-wells or even a plurality of reservoirs each containing a multitude of micro-wells simultaneously. Additionally, a high degree of parallelization and a high throughput is achieved, since the inventive approach allows to have micro-wells everywhere on the base and / or on the inner walls of a reservoir.

[0028] Accordingly, an aspect of the present application is directed to a method for generating isolated liquid subvolumes in a plurality of micro-wells of a reservoir. The reservoir comprises a base, at least one sidewall and a single opening at least partially spanning the base. The plurality of micro-wells is arranged on the base and / or on the at least one sidewall. The method comprises introducing a sample liquid into the reservoir through the single opening; and introducing a sealing material into the reservoir through the single opening to seal subvolumes of the sample liquid in the plurality of micro-wells using a movement of the sealing material towards the base due to a body force applied to the sealing material. The sealing material, for example, can be either a non-miscible liquid, i.e. non-miscible with the sample liquid, or a solid material. After the sealing material has been introduced in the reservoir, for example, all micro-wells of the plurality of micro-wells are covered by the sealing material, e.g., the sealing material isolates the subvolumes of the sample liquid in the plurality of micro-wells from a bulk sample liquid volume, i.e. the sealing material is arranged between the subvolumes of the sample liquid in the plurality of micro-wells and the bulk sample liquid volume.

[0029] According to an embodiment, the body force comprises at least one of a gravitational force, a centrifugal force and a magnetic force, i.e. the body force may represent a gravitational force, a centrifugal force or a magnetic force or may represent a combination of forces, like a combination of the gravitational force and the centrifugal force or a combination of the gravitational force and the magnetic force or a combination of the centrifugal force and the magnetic force or a combination of all three forces. The body force is a force that acts throughout the volume of the sealing material, as opposed to a surface force or a convective force applied by a pumping system, which are not acting everywhere on the materials. Body forces result from long-range field interactions, such as gravitational, rotational or magnetic fields, and affect every part of the sealing material directly and simultaneously. For example, a gravitational force acts on every part of a body, i.e. the sealing material due to the earth's

[0030] I I24EM02-2024323325. DOCX mh gravity or other gravitational fields; a magnetic or electromagnetic force acts on charged particles within a body affecting the entire material, i.e. the sealing material, and a centrifugal force may be experienced by objects in a rotating reference frame, acting throughout the volume of the object, i.e. the sealing material. The body force acts uniformly throughout the volume of the sealing material affecting every particle within the sealing material, not just those at the surface. In case of the body force comprising a magnetic force, the sealing material may comprise ferromagnetic properties. In case of the body force comprising a gravitational force and / or a centrifugal force, the sealing material may have a higher density than the sample liquid. In case of the body force comprising a gravitational force and a magnetic force or a centrifugal force and a magnetic force or a gravitational force, a magnetic force and a centrifugal force, the sealing material may have a higher density than the sample liquid and may comprise ferromagnetic properties.

[0031] According to an embodiment, the sample liquid is introduced into the reservoir through the single opening to arrange the subvolumes of the sample liquid in the plurality of micro-wells, i.e. all micro-wells of the plurality of micro-wells are directly filled by the sample liquid. For example, the method involves adding enough, i.e. a predefined amount of, sample liquid into the reservoir to fill and cover all micro-wells. This has the advantage that a type of the sealing material and an introduction method for the sealing material, can be freely chosen simplifying the generation of isolated liquid subvolumes. This is especially advantageous in cases where the sealing material is a non-miscible liquid, since it allows the sealing material to be simply added after the insertion of the sample liquid without having to worry about filling a micro-well with the sealing material before it is filled with the sample liquid. Thus, an easy and efficient generation of isolated liquid subvolumes in the plurality of micro-wells is achieved.

[0032] According to an alternative embodiment, upon introducing of the sample liquid into the reservoir through the single opening, the plurality of micro-wells are not, or at least not partially, filled with the sample liquid. The movement of the sealing material towards the base due to the body force lifts the sample liquid in the direction of the single opening to arrange subvolumes of the sample liquid in the plurality of micro-wells (i.e. in the micro-wells of the plurality of microwells, which are not yet filled when the sample liquid is introduced in the reservoir) before sealing the respective subvolumes of the sample liquid in the plurality of micro-wells with the sealing material. This approach allows to introduce into the reservoir just as much sample liquid as is required to fill the plurality of micro-wells, i.e. a minimum amount of sample liquid is defined by a volume that is made up of the volumes of all micro-wells of the plurality of microwells. This increases an efficiency in generating isolated liquid subvolumes, reduces a subsampling error and saves costs for the sample liquid.

[0033] I I24EM02-2024323325. DOCX mh For example, in case of the plurality of micro-wells being arranged on the at least one sidewall at a height (with respect to the base) that is above a filling level of the sample liquid within the reservoir, the plurality of micro-wells are not filled when introducing the sample liquid into the reservoir through the single opening. For example, in case of the plurality of micro-wells being arranged on the base and on the at least one sidewall or only on the at least one sidewall, the plurality of micro-wells can at least partially not be filled with the sample liquid by introducing a defined amount of sample liquid into the reservoir through the single opening, so that a filling level of the sample liquid does not reach all micro-wells of the plurality of micro-wells. In the case where the plurality of micro-wells being arranged only on the base or on the base and on the at least one sidewall, the plurality of micro-wells can at least partially not be filled with the sample liquid by using a base which is inclined with respect to the horizontal, so that a filling level of the sample liquid does not reach all micro-wells of the plurality of micro-wells when introducing a defined amount of the sample liquid into the reservoir through the single opening.

[0034] In the embodiments of not or at least partially not filling the plurality of micro-wells with the sample liquid at the introduction of same into the reservoir, the sealing material is preferable a liquid material. In all of the above described different arrangements of the plurality of microwells it is thus possible to introduce the sealing material using an, e.g., hollow or tubular, applicator comprising an inlet and an outlet. The outlet of the applicator may be arranged within the sample liquid when introducing the sealing material, i.e. between the base and a filling level of the sample liquid. In case of using an inclined base, the outlet of the applicator is preferably arranged in proximity to the base in a lowest region thereof with respect to the horizontal. The applicator, for example, is configured to transport the sealing material from the single opening through the reservoir towards the base of the reservoir using the body force. According to an embodiment, the applicator may be a pipetting tip configured to transport the sealing material from the single opening through the reservoir towards the base, wherein the sealing material sinks from the outlet of the applicator towards the base due to the body force. In the special case of using an inclined base and the plurality of micro-wells being arranged on the base, the sealing material can be introduced into the reservoir by letting the sealing material flow along the at least one sidewall to a lowest region of the base with respect to the horizontal using the body force. The sealing material, for example, does not come into contact with the base before coming into contact with the sample liquid when being introduced in this way.

[0035] In case of the sealing material being a liquid, it may have a smaller contact angle to the base and / or to the at least one sidewall of the reservoir than the sample liquid. By using a sealing liquid with a smaller contact angle than the sample liquid an adhesion of the sealing liquid on

[0036] I I24EM02-2024323325. DOCX mh the solid surface of the reservoir between individual micro-wells of the plurality of micro-wells is supported. This facilitates a displacement of the bulk sample liquid from the openings of the micro-wells and an isolation of the micro-wells from each other without enclosing any air bubbles and increases the stability of the sealing layer.

[0037] According to an embodiment, the sealing material is a liquid and introducing the sealing material comprises using a tubular applicator comprising an inlet and an outlet. The tubular applicator is configured to transport the sealing material using (e.g., only) the body force from the single opening through the reservoir towards the base of the reservoir. The tubular applicator, for example, is fixed to the reservoir, e.g., by structures connecting the tubular applicator with the at least one sidewall and / or the base. Alternatively, the tubular applicator may be connectable to the single opening of the reservoir in form of a lid (e.g., a cap or cover) comprising the tubular applicator. The usage of the tubular applicator improves a control of a position and velocity of the introduction of the sealing material.

[0038] Optionally, the applicator comprises a membrane closing the outlet of the applicator. The movement of the sealing material towards the base through the applicator deforms the membrane to seal the subvolumes of the sample liquid in the plurality of micro-wells. For example, using the body force the sealing material is transported / channeled through the cavity of the tubular applicator towards the base and pressed against the membrane to deform same, so that the membrane covers the plurality of micro-wells. The applicator comprising a membrane is preferably used in connection with a reservoir with the plurality of micro-wells being arranged only on the base, e.g., so that the membrane can be pressed against the base for covering the plurality of micro-wells arranged on the base. However, this applicator is also appropriate for reservoirs with at least some or all micro-wells of the plurality of micro-wells being arranged on the at least one sidewall. For example, the membrane may be deformable by the sealing material, so that the membrane presses against the at least one sidewall covering the plurality of micro-wells arranged on the at least one sidewall.

[0039] In case of using an applicator with a membrane, the subvolumes of the sample liquid can be recovered from the plurality of micro-wells by removing the sealing material from the applicator, e.g., through the inlet of the applicator, so that the membrane retracts. The membrane, for example, is reversible deformable. Due to the retracting movement of the membrane, same lifts from the base and / or the at least one sidewall, so that the subvolumes of the sample liquid in the plurality of micro-wells are no longer sealed. The membrane allows to easily recover the sample liquid from the micro-wells for subsequent analysis.

[0040] I I24EM02-2024323325. DOCX mh In case of not using an applicator with a membrane and the sealing material being a liquid, the subvolumes of the sample liquid can be recovered from the plurality of micro-wells by applying a pressure, e.g., in addition to the body force, or by increasing the body force further, pushing the sealing material into the plurality of micro-wells and releasing the subvolumes of the sample liquid from the plurality of micro-wells. Due to the sealing material having a higher density than the sample liquid, the released sample liquid rises through the sealing material in the direction of the single opening, so that the released sample liquid floats on top of the sealing material and can be recovered from the reservoir through the single opening. Thus, an easy recovery of the sample liquid from the micro-wells for subsequent analysis is achieved.

[0041] Alternatively, in case of not using an applicator with a membrane and the sealing material being a liquid, the subvolumes of the sample liquid can be recovered from the plurality of microwells by removing the sealing material from the reservoir through the single opening; introducing a recovery liquid into the reservoir through the single opening to cover the plurality of micro-wells; and waiting for a predetermined time interval in which the sample liquid diffuses into the recovery liquid. The recovery liquid comprising the sample liquid diffused into the recovery liquid may be recovered from the reservoir through the single opening of the reservoir. Thus, an easy recovery of the sample liquid from the micro-wells for subsequent analysis is achieved.

[0042] Alternatively, to the sealing material being a liquid, the sealing material can be a solid material. Optionally, the solid material is soft, like rubber material or silicon material. The sealing material, for example, can be introduced in the reservoir via a solid sealing member comprising the sealing material. For example, the solid sealing member sinks due to the body force through the sample liquid towards the base and seals micro-wells arranged on the base of the reservoir. The usage of a solid material as sealing material has the advantage that same can be easily removed from the reservoir and thus simplifies a recovery of the sample liquid from the micro-wells.

[0043] According to an embodiment, when seen in a direction from the single opening to the base of the reservoir, the solid sealing member has a smaller cross section than the reservoir to allow the sample liquid to flow through a gap between the solid sealing member and the at least one sidewall towards the single opening when the solid sealing member, i.e. the sealing material, moves towards the base. Additionally or alternatively, the solid sealing member has one or more through-holes to allow the sample liquid to flow through the one or more through-holes towards the single opening when the solid sealing member, i.e. the sealing material, moves

[0044] I I24EM02-2024323325. DOCX mh towards the base. This enables an easy and efficient introduction of the sealing material into the reservoir.

[0045] Independent of whether the sealing material is a liquid material or a solid material, the introducing of the sealing material into the reservoir may comprises applying the sealing material on top of the sample liquid, wherein the sealing material sinks through the sample liquid due to the body force to the base of the reservoir.

[0046] In all of the embodiments it is possible to use a reservoir in which the base of the reservoir is inclined with respect to the horizontal, e.g., by tilting the reservoir, so that its base is inclined, or by using a reservoir with an integrated / built-in inclined base. In case of using a solid material as sealing material, the solid sealing member comprising the sealing material may comprise an inclined surface having the same angle with respect to the horizontal as the inclined base. In this case, the sealing material may be introduced into the reservoir through the single opening so that the inclined surface of the solid sealing member faces the inclined base of the reservoir. The usage of an inclined base is however especially advantageous in combination with the sealing material being a liquid material. In this case the sealing material moves towards the base due to the body force, whereby a filling level of the sealing material rises within the reservoir displacing the sample liquid, i.e. lifting the sample liquid. The inclined base might enable an easier and more reliable displacement of the sample liquid on top of the microwells by the sealing material. The inclined base reduces the risk of enclosing sample liquid between multiple micro-wells and thus reduces a risk of cross-talk between individual microwells.

[0047] A further aspect of the present application is directed to an apparatus for generating isolated liquid subvolumes in a plurality of micro-wells of a reservoir. The apparatus comprises the reservoir with a base, at least one sidewall and a single opening spanning at least partially the base. The plurality of micro-wells is arranged on the base and / or on the at least one sidewall. Additionally, the apparatus may comprise an applicator for introducing a sealing material into the reservoir through the single opening to seal subvolumes of the sample liquid in the plurality of micro-wells using a movement of the sealing material towards the base due to the body force, wherein the sealing material has a higher density than the sample liquid.

[0048] According to this aspect of the present application the difficulty that the usage of fluidic channels causes long process times or requires high system pressure and increases the risk of losing sample material has been overcome by providing a reservoir with a single opening and using an applicator for introducing the sealing material into the reservoir through the single

[0049] II24EM02-2024323325.DOCX mh opening. This is based on the idea that the sample liquid can be directly introduced in the reservoir without the need of channels guiding the sample liquid to the reservoir or along the micro-wells for filling same (i.e. the reservoir does not need to be designed as a channel, so that the sample liquid flows from an inlet over the plurality of micro-wells to an outlet), so that a probability of sample-molecules or sample-particles being adsorbed at surfaces is reduced. Further, the inventors found, that it is possible to efficiently introduce a sealing material having a higher density than the sample liquid into the reservoir with the applicator using a body force, wherein the applicator enables to introduce the sealing material near the micro-wells and the body force moves the sealing material to the base so that a bulk sample liquid outside the micro-wells is efficiently displaced by the sealing material but not the sample liquid subvolumes within the micro-wells. The applicator allows to generate isolated liquid subvolumes in the plurality of micro-wells without the need of designing the reservoir as a channel, so that the sealing material flows from an inlet over the plurality of micro-wells to an outlet for sealing the subvolumes. Thus, the inventive apparatus allows to efficiently generate isolated liquid subvolumes in the plurality of micro-wells without the usage of flow-channels for transporting the sample liquid to the reservoir or within the reservoir along the micro-wells.

