Method for arranging microspheres in non-aqueous liquid phase

Abstract

The present invention relates to a method for aligning microspheres in a non-aqueous, water-immiscible liquid phase within an incubation chamber and for not contacting or exposing such microspheres to a gas phase. The invention also relates to a method for incubating microspheres, and to a method for detecting and optically detecting microspheres. In addition, the invention also relates to a perforated plate configured for use in such a method. In addition, the present invention relates to a device for aligning microspheres in a non-aqueous, water-immiscible liquid phase within an incubation chamber and for preventing such microspheres from contacting or exposing to the gas phase. In addition, the invention also relates to a device for incubating the microspheres and a device for detecting and optically detecting the microspheres.

Description

Technical Field

[0001] The present invention relates to a method for arranging microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber and for keeping such microspheres from contacting or being exposed to a gas phase. In addition, the present invention relates to a multiwell plate configured for use in the method. In addition, the present invention relates to a device for arranging microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber and for keeping such microspheres from contacting or being exposed to a gas phase. Background Art

[0002] Microspheres are often and sometimes referred to herein as "nanoreactor beads", a new type of molecular biology reagent that provides a wide range of applications, such as nucleic acid extraction, digital nucleic acid amplification such as digital polymerase chain reaction and detection. In its simplest configuration, such microspheres or "nanoreactor beads" contain hydrogels that provide a defined volume and act as a reaction space for biochemical reactions to occur. In nucleic acid analysis, hydrogels are used to absorb reaction components (such as buffers, amplification reagents, etc.) and target materials to be amplified. After the reagent mixture is loaded into the hydrogel, the microspheres are encapsulated in the oil phase, so that the respective volumes of each microsphere act as separate reaction spaces and do not cross-react with other microspheres / nanoreactors. At this stage, the oil-encapsulated microspheres can then be exposed to suitable amplification reaction conditions, such as specific temperature profiles to which the microspheres are exposed.

[0003] Since each microsphere provides a separate volume, it is important to ensure that conditions are consistent across all microspheres. Typically, this is achieved by incubating the entire vessel / container in which the microspheres reside until equilibrium conditions (e.g., equilibrium temperature) are reached. However, as reflected in the classic digital PCR thermal cycling protocol, this approach is rather slow.

[0004] In addition, due to the difference between the oil phase and the microspheres and due to the immiscibility between the oil phase and the water phase, the microspheres containing the water phase are concentrated and tend to agglomerate due to the forces generated at elevated temperatures. In addition, the microspheres may accumulate at the interface formed between the oil phase and the surrounding air, which may cause drying artifacts.

[0005] Once the amplification reaction is complete, the microspheres are examined for the presence of amplified targets, which requires appropriate optical detection equipment. However, in situ detection is cumbersome and requires complex techniques such as light sheet imaging (Liao et al., PNAS USA, 2020, 117 (41), pp. 25628-25633). Alternatively, for detection after amplification, the microspheres can be transferred to a reader similar to a flow cytometer (Hindson et al., Anal. Chem., 2011, 83(22): pp. 8604-8610; Kiss et al. Anal. Chem. 2009, 80(23): pp.8975-8981). Alternatively, to circumvent the above problems, microfluidic technologies have been developed to generate and contain droplets in microfluidic devices, providing ordered arrays of droplets for uniform exposure under the same conditions and simplifying readout by imaging droplets arranged in two dimensions (Madic et al., Biomol. Detect. Quantif. 2016,10: pp. 34-46).

[0006] Therefore, there is still a need in the art to reduce the complexity of disposable use and provide a cost-effective and safe method for containing microspheres / nanoreactor beads in an arrangement suitable for amplification and detection. There is also a need in the art to provide a method that avoids the above-mentioned shortcomings and problems associated with the prior art. In particular, there is a need to provide a method for arranging microspheres in a suitable arrangement and preventing such microspheres from contacting or being exposed to a gas phase. Summary of the invention

[0007] In a first aspect, the invention relates to a method for arranging microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber and for keeping such microspheres from contacting or being exposed to a gas phase, the method comprising the steps of: a) providing, in any order, a suspension comprising magnetic microspheres suspended in a non-aqueous, water-immiscible liquid phase and an incubation chamber; The incubation chamber has a closed bottom, an open top, at least one wall and a defined volume; the open top is spaced a defined distance from the closed bottom; the chamber is sized to accommodate a defined volume of the suspension therein; The magnetic microspheres have a first specified density; the non-aqueous, water-immiscible liquid phase has a second specified density, wherein the first specified density < the second specified density; and wherein, preferably, the suspension has a specified concentration of microspheres; b) filling the incubation chamber with the suspension by placing the suspension into the incubation chamber such that a first interface is formed in the incubation chamber between the non-aqueous, water-immiscible liquid phase and a gas phase, the gas phase surrounding the incubation chamber, at the open top or at a first position along the defined distance between the open top and the closed bottom; c) removing the magnetic microspheres from the first interface or keeping them removed from the first interface by attracting the magnetic microspheres to the closed bottom or to the at least one wall of the incubation chamber, wherein the attraction is achieved by applying a magnetic force through the closed bottom or through the at least one wall of the incubation chamber; d) placing a solid surface at the open top of the incubation chamber or at a location along the specified distance between the open top and the closed bottom of the incubation chamber while maintaining the magnetic microspheres attracted to the closed bottom or the at least one wall of the incubation chamber by continuously applying a magnetic force through the closed bottom or the at least one wall; wherein such placement is performed such that the solid surface is in contact with the liquid phase and such that a second interface is formed between the non-aqueous, water-immiscible liquid phase and the solid surface at the open top or at said location, wherein the second interface replaces or eliminates the first interface so that the gas phase is not in contact with the liquid phase; e) releasing the microspheres from the bottom or from the at least one wall by ceasing to apply a magnetic force through the bottom or through the at least one wall of the incubation chamber, allowing magnetic microspheres to assemble at the second interface.

[0008] In one embodiment, steps b) and c) are performed simultaneously or in a temporally overlapping manner.

[0009] In one embodiment, the applying of the magnetic force through the closed bottom or the at least one wall of the incubation chamber in step c) occurs by placing a magnet near the bottom or the at least one wall of the incubation chamber or vice versa, so that the magnetic force is applied through the bottom or the at least one wall by means of the magnet, wherein, preferably, in step c), the bottom or the at least one wall of the incubation chamber is in physical contact with the magnet, or in contact with the magnet in such a way that the magnet can apply the magnetic force therethrough.

[0010] In one embodiment, by step d), the incubation chamber becomes closed because the open top is converted into a closed top by the solid surface.

[0011] In one embodiment, in step d), the solid surface is placed at the open top of the incubation chamber or at a position along the defined distance between the open top and the closed bottom of the incubation chamber by: - placing the cover only at the open top of the incubation chamber or at the position, - Alternatively, a separate solid surface, such as a plate, which is not a cover, is first placed at or at said open top of said incubation chamber and then the cover is placed on top of said separate solid surface.

[0012] In one embodiment, the continuous application of the magnetic force in step d) occurs by holding a magnet near the bottom or at least one wall of the incubation chamber, or vice versa, so that the magnetic force is continuously applied through the bottom or through the at least one wall by means of the magnet, wherein, preferably, in step d), the bottom or the at least one wall of the incubation chamber is in physical contact with the magnet, or at least in such a way that the magnet can apply the magnetic force therethrough.

[0013] In one embodiment, step e) is performed by removing the magnet from the vicinity of the bottom or the at least one wall, or vice versa, thereby preventing the magnetic force from being applied through the bottom of the incubation chamber or through the at least one wall.

[0014] In one embodiment, the arrangement of the microspheres at the second interface in step e) is achieved by actively generating convection, preferably thermal convection or mechanical convection, in the incubation chamber.

[0015] In one embodiment, step e) is achieved by exposing the bottom of the incubation chamber to a temperature control device, preferably by bringing the bottom of the incubation chamber into thermal contact with the temperature control device, more preferably by bringing the bottom of the incubation chamber into physical contact with the temperature control device.

[0016] In one embodiment, the method is a method of arranging the microspheres in a microsphere layer along the second interface, and wherein the solid surface placed at the open top of the incubation chamber or at the specified distance along the open top and the closed bottom of the incubation chamber in step d) is a planar solid surface.

[0017] In one embodiment, the layer is a monolayer of microspheres along the second interface.

[0018] In one embodiment, the packing of the microspheres in the layer is selected from random packing, regular packing and close packing, in particular hexagonal close packing.

[0019] In one embodiment, the packing of the microspheres in the layer is close packed, particularly hexagonal close packed.

[0020] In one embodiment, the number of magnetic microspheres filled into the incubation chamber in step b) is selected so that it does not exceed the maximum number of microspheres that can be arranged in a layer along the second interface, the layer having as dense a packing as possible; and / or so that when arranged in the layer in as dense a packing as possible, the area covered by the magnetic microspheres is equal to or less than the area provided by the planar solid surface in contact with the liquid phase.

