Method for disposing microspheres in a non-aqueous liquid phase

Abstract

The present invention relates to a method for disposing microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber, without such microspheres coming into contact with or being exposed to a gas phase. It also relates to a method for incubating microspheres, and a method for detecting and optically probing microspheres. The present invention further relates to a multi-well plate configured for use in such method(s). The present invention further relates to an apparatus for disposing microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber, without such microspheres coming into contact with or being exposed to a gas phase. The present invention further relates to an apparatus for incubating microspheres and an apparatus for detecting and optically probing microspheres.

Description

[Technical field]

[0001] The present invention relates to a method for disposing microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber, without such microspheres coming into contact with or being exposed to a gas phase, and further relates to a multi-well plate adapted for use in such a method.The present invention further relates to an apparatus for disposing microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber, without such microspheres coming into contact with or being exposed to a gas phase. [Background technology]

[0002] Microspheres, generally and sometimes referred to herein as "nanoreactor beads", are an emerging class of molecular biology reagents with a wide range of applications, such as nucleic acid extraction, digital nucleic acid amplification, e.g., digital polymerase chain reaction and detection. In their simplest configuration, such microspheres or "nanoreactor beads" comprise a hydrogel that provides a defined volume that acts as a reaction space for a biochemical reaction to occur. In the context of nucleic acid analysis, the hydrogel is used to capture the reaction components (e.g., buffers, amplification reagents, etc.) and the target material to be amplified. Upon loading the hydrogel with the reagent mix, the microspheres are enveloped in an oil phase, such that the volume of each individual microsphere acts as an isolated reaction space that does not cross-react with other microspheres / nanoreactors. At this stage, the oil-encased microspheres can then be exposed to suitable amplification reaction conditions, such as the specific temperature profile to which the microspheres are exposed.

[0003] Since each individual microsphere provides an isolated volume, it is important to ensure uniform conditions 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 within such vessel / container. However, this approach is quite time-consuming, as reflected by classical digital PCR thermocycling protocols.

[0004] Furthermore, due to the difference between the oil phase and the microspheres and the immiscibility between the oil phase and the water phase, the microspheres containing the water phase tend to concentrate and coalesce at high temperatures due to the resulting forces. Furthermore, the microspheres tend to accumulate at the interface formed between the oil phase and the surrounding atmosphere, which can result in drying artifacts.

[0005] Once the amplification reaction is complete, the microspheres are inspected for the presence of amplified target, which is done by a suitable optical detection device. However, in situ detection is laborious and requires complex techniques such as light sheet imaging (Liao et al., PNAS USA, 2020, 117(41), pp. 25628-25633). Alternatively, the microspheres may be transferred to a reader similar to a flow cytometer to detect when amplification is complete (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 aforementioned problems, microfluidic techniques have been developed to generate and contain droplets in microfluidic devices, providing regularly arranged arrays of droplets for uniform exposure to identical conditions and easy readout by imaging the droplets arranged in two dimensions (Madic et al., Biomol. Detect. Quantif. 2016,10:pp.34-46). Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there remains a need in the art to reduce the complexity of disposables and to provide a cost-effective and safe method for housing microspheres / nanoreactor beads in a suitable configuration for amplification and detection. There is also a need in the art to provide a methodology that avoids the aforementioned shortcomings and problems associated with the prior art. In particular, there is a need to provide a method for placing microspheres in a suitable configuration such that such microspheres are not in contact with or exposed to the gas phase. [Means for solving the problem]

[0007] In a first aspect, the present invention provides a method for disposing microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber such that such microspheres are not in contact with or exposed to a gas phase, said method comprising: 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 having a closed bottom, an open top, at least one wall, and a defined volume; the open top being spaced a defined distance from the closed bottom; the chamber being dimensioned to accommodate a defined volume of the suspension therein; the magnetic microspheres have a first defined density; the non-aqueous water-immiscible liquid phase has a second defined density, the first defined density < the second defined density; preferably, the suspension has a defined concentration of microspheres; b) filling the incubation chamber with the suspension by placing the suspension within the incubation chamber, such that a first interface between the non-aqueous water-immiscible liquid phase and a gas phase surrounding the incubation chamber is formed within the incubation chamber at the open top or at a first location 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 them to the closed bottom or to the at least one wall of the incubation chamber, wherein the attraction is achieved by exerting a magnetic force across the closed bottom or across the at least one wall of the incubation chamber; d) while maintaining the magnetic force across the closed bottom or the at least one wall of the incubation chamber, placing a solid surface at the open top of the incubation chamber or at a location along the defined distance between the open top and the closed bottom of the incubation chamber 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 the location, such second interface replacing or eliminating the first interface, thereby preventing the gas phase from contacting the liquid phase; e) causing the magnetic microspheres to collect at the second interface by ceasing the application of magnetic force across the bottom or across the at least one wall of the incubation chamber, thereby releasing the microspheres from the bottom or the at least one wall; The present invention relates to a method comprising the steps of: [Brief description of the drawings]