[0050] This apparatus is based on the same considerations as the above described method. The aspects described with regard to the method also represent a description of the corresponding apparatus, where a method step or a feature of a method step corresponds to a block or device of the apparatus, i.e., aspects described in the context of a method step may represent a description of a corresponding block or item or feature of a corresponding apparatus.

[0051] According to an embodiment, the applicator is a tubular applicator coupled to the reservoir, e.g., by structures coupling the applicator to the at least one sidewall and / or the base or by a removable lid comprising the applicator, wherein the removable lid may be configured to be coupled to the reservoir, so that the applicator reaches through the single opening into the reservoir. The applicator comprises an inlet and an outlet. The applicator is configured to transport the sealing material using the body force from the single opening through the reservoir towards the base of the reservoir. The applicator enables a high efficiency in sealing the subvolumes of the sample liquid in the plurality of micro-wells, since the outlet of the applicator can be positioned near the micro-wells displacing the excess sample liquid outside of the micro-wells more quickly.

[0052] Optionally, the applicator comprises a membrane closing the outlet of the applicator. The membrane is deformable by the sealing material. The deformable membrane increases an efficiency in sealing the subvolumes of the sample liquid in the plurality of micro-wells and

[0053] I I24EM02-2024323325. DOCX mh simplifies a recovery of the subvolumes of the sample liquid in the plurality of micro-wells, since it can easily be retracted.

[0054] According to an embodiment, the plurality of micro-wells is arranged on the base and wherein the base is inclined with respect to the horizontal. The inclined base enables an easier and more reliable displacement of the sample liquid on top of the micro-wells by the sealing material. The inclined base reduces the risk of enclosing sample liquid between multiple microwells and thus reduces a risk of cross-talk between individual micro-wells.

[0055] Optionally, the applicator is a tubular applicator comprising an inlet and an outlet. The outlet of the applicator is arranged in proximity to the inclined base in a lowest region thereof with respect to the horizontal. The applicator is configured to transport the sealing material using the body force from the single opening through the reservoir towards the base of the reservoir. The special positioning of the applicator minimizes the risk of enclosing sample liquid between multiple micro-wells. This is especially advantageous in case of the micro-wells being arranged on the base. This is based on the finding that the sealing material starts spreading over the inclined surface from the lowest point, displacing surplus sample liquid easily in direction of the single opening. When the sealing material moves towards the base due to the body force, a filling level of the sealing material within the reservoir rises gradually sealing the micro-wells. This enables an easier and more reliable displacement of the sample liquid on top of the microwells. This arrangement can be beneficial to achieve a higher yield of sealed micro-wells.

[0056] Alternatively, the applicator comprises an inlet and an outlet and the outlet of the applicator is arranged in proximity to the at least one sidewall in proximity to the single opening for letting the sealing material flow along the at least one sidewall to a lowest region of the inclined base with respect to the horizontal. The inventors found that this controlled flow of the sealing material along the sidewall also minimizes the risk of enclosing sample liquid between multiple micro-wells. This is based on the finding that the sealing material is reliably guided along the at least one sidewall to the lowest point of the reservoir due to the body force and due to the sealing material having a higher density than the sample liquid, so that a filling level of the sealing liquid rises and minimizes the risk of enclosing sample liquid between multiple microwells. Thus, a high yield of sealed micro-wells can be achieved.

[0057] According to an embodiment the at least one sidewall is tapered towards the base. The at least one sidewall comprises a plurality of micro-steps in a direction from the single opening towards the base and each of the micro-steps is partitioned into the plurality of micro-wells by partition walls. Each of the plurality of micro-wells has the form of triangular prism. Such a shape can

[0058] II24EM02-2024323325.DOCX mh have some significant advantages in practice, because the micro-wells can be produced on a tapered wall of the reservoir by injection molding more easily than on parallel walls.

[0059] According to an embodiment, the apparatus comprises further reservoirs. Each of the further reservoirs has a base, at least one sidewall and a single opening spanning at least partially the respective base and a plurality of micro-wells arranged on the respective base and / or on the respective at least one sidewall. Additionally, the apparatus comprises a substrate on which the reservoir and the further reservoirs are arranged or in which the reservoir and the further reservoirs are formed. This enables a high degree of parallelization, i.e. more different samples per unit area e.g. per assay plate, for increased throughput.

[0060] A further aspect of the present application is directed to an apparatus for generating isolated liquid subvolumes in a plurality of micro-wells of a reservoir. The apparatus comprises the reservoir with a base, at least one sidewall and a single opening spanning at least partially the base. The plurality of micro-wells is arranged on the base and / or on the at least one sidewall and the base may be inclined with respect to the horizontal. The inclined base reduces the risk of enclosing sample liquid between multiple micro-wells and thus reduces a risk of cross-talk between individual micro-wells. This apparatus is based on the same considerations as the above described apparatus and can, by the way, be completed with all features and functionalities, which are also described with regard to the above described apparatus.

[0061] Brief Description of the Drawings

[0062] The drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the invention. In the following description, various embodiments of the invention are described with reference to the following drawings, in which:

[0063] Fig. 1 shows a block diagram of an embodiment of a method for generating isolated liquid subvolumes;

[0064] Fig. 2 shows an embodiment of an assay plate and of a reservoir with micro-wells; Fig. 3 shows a schematic drawing of an embodiment of a method for generating isolated liquid subvolumes;

[0065] Fig. 4 shows an embodiment of a method for first sealing the microwells (left) and then recovering sample liquid from the micro wells by further increasing the body force acting on the sealing material (right);

[0066] Fig. 5 shows an embodiment of generating isolated liquid subvolumes using an applicator;

[0067] Fig. 6 shows an embodiment of a reservoir with micro-wells on its sidewall;

[0068] II24EM02-2024323325.DOCX mh Fig. 7 shows an embodiment of a method for generating isolated liquid subvolumes using an applicator;

[0069] Fig. 8 shows an embodiment of a reservoir with inclined base, wherein an applicator is used for the sealing process;

[0070] Fig. 9 shows an embodiment of a reservoir with inclined base, wherein the sealing process is performed without using an applicator;

[0071] Fig. 10 shows a schematic view of a solid sealing member for a reservoir with inclined base;

[0072] Fig. 11 shows an embodiment of a sealing procedure using a solid sealing material; Fig. 12 shows an embodiment of a sealing procedure using an applicator with a membrane;

[0073] Fig. 13 shows a comparison between a sealing process with and without using an applicator with a membrane;

[0074] Fig. 14 shows an embodiment of a tapered shaped reservoir;

[0075] Fig. 15 shows experimental results with micro structured foil;

[0076] Fig. 16 shows fluorescent microscopic images of filled and sealed micro-wells in a photobleaching experiment; and

[0077] Fig. 17 shows fluorescent microscopic images of two different successful implementations of the invention, one using inverted pyramidal wells and on using inverted conical cylindrical wells.

[0078] Detailed Description of the Embodiments

[0079] Equal or equivalent elements or elements with equal or equivalent functionality are denoted in the following description by equal or equivalent reference numerals or are identified with the same name, and a repeated description of elements provided with the same reference number or being identified with the same name is typically omitted, even if occurring in different figures. Hence, descriptions provided for elements having the same or similar reference numbers or being identified with the same names are mutually exchangeable or may be applied to one another in the different embodiments.

[0080] In the following description, a plurality of details is set forth to provide a more thorough explanation of embodiments of the present invention. However, it will be apparent to those skilled in the art that embodiments of the present invention may be practiced without these specific details. In other instances, well-known structures and devices are shown in block diagram form rather than in detail in order to avoid obscuring embodiments of the present invention. In addition, features of the different embodiments described herein after may be combined with each other, unless specifically noted otherwise.

[0081] I I24EM02-2024323325. DOCX mh For facilitating the description of the different embodiments, some of the figures comprise a Cartesian coordinate system x, y, z, wherein the x-y-plane corresponds, i.e. is parallel, to a horizontal (= a reference plane = x-y-plane), wherein the direction vertically up with respect to the reference plane (x-y-plane) corresponds to the “+z” direction, and wherein the direction vertically down with respect to the reference plane (x-y-plane) corresponds to the “-z” direction. In the following description, the term “lateral” means a direction parallel to the x- and / or y-direction, i.e. parallel to the x-y-plane, wherein the term “vertical” means a direction parallel to the z-direction.

[0082] Herein described surface tension forces (interfacial tension forces) may relate to surface tensions at interfaces (interfacial tension), i.e. at a liquid-liquid interface, a liquid-solid interface and / or a liquid-gas interface. In the present invention the mentioned surface tension forces may mainly relate to interfacial tensions at interfaces between two liquids or between a liquid and a solid material.

[0083] Fig. 1 shows a block diagram of a method 100 for generating isolated liquid subvolumes in a plurality of micro-wells (e.g., micro-cavities, micro-dents or micro-recesses) of a reservoir comprising a base, at least one sidewall and a single opening at least partially spanning the base. The plurality of micro-wells is arranged on the base and / or on the at least one sidewall of the reservoir.

[0084] The method comprises introducing 110 a sample liquid into the reservoir through the single opening and introducing 120 a sealing material into the reservoir through the single opening to seal subvolumes of the sample liquid in the plurality of micro-wells using a movement of the sealing material towards the base due to a body force applied to the sealing material. Due to the movement of the sealing material towards the base, the sealing material displaces the sample liquid arranged outside the plurality of micro-wells and seals the subvolumes of the sample liquid within the plurality of micro-wells.

[0085] The body force exemplarily comprises at least one of a gravitational force, a centrifugal force and a magnetic force. The body force is a force that acts throughout the volume of the sealing material and not only on the surface of the sealing material. In case of the body force comprising a magnetic force, the sealing material may comprise ferromagnetic properties. In view of the present invention it is sufficient, if the body force acts only throughout the volume of the sealing material. This can, for example, be the case for moving a magnetic sealing material with the aid of a magnetic field, if the sample liquid and the material of the reservoir

[0086] I I24EM02-2024323325. DOCX mh have no magnetic properties. In this case, the body force representing a magnetic force does not act on the reservoir and the sample liquid, but the plurality of micro-wells are still sealed. In case the body force comprises, additionally or alternatively, a gravitational force and / or a centrifugal force, same acts on the sealing material, but also on the reservoir and the sample liquid. But also in this case, the plurality of micro-wells are efficiently sealed by the sealing material, provided that the sealing material has a higher density than the sample liquid. It is, therefore, irrelevant whether the body force acts only on the sealing material or also on additional materials or components of a herein described apparatus. It is only important that the body force acts at least on the sealing material.

[0087] The method 100 allows different types of reservoirs, i.e. reservoirs with different shapes. For example, the base 20 can have a circular shape or oval shape, so that the reservoir 10 comprises one single sidewall 30, e.g., see Fig. 2. Alternatively, the reservoir 10 may comprise a base 20 with the shape of a circular segment (like the shape of a semicircle) and two sidewalls 30, or a base 20 with a shape having three or more straight sides (like a triangular shape, a square shape, a rectangular shape or a polygonal shape) and three or more sidewalls 30. Optionally the at least one sidewall 30 is at least partially tapered. Further, it is not necessary that the base 20 of the reservoir 10 is parallel to the horizontal, as shown in Fig. 2. Instead, it is also possible that the base 20 is curved (like a spherical shape), e.g., see Fig. 14, or inclined with respect to the horizontal, e.g., see Fig. 8-9.

[0088] The method 100 uses a reservoir 10 comprising one single opening 40, which may span the complete base 20, see Fig. 2, Fig. 3 and Fig. 14, or which may only partially span the base 20, e.g., a projection of the single opening 40 onto the base 20 may have a smaller area than the base 20, see Fig. 4 to 9 and Fig. 11-12. The single opening 40 may function as inlet and optionally, e.g. if necessary (e.g., if it is desired to recover the sample liquid and / or the sealing material from the reservoir 10), as outlet. Apart from the single opening 40, the reservoir 10 comprises no further openings. The sample liquid can be directly inserted into the reservoir 10 through the single opening 40, e.g. using pipetting or using pouring, without using guiding channels for guiding the sample liquid to the single opening 40 and to the base 20. Within the reservoir 10 the sample liquid may get in contact with the base 20 and the at least one sidewall 30 of the reservoir 10, but with no other surface of the reservoir 10.

[0089] Fig. 2 shows exemplarily a reservoir 10 with the plurality of micro-wells 50 being arranged on the base 20. Alternatively or additionally it is possible that the plurality of micro-wells 50 are arranged on the at least one sidewall 30. The herein described methods 100 generate isolated

[0090] I I24EM02-2024323325. DOCX mh liquid subvolumes, i.e. individual compartments from the sample liquid, in the plurality of microwells 50.

[0091] As briefly mentioned above, the scope of this invention is to produce a large number of individual compartments (typically thousands to millions) from a bulk liquid volume, i.e. from the sample liquid, that are physically separated from each other (i.e. isolated compartments; i.e. isolated subvolumes of the sample liquid). Furthermore, the invention provides a solution for processing a large number of different samples in parallel, e.g., see the assay plate 200 in Fig. 2. Similar like regular micro biological assays are processed often in so called standardized micro titer plates (MTP) - the handling of which can be highly automated to increase throughput - the present invention discloses also an assembly of elements according to the invention on which many different liquid samples can be processed in parallel. This so called assay plate 200 features a number of N reservoirs 10, in each of which the compartmentalization into a large number of M micro compartments (i.e. each reservoir 10 comprises M micro-wells 50) can be carried out in parallel, i.e. the assay plate 200 comprises one or more of the herein described reservoirs 10. In total, the assay plate 200 thus features N x M micro compartments. The assay plate 200 itself can be made fully compatible to the MTP standard and automatic handling equipment. This enables automatic processing of the assay plate 200 with conventional liquid handling instrumentation. Typical numbers for N and M can be in the range N = 1,10, 24, 96,... , 6.144 and M = 100, 1.000, ... , 1010. However, it is also possible to provide an assay plate featuring more than 6.144 reservoirs 10 and / or featuring reservoirs with more than 1010micro-wells 50.

[0092] As shown in Fig. 2, the assay plate 200 may comprise a substrate 210 on which one or more herein described reservoirs 10 are arranged. Alternatively, it is also possible that the one or more herein described reservoirs 10 are formed within the substrate 210, e.g., see Fig. 9. Optionally, the assay plate 200 may further comprise a cover 220 or lid covering all of the one or more reservoirs 10.

