Microspheres Comprising a First Material and a Second Material

Abstract

The present invention relates to a microsphere comprising a first material, preferably a first polymer, and a second material, preferably a second polymer. The present invention also relates to a liquid phase comprising a plurality of such microspheres. Furthermore, the present invention relates to a method for capturing nucleic acid from a sample and a method for enhancing nucleic acid from a sample. The present invention also relates to the use of a plurality of microspheres in an assay for the detection of multiple nucleic acid analytes. Furthermore, the present invention relates to the use of a plurality of microspheres in an assay for the detection of a single nucleic acid analyte in multiple samples.

Description

[Technical Field]

[0001] The present invention relates to a microsphere comprising a first material, preferably a first polymer, and a second material, preferably a second polymer. The present invention also relates to a liquid phase comprising a plurality of such microspheres. The present invention further relates to methods for capturing nucleic acids from a sample and methods for enhancing nucleic acids from a sample. The present invention also relates to the use of a plurality of microspheres in an assay for the detection of multiple nucleic acid analytes. The present invention further relates to the use of a plurality of microspheres in an assay for the detection of a single nucleic acid analyte in multiple samples.

[0002] Chitosan has been used as a polymer to capture DNA in aqueous systems (Cao et al. 2006, Anal. Chem., vol. 78, pp. 7222-7228). A completely aqueous DNA purification protocol was established using a microchip with channels coated with chitosan polymer. A similar protocol has also been successfully used for RNA (Hagan et al. 2009, Anal. Chem., vol. 81, pp. 5249-5256; and Zhu et al., 2020, Micromaschines, vol. 11, p. 186, doi: 10.3390 / mi11020186). Chitosan-coated nylon membranes have been shown to concentrate DNA from highly dilute solutions and are a suitable matrix for subsequent in situ PCR on the membrane (Schlappi et al., 2016, Anal. Chem., vol. 88, pp. 7647-7653). Microspheres containing agarose and non-crosslinked gelatin are described in International Patent Applications WO 2021 / 122563 and WO 2021 / 122579.

[0003] There is a need in the art for improved nanoreactor beads that allow for efficient capture and / or amplification of nucleic acids. There is also a need in the art for improved beads that are easier to handle and particularly suitable for use in capture and / or amplification protocols. Furthermore, there is a need in the art to provide improved beads that are versatile for use in multiplex assays, for example, involving multiple analytes and / or multiple samples. There is also a need in the art to provide improved beads that are easier to individually locate and / or identify and / or determine whether they are fused with other beads, particularly during digital amplification and / or detection processes. There is also a need in the art to provide improved beads that allow for identification and / or detection of multiple different beads via different detection channels.

[0004] In a first aspect, the present invention relates to a microsphere comprising a first material and a second material, wherein the first material is capable of forming a porous hydrogel when exposed to or containing an aqueous solution and has a melting temperature ranging from 40°C to 90°C; and wherein the second material forms a network within the porous hydrogel or the second material is attached to the first material, and both materials together form a network within the porous hydrogel; the second material has a pKa value and is capable of coagulating in an aqueous environment where the pH is above the pKa value; the network is capable of shrinking when exposed to a temperature above the melting temperature of the first material; and the microsphere further comprises at least one of a) a fluorescent label and b) magnetic particles.

[0005] In one embodiment, the first material is a first polymer and the second material is a second polymer, oligomer, or monomer; - when said second material is a second polymer, said second polymer is a polymer crosslinked with itself or with said first polymer; When said second material is an oligomer or a monomer, said oligomer or said monomer is attached to said first polymer.

[0006] In one embodiment, said second material, preferably said second polymer or said oligomer or said monomer, is further capable of binding nucleic acids at a pH below said pKa value, preferably said pKa value of said second material, preferably said second polymer or said oligomer or said monomer is less than 6.

[0007] In one embodiment, the fluorescent label is selected from: a) fluorescent particles with a size ranging from 50 nm to 10 μm, preferably from 100 nm to 5 μm, more preferably from 1 μm to 5 μm, even more preferably from 1 μm to 3 μm; b) non-particulate fluorescent dyes attached to the first and / or second material, preferably the first and / or second polymer / oligomer / monomer; and c) a combination of a) and b).

[0008] In one embodiment, the size of the magnetic particles ranges from 50 nm to 10 μm, preferably from 100 nm to 5 μm, more preferably from 1 μm to 5 μm, and even more preferably from 1 μm to 3 μm.

[0009] In one embodiment, said first material, preferably said first polymer, comprises a binding member that allows reversible and pH-independent immobilization of one or several amplification primers to said first material, preferably said first polymer, and preferably for such reversible and pH-independent immobilization said binding member specifically interacts with a conjugate attached to said one or several amplification primers; more preferably said microspheres further bind to said first material, preferably said first polymer, via said specific interaction between said binding member on said first material, preferably said first polymer, and said conjugate on said one or several amplification primers. or one or several amplification primers having a conjugate attached and immobilized to the first polymer; even more preferably, the binding member is selected from avidin, streptavidin and derivatives thereof, and the conjugate is selected from desthiobiotin, iminobiotin, biotin with a cleavable spacer arm, selenobiotin, oxybiotin, homobiotin, norbiotin, diaminobiotin, biotin sulfoxide, biotin sulfone, epibiotin, 5-hydroxybiotin, 2-thiobiotin, azabiotin, carbobiotin, methylated derivatives of biotin, and ketone biotin; or vice versa. It should be noted that the binding member and the conjugate are chosen to interact, i.e., to bind to each other in a reversible manner.

[0010] In one embodiment, the first material is a first polymer and is selected from agarose, gelatin, hyaluronan, elastin, elastin-like polypeptides, thermoresponsive polymers having an upper critical solution temperature (UCST) (e.g., selected from poly(N-acryloylglycinamide) (PNAGA), poly(allylamine)-co-poly(allylurea) and derivatives thereof), poly(methacrylamide), poly(N-acryloylaspartamide), poly(N-methacryloylglutamine amide), poly(acrylamide)-co-(acrylonitrile), poly(sulfobetaine), poly(phosphorylcholine); and other polymers capable of forming porous hydrogels when exposed to or containing aqueous solutions and having a melting temperature ranging from 40° C. to 90° C. and the second material is either a second polymer selected from chitosan and its derivatives, gelatin and its derivatives, methylcellulose, poly(N-isopropylacrylamide) (pNIPAM), poly(ethyleneimine), poly(2-dimethyl(aminoethyl)methacrylate), poly(lysine), poly(histidine), poly(arginine) and a polymer backbone having a basic amino acid attached or incorporated as part of such backbone; or the second material is an oligomer (preferably selected from oligopeptides, which oligopeptides comprise or consist of basic amino acids such as histidine, lysine and arginine); or the second material is a monomer (selected from basic amino acids such as histidine, lysine and arginine), provided that when said first and second materials are first and second polymers, respectively, said polymers are different; Preferably, the first material is a first polymer that is non-crosslinked, and the second material is a second polymer that is crosslinked; or equally preferably, the first material is a first polymer and the second material is a second polymer crosslinked with the first polymer; more preferably, the first polymer is agarose and the second polymer is crosslinked chitosan.

[0011] In one embodiment, the microspheres additionally contain an aqueous solution such that the first material, preferably the first polymer, is in the form of a porous hydrogel.

[0012] In one embodiment, the microsphere further comprises a nucleic acid that is not an oligonucleotide primer, - bound to the second material, preferably the second polymer, when the aqueous solution has a pH below the pKa value of the second material, preferably the second polymer; and - when the aqueous solution has a pH equal to or greater than the pKa value of the second material, preferably the second polymer, the first material, preferably the first polymer, is still retained within microspheres beside or within the porous hydrogel, but is not necessarily bound to or is no longer bound to the second material, preferably the second polymer.

[0013] Examples of such nucleic acids that are not oligonucleotide primers and that may be bound to a second material or that may be retained by the microspheres without being bound to a second material include nucleic acids from a sample (such nucleic acids may be microbial or viral nucleic acids), or more generally, target nucleic acids whose presence is intended to be tested or detected.