[0008] Furthermore, see the following figure: [Figure 1]FIG. 1 shows a schematic flow diagram of an embodiment of a method for placing microspheres in a non-aqueous water-immiscible liquid phase in a single incubation chamber, without such microspheres contacting or being exposed to a gas phase. The microspheres are initially located at the interface between the non-aqueous water-immiscible liquid phase and the gas phase surrounding the incubation chamber. The microspheres are then attracted to the bottom by a magnet, and a solid surface is placed at the top of the incubation chamber or at a position within the incubation chamber, i.e., along a defined distance between the open top and closed bottom of the incubation chamber, so that the solid surface comes into 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 each position within the incubation chamber where the solid surface is placed, and there is no longer an interface between the liquid phase and the gas phase, and thus the interface is replaced or eliminated by another interface between the solid surface and the liquid phase. The microspheres are then released by removing the magnet. The incubation chamber is then placed under temperature control for the subsequent reaction. It should be noted that in this figure, the solid surface is or may be curved, so that the microspheres are not arranged in layers, but randomly. However, even with such a surface, an interface with the surrounding gas phase, e.g. air, is excluded. The letters in FIG. 1 correspond to the respective steps of the method of disposing microspheres according to the invention as follows: letter i) in FIG. 1 corresponds to the combination of steps a) and b) of the method of disposing microspheres according to the invention; letter ii) in FIG. 1 corresponds to step c) of the method of disposing microspheres according to the invention, and letter iii) in FIG. 1 corresponds to step d) of the method of disposing microspheres according to the invention; letter iv) in FIG. 1 corresponds to step e) of the method according to the invention, but with the additional limitation that an additional (optional) thermal control element is shown in FIG. 1. However, it is clear to the skilled person that the method of disposing microspheres according to the invention also works without such thermal control.However, in some cases, such thermal control may be preferred because it allows for more convection and therefore easier placement of the microspheres along the second interface. [Diagram 2] 2 shows the same schematic flow diagram of an embodiment of a method for disposing microspheres in a non-aqueous, water-immiscible liquid phase such that such microspheres are not in contact with or exposed to a gas phase, but this time for multiple incubation chambers such as those found in a multi-well plate, i.e., multiple wells. Each letter in FIG. 2 is the same as in FIG. 1. [Diagram 3] FIG. 3 shows a schematic flow diagram of an embodiment of a method for disposing microspheres in a non-aqueous water-immiscible liquid phase in an incubation chamber, without such microspheres coming into contact with or being exposed to a gas phase, using a solid surface placed on top of the incubation chamber or at a position within the incubation chamber, i.e. along a defined distance between the open top and closed bottom of the incubation chamber, however, in contrast to FIGS. 2 and 3, the solid surface employed here is a planar solid surface. This allows the microspheres to be disposed in a planar layer along such a planar solid surface. The planar solid surface may for example be the lid of the incubation chamber. Such an embodiment is particularly interesting for optically probing and further investigating the microspheres after disposing and after having undergone a biochemical reaction such as an amplification reaction. This is additionally indicated in letter iv) of FIG. 3 by a schematic (optional) optical detector. However, such optional optical detector is not necessarily part of the disposing method according to the invention. Such optical detector is part of the detection and optical probing method according to the invention. [Figure 4]Figure 4 shows a schematic flow diagram of an embodiment of a method for disposing microspheres in a non-aqueous, water-immiscible liquid phase, this time in multiple incubation chambers such as those found in the wells of a multi-well plate, such that such microspheres are not in contact with or exposed to the gas phase. Similar to Figure 3, the method employs a planar solid surface that forms a second interface along which the microspheres can be disposed, allowing them to be arranged in a layer, thus facilitating subsequent optical probing and interrogation. The solid surface may be, for example, the lid of a multi-well plate. [Diagram 5] Figure 5 is similar to the embodiment shown in Figure 4, except that this embodiment additionally employs a separate solid planar surface separate from the lid, which serves as a planar solid surface for forming a second interface along which the microspheres are placed. The separate solid planar surface may be a flat plate, for example a cover glass. Such an embodiment is particularly useful in that it allows for a separation of the function of sealing the multiwell plate, achieved by the lid in letter iii-2) of Figure 5, from the function of providing an interface along which the microspheres are placed, as seen in letter iii-1) of Figure 5. This allows for wider manufacturing tolerances, thus keeping disposable costs low and overwhelmingly compensating for the additional part, i.e. an additional flat plate, for example a cover glass. [Figure 6]FIG. 6 shows a fluorescence image of an embodiment of a multiwell plate with microspheres, where the microspheres form a close-packed layer. More specifically, FIG. 6 shows a fluorescence image of a miniwell plate (i.e., a specific multiwell plate with only 6 wells) with microspheres representing "nanoreactor beads". Imaging was performed after thermocycling according to the following thermal protocol: 50° C. for 10 min (reverse transcriptase step), 80° C. for 2 min (system preheat), 98° C. for 2 min (initial denaturation), 98° C. for 30 s and 57° C. for 30 s for 40 cycles. FIG. 6A: Fluorescence image acquired with a FAM fluorescence filter set of microspheres ("nanoreactor beads") processed on a 6-well miniwell plate. All beads are stained with FAM dye for bead recognition and automated image segmentation. The wells are densely packed with nanoreactor beads. A close-up of the marked area shows a self-assembled monolayer of hexagonally packed nanoreactor beads. Figure 6B: The same beads as shown in A were fitted with a primer set specific for a target in MS2 phage RNA. Additionally, a TaqMan probe labeled with Cy5 dye was used to allow detection of target-specific amplification of a target present on a subset of beads. A full fluorescence image of a 6-well miniwell plate acquired using a Cy5 fluorescence filter set is shown on the left. A close-up of the labeled area on the right shows densely packed dark and light nanoreactor beads. The light signal arises from amplicon formation during thermocycling and indicates the presence of the target on each nanoreactor bead. [Figure 7]Figure 7 shows a layered arrangement of microspheres, however the packing is random packing and there are defects in the layer / monolayer of microspheres. More specifically, Figure 7 shows a fluorescence image of a miniwell plate with microspheres representing "nanoreactor beads". Imaging was performed after thermocycling according to the following thermal protocol: 98°C for 2 min (initial denaturation) 45 cycles of 98°C for 5 s and 59°C for 15 s Figure 7A: Fluorescence image acquired with a Cy5 fluorescence filter set. For bead recognition and automatic image segmentation, all microspheres (= "beads" or "nanoreactor beads") are stained with Cy5 dye. The wells are only partially occupied by nanoreactor beads. Nevertheless, a magnification of the marked area shows a monolayer of nanoreactor beads. Figure 7B: FAM channel: signal of a FAM-TaqMan probe targeted to RPP30 with genomic DNA as template The same beads as shown in A, equipped with a primer set specific for a target represented in the human RPP30 gene. The beads also contained target-specific TaqMan probes labeled with FAM dye. A full fluorescence image of a 6-well miniwell plate, acquired using a FAM fluorescence filter set, is shown on the left. A magnification of the labeled area on the right shows the same beads in dark and light colors, respectively. The light signal here indicates PCR amplification of the molecular target. [Figure 8] Figure 8a) shows an exploded view of an embodiment of a disassembled multi-well plate, referred to in the figure as a "mini-well plate", with multiple wells, e.g. 6 wells, a sealing rim, and a separate flat plate, e.g. a cover slip, and a lid. The lid has a gasket that allows sealing of the well plate, and also contains a light-transmissive detection area as well as a flat cover slip to allow subsequent optical detection. Figure 8b) shows an isometric view of the closed assembly of the multi-well plate of a), including the flat plate (= cover slip) and the lid. [Figure 9]Figure 9a) shows a top view of an embodiment of an assembly of a multiwell plate, a flat plate, e.g. a cover slip, and a lid. Line A indicates the cut of the cross section shown in Figure 9b). Also shown in Figure 9b) is a detail circled on the right and shown in more detail in Figure 9c), which shows the respective relative positions of the lid and the multiwell plate including the sealing rim, and the gasket. The lid is locked in place by a locking means, e.g. a "snap lock". Sealing is achieved by pressing the gasket against the planar surface of the sealing rim of the multiwell plate. [Figure 10] FIG. 10a) shows a top view of an embodiment of an apparatus for positioning microspheres in a non-aqueous, water-immiscible liquid phase in a multi-well plate such that such microspheres are not in contact with or exposed to the gas phase. Such an apparatus may be referred to herein as a "magnet rack." Line A in FIG. 10a) shows a cut plane of the cross section shown in FIG. 10b), which shows a cross section of a magnet rack in which the rack body comprises magnets and is mounted on a base plate, such as a metal base plate. The body features positioning means that aid in proper placement of a multi-well plate on the rack. FIG. 10c) shows an isometric view of the magnet rack. [Figure 11] Fig. 11a) shows a top view of an embodiment of a magnet rack with an assembled multiwell plate. Line A shows the cut of the cross section shown in Fig. 11b), which is a cross section of the magnetic rack with a multiwell plate assembly attached. Fig. 11c) shows an isometric view of the magnet rack with a multiwell plate attached. Push labels on the plate indicate the operation of the unit. By pushing both labeled parts, the locking means moves to the released position. After the multiwell plate, guided by the positioning means, is properly placed on the rack, the locking means is released and snaps into place, capturing the parts protruding from the body of the multiwell plate. [Figure 12]FIG. 12 shows an embodiment of a device for incubating microspheres in a multiwell plate, such device having a body and a receptacle located in such body to receive such a multiwell plate. Furthermore, the device is provided with a flap that can be switched between an open and a closed state. When the flap is open, the device is ready to receive a multiwell plate. Line B in FIG. 12a) shows a cut of the cross section shown in FIG. 12b) showing the presence of a temperature control unit, for example a thermoelectric module with a thermal interface at the bottom of the device's receptacle, as well as a cooler and an interface. The thermoelectric module is a specific embodiment of a temperature control device that can be used according to the invention. FIG. 12c) shows an isometric view of the device with the flap open. [Figure 13] FIG. 13 shows the same instrument / apparatus as FIG. 12, but this time including a multi-well plate. [Figure 14] Figure 14 shows the same device as Figures 12 and 13, but this time with the flaps closed, also showing the release of the flaps and the release button which enables their opening. [Figure 15] Figure 15 shows an embodiment of a device for detecting microspheres placed in a non-aqueous water-immiscible liquid phase in multiple incubation chambers in a multi-well plate. In this embodiment, detection is performed by fluorescence detection. Line A in Figure 15a) shows the cut of the cross section shown in Figure 15b). Figure 15b) further shows part of the optical system used, including the imaging lens, the (fluorescence) filter setup and the imaging detector, as well as the electronic interface of this device to the device / instrument incubating said microspheres. Figure 15c) shows an isometric view of the instrument / apparatus, explicitly showing the loading bay of the incubating instrument in the form of an open space / slot into which the incubating instrument is inserted. [Figure 16] FIG. 16 shows both the detection device and the incubation device, where in FIG. 16a) the incubation device has not yet been moved to the loading bay of the detection device, whereas in FIG. 16b) it has been moved. [Figure 17] Figure 17 shows the combination of the devices of Figure 16, where the incubation device is inserted into the detection device. Line B in Figure 17a) shows the cut of the cross section shown in Figure 17b). From Figure 17b) it can be seen that the multi-well plate is also included, as well as any microspheres appropriately placed therein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] In one embodiment, steps b) and c) are performed simultaneously or overlapping in time.

[0010] In one embodiment, exerting a magnetic force over the closed bottom or at least one wall of the incubation chamber in step c) is performed by either placing a magnet in the vicinity of the bottom or at least one wall of the incubation chamber or vice versa, such that the magnet exerts a magnetic force over the entire bottom or at least one wall, preferably in step c) the bottom or at least one wall of the incubation chamber is in physical contact with the magnet or in such contact that the magnet can exert a magnetic force over its entirety.

[0011] In one embodiment, step d) results in said incubation chamber becoming closed, in that said solid surface converts said open top into a closed top.