[0093] An apparatus for producing separated, closed liquid compartments from a bulk liquid volume, e.g., from the sample liquid 60, comprises at least one reservoir 10 for receiving and holding the sample liquid 60 to be compartmentalized. The reservoir 10 comprises at least one micro structured surface area that is formed by at least one micro-well 50. For example, the reservoir 10 comprises a micro structured surface area on its base 20 and / or a micro structured surface area on its at least one sidewall 30. To seal the micro-wells 50 a sealing material 70 that is not miscible with the sample liquid 60, e.g., a solid material or a liquid material, is used that is optionally also considered to be part of the apparatus. This so called sealing material 70 has

[0094] I I24EM02-2024323325. DOCX mh preferably a larger density than the sample liquid 60 and / or a smaller contact angle on the micro structured surface area than the sample liquid 60. As an optional feature, the apparatus might also feature at least one geometric feature referred to as sealing material applicator, e.g., see Fig. 5, Fig. 7, Fig. 8 and Fig. 12, that is used to guide the sealing material 70 to a specific position on the micro structured surface area and to control the flow velocity by which the sealing material 70 being a liquid is spreading onto the micro structured surface area to close-off the individual micro-wells 50.

[0095] As mentioned above, the sealing material 70 can be either a liquid that is not miscible with the sample liquid 60 or it can be a solid material. In any case, the density of the sealing material 70 is preferably larger than the density of the sample liquid 60, such that when the sealing material 70 is introduced into the opening 40 of the reservoir 10, it will sink due to gravity, thereby displacing the bulk sample liquid 60. Eventually, the sealing material 70 is reaching the micro structured surface area, closing-off the individual micro-wells 50 from the bulk sample liquid 60 and isolating them from each other. If the sealing material is a liquid, its density can be considered homogenous and preferably larger than the density of the sample liquid 60. If the sealing material 70 is a solid material, the density is not necessarily homogeneous, as the material does not need to have homogeneous properties all over its volume. There might be regions in the solid sealing body that have larger or smaller density than the bulk sample liquid 60. In this case it is advantageous to use a solid sealing body, i.e. a solid sealing member, having in average a higher density than the sample liquid 60 and a shape that does not prevent sinking.

[0096] The apparatus for producing separated, closed liquid compartments from a bulk liquid volume is particularly useful, if several of the individual units (i.e. reservoirs 10 with micro structured surface area) are arranged together on a support, i.e. the substrate 210, to enable the processing of several different assay liquids in parallel. Without loss of generality, Fig. 2 shows on the left exemplarily an apparatus that has a multitude of cylindrical reservoirs 10 arranged in an array that fits the layout of a conventional micro titer plate (MTP). Such an apparatus may herein be referred to as assay plate 200. A close-up of an individual reservoir 10 featuring a micro structured surface area at the bottom, i.e. at the base 20, is shown on the right in Fig. 2. In other words, Fig. 2 shows on the left a sketch of an assay plate 200 according to the present invention, featuring a plurality of reservoirs 10 arranged on a common support and on the right a detailed view of a reservoir 10 with the features according to present invention, including a close-up cross sectional view of the micro structured surface area exhibiting a plurality of micro-wells 50.

[0097] I I24EM02-2024323325. DOCX mh The micro structured surface area can be anywhere inside the reservoir 10. In practice, the number of micro-wells 50 constituting this micro structured surface area will be rather large such as 10, 100, 1000 up to 1010micro-wells 50 in one single reservoir 10. The optimal position depends on the orientation of the reservoir 10 in relation to the direction of the body force when the sealing material 70 is supplied, the type and amount of liquids that are used and whether additional means to control the spreading of the sealing material 70, like a sealing material applicator are used. For simplicity, let’s consider for the moment an example like shown in Fig. 2, where the micro structured surface area is located at the base 20 of the reservoir 10. Later on, further examples will be presented, where the micro structured surface area is not located at the base 20 of the reservoir 10, and it will be explained how the sealing material 70 can be applied to these different designs to achieve proper operation.

[0098] As will be described in more detail with regard to Fig. 3, the basic working principle of liquid compartmentalization, i.e. of generating isolated liquid subvolumes 602, relies on the confinement of a liquid subvolume 602between the walls of an open micro-well 50 that is covered by a sealing material 70, e.g., a non-miscible liquid or a solid material (e.g., a soft and / or elastic material). The sealing material 70 is applied, e.g., introduced 120, after the bulk assay liquid, i.e. the sample liquid 60, has been introduced 110 into the reservoir 10 and has filled the micro-wells 50 contained therein. The large number of isolated compartments are thus formed by a large number of micro-wells 50 filled with the assay liquid, i.e. the sample liquid 60, that are afterwards closed by a layer of sealing material 70. The sealing material 70 breaks the fluidic connections between the various micro-wells 50 that is established during filling of the micro-wells 50 in parallel from the bulk liquid 6O1. Thus, chemical cross-talk between the individual compartments that are formed this way is prevented, which is the prerequisite for using the isolated compartments in digital assays.

[0099] The method 100 for generating isolated liquid compartments may comprise the following four steps that are carried out consecutively:

[0100] 1. An apparatus as described herein, comprising at least one reservoir 10 featuring a micro structured surface area with at least one micro-well 50 inside the reservoir 10 (exemplarily shown on the base 20 in Fig. 3) is provided 105, e.g., empty of any liquids.

[0101] 2. A certain volume of sample liquid 60, e.g., bulk assay liquid, is introduced 110, e.g., supplied, to the reservoir 10 (e.g. by a pipette). Typically, the volume of the sample liquid 60 will be at least equal to the volume of all micro-wells 50 to be filled. Otherwise, less isolated liquid subvolumes 602, e.g., liquid compartments, will be produced than micro-wells 50 are available in the reservoir 10. The volume of the sample liquid 60 can of course also be larger than the volume of all the micro-wells 50, but it should be

[0102] I I24EM02-2024323325. DOCX mh significantly less than the total volume that the reservoir 10 is able to hold, in order to leave some remaining volume for the sealing material 70 in the reservoir 10.

[0103] 3. A certain volume of sealing material 70 is introduced 120, e.g., supplied, to the reservoir 10. This step is shown right after the application of the sealing material 70, before it has settled to the bottom, i.e. the base 20, due to the body force. For doing so an optional sealing material applicator, e.g., see Fig. 5, Fig. 7, Fig. 8 and Fig. 12, might be used to better control the position and velocity of the application of the sealing material 70. The volume of sealing material 70 should be at least sufficient to cover all the microwells 50, and it should be no larger than the volume that the reservoir 10 can hold minus the volume of the sample liquid 60 already supplied to the reservoir 10 to avoid spill-over.

[0104] 4. A body force may be provided to move the sealing material 70 towards the micro structured surface area and to press it onto the micro-wells 50. After a certain waiting time the system will have reached equilibrium (see numeral 130 in Fig. 3), whereby the bulk sample liquid 6O1 above the micro-wells 50 will have been displaced by the sealing material 70, thereby sealing the sample liquid 60 filled micro-wells 50 individually. In certain cases, gravity might provide a sufficiently large force to the sealing material 70 that achieves the desired effect, spontaneously (due to the density difference of the materials). If gravity is not present or not sufficient, the body force might be also provided by centrifugation of the reservoir and / or by providing an external magnetic field. When the system has reached equilibrium, any excess bulk sample liquid 6O1 that was not confined in the micro-wells 50 will be floating on the top of the sealing material 70 and might be recovered from there through the reservoir opening 40.

[0105] In other words, Fig. 3 shows a sketch of the situation inside the reservoir 10 at different stages of the inventive method 100. Below the reservoir 10 a close-up cross-sectional view of the micro-wells 50 is shown for each of the stages, namely providing 105 an empty reservoir 10 (i.e. inventive apparatus) with a micro structured surface area at the base 20; introducing 110, e.g., filling, into the reservoir 10 the sample liquid 60 (e.g. provided by a pipette through the reservoir opening 40) that leads to spontaneous filling of the micro-wells 50; introducing 120 the sealing material 70 into the reservoir 10, e.g., added on top of the sample liquid 60 (e.g. provided by a pipette through the reservoir opening 40); waiting for the sealing material 70 to settle to the base 20 of the reservoir 10 due to the body force and displacing the sample liquid 60 on top of the micro-wells 50, thereby closing-off the micro-wells 50 at the base 20 with the remaining assay liquid, i.e. the subvolumes 6O2 of the sample liquid 60, inside.

[0106] I I24EM02-2024323325. DOCX mh It is to be noted that Fig. 2 and Fig. 3 show examples of reservoirs 10 with a single opening 40 spanning the complete base 20. However, it is also possible that the respective opening 40 does not completely span the base 20, e.g., as exemplarily shown in Fig. 4 to 9 and Fig. 11-12.

[0107] A large number of micro-wells 50 is present inside the reservoir 10 (e.g. at the bottom surface, i.e. the base 20, of the reservoir 10, or additionally or alternatively at the at least one sidewall 30 of the reservoir 10). Such reservoirs 10 with micro-wells 50 and a single opening 40 can be fabricated by polymer replication technology (see [2001_Becker]) at low cost as single-use consumables. The micro-wells 50 may have dimensions in the order of several micrometers up to a few hundred of micrometers, depending on the volume requirement for the individual compartments. The design of these micro-wells 50 with regard to shape and depth, preferably obeys following basic design rule that states that the so called Concus-Finn condition [1969_Concus] [2005_Seemann] has to be valid for at least one corner of the micro-well’s inner surface (i.e. 0 + a < 90°, whereby 0 is the contact angle of the assay liquid and 2a is the opening angle of the corner). Briefly, this means that intersecting surfaces in the micro-well 50 must enclose a sufficiently small corner angle of 2a for a given contact angle 0 of the sample liquid 60. Otherwise, there is a potential risk that the micro-wells 50 are not filling spontaneously when the sample liquid 60 is introduced into the reservoir 10 and supplied to the one or more micro structured surfaces, i.e. the base 20 and / or the at least one sidewall 30 on which the plurality of micro-wells 50 are arranged.

[0108] The micro-wells 50, for example, are sealed by adding a volume of non-miscible liquid or a sheet a soft but solid material to the reservoir 10 that settles on the individual micro-wells 50 due to the body force and thereby closes-off the micro-wells 50 from their neighbors and the bulk liquid. The body force may comprise a gravitational force and the sealing material may sink to the base 20 through the sample liquid 60 due to its preferably higher density compared to the sample liquid 60. Additionally, or alternatively to the gravitational force, the body force may comprise a centrifugal force (e.g., by inserting the reservoir 10 into a centrifuge, wherein a centrifugal force applied by the centrifuge shows from the single opening 40 of the reservoir 10 to the base 20 of the reservoir 10) and / or a magnetic force (e.g., wherein the sealing material comprises ferromagnetic properties and a magnetic field moves the sealing material 70 from the single opening 40 of the reservoir 10 to the base 20 of the reservoir 10). If the sealing material 70 itself is a liquid, the liquid connection between the micro-wells 50 is disrupted in the sense that no sample liquid 60 and no molecules dissolved in the sample liquid 60 can leave from the micro-well 50, sealed by the liquid / liquid interface. If the sealing material 70 is a sheet of soft but solid material the micro-wells 50 are physically separated. The physical

[0109] I I24EM02-2024323325. DOCX mh mechanism that drives the settling of the sealing material on the micro-wells 50 is the body force, potentially supported by surface tension in addition. Therefore, it would be advantageous, if the sealing material 70, in case of being a liquid, i.e., a sealing liquid, has a smaller contact angle 0 on the micro structured surface than the sample liquid 60. For example, the body force pushes the sealing liquid onto the micro-wells 50 and the low contact angle supports adhesion of the sealing liquid on the surface between individual micro-wells 50. By this the bulk assay liquid 6O1 is displaced from the opening of the micro-wells 50 and the microwells 50 are isolated from each other without enclosing any air bubbles. Depending on 1. the properties of the micro-wells 50 (material, design, surface properties, size, etc.), 2. the properties of the sample liquid 60 as well as 3. the properties of the sealing liquid, the sealing happens spontaneously (driven by the body force and the density difference between the sealing liquid and the sample liquid 60) when a sufficiently large volume of the sealing liquid is provided to the reservoir 10.

[0110] In order to facilitate the sealing or to improve the yield of properly sealed micro-wells 50 in case of the sealing material 70 comprising a solid material, the sealing material 70 may have a smaller cross section than the base 20 (or than the opening 40, in case of the opening 40 not completely spanning the base 20), so that the sample liquid 60 can flow between the sealing material 70 and the at least one sidewall 30 towards the single opening 40 when the sealing material 70 moves towards the base 20. Additionally, or alternatively, the solid sealing material 70 may comprise a plurality of through holes through which the sample liquid 60 can flow towards the single opening 40 when the solid sealing material 70 moves towards the base 20.

[0111] In order to facilitate the sealing or to improve the yield of properly sealed micro-wells 50 in case of the sealing material 70 being a liquid, the supply and spreading of the sealing liquid can be controlled by various means. For example a specific fluidic applicator can be used to precisely determine the position and the flow rate of the sealing liquid spreading onto the micro structured surface, e.g., e.g., see Fig. 5, Fig. 7, Fig. 8 and Fig. 12. Also the direction and the magnitude of the acceleration (e.g. caused by gravity, centrifugation and / or magnetism) by which the higher density sealing material 70 is propelled towards the base 20 can be modified. For example, the orientation of the base 20 towards the direction of gravity and / or increasing the gravitational effect by centrifugation can be means to influence the dynamics of the spreading of the sealing material 70. Whether or not certain means are necessary depends on the specific properties of the liquids as well as on the design of the reservoir 10 and the micro structured surface. Several examples of different possible embodiments will be given below.

[0112] I I24EM02-2024323325. DOCX mh In case of the sealing material 70 being a liquid there might be a risk that the sealing liquid accidentally displaces sample liquid 60 within the micro-wells 50 due to its higher density and optionally also smaller contact angle. The inventors found that this is mainly a problem for larger wells, e.g. with dimensions of several millimeters. Due to the small dimensions of the micro-wells 50, the subvolumes 6O2 of the sample liquid 60 within the plurality of micro-wells 50 are more strongly bond to a surface of the reservoir 10 than the bulk sample liquid 6O1 outside the plurality of micro-wells 50. For this reason, the movement of the sealing material 70 towards the base 20 displaces only the bulk sample liquid 6O1 outside the plurality of microwells 50 and not the subvolumes 602of the sample liquid 60 within the plurality of micro-wells 50.