[0014] In one embodiment, the aqueous solution is one of the following: a) a first composition for washing and / or facilitating binding of nucleic acids to the second material (e.g., the second polymer), wherein the first composition has a pH value below the pKa value of the second material (e.g., the second polymer), or more preferably, the first composition comprises a buffer that buffers to a pH range below the pKa value of the second material (e.g., the second polymer); or b) A second composition for promoting and performing nucleic acid amplification, said second composition comprising a buffer for buffering to a pH range suitable for performing nucleic acid amplification, mono-nucleoside-triphosphates, an amplification enzyme such as a suitable nucleic acid polymerase (e.g., Taq polymerase), optionally one or more amplification primers or one or more sets of amplification primers if not already immobilized on said first material (preferably said first polymer); and optionally a nucleic acid dye for detection of an amplification product such as an amplified nucleic acid; and / or optionally a molecular probe (e.g., TaqMan probe or molecular beacon), respectively; said pH range suitable for performing nucleic acid amplification being higher than the pKa value of said second material (e.g., said second polymer).

[0015] In one embodiment, the second material, preferably the second polymer, is in a non-aggregated form, which may be particularly true if the respective microspheres are not exposed to pH values ​​equal to or greater than the pKa value of the second material.

[0016] In one embodiment, the network within the porous hydrogel is not contracted, which may be particularly true if each microsphere has not been subjected to heat treatment by heating above the melting temperature of the first material, preferably the first polymer.

[0017] In a further embodiment, the second material, preferably the second polymer, is in a non-aggregated form and the network within the porous hydrogel of the microspheres is not contracted, which may be particularly true if each microsphere has not been exposed to a pH value equal to or greater than the pKa value of the second material and if each microsphere has not been subjected to a heat treatment by heating to a temperature above the melting temperature of the first material, preferably the first polymer.

[0018] In another embodiment, the second material, preferably the second polymer, is in an aggregated form, which may be particularly true when each microsphere is exposed to a pH value equal to or greater than the pKa value of the second material.

[0019] In one embodiment, the network within the porous hydrogel is contracted, which may be particularly true if each microsphere is subjected to a heat treatment by heating to a temperature above the melting temperature of the first material, preferably the first polymer.

[0020] In a further embodiment, the second material, preferably the second polymer, is in a coagulated form and the network of microspheres within the porous hydrogel is contracted, which may be particularly the case when each microsphere is exposed to a pH value equal to or greater than the pKa value of the second material and when each microsphere is subjected to a heat treatment by heating to a temperature above the melting temperature of the first material, preferably the first polymer.

[0021] In a further aspect, the present invention relates to a liquid phase, e.g., an oil phase, immiscible with water and aqueous solutions, said liquid phase comprising a plurality of microspheres of a second material, preferably a second polymer, in an unaggregated form and / or an uncollapsed network within said hydrogel.

[0022] In a further aspect, the present invention relates to a liquid phase, e.g., an oil phase, immiscible with water and aqueous solutions, said liquid phase comprising a plurality of microspheres in which a second material, preferably a second polymer, is in a coagulated form and / or a collapsed network within said hydrogel.

[0023] In a further aspect, the present invention relates to a method for capturing and / or concentrating nucleic acids from a nucleic acid or sample, said method comprising any of the following: a) providing a plurality of microspheres as defined in any one of claims 1 to 8 (i.e., a plurality of microspheres that do not (yet) contain one or several nucleic acids to be captured, enriched, amplified and / or detected) in a first composition for promoting binding of nucleic acids to the second material, preferably the second polymer, or exposing the microspheres to such a first composition; the first composition has a pH value below the pKa value of the second material (e.g., the second polymer), or preferably, the first composition comprises a buffer that buffers to a pH range below the pKa value of the second material (e.g., the second polymer); thereby allowing the microspheres to equilibrate with and incorporate the first composition; preferably, the pKa value of the second material (e.g., the second polymer) is greater than 6; and b) exposing the microspheres from step a) to a sample containing or suspected of containing nucleic acid, whereby the nucleic acid, if present, can bind to the second material (e.g., the second polymer), the sample preferably also containing a buffer that buffers the pH range to a range below the pKa value of the second material (e.g., the second polymer), thereby capturing nucleic acid in the sample, if present; or a') exposing a plurality of microspheres as defined in any one of claims 1 to 8 to a sample containing or suspected to contain nucleic acid, thereby allowing said nucleic acid to bind to said second material (e.g. said second polymer), if present, said sample having a pH below the pKa value of said second material (e.g. said second polymer), preferably said sample comprising a buffer that buffers the pH range to below the pKa value of said second material (e.g. said second polymer); thereby capturing nucleic acid in said sample, if present, as a result of such exposure.

[0024] In a further aspect, the present invention relates to a method for enriching nucleic acids from a sample, said method comprising carrying out a method for capturing and / or concentrating nucleic acids as defined herein in accordance with the present invention; and subsequently carrying out any of the following steps: (Option A): c) exposing the microspheres from step b) or step a') to a second composition for promoting and performing nucleic acid amplification, the second composition comprising a second buffer buffering the microspheres to a pH range suitable for performing nucleic acid amplification, mono-nucleoside triphosphates, an amplification enzyme such as a suitable nucleic acid polymerase (e.g., Taq polymerase), and, if the microspheres do not already comprise one or more amplification primers or one or more sets of amplification primers immobilized on the first material, preferably the first polymer, via specific interactions between binding members on the first material, preferably the first polymer, and conjugates on the one or more amplification primers; thereby allowing the microspheres to equilibrate with and incorporate the second composition for promoting and performing nucleic acid amplification; further, the second composition optionally comprises a nucleic acid dye and / or a respective molecular probe (e.g., TaqMan probe or molecular beacon) for detection of amplification products such as amplified nucleic acids; preferably, the pH range suitable for performing nucleic acid amplification is higher than the pKa value of the second material (e.g., the second polymer); d) subjecting the microspheres of step c) to an amplification protocol within the composition to promote and effect nucleic acid amplification, such protocol comprising, or preceded by, or followed by at least one step of heating the suspension to a temperature above the melting temperature of the first material, preferably the first polymer, and preferably comprising repeatedly and cyclically raising and lowering the temperature to which the suspension of microspheres is exposed to a level that allows amplification of nucleic acids (if present in the microspheres) that correspond to the sequences of the one or more amplification primers or one or more sets of amplification primers; Or (option B): e) exposing the microspheres from step b) or step a') to a second composition for promoting and performing nucleic acid amplification, the second composition comprising a second buffer buffering the microspheres to a pH range suitable for performing nucleic acid amplification, mono-nucleoside triphosphates, an amplification enzyme such as a suitable nucleic acid polymerase (e.g., Taq polymerase), and, if the microspheres do not already comprise one or more amplification primers or one or more sets of amplification primers immobilized on the first material, preferably the first polymer, via specific interactions between binding members on the first material, preferably the first polymer, and conjugates on the one or more amplification primers; thereby allowing the microspheres to equilibrate with and take up the second composition for promoting and performing nucleic acid amplification; further, the second composition optionally comprises a nucleic acid dye and / or a respective molecular probe (e.g., TaqMan probe or molecular beacon) for detection of amplification products such as amplified nucleic acids; preferably, the pH range suitable for performing nucleic acid amplification is higher than the pKa value of the second material (e.g., the second polymer); f) transferring the microspheres from step e) to a liquid phase immiscible with water and aqueous solutions, e.g., an oil phase, thereby producing a suspension of the microspheres separated from each other by the water-immiscible liquid phase, e.g., an oil phase; and g) subjecting the microspheres of step f) to an amplification protocol, which protocol comprises or is preceded or followed by at least one step of heating the suspension to a temperature above the melting temperature of the first material, preferably the first polymer, and which preferably comprises repeatedly and cyclically raising and lowering the temperature to which the suspension of microspheres is exposed to a level that allows amplification of nucleic acids (if present in the microspheres) that correspond to the sequences of the one or more amplification primers or one or more sets of amplification primers, and As a result of heating to a temperature above the melting temperature of the second material in the microsphere, preferably the second polymer, shrinks, thereby creating a shrunken core within each microsphere, and fluorescent labels and / or magnetic particles, if present or contained within each microsphere, accumulate within the shrunken core, thereby facilitating detection and / or further manipulation of each microsphere; preferably, i) the fluorescent labels and / or ii) the magnetic particles accumulated within the shrunken core of the microsphere are used to locate and / or identify an individual microsphere or multiple microspheres.