[0012] In one embodiment, in step d), the solid surface is - placing only a lid on the open top or location of the incubation chamber; - or first placing a separate solid surface that is not a lid, such as a flat plate, on the open top or at the location of the incubation chamber, and then placing a lid on top of the separate solid surface. by being positioned at the open top of the incubation chamber or at a location along the defined distance between the open top and the closed bottom of the incubation chamber.

[0013] In one embodiment, continuing to exert the magnetic force in step d) is performed by either holding a magnet in the vicinity of the bottom or at least one wall of the incubation chamber or vice versa, such that the magnet continues to exert a magnetic force over the entire bottom or at least one wall, preferably in step d) the bottom or at least one wall of the incubation chamber is in physical contact with the magnet or at least in such contact that the magnet can exert a magnetic force over its entirety.

[0014] 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 ceasing the exertion of a magnetic force across the bottom or across the at least one wall of the incubation chamber.

[0015] In one embodiment, the assembly of said microspheres at said second interface in step e) is promoted by actively generating convection, preferably thermal or mechanical convection, within said incubation chamber.

[0016] In one embodiment, step e) is facilitated 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.

[0017] In one embodiment, the method is a method of disposing the microspheres in a layer along the second interface, and the solid surface placed in step d) at the position along the open top of the incubation chamber or the specified distance between the open top and the closed bottom of the incubation chamber is a planar solid surface.

[0018] In one embodiment, the layer is a monolayer of microspheres along the second interface.

[0019] In one embodiment, the packing of the microspheres in said layer is selected from random packing, ordered packing, and close packing, in particular hexagonal close packing.

[0020] In one embodiment, the packing of the microspheres in said layer is close-packed, in particular hexagonal close-packed.

[0021] In one embodiment, the number of magnetic microspheres filled into the incubation chamber in step b) is selected so as not to exceed the maximum number of microspheres that can be arranged in the most densely packed layer possible along the second interface; and / or so that when arranged in the most densely packed layer possible, the magnetic microspheres cover an area less than or equal to the area provided by the planar solid surface in contact with the liquid phase.

[0022] In one embodiment, the microspheres are monodisperse, preferably with a coefficient of variation (CV) <15%, more preferably CV <10%, even more preferably CV <5%.

[0023] In one embodiment, the solid surface, preferably the planar solid surface, comprises a transparent region, allowing optical probing and detection of the liquid phase and any microspheres located beneath said region.

[0024] In one embodiment, the incubation chambers are wells in a multiwell plate, the multiwell plate having a plurality of such incubation chambers arranged in the multiwell plate, preferably regularly spaced apart in the multiwell plate, as defined in any of the above claims.

[0025] In one embodiment, the method is performed using two or more wells in the multiwell plate.

[0026] In a further aspect, the present invention also provides a method for incubating microspheres in a non-aqueous water-immiscible liquid phase in an incubation chamber or in a multi-well plate, said method comprising: f) carrying out the method for positioning microspheres according to the invention, preferably according to an embodiment in which said solid surface is planar and contains transparent areas allowing optical probing and detection; g) performing an incubation reaction with the microspheres, optionally involving changing the temperature of the microspheres one or several times; The present invention relates to a method comprising the steps of:

[0027] In a further aspect, the present invention provides a method for detecting and optically probing microspheres in a non-aqueous water-immiscible liquid phase in an incubation chamber or in a multi-well plate, said method comprising: h) carrying out the method according to claim 16 or, when dependent on claim 16, the method according to any of claims 17 to 18; Optionally, i) performing an incubation reaction with said microspheres, optionally involving changing the temperature of said microspheres one or several times; k) detecting and optically probing said microspheres through said transparent area of ​​said planar solid surface, preferably using a suitable detection module; The present invention relates to a method comprising the steps of:

[0028] In yet another aspect, the invention provides a multiwell plate adapted for use in a method according to the invention, such as a method for positioning microspheres, a method for incubating microspheres, or a method for detecting and optically probing microspheres, said multiwell plate comprising: - a flat 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 dimensioned to accommodate a defined volume of a suspension comprising a defined number of magnetic microspheres or magnetic microspheres suspended in a non-aqueous water-immiscible liquid phase therein; said wells being embedded in said planar surface of said flat body and configured such that said planar surface is contacted by a planar solid surface such that each well is closed by such planar solid surface; - a sealing rim at the periphery of the flat body, the sealing rim surrounding and projecting from the flat body of the multiwell plate; - a lid configured to be mounted and placed on said multiwell plate, in particular on said flat body, and dimensioned to cover and seal said multiwell plate, in particular to cover and seal said flat body, said lid having an underside which is a planar solid surface; having a periphery and comprising a gasket on its periphery which interacts with said sealing rim of said flat body and seals said multiwell plate, in particular on said flat body, when said lid is placed on said multiwell plate, in particular on said flat body; - optionally a plate having a lower surface that is a planar solid surface, the plate being configured to be placed on said flat body and dimensioned to cover said flat body; The present invention relates to a multi-well plate comprising:

[0029] In one embodiment of a multi-well plate, each well is closed by a planar solid surface, such planar solid surface being provided either by the underside of the lid or by the underside of the plate, if present.

[0030] In one embodiment of the multiwell plate, the multiwell plate further comprises a plate having a lower surface that is a planar solid surface, the plate configured to be placed on the flat body and dimensioned to cover the flat body.

[0031] In embodiments, a plate is provided and, if present, is provided separately from the lid and does not form part of such lid. For example, if the lid has a multi-layer structure, the plate does not form part of the multi-layer structure and is not any layer thereof. Thus, in embodiments in which a plate is provided and present in the multi-well plate, the plate is not an integral part of the lid and is provided separately from the lid.

[0032] In one embodiment of a multi-well plate, the lid and, if present, the plate (each) comprises (includes) a region covering multiple wells, which region is optically transparent to allow optical probing and detection of any of the wells located beneath the region(s).

[0033] In one embodiment, the multiwell plate further comprises a locking means configured to lock the lid in position over the multiwell plate such that the multiwell plate is sealed, and optionally also the flat plate, if present, over the flat body of the multiwell plate.

[0034] In one embodiment of the multi-well plate, said flat body is made of or comprises a plastic, preferably a plastic having elemental carbon as an additive, more preferably a plastic having more than 50% by weight of elemental carbon as an additive, even more preferably said plastic is a polycarbonate having more than 50% by weight of elemental carbon as an additive.

[0035] In a further aspect, the present invention also provides an apparatus for disposing microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber such that such microspheres are not in contact with or exposed to a gas phase, the apparatus comprising: - a receptacle for receiving an incubation chamber or a multiwell plate; said receptacle having a bottom surface and at least one positioning means for positioning said incubation chamber or said multiwell plate on said bottom surface, said positioning means protruding from such bottom surface, said receptacle having dimensions such that said incubation chamber or said multiwell plate is received therein with a snug fit; said incubation chamber having a closed bottom surface, an open top surface, at least one wall and a defined volume; and said multiwell plate is as defined herein; - a magnet or set of magnets located on and integrated into the bottom surface of the receptacle or located on and integrated into the positioning means, the magnet or magnets being configured to exert a magnetic force across the closed bottom of the incubation chamber, or across the bottom of each well of the multiwell plate, or across the at least one wall of the incubation chamber, or across the at least one wall of each well of the multiwell plate; - optionally, locking means, preferably on the bottom surface within the receptacle, for locking the incubation chamber or the multiwell plate in the receptacle; The present invention relates to an apparatus comprising:

[0036] In preferred embodiments, the locking means are operable to reversibly lock and / or release the incubation chamber or multiwell plate, in particularly preferred embodiments, the locking means are operable to interact with the positioning means to reversibly engage the positioning means with the incubation chamber or multiwell plate and / or to release the positioning means.

[0037] It should be noted that the locking means allows easier and more robust handling of any incubation chamber or multi-well plate locked to the device, for example inversion or shaking of the incubation chamber or multi-well plate when locked by the locking means can be easily achieved.

[0038] In one embodiment, said locking means comprises one or several push buttons, preferably located adjacent to said receptacle, more preferably located symmetrically adjacent to said receptacle.

[0039] In yet another aspect, the present invention also provides an apparatus for incubating microspheres in a non-aqueous water-immiscible liquid phase in an incubation chamber or in a multi-well plate, said apparatus comprising: - a body having a top surface and a receptacle configured to receive a multi-well plate as defined herein, said receptacle being located on said top surface, said receptacle having a bottom; - a temperature control device located and / or integrated into the bottom of the receptacle and configured to be in thermal and / or physical contact with a bottom of an incubation chamber or a bottom of each well of a multi-well plate when inserted into the receptacle; - a lid or flap attached to the body and covering a top surface of the body and configured to be placed over the receptacle and over the incubation chamber or the multiwell plate when placed in the receptacle of the device, the lid or flap being further configured to lock the incubation chamber or the multiwell plate in the receptacle when the lid or flap is placed over the receptacle, and further comprising an optically transparent area that becomes overlying the receptacle, thereby allowing optical probing of the incubation chamber or the multiwell plate present in the receptacle; The present invention relates to an apparatus comprising:

[0040] In one embodiment, the incubating apparatus further comprises within said receptacle an incubation chamber or a multi-well plate as defined herein.