[0113] Nevertheless, it might be advantageous to additionally consider for the sealing process the Bond number Bo. In order to prevent the sealing liquid from displacing sample liquid 60 within the micro-wells 50, it is proposed to have a Bond number, which is much smaller than 1, i.e. Bo « 1. The inventors found that Bond numbers of Bo < 10, Bo < 1, Bo < 0.1, Bo < 0.01 or even smaller ones are advantageous. The Bond number, which traditionally compares gravitational forces to surface tension forces, can be generalized to consider other forms of acceleration besides gravity. The Bond number is essentially a ratio that compares a body force (such as a gravitational force, a centrifugal force or a magnetic force) to a capillary force (surface tension). While the classical Bond number uses gravitational acceleration, it can be modified to account for any form of acceleration:

[0114]

[0115] wherein Ap is the density difference between the two fluids, i.e. between the sample liquid 60 and the sealing liquid; a is the acceleration acting on the system (caused by the body force e.g. gravity, centrifugation and / or a magnetic field); R is the radius of an opening of a microwell 50 of the plurality of micro-wells 50; and a is the surface tension (interfacial tension) between the two fluids i.e. between the sample liquid 60 and the sealing liquid.

[0116] As mentioned above, it would be advantageous, if the dimensions of the micro-wells 50 are in an appropriate relation to the rheological properties of the sample liquid 60 and the sealing liquid, in order to prevent that sealing liquid enters into the micro-wells 50 during the sealing process. In particular, this means the micro-wells 50 must not be too large, which can be expressed quantitatively in terms of the so called Bond number Bo which has to be sufficiently small. Preferably, values of Bo < 10, Bo < 1, Bo < 0.1, Bo < 0.01 or even smaller ones may be used, whereby the characteristic length in the formula is given approximately by the diameter of the micro-wells 50. For sufficiently small Bond numbers not enough hydrostatic energy is available from the density difference between both liquids, i.e. the sample liquid 60 and the I I24EM02-2024323325. DOCX mh sealing liquid, to form a free droplet of sample liquid 60 in the surrounding sealing liquid. Therefore, the sample liquid 60 cannot leave the micro-well 50 and the sealing liquid cannot enter the micro-well 50. Thus, by considering the Bond number, the size of the micro-wells 50 can be designed appropriately for a given combination of liquids to prevent the liquid with higher density, i.e. the sealing liquid, from entering the micro-wells 50 during the sealing process. Of course, also the contact angle and the viscosity may affect the sealing process, but their influence is minor as compared to the density, surface tension and the dimensions of the micro-wells 50. As a rule of thumb, one may consider that if issues of sealing liquid entering the micro-wells 50 are observed, one may decrease the diameter of the micro-wells 50 further, which makes the Bond number decrease as well and makes it harder or even impossible for sealing liquid to enter into the micro-wells 50.

[0117] It should be noted that Bo is also dependent on the acceleration factor a, which means that for a given configuration of micro-wells 50 and liquids, Bo can be increased to a level that allows for releasing the sample liquid from the micro-wells 50 (e.g. by centrifugation). The sample liquid 60 is then readily replaced by the sealing liquid filling the micro-wells 50. At sufficiently high acceleration, the required hydrostatic energy can be provided that is necessary to create the surface of a droplet with sample liquid 60 in the sealing liquid. How large the required energy is, depends primarily on the volume of the droplets and therefore on the size respectively the volume of the micro-wells 50. More energy is required for small micro-wells 50 than for larger ones. Whether droplets can detach from the micro-wells 50 depends on the combination of a volume of the micro-wells 50, a density of the liquids i.e. of the sample liquid 60 and the sealing liquid, and an acceleration (e.g. caused by gravitation, centrifugation or magnetism). Below a procedure is described for using this effect to deliberately remove the sample liquid 60 from the micro-wells 50 by increasing the acceleration factor.

[0118] In the following three examples using gravity as body force (with a gravitational acceleration of g = 9.81^) are provided, in which isolated liquid subvolumes of the sample liquid 60 can be sealed without displacing subvolumes of the sample liquid 60 from micro-wells 50. The examples are provided for an aqueous sample liquid with a density of p = 1000.0 and a

[0119]

[0120] surface tension of a = 72.8— in combination with a sealing liquid with density of p = 1850.0^.

[0121] F 000000029

[0122] F 00029

[0123]

[0124] I I24EM02-2024323325. DOCX mh

[0125] For micro-wells 50 with R=50pm opening: Bo =

[0126]

[0127] = 0.00029

[0128] 0.0728 —

[0129] s

[0130] The three examples using micro-wells with an opening diameter between 1 and 100pm cover the application-related relevant area. For the three examples the requirement of Bo «1 is met.

[0131] Apart from generating isolated liquid subvolumes 6O2 in a plurality of micro-wells 50, it can, in some cases, be beneficial to recover (see Fig. 4) the sample liquid, see 6O2, from the micro wells 50 after a certain time to conduct further analysis on the liquids. For example, when first an amplification of DNA is performed in the micro compartments, i.e. in the isolated liquid subvolumes 6O2, and later the analysis of the amplified DNA should be conducted by means of NGS, all the liquids or parts of the liquid have to be extracted from the micro-wells 50 and have to be pooled together to form a NGS library. The presented apparatus (comprising a herein described reservoir 10) allows for easy recovery of liquids from the micro-wells 50 in such applications. The method described with regard to Fig. 4 leads to a release of the liquids from the micro-wells 50, provided that a sealing liquid 70a, i.e. a sealing material 70 being a liquid material, is used to cover the micro-wells 50 (a solid sealing material will not work for this purpose). In such cases, the sample liquid, see 6O2, confined in the micro-wells 50 can be released by further increasing the hydrostatic pressure on the liquids inside the reservoir 10, for example, by increased centrifugation or magnetic field, beyond the point where the so called Bond number Bo becomes significantly larger than one (Bo » 1). In other words, the method comprises applying 140 a body force 142, so that the so called Bond number Bo becomes larger than one (e.g., so that Bo > 1, Bo > 10, Bo > 100 or Bo > 1000) to recover the subvolumes 6O2 of the sample liquid within the micro-wells 50, e.g., by displacing the subvolumes 6O2 of the sample liquid within the micro-wells 50 with the sealing liquid 70a using a movement of the sealing liquid 70a towards the base 20 due to the body force 142.

[0132] In this case, the hydrostatic pressure phyd exerted by the liquid column is larger than the pressure p<yropinside a droplet 144 of sample liquid suspended in the sealing liquid 70a.

[0133]

[0134] Hereby, p is the density of the sealing liquid 70a, the acceleration of the liquid column is G x g (where G is a constant factor determined by the centrifugation conditions and / or the magnetic conditions and / or any additional hydrostatic pressure applied to the liquid column (e.g. by means of a pneumatic pressure source) and g is the gravitational acceleration) and h is the filling height of the sealing liquid 6O1 on top of the micro-wells 50. The interfacial tension between the sealing liquid 70a and the sample liquid is denoted by o and r is the radius of a droplet 144 that is formed by the liquid volume confined in one micro-well 50.

[0135] I I24EM02-2024323325. DOCX mh Fig. 4 shows schematically the recovery of the isolated liquid subvolumes 6O2 of sample liquid from the plurality of micro-wells 50 of a herein described reservoir 10. Fig. 4 shows the recovery exemplarily for a reservoir 10 with a single opening 40, which does not completely span the base 20, and with the plurality of micro-wells 50 being arranged on the base 20. However, any other herein described reservoir 10 could be used instead. E.g., a single opening 40 completely spanning the base 20 is allowed. The method is also suitable for a reservoir 10 with microwells 50 on its at least one sidewall 30.

[0136] Fig. 4 shows cross sections of the micro-wells 50 for different levels of acceleration. For example, on the left, for Bo < 1 (e.g. under influence of gravity, i.e. the body force used for sealing the subvolumes 602of sample liquid within the micro-wells 50 is a gravitational force; However, as explained above, any other body force or combination of body forces is also possible) the sealing liquid 70a covers the micro-wells 50 and seals the sample liquid, see 602, inside, while a supernatant of sample liquid, see60i, is floating on top of the sealing liquid 70a. On the right, for Bo » 1 (e.g. using additionally to gravity centrifugation or a magnetic field to achieve at least the minimum required acceleration level G x g) the sample liquid is able to form droplets 144 inside the sealing liquid 70a which are rising up from the micro-wells 50 due to the smaller density of the sample liquid. Eventually the rising droplets 144 merge with the supernatant, see 6O1, on top of the sealing liquid 70a and can be recovered from there.

[0137] The condition expressed by the equation phyd= p ■ G x g ■ h » ^ = Pdrop and by Bo » 1 thus means that only at the increased acceleration level of G x g sufficient energy is provided to the system that droplets 144 of sample liquid can form inside the sealing liquid 70a. This is however a pre-requisite for the sample liquid being able to leave the micro-wells 50 and to being replaced by sealing liquid 70a inside the micro-wells 50. In some configurations, only a part of the sample liquid, see 602, inside the micro-wells 50 might be replaced by sealing liquid 70a, leaving a remaining part of the sample liquid still inside the micro-wells 50. At lower acceleration levels the replacement of sample liquid by sealing liquid is not possible due to the energy limitation (i.e. the system has not sufficient energy to release the sample liquid from the micro-wells 50, as formation of droplets of sample liquid inside the sealing liquid requires a certain minimum energy given by the equation phyd= p ■ G x g ■ h » ^ = Pdrop) ■

[0138] The release of the sample liquid from the sealed micro-wells 50 that have been generated by following the steps 1. -4. described above with regard to Fig. 3 (or any other herein described method that leads to the same fluidic situation), is achieved by applying following steps 5. -6.:

[0139] I I24EM02-2024323325. DOCX mh 5. An additional hydrostatic pressure, is provided onto the liquids inside the reservoir 10 until Bo » 1. This pressure might be produced by centrifugation, a magnetic field or by any other suitable means.

[0140] 6. Wait until the droplets 144 have risen to the top surface of the sealing liquid 70a and have merged into the bulk sample liquid 6O1 contained in the reservoir 10. Recover the sample liquid from there through the opening 40 of the reservoir 10 (e.g. by a pipette), carefully to avoid aspiration of sealing liquid 70a.

[0141] It should be noted that the described method for releasing liquids from the micro-wells 50 works not only for the specific example presented above, where the micro structured surface is located at the bottom of the reservoir. The method works as well for other embodiments of the apparatus, regardless where the micro-wells 50 are located inside the reservoir.

[0142] In the following further examples (see Fig. 5-9) for generating isolated liquid subvolumes 602in a plurality of micro-wells 50 are discussed, which may all lead to the same fluidic situation as described on the left of Fig. 4. For example, in each of Fig. 5-9, the depiction shown on the rightmost side of the respective figure may correspond to the same fluidic situation as described on the left of Fig. 4.

[0143] In the following, further examples of implementation of the invention will be presented. These examples do not provide an exhaustive collection and other implementations of the inventive apparatus and method are possible that are not explicitly shown here. Specific designs of the micro structured surface area / micro-wells 50 and certain shapes of the at least one sidewall 30 of the reservoir 10 can be beneficial for guiding the sealing material 70 to achieve a high yield of sealed compartments, i.e. subvolumes 602of sample liquid 60. Also variations of the method for filling the reservoir 10 and supporting the sealing process by sequences of centrifugation or providing additional pressure sources, like a magnetic field, might be implemented.

[0144] An embodiment according to Fig. 5, for example, is directed to using an applicator 80 for controlling the sealing process in case of the sealing material 70 being a liquid, i.e. a sealing liquid 70a. Fig. 5 shows a sketch of a sealing material applicator 80 positioned inside a herein described reservoir 10 and a time sequence (from left to right) of how the sample liquid 60 is supplied and then the sealing liquid 70a is added through the applicator 80 (e.g. by a pipette).

[0145] It is to be noted that Fig. 5 shows an example of a reservoir 10 with a single opening 40 that does not completely span the base 20. However, it is also possible that the respective opening 40 does completely span the base 20, e.g., as exemplarily shown in Fig. 2 and Fig. 3.

[0146] I I24EM02-2024323325. DOCX mh An optional sealing material applicator 80 might be used in combination with a herein described reservoir 10 to better control the position and velocity by which the sealing liquid 70a settles on the micro-wells 50 to close them off. The sealing material applicator 80, i.e. a tubular applicator, comprises a fluidic duct, tube or channel with a first opening, i.e. an inlet 82, where the sealing liquid 70a can be supplied and a second opening, i.e. an outlet 84, where the sealing liquid 70a can exit. The outlet 84, herein also denoted as exit opening, is positioned in proximity of the micro well array, i.e. the plurality of micro-wells 50, at a position from which the sealing liquid 70a can spread over the micro well array in a defined way. For example, the exit opening can be close to the bottom, i.e. the base 20, in the middle of the reservoir 10 like depicted in Fig. 5 to cover the micro structured surface area at the base 20 of the reservoir 10, starting from the center of the micro structured surface area and covering it in radial direction towards the sidewall 30 of the reservoir 10. The spreading of the sealing liquid 70a is driven by gravity (due to the higher density of the sealing liquid 70a) and / or other body forces and proceeds from the outlet 84 of the sealing material applicator 80 in the center towards the sidewall 30 of the reservoir 10 in radial direction and covers the micro structured surface area, i.e. an area on the base 20 comprising the plurality of micro-wells 50.

[0147] Apart from using gravity for transporting the sealing liquid 70a through the applicator 80 from the opening 40 of the reservoir 10 to the base 20 of the reservoir 10, it is possible to use additionally body forces, e.g. a centrifugal force and / or a magnetic force,. For example, the applicator 80 may comprise a capillary tube, which restricts the flow velocity of the sealing liquid 70a through the applicator 80 due to gravity, for which reason, additionally, a centrifugal force and / or a magnetic force (showing from the opening 40 in a direction towards the base 20) may be applied, so that the sealing liquid 70a moves efficiently towards the base 20.

[0148] The purpose of the controlled supply of the sealing liquid 70a in terms of position and spreading velocity is to achieve a high yield of properly sealed micro-wells 50. Even in cases where a specific combination of sealing liquid 70a and sample liquid 60 does not lead to a spontaneous sealing of the micro-wells 50 with sufficiently high yield, the controlled supply of the sealing liquid 70a can lead to a significant improvement. In cases where the sealing liquid 70a and sample liquid 60 have optimum properties, the spontaneous sealing can take place without using a sealing material applicator 80 and even regardless of where and how fast the sealing liquid 70a is supplied to the reservoir 10. Therefore, the sealing material applicator 80 is to be considered as an optional part of the apparatus according to the present invention.

[0149] I I24EM02-2024323325. DOCX mh As shown in Fig. 5, it is recommended to introduce 110 the sample liquid 60 through the single opening 40 of the reservoir 10 without using the applicator 80 (However, it may also be possible to use the applicator 80). This has the advantage that the sample liquid 60 does not need to flow through the channel of the applicator 80 and thus, a risk of losing sample molecules or sample particles due to adsorption at an inner surface of the applicator 80 is reduced. Therefore, it is proposed to insert the applicator 80 through the opening 40 of the reservoir 10 not until the sealing liquid 70a is introduced 120 or to couple the applicator 80 to the reservoir 10, so that the sample liquid 60 can be introduced 110 through a gap between the applicator 80 and an edge of the opening 40.