[0021] In one embodiment, the microspheres are monodisperse, preferably with a coefficient of variation (CV) < 15%, more preferably CV < 10%, even more preferably CV < 5%.

[0022] In one embodiment, the solid surface, preferably the planar solid surface, comprises regions that are transparent and allow optical probing and detection of the liquid phase and any microspheres located beneath the regions.

[0023] In one embodiment, the incubation chambers are wells in a multiwell plate, and wherein the multiwell plate has a plurality of such incubation chambers as defined in any one of the preceding claims, arranged in the multiwell plate, preferably regularly spaced apart.

[0024] In one embodiment, the method is performed using two or more wells in the multi-well plate.

[0025] In another aspect, the present invention also relates to a method for incubating microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber or a multiwell plate, the method comprising the steps of: f) carrying out a method for arranging microspheres according to the invention, preferably a method according to one embodiment, wherein the solid surface is planar and comprises transparent areas allowing for optical probing and detection; g) performing an incubation reaction with the microspheres, wherein, optionally, the incubation reaction involves one or more changes in the temperature of the microspheres.

[0026] In another aspect, the present invention relates to a method for detecting and optically probing microspheres in a non-aqueous, water-immiscible liquid phase within an incubation chamber or a multiwell plate, the method comprising the steps of: h) carrying out a method according to claim 16, or carrying out a method according to any one of claims 17-18 when any one of claims 17-18 is dependent on claim 16; Optionally, i) performing an incubation reaction with the microspheres, wherein, optionally, the incubation reaction involves one or more temperature changes of the microspheres; k) detecting and optically probing the microspheres through the transparent area of ​​the planar solid surface, preferably using a suitable detection module.

[0027] In a further aspect, the present invention relates to a multiwell plate configured for use in a method according to the present invention, such as a method for arranging microspheres, a method for incubating microspheres or a method for detecting and optically probing microspheres, the multiwell plate comprising: - a planar body having a planar surface and comprising a plurality of wells, each well having a closed bottom, an open top, at least one wall and a fixed volume; each well being sized to accommodate a specified number of magnetic microspheres or a specified volume of a suspension comprising the magnetic microspheres suspended in a non-aqueous, water-immiscible liquid phase therein; wherein the wells are embedded in the planar surface of the planar body, the planar surface being configured to contact a planar solid surface such that each well is closed by the planar solid surface; - a sealing edge at the periphery of the planar body, the sealing edge surrounding the planar body of the perforated plate and protruding therefrom; - a cover configured to be attached to and placed on top of the multiwell plate, in particular on top of the planar body, and sized to cover and seal the multiwell plate, in particular to cover and seal the planar body, the lower surface of the cover being a planar solid surface; the cover having a periphery and comprising a gasket at its periphery, the gasket interacting with the sealing edge of the planar body and sealing the multiwell plate, in particular the sealed planar body, when the cover is placed on top of the multiwell plate, in particular on top of the planar body; and - Optionally, the lower surface is a flat plate of a planar solid surface, the plate being configured to be placed on top of the flat body and being sized to cover the flat body.

[0028] In one embodiment of the multiwell plate, each well is closed by a planar solid surface provided by the lower surface of the cover or the lower surface of the plate, if present.

[0029] In one embodiment of the multiwell plate, the multiwell plate further comprises a flat plate whose lower surface is a planar solid surface, the flat plate being configured to be placed on top of the planar body and being sized to cover the planar body.

[0030] In embodiments where a flat plate is provided and present, the flat plate is provided separately from the cover and does not form part of the cover. For example, if the cover has a multi-layer structure, the flat plate does not form part of the multi-layer structure and is not any layer therein. Thus, in embodiments where a flat plate is provided and present in the multi-well plate, the flat plate is not an integral part of the cover and is provided separately from the cover.

[0031] In one embodiment of a multi-well plate, the cover and the plate (if present) (each) comprise an area covering the plurality of wells, which area is optically transparent and allows optical probing and detection of any wells located beneath said area.

[0032] In one embodiment, the multiwell plate further comprises a locking device configured to lock the cover in position on top of the multiwell plate, and optionally also configured to lock the plate (if present) on top of the flat body of the multiwell plate, such that the multiwell plate is sealed.

[0033] In one embodiment of the porous plate, the flat body is made of or comprises the following material: plastic, preferably, plastic with elemental carbon as an additive, more preferably, plastic with elemental carbon as an additive in a proportion of >50 wt.%, even more preferably, the plastic is polycarbonate with elemental carbon as an additive in a proportion of >50 wt.%.

[0034] In another aspect, the present invention also relates to an apparatus for arranging microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber and for keeping such microspheres from contacting or being exposed to a gas phase, the apparatus comprising: - a receptacle for receiving an incubation chamber or a multiwell plate; the receptacle having a bottom surface and at least one positioning device for positioning the incubation chamber or the multiwell plate on the bottom surface, the positioning device protruding from the bottom surface, the receptacle having a size to tightly accommodate the incubation chamber or the multiwell plate; the incubation chamber having a closed bottom, an open top, at least one wall and a defined volume; the multiwell plate is as defined herein; - a magnet or a set of magnets located at and incorporated into the bottom surface of the container or located at and incorporated into the positioning device, the magnet being configured to apply a magnetic force through the closed bottom of the incubation chamber, or through the bottom of each well in the multiwell plate, or through the at least one wall of the incubation chamber, or through the at least one wall of each well in the multiwell plate; - Optionally, locking means for locking the incubation chamber or the multiwell plate in the container, preferably on the bottom surface of the container.

[0035] In a preferred embodiment, the locking device is operable to reversibly lock and / or release the incubation chamber or multi-well plate. In a particularly preferred embodiment, the locking device is operable to interact with the positioning device to reversibly achieve the engagement of the positioning device with the incubation chamber or multi-well plate, and / or the release of the positioning device from it.

[0036] It should be noted that the locking device allows simple and stable operation of any incubation chamber or multiwell plate locked in the device. For example, when locked by the locking device, the overturning of the incubation chamber or multiwell plate or its shaking becomes easy and feasible.

[0037] In one embodiment, the locking means comprises one or more buttons, which are preferably arranged adjacent to the container, more preferably symmetrically arranged adjacent to the container.

[0038] In another aspect, the present invention also relates to a device for incubating microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber or a multi-well plate, the device comprising: - a body having an upper surface and a container configured to receive a multiwell plate as defined herein, the container being located on the upper surface, the container having a bottom; - a temperature control device located at and / or incorporated into the bottom of the container and configured to be in thermal and / or physical contact with the bottom of the incubation chamber or the bottom of each well of a multi-well plate when inserted into the container; and - A cover or flap attached to the body and configured to cover the upper surface of the body and to be placed on top of the container and the incubation chamber or the multiwell plate when placed in the container of the device; wherein the cover or flap is also configured to lock the incubation chamber or the multiwell plate in the container when the cover or flap is placed on top of the container, wherein the cover or flap also includes an optically transparent area that becomes located on the top of the container and thereby allows optical detection of the incubation chamber or the multiwell plate present in the container.

[0039] In one embodiment, the means for incubation further comprises in said container an incubation chamber or a multiwell plate as defined herein.

[0040] In another aspect, the present invention relates to a device for detecting and optically probing microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber or a multiwell plate, said incubation chamber or said multiwell plate being located in a device for incubation as defined herein, said device for detecting comprising: - a housing comprising a detection module comprising an image detector, one or more filters and at least one imaging lens; - a loading zone configured to receive and accommodate a device for incubation as defined herein, said loading zone comprising an interface for electrically connecting to the device for incubation as defined herein, wherein said loading zone is located within said housing such that when said detection module is present in said loading zone, said detection module can be used to detect and optically detect said device for incubation, preferably can be used to detect and optically detect an incubation chamber or a multiwell plate located in said device for incubating microspheres; wherein the detection and optical detection is performed through said optically transparent area of ​​said cover or flap of said device for incubation; and wherein said optically transparent area of ​​said cover or flap of said device for incubation is aligned with: said optically transparent area of ​​said cover and said flat surface of said multiwell plate, if such an incubation chamber or multiwell plate is present in said device for incubation, the image detector, a filter from the one or more filters, and The at least one imaging lens; Optical detection is allowed to occur through the transparent area of ​​the cover or flap of the device for incubation and through the optically transparent area of ​​the cover and the flat plate of the multiwell plate if present in the device for detecting.

[0041] The inventors have surprisingly discovered that it is possible to arrange the microspheres in a non-aqueous, water-immiscible liquid phase within an incubation chamber and prevent such microspheres from contacting or being exposed to a gas phase. According to an embodiment of the invention, this is done by taking advantage of the buoyancy or density difference between the microspheres and the liquid phase in which they are suspended, and by imparting magnetic properties to such microspheres (i.e., by making them magnetic), and using this magnetism to manipulate the microspheres within the incubation chamber, for example by attracting them to the bottom or walls of the incubation chamber, while placing a solid surface (e.g., a solid surface of a substrate) at the open top of the incubation chamber so that the solid surface is in contact with the liquid phase, thereby removing the gas phase (which was previously in contact with the liquid phase) from contact with the liquid phase. In this way, any interface between the liquid phase and the gas phase surrounding the incubation chamber is removed or excluded and replaced by another interface, which is the interface between the solid phase and the liquid phase.