[0025] In the above scheme of steps, Option A represents an embodiment of a method for amplifying nucleic acids from a sample, in which the microspheres are not necessarily separated from one another and serve primarily the purpose of capturing and concentrating nucleic acids, with amplification being carried out in a bulk volume surrounding the microsphere(s).

[0026] Option B represents an embodiment of a method for amplifying nucleic acids from a sample in which the microspheres are necessarily separated from one another, each of which serves as a separate reaction space / volume in which an individual amplification reaction can occur. This embodiment is particularly useful for performing nucleic acid amplification in a digital or homogenous format.

[0027] In one embodiment of the method for amplifying nucleic acids from a sample, the method further comprises the steps of: h) separating the amplified nucleic acid (if present) said composition for promoting and performing nucleic acid amplification (Option A); or detecting in said microspheres (option B); The amplification protocol results in an optically detectable signal, and an optically detectable signal is generated. said composition for promoting and performing nucleic acid amplification (Option A); or in the space surrounding the contracted core of each microsphere (Option B); and detection of said optically detectable signal indicates the presence of amplified nucleic acid.

[0028] In one embodiment, an individual microsphere or multiple microspheres are located and / or identified using i) the fluorescent label and / or ii) the magnetic particles accumulated within the contracted core of the microsphere.

[0029] In one embodiment, fused microspheres are distinguished from non-fused microspheres using i) the fluorescent label and / or ii) the magnetic particles accumulated within the contracted core of the microsphere; and / or iii) the presence of more than one contracted core in fused microspheres; and / or iv) the presence of only one contracted core in non-fused microspheres.

[0030] In a further aspect, the present invention relates to the use of a plurality of microspheres as defined herein in an assay for the detection of multiple nucleic acid analytes, said use comprising: carrying out a method for amplifying nucleic acids from a sample according to the present invention as defined herein; and said use comprising different microspheres, each microsphere comprising a different fluorescent label and being specific for the detection of a particular nucleic acid analyte by comprising a specific amplification primer or a specific set of such amplification primers for the amplification of said particular nucleic acid analyte, and said different microspheres being detectably distinguishable from one another by means of said fluorescent labels, and each microsphere being specific for a different nucleic acid analyte.

[0031] In a further aspect, the present invention relates to the use of a plurality of microspheres as defined in the present invention in an assay for the detection of a single nucleic acid analyte in multiple samples, said use comprising: carrying out a method for amplifying nucleic acid from a sample according to the present invention as defined herein, and said use comprising different microspheres, each microsphere comprising a different fluorescent label and further comprising an amplification primer or a set of such amplification primers for amplification of said single nucleic acid analyte, each microsphere being specific for a particular sample by being separately exposed to such particular sample in step b) or a'), said different microspheres being detectably distinguishable from one another by means of said fluorescent labels, and each microsphere being specific for the same nucleic acid analyte and for a different sample.

[0032] The present inventors have devised a new type of microsphere that is particularly suited to nucleic acid capture and / or amplification protocols and can be easily and individually identified and handled. The microspheres described in embodiments of the present invention allow efficient binding / capture of nucleic acids from a sample by each microsphere and subsequent concentration / concentration within the microsphere. The microspheres described in embodiments of the present invention exhibit thermoresponsive behavior, which is particularly beneficial, for example, in protocols requiring identification and / or location and / or detection of individual microspheres. Without wishing to be bound by any theory, the present inventors believe that concentration / concentration provides improved access / accessibility of any target nucleic acids to amplification / detection reagents provided within the beads. Similarly, because the microspheres described in the present invention also contain at least one a) fluorescent label and b) magnetic particles, any such fluorescent label and / or magnetic particles are concentrated within a small volume, i.e., within the interior of the microsphere (sometimes referred to herein as the "core"), which contains a second crosslinked material, which may be a (second) polymer in aggregated form. Thus, the remainder of the volume provided by the microsphere is fully available for such detection / amplification reactions, while the fluorescent labels and / or magnetic particles are concentrated within a small volume and do not interfere with such detection / amplification reactions. Furthermore, because any fluorescent labels and / or magnetic particles present within the microspheres are removed from the region outside the reaction volume defined by the microspheres, it is possible to read the target-specific signal in the same fluorescent channel used for the specific labeling of the microspheres (i.e., the code used to identify and designate individual microspheres). Because any target-specific signal (i.e., the signal from the amplified nucleic acid target) is localized in this region outside the reaction volume defined by the microspheres, this means that the fluorescent label signal (=code) for the microspheres is physically and spatially separated from any target-specific signal, thereby enabling differentiation between the two signals.

[0033] The microspheres according to embodiments of the present invention are "hybrid" microspheres in the sense that they comprise a first material, preferably a first polymer, and a second material, preferably a second polymer, wherein the first material, preferably the first polymer, is non-crosslinked and the second material, preferably the second polymer, is crosslinked. As used in this context, when a second material, e.g., a second polymer, is referred to as being crosslinked, this means that such second material, e.g., the second polymer, is itself crosslinked or crosslinked to the first material, e.g., the first polymer. Various statements are made below with respect to exemplary embodiments including a first polymer as the "first material" and a second polymer as the "second material." However, such statements equally apply to embodiments in which the second material is an oligomer or monomer attached to the first material, e.g., the first polymer.

[0034] As used herein, the term "attached" refers to the association of one entity with another entity, typically involving at least one covalent bond between such entities.

[0035] In a preferred embodiment, the term "crosslinked" refers to the property of a second material, e.g., a second polymer, being "crosslinked" in the sense that there are one or several covalent bonds that bind or link the second polymer to itself or to the first material, e.g., the first polymer. In other words, such "chemical crosslinks" refer to the intermolecular bonding of two or more molecules of said second polymer (type) by one or several covalent bonds, or the intermolecular bonding of a single molecule of said second polymer (type) to itself by one or several covalent bonds, i.e., different stretches of such molecular chains of said second polymer (type) are bonded to each other by one or several covalent bonds, or are bonded to the intermolecular bonding of one or more molecules of the second polymer (type) to one or more molecules of said first polymer (type) by one or several covalent bonds. In embodiments where the second material is an oligomer or monomer, such oligomer or monomer is attached to the first polymer.

[0036] When microspheres according to embodiments of the present invention are used in protocols in which they are contained in a water-immiscible liquid phase, such as an oil phase, and such protocols include, preceded by, or followed by at least one step of heating the microspheres to a temperature above the melting temperature of the first polymer, the first polymer undergoes a phase transition from a gel to a soluble state, and the reaction compartments provided by each microsphere can be considered droplets. Because droplets occasionally fuse, this represents a potential problem in handling, manipulation, and / or processing, and because different microspheres, i.e., droplets, may be specific for different target analytes, different capture / amplification reactions may become intermingled as a result of such fusion. However, when microspheres according to embodiments of the present invention contain a crosslinked second polymer (or oligomers or monomers attached to the first polymer), exposing the microsphere to a temperature above the melting temperature of the first polymer results in the formation of a contracted network of the second polymer, which becomes a compact structure that can be easily recognized within the volume of the microsphere (similar to the yolk in an egg). Such compact structures formed by the contracted networks are sometimes referred to herein as the "cores" of the microspheres. Furthermore, the contracted networks of the second polymer ("cores") also concentrate any fluorescent labels and / or magnetic particles present within the microspheres, thus providing a robust means for identifying and / or locating and / or detecting individual microspheres. They also provide an excellent means for controlling microsphere fusion and identifying any fused microspheres, since such fused microspheres contain as many contracted networks as the microspheres form part of such fusion. These contracted networks (="cores") remain individually distinguishable within what is considered a single microsphere volume, and thus such fused microspheres can be easily distinguished from non-fused microspheres.

[0037] The term "microsphere," as used herein, refers to a spheroid, or a substantially spherical or ellipsoidal or other rounded, e.g., ovoid, body comprising a first material, preferably a first polymer, and a second material, preferably a second polymer, as defined herein, and having an average diameter ranging from 20 μm to 500 μm, preferably from 20 μm to 200 μm, more preferably from 20 μm to 100 μm.