[0150] As shown in Fig. 5, the applicator 80 reaches through the opening 40 of the reservoir 10 and ends in direct proximity to the base 20 of the reservoir 10 to transport the sealing liquid 70a using a body force from the single opening 40 through the reservoir 10 towards the base 20 of the reservoir 10. In order to avoid obscuring embodiments of the present invention the applicator 80 is shown as an applicator floating within the reservoir 10. However, according to an embodiment, a person may hold the applicator 80 when introducing 120 the sealing liquid 70a. According to an alternative embodiment, the applicator 80 may be coupled to the reservoir 10. For example, the applicator 80 may be fixed to the at least one sidewall 30 of the reservoir 10 and / or to the base 20 of the reservoir 10, e.g., using structures, like bars, connecting the tubular applicator 80 with the at least one sidewall 30 and / or the base 20. According to an alternative example, the applicator 80 may be coupled to the at least one sidewall 30 of the reservoir 10 and / or to the base of the reservoir 10 using a removable lid or cap comprising the channel with the inlet 82 and the outlet 84. The removable lid or cap may be coupled to the reservoir 10 at the opening 40, so that the applicator 80 reaches through the opening 40 of the reservoir 10 and ends in direct proximity to the base 20 of the reservoir 10.

[0151] Even though Fig. 5 shows the usage of the applicator 80 exemplarily for a reservoir 10 with the plurality of micro-wells 50 being arranged on the base 20, it is possible that additionally, or alternatively, the plurality of micro-wells 50 are arranged on the at least one sidewall 30. The only difference in the method for generating the isolated liquid subvolumes in the plurality of micro-wells 50 is, that in case of the plurality of micro-wells 50 being arranged only on the base 20, it is enough to introduce 120 an amount of sealing liquid 70a into the reservoir 10 that is enough to cover the base 20, and in case of the plurality of micro-wells 50 being arranged on the at least one sidewall 30 and optionally also on the base 20, the amount of sealing liquid 70a introduced 120 into the reservoir 10 has to be enough, so that a filling level of the sealing liquid 70a within the reservoir 10 is high enough to cover the micro structured surface area on

[0152] I I24EM02-2024323325. DOCX mh the at least one sidewall 30, i.e. to cover an area on the at least one sidewall 30 comprising at least some of the plurality of micro-wells 50 (cf. Fig. 7).

[0153] Fig. 6 and Fig. 7, for example, shows an embodiment of an apparatus comprising a reservoir 10, where a micro structured surface area, i.e. an area in which one or more of the plurality of micro-wells 50 are arranged, is located on the inner sidewall 30 of the cylindrical reservoir 10. The method for generating isolated liquid compartments using such an apparatus may be performed as described with regard to Fig. 1, Fig 3 or Fig. 5, wherein it can be carried out with (cf. Fig. 7) or without (cf. Fig. 6) using a sealing material applicator 80. However, if the microwells 50 are located on the sidewall 30 of the reservoir 10 the mode of action can be slightly different than for the case where the micro-wells 50 are located on the bottom surface, i.e. the base 20 (cf. Fig. 3 and Fig. 5). In the case of the example with micro structures, e.g., the microwells 50, on the sidewall 30 of the reservoir 10, as considered here, the sealing liquid 70a is flowing parallel to the micro structured surface area and thus parallel to the openings of the micro-wells 50 as the liquid level of the sealing liquid 70a rises when more and more sealing liquid 70a is added, i.e. introduced 120, to the reservoir 10. In other words, when introducing 120 the sealing liquid 70a into the reservoir 10 to seal subvolumes of the sample liquid 60 in the micro-wells 50 using a movement of the sealing liquid 70a towards the base 20 due to a body force applied to the sealing liquid 70a, the sealing liquid 70a settles on the base 20 and a filling level of the sealing liquid 70a within the reservoir 10 rises parallel to the micro structured surface area on the sidewall 30. The rising filling level of the sealing liquid 70a displaces a bulk sample liquid 6O1 outside the micro-wells 50 and seals the micro-wells 50 arranged on the sidewall 30. This can be beneficial to displace the sample liquid 60, e.g., assay liquid, on top of the micro well openings in a safe and efficient way to generate a high yield of isolated compartments, i.e. subvolumes of the sample liquid 60. If the sealing liquid 70a is approaching the micro structured surface area (i.e. the one arranged on the sidewall 30 in case of a further micro structured surface area being arranged on the base 20) from a perpendicular direction, like it is the case when using an apparatus according to fig. 2 and using no sealing liquid applicator (cf. fig. 5), the likelihood of enclosing sample liquid 60 on top of the microwells 50 is higher and the yield of correctly sealed micro-wells 50 can be lower. Therefore, placing a micro structured surface area on at least one sidewall 30 of the reservoir 10 can have functional advantages over placing it at the bottom, i.e. the base 20.

[0154] Because a sidewall surface area of a reservoir 10 is in practice usually larger than the bottom area and thus can accommodate more micro-wells 50 in total, placing the micro-wells 50 onto the sidewalls 30 can provide a higher number of micro-wells 50 and additional advantages in certain applications. On the other hand, fabrication of micro structured surface areas on the

[0155] II24EM02-2024323325.DOCX mh sidewalls 30 of a reservoir 10 is more challenging by polymer replication technologies than on the base 20 of the reservoir 10. Therefore, the proper choice of the size and location of the micro structured surface area and the total number of micro-wells 50 will depend on many considerations regarding functional aspects, fabrication technology and cost as well as application related considerations. The inventive method described with regard to Fig. 1 or Fig. 3 works regardless of the location of the micro structured surface area inside the reservoir 10, and the apparatus given as example in fig. 2 can be combined with the examples shown in fig. 6 and I or fig. 7 to yield an apparatus that has micro structured surface areas at the bottom, i.e. the base 20, as well as on the at least one sidewall 30 of the reservoir 10.

[0156] Fig. 6 shows a sketch of a reservoir 10 with a micro structured surface area at a sidewall 30 and a time sequence of operation of such a reservoir according to the present invention (from left to right): First the assay liquid, i.e. the sample liquid 60, is introduced 110 through the reservoir inlet, i.e., the single opening 40, (e.g. by a pipette), then the sealing material, e.g., the sealing liquid 70a, is added continuously through the reservoir inlet (e.g. by a pipette) and rises inside such reservoir 10 and thereby covers the micro structured surface area on the sidewall 30 and seals the assay liquid inside the micro-wells 50.

[0157] Fig. 7 shows a sketch of a reservoir 10 with a micro structured surface area at a sidewall 30 and with a sealing material applicator 80 in the center of the reservoir 10 and a time sequence of operation of such a reservoir 10 according to the present invention (from left to right): First the assay liquid, i.e. the sample liquid 60, is introduced 110 through the reservoir opening 40 or through the sealing material applicator 80 (e.g. using a pipette), then the sealing material, e.g., the sealing liquid 70a, is added, i.e. introduced 120, continuously through the sealing material applicator 80 (e.g. by a pipette) and rises inside the reservoir 10 and thereby covers the micro structured surface area on the sidewall 30 and seals the assay liquid inside the micro-wells 50.

[0158] The amount of sample liquid 60 that is used to operate the apparatus according to the described method can be freely chosen. In the examples shown in Fig. 6 and Fig. 7 different volumes of sample liquid 60 are supplied to the reservoir 10: In the case shown in fig. 6 first as much sample liquid 60 is introduced 110 into the reservoir 10, so that the micro structured surface area on the sidewall 30 is fully covered and all the micro-wells 50 are filled, before the sealing liquid 70a is supplied, i.e. introduced 120. When the sealing liquid 70a is supplied afterwards, the excess sample liquid 6O1 that is not trapped in the micro-wells 50 is lifted upwards with the rising level of the sealing liquid 70a and forms a supernatant that might be even recovered for further analysis. However, it is even more efficient to use less sample liquid

[0159] I I24EM02-2024323325. DOCX mh 60 to achieve a lower sub-sampling error and to save on costs for the sample liquid 60 like depicted in the cases sketched in fig. 7. Theoretically, only as little sample liquid 60 has to be provided to the reservoir 10 as it is needed to fill the micro-wells 50, only (i.e. a combined volume of all micro-wells 50 of the plurality of micro-wells 50 may define a minimum amount of sample liquid 60, which is to be introduced 110 into the reservoir 10). When this volume is provided to the reservoir 10, it forms a liquid layer at the base 20. The thin layer of sample liquid 60 at the base 20 of the reservoir 10 will lead to filling of the micro-wells 50 only in the lower part of the reservoir 10, i.e. the micro-wells 50 arranged on the base 20 (if any) and if the filling level of the sample liquid is high enough to reach a micro structured surface area on the sidewall 30, a lower part of the micro structured surface area on the sidewall 30 were the micro-wells 50 are in fluidic contact with the layer of sample liquid 60. But as more and more sealing liquid 70a is introduced 120, the layer of sample liquid 60 is lifted upwards and thereby reaches also the higher parts of the micro structured surface area on the sidewall 30. The micro-wells 50 are thus filled just in time with sample liquid 60, before the sealing liquid 70a follows to seal the micro-wells 50. When the top end of the micro structured surface area is reached the sample liquid 60 might be used up completely. In practice however, it is recommended to add a safety margin to this minimum required volume. This leads to a small amount of residual sample liquid 60 that remains floating on top of the sealing liquid 70a (like in fig. 7), when the level of sealing liquid 70a has reached or exceeded the top end of the micro structured surface area on the sidewall 30. The difference between the examples shown in fig. 6 and fig. 7 regarding the sample liquid 60 is the different volume of sample liquid 60 that is initially supplied. In the example in fig. 6, a sufficient amount is supplied that the micro-wells 50 are filled before the sealing liquid 70a is added. In fig. 7, a much smaller amount is supplied, such that at least some micro-wells 50 are only filled during addition of the sealing liquid 70a while the sample liquid 60 is lifted upwards by the rising liquid level. The volume of sample liquid 60 actually used according to the inventive method can be anywhere between these two extremes (or even larger), and the choice of volume is independent whether a sealing material applicator 80 is used (like in fig. 7) or not (like in fig. 6).

[0160] As alternative to the method for releasing / recovering the sample liquid 60 from the micro-wells 50, as presented with regard to Fig. 4, the present example allows for recovery of the molecular content of the encapsulated liquid volumes, i.e. the subvolumes of the sample liquid, from the micro-wells 50 by a different method, because the micro-wells 50 are not located at the base 20 of the reservoir 10 (but this different method is also suitable for reservoirs 10 with microwells on the base 20). This method is described in the following: The release of the molecular content from the sealed micro-wells 50 that have been generated, e.g., by following the steps

[0161] I I24EM02-2024323325. DOCX mh 1. -4. described above with regard to Fig. 3 (or any other method that leads to the same fluidic situation), can be achieved by applying following steps 5. - 8.:

[0162] 5. The sealing liquid 70a is removed from the reservoir 10 through the reservoir opening 40 (e.g. by pipetting). Note: A minor part of sealing liquid 70a might not be accessible and might remain inside the reservoir 10, still. This will not impair the proper functioning of the method.

[0163] 6. The reservoir 10 is filled with a liquid that is optionally miscible with the sample liquid 60 inside the micro-wells 50 (e.g. a suitable buffer solution) up to the top end of the micro structured area.

[0164] 7. As an optional step the reservoir 10 can be centrifuged or exposed to a magnetic field to remove the film of sealing liquid 70a that might still cover the micro-wells 50. Due to the larger density of the sealing liquid 70a, a liquid film of sealing liquid 70a covering the side walls 30 will flow to the base 20 at sufficiently high acceleration (due to centrifugation or a magnetic field). Whether or not such a centrifugation step or magnetic field application step is necessary depends, amongst others, on the density of the sealing liquid 70a, its viscosity, its surface tension and its contact angle on the micro structured surface area. The sealing liquid 70a might even flow spontaneously to the base 20 of the reservoir 10 by gravity, leaving the open micro-wells 50 behind.

[0165] 8. After a short incubation time, the miscible liquid is aspirated through the reservoir opening 40 (e.g. by pipetting) from the reservoir 10. The aspirated liquid contains now the molecular content that was encapsulated in the micro-wells 50 before.

[0166] The working principle of this method relies on the diffusion of the molecular content out of the micro-wells 50 into the bulk liquid solution, once the layer of sealing liquid 70a is removed from the opening of the micro-wells 50 (either by gravity or by using centrifugation or a magnetic field as described in step 7). The liquid connection established between the micro-wells 50 and the bulk liquid volume in the reservoir 10 allows for free diffusion of the molecular content throughout the whole reservoir 10. Over time, a homogeneous concentration of the molecular content present in the micro-wells 50 before will be established in the bulk liquid volume. The duration of this process depends on the diffusion constant of the molecules and typically will take minutes to hours for typical entities relevant for molecular biology applications.

[0167] It should be noted that the method for releasing the liquid volume from the micro-wells 50 described with regard to Fig. 4 will also work on the apparatus with micro structured surface area on the sidewall 30. The method described above for recovery of the molecular content of the encapsulated liquid volumes from the micro-wells 50 provides an additional option for the

[0168] I I24EM02-2024323325. DOCX mh present implementation example and not an alternative to the general method described with regard to Fig. 4.

[0169] The examples presented in the following with regard to Fig. 8 and Fig. 9 combine the advantages of the previous examples in terms of having a micro structured surface area at the base 20 of the reservoir 10 like shown in fig. 3 (to enable read-out by a microscope or plate reader) as well as allowing the sealing material, e.g., a sealing liquid 70a, to spread over the micro-wells 50 from the side, like shown in fig. 7 (to enable easier and more reliable displacement of the sample liquid 60 on top of the micro-wells 50). This advantageous implementation can be achieved by an inclined bottom, i.e. an inclined base 20, of the reservoir 10 that is equipped with the micro structured surface area, i.e. an area comprising at least some of the plurality of micro-wells 50, in combination with a sealing material applicator 80 that guides the sealing liquid 70a to the lowest point of the inclined base 20, like shown in fig. 8, or without the sealing material applicator 80, like shown in Fig. 9.

[0170] In case of using the sealing material applicator 80, the outlet 84 of the applicator 80 is arranged in proximity to the base 20 in a lowest region thereof with respect to the horizontal. A horizontal is herein understood as line or plane that is parallel to a horizon or ground. The horizontal is perpendicular to the vertical direction (z-axis), which aligns with the force of gravity, e.g., the horizontal may be a plane parallel to an xy-plane or a line within the xy-plane. In geometry and everyday language, "horizontal" refers to anything that lies flat or level with respect to the ground, without any inclination or tilt. The horizontal may herein be understood as a plane perpendicular to the direction of the gravitational force, i.e. the force of gravity.