[0042] Examples of microspheres suitable for use in the context of the present invention are, for example, microspheres as described in previous patent applications of the applicant, for example European patent application No. 21 206 745.8 filed on November 5, 2021; or as described in International Patent Application No. PCT / EP2017 / 084370 filed on December 22, 2017 (published as WO 2018 / 122162), or as described in International Patent Application Nos. PCT / EP2020 / 086171 and PCT / EP2020 / 086194 (published as WO 2021 / 122563 and WO 2021 / 122579, respectively). In broad terms, microspheres according to the present invention are microspheres comprising a hydrogel and comprising a volume for receiving an aqueous solution and for providing a reaction space in which a suitable chemical or biochemical reaction can occur.

[0043] As used herein, the term "microsphere" refers to a spherical body, or a substantially spherical or ellipsoidal or other regular rounded body, such as an egg-shaped body, which contains a hydrogel and provides a reaction space within its volume. Generally, the "microsphere" according to the present invention preferably has an average diameter of 20 μm to 500 μm, preferably 20 μm to 200 μm, more preferably 20 μm to 100 μm along its longest extension direction.

[0044] By incorporating smaller magnetic particles into such microspheres, such microspheres can be made magnetic. Incorporating such magnetic particles into microspheres allows such microspheres to be attracted by a magnet (permanent magnet or electromagnet) placed near the microspheres. As used herein, the term "magnetic particles" refers to particles that substantially exhibit non-diamagnetic behavior, thereby allowing such particles to be attracted by a magnet. In one embodiment, such magnetic particles are ferromagnetic particles. In another embodiment, such magnetic particles are paramagnetic particles. In another embodiment, such magnetic particles are ferrimagnetic particles. In yet another embodiment, such magnetic particles are superparamagnetic particles. In a preferred embodiment, such magnetic particles are ferromagnetic particles or superparamagnetic particles. As used herein, the term "magnetic particles" is intended to exclude any particles having diamagnetic behavior. As used herein, "magnetic particles" preferably have a sufficiently small size and an average diameter or average length extension in one dimension to allow such magnetic particles to be incorporated into the microspheres according to the present invention. In an embodiment of the present invention, the size of such magnetic particles and the average diameter or average length in one dimension extend in the range of 50 nm to 10 μm, preferably 100 nm to 5 μm, more preferably 1 μm to 5 μm, even more preferably 1 μm to 3 μm. Preferably, the size of the magnetic particles and the average diameter or average length in one dimension extend so that they can be easily accommodated in the microspheres according to the present invention. In many cases, the microspheres according to embodiments of the present invention may include a hydrogel that forms a porous network, and for such a network, the size of the magnetic particles and the average diameter or average length in one dimension extend so that they are larger than the average mesh or pore size of the microsphere network.

[0045] According to an embodiment of the present invention, such magnetic microspheres can be filled with appropriate reagents for carrying out chemical or biochemical reactions, and such reagents will be incorporated into the volume of the microspheres as an aqueous mixture and will be contained therein. Subsequently, the microspheres are suspended in a non-aqueous, water-immiscible liquid phase and filled as a suspension into an incubation chamber having a closed bottom, an open top, at least one wall (e.g., a circumferential wall) and a specified volume. If the incubation chamber has one wall, it can be a circumferential wall, so that the incubation chamber has a circular cross-section. If it has multiple walls, the corresponding cross-section will be triangular, square, rectangular, etc., depending on the number of walls of the incubation chamber.

[0046] The non-aqueous, water-immiscible liquid phase is selected so that its density is greater than the density of the magnetic microspheres (including any aqueous mixture contained in the microspheres). In other words, preferably, the magnetic microspheres, including any aqueous mixture contained in the microspheres, have a first specified density, which is less than the density of the non-aqueous, water-immiscible liquid phase, i.e., the "second specified density". The smaller microsphere density will cause the microspheres to generate buoyancy in the non-aqueous, water-immiscible liquid phase. Examples of suitable non-aqueous, water-immiscible liquid phases are oils, particularly mineral oils, fluorocarbon (FC) oils, perfluorocarbon (PFC) oils, perfluoropolyether (PFPE) oils, and hydrofluoroether (HFE) oils. An example of a suitable hydrofluoroether oil is Novec 7500®, for example, commercially available from 3M Deutschland GmbH of Neuss, Germany. In some embodiments, the non-aqueous, water-immiscible liquid phase may be or be supplemented with additives, such as emulsifiers, surfactants, and / or stabilizers.

[0047] Subsequently, according to an embodiment of the method of the present invention, a suspension of microspheres is filled into an incubation chamber, resulting in the formation of a first interface between a non-aqueous, water-immiscible liquid phase and a gas phase surrounding the incubation chamber. This first interface is formed at the open top or at a first position along the specified distance between the open top and the closed bottom. The position where this first interface is formed depends on whether the incubation chamber is completely or only partially filled with the suspension of the magnetic microspheres in a non-aqueous, water-immiscible liquid phase in step b). If the incubation chamber is completely filled, i.e., to its open top, the first interface will be formed at the open top. If the incubation chamber is only partially filled, i.e., not to its open top, the first interface will be formed at a first position along the specified distance between the open top and the closed bottom. The exact position of this first position depends on the amount of suspension filled into the incubation chamber. However, typically, this first position is located within the incubation chamber and is therefore located below the open top of the incubation chamber. Therefore, this first position will be different from the open top of the incubation chamber.

[0048] In the absence of a magnet near the incubation chamber / the magnetic microspheres in such incubation chambers, these microspheres will tend to move to the first interface between the non-aqueous, water-immiscible liquid phase and the gas phase surrounding the incubation chamber. As a result, the magnetic microspheres will be at least partially exposed to the gas phase. However, if a magnetic force is applied through the closed bottom of the incubation chamber or through the wall of the incubation chamber, the magnetic microspheres will be attracted thereto, and can remain attracted thereto as long as such a magnetic force is applied. Therefore, effectively, they will be removed from the first interface and will be fully immersed in the non-aqueous, water-immiscible liquid phase. According to an embodiment of the present invention, while the magnetic microspheres remain attracted to the bottom or wall of the incubation chamber, a solid surface is then placed at the open top of the incubation chamber or at a position along the open top and the closed bottom of the incubation chamber at a specified distance, wherein such placement is performed so that the solid surface is in contact with the liquid phase and a second interface is formed at the open top or at a position along the open top and the closed bottom of the incubation chamber, the second interface being the interface between the non-aqueous, water-immiscible liquid phase and the solid surface. As a result of such placement, such a second interface replaces or eliminates the first interface (between the non-aqueous, water-immiscible liquid phase and the gas phase surrounding the incubation chamber), thereby eliminating contact between the gas phase and the liquid phase, and therefore also eliminating contact with the microspheres. Typically, the position at which the solid surface is placed in step d) depends on the position of the first interface (i.e., the interface between the non-aqueous, water-immiscible liquid phase and the gas phase surrounding the incubation chamber). If the first interface is located at the open top of the incubation chamber (because the incubation chamber has been completely filled to its open top with the suspension in step b), the solid surface is placed at the open top in step d). However, if the first interface is located at a first position in the incubation chamber, which first position is different from the top opening (because the incubation chamber is only partially filled with the suspension in step b), the solid surface is preferably placed at a position matching the first position, i.e., the position of the first interface, or below the first position of the first interface in step d). Therefore, in some embodiments, the position where the solid surface is placed in step d) may be the first position. Alternatively, in step d), the solid surface may also be placed at a position in the incubation chamber that is lower than the first position, i.e., lower than the first interface, to absolutely ensure that the second interface formed replaces or eliminates the first interface. Regardless of where the solid surface is placed in step d), step d) should be performed in such a way that, as a result of step d), the solid surface is in contact with the liquid phase and a second interface is formed between the non-aqueous, water-immiscible liquid phase and the solid surface at the open top or at the location where the solid surface was placed in step d), wherein the second interface replaces or eliminates the first interface, thereby preventing the gas phase from contacting the liquid phase.

[0049] According to an embodiment of the present invention, the size of the solid surface placed at the open top of the incubation chamber or at a position along the specified distance between the open top of the incubation chamber and the closed bottom is selected so that it will completely cover the liquid phase and / or the open top of the incubation chamber to form a second interface that replaces or eliminates the first interface (between the non-aqueous, water-immiscible liquid phase and the gas phase surrounding the incubation chamber). More specifically, in some embodiments, the size of the solid surface placed at the open top of the incubation chamber or at a position along the specified distance between the open top of the incubation chamber and the closed bottom is selected so that it completely covers the liquid phase and / or the open top of the incubation chamber and has a cross-section that allows the formation of a second interface between the non-aqueous, water-immiscible liquid phase and the solid surface, which second interface replaces or eliminates the first interface, so that the gas phase (surrounding the incubation chamber) does not contact the non-aqueous, water-immiscible liquid phase. In a particularly preferred embodiment, the cross-section of the solid surface is selected so that it allows the solid surface to be inserted into the incubation chamber and / or to fit tightly against the wall of the incubation chamber.