[0041] In yet another aspect, the present invention relates to an apparatus for detecting and optically probing microspheres in a non-aqueous water-immiscible liquid phase in an incubation chamber or in a multi-well plate, said incubation chamber or said multi-well plate being located in an incubating apparatus as defined herein, said detecting apparatus comprising: - a housing comprising a detection module, said detection module comprising an image detector, one or several filters, and at least one imaging lens; A loading bay configured to receive and house an incubating device as defined herein, said loading bay comprising an interface for electronically connecting to an incubating device as defined herein, said loading bay being located within said housing such that said incubating device can be detected and optically probed when present within said loading bay using a detection module, preferably such that an incubation chamber or multi-well plate located within said device for incubating microspheres can be detected and optically probed; detection and optical probing is performed through said optically transparent area of ​​said lid or flap of said incubating device; said optically transparent area of ​​said lid or flap of said incubating device being: the optically transparent region(s) of the lid and the plate of the multiwell plate, if such an incubation chamber or multiwell plate is present in the incubating device; said image detector; one of said one or more filters, and said at least one imaging lens Aligned with a loading bay such that optical probing occurs through the transparent area of ​​the lid or flap of the incubating device and, if present in the detecting device, through the optically transparent area(s) of the lid and the flat plate of the multiwell plate; The present invention relates to an apparatus comprising:

[0042] The inventors have surprisingly found that it is possible to place microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber and prevent such microspheres from contacting or being exposed to the gas phase. According to an embodiment of the present invention, this is achieved by taking advantage of the buoyancy or density difference between the microspheres and the liquid phase in which they are suspended, and by endowing such microspheres with magnetic qualities (i.e. making them magnetic) and using such magnetic qualities to handle the microspheres in the incubation chamber, for example by attracting them to the bottom or wall of the incubation chamber, while at the same time placing a solid surface (e.g. of a substrate) at the open top of the incubation chamber such that the solid surface is in contact with the liquid phase, thereby removing the gas phase (previously in contact with the liquid phase) from contacting the liquid phase. In doing so, any interface between said liquid phase and the gas phase surrounding the incubation chamber is removed or eliminated and replaced by another interface, which is the interface between the solid surface and the liquid phase.

[0043] Examples of microspheres suitable for use in the context of the present invention are, for example, those described in previous patent applications by the applicant, for example those described in European Patent Application No. 21 206 745.8, filed November 5, 2021; or those described in International Patent Application No. PCT / EP2017 / 084370, filed December 22, 2017 (published as WO 2018 / 122162), or those described in International Patent Applications Nos. PCT / EP2020 / 086171 and PCT / EP2020 / 086194 (published as WO 2021 / 122563 and WO 2021 / 122579, respectively). Broadly speaking, the microspheres according to the present invention are microspheres that contain a hydrogel and include a volume for receiving an aqueous solution and for providing a reaction space in which a suitable chemical or biochemical reaction can take place.

[0044] As used herein, the term "microsphere" is intended to refer to spheroids or substantially spherical or ellipsoidal or other regular circular shapes, e.g., ovoids, that contain a hydrogel and provide a reaction space within their volume. Typically, "microspheres" according to the present invention preferably have an average diameter along their longest extension in the range of 20 μm to 500 μm, preferably 20 μm to 200 μm, more preferably 20 μm to 100 μm.

[0045] Such microspheres can be made magnetic by incorporating smaller magnetic particles into them. Incorporation of such magnetic particles into the microspheres allows such microspheres to be attracted by a magnet (permanent magnet or electromagnet) placed in the vicinity of the microsphere. As used herein, the term "magnetic particles" is meant to refer to particles that exhibit essentially non-diamagnetic behavior, thus 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 or superparamagnetic particles. As used herein, the term "magnetic particles" is meant to exclude any particles that have diamagnetic behavior. As used herein, "magnetic particles" preferably have a size and average diameter or average elongation in one dimension that is small enough for such magnetic particles to be incorporated into the microspheres of the present invention. In an embodiment of the present invention, such magnetic particles have a size and average diameter or average one-dimensional extension 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 and average diameter or average one-dimensional extension of the magnetic particles are selected so that they can be easily accommodated in the microspheres according to the present invention. In many cases, the microspheres according to the present invention may contain a hydrogel that forms a porous network, for which the size and average diameter or average one-dimensional extension of the magnetic particles are selected to be larger than the average mesh or pore size of the network of the microsphere.

[0046] According to an embodiment of the present invention, such magnetic microspheres may be filled with suitable reagents for carrying out chemical or biochemical reactions, which are incorporated and thereby contained in the volume(s) of said microspheres as an aqueous mixture. They are then 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 (which may be, for example, a peripheral wall) and a defined volume. If the incubation chamber has one wall, this may be a peripheral wall, and therefore the incubation chamber has a circular (like) cross section. If it has several walls, the respective cross sections are triangular, square, rectangular, etc., depending on the number of walls of said incubation chamber.

[0047] The non-aqueous water-immiscible liquid phase is selected to have a density greater than that of the magnetic microsphere, including any aqueous mixture contained in the microsphere. In other words, the magnetic microsphere, including any aqueous mixture contained in the microsphere, preferably has a first specified density that is less than the density of the non-aqueous water-immiscible liquid phase, i.e., the "second specified density". The lower density of the microsphere creates buoyancy of the microsphere in the non-aqueous water-immiscible liquid phase. Examples of suitable non-aqueous water-immiscible liquid phases are oils, especially 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®, which is commercially available, for example, from 3M Deutschland GmbH (Neuss, Germany). In some embodiments, the non-aqueous water-immiscible liquid phase may be or is supplemented with additives such as emulsifiers, surfactants, and / or stabilizers.

[0048] Then, according to an embodiment of the method according to the invention, the incubation chamber is filled with a suspension of microspheres, so that a first interface is formed between the non-aqueous water-immiscible liquid phase and the gas phase surrounding the incubation chamber. Such a first interface is formed at the open top or at a first position along the defined distance between the open top and the closed bottom. The position at which such a first interface is formed depends on whether in step b) the incubation chamber is completely filled or only partially filled with the suspension of magnetic microspheres in a non-aqueous water-immiscible liquid phase. If the incubation chamber is completely filled, i.e. up to its open top, the first interface is formed at the open top, and if the incubation chamber is only partially filled, i.e. not up to its open top, the first interface is formed at a first position along the defined distance between the open top and the closed bottom. The exact position of such a first position depends on the amount of suspension filled into the incubation chamber. Typically, however, such a first location will be located within an incubation chamber and therefore below the open top of the incubation chamber, and therefore distinct from the open top of the incubation chamber.

[0049] In the absence of a magnet in the vicinity of the incubation chamber / magnetic microspheres located within such incubation chamber, these microspheres tend to move towards 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 are at least partially exposed to said gas phase. However, if a magnetic force is applied across the closed bottom of the incubation chamber or across the incubation chamber walls, the magnetic microspheres are attracted thereto and can remain attracted thereto as long as such magnetic force is applied. Thus, in effect, they are removed from said first interface and completely submerged in said non-aqueous water-immiscible liquid phase. According to an embodiment of the invention, while the magnetic microspheres remain attracted to the bottom or wall(s) of the incubation chamber, a solid surface is then placed at the open top of the incubation chamber or at a position along a defined distance between the open top and the closed bottom of the incubation chamber, such that the solid surface is in contact with the liquid phase and that at said open top or at said position along the defined distance between the open top and the closed bottom of the incubation chamber, a second interface is formed, which is an interface between a non-aqueous water-immiscible liquid phase and the solid surface. As a result of such placement, such second interface replaces or eliminates the first interface (between the non-aqueous water-immiscible liquid phase and the gas phase surrounding the incubation chamber), whereby the gas phase is no longer in contact with the liquid phase and therefore also 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 in step b) the incubation chamber is completely filled with the suspension up to its open top), then in step d) a solid surface is placed on said open top.However, if the first interface is located at a first position in the incubation chamber, which first position is different from the open top (because in step b) the incubation chamber is only partially filled with the suspension), then in step d) the solid surface is preferably placed at the first position, i.e. either at a position that coincides with the position of the first interface or at a position that is below the first position of the first interface. Thus, in some embodiments, such a position at which the solid surface is placed in step d) may be the first position. Alternatively, in step d), the solid surface may be placed at a position below the first position in the incubation chamber, i.e. below the position of the first interface, in order to absolutely ensure that the second interface formed replaces or eliminates the first interface. Regardless of the location where the solid surface is placed in step d), step d) should be performed such that as a result of such step d), said solid surface comes into contact with said liquid phase such that a second interface is formed between said non-aqueous water-immiscible liquid phase and said solid surface at said open top or at said location where the solid surface is placed in step d), such second interface displaces or eliminates said first interface, thereby preventing said gas phase from contacting said liquid phase;