[0171] It should be noted in this regard, that the base 20 of the reservoir 10 is inclined with respect to the direction of gravity and not necessarily in relation to the walls 30 of the reservoir 10 or a surface of an assay plate 200 comprising a plurality of reservoirs 10. To achieve an inclined base 20 of the reservoir 10 for the purpose of carrying out the inventive method, it can be sufficient to place the apparatus described as example with regard to Fig. 2 on an inclined surface (e.g. by using an inclined support 230 to hold the assay plate 200, like shown in Fig. 9). This way, the direction of gravity (shown in Fig. 9 by the arrow 300) is not perpendicular to the micro structured surface area at the base 20 of the reservoirs 10, but points at an inclination angle other than 90° on the micro structured surface area (e.g. 80°, 70°, ..., 40°, 30°). It should be noted, that inclination of the apparatus could be achieved by other means than an inclined surface, see inclined support 230 in Fig. 9, e.g. by hand or by an automated system (e.g. a robot arm).

[0172] I I24EM02-2024323325. DOCX mh With the inclined base 20 the sealing liquid 70a starts spreading over the inclined surface, i.e. the inclined base 20, from the lowest point sidewise, displacing surplus assay liquid, see 6O1 in Fig. 8 and Fig. 9, easily to the top and in direction of the flow. This arrangement can be beneficial to achieve a higher yield of sealed micro-wells 50.

[0173] The operation of the apparatus is otherwise the same like described already with regard to Fig. 3 and Fig. 4. The individual steps of filling the reservoir 10 with the different liquids using an applicator 80 is shown in Fig. 8 and without using an applicator 80 is shown in Fig. 9.

[0174] Fig. 8 shows a sketch of a reservoir 10 with micro structures, e.g., the micro-wells 50, at the inclined bottom surface, i.e. the base 20, and a sealing material applicator 80 at the rim of the reservoir opening 40, e.g., the inlet 82 of the applicator 80 is arranged at the rim of the reservoir opening 40. Fig. 8 shows a time sequence of operation of such a reservoir 10 according to the present invention (from left to right): First the sample liquid 60 is introduced 110 through the reservoir opening 40 or through the sealing material applicator 80 (e.g. using a pipette), then the sealing material, e.g. the sealing liquid 70a, is added, e.g., introduced 120, continuously through the sealing material applicator 80 (e.g. using a pipette) and rises inside the reservoir 10 and thereby covers the micro structured surface area on the inclined base 20 and seals the sample liquid 60 inside the micro-wells 50.

[0175] Fig. 9 shows a sketch of an assay plate 200 according to the present invention with microwells 50 at the base 20 of the reservoir 10 positioned on an inclined support 230, in order that gravity (indicated by arrow 300) is not acting perpendicular onto the base 20 of the reservoir 10, but at a reduced angle. Fig. 9 shows a time sequence of operation of such a reservoir 10 according to the present invention (from left to right): First the sample liquid 60 is introduced 110 through the reservoir opening 40 (e.g. using a pipette), then the sealing liquid 70a is added, e.g., introduced 120, at the lowest point of the reservoir inlet, i.e. opening 40, to the wall 30 of the reservoir 10 continuously (e.g. using a pipette), such that the sealing liquid 70a runs down the wall 30 of the reservoir 10, accumulates at the base 20 of the reservoir 10 and thereby covers the micro structured surface area on the inclined base 20 and seals the sample liquid 60 inside the micro wells 50.

[0176] In any of this inclined configurations (cf. Fig. 9), the sealing liquid 70a can be easily applied to the lowest part of the inclined micro structured surface area, e.g., to the lowest part of the inclined base 20 on which micro-wells 50 are arranged, by adding, e.g., introducing 120, the sealing liquid 70a at the lowest point (with respect to the horizontal) of the single opening 40 to the wall 30 of the reservoir 10 continuously and letting it run down the wall 30 of the reservoir

[0177] I I24EM02-2024323325. DOCX mh 10. Using this technique - as shown in fig. 9 - no sealing liquid applicator 80 is needed, which reduces costs for production of the apparatus, and makes the handling of the sealing liquid 70a as simple as possible. The inventors found that pipetting the sealing liquid 70a to a wall 30 of a reservoir 10 results in that, that the sealing liquid 70a smoothly runs down into the reservoir 10. Therefore, the example presented in fig. 9 is easy to implement and results in an efficient and reliable sealing of the subvolumes of sample liquid within the micro-wells 50.

[0178] Fig. 8 and Fig. 9 show both examples of reservoirs 10 with the plurality of micro-wells 50 being arranged on the base 20. However, it is in both cases also possible that additionally (or alternatively) at least some micro-wells 50 are arranged on the sidewall 30 of the reservoir 10. Especially in the example shown in Fig. 8 with the applicator 80 the positioning of micro-wells 50 within the reservoir 10 is not restricted. In case of using the method shown in Fig. 9 it is recommended to position the micro-wells 50 on a side of the sidewall 30 opposite to the side on which the sealing liquid 70a is to run down to the lowest point of the reservoir 10, so that it is also possible to not directly fill all micro-wells 50 when introducing 110 the sample liquid 60, as described with regard to Fig, 7, since otherwise the sealing liquid 70a may fill the microwells 50 on the sidewall 30 before same are filled with the sample liquid 60.

[0179] Additionally, it may be noted that, instead of using the applicator 80 in the embodiment of Fig. 8 or instead of letting the sealing liquid 70a run down the sidewall 30 in the embodiment of Fig. 9, the sealing procedure may be performed as described with regard to Fig. 6 by introducing 120 the sealing liquid 80a on top of the sample liquid 60, so that same sinks through the sample liquid 60 due to the body force to the inclined base 20 of the reservoir 10. In this case all microwells 50 (independent of whether they are arranged on the inclined base 20 and / or on the at least one sidewall 30) should be filled with sample liquid 60 before introducing 120 the sealing liquid 70a.

[0180] In Fig. 8 and in Fig. 9 the sealing material is described as a sealing liquid 70a. However, it is alternatively possible to use as sealing material a solid material, e.g., as will be described with regard to Fig. 11. In case of having a reservoir 10 with a manufactured inclined base 20, as shown in Fig. 8, the sealing material 70 can represent a solid sealing member with an inclined surface 72 (see Fig. 10), wherein the inclined surface 72 has the same inclination as the base 20 of the reservoir 10. In case of tilting a reservoir 10, so that same has an inclined base 20, as shown in Fig. 9, the sealing material 70 can represent a solid sealing member as will be described with regard to Fig. 11. In both cases the sealing procedure may function as described in the following with regard to Fig. 11.

[0181] I I24EM02-2024323325. DOCX mh As will be described with regard to Fig. 11 in more detail, the sealing material 70 shown in Fig. 10 may have dimensions smaller than the opening 40 or than a reservoir inner and / or may comprise through-holes for facilitating the sealing procedure and increasing an efficiency.

[0182] In case of using the sealing member shown in Fig. 10 for the sealing procedure in the embodiment of Fig. 8, the applicator 80 will be omitted.

[0183] Fig. 11 shows a sketch of a reservoir 10 with a micro structured surface area, i.e. an area comprising the plurality of micro-wells 50, at the base 20. Fig. 11 shows a time sequence of operation of such a reservoir 10 according to the present invention (from left to right): First the sample liquid 60 is introduced 110 through the reservoir opening 40 (e.g. by a pipette), then the sealing member (e.g., an example for the solid sealing material 70; e.g., made from sealing material 70) is introduced 120 through the reservoir opening 40. Due to a body force (e.g., a gravitational force, a centrifugal force and / or a magnetic force) the sealing member, see 70, sinks to the base 20 and seals the micro-wells 50 by displacing the sample liquid 60 from top of the micro-wells 50.

[0184] The embodiment presented with regard to Fig. 11 uses a solid (optionally soft) material preferably in shape of the reservoir 10 as sealing material (e.g. a disc, cf. fig. 11). In contrast to the previous examples, the sealing material 70 - being a solid - is not able to flow, but it still can settle on the micro-wells 50 to close them-off due to the body force and its higher density. In addition to the required higher density of the sealing material 70, the shape of the so called sealing member made out of sealing material 70 needs to prevent floating. In this example this is achieved by a flat shape similar to the reservoir’s cross-section. Furthermore, the sealing member must not fit too tight into the reservoir 10 to prevent getting stuck during settling. In case the opening 40 of the reservoir 10 does not completely span the base 20, the sealing member has to fit through the opening 40, i.e. a cross-section of the sealing member has to be smaller than an area spanned by the opening 40. Potentially, the sealing member can be equipped with some holes connecting the lower side of the sealing member with its upper side, in order to leave way for the displaced sample liquid 60 to rise to the top of the reservoir 10. Otherwise, the assay liquid, i.e. the sample liquid 60, can rise up along the sidewall 30 of the reservoir 10 through the slit between the sealing member and the reservoir wall, i.e. the sidewall 30. A sketch of the apparatus according to this example is provided in fig. 11 along with a time sequence of its operation.

[0185] The usage of the solid sealing material 70 has the additional advantage that the sample liquid 60 from the micro-wells 50 can be easily recovered by removing the sealing material 70 from

[0186] I I24EM02-2024323325. DOCX mh the reservoir 10. The sample liquid 60 diffuses from the micro-wells 50 into the bulk sample liquid and can be aspirated through the reservoir opening 40 (e.g. by pipetting) from the reservoir 10.

[0187] As described above with regard to Fig. 5, Fig. 7 and Fig. 8, a herein described apparatus may comprise an applicator 80 (e.g., a herein described method may use an applicator 80). Optionally, as will be described in the following with regard to Fig. 12 and Fig. 13, the applicator may comprise a membrane closing its outlet 84.

[0188] Fig. 12 shows on the left a cross-section of a fluidic applicator 80 where the applicator outlet 84 is closed with an elastic membrane 86, e.g. of solid material, such that the applicator 80 can be filled with a hydraulic liquid (an example for a sealing material being a sealing liquid 70a). Fig. 12 shows on the right a time sequence of operation of such an applicator 80 to seal the micro-wells 50 on the base 20 of the reservoir 10 according to the present invention (from left to right): First the sample liquid 60 is introduced 110 through the reservoir opening 40 (e.g. by a pipette), then the sealing liquid 70a is introduced 120 through the applicator inlet 82. Due to a body force (e.g., gravity and / or centrifugation and / or magnetism) the sealing liquid 70a presses the elastic membrane 86 to the base 20 and seals the micro-wells 50 by displacing the sealing liquid 6O1 from top of the micro-wells 50. Hereby, the sealing liquid 70a remains separated from the sample liquid 60 by the elastic membrane 86 at all times and does not get in contact with the sample liquid 60. As show on the far right of Fig. 12, subvolumes 6O2 of the sample liquid 60 are sealed by the membrane 86 within the micro-wells 50, wherein the membrane 86 is pressed to the base 20 of the reservoir 10 by the sealing liquid 70a.

[0189] The embodiment uses a soft but solid material to form the elastic membrane 86 at the outlet 84 of the fluidic applicator 80. This membrane 86 at the end of the applicator 80 is brought into close proximity to the micro-wells 50 to be sealed at the base 20 of the reservoir 10, similarly to previous examples. Then the sealing liquid 70a is supplied, see 120, to the applicator inlet 82. This sealing liquid 70a has a higher density than the sample liquid 60 and is moved within the applicator 80 towards the base 20 using the body force. The pressure building up in the sealing liquid 70a will lead to a deformation respectively inflation of the elastic membrane 86. As the membrane protrudes form the lower end, i.e. the outlet 84, of the applicator 80, it touches the micro structured surface area at the base 20 of the reservoir 10 and thereby seals the micro-wells 50. The sealing liquid 70a remains surrounded by the elastic membrane 86 at all times and does not get into contact with the sample liquid 60.

[0190] I I24EM02-2024323325. DOCX mh In contrast to previous examples, this example discloses a combination of a liquid and a solid material to achieve the sealing of the micro-wells 50. This can be advantageous, because a solid material can provide a more complete sealing, while a liquid material (in this case the hydraulic liquid) provides easier handling in laboratory practice than a fully solid sealing member (cf. example of Fig. 11). Similar to the example shown in Fig. 5, it is also in the present example advantageous to place the applicator 80 in the center of the reservoir 10, in order to enable the elastic membrane to spread from the center evenly in radial direction until all microwells 50 are closed (this positioning might also be advantageous for the embodiment with the inclined base 20, cf. Fig. 8). Thereby displacing the excess assay liquid, see 6O1, to the side where it can rise upwards in the reservoir 10.

[0191] At the bottom of Fig. 12 a close-up view of the sealing process using an elastic membrane 86 and a hydraulic liquid is shown. Fig. 13 shows a comparison between the sealing process with a membrane 86 (see the top row) and without a membrane 86 (see the lower row), wherein the sealing process without the membrane 86 uses a sealing liquid 70a with higher density and smaller contact angle than the sample liquid 60. In both cases subvolumes 6O2 of sample liquid 60 are efficiently sealed within the micro-wells 50. It should be noted, that filling of the elastic membrane 86 and thus, sealing of the micro-wells 50, could also be achieved by pressurizing the elastic membrane with gas.

[0192] Fig. 13 shows a close-up cross sectional view of the sealing process (time sequence from left to right, arrows indicate the flow direction of the immiscible liquids, e.g., of the sealing liquid 70a (see 122) and of the sample liquid 60 (see 124)). On the top of Fig. 13 the sealing process is show for the case of using a sealing material applicator 80 with elastic membrane 86 and a hydraulic liquid like in the present example and on the bottom of Fig. 13 the sealing process is shown for the case of using a liquid sealing material 70a that has a higher density and optionally a lower contact angle than the sample liquid 60.

[0193] The usage of an applicator 80 with a membrane 86 at its outlet 84 has the additional advantage that the sample liquid 60 from the micro-wells 50 can be easily recovered by removing the sealing material 70 through the inlet 82 from the applicator 80, so that the membrane 86 retracts. The sample liquid 60 diffuses from the micro-wells 50 into the bulk sample liquid and can be aspirated through the reservoir opening 40 (e.g. by pipetting) from the reservoir 10.

[0194] The previous examples featured a cylindrical reservoir 10 with a round base 20 and a cylindrical sidewall 30. However, as already mentioned, it is possible to use reservoirs 10 with

[0195] I I24EM02-2024323325. DOCX mh other shapes. Fig. 14 shows exemplarily an embodiment of such a reservoir 10 with a different shape, namely a reservoir 10 with a micro structured surface area on a tapered sidewall 30.