[0050] In some embodiments, wherein the incubation chambers are wells in a multi-well plate, preferably, the first interface is located at the open top of each respective well (because, preferably, each well has been completely filled to its open top by the suspension in step b). In such embodiments, in step d), a solid surface is placed at the open top of each respective well. In some such embodiments, the solid surface may be provided separately for each well, or, alternatively, may be provided together as a single solid surface for all wells. In these embodiments, preferably, the solid surface is placed at the open top of the respective well by: - or simply placing the cover of the multiwell plate at the open top of the respective well, - or first placing a separate solid surface (eg a flat plate) which is not a cover at the open top of the respective well and then placing the cover of the multiwell plate on top of the separate solid surface.

[0051] Finally, according to an embodiment of the present invention, in step e), the microspheres are released therefrom and allowed to assemble at the second interface by ceasing to apply the magnetic force through the bottom of the incubation chamber or by applying the magnetic force through the wall of the incubation chamber, so as to allow the magnetic microspheres to assemble at the second interface due to their lower density compared to the density of the non-aqueous, water-immiscible liquid phase.

[0052] According to an embodiment of the present invention, the term "magnet" refers to any material or object or body that generates a magnetic field capable of exerting a magnetic force on a magnetic material. The magnet according to an embodiment of the present invention may be a permanent magnet, such as a ferromagnetic material, or may be an electromagnet that can be turned on and off, so that steps c) and d) of applying a magnetic force, and step e) of stopping applying a magnetic force can be easily implemented.

[0053] It should be noted that, depending on the shape, form, dimensions and other qualities of the solid surface placed at the open top of the incubation chamber or at a specified distance along the open top and closed bottom of the incubation chamber and forming the second interface in step d), the microspheres can be arranged accordingly. For protocols involving simple incubation of microspheres, for example for nucleic acid amplification in such microspheres without subsequent detection, it is sufficient to simply remove the microspheres from the first interface and thereby exclude them from the gas phase surrounding the incubation chamber (or the wells in the multi-well plate) in step c). In such an embodiment, the solid surface placed in step d) can be a surface of any shape and size, as long as the surface can remove the microspheres from the gas phase (i.e., the first interface) and prevent the microspheres from contacting it by forming a second interface that replaces or eliminates the first interface. For example, a curved solid surface of appropriate size is very suitable for this purpose. However, if the microspheres are used in protocols involving not only nucleic acid amplification but also simultaneous or subsequent detection of signals generated in the microspheres and / or imaging of such signals, it is important that these microspheres are arranged in a layer (preferably a single layer) so that the microspheres can be optically detected, imaged and / or optically studied. This arrangement in a layer, preferably a monolayer, can be achieved by a solid surface which is a planar solid surface.

[0054] In one embodiment, the method according to the present invention is a method for arranging microspheres in a layer of microspheres along the second interface. In a preferred embodiment, such a layer is a monolayer of microspheres along the second interface. When the microspheres are arranged in a layer or monolayer according to an embodiment of the present invention, their stacking can be appropriately selected and customized. In one embodiment, the stacking of microspheres in such a layer or monolayer is selected from random stacking, regular stacking and close stacking, in particular hexagonal close stacking. In a preferred embodiment, the stacking of microspheres in a layer or monolayer is close stacking, more preferably hexagonal close stacking.

[0055] In particular, in those embodiments where the microspheres are arranged into a layer or monolayer of microspheres along the second interface, preferably, the solid surface placed at the open top of the incubation chamber or at a second position at a defined distance along the open top and closed bottom of the incubation chamber in step d) is a planar solid surface. In particular, in those embodiments involving a planar solid surface, it is possible to achieve an arrangement of the magnetic microspheres into a layer, preferably a monolayer.

[0056] As used herein, the term "planar surface" or "planar solid surface" refers to a surface that is substantially flat and has a planar or approximately planar appearance. In some embodiments, such a planar solid surface may be a smooth surface. However, such a term "planar surface" or "planar solid surface" should also be interpreted as still allowing a certain degree of surface roughness in such a plane. For the sake of clarity, a "planar surface" or "planar solid surface" may also be such a surface, which may include certain structures designed to promote the specific stacking of the microspheres in the layer or monolayer formed along the planar surface (and the second interface). Moreover, for example, such a "planar surface" or "planar solid surface" may have a certain degree of patterning, for example, to achieve or promote a specific stacking of the microspheres in the layer or monolayer.

[0057] In addition to the effect that the planar solid surface (and possible structures or patterns thereon) may have on the packing of the microspheres, the packing of the microspheres in the layer arrangement can also be customized by selecting an appropriate concentration of microspheres in the non-aqueous, water-immiscible liquid phase (related to the area / size of the solid planar surface), and / or by selecting the number of magnetic microspheres (related to the area / size of the solid planar surface) filled into the incubation chamber in step b). In one embodiment, the number of magnetic microspheres filled into the incubation chamber in step b) is selected so that it is lower, preferably significantly lower than the maximum number of microspheres that can be arranged in the layer along the second interface, and the layer has the most dense packing. This will result in random packing or regular packing of the microspheres in the layer. In another embodiment, the number of magnetic microspheres filled into the incubation chamber in step b) is selected so that it does not exceed the maximum number of microspheres that can be arranged in the layer along the second interface, and the layer will have the most dense packing, but it is close to this maximum number or almost equal to this maximum number, or in fact (theoretically) the maximum number of microspheres that can be arranged in the layer along the second interface. This will result in a close packing or even hexagonal close packing of the microspheres in the layer. In one embodiment, the number of magnetic microspheres filled into the incubation chamber in step b) is selected so that when arranged in a layer as close as possible, the area covered by the magnetic microspheres is equal to or smaller than the area provided by the solid surface in contact with the liquid phase.

[0058] On the other hand, if the number of magnetic microspheres filled into the incubation chamber in step b) is chosen such that it exceeds the maximum number of microspheres that can be arranged in a layer along the second interface (the layer having the densest packing), there will not be a simple layer of microspheres, in particular a monolayer of microspheres, arranged along the second interface, but the microspheres will be arranged in a three-dimensional irregular bulk volume. This is because such a (high or excessive) number of microspheres can only be arranged along the second interface by accommodating such a (high or excessive) number of microspheres in a three-dimensional volume, and not in a monolayer or, more precisely, a monolayer of microspheres.

[0059] In another aspect, the present invention also relates to a multiwell plate configured for use in a method according to an embodiment of the present invention as defined herein. According to an embodiment of the present invention, the multiwell plate comprises: - a planar body having a planar surface and comprising a plurality of wells, each well having a closed bottom, an open top, at least one wall and a defined volume; each well being sized to accommodate a defined number of magnetic microspheres or a defined volume of a suspension comprising the magnetic microspheres suspended in a non-aqueous, water-immiscible liquid phase therein; wherein the wells are embedded in the planar surface of the planar body, the planar surface being configured to contact a solid surface, preferably a planar solid surface, such that each well is closed by the solid surface, preferably by the planar solid surface; - a sealing edge at the periphery of the planar body, the sealing edge surrounding the planar body of the perforated plate and protruding therefrom; - a cover configured to be attached to and placed on top of the multiwell plate, in particular on top of the planar body, and having dimensions designed to cover and seal the multiwell plate, in particular to cover and seal the planar body, the lower surface of the cover being a solid surface, preferably a planar solid surface, the cover having a periphery and comprising a gasket at its periphery, the gasket interacting with the sealing edge of the planar body and sealing the multiwell plate, in particular to seal the planar body, when the cover is placed on top of the multiwell plate, in particular on top of the planar body; and - Optionally, the lower surface is a flat plate of a planar solid surface, the plate being configured to be placed on top of the flat body and being sized to cover the flat body.

[0060] It should be noted that, in the embodiment that there is the flat plate with lower surface, it is usually placed on the top of flat body and size is designed to cover described flat body, and by being placed on this flat top, described cover is configured to be placed on the top of porous plate, preferably on the top of described flat body.In a preferred embodiment, the flat plate that is placed on the flat body top of porous plate has enough density / weight, makes under the influence of gravity, makes it be placed on the top of the flat body of porous plate, and even not contact and fix with the cover of described porous plate that is placed on the top of porous plate, preferably the top of flat body, also can remain there.For example, this flat plate can be made by giving it enough density / weight and make it be placed on the top of the flat body of porous plate and even not contacted by the cover of described porous plate and not fixed by the cover of described porous plate and remain there.The example of this suitable material for flat plate is glass or quartz.In such embodiment, even cover is placed on the top of described porous plate and also effectively is placed on the top of described flat plate, but it does not need to contact and in fact does not contact flat plate, so that this flat plate is placed and remain on the top of the flat body of porous plate.