[0050] According to embodiments of the invention, a solid surface placed at the open top of an incubation chamber or at a position along a defined distance between the open top and the closed bottom of the incubation chamber has its dimensions selected to completely cover said liquid phase and / or said open top of the incubation chamber so as to form a second interface that replaces or excludes the first interface (between the non-aqueous water-immiscible liquid phase and the gas phase surrounding the incubation chamber). More specifically, in some embodiments, a solid surface placed at the open top of an incubation chamber or at a position along a defined distance between the open top and the closed bottom of the incubation chamber has its dimensions selected to completely cover said liquid phase and / or said 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 said solid surface, which second interface replaces or excludes the first interface, thereby preventing the gas phase (surrounding the incubation chamber) from contacting the non-aqueous water-immiscible liquid phase. In a particularly preferred embodiment, the cross-section of the solid surface is selected such that it can be inserted into the incubation chamber and / or fit snugly into the wall(s) of the incubation chamber.

[0051] In some embodiments, when the incubation chambers are wells in a multi-well plate, the first interface is preferably located at the open top of each respective well (because in step b) each well is preferably completely filled with the suspension up to its open top). 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 may be provided as a single solid surface for all wells together. In these embodiments, the solid surface is - placing only the lid of said multi-well plate onto the open top(s) of each of said well(s); or - or first placing a separate solid surface that is not a lid, such as a flat plate, on the open top(s) of each of the well(s) and then placing a lid of the multiwell plate on top of the separate solid surface. in said open top(s) of said respective well(s).

[0052] Finally, according to an embodiment of the present invention, in step e), the magnetic microspheres are aggregated at the second interface by ceasing to exert the magnetic force across the bottom of the incubation chamber or across the walls of the incubation chamber, whereby the microspheres become free therefrom and they aggregate at the second interface, since their density is lower compared to the density of the non-aqueous water-immiscible liquid phase.

[0053] According to embodiments of the present invention, the term "magnet" is meant to refer to any material or object or body that produces a magnetic field capable of exerting a magnetic force on magnetic materials. The magnet according to embodiments of the present invention may be a permanent magnet, such as a ferromagnetic material, or an electromagnet that can be switched on and off, thereby facilitating steps c) and d) of exerting a magnetic force and step e) of stopping the exertion of the magnetic force.

[0054] It should be noted that the microspheres can be positioned accordingly depending on the shape, form, dimensions and other qualities of the solid surface that is placed in step d) at the open top of the incubation chamber or at a position along a defined distance between the open top and the closed bottom of the incubation chamber and forms the second interface. For protocols involving simple incubation of microspheres, e.g., nucleic acid amplification is performed in such microspheres without subsequent detection, it is sufficient to simply remove the microspheres from the first interface in step c) and thereby exclude them from the gas phase surrounding the incubation chamber (or the wells in a multi-well plate). In such embodiments, the solid surface placed in step d) can be of any shape and dimensions, as long as it removes the microspheres from the gas phase, i.e., the first interface, and prevents them from coming into contact with it by forming a second interface that replaces or excludes the first interface. For example, a solid curved surface of suitable dimensions is perfectly suited for such purposes. However, when the microspheres are used in protocols that involve not only nucleic acid amplification but also simultaneous or subsequent detection of signals generated within the microspheres and / or imaging of such signals, it is important that these microspheres are preferably arranged in a layer, preferably a monolayer, that allows optical probing, imaging and / or optical interrogation of said microspheres. Such a layer, preferably a monolayer, arrangement can be achieved by a solid surface that is a planar solid surface.

[0055] In one embodiment, the method according to the present invention is a method for arranging microspheres in layers of microspheres along the second interface. In a preferred embodiment, such layers are monolayers of microspheres along the second interface. When arranging microspheres in layers or monolayers according to the embodiment of the present invention, their packing can be appropriately selected and adjusted. In one embodiment, the packing of such layered or monolayered microspheres is selected from random packing, regular packing, and close packing, in particular hexagonal close packing. In a preferred embodiment, the packing of layered or monolayered microspheres is close packing, more preferably hexagonal close packing.

[0056] In particular, in embodiments where the microspheres are arranged in layers or monolayers of microspheres along the second interface, it is preferred that the solid surface placed in step d) at the open top of the incubation chamber or at the second location along the defined distance between the open top and closed bottom of the incubation chamber is a planar solid surface. In particular, in those embodiments involving a planar solid surface, the magnetic microspheres can be arranged in layers, preferably in a monolayer.

[0057] As used herein, the term "planar surface" or "planar solid surface" is meant to refer to a surface that is essentially flat and has a planar or approximating planar appearance. In some embodiments, such planar solid surfaces may be smooth. However, such terms "planar surface" or "planar solid surface" should be interpreted as still allowing for some degree of surface roughness in such planes. For clarity, a "planar surface" or "planar solid surface" may also be a surface that may include a certain structure designed to facilitate specific packing of the layer or monolayer of the microspheres formed along the planar surface (and second interface). Also, for example, such a "planar surface" or "planar solid surface" may have some degree of patterning, for example, to achieve or facilitate specific packing of the layer or monolayer of the microspheres.

[0058] Apart from the influence 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 layered arrangement can also be adjusted by selecting an appropriate concentration of the microspheres in the non-aqueous water-immiscible liquid phase in relation to the area / size of the solid planar surface, and / or by selecting the total number of magnetic microspheres filled in the incubation chamber in step b) in relation to the area / size of the solid planar surface. In one embodiment, the number of magnetic microspheres filled in the incubation chamber in step b) is selected to be below, preferably significantly below, the maximum number of microspheres that can be arranged in the closest possible packed layer along the second interface. As a result, such a layer is randomly or regularly packed with microspheres. In another embodiment, the number of magnetic microspheres filled into the incubation chamber in step b) is selected to be not more than the maximum number of microspheres that can be arranged in the closest possible layer along the second interface, but close to or approximately equal to such maximum number, or indeed to be the maximum number of microspheres that can be (theoretically) arranged in a layer along the second interface. As a result, the microspheres are close-packed, or even hexagonally close-packed, in such a layer. In one embodiment, the number of magnetic microspheres filled into the incubation chamber in step b) is selected to cover an area equal to or smaller than the area provided by said solid surface in contact with the liquid phase when the magnetic microspheres are arranged in the closest possible layer.

[0059] On the other hand, if the number of magnetic microspheres filled into the incubation chamber in step b) is selected to exceed the maximum number of microspheres that can be arranged in the closest possible packed layer along the second interface, there will not be a simple layer of microspheres arranged along the second interface, in particular a monolayer of microspheres, but rather the microspheres will be arranged in a three-dimensional irregular bulk volume, since such a (large or excessive) number of microspheres can only be arranged along the second interface by accommodating them in a monolayer of microspheres, or more precisely not in a monolayer, but rather in a three-dimensional bulk volume.

[0060] In another aspect, the present invention also relates to a multi-well plate configured for use in the methods according to embodiments of the present invention as defined herein. According to an embodiment of the present invention, such a multi-well plate comprises: - a flat 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 dimensioned to accommodate a defined volume of a suspension comprising a defined number of magnetic microspheres or magnetic microspheres suspended in a non-aqueous water-immiscible liquid phase therein; said wells being embedded in said planar surface of said flat body, said planar surface being configured to contact a solid surface, preferably a planar solid surface, such that each well is closed by such solid surface, preferably such a planar solid surface; - a sealing rim at the periphery of the flat body, the sealing rim surrounding and projecting from the flat body of the multiwell plate; - a lid configured to be mounted and placed on said multiwell plate, in particular on said flat body, and dimensioned to cover and seal said multiwell plate, in particular on said flat body, said lid having a lower surface which is a solid surface, preferably a planar solid surface, having a periphery and comprising a gasket on its periphery which interacts with said sealing rim of said flat body and seals said multiwell plate, in particular on said flat body, when said lid is placed on said multiwell plate, in particular on said flat body; - optionally a plate having a lower surface that is a planar solid surface, the plate being configured to be placed on said flat body and dimensioned to cover said flat body; Equipped with.