[0196] Fig. 14 shows a cross sectional view of a round reservoir 10 with an at least partly tapered sidewall 30 similar to a PCR-tube. On the far left of Fig. 14 a side view, i.e. section through the axis of symmetry (z-axis), is shown with magnified details of the micro-wells 50 next to it. In the middle a top view, i.e. a section perpendicular to the axis of symmetry (z-axis), is shown with magnified details of the micro-wells 50 next to it and on the far right of Fig. 14 a 3D-sketch of the shape of the volume enclosed by a single micro-well 50 is shown.

[0197] The example shown in Fig. 14 relates to a reservoir 10, where the sidewall 30 of the reservoir 10 is not parallel to the axis of symmetry (z-axis), but the reservoir 10 has - at least in parts -a conical shape with a at least partially tapered sidewall 30. Such shape can have some significant advantages in practice, because 1. many laboratory instruments are designed to work with reservoirs 10 of such shape (e.g. similar to a PCR-tube or PCR-cycler, shakers, cooling blocks etc.) 2. the micro-wells 50 can be produced on the tapered wall 30 of the reservoir 10 by injection molding more easily than on parallel walls, e.g., more easily than on a sidewall parallel to the force of gravity. This is due to the fact, that the molded part needs to be released from the tool after solidification, and this is only possible if the design has no recess with respect to the demolding direction. The tapering of the walls 30 thus allows for release of the injection molding tool through the reservoir opening 40 easily, provided that the micro structures, e.g., the micro-wells 50, do not form any recess, which is not completely open in the direction of the reservoir opening 40. An example of such an injection moldable design of a round reservoir with tapered walls is shown in fig. 14. The inserts show magnified closeup views of the cross sections to present the detailed shape of the micro-wells 50. The microwells 50 in this example have a triangular shape, where the longer side of the triangle is parallel to the demolding direction (given by the symmetry axis), e.g. a direction perpendicular to the reservoir opening 40, and the shorter side walls are directed in radial, respectively tangential direction. The shape of the resulting micro-well volume is sketched on the far right of Fig. 14. Any other shape that features no recess with respect to the demolding direction and can be fabricated by tooling technology is of course also possible.

[0198] It is to be noted that even though Fig. 4 to 9 and Fig. 11-12 show embodiments of reservoirs 10 with a single opening 40 that does not completely span the base 20, each of the reservoirs 10 could alternatively be realized with a single opening 40 spanning the complete base 20.

[0199] II24EM02-2024323325.DOCX mh Additionally, it may be noted that herein a plurality of examples are described, which use a magnetic field or a magnetic force. In this cases it is recommended to use a sealing material 70 with ferromagnetic properties.

[0200] As described herein, the body force comprises at least one of a gravitational force, a centrifugal force and a magnetic force. The advantage of the herein described apparatus and methods is that an efficient and controlled sealing process for generating isolated sample liquid subvolumes can already be achieved using gravity alone due to the sealing material 70 having a higher density than the sample liquid 60. The sealing process can be even more efficient, if a centrifugal force and / or a magnetic force is used as the body force applied to the sealing material 70. In this regard the inventors found that the higher density of the sealing material 70 compared to the density of the sample liquid 60 has the advantage, that it is possible to stop applying the centrifugal force and / or the magnetic force after the sealing process and still have isolated subvolumes of the sample liquid, since gravity holds the sealing material 70 in place.

[0201] Proof-of-Principle experiments have been carried out to demonstrate the working principle according to this invention. Fig. 15 shows experimental results with micro structured foil. A micro structured foil was used, featuring a multitude of micro-wells 50 in shape of inverted pyramids with opening angle 54,7° and a side length of the pyramid base of 72 pm. Pieces of the foil where cut and placed on the bottom of the reservoirs of a regular 12 well MTP. The reservoirs where filled with PCR master mix of different vendors that was spiked with a fluorescent dye (Rhodamin B) to increase the optical contrast. Then different sealing liquids where applied by a pipette to the individual reservoirs. Various ways to apply the sealing liquids where tested. Amongst others the configuration shown in fig. 9, where the reservoir 10 was tilted and the sealing liquid 70a was applied by a pipette to run down the wall 30 of the reservoir 10 to the lowest point of the reservoir 10, has been tested. It turned out that the size of the wells 50, the type and combination of liquids and how the sealing liquid 70a is applied, do significantly influence the yield of successfully sealed micro-wells 50. Some micro-wells 50 where always sealed, but for certain combinations of materials and parameters the yield was much higher than for others, such that nearly all wells 50 where sealed.

[0202] Fig. 15 shows fluorescent microscopic images of filled and sealed micro-wells 50 with an opening of approximately 72 x 72 pm2. On the left the micro-wells 50 are covered with a sealing liquid of type A that was resulting in a poor yield and on the right the micro-wells 50 are covered with a sealing liquid of type B that was resulting in a yield close to 100%.

[0203] II24EM02-2024323325.DOCX mh Fluorescent microscopic images of the resulting sealed micro-well arrays are shown in Fig. 15 as example. The PCR master mix is visible in green color, while the sealing liquid is transparent and the walls of the micro-wells 50 appear dark. The rims between the micro-wells 50 that are supposed to be wetted by the sealing liquid appear lighter than the walls inside the micro-wells 50. It seems like the sealing liquid intrudes to some extent into the pyramid shaped micro-wells 50, but an encapsulated volume of green assay liquid is clearly visible at the bottom of the micro-wells 50.

[0204] In order to prove whether the fluidic communication between neighboring micro-wells is indeed completely interrupted, a bleaching experiment was carried out as follows (see Fig. 16): A sample with even smaller micro-wells with an opening of approximately 20 pm in diameter was illuminated for 15 min at 484 nm wavelength by the light source of the microscope. Obviously, the illumination has led to bleaching of the green fluorescent intensity in those micro-wells that have been exposed to the illumination (like shown by the bleached spot visible in the left image in Fig. 16). Subsequently, another image was acquired after 60 min to observe the intensity of the bleached spot (cf. the right image of Fig. 16). It can be expected that if fluidic communication between the micro-wells would be possible, the green fluorescent intensity would be restored over time. Because unbleached molecules of the fluorescent dye could diffuse into the bleached area from the neighboring wells. Obviously, this is not the case. Even after 60 minutes, the size and intensity of the bleached spot is basically unchanged. While the diffusion coefficient of Atto 488 of approx. 400 pm2 / s suggests a diffusion length of 400 pm2 / s-3600s = 1200 pm during the observation time. For the given size of the microwells this means that the fluorescent dye should have been able to diffuse over a distance of approximately 48 micro-wells (having a 25 pm distance between the centers of two neighboring micro-wells). As the bleaching spot is approximately 20 to 25 micro-wells in diameter, diffusion should have been able to drive the fluorescent dye across the whole bleaching spot, which must have led to a full elimination of the bleaching spot and an observable change in the fluorescent intensity inside. As the observed fluorescent intensity and size of the bleaching spot remained constant, this leads to the conclusion that un-bleached fluorescent dye could not diffuse into the bleached area from the neighboring micro-wells, indicating that the fluidic communication between the wells was interrupted by the sealing liquid. This experiment proves the working principle of the inventive apparatus and method to generate well isolated micro compartments.

[0205] Fig. 16 shows fluorescent microscopic images of filled and sealed micro-wells with a round opening of approximately 20 pm in diameter after bleaching of the fluorescent dye at the

[0206] I I24EM02-2024323325. DOCX mh position indicated by the arrow and circle. The image on the left has been taken directly after 15 min of bleaching, the image on the right has been taken 60 minutes later.

[0207] Fig. 17 shows fluorescent microscopic images of filled and sealed micro-wells 50 with two different geometries in a similar size. The image on the left shows micro-wells created by inverted pyramids with opening angle 54,7° and a side length of the pyramid base of 25 pm. The image on the right shows micro-wells created by inverted conical cylinders with opening angle 80° and a diameter of 20 pm at the base. Both geometry types were filled and sealed using comparable experimental setups, demonstrating the high level of geometry independence when using the presented invention.

[0208] One huge advantage of the present invention is the high number and high density of liquid compartments that can be generated per surface area. While the state of the art technologies waste a lot of space for channels and chambers that are needed to flow the liquids to the compartments, the apparatus according to the present invention can be designed to have micro wells everywhere on the inner walls of the reservoirs. While common systems for 96 samples on the footprint of a standard micro well plate can only achieve approx. 8.000 liquid compartments per sample (e.g. QIAcuity, having 96 x 8.000 = 768.000 compartments in total) and others for 24 samples can achieve approx. 100.000 liquid compartments per sample on the footprint of standard micro well plate (e.g. Roche Digital Light Cycler, having 24 x 100.000 = 2.4 Mio. compartments in total); the apparatus according to the present invention can achieve up to 500.000 liquid compartments of about 1 pL volume on the bottom of each reservoir for 96 samples on the footprint of a standard micro well plate (i.e. assay plate according to present invention, having 96 x 500.000 = 48 Mio. compartments in total, or even more, if the walls of the reservoir are equipped with micro wells, too). So, the integration density of the compartments in the apparatus according to the present invention is significantly higher.

[0209] Another advantage is the simpler design of the assay plate in contrast to microfluidic chips and plates that are used by state of the art technologies. Due to the open design of the reservoir that can consist of one layer of polymer material, only (instead of a microfluidic chip with at least two layers), the fabrication costs for the assay plate are much lower than for the multilayered microfluidic chips used by state-of-the art technologies.

[0210] It is also advantageous that the present invention does not need any external drive mechanism to seal the liquid compartments in principle, since using gravity as body force is already efficient. This simplifies the instrumentation that is needed to operate the apparatus. In fact, the assay plate according to the present invention (particularly the example shown in fig. 9)

[0211] II24EM02-2024323325.DOCX mh can be operated by standard laboratory equipment (i.e. pipettes, centrifuges, PCR cyclers, plate readers and liquid handling robots), while the state-of-the art technologies rely on specific instruments that are customized to the requirements of the specific microfluidic chip.

[0212] Another advantage of the present invention is that the liquid can be recovered from the isolated liquid compartments easily, like described with regard to Fig. 4 or Fig. 6 / 7 or Fig. 11 or Fig. 12. For many of the state-of-the art technologies this is not possible at all.

[0213] None of the state-of-the art technologies teaches to produce compartments that are formed by solid walls (micro wells) in combination with a sealing material, e.g. a liquid material or a solid material, whereby the sealing material is settling spontaneously due to its larger density -without any additional driving mechanisms - onto the micro wells to seal them and isolate them using gravity.

[0214] State-of-the art technologies always require either 1. a pressure source to flow the sealing liquid actively over the micro wells by means of fluidic pressure or negative pressure to isolate the micro wells [2012_Kan] [US20120196774A1_Quanterix], or 2. use mechanical mechanisms and high forces to press a solid material (e.g. foil) onto the open micro wells to produce the sealing [US20190217298A1_QIAGEN [US10385383B2_ThermoFisher], The present invention is presenting a completely novel technical solution that is neither requiring external pressure sources nor high mechanical forces to achieve the sealing of the micro wells.

[0215] Furthermore the present invention does not require any channels or fluidic ducts [US20190217298A1_QIAGEN] [2014_Zhu] [EP3714977A1_Roche] [US20220305493A1_Bosch] for guiding the sealing liquid to the individual micro wells. In contrast to other technical solutions where a sealing liquid is used, the sealing liquid in the present invention is not guided through a channel or duct towards the micro wells, but a sufficiently large volume of sealing liquid is applied that all micro wells are covered as the sealing liquid spreads due to gravity and takes on its stationary position as sealing layer of the micro wells governed by general fluid dynamic principles spontaneously (described by the Navier-Stokes-Equation). The pressure values required for flowing the sealing material into place are significantly lower than for state-of-the art technologies. For this reason the liquid flow can be achieved by gravity and the density difference of the liquids, only.

[0216] Only one disclosure according to the state-of-the art [US20220305493A1_Bosch] teaches to optionally use a sealing material (commonly termed “oil” in other publications) that has higher density than the assay liquid, at all. However, in said disclosure the higher density of the sealing liquid is not driving the flow of the sealing liquid, but is just mentioned as an optional,

[0217] I I24EM02-2024323325. DOCX mh yet not essential means, to generate a more even flow field, respectively a more flat meniscus. The present invention is the only technical solution that is not necessarily requiring any additional external driving force to seal the micro wells, but exploits the density difference of the materials in combination with gravity and / or other body forces as driving force to move the sealing material into position.

[0218] None of the state-of-the art technologies teaches to use a sealing material applicator, like described as optional part of the apparatus, to support the even distribution of the sealing material for efficient and secure closing of the micro wells without enclosing bubbles. In contrast to micro channels used in other technologies, the sealing material applicator in this invention is optional and not essential.

[0219] None of the state-of-the art technologies teaches how to recover the volume from the micro wells for subsequent analysis. The present invention provides this novel functionality when the acceleration of the sealing liquid towards the micro wells is increased e.g. through centrifugal forces beyond a specific acceleration threshold, where Bo » 1 , as described in detail above.

[0220] An embodiment relates to an apparatus for producing separated, closed liquid compartments from a bulk liquid volume, comprising

[0221] 1. at least one reservoir for receiving and holding the bulk liquid volume,

[0222] 2. said reservoir having inside a micro structured surface with at least one micro well, and 3. a non-miscible sealing material to seal the at least one micro well,

[0223] characterized by the sealing material having a higher density than the bulk liquid.

[0224] Another embodiment relates to a method for producing separated, closed liquid compartments from a bulk liquid volume, comprising

[0225] 1. provide an apparatus, as described herein, empty of any liquids

[0226] 2. supply a certain volume of bulk liquid to the reservoir (e.g. by a pipette)

[0227] 3. supply a certain volume of sealing material to the reservoir

[0228] 4. provide a body force to the whole apparatus (e.g. by gravity or centrifugation or magnetism) to flow or move the sealing material onto the micro structured surface 5. wait until the sealing material has spread over the micro structured surface and has settled onto the rims of the individual micro wells to close the at least one well.

[0229] The apparatus and / or the method may comprise one or more of the following:

[0230] • Include requirement for sealing liquid (instead of material)

[0231] • Include requirement for higher density and lower contact angle of the sealing liquid in comparison to the assay liquid.

[0232] I I24EM02-2024323325. DOCX mh • Include a mandatory use of a sealing material applicator.

[0233] • Include the requirement for Bo < 1 (e.g., in the method)

[0234] • Include the requirement of using a centrifugation step (e.g., in the method)

[0235] • Include the requirement that the design of the micro wells has to meet the Concus-Finn condition

[0236] According to an embodiment the reservoir made from a solid material (e.g. a polymer material) features at least one micro well that can be sealed from one side by the sealing material (e.g. a non-miscible liquid or a soft and elastic solid material).