[0061] Yet, in other embodiments, the cover is configured to be placed on the top of the multiwell plate by actually contacting the flat plate at the top thereof, preferably placed on the top of the flat body, so that the flat plate is placed on and in contact with the flat body of the multiwell plate, and further (on its other side) contacts with the cover placed on the top of the flat plate. In such embodiments, the flat plate is effectively in contact with the cover on one side, and contacts with the flat body of the multiwell plate on the other side. In such embodiments, the cover also helps to fix the flat plate on the flat body of the multiwell plate by contacting the flat plate.

[0062] It should be noted that in the embodiment where a flat plate is provided and present, the flat plate is provided separately from the cover and does not form an integral part thereof. In particular, it is not any layer or component of the cover, but is an entity completely separate from the cover, and the cover and the flat plate are separate components from each other.

[0063] Multiwell plates according to embodiments of the present invention are particularly suitable for arranging microspheres in a non-aqueous, water-immiscible liquid phase within the wells of the multiwell plate and for keeping such microspheres from contacting or being exposed to a gas phase.

[0064] In another aspect, the invention relates to a device for arranging microspheres in a non-aqueous, water-immiscible liquid phase within some or all of the wells of a multiwell plate according to the invention, and for keeping such microspheres out of contact with or exposure to a gas phase.

[0065] According to an embodiment of the present invention, the device comprises: - a container for receiving an incubation chamber or a multi-well plate; the container having a bottom surface and at least one positioning device for positioning the incubation chamber or the multi-well plate on the bottom surface, the positioning device protruding from the bottom surface, the container having a size for tightly accommodating the incubation chamber or the multi-well plate; the incubation chamber having a closed bottom, an open top, at least one wall and a specified volume; the multi-well plate is as defined in the embodiments herein; - a magnet or a set of magnets located at and incorporated into the bottom surface of the container or located at and incorporated into the positioning device, the magnet being configured to apply a magnetic force through the closed bottom of the incubation chamber, or through the bottom of each well in the multiwell plate, or through the at least one wall of the incubation chamber, or through the at least one wall of each well in the multiwell plate; - Optionally, locking means for locking, preferably reversibly locking, the incubation chamber or the multiwell plate in the container, preferably on the bottom surface in the container.

[0066] Furthermore, in another aspect, the present invention relates to a device for incubating microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber or a multiwell plate, the device comprising: - a body having an upper surface and a container configured to receive a multiwell plate according to an embodiment of the invention defined herein, the container being located on the upper surface, the container having a bottom; - a temperature control device located at and / or incorporated into the bottom of the container and configured to be in thermal and / or physical contact with the bottom of the incubation chamber or the bottom of each well of a multi-well plate when inserted into the container; and - A cover or wing attached to the body and configured to cover the upper surface of the body and to be placed on top of the container and the incubation chamber or the multiwell plate when placed in the container of the device; wherein the cover or wing is also configured to lock the incubation chamber or the multiwell plate in the container when the cover or wing is placed on top of the container, wherein the cover or wing also includes an optically transparent area that becomes located on the top of the container and thereby allows optical detection of the incubation chamber or the multiwell plate present in the container.

[0067] In one embodiment, the device for incubating microspheres in a non-aqueous, water-immiscible liquid phase further comprises in said container an incubation chamber or a multiwell plate according to the invention as defined herein.

[0068] In another aspect, the present invention relates to a device for detecting and optically probing microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber or a multiwell plate, said incubation chamber or said multiwell plate being located in a device for incubation according to the invention as defined herein, said device for detecting comprising: - a housing comprising a detection module comprising an image detector, one or more filters and at least one imaging lens; - a loading zone configured to receive and accommodate a device for incubation according to the present invention, as defined herein, comprising an interface for electrical connection to a device for incubation according to the present invention, as defined herein, wherein the loading zone is located within the housing such that when the detection module is present in the loading zone, the detection module can be used for detection and optical detection of the device for incubation, preferably for detection and optical detection of an incubation chamber or a multiwell plate located in the device for incubating microspheres; wherein the detection and optical detection is performed through the optically transparent area of ​​the cover or flap of the device for incubation; and wherein the optically transparent area of ​​the cover or flap of the device for incubation is aligned with: said optically transparent area of ​​said cover and said flat surface of said multiwell plate, if such an incubation chamber or multiwell plate is present in said device for incubation, the image detector, a filter from the one or more filters, and The at least one imaging lens; Optical detection is allowed to occur through the transparent area of ​​the cover or flap of the device for incubation and through the optically transparent area of ​​the cover and the flat plate of the multiwell plate if present in the device for detecting.

[0069] In a further aspect, the present invention also relates to the use of elemental carbon, in particular graphite, for incorporation into plastic materials to increase the thermal conductivity of such plastic materials and thereby make such plastic materials particularly suitable for the manufacture of incubation chambers or multiwell plates for carrying out incubation reactions, such as nucleic acid amplification reactions, involving one or more temperature changes.

[0070] In yet another aspect, the present invention relates to the use of elemental carbon, in particular graphite, for increasing the thermal conductivity of an incubation chamber or a porous plate, wherein such use involves incorporating the elemental carbon (e.g. graphite) into the plastic material from which the incubation chamber or the porous plate is made. In a particularly preferred embodiment of such use, the plastic material from which the incubation chamber and / or the porous plate is made is polycarbonate.

[0071] In another aspect, the present invention relates to the use of elemental carbon, in particular graphite, for the manufacture of an incubation chamber or a multi-well plate, the incubation chamber or the multi-well plate being configured for carrying out an incubation reaction, such as a nucleic acid amplification reaction, which involves one or more temperature changes of the incubated sample, wherein such manufacture comprises incorporating the elemental carbon into the plastic material from which the incubation chamber or the multi-well plate is manufactured. In one embodiment of such use, the incubation chamber or the multi-well plate is configured to carry out an incubation reaction involving one or more temperature changes of the incubated sample in an accelerated manner, by incorporating the elemental carbon into the plastic material, compared to carrying out the same incubation reaction in an incubation chamber or multi-well plate manufactured from a plastic material into which the elemental carbon (e.g. graphite) is not incorporated.

[0072] In one embodiment of this use, an incubation chamber or multiwell plate configured to perform an incubation reaction (e.g., a nucleic acid amplification reaction involving one or more temperature changes of an incubated sample) is configured to perform an incubation reaction involving one or more temperature changes of an incubated sample in the following manner: each temperature change has a temperature difference of 20°C to 25°C achieved within <5 seconds, preferably each temperature change has a temperature difference of 20°C to 25°C achieved within <3.5 seconds.

[0073] On the other hand, the invention further relates to an incubation chamber or a multi-well plate for implementing an incubation reaction (e.g. a nucleic acid amplification reaction, which involves one or more temperature changes), the incubation chamber or the multi-well plate being made of a plastic into which elemental carbon, particularly graphite, has been incorporated. In a preferred embodiment of this aspect, the plastic material of the incubation chamber and / or the multi-well plate is polycarbonate. Compared with carrying out the same incubation reaction in an incubation chamber or a multi-well plate made of a plastic material not incorporating elemental carbon (e.g. graphite), the incubation chamber or the multi-well plate according to this aspect of the invention is configured to implement an incubation reaction involving the incubation sample carrying out one or more temperature changes in an accelerated manner.

[0074] An example of a suitable plastic material into which elemental carbon has been incorporated is "Makrolon TC621 ® ” which is commercially available from Covestro AG and can be used in this aspect of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] In addition, reference is made to the accompanying drawings, wherein: Figure 1A schematic flow chart of an embodiment of a method for arranging microspheres in a non-aqueous, water-immiscible liquid phase in a single incubation chamber and for preventing such microspheres from contacting or being exposed to a gas phase is shown. The microspheres are initially located at the interface between a non-aqueous, water-immiscible liquid phase and a gas phase surrounding the incubation chamber. Thereafter, the microspheres are attracted to the bottom by a magnet, and a solid surface is placed at the top of the incubation chamber or at a certain position in the incubation chamber, i.e., at a position at a prescribed distance between the open top and the closed bottom of the incubation chamber, so that the solid surface is in contact with the liquid phase, and another interface is formed between the non-aqueous, water-immiscible liquid phase and the solid surface at the open top of the incubation chamber or at a corresponding position where the solid surface is placed in the incubation chamber, and an interface is no longer present between the liquid phase and the gas phase, so that the interface is replaced or eliminated by another interface between the solid surface and the liquid phase. Thereafter, the microspheres are released by removing the magnet. The incubation chamber is then placed under thermal control for subsequent reactions. It should be noted that in this figure, the solid surface is or may be a curved surface, so the arrangement of the microspheres occurs in an irregular manner rather than forming a layer. However, such a surface also does not include an interface with a surrounding gas phase, such as air.

[0076] Figure 1 The letters in correspond to the steps of the method for arranging microspheres of the present invention as follows: Figure 1 The letter i) corresponds to the combination of steps a) and b) of the method for arranging microspheres according to the present invention. Figure 1 The letter ii) corresponds to step c) of the method for arranging microspheres according to the present invention, and Figure 1 Letter iii) corresponds to step d) of the method for arranging microspheres according to the present invention. Figure 1 The letter iv) corresponds to step e) of the method according to the invention, with the additional limitation that Figure 1 . However, it is clear to a person skilled in the art that the method for aligning microspheres according to the present invention can also work without such thermal control. However, in some cases, such thermal control may be preferred because it allows additional convection and thus facilitates the alignment of the microspheres along the second interface.