[0061] It should be noted that in the embodiment where a flat plate with a lower surface is present, it is typically placed on a flat body and has dimensions to cover said flat body, and said lid is configured to be placed on top of the multiwell plate, preferably on said flat body, by being placed on such a flat plate. In a preferred embodiment, the flat plate placed on the flat body of the multiwell plate has sufficient density / weight to allow it to be placed on the flat body of the multiwell plate under the influence of gravity, to come into contact with the lid of said multiwell plate, which is placed on the flat body, preferably on said flat body, and to remain there without being fixed by said lid. For example, such a flat plate can be made of a material that gives it sufficient density / weight to allow it to be placed on the flat body of the multiwell plate, to come into contact with the lid of said multiwell plate, which is placed on the flat body, and to remain there without being fixed by said lid. Examples of materials suitable for such a flat plate are glass or quartz. In such an embodiment, even though a lid is placed over the multiwell plate and in fact over the flat plate, it does not need to, and in fact does not, contact the flat plate in order for such a flat plate to rest on and remain on the flat body of the multiwell plate.

[0062] However, in other embodiments, the lid is configured to be placed on top of the multiwell plate, preferably on said flat body, by actually contacting the plate on it, such that the plate rests on and contacts the flat body of the multiwell plate, and also contacts (on its opposite side) the lid, which is placed on top of the plate. In such embodiments, the plate is in effect in contact with the lid on one side, and itself in contact with the flat body of the multiwell plate on the other side. In such embodiments, the lid also serves to fix the plate on the flat body of the multiwell plate by contacting the plate.

[0063] It should be noted that in those embodiments in which a plate is provided and present, said plate is provided separately from said lid and does not form an integral part thereof, in particular it is not any layer or component of said lid but is an entirely separate entity from said lid, both said lid and said plate being separate components from each other.

[0064] Multiwell plates according to embodiments of the present invention are particularly suitable for disposing microspheres in a non-aqueous, water-immiscible liquid phase within the wells of the multiwell plate and for preventing such microspheres from contacting or being exposed to a gas phase.

[0065] In a further aspect, the present invention relates to an apparatus for disposing microspheres in a non-aqueous, water-immiscible liquid phase in some or all of the wells of a multi-well plate according to the present invention, such that such microspheres are not in contact with or exposed to a gas phase.

[0066] According to an embodiment of the invention, such an apparatus comprises: - a receptacle for receiving an incubation chamber or a multiwell plate; said receptacle having a bottom surface and at least one positioning means for positioning said incubation chamber or said multiwell plate on said bottom surface, said positioning means protruding from such bottom surface and having dimensions to receive said incubation chamber or said multiwell plate in said receptacle with a snug fit; said incubation chamber having a closed bottom surface, an open top surface, at least one wall and a defined volume; and said multiwell plate as defined in the embodiments of the present specification; - a magnet or set of magnets located at and integrated into the bottom surface of the receptacle or located at and integrated into the positioning means, the magnet or magnets being configured to exert a magnetic force across the closed bottom of the incubation chamber, or across the bottom of each well of the multiwell plate, or across the at least one wall of the incubation chamber, or across the at least one wall of each well of the multiwell plate; - optionally locking means, preferably on the bottom surface within said receptacle, for locking, preferably reversibly, said incubation chamber or said multi-well plate in said receptacle; Equipped with.

[0067] In yet another aspect, the present invention provides an apparatus for incubating microspheres in a non-aqueous water-immiscible liquid phase in an incubation chamber or in a multi-well plate, said apparatus comprising: - a body having a top surface and a receptacle configured to receive a multi-well plate according to embodiments of the invention defined herein, said receptacle being located on said top surface, said receptacle having a bottom; - a temperature control device located and / or integrated into the bottom of the receptacle and configured to be in thermal and / or physical contact with a bottom of an incubation chamber or a bottom of each well of a multi-well plate when inserted into the receptacle; - a lid or flap attached to the body and covering a top surface of the body and configured to be placed over the receptacle and over the incubation chamber or the multiwell plate when placed in the receptacle of the device, the lid or flap being further configured to lock the incubation chamber or the multiwell plate in the receptacle when the lid or flap is placed over the receptacle, and further comprising an optically transparent area that becomes overlying the receptacle, thereby enabling optical probing of the incubation chamber or the multiwell plate present in the receptacle; The present invention relates to an apparatus comprising:

[0068] In one embodiment, the apparatus for incubating microspheres in a non-aqueous water-immiscible liquid phase further comprises within said receptacle an incubation chamber or multi-well plate according to the invention as defined herein.

[0069] In yet another aspect, the present invention relates to an apparatus for detecting and optically probing microspheres in a non-aqueous water-immiscible liquid phase in an incubation chamber or in a multiwell plate, said incubation chamber or said multiwell plate being located in an incubating device according to the invention as defined herein, said detecting device comprising: - a housing comprising a detection module, said detection module comprising an image detector, one or several filters, and at least one imaging lens; - a loading bay configured to receive and house an incubating device according to the invention as defined herein, said loading bay comprising an interface for electronically connecting to an incubating device according to the invention as defined herein, said loading bay being located within said housing such that said incubating device can be detected and optically probed when present within said loading bay using a detection module, preferably such that an incubation chamber or a multiwell plate located within said device for incubating microspheres can be detected and optically probed; detection and optical probing is performed through said optically transparent area of ​​said lid or flap of said incubating device; said optically transparent area of ​​said lid or flap of said incubating device is the optically transparent region(s) of the lid and the plate of the multiwell plate, if such an incubation chamber or multiwell plate is present in the incubating device; said image detector; one of said one or more filters, and said at least one imaging lens Aligned with a loading bay such that optical probing occurs through the transparent area of ​​the lid or flap of the incubating device and, if present in the detecting device, through the optically transparent area(s) of the lid and the flat plate of the multiwell plate; The present invention relates to an apparatus comprising:

[0070] In yet another aspect, the present invention also relates to the use of elemental carbon, in particular graphite, for incorporation into plastic materials in order to increase the thermal conductivity of such materials and thereby make such plastic materials particularly suitable for the manufacture of incubation chambers or multi-well plates for carrying out incubation reactions, such as nucleic acid amplification reactions, involving one or several temperature changes.

[0071] 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 multiwell plate, comprising the incorporation of said elemental carbon, such as graphite, into the plastic material from which said incubation chamber or said multiwell plate is made. In a particularly preferred embodiment of such use(s), the plastic material from which the incubation chamber and / or the multiwell plate is made is polycarbonate.

[0072] In a further aspect, the invention relates to the use of elemental carbon, in particular graphite, for the manufacture of an incubation chamber or a multiwell plate configured for carrying out an incubation reaction, such as a nucleic acid amplification reaction, involving one or several temperature changes of an incubated sample, said manufacture comprising the incorporation of said elemental carbon into the plastic material from which said incubation chamber or said multiwell plate is made. In one embodiment of such a use, the incubation chamber or multiwell plate is configured, due to the incorporation of said elemental carbon into the plastic material, to carry out an incubation reaction, involving one or several temperature changes of an incubated sample, in an accelerated manner, as compared to the same incubation reaction carried out in an incubation chamber or multiwell plate not incorporating elemental carbon, e.g. graphite, into the plastic material.

[0073] In one embodiment of such a use, the incubation chamber or multi-well plate configured for carrying out an incubation reaction, such as a nucleic acid amplification reaction, involving one or several temperature changes of the incubated sample is configured for carrying out such an incubation reaction in a manner that involves one or several temperature changes of the incubated sample, where a temperature difference of 20°C to 25°C is achieved over a time period of less than 5 seconds per individual temperature change, preferably where a temperature difference of 20°C to 25°C is achieved over a time period of less than 3.5 seconds per individual temperature change.

[0074] In a further aspect, the present invention also relates to an incubation chamber or multiwell plate for performing incubation reactions, such as nucleic acid amplification reactions, involving one or several temperature changes, which is made of a plastic with elemental carbon, in particular graphite, incorporated therein. In a preferred embodiment of this aspect, the plastic material from which the incubation chamber and / or multiwell plate is made is polycarbonate. Such an incubation chamber or multiwell plate according to this aspect of the invention is configured to accelerate incubation reactions involving one or several temperature changes of the incubated samples, compared to the same incubation reactions performed in an incubation chamber or multiwell plate not incorporating elemental carbon, e.g. graphite, in its plastic material.