[0237] According to an embodiment the sealing material has a higher density than the bulk assay liquid to be separated and compartmentalized into the micro wells.

[0238] According to an embodiment the sealing material has a smaller contact angle than the bulk assay liquid (optional feature).

[0239] The herein discussed sealing material applicator is an optional feature.

[0240] Increasing the acceleration by centrifugation or by applying a magnetic field or any other body force is an optional feature.

[0241] An apparatus according to a herein described embodiment, wherein the sealing material has a smaller contact angle on the micro structured surface than the bulk liquid.

[0242] An apparatus according to a herein described embodiment featuring in addition a sealing material applicator for controlled supply of the sealing material, wherein the applicator consists of a fluidic channel through which the liquid sealing material is supplied at the one end of the channel and the other, open end of the fluidic channel that is placed in close proximity to the micro structured surface.

[0243] An apparatus according to a herein described embodiment featuring in addition an applicator for controlled supply of a soft and elastic sealing material, wherein the applicator consists of a fluidic channel having one open end through which a fluid pressure can be applied and the other end closed by the soft and elastic sealing material that is placed in close proximity to the micro structured surface.

[0244] II24EM02-2024323325.DOCX mh An apparatus according to a herein described embodiment, wherein the micro structured surface is located at the bottom of the reservoir.

[0245] An apparatus according to a herein described embodiment, wherein the micro structured surface is located at the walls of the reservoir.

[0246] An apparatus according to a herein described embodiment, wherein the micro structured surface is located at the bottom and at the walls of the reservoir.

[0247] An apparatus according to a herein described embodiment, wherein the micro structured surface features approximately 10, 100, 1000, 10.000, 105, 106..., 109, 101° micro wells, each holding a liquid volume of approximately 1pl, 100 nl, 10 nl, 1 nl, 100 pl, 10 pl ..., 10 fl, 1 fl. However, it is also possible that the micro structured surface features more than 1010microwells and / or micro-wells holding more than 1 l or less thanl fl liquid volume.

[0248] An apparatus according to a herein described embodiment, wherein the micro wells have different sizes and / or volumes.

[0249] An apparatus according to a herein described embodiment, wherein a plurality of reservoirs are arranged in a regular array to form a assay plate with 12, 24, 48, 96, 384, 1.536, 3.456 or 6.144 separated reservoirs, each of which exhibiting the features of a herein described reservoir.

[0250] An embodiment relates to tilting the assay plate with micro structured surface at the bottom to achieve the operation principle shown in fig. 9.

[0251] An embodiment relates to a method for releasing the liquid from the separated compartments by increasing Bo » 1 by additional pressure supplied to the reservoirs (e.g. by centrifugation or by applying a magnetic field)

[0252] Although some aspects have been described as features in the context of an apparatus it is clear that such a description may also be regarded as a description of corresponding features of a method, e.g., where a block or device corresponds to a method step or a feature of a method step. It is further to be noted that methods disclosed in the specification or in the claims may be implemented by a device having means for performing each of the respective steps of these methods. Analogously, some aspects have been described as features in the context of a method, but it is clear that such a description may also be regarded as a description of

[0253] I I24EM02-2024323325. DOCX mh corresponding features concerning the functionality of an apparatus. Some or all of the method steps may be executed by (or using) a hardware apparatus, like for example, a microprocessor, a programmable computer or an electronic circuit or a liquid handling robot or a centrifuge. In some embodiments, one or more of the most important method steps may be executed by such an apparatus. Analogously, an apparatus may comprise a hardware apparatus, like for example, a microprocessor, a programmable computer or an electronic circuit or a liquid handling robot or a centrifuge, configured to perform one or more method steps.

[0254] Furthermore, in some embodiments a single step may include or may be broken into multiple sub steps. Such sub steps may be included and part of the disclosure of this single step unless explicitly excluded.

[0255] In the foregoing detailed description, it can be seen that various features are grouped together in embodiments for the purpose of streamlining the disclosure. This method of disclosure is not to be interpreted as reflecting an intention that the claimed embodiments require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter may lie in less than all features of a single disclosed embodiment. Thus, the following claims are hereby incorporated into the Detailed Description, where each claim may stand on its own as a separate embodiment. While each claim may stand on its own as a separate embodiment, it is to be noted that - although a dependent claim may refer in the claims to a specific combination with one or more other claims - other embodiments may also include a combination of the dependent claim with the subject matter of each other dependent claim or a combination of each feature with other dependent or independent claims. Such combinations are proposed herein unless it is stated that a specific combination is not intended. Furthermore, it is intended to include also features of a claim to any other independent claim even if this claim is not directly made dependent to the independent claim.

[0256] Although specific embodiments have been illustrated and described herein, it will be appreciated by those of ordinary skill in the art that a variety of alternate and / or equivalent implementations may be substituted for the specific embodiments shown and described without departing from the scope of the present embodiments. This application is intended to cover any adaptations or variations of the specific embodiments discussed herein. Therefore, it is intended that the embodiments be limited only by the claims and the equivalents thereof and not by the specific details presented byway of description and explanation of the examples herein.

[0257] I I24EM02-2024323325. DOCX mh References

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[0260] [2017_Zhang] Zhang et al., Digital Bioassays: Theory, Applications, and Perspectives,

[0261] Anal. Chem., 89, 1, 92-101 (2017)

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[0272] PNAS, 102 (6) 1848-1852 (2005)

[0273] I I24EM02-2024323325. DOCX mh

Claims

Claims1. Method (100) for generating isolated liquid subvolumes (6O2) in a plurality of microwells (50) of a reservoir (10) comprising a base (20), at least one sidewall (30) and a single opening (40) at least partially spanning the base (20), wherein the plurality of micro-wells (50) is arranged on the base (20) and / or on the at least one sidewall (30), the method (100) comprising:introducing (110) a sample liquid (60) into the reservoir (10) through the single opening (40); andintroducing (120) a sealing material (70, 70a) into the reservoir (10) through the single opening (40) to seal subvolumes (602) of the sample liquid (60) in the plurality of microwells (50) using a movement of the sealing material (70, 70a) towards the base (20) due to a body force applied to the sealing material (70, 70a).

2. Method (100) of claim 1, wherein the body force comprises at least one of a gravitational force, a centrifugal force and a magnetic force.

3. Method (100) of claim 1 or claim 2, wherein the body force comprises a magnetic force and wherein the sealing material (70, 70a) comprises ferromagnetic properties.

4. Method of any of claims 1 to 3, wherein the body force comprises a gravitational force and wherein the sealing material (70, 70a) has a higher density than the sample liquid (60).

5. Method (100) of any of claims 1 to 4, wherein the sample liquid (60) is introduced into the reservoir (10) through the single opening (40) to arrange the subvolumes (602) of the sample liquid (60) in the plurality of micro-wells (50).

6. Method (100) of any of claims 1 to 5, wherein the sealing material (70, 70a) is a liquid having a smaller contact angle to the base (20) and / or to the at least one sidewall (30) than the sample liquid (60).

7. Method (100) of any of claims 1 to 5, wherein the sealing material (70, 70a) is a solid material and the sealing material (70, 70a) is introduced in the reservoir (10) via a solid sealing member comprising the sealing material (70, 70a).II24EM02-2024323325.DOCX mh8. Method (100) of claim 7,wherein, when seen in a direction from the single opening (40) to the base (20) of the reservoir (10), the solid sealing member has a smaller cross section than the reservoir (10) to allow the sample liquid (60) to flow through a gap between the solid sealing member and the at least one sidewall (30) towards the single opening (40) when the sealing material (70, 70a) moves towards the base (20),and / orwherein the solid sealing member has one or more through-holes to allow the sample liquid (60) to flow through the one or more through-holes towards the single opening (40) when the sealing material (70, 70a) moves towards the base (20).

9. Method (100) of any of claims 1 to 8, wherein introducing the sealing material (70, 70a) comprises applying the sealing material (70, 70a) on top of the sample liquid (60), wherein the sealing material (70, 70a) moves through the sample liquid (60) due to the body force to the base (20) of the reservoir (10).

10. Method (100) of any claim 1 to 6, wherein the sealing material (70, 70a) is a liquid and introducing the sealing material (70, 70a) comprises using a tubular applicator (80) comprising an inlet (82) and an outlet (84), wherein the tubular applicator (80) is configured to transport the sealing material (70, 70a) using the body force from the single opening (40) through the reservoir (10) towards the base (20) of the reservoir (10).

11. Method (100) of claim 10, wherein the applicator (80) comprises a membrane (86) closing the outlet (84), wherein the membrane (86) is deformed by the movement of the sealing material (70, 70a) towards the base (20) to seal the subvolumes (602) of the sample liquid (60) in the plurality of micro-wells (50).

12. Method (100) of claim 10,wherein the plurality of micro-wells (50) is arranged on the at least one sidewall (30), wherein, upon introducing of the sample liquid (60), the plurality of micro-wells (50) are not or at least partially not filled with the sample liquid (60), andwherein the movement of the sealing material (70, 70a) towards the base (20) due to the body force lifts the sample liquid (60) in the direction of the single opening (40) to arrange the subvolumes (602) of the sample liquid (60) in the plurality of micro-wellsI I24EM02-2024323325. DOCX mh(50) before sealing the subvolumes (6O2) of the sample liquid (60) in the plurality of micro-wells (50).

13. Method (100) of any claim 1 to 12, further comprising using the reservoir (10) in which the base (20) of the reservoir (10) is inclined with respect to the horizontal.

14. Method (100) of claim 13 when referring back to claim 9, wherein the plurality of microwells (50) is arranged on the base (20) and wherein the outlet (84) of the applicator (80) is arranged in proximity to the base (20) in a lowest region thereof with respect to the horizontal.

15. Method (100) of claim 13 when referring back to any of claims 1 to 6, wherein the plurality of micro-wells (50) is arranged on the base (20) and wherein the sealing material (70, 70a) is a liquid and introduced into the reservoir (10) by letting the sealing material (70, 70a) flow along the at least one sidewall (30) to a lowest region of the base (20) with respect to the horizontal.

16. Method (100) of claim 14 or claim 15, wherein, upon introducing of the sample liquid (60), the plurality of micro-wells (50) are not or at least partially not filled with the sample liquid (60), andwherein the movement of the sealing material (70, 70a) towards the base (20) due to the body force lifts the sample liquid (60) in the direction of the single opening (40) to arrange the subvolumes (6O2) of the sample liquid (60) in the plurality of micro-wells (50) before sealing the subvolumes (6O2) of the sample liquid (60) in the plurality of micro-wells (50).

17. Method (100) of any of claims 1 to 6 and 9 to 16, wherein the sealing material (70, 70a) is a liquid; andwherein the method (100) further comprises recovering the subvolumes (602) of the sample liquid (60) from the plurality of micro-wells (50) by applying a force with a direction from the single opening (40) to the base (20) pushing the sealing material (70, 70a) into the plurality of micro-wells (50) and releasing the subvolumes (602) of the sample liquid (60) from the plurality of micro-wells (50).

18. Method (100) of any of claims 1 to 6 and 9 to 16, wherein the sealing material (70, 70a) is a liquid; andII24EM02-2024323325.DOCX mhwherein the method (100) further comprises recovering the subvolumes (6O2) of the sample liquid (60) from the plurality of micro-wells (50) byremoving the sealing material (70, 70a) from the reservoir (10) through the single opening (40);introducing a recovery liquid into the reservoir (10) through the single opening (40) to cover the plurality of micro-wells (50); andwaiting for a predetermined time interval in which the sample liquid (60) diffuses into the recovery liquid.

19. Apparatus for generating isolated liquid subvolumes (6O2) in a plurality of micro-wells (50) of a reservoir (10), the apparatus comprising:the reservoir (10) with a base (20), at least one sidewall (30) and a single opening (40) spanning at least partially the base (20), wherein the plurality of micro-wells (50) is arranged on the base (20) and / or on the at least one sidewall (30); andan applicator (80) for introducing a sealing material (70, 70a) into the reservoir (10) through the single opening (40) to seal subvolumes (6O2) of the sample liquid (60) in the plurality of micro-wells (50) using a movement of the sealing material (70, 70a) towards the base (20) due to a body force applied to the sealing material (70, 70a).

20. Apparatus of claim 19, wherein the plurality of micro-wells (50) is arranged on the base (20) and wherein the base (20) is inclined with respect to the horizontal.

21. Apparatus of claim 20,wherein the applicator (80) is a tubular applicator (80) comprising an inlet (82) and an outlet (84);wherein the outlet (84) of the applicator (80) is arranged in proximity to the base (20) in a lowest region thereof with respect to the horizontal; andwherein the applicator (80) is configured to transport the sealing material (70, 70a) using the body force from the single opening (40) through the reservoir (10) towards the base (20) of the reservoir (10).

22. Apparatus of claim 20,wherein the applicator (80) comprises an inlet (82) and an outlet (84); andI I24EM02-2024323325. DOCX mhwherein the outlet (84) of the applicator (80) is arranged in proximity to the at least one sidewall (30) near the single opening (40) for letting the sealing material (70, 70a) flow along the at least one sidewall (30) to a lowest region of the base (20) with respect to the horizontal.

23. Apparatus of claim 19, wherein the applicator (80) is a tubular applicator (80) coupled to the reservoir (10); wherein the applicator (80) comprises an inlet (82) and an outlet (84); and wherein the applicator (80) is configured to transport the sealing material (70, 70a) using the body force from the single opening (40) through the reservoir (10) towards the base (20) of the reservoir (10).

24. Apparatus of claim 23, wherein the applicator (80) comprises a membrane (86) closing the outlet (84) of the applicator (80); and wherein the membrane (86) is deformable by the sealing material (70, 70a).

25. Apparatus of claim 19, wherein the at least one sidewall (30) is tapered towards the base (20), wherein the sidewall (30) comprises a plurality of micro-steps in a direction from the single opening (40) towards the base (20) and each of the micro-steps is partitioned into the plurality of micro-wells (50) by partition walls, wherein each of the plurality of micro-wells (50) has the form of triangular prism.

26. Apparatus of any of claims 19 to 25, comprisingfurther reservoirs (10), wherein each of the further reservoirs (10) has a base (20), at least one sidewall (30) and a single opening (40) spanning at least partially the base (20) and a plurality of micro-wells (50) arranged on the base (20) and / or on the at least one sidewall (30);a substrate (210) on which the reservoir (10) and the further reservoirs (10) are arranged or in which the reservoir (10) and the further reservoirs (10) are formed.I I24EM02-2024323325. DOCX mh

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