[0077] Figure 2 The same schematic flow diagram of an embodiment of a method for arranging microspheres in a non-aqueous, water-immiscible liquid phase and for keeping such microspheres out of contact with or exposure to a gas phase is shown, but this time for multiple incubation chambers, such as in the case of a multi-well plate, i.e., multiple wells. Figure 2 The letters in Figure 1 The corresponding letters in are the same.

[0078] Figure 3A schematic flow diagram of an embodiment of a method for arranging microspheres in a non-aqueous, water-immiscible liquid phase within an incubation chamber and for keeping such microspheres from contacting or being exposed to a gas phase is shown, wherein the method employs a solid surface that is placed at the top of the incubation chamber or at a location within the incubation chamber, i.e., at a defined distance along the open top and closed bottom of the incubation chamber, however, wherein the solid surface is placed at a location that is not in contact with the gas phase. Figure 2 and 3 The difference is that the solid surface used here is a planar solid surface. This allows the microspheres to be arranged in a planar layer along such a planar solid surface. The planar solid surface can be, for example, the cover of an incubation chamber. Such an embodiment is particularly interesting, in which the microspheres, after being arranged and after undergoing a biochemical reaction, such as an amplification reaction, will be optically detected and further studied. This is also achieved by means of a schematic (optional) optical detector in Figure 3 However, this optional optical detector is not necessarily part of the arrangement method according to the invention. This optical detector is part of the method for detection and optical detection according to the invention.

[0079] Figure 4 A schematic flow diagram of an embodiment of a method for arranging microspheres in a non-aqueous, water-immiscible liquid phase and for keeping such microspheres from contacting or exposing them to a gas phase is shown, this time in a plurality of incubation chambers, such as wells in a multi-well plate. Figure 3 Likewise, the method employs a planar solid surface forming a second interface along which the microspheres can be arranged, thereby allowing them to be arranged in a layer arrangement, thereby facilitating subsequent optical probing and investigation.The solid surface may be, for example, the lid of a multiwell plate.

[0080] Figure 5 Similar to Figure 4 The embodiment shown, except that this embodiment additionally employs a separate solid planar surface separate from the cover, which serves as a planar solid surface to form a second interface at which the microspheres are arranged. The separate solid planar surface can be a flat plate, such as a cover slip. This embodiment is particularly useful because it allows the microspheres to be placed through the cover. Figure 5 The function of sealing the multi-well plate performed by the cover in letter iii-2) is similar to that of Figure 5 The functional separation provided for aligning the microspheres along this interface is shown as letter iii-1). This allows for wider manufacturing tolerances, thereby reducing the one-time cost, which more than makes up for the cost of additional components, i.e. additional plates such as cover glasses.

[0081] Figure 6 Shown is a fluorescent image of an embodiment of a multiwell plate equipped with microspheres, where the microspheres form a tightly packed layer.

[0082] More specifically, Figure 6 Shown is a fluorescence image of a MiniWellPlate (i.e. a specific multiwell plate with only 6 wells) equipped with microspheres representing "nanoreactor beads". Imaging was performed after thermal cycling according to the following thermal protocol: • 10 min at 50 °C (reverse transcriptase step) • 2 min 80 ℃ (system preheating) • 2 min at 98 °C (initial denaturation) • 40 cycles, 30s 98℃ & 30s 57℃ Figure 6 A: Fluorescence images of microspheres ("nanoreactor beads") after processing on a mini-well plate with 6 wells using a FAM fluorescence filter set. All beads were stained with FAM dye for bead identification and automatic image segmentation. The wells were densely packed with nanoreactor beads. A magnified view of the marked area shows a self-assembled monolayer of hexagonally packed nanoreactor beads.

[0083] Figure 6 B: The same beads shown in A. were equipped with primer sets specific for targets in MS2 phage RNA. In addition, a TaqMan probe labeled with Cy5 dye was used to detect target-specific amplification of targets present in some of the beads. An overview fluorescence image of a mini-well plate with six wells acquired using a Cy5 fluorescence filter set is shown on the left. A magnified view of the marked area on the right shows densely packed dark and light nanoreactor beads. The light signal is generated by amplicon formation during thermal cycling, indicating the presence of the target on the corresponding nanoreactor bead.

[0084] Figure 7 The arrangement of microspheres in layers is shown, but the packing is random and there are defects in the layers / monolayers of microspheres. More specifically, Figure 7 Shown is a fluorescence image of a mini-well plate equipped with microspheres representing "nanoreactor beads". Imaging was performed after thermal cycling according to the following thermal protocol: • 2 min at 98 °C (initial denaturation) • 45 cycles, 5 s at 98 °C & 15 s at 59 °C Figure 7 A: Fluorescence image acquired using the Cy5 fluorescence filter set. All microspheres (= "beads" or "nanoreactor beads") were stained with Cy5 dye for bead identification and automatic image segmentation. The nanoreactor beads only occupy part of the well. Nevertheless, the magnified view of the marked area shows a monolayer of nanoreactor beads.

[0085] Figure 7 B: FAM channel: Signal of FAM-TaqMan probe using genomic DNA as template and RPP30 as target The same beads as shown in A., equipped with a primer set specific for the target represented by the human RPP30 gene. The beads also contain a target-specific TaqMan probe labeled with FAM dye. An overview fluorescent image of a mini-well plate with six wells acquired using a FAM fluorescent filter set is shown on the left. The enlarged view of the marked area on the right shows the same dark and bright beads, respectively. The bright signal here indicates PCR amplification of the molecular target.

[0086] Figure 8 a) shows an exploded view of an embodiment of a disassembled multi-well plate, referred to in the figure as a "mini-well plate", having a plurality of wells (e.g., 6 wells), a sealed edge and a separate flat plate (e.g., a cover glass) and a cover. The cover has a gasket for sealing the well plate and also includes a light-permeable detection area, like a flat cover glass, for subsequent optical detection. Figure 8 b) is an isometric view of the closing assembly of the multiwell plate of a), including the plate (=cover glass) and the lid.

[0087] Fig. 9 a) shows a top view of an embodiment of an assembly of a multiwell plate, a flat plate (e.g. a cover glass) and a cover. Line A indicates Fig. 9 b) Cut surface of the cross section shown. Fig. 9 b) also shows the details circled on the right and Fig. 9 c) shows in more detail that the Fig. 9 c) shows the cover and multiwell plate, including the sealing edge and the corresponding positioning of the gasket. The cover is locked in place by a locking device, such as a "snap-lock". The seal is achieved by pressing the gasket against the flat surface of the sealing edge of the multiwell plate.

[0088] Fig.10 a) shows a top view of an embodiment of a device for arranging microspheres in a non-aqueous, water-immiscible liquid phase within a porous plate and for keeping such microspheres from contact with or exposure to a gas phase. Such devices are sometimes also referred to herein as "magnetic racks". Line A in Figure a) indicates the cutting plane of the cross-section shown in Figure b), which shows a cross-section of a magnetic rack in which the main frame body is equipped with magnets and mounted on top of a substrate (e.g., a metal substrate). The main body has positioning means to support the correct placement of the porous plate on the rack. Figure c) shows an isometric view of the magnetic rack.

[0089] Fig.11a) shows a top view of an embodiment of a magnetic stand with an assembled multiwell plate. Line A indicates Fig.11 b) A cut surface of the cross section shown, which is a cross section of the magnetic rack with the multiwell plate assembly mounted. Fig.11 c) shows an isometric view of a magnetic rack with a mounted multiwell plate. Push-labels on the plate indicate the operation of the unit. By pushing the two marked parts, the locking device moves to the release position. After the multiwell plate guided by the positioning device is correctly placed on the rack, the locking device is released and snaps into place, locking the part protruding from the body of the multiwell plate.

[0090] Fig.12 An embodiment of a device for incubating microspheres in a multiwell plate is shown, such device having a body and a container located in the body for receiving the multiwell plate. In addition, the device includes a flap that can be switched between an open and a closed state. When the flap is open, the device is ready to receive the multiwell plate. Fig.12 Line B in a) indicates Fig.12 b) is the cross-sectional cut surface shown in the figure. Fig.12 b) shows the presence of a temperature control unit, such as a thermoelectric module with a thermal interface at the bottom of the container of the device, as well as a cooler and the interface. A thermoelectric module is a specific embodiment of a temperature control device that can be used according to the invention. Fig.12 c) shows an isometric view of the instrument with open flaps.

[0091] Fig.13 Shown with Fig.12 Same instrument / setup, but this time including a multiwell plate.

[0092] Fig.14 Shown with Fig.12 and Fig.13 The same device as in FIG. 1 , but this time the flaps are closed. Also shown in this figure is a release button that allows the flaps to be released and opened.