[0075] An example of a suitable plastic material incorporating elemental carbon is "Makrolon TC621®", available from Covestro AG, which may be used in this aspect of the invention. EXAMPLES

[0076] Furthermore, reference is made to the examples to illustrate, but not to limit, the present invention.

[0077] Performing transcription and polymerase chain reaction within bead-microspheres in multiwell plates and subsequently detecting the bead-microsphere monolayer

[0078] Loading of Bead Microspheres Polymerase chain reaction (PCR) reagents were loaded onto hydrated bead microspheres incorporating magnetic particles (40 μL bead bed, 100 μm bead diameter, see Table) in an aqueous environment by incubating with agitation for 5 min at room temperature using an equal volume of a 2x concentrated master mix (see Table), both containing template and the corresponding primer-probe set.

[0079] [Table 1]

[0080] Suspension and Dispersion of Bead Microspheres The bead-microspheres were gently sedimented for 1 min at 300 rpm using a centrifuge with a swing-out rotor, the supernatant was discarded, and then 100 μL of separation oil (Novec 7500™ with 5% PicoSurf, SphereFluidics) was added. The tube with the prepared mixture was vigorously stirred for 15 s with a Minilys personal homogenizer (Bertin instruments, medium speed) to form a stable suspension of the aqueous bead-microspheres in the fluorophilic oil, a process that was facilitated by the inclusion of a surfactant. As the density of the applied oil is about 1,6 g / mL, the aqueous bead-microspheres float on the surface of the oil. To minimize the amount of microemulsion (emulsified water and air in oil) that occurs concomitantly with the suspension process, the entrapped microspheres were washed three times with separation oil.

[0081] Loading and Assembling the Multiwell Cartridge The multiwell plate was placed on a loading rack with magnets under each well position and filled with 740 μL of packing oil (Novec 7500™ with 0.5% PicoSurf). Then, 3 μL of loaded bead microspheres were pipetted into each well. The magnets of the loading rack held the superparamagnetic bead microspheres in place in their individual wells. A custom-made bead plate (23×29 mm, 500 μm thick cover glass) was placed upright on one side of the multiwell plate and applied to the multiwell plate by gently tilting the cover glass over the entire plate. The vitreous bead plate was submerged in the volume of packing oil. This separated the wells and covered them without any air bubbles in the wells. A lid with a gasket on the sealing rim was applied to the multiwell plate as well and closed with strong pressure to ensure snap locks on both sides. The lid did not come into direct contact with the bead plate while the packing oil was in the gap.

[0082] Installation and Thermocycling In this state, the cartridges became liquid-tight and the bead microspheres were captured in their respective wells. Therefore, the complete cartridge could be removed from the loading rack, processed, and advanced to both the thermal control and optical detection units. The multiwell plate was placed in a thermocycling chamber consisting of a Peltier element, electronic control, and cooling unit. When the multiwell plate was mounted in this chamber, a mechanical force was applied to the plate and lid, which pressed them together, resulting in a tight fit of the squeeze-tight gasket and an airtight seal. PCR cycling was performed after the steps of reverse transcription (if RTase is present: 10 min, 50° C.), preheating (2 min, 80° C.), and initial denaturation (2 min, 98° C.) as follows: MS2 40× cycles of 98° C. for 30 s and 57° C. for 30 s, RPP30 45× cycles of 98° C. for 5 s and 59° C. for 15 s. The heat applied during the initial denaturation PCR step partially melted the gasket, allowing the heat seal of the cartridge to last longer. After the final PCR step, the cartridge was actively cooled to 20°C and ready for image acquisition.

[0083] Imaging Images of each channel were acquired at each chamber position, resulting in a total of 2 × 25 images. One channel is required for the specific amplified signal (see table) and the second channel for segmentation of the bead-microspheres. Automated image acquisition was triggered by the BLINK toolbox software and performed by using an upright fluorescence microscope (Zeiss AxioZoom) equipped with a 20× objective (field of view 5.633 × 3.538 mm) and a SOLA SE V-nIR light engine (Lumencor). The microscope was further equipped with an automated XY stage fitted with three fluorescent filter sets (Cy5 ET F46-006, Cy3 ET F46-004, FITC HC F36-502, AHF Analysentechnik) and a thermocycler equipped with reaction chambers.

[0084] [Table 2]

[0085] Bead segmentation and end-point analysis All acquired images are subjected to an automated multifaceted image processing algorithm developed by BLINK to detect and segment individual nanoreactor beads. Then, features such as the fluorescence signal in each channel, the position, diameter / volume, etc. of all segmented nanoreactors are collected at the individual level to enable extensive statistics. Experimental data are processed with the open-source software Jupyter.

[0086] The features of the invention disclosed in this specification, in the claims and / or in the accompanying drawings may, both individually and in any combination thereof, be material for realizing the invention in diverse forms thereof.

Claims

1. A method for arranging microspheres in a non-aqueous, water-immiscible liquid phase within an incubation chamber, such that such microspheres are neither in contact with nor exposed to a gas phase, wherein the method is: a) A step of providing a suspension containing magnetic microspheres suspended in a non-aqueous, water-immiscible liquid phase and an incubation chamber in any order; The incubation chamber has a closed bottom, an open top, at least one wall, and a specified volume; the open top is spaced a specified distance from the closed bottom; and the chamber is sized to accommodate a specified volume of the suspension within it; 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; preferably, the suspension has microspheres of a specified concentration; b) A step of filling the incubation chamber with the suspension by placing the suspension inside the incubation chamber, thereby forming a first interface between the non-aqueous, water-immiscible liquid phase and the gas phase surrounding the incubation chamber at the open top or at a first position along the specified distance between the open top and the closed bottom within the incubation chamber; c) a step of removing the magnetic microspheres from the first interface or maintaining the state of being removed from the first interface by attracting the magnetic microspheres to the closed bottom of the incubation chamber or to the at least one wall, wherein the attraction is achieved by applying a magnetic force to the entire closed bottom of the incubation chamber or to the at least one wall; d) A step of installing a solid surface in the open top of the incubation chamber or at a position along the specified distance between the open top and the closed bottom of the incubation chamber, while maintaining the state in which the magnetic microspheres are attracted to the closed bottom or at least one wall of the incubation chamber by continuously applying a magnetic force to the closed bottom or at least one wall of the incubation chamber, wherein such installation is carried out so as to bring the solid surface into contact with the liquid phase, and such a second interface is formed between the non-aqueous, water-immiscible liquid phase and the solid surface in the open top or at the position, and such a second interface replaces or eliminates the first interface, thereby preventing the gas phase from coming into contact with the liquid phase; e) The step of stopping the magnetic force from being applied to the entire bottom of the incubation chamber or to the entire wall of at least one wall, thereby causing the magnetic microspheres to gather at the second interface and thereby freeing the microspheres from the bottom surface or to at least one wall, A method that includes this.

2. The method according to claim 1, wherein steps b) and c) are performed simultaneously or overlapping in time.

3. The method according to claim 1, wherein in step c), applying a magnetic force to the closed bottom or the at least one wall of the incubation chamber is done by placing a magnet near the bottom or the at least one wall of the incubation chamber, or vice versa, so that the magnet applies a magnetic force to the bottom or the at least one wall, 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 a magnetic force to the entire surface.

4. The method according to claim 1, wherein the incubation chamber becomes closed at the point in step d) where the open upper part is converted into a closed upper part by the solid surface.

5. In step d), the solid surface is - Install only a lid on the open top or position of the incubation chamber, Alternatively, a separate solid surface, such as a flat plate, may be placed first on the open top or position of the incubation chamber, and then the lid may be placed on top of the separate solid surface. The method according to claim 1, wherein the device is installed at the open upper part of the incubation chamber or at a position along the specified distance between the open upper part and the closed bottom of the incubation chamber.

6. The method according to claim 1, wherein in step d), the continued application of the magnetic force is performed by holding the magnet near the bottom of the incubation chamber or the at least one wall, or vice versa, so that the magnet continues to apply a magnetic force to the entire bottom or the at least one wall, preferably in step d), the bottom of the incubation chamber or the at least one wall is in physical contact with the magnet or in contact with it in such a way that the magnet can apply a magnetic force to the entire surface.

7. The method according to claim 1, wherein step e) is performed by removing the magnet from the bottom or the vicinity of the at least one wall, or vice versa, thereby stopping the magnetic force from being exerted over the entire bottom of the incubation chamber or over the at least one wall.

8. The method according to claim 1, wherein the aggregation of the microspheres at the second interface in step e) is facilitated by actively generating convection, preferably thermal convection or mechanical convection, within the incubation chamber.