[0038] According to an embodiment of the present invention, the inventors provide a microsphere (and a plurality of such microspheres) comprising a first material, preferably a first polymer, and a second material, preferably a second polymer, wherein the first polymer is a non-crosslinked polymer capable of forming a porous hydrogel when exposed to or containing an aqueous solution, the melting temperature of the first polymer being in the range of 40°C to 90°C; the second polymer is a crosslinked polymer having a pKa value and capable of coagulating in an aqueous environment where the pH is equal to or greater than the pKa value; the second polymer forms a network that can shrink when exposed to a temperature equal to or greater than the melting temperature of the first polymer; and the microsphere further comprises at least one of a) a fluorescent label and b) magnetic particles. In a preferred embodiment, the microsphere comprises both a) a fluorescent label and b) magnetic particles.

[0039] In a preferred embodiment, the term "crosslinked" refers to the property of a second polymer being "crosslinked" in the sense that there are one or several covalent bonds that bind or link the second polymer to itself or to a first material, e.g., the first polymer. In other words, such "chemical crosslinking" refers to the intermolecular bonding of two or more molecules of said second polymer (type) by one or several covalent bonds, or the intermolecular bonding of a single molecule of said second polymer (type) to itself by one or several covalent bonds, i.e., different stretches of such molecular chains of said second polymer (type) are bonded to each other by one or several covalent bonds, or the intermolecular bonding of one or more molecules of the second polymer (type) to one or more molecules of said first polymer (type) by one or several covalent bonds. When a first polymer is referred to herein as preferably "non-crosslinked," this means that such first polymer is not, as such, crosslinked with itself. Such a non-crosslinked first polymer may nevertheless be part of a network in which a second polymer is crosslinked in the above sense.

[0040] In embodiments described herein, the second material, preferably the second polymer, has a pKa value and can coagulate in an aqueous environment at a pH value equal to or greater than its pKa value. More preferably, the second polymer is a crosslinked polymer, forming a network that can shrink when exposed to a temperature equal to or greater than the melting temperature of the first polymer. Exposing the microspheres described herein to elevated temperatures (such elevated temperatures equal to or greater than the melting temperature of the first material, e.g., the first polymer) causes the network formed by the second material, e.g., the second polymer, or the second and first materials together, to shrink and form a core, i.e., a limited volume within the total volume of the microsphere, which is smaller than the total volume of the microsphere. Without wishing to be bound by any theory, the inventors believe that in the formed network, stretches and regions of the second material, e.g., the second polymer, become entangled with stretches and regions of the first material, e.g., the first polymer, and upon network shrinkage, these entangled regions and stretches of the first material, e.g., the first polymer, within the contracted network are also drawn in and concentrated. Such contracted networks thus form a densely concentrated core within the microsphere.

[0041] According to an embodiment of the present invention, when the first material is a first polymer, the first polymer is preferably a non-crosslinked polymer, meaning that it is not crosslinked with itself, i.e., it is not crosslinked in the sense that it does not have one or several covalent bonds that intermolecularly or intramolecularly bind or link the first polymer to itself. However, in such an embodiment, when the second material is also a polymer, i.e., a second polymer crosslinked with the first polymer, this can mean that the first polymer is also effectively "crosslinked" by being linked to the second polymer. However, in any case, when used in the context of a first polymer, the term "non-crosslinked" is meant to describe a scenario in which such a first polymer is neither crosslinked with itself nor crosslinked with itself.

[0042] A preferred first polymer is agarose. A preferred second polymer is chitosan. As used herein, the term "agarose" is intended to include derivatives of agarose, so long as such derivatives are non-crosslinked (as defined above), can form porous hydrogels when exposed to or contain aqueous solutions, and have a melting temperature ranging from 40°C to 90°C. As used herein, the term "chitosan" refers to a linear polysaccharide composed of randomly distributed β-(1→4)-linked D-glucosamine (deacetylated units) and N-acetyl-D-glucosamine (acetylated units). When such "chitosan" is the second polymer described in embodiments of the present invention, it is capable of coagulating in an aqueous environment at a pH above its pKa value and is preferably crosslinked as defined above. The term "chitosan" is also meant to include its acid adducts and / or acid salts, such as chitosan HCl, chitosan glutamate, chitosan lactate, chitosan acetate, and its carboxymethylated forms, such as N-carboxymethylchitosan.

[0043] Furthermore, the microspheres according to embodiments of the present invention further comprise at least one of a) a fluorescent label and b) magnetic particles, preferably both a) a fluorescent label and b) magnetic particles, so that upon contraction of the network formed by the second polymer or the second polymer together with the first polymer, the fluorescent label and / or magnetic particles are also concentrated within the contracted network (core). Without wishing to be bound by any theory, the inventors believe that the contracted network has an average pore size small enough to "attract" the fluorescent label and / or magnetic particles because of their larger size. Without wishing to be bound by any theory, such contraction is therefore believed to have multiple effects: first, it provides available volume within which amplification and / or reaction and / or detection can occur within the remainder of the microsphere; and second, it provides concentration of any fluorescent label and / or magnetic particles within the contracted network, thereby facilitating detection (via the fluorescent label) or mechanical handling (via the magnetic particles) of the microsphere.

[0044] As used herein, the term "fluorescent label" refers to fluorescent particles ranging in size from 50 nm to 10 μm, preferably from 100 nm to 5 μm, more preferably from 1 μm to 5 μm, and even more preferably from 1 μm to 3 μm; and non-particulate fluorescent dyes attached to the first and / or second material, preferably to the first and / or second polymer, more preferably to the second polymer. In either case, i.e., whether the fluorescent label is particulate or non-particulate, contraction of the network induced by exposing the microsphere to a temperature above the melting temperature of the first material, preferably the first polymer, will lead to concentration of the fluorescent label and / or magnetic particles within the core, i.e., the contracted network formed by the second polymer alone or in combination with the first polymer. Thus, in preferred embodiments comprising fluorescent particles, such fluorescent particles have a size greater than the average mesh or pore size of the contracted network.

[0045] As used herein, the term "magnetic particles" refers to particles that exhibit magnetic behavior, thus allowing the 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 another embodiment, such magnetic particles are superparamagnetic particles. In a preferred embodiment, such magnetic particles are ferromagnetic or paramagnetic particles. As used herein, the term "magnetic particles" is meant to exclude diamagnetic particles. As used herein, "magnetic particles" have a size and one-dimensional average diameter or average long extension ranging from 50 nm to 10 μm, preferably from 100 nm to 5 μm, more preferably from 1 μm to 5 μm, and even more preferably from 1 μm to 3 μm. Preferably, the size and one-dimensional average diameter or long extension of the magnetic particles are selected so that they are larger than the average mesh or pore size of the contracted network, and so that contraction of the network induced by exposing the microspheres to a temperature above the melting temperature of the first material, preferably the first polymer, leads to a concentration of the magnetic particles within the core, i.e., the contracted network formed by the second polymer alone or in combination with the first polymer.

[0046] It follows from the above that the average pore size of the contracted network is less than 50 nm, preferably less than 25 nm, more preferably less than 15 nm, and even more preferably less than 10 nm.

[0047] As used herein, the phrase "said microspheres comprise at least one of a) fluorescent labels and b) magnetic particles" is also meant to contemplate or include scenarios in which such microspheres comprise more than one (type) fluorescent label and / or more than one (type) magnetic particle. In one embodiment of the microparticles according to the present invention, the microparticles comprise at least two different fluorescent labels.