[0093] Fig.15 An embodiment of a device for detecting microspheres in a non-aqueous, water-immiscible liquid phase arranged in a plurality of incubation chambers in a multi-well plate is shown. In this embodiment, detection is performed by fluorescence detection. Fig.15 Line A in a) indicates Fig.15 b) Cut surface of the cross section shown. Fig.15 b) Also shown are parts of the optics used, including the imaging lens, the (fluorescence) filter arrangement and the imaging detector, as well as the electronic interface of this arrangement with the device / instrument used to incubate the microspheres. Fig.15c) shows an isometric view of the instrument / device and explicitly shows the loading bay of the instrument for incubation in the form of an open space / slot into which the device for incubation is inserted.

[0094] Fig.16 A device for detection and a device for incubation are shown, wherein Fig.16 In a), the incubation device has not yet been moved into the loading area of ​​the detection device, and Fig.16 b) it has been moved into the loading area.

[0095] Fig.17 Shows Fig.16 A combination of devices, wherein the device for incubation has been inserted into the device for detection. Fig.17 Line B in a) indicates Fig.17 b) is the cut surface of the cross section shown. Fig.17 b) It can be seen that the multiwell plate and any microspheres appropriately arranged therein are also included.

[0096] Furthermore, the embodiments referred to are intended to illustrate the present invention rather than to limit the present invention.

[0097] Example Transcription and polymerase chain reaction in beads in multiwell plates followed by detection of beads monolayers Bead microsphere loading Hydrated beads (40 µl bead bed, 100 μm bead diameter, see table) each doped with magnetic particles were loaded with polymerase chain reaction (PCR) reagents (see table) using equal volumes of a double concentrated master mix supplemented with template and corresponding primer probe sets in an aqueous environment by incubation with agitation for 5 min at room temperature.

[0098] Table 1: Beads and PCR mix components

[0099] Suspension and dispersion of beads The beads were gently pelleted using a centrifuge with a swing-out rotor at 300 rpm for 1 min, the supernatant discarded, and 100 µl of separation oil (Novec 7500™, including 5% PicoSurf, SphereFluidics) was added. The tube containing the prepared mixture was vigorously stirred in a Minilys personal homogenizer (Bertin instruments, medium speed) for 15 s to force the aqueous beads to form a stable suspension in the fluorophilic oil, a process facilitated by the included surfactant. Since the density of the applied oil was ~1.6 g / ml, the aqueous beads floated to the surface of the oil. The engulfed beads were washed three times with separation oil to minimize the amount of microemulsion (water and air emulsified in the oil) that occurred during the suspension process.

[0100] Loading and assembly of multiwell boxes The multiwell plate is placed on a loading rack containing a magnet under each well position and filled with 740 µl of filling oil (Novec 7500™ with 0.5% PicoSurf). Subsequently, 3 µl of engulfed beads are pipetted into each well. The magnet of the loading rack holds the superparamagnetic beads in the individual wells. A custom-made bead plate (cover glass 23×29 mm, 500µm thickness) is applied to the multiwell plate in the following manner, by placing it upright on one side of the plate and slowly tilting the cover glass to cover the entire plate. The transparent (vitreous) bead plate is immersed in the filling oil volume. Thus, the wells are separated and covered without any air bubbles inside the wells. A cover containing a gasket at the sealing edge is applied to the multiwell plate in the same manner and closed by applying force to ensure that the spring locks on both sides are locked. The cover is not in direct contact with the bead plate when the filling oil is in the gap.

[0101] Mounting and Thermal Cycling In this state, the cartridge is liquid-tight and the beads are trapped in their respective wells. Therefore, the entire cartridge can be removed from the loading rack and sent to the thermal control unit and optical detection unit for processing. The multiwell plate is placed in a thermal cycling chamber consisting of a Peltier element, electronic control and cooling unit. While the multiwell plate is installed in this chamber, the mechanical force applied presses the multiwell plate and the lid together, which squeezes the seal gasket firmly and improves the gas tightness. PCR cycling is performed after reverse transcription (if RTase is present: 10 min, 50 °C), preheating (2 min, 80 °C) and initial denaturation (2 min, 98 °C) steps as follows: MS2 40× cycles 30 s 98 °C & 30 s 57 °C, RPP30 45× cycles 5 s 98 °C & 15 s59 °C. The heat applied in the initial denaturation PCR step causes the gasket to partially melt, thus creating an additional durable heat seal to the cartridge. After the final PCR step, the cartridge was actively cooled to 20 °C in preparation for image acquisition.

[0102] Imaging An image of each channel was acquired at each chamber position, for a total of 2 × 25 images. One channel was required for a specific amplification signal (see table) and the second channel for bead-microsphere segmentation. Automated image acquisition was triggered by the BLINK toolbox software and was achieved by using an upright fluorescence microscope (Zeiss AxioZoom) equipped with a 20x objective (field of view 5.633 × 3.538 mm) and a SOLA SE V-nIR light engine (Lumencor). The microscope was also equipped with three fluorescence filter sets (Cy5ETF46-006, Cy3 ET F46-004, FITC HC F36-502, AHF Analysentechnik) and an automated xy stage, where a thermal cycler with reaction chambers was mounted.

[0103] Table 2: Channel Overview

[0104] Bead segmentation and endpoint analysis All acquired images were subjected to an automated multifaceted image processing algorithm developed by BLINK to detect and segment individual nanoreactor beads. The fluorescence signal, position, diameter / volume and other features in each channel of all segmented nanoreactors were subsequently collected at various levels to perform extensive statistics. The experimental data were processed by the open source software Jupyter.

[0105] The features of the invention disclosed in the description, the claims and / or the drawings may, alone or in any combination thereof, be material for realizing the invention in various forms.

Claims

1. A method for arranging microspheres in a non-aqueous, water-immiscible liquid phase within an incubation chamber and for keeping such microspheres from contacting or being exposed to a gas phase, the method comprising the steps of: a) providing, in any order, a suspension comprising magnetic microspheres suspended in a non-aqueous, water-immiscible liquid phase and an incubation chamber; The incubation chamber has a closed bottom, an open top, at least one wall and a defined volume; the open top is spaced a defined distance from the closed bottom; the chamber is sized to accommodate a defined volume of the suspension therein; The magnetic microspheres have a first specified density; the non-aqueous, water-immiscible liquid phase has a second specified density, wherein the first specified density is less than the second specified density; and wherein, Preferably, the suspension has a specified concentration of microspheres; b) filling the incubation chamber with the suspension by placing the suspension into the incubation chamber such that a first interface is formed in the incubation chamber between the non-aqueous, water-immiscible liquid phase and a gas phase, the gas phase surrounding the incubation chamber, at the open top or at a first position along the defined distance between the open top and the closed bottom; c) removing the magnetic microspheres from the first interface or keeping them removed from the first interface by attracting the magnetic microspheres to the closed bottom or to the at least one wall of the incubation chamber, wherein the attraction is achieved by applying a magnetic force through the closed bottom or through the at least one wall of the incubation chamber; d) placing a solid surface at the open top of the incubation chamber or at a location along the specified distance between the open top and the closed bottom of the incubation chamber while maintaining the magnetic microspheres attracted to the closed bottom or the at least one wall of the incubation chamber by continuously applying a magnetic force through the closed bottom or the at least one wall; wherein such placement is performed such that the solid surface is in contact with the liquid phase and such that a second interface is formed between the non-aqueous, water-immiscible liquid phase and the solid surface at the open top or at said location, wherein the second interface replaces or eliminates the first interface so that the gas phase is not in contact with the liquid phase; e) releasing the microspheres from the bottom or from the at least one wall by ceasing to apply a magnetic force through the bottom or through the at least one wall of the incubation chamber, allowing the magnetic microspheres to assemble at the second interface. 2 . The method according to claim 1 , wherein steps b) and c) are performed simultaneously or in a temporally overlapping manner.

3. A method according to any one of the preceding claims, wherein the applying of the magnetic force through the closed bottom or the at least one wall of the incubation chamber in step c) occurs by placing a magnet near the bottom or the at least one wall of the incubation chamber or vice versa, so that by means of the magnet, a magnetic force is applied through the bottom or the at least one wall, wherein, Preferably, in step c), the bottom or the at least one wall of the incubation chamber is in physical contact with the magnet, or in contact with the magnet in such a way that the magnet can exert a magnetic force therethrough.

4. The method according to any one of the preceding claims, wherein by step d), the incubation chamber becomes closed, wherein the open top is transformed into a closed top by the solid surface.

5. The method according to any one of the preceding claims, wherein in step d), the solid surface is placed at the open top of the incubation chamber or at a position along the defined distance between the open top and the closed bottom of the incubation chamber by: - placing the cover only at the open top of the incubation chamber or at the position, - Alternatively, a separate solid surface, such as a plate, which is not a cover, is first placed at said open top or at said location of said incubation chamber and then the cover is placed on top of said separate solid surface.