9. The method according to claim 1, wherein step e) is facilitated by exposing the bottom of the incubation chamber to a temperature control device, preferably by thermally contacting the bottom of the incubation chamber with the temperature control device, and more preferably by physically contacting the bottom of the incubation chamber with the temperature control device.

10. The method according to claim 1, wherein the method is a method of arranging the microspheres in layers along the second interface, and in step d), the solid surface installed at the open upper part of the incubation chamber or at the position along the specified distance between the open upper part and the closed bottom of the incubation chamber is a planar solid surface.

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

12. The method according to claim 10, wherein the packing of microspheres in the layer is selected from random packing, regular packing, and close packing, particularly hexagonal close packing.

13. The method according to claim 10, wherein the packing of microspheres in the layer is selected from close packing, particularly hexagonal close packing.

14. The method according to claim 10, wherein the number of magnetic microspheres filling the incubation chamber in step b) is selected such that it does not exceed the maximum number of microspheres that can be arranged in layers packed as closely as possible along the second interface; and / or, if arranged in layers packed as closely as possible, the magnetic microspheres are selected to cover an area less than or equal to the area provided by the planar solid surface in contact with the liquid phase.

15. The method according to claim 1, wherein the microspheres are preferably monodisperse having a coefficient of variation (CV) < 15%, more preferably CV < 10%, and even more preferably CV < 5%.

16. The method according to claim 1, wherein the solid surface, preferably the planar solid surface, includes a transparent region, enabling optical probing and detection of any microspheres located beneath the liquid phase and the region.

17. The method according to claim 1, wherein the incubation chamber is a well in a multiwell plate, and the multiwell plate has a plurality of such incubation chambers arranged in the multiwell plate as defined in claim 1, preferably arranged regularly spaced apart on the multiwell plate.

18. The method according to claim 17, wherein the method is carried out using two or more wells in the multiwell plate.

19. A method for incubating microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber or a multi-well plate, wherein the method is: f) A step of carrying out the method according to any one of claims 1 and 16 to 18; g) An incubation reaction is carried out using the microspheres, and optionally, the incubation reaction involves changing the temperature of the microspheres once or several times. A method that includes this.

20. A method for detecting and optically probing microspheres in a non-aqueous, water-immiscible liquid phase within an incubation chamber or a multi-well plate, wherein the method is: h) The method according to claim 16, or, if dependent on claim 16, the step of carrying out the method according to any one of claims 17 to 18, Optionally, i) an incubation reaction is carried out using the microspheres, and optionally, the incubation reaction involves changing the temperature of the microspheres once or several times. k) A step of detecting the microspheres through the transparent region of the planar solid surface using a preferably suitable detection module and optically probing them, A method that includes this.

21. A multiwell plate configured for use in the method of claim 1, wherein the multiwell plate is: - A flat body having a plane and comprising a plurality of wells, each well having a closed bottom, an open top, at least one wall, and a specified volume; each well being sized to accommodate a specified number of magnetic microspheres, or a specified volume of suspension containing magnetic microspheres suspended in a non-aqueous, water-immiscible liquid phase therein; the wells being embedded in the plane of the flat body, and the plane being in contact with a planar solid surface, so that each well is closed by such a planar solid surface; - A sealing rim on the outer circumference of the flat body, the sealing rim surrounding the flat body of the multiwell plate and protruding therefrom; - A lid configured to be mounted and installed on the multiwell plate, and having dimensions such as to cover and seal the multiwell plate, having a bottom surface which is a flat solid surface; a lid having an outer circumference, and having a gasket on its outer circumference which interacts with the sealing rim of the flat body when the lid is installed on the multiwell plate, and seals the multiwell plate; - Optionally, a flat plate having a bottom surface which is a planar solid surface, configured to be placed on the flat body, and having dimensions such that it covers the flat body, A multiwell plate equipped with the following features.

22. The multiwell plate according to claim 21, wherein each well is closed by a planar solid surface, and such planar solid surface is provided by either the lower surface of the lid or, if present, the lower surface of the plate.

23. The multiwell plate according to claim 21, further comprising a flat plate having a bottom surface which is a planar solid surface, configured to be installed on the flat body, and having dimensions such that it covers the flat body.

24. The multiwell plate according to claim 21, wherein the lid and, if present, the plate include (singular) (or including (plural)) regions that cover a plurality of wells, the regions being optically transparent, and enabling optical probing and detection of any of the wells located beneath the region(s).

25. The multiwell plate according to claim 21, further comprising locking means configured to lock the lid in a position on the multiwell plate so that the multiwell plate is sealed, and optionally the flat plate, if present, also configured to lock on the flat body of the multiwell plate.

26. The multiwell plate according to claim 21, wherein the flat body is made of or contains a plastic, preferably a plastic having elemental carbon as an additive, more preferably a plastic having more than 50% by weight of elemental carbon as an additive, and even more preferably the plastic is a polycarbonate having more than 50% by weight of elemental carbon as an additive.

27. An apparatus for arranging microspheres in a non-aqueous, water-immiscible liquid phase within an incubation chamber, such that the microspheres are neither in contact with nor exposed to a gas phase, wherein the apparatus: - A receptacle for receiving an incubation chamber or a multiwell plate; the receptacle having a bottom surface and at least one positioning means for positioning the incubation chamber or the multiwell plate on the bottom surface, wherein the positioning means protrudes from such bottom surface, and the receptacle has dimensions such that the incubation chamber or the multiwell plate is slidably fitted therein; the incubation chamber having a closed bottom surface, an open top surface, at least one wall, and a specified volume; and the multiwell plate having a receptacle as defined in claim 21; - A magnet or set of magnets located on the bottom surface of the receptacle and incorporated therein, or located on the positioning means and incorporated therein, wherein the magnet (single) or magnet (multiple) is configured to exert a magnetic force over the entire closed bottom of the incubation chamber, or over the entire bottom of each well of the multiwell plate, or over the at least one wall of the incubation chamber and over the at least one wall of each well of the multiwell plate; - Optionally, preferably on the bottom surface within the receptacle, locking means for locking the incubation chamber or the multiwell plate within the receptacle, A device equipped with the following features.

28. An apparatus for incubating microspheres in a non-aqueous, water-immiscible liquid phase in an incubation chamber or a multi-well plate, wherein the apparatus includes: - A body having an upper surface and a receptacle configured to receive the multiwell plate described in claim 21, wherein the receptacle is located on the upper surface and the receptacle is located on the bottom of the body; - A temperature control device located at and / or incorporated into the bottom of the receptacle and configured to make thermal and / or physical contact with the bottom of the incubation chamber or the bottom of each well of the multiwell plate when inserted into the receptacle; - A lid or flap attached to the main body, covering the upper surface of the main body, and configured to be placed on the receptacle and, when the device is installed inside the receptacle, on the incubation chamber or the multiwell plate, further configured to lock the incubation chamber or the multiwell plate inside the receptacle when the lid or flap is installed on the receptacle, and further includes an optically transparent area that becomes located above the receptacle, thereby enabling optical probing of the incubation chamber or multiwell plate present inside the receptacle. A device equipped with the following features.

29. The apparatus according to claim 28, further comprising an incubation chamber or multiwell plate according to claim 21 within the receptacle.

30. An apparatus for detecting and optically probing microspheres in a non-aqueous, water-immiscible liquid phase within an incubation chamber or a multiwell plate, wherein the incubation chamber or the multiwell plate is located within the incubation apparatus described in claim 28, and the detection apparatus is: - A housing comprising a detection module, wherein the detection module comprises an image detector, one or more filters, and at least one imaging lens; A loading bay configured to receive and house the incubation apparatus according to claim 28, wherein the loading bay comprises an interface for electronically connecting to the incubation apparatus according to claim 28, and the loading bay is positioned within the housing so that an incubation chamber or multiwell plate located within the apparatus for incubating microspheres can be detected and optically probed, preferably using a detection module, when the incubation apparatus is located within the loading bay; detection and optical probing are performed through the optically transparent area of ​​the lid or flap of the incubation apparatus; the optically transparent area of ​​the lid or flap of the incubation apparatus is - If such an incubation chamber or multiwell plate is present in the incubation apparatus, the optically transparent area(s) of the lid and the flat plate of the multiwell plate, - The image detector, - One of the one or more filters mentioned above, and - The at least one imaging lens They lined up, As a result, optical probing is performed through the transparent area of ​​the lid or flap of the incubation device, and, if present in the detection device, through the optically transparent area(s) of the lid and the flat plate of the multiwell plate in the loading bay, A device equipped with the following features.

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