[0048] Microspheres according to embodiments of the present invention are sometimes referred to herein interchangeably as "beads" or "nanoreactor beads." Without wishing to be bound by any theory, microspheres according to embodiments of the present invention have: a) the ability to undergo a phase transition from a gel state to a soluble state because the first polymer is uncrosslinked and has a melting temperature ranging from 40°C to 90°C; and b) the ability to undergo internal reorganization to the extent that the second polymer and the network formed thereby contract when exposed to temperatures above the melting temperature of the first polymer, thereby leading to internal compartmentalization within the microsphere when exposed to temperatures above the melting temperature of the first polymer, with the contracted network forming a high-density region, volume, or "core" within a larger (less dense) volume than that provided by the microsphere itself. Such internal compartmentalization in the form of such a "core" can be detected visually, and such detection is facilitated by the concentration and / or concentration of fluorescent labels and / or magnetic particles within the core. At the same time, and particularly in embodiments of the invention in which the microspheres are located in a water-immiscible liquid phase (i.e., the microspheres are separated from other microspheres by such a water-immiscible phase), such internal compartmentalization creates a space surrounding the core in which an amplification reaction and subsequent detection can occur. Microspheres according to embodiments of the invention are sometimes referred to herein interchangeably as "nanoreactor beads" or "nanoreactor microspheres," because they provide and limit a reaction volume in which target nucleic acid binding and / or amplification can occur, and the nanoreactor beads or microspheres can simultaneously be easily handled and detected. Because the reaction volumes of nanoreactor beads or nanoreactor microspheres are in the nanoliter range, they are sometimes simply referred to as "nanoreactors." For example, the microspheres have average diameters of 500 micrometers, 100 micrometers, and 20 micrometers, respectively, and volumes of 65 nanoliters, 0.5 nanoliters, and 0.004 nanoliters, respectively.

[0049] In a preferred embodiment, the amplification primer, i.e., the analyte-specific reagent, is immobilized on the first material, preferably the first polymer, in a reversible and pH-independent manner. "Reversible" or "reversibly immobilized," as used herein in the context of "reversibly immobilizing" one entity on another, preferably refers to a type of attachment in which, under appropriate conditions, the attachment between the entities can be "undone," but also allows the entities to reattach to each other under appropriate conditions. Removal of reversible immobilization typically occurs and can be achieved in a manner that leaves the entities themselves, particularly their structure and binding ability, unchanged, allowing repeated cycles of attachment (i.e., binding) and debinding of the two entities to each other. A typical example of such reversible attachment is the hybridization of two complementary nucleic acid strands under appropriate conditions, e.g., hybridization conditions, where the resulting duplex can be reversible when exposed to a different set of conditions, e.g., elevated temperature, resulting in the duplex melting and becoming single-stranded again. When cooled, the duplexes reanneal again, thus demonstrating the reversible nature of the attachment involved. An example of an attachment that is not "reversible" in the above sense is the binding between wild-type biotin and wild-type streptavidin, which is one of the strongest non-covalent binding events known and cannot be reversed unless one or both entities are structurally altered or perhaps disrupted, for example, by denaturation. The reversible nature of the immobilization of amplification primers on each first material, e.g., first polymer, according to embodiments of the present invention facilitates customized production of microparticles according to user needs and also allows each library of microparticles to be adapted to a wide variety of different methods. Individualized and / or customized libraries can thus be easily created according to each point-of-care environment and its needs. Furthermore, the reversible attachment of the analyte-specific reagent allows for the detachment of the analyte-specific reagent in an environment where this is desired (e.g., in a method protocol requiring free availability (freely diffusible) of the analyte-specific reagent, where such analyte-specific reagent should be available (and freely diffusible) for subsequent reactions). Preferably, such reversible immobilization occurs via a first material, e.g., a first polymer, having a binding member that allows for reversible immobilization of the amplification primer on the first material. In a preferred embodiment, such binding member is attached to the first material, e.g., the first polymer, of the microsphere. Such binding member is designed and intended to bind to a conjugate that is, in turn, conjugated to the amplification primer. Therefore, in this preferred embodiment, there is an interaction between the binding member attached to the first material of the microsphere on the one hand and the conjugate conjugated to the amplification primer on the other hand. In a preferred embodiment, the binding member and the conjugate are selected to interact in a reversible manner, i.e., bind to each other. As an example, the member attached to the first material can be streptavidin or a derivative thereof, and the conjugate conjugated to the amplification primer can be biotin or a biotin derivative such as desthiobiotin, or vice versa (i.e., the reagent-binding molecule is biotin or a biotin derivative such as desthiobiotin, and the conjugate is streptavidin or a derivative thereof), as long as the interaction between the two entities is reversible and the binding can be reversed by a change, such as an increase in temperature.For example, a nucleic acid primer (or a set of nucleic acid primers and, optionally, a detection probe) can be easily conjugated to desthiobiotin or even commercially provided in such a desthiobiotinylated form. On the other hand, the first material may have an attached binding member, which may be streptavidin. It is extremely simple and easy for an end user to select a set of desthiobiotin-primers specific to an analyte of interest and reversibly attach / immobilize them to the first material of a streptavidin-attached microsphere under ambient conditions. The binding event between biotin derivatives and streptavidin under ambient conditions is easily achieved by simply exposing a microsphere containing a first material with streptavidin as a binding member to a solution of such (desthio)biotin derivative-labeled primers under appropriate conditions. The immobilization can be easily reversed, for example, by simply increasing the temperature.

[0050] The presence of two different polymers makes the microspheres described in this invention "hybrid" because they exhibit thermoresponsive behavior, and therefore they may also be referred to as "hybrid thermoresponsive" microspheres or beads. [Brief explanation of the drawings]

[0051] [Figure 1] FIG. 1 illustrates a possible approach for the synthesis of microspheres ("bead-synthesis") according to an embodiment of the present invention. [Figure 2] FIG. 2 shows various examples for the synthesis of microspheres (“beads”) comprising agarose as the first material / first polymer and chitosan as the second material / second polymer. [Figure 3]3 illustrates an embodiment comprising microspheres ("beads") to which an analyte-specific reagent (e.g., an amplification primer specific for a particular target) is attached / immobilized to a first material through a specific interaction between a binding member on the first material and a conjugate on the analyte-specific reagent (e.g., an amplification primer specific for a particular target), the analyte-specific reagent having attached thereto a conjugate that specifically interacts with the binding member on the first material. Also, while indicating different stages, the term "template bead" refers to a microsphere that contains only the first material and no second material (yet); the second material is added only in a further step, thereby generating a "precursor bead." To such a "precursor bead," an analyte-specific reagent is then added, thereby transforming the "precursor bead" into an "analyte-specific bead." [Figure 4] FIG. 4 shows exemplary characteristic behavior of microspheres (beads) according to the embodiment of the present invention produced in FIG. 3 as a function of the temperature and pH to which they are exposed. [Figure 5] FIG. 5 shows a schematic diagram of a microsphere according to an embodiment of the invention before and after being used in an amplification reaction. [Figure 6] FIG. 6 shows a schematic workflow involving microspheres (also referred to herein as "nanoreactor beads") according to an embodiment of the present invention. [Figure 7] Figure 7 shows fluorescence images of microspheres with fluorescent labels attached to a second polymer according to the present invention in the presence and absence of high concentrations of chaotropic salt. The beads exhibit comparable fluorescence, indicating the stability of the resulting microsphere matrix. [Figure 8] Figure 8 shows photographs of 1% dyed chitosan solution mixed with buffers of increasing pH from pH 2.0 to pH 9.0. [Figure 9] Figure 9 shows fluorescence images of microspheres incubated at pH 5.0 and pH 8.9, respectively, and exposed to 25°C and 98°C, respectively. [Figure 10]FIG. 10 shows an embodiment of a microsphere according to the invention imaged at room temperature after incubation at room temperature and at 95° C., where the microsphere contains carboxyfluorescein (FAM) as a fluorescent label or cyanine-5 (Cy5) as a fluorescent label, and these labels are detected in two different channels. [Figure 11] FIG. 11 shows an embodiment in which each microsphere is assigned a different type of microsphere based on their presence concentrated in the core of each fluorescent microparticle. [Figure 12] FIG. 12 shows the fluorescent signal intensity of the outer microsphere compartment of the same microspheres shown in FIG. 11, demonstrating amplification of molecular targets by PCR in some microspheres and lack of amplification in others. [Figure 13] Figure 13 shows a standard calibration curve of gDNAcp output vs. gDNAcp input (Panel A), and Figure 13 Panel B shows an image of a completed PCR protocol for the same chamber using two different fluorescence channels (green, lambda excitation 1 = 550 nm) (red channel, lambda excitation 2 = 635 nm), whereby the green channel is used to recognize all microspheres (segmentation, thus all microspheres are labeled), and the red channel is used to detect molecular targets by digital PCR (bright microspheres). [Figure 14] Figure 14 shows three images of the completed PCR protocol in the chamber using three different fluorescence channels: one for label identification (red: lambda excitation 1 = 650 nm), one for detection of HCV-specific amplification signals in several microspheres indicating the number of targets in the analyzed sample (blue: lambda excitation 2 = 470 nm), and one for application of an image segmentation algorithm to identify each microsphere analyzed (green: lambda excitation 3 = 550 nm). [Figure 15]Figure 15 shows monitoring of microsphere digital PCR reactions with detection of Staphylococcus aureus (S. aureus) genomic DNA; panel A shows the field of view of the Cy5 channel after amplification (PCR signal, light and dark microspheres are clearly visible); a Cy3 image taken before PCR reveals analyte-specific microspheres; and a FAM image shows control beads. Panel B shows real-time curves of Cy5 fluorescence of individual beads shown in the inset and the average intensity of PCR-positive and -negative microspheres. Panel C shows the density distribution of Ct values ​​estimated from all 618 valid beads of the positive microsphere type.

[0052] More specifically, referring again to Figures 1-6, Figure 1 illustrates a possible approach for synthesizing microspheres (beads) according to the present invention. As shown in Figure 1, hydrogel beads are produced using a first material as described herein. The first material, when exposed to or containing an aqueous solution, is capable of forming a porous hydrogel, having a melting temperature ranging from 40°C to 90°C. Such hydrogel beads, which contain only the first material as described herein but not the second material, may also be referred to herein as "templated hydrogel beads" or "templated hydrogel microspheres." Starting from such templated hydrogel beads, one alternative for producing microspheres according to the present invention involves adding a second material as described herein, followed by a crosslinker. Addition of such a crosslinker, for example, results in crosslinking of the second material to itself and / or the first material.

[0053] Crosslinking chemicals are known to those skilled in the art and typically involve primary amine, carboxyl, sulfhydryl, and / or carbonyl groups on each polypeptide chain. Suitable crosslinking agents are diverse and are summarized, for example, in Thermofisher's "Crosslinking Reagents Technical Handbook," available for download at https: / / tools.thermofisher.com / content / sfs / brochures / 1602163-crosslinking-reagents-handbook.pdf. Examples of suitable crosslinking agents include bis-NHS-PEG (bis-N-succinimidyl-(pentaethylene glycol) ester) and glutaraldehyde. Other crosslinking agents include carbodiimides, imidoesters, maleimides, haloacetyl groups, pyridyl disulfides, hydrazides, alkoxyamines, diazirines, and / or aryl azides.

[0054] An example of this first alternative route for the synthesis of microspheres according to embodiments of the present invention is described in Example 1 of the present application.

[0055] An alternative route for synthesizing the microspheres described in the present invention also begins with a template hydrogel microsphere containing only the first material, which is then activated. Activation in this context refers to a process in which a suitable coupling reagent is added to the first material, thereby activating the first material and allowing it to react with the second material. An example of such an activation reaction is the activation of the OH groups of agarose, the first material / first polymer, using 1,1'-carbonyldiimidazole (also abbreviated as CDI).

[0056] An example of this alternative route for the synthesis of microspheres according to embodiments of the present invention is described in Example 2. This alternative route is also particularly suitable when the second material is not a polymer but an oligomer or monomer, such that such oligomer or monomer is attached to the first material.

[0057] Figure 2 shows an example of an alternative synthetic route to Figure 1, using agarose as the first material and chitosan as the second material. The "crosslinking" alternative is shown at the top of the figure, and the "activation" alternative is shown at the bottom of the figure.

[0058] Figure 3 shows a schematic diagram disclosing details of the case where the microspheres according to an embodiment of the present invention also contain analyte-specific reagents, such as amplification primers. The process begins with so-called "template beads" or "template microspheres," which contain only the first material according to the present invention, which in this example already contains fluorescent labels and (para)magnetic particles. Furthermore, the first material contains a binding member that enables reversible and pH-independent immobilization of one or several amplification primers (= "analyte-specific reagents"), and this binding member specifically interacts with a conjugate attached to said one or several amplification primers. This reversible and pH-independent immobilization of one or several amplification primers can be achieved by appropriate selection of the binding member on the first material and the conjugate attached to the amplification primers, as further explained above. The "template beads" are converted into "precursor beads" by adding a second material, such as chitosan, to the template beads or template microspheres and immersing them in the template beads. The precursor beads, in contrast to the "template beads," also contain a second material (e.g., chitosan) that can now be crosslinked to itself and / or the first material. An analyte-specific reagent is then added, and they become attached via the specific interaction between the binding members on the first material and the conjugates on the analyte-specific reagent itself. The result of this process is a microsphere / bead specific for a particular analyte.

[0059] Figure 4 shows typical features of analyte-specific microspheres obtained from the process described in Figure 3, which are also referred to as "nanoreactor beads." However, it should be noted that the presence of analyte-specific reagents reversibly attached to the first material / first polymer is optional in the sense that these analyte-specific reagents can also be added separately at a later stage, i.e., when the amplification reaction is performed, for the subsequent amplification reaction. However, returning to Figure 4, the embodiments shown herein already have these reversibly attached analyte-specific reagents, but in a pH-independent manner, all components, including the first and second materials (e.g., agarose and chitosan, respectively), distributed throughout the microsphere. When the temperature is increased above the melting point of the first material (e.g., agarose), the network contracts, forming a contracted network containing the second material alone or the second material together with the first material. When the pH value is increased above the pKa value of the second material, the second material also coagulates, thereby promoting network contraction. Because the pore size of the contracted network is smaller than the size of the fluorescent label and / or (para)magnetic particles, they are concentrated / aggregated within the contracted core. However, at the same time, raising the temperature above the melting temperature of the first material causes the first material to liquefy, thereby creating or generating a space surrounding the contracted core, in which a solution exists. This solution may contain, for example, an analyte-specific reagent released by the temperature increase. Furthermore, if the microsphere contains or contains a nucleic acid of interest, such as a target nucleic acid (such nucleic acid, however, is not shown in Figure 4), the target nucleic acid will initially be concentrated and / or bound to the second material due to the nucleic acid-binding ability of the second material at a pH lower than the pKa of the second material. Once the pH value is raised above the pKa of the second material, the nucleic acid of interest will then be released from its binding state with the second material and released into the space surrounding the contracted core.

[0060] In a second step, shown in Figure 4, the temperature is again reduced to below the melting temperature of the first material, so that the first material solidifies again, but the aggregated / shrunken core remains in place, along with the fluorescent label and magnetic particles. This characteristic behavior of the microspheres according to embodiments of the present invention applies particularly when such microspheres are isolated in a water-immiscible liquid phase, as shown in Figure 4, such that such microspheres exist as solid or semi-solid particles below the melting point of the first material, and as liquid droplets with a solid, shrunken core above the melting temperature of the first material.

[0061] FIG. 5 shows a characteristic appearance of an embodiment of a microsphere according to the present invention, e.g., the microsphere manufactured in FIG. 3 and shown in FIG. 4, i.e., containing an analyte-specific reagent, e.g., an amplification primer. In such a microsphere prior to use in an amplification reaction, the structure is as shown in FIGS. 4 and 3, i.e., all components are distributed throughout the microsphere, and the analyte-specific reagent is immobilized to the first material via specific interactions between the binding members on the first material and the binding entities attached to the analyte-specific reagent. During and after use, the microsphere is exposed to and / or subjected to a high-temperature process involving a temperature higher than the melting point / temperature of the first material, resulting in the second material shrinking and forming a shrunken core with the fluorescent label and magnetic particles concentrated therein. For such an amplification reaction, the microsphere is also exposed to a sample containing or suspected to contain nucleic acid, particularly a specific target nucleic acid. If such a specific target nucleic acid is present in such a sample, the sample is amplified during the amplification reaction according to the amplification protocol, and the amplified nucleic acid will be located within the space surrounding the shrunken core.

[0062] Figure 6 shows a schematic workflow that can be implemented with microspheres according to the "nanoreactor bead" embodiment of the present invention. The beads / microspheres shown in this figure do not show components contained within such microspheres (as shown in previous figures), such as fluorescent labels, magnetic particles, or any analyte-specific reagents that are initially attached to the first material or that will instead be added during the amplification protocol; only the microspheres containing the first and second materials are shown in Figure 6. Such microspheres are exposed to a sample containing or suspected to contain nucleic acids in a buffer solution having a pH lower than the pKa of the second material. In a preferred embodiment, the pKa of the second material is greater than 6, typically at a low pH value in the acidic range. As a result of this pH value, the second material becomes protonated / ionized, thereby becoming positive and binding to any (negatively charged) nucleic acids contained in the sample. As a result, the microsphere contains nucleic acids bound thereto and within it, because the second material, which has this affinity for nucleic acids and is contained in the microsphere, is distributed throughout the microsphere. (Because such microspheres contain both the first and second materials, they are sometimes referred to in this figure as "hybrid" microspheres or "hybrid" beads.) The nucleic acid-binding microspheres are not shown. In a subsequent step, the microspheres are washed and then filled with a composition for amplification. In a preferred embodiment, the washing composition is also a composition for promoting binding of nucleic acids to the second material, and preferably such a washing or promoting composition has a pH value still higher than the pKa value of the second material.

[0063] In the "loading" step, the microspheres are exposed to (e.g., soaked in, immersed in, etc.) a further composition for promoting and carrying out a nucleic acid amplification reaction and detection. In a preferred embodiment, such a nucleic acid amplification reaction comprises a buffer that buffers the reaction to a pH range appropriate for carrying out nucleic acid amplification. Furthermore, such a composition preferably also comprises mononucleoside triphosphates, an amplification enzyme, and optionally one or more amplification primers or one or more sets of amplification primers (if such primers are not already immobilized on the first material). Furthermore, such a composition for promoting and carrying out a nucleic acid amplification reaction and / or detection may comprise a nucleic acid dye for detecting the amplification product, and may optionally comprise a molecular probe, such as TaqMan or a molecular beacon, respectively. Preferably, in many embodiments, the pH value of such a composition is higher than the pKa value of the second material. Once the microspheres have been loaded with the composition for promoting and carrying out nucleic acid amplification, they are transferred to a water-immiscible liquid phase, such as an oil phase, so that the microspheres are, as it were, isolated and exist as droplets of aqueous solution within the surrounding water-immiscible phase. Once this occurs, the microspheres are subjected to an amplification protocol that includes, precedes, or follows at least one step in which the microspheres (and suspensions containing such microspheres) are heated to a temperature above the melting temperature of the first material. As a result of such a heating step, the first material liquefies and the second material shrinks, resulting in phase separation within the microsphere and creating a shrunken core surrounded by spaces where amplification / detection reactions can or will occur. Fluorescent labels concentrated within the core allow for identification / detection of the microspheres.Similarly, magnetic particles concentrated within the core allow for efficient handling / manipulation of such microspheres, and depending on the presence or absence of other fluorescent dyes (e.g., attached to the first material), the presence or absence of molecular probes, a number of different channels of the detection scheme / device / system may be used for: locating and / or identifying individual microspheres; distinguishing fused microspheres from non-fused microspheres; distinguishing one microsphere from another; detecting and distinguishing different amplification products (if present); detecting and distinguishing the presence or absence of one or several amplification products in one or several samples being analyzed.

[0064] The microspheres described in embodiments of the present invention are highly versatile in that there is no upper limit as to the number of different fluorescent labels that can be used (as well as the number of non-particulate fluorescent dyes that can be used).

[0065] Furthermore, if the microspheres also contain non-particulate fluorescent dyes somehow associated with the first material, additional coding possibilities arise, since then not only the "color" or rather the mixture of "colors" of the contracted core of each microsphere, but also the color of the surrounding space (i.e., the periphery of such core) can be used for differentiation.

[0066] Furthermore, reference is made to the following examples, which are given to illustrate, but not to limit, the present invention: [Example]

[0067] Example 1 Preparation of crosslinked nanoreactor beads by linking molecules of a second polymer together 2. Preparation of Agarose Suspension with Coating and Paramagnetic Particles Component 1: SeaKem ME Agarose (Lonza) Component 2: 1 μm coated fluorescent particles (Thermo, λex=480 nm, λem=520 nm) Component 3: 1μm Magnefy COOH magnetic particles (Bangslabs)

[0068] Component 1 ("First Material", "First Polymer") was weighed and dissolved in nuclease-free water (Roth) to prepare a 1% (w / v) homogeneous solution. This was achieved by brief initial vortexing (20', 2000 rpm) and heating to 95°C for 10 minutes at 1200 rpm in a thermomixer (Eppendorf). Unwanted impurities were removed using a 5 μm syringe filter (Whatman). The temperature was then reduced to 80°C for combining the agarose with the other components.

[0069] Preparation of component 1 begins with washing three times with 10 volumes of nuclease-free water (Roth). This step removes unwanted impurities in the storage buffer. To prevent aggregation, the particles are sonicated for 15 minutes at 23°C.

[0070] All components are stored in polypropylene tubes at 80°C / 1200 rpm in a thermomixer (Eppendorf) until further processing.

[0071] Typical final concentrations are as follows:

[0072] [Table 1]

[0073] Agarose can also be modified with fluorescent dyes; typical protocols are listed below.

[0074] Staining of agarose with Cy3 Materials used: Agarose (A9793; SeaKem ME) Cy3 mono NHS ester (GE) 50mM potassium phosphate buffer, pH 8.3

[0075] 5 ml of 1.5% agarose solution is prepared in 50 mM potassium phosphate buffer (pH 8.3). To do this, 75 mg of agarose is mixed with 5 ml of buffer and incubated at 90°C for 20 min. After cooling to room temperature and allowing the solution to solidify, the agarose is mechanically crushed and mixed with 1.6 mg of dye solution dissolved in 50 mM potassium phosphate buffer (pH 8.3). The final volume of the staining solution is 10 ml. The incubation time for staining is 120 min. During this time, the mixture is rotated at 30 rpm on a rotating wheel at room temperature. The agarose is then washed several times with distilled water until no free dye remains in the supernatant. Finally, the agarose is dried in a Speedvac and is ready for bead preparation.

[0076] Generation of monodisperse templated nanoreactor beads Monodisperse templated nanoreactor beads ("microspheres") are generated in a one-step process using a simple flow-focusing device with the described components, agarose, chitosan, and coated and magnetic particles, on a modified μEncapsulator system (Dolomite microfluidics). Specifically, a fluorophilic standard junction tip (100 μm) is used with a four-way linear connector and a tip interface H, which interfaces the fluidic connection between the tubing and the tip. Two Mitos P-Pumps deliver the agarose / particle suspension and an emulsifier-containing carrier oil, e.g., PicoSurf (Sphere Fluidics, 2.5–5% in Novec 7500, 3M).

[0077] This prefabricated system utilizes a modified heating device installed on a hot plate. This allows the driving solution to be heated while maintaining the agarose / chitosan / particle suspension in a liquid state and ensuring a stable temperature when the carrier oil and component suspension come into contact at the chip junction. A heated cover for the agarose / chitosan / particle suspension reservoir prevents unwanted evaporation. A magnetic stir bar (controlled at 300-600 rpm by the hot plate) is used to keep the suspension intact.

[0078] For bead production, the heating temperature is set to 75 °C. The fluid lines are primed for 1 min at 2000 mbar using Flow Control Software. The flow rate is adjusted to 10-50 μl / min for stable droplet formation. Parameters such as diameter are monitored using Dolomite Flow Control Advanced Software.

[0079] Monodisperse beads are collected through a 200 μm PTFE tube connected to the outlet of the microfluidic chip. The beads are cooled in the tube and finally collected in a tube (e.g., 15 ml, BD) and placed on ice to initiate solidification of the hybrid hydrogel. To prevent loss of the aqueous phase of the beads at the oil-air interface, the sample is overlaid with 500–1000 μl of nuclease-free water (Roth). The template nanoreactor beads are cooled to 4°C and allowed to stand for at least 1 h to form a stable hybrid polymer scaffold.

[0080] Recovery of templated nanoreactor beads from oil suspension The solidified template nanoreactor beads accumulate on top of the emulsion oil in the collection tube. The PicoSurf oil is carefully removed with a pipette, taking care not to dislodge the beads. The PicoSurf residue is further diluted with solvent Novec 7500 (3 M, 1:1 volume of collection beads). 1H,1H,2H,2H-Perfluorooctanol (PFO, Sigma, 1:4 volume of collection beads) is then added to break up the emulsion. Nuclease-free water (Roth, 3:1 volume of collection beads) is also added. The beads now float in the aqueous phase. To accelerate this process, the tube is vortexed for 5 s and centrifuged at 2500 rpm for 5 s.

[0081] Finally, carefully pipette the beads from the aqueous layer of the tube and transfer them to a new tube. Repeat this process to remove any remaining fluorocarbon oil and detergent. Wash the collected beads once with nuclease-free water (Roth, 3:1 volume of collected beads).

[0082] Cross-linking of chitosan to generate cross-linked nanoreactor beads A 2% chitosan solution was prepared and roughly mixed with the suspension containing approximately 10,000 template nanoreactor beads. Bis-N-succinimidyl-(pentaethylene glycol) ester (BIS-NHS-PEG; SIGMA-ALDRICH) was used as the chitosan crosslinker. A linker solution was prepared by dissolving 1 mg of solid BIS-NHS-PEG in 1 mL of distilled water, and 250 μL of this solution was added to the suspension. The suspension was vortexed. The vortexed suspension was allowed to stand for 30 seconds. 20 mL of PicoSurf oil (Sphere Fluidics) was added to the suspension, and the suspension was immediately placed in a Bead Ruptor device (OMNI) and shaken three times for 5 seconds at 4 m / s. The bead-in-oil suspension was then placed in a refrigerator and left to stand overnight at 4°C. The crosslinked templated nanoreactor beads are recovered from the PicoSurf oil following the protocol described for recovery of templated nanoreactor beads.

[0083] If the chitosan itself is to be labeled with a non-particulate fluorescent dye, a typical protocol is listed below, which may be carried out prior to using such labeled chitosan in the crosslinked nanoreactor bead generation protocol described above:

[0084] Staining of chitosan with Cy5 Materials used: Chitosan HCl (Heppe Medical chitosan GmbH). Cy5 mono NHS ester (GE) Distilled water

[0085] Chitosan HCl (1 g) was dissolved in distilled water to prepare a 2% solution. 1.6 mg of Cy5 mono-NHS ester was dissolved in 100 μl of distilled water and added to the chitosan solution. After brief stirring, the solution was incubated overnight at room temperature on a rotating wheel at 30 rpm. The solution was then dialyzed against distilled water for a total of 48 h, with three changes of water. The dyed chitosan polymer was then concentrated to a 2% solution in a Speedvac.

[0086] Example 2 Preparation of crosslinked nanoreactor beads by linking first and second polymer molecules The following embodiment demonstrates the creation of hybrid hydrogel beads consisting of an agarose scaffold and a capture matrix composed of a second material: chitosan polymer, which is applied as an ionizable capture matrix for nanoreactor beads. In this example, chitosan is crosslinked to the agarose matrix; crosslinking is achieved by binding chitosan to chemically activated agarose polymers that comprise the matrix of pre-fabricated hydrogel beads. As a result, chitosan molecules bond with agarose molecules, forming bonds between different chitosan molecules. In addition to classical chitosan, various water-soluble derivatives are available and can be used interchangeably: -Chitosan HCl -Chitosan Glutamate (Glutamat) -Carboxymethyl chitosan -Chitosan lactate -Chitosan acetate (Heppe Medical chitosan GmbH)

[0087] Generation of monodisperse agarose beads Monodisperse beads composed of agarose are produced using a modified μEncapsulator system (Dolomite microfluidics). Solidified agarose beads are recovered from the emulsion oil. Excess emulsion oil is carefully removed with a pipette. After adding 1H,1H,2H,2H-perfluorooctanol and distilled water, the emulsion is broken down, and the beads are then transferred to the aqueous phase. After washing the agarose beads several times with distilled water, the beads are available for further treatment with chitosan.

[0088] Reaction of activated agarose beads with cross-linked chitosan The vial containing the coupling reagent, 1,1'-carbonyldiimidazole (CDI; Sigma), was allowed to warm to room temperature before opening to prevent condensation. The reaction proceeds in two steps:

[0089] Step 1: Activation of OH groups of agarose Centrifuge the agarose bead suspension at 700 rpm for 1 min and discard the supernatant. Dissolve CDI in 2 mM HCl to obtain a 200 mM solution. Next, mix the beads with the CDI solution in a 1:1 ratio and react in 1 mM HCl. Incubate the reaction tube at room temperature on a rotating wheel at 30 rpm for 10 min.

[0090] After the reaction, the beads were washed by centrifugation at 700 rpm for 2 minutes. The supernatant was removed, and the recovered beads were resuspended in an equal volume of distilled water and mixed. This washing procedure was repeated twice, and the activated beads were prepared for subsequent chitosan conjugation.

[0091] The activation reaction is carried out at a pH below 5.0. Alternatives to 1 mM HCl for adjusting the pH include, for example: - 20 mM acetate buffer; pH 4.0 -20mM citrate buffer; pH4.0 -5mM malonic acid; pH 3.0

[0092] Step 2: Coupling of chitosan to pre-formed precursor beads containing activated agarose polymer A 1% chitosan solution is prepared in distilled water. In the second step, the activated beads and the 1% chitosan solution are immediately mixed in a 1:1 ratio. This mixture is incubated at room temperature on a rotating wheel at 30 rpm for 30 min. After the reaction, the beads are washed by centrifugation at 700 rpm for 2 min. The supernatant is removed, and the beads are resuspended in an equal volume of distilled water and mixed. This washing procedure is repeated at least 10 times to ensure that there is no free chitosan in the supernatant.

[0093] The stability of the cross-linked polymer beads is checked using Cy5-stained chitosan and after adding a high concentration (3.25 M) of chaotropic salt (eg, guanidinium HCl). The images in Figure 7 demonstrate the stability of the coupling achieved under chaotropic conditions.

[0094] Characterization of chitosan solubility at different pH values This section attempts to explain the specific behavior of the microspheres described in this invention.

[0095] Materials used: Chitosan-HCl (Heppe Medical chitosan GmbH) for better visibility Use Cy5-conjugated chitosan (see protocol above) Malonic acid MES (2-(N-morpholino)ethanesulfonic acid) buffer Potassium phosphate buffer

[0096] 250 μl of 1% dyed chitosan solution is mixed with buffers of increasing pH and incubated at room temperature for 10 min. The solution is then centrifuged at 2000 rpm for 2 min. The results are shown in Figure 8 and the buffers used were:

[0097] 1 50mM malonic acid; pH 2.0 2 50mM malonic acid; pH 3.0 3 50mM malonic acid; pH 4.0 4 50mM MES buffer; pH5,0 5 50mM MES buffer; pH6,0 6 50mM MES buffer; pH7,0 7 50mM potassium phosphate buffer; pH 8.0 8 50mM potassium phosphate buffer; pH 9.0

[0098] The results show that at pH below 6 the solution remains intact, but above the pKa of chitosan, pH 7 and above, polymer flocculation occurs.

[0099] Without wishing to be bound by any theory, it is believed that this pH dependent aggregation / aggregation of chitosan polymers is also observed in hybrid beads and forms an important part of the present invention.

[0100] For the following experiments, two different buffers were used: Buffer 1: 200mM MES buffer; pH5.0, 100mM KCl Buffer 2: 200 mM Tris / HCl; pH 8.9, 100 mM KCl

[0101] 50 μl of the beads prepared in Example 2 are mixed with 50 μl of Buffer 1, and 50 μl of the same beads are mixed with 50 μl of Buffer 2. After 1 minute of incubation, the beads are centrifuged at 700 rpm for 1 minute to allow aggregation. The supernatant is removed. The beads are then transferred to a