6. A method according to any one of the preceding claims, wherein the continuous application of the magnetic force in step d) occurs by keeping a magnet near the bottom or at least one wall of the incubation chamber or vice versa, so that the magnetic force is continuously applied through the bottom or through the at least one wall by means of the magnet, wherein, Preferably, in step d), the bottom or the at least one wall of the incubation chamber is in physical contact with the magnet, or at least in such a way that the magnet can exert a magnetic force therethrough.

7. A method according to any one of the preceding claims, wherein step e) is performed by removing the magnet from the vicinity of the bottom or the at least one wall, or vice versa, thereby preventing the magnetic force from being applied through the bottom or through the at least one wall of the incubation chamber.

8. The method according to any one of the preceding claims, wherein the assembly of the microspheres at the second interface in step e) is achieved by actively generating convection, preferably thermal or mechanical convection, in the incubation chamber.

9. The method according to any of the preceding claims, wherein step e) is achieved by exposing the bottom of the incubation chamber to a temperature control device, preferably by bringing the bottom of the incubation chamber into thermal contact with the temperature control device, more preferably by bringing the bottom of the incubation chamber into physical contact with the temperature control device.

10. The method according to any one of the preceding claims, wherein the method is a method of arranging the microspheres in a microsphere layer along the second interface, and wherein the solid surface placed at the open top of the incubation chamber or at the position along the specified distance between the open top and the closed bottom of the incubation chamber in step d) is a planar solid surface.

11. The method of claim 10, wherein the layer is a monolayer of microspheres along the second interface.

12. The method according to any one of claims 10-11, wherein the packing of the microspheres in the layer is selected from random packing, regular packing and close packing, in particular hexagonal close packing.

13. The method according to any one of claims 10 to 12, wherein the packing of the microspheres in the layer is close packed, in particular hexagonal close packed.

14. The method according to any one of claims 10 to 13, wherein the number of the magnetic microspheres filled into the incubation chamber in step b) is selected so that it does not exceed the maximum number of microspheres that can be arranged in a layer along the second interface, the layer having as dense a packing as possible; and / or so that when arranged in the layer in as dense a packing as possible, the area covered by the magnetic microspheres is equal to or less than the area provided by the planar solid surface in contact with the liquid phase.

15. The method according to any one of the preceding claims, wherein the microspheres are monodisperse, preferably with a coefficient of variation (CV) < 15%, more preferably CV < 10%, even more preferably CV < 5%.

16. A method according to any one of the preceding claims, wherein the solid surface, preferably the planar solid surface, comprises areas that are transparent and allow optical probing and detection of the liquid phase and any microspheres located beneath said areas.

17. A method according to any one of the preceding claims, wherein the incubation chambers are holes in a multiwell plate, and wherein the multiwell plate has a plurality of such incubation chambers as defined in any one of the preceding claims, the incubation chambers being arranged in the multiwell plate, preferably regularly spaced apart in the multiwell plate.

18. The method of claim 17, wherein the method is performed using two or more wells in the multi-well plate.

19. A method for incubating microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber or a multiwell plate, the method comprising the steps of: f) carrying out the method according to any of the preceding claims, preferably according to any of claims 16 to 18; g) performing an incubation reaction with the microspheres, wherein: Optionally, the incubation reaction involves one or more changes in the temperature of the microspheres.

20. A method for detecting and optically probing microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber or in a multiwell plate, the method comprising the steps of: h) carrying out a method according to claim 16, or carrying out a method according to any one of claims 17-18 when any one of claims 17-18 is dependent on claim 16; Optionally, i) incubating with the microspheres, wherein: Optionally, the incubation reaction involves one or more temperature changes of the microspheres; k) detecting and optically probing the microspheres through the transparent area of ​​the planar solid surface, preferably using a suitable detection module.

21. A multiwell plate configured for use in the method according to any one of claims 1 to 20, comprising: - a planar body having a planar surface and comprising a plurality of holes, each hole having a closed bottom, an open top, at least one wall and a defined volume; Each well is sized to accommodate a specified number of magnetic microspheres or a specified volume of a suspension comprising the magnetic microspheres suspended in a non-aqueous, water-immiscible liquid phase therein; wherein the holes are embedded in the planar surface of the planar body, the planar surface being configured to contact a planar solid surface such that each hole is closed by the planar solid surface; - a sealing edge at the periphery of the planar body, the sealing edge surrounding the planar body of the perforated plate and protruding therefrom; - a cover configured to be attached to and placed on top of the multiwell plate and sized to cover and seal the multiwell plate, the lower surface of the cover being a planar solid surface; the cover having an outer periphery and including a gasket at its outer periphery, the gasket interacting with the sealing edge of the planar body and sealing the multiwell plate when the cover is placed on top of the multiwell plate; as well as - Optionally, the lower surface is a flat plate of a planar solid surface, the plate being configured to be placed on top of the flat body and being sized to cover the flat body.

22. A multiwell plate according to claim 21, wherein each well is enclosed by a planar solid surface provided by the lower surface of the cover or the lower surface of the plate, if present.

23. The multiwell plate of any one of claims 21-22, further comprising a flat plate having a lower surface that is a planar solid surface, the flat plate being configured to be placed on top of the planar body and being sized to cover the planar body.

24. A multiwell plate according to any one of claims 21-23, wherein the cover and the plate, if present, (each) comprise an area covering a plurality of wells, the area being optically transparent and allowing optical probing and detection of any wells located beneath the area.

25. A porous plate according to any one of claims 21-24, further comprising a locking device configured to lock the cover in position at the top of the porous plate, and optionally configured to also lock the flat plate, if present, to the top of the flat body of the porous plate so that the porous plate is sealed.

26. A porous plate according to any one of claims 21-25, wherein the flat body is made of or comprises the following material: plastic, preferably plastic with elemental carbon as an additive, more preferably plastic with elemental carbon as an additive in a proportion of >50 wt.%, even more preferably the plastic is polycarbonate with elemental carbon as an additive in a proportion of >50 wt.%.

27. An apparatus for arranging microspheres in a non-aqueous, water-immiscible liquid phase within an incubation chamber and for keeping such microspheres from contacting or being exposed to a gas phase, the apparatus comprising: - Containers for receiving incubation chambers or multiwell plates; The container has a bottom surface and at least one positioning device for positioning the incubation chamber or the multi-well plate on the bottom surface, the positioning device protruding from the bottom surface, and the container has a size that can tightly accommodate the incubation chamber or the multi-well plate; the incubation chamber has a closed bottom, an open top, at least one wall and a specified volume; the multi-well plate is as described in any one of claims 21-26; - a magnet or a set of magnets located at and incorporated into the bottom surface of the container or located at and incorporated into the positioning device, the magnet being configured to apply a magnetic force through the closed bottom of the incubation chamber, or through the bottom of each well in the multiwell plate, or through the at least one wall of the incubation chamber, or through the at least one wall of each well in the multiwell plate; - Optionally, locking means for locking the incubation chamber or the multiwell plate in the container, preferably on the bottom surface of the container.

28. A device for incubating microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber or a multiwell plate, the device comprising: - a body having an upper surface and a container configured to receive a multiwell plate according to any one of claims 21 to 26, the container being located on the upper surface, the container having a bottom; - a temperature control device located at and / or incorporated into said bottom of said container and configured to be in thermal and / or physical contact with the bottom of the incubation chamber or the bottom of each well of a multi-well plate when inserted into said container; as well as - A cover or wing attached to the body and configured to cover the upper surface of the body and, when placed in the container of the device, be placed on top of the container and the incubation chamber or the multiwell plate; wherein the cover or wing is also configured to lock the incubation chamber or the multiwell plate in the container when the cover or wing is placed on top of the container, wherein the cover or wing also includes an optically transparent area that becomes located on the top of the container and thereby allows optical detection of the incubation chamber or the multiwell plate present in the container.

29. The device according to claim 28, further comprising an incubation chamber or a multiwell plate according to any one of claims 21-26 in the container.

30. A device for detecting and optically probing microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber or a multiwell plate, the incubation chamber or the multiwell plate being located in the device for incubation according to claim 28, the device for detecting comprising: - a housing comprising a detection module comprising an image detector, one or more filters and at least one imaging lens; - a loading zone configured to receive and accommodate the device for incubation according to any one of claims 28-29, the loading zone comprising an interface for electrically connecting to the device for incubation according to any one of claims 28-29, wherein the loading zone is located within the housing so that when the detection module is present in the loading zone, the detection module can be used to detect and optically detect the device for incubation, preferably can be used to detect and optically detect an incubation chamber or a multiwell plate located in the device for incubating microspheres; wherein the detection and optical detection are performed through the optically transparent area of ​​the cover or wing of the device for incubation; and wherein the optically transparent area of ​​the cover or wing of the device for incubation is aligned with: said optically transparent area of ​​said cover and said flat surface of said multiwell plate, if such an incubation chamber or multiwell plate is present in said device for incubation, the image detector, a filter from the one or more filters, and The at least one imaging lens; Optical detection is performed through the transparent area of ​​the cover or flap of the device for incubation and through the optically transparent area of ​​the cover and the flat plate of the multiwell plate if present in the device for detection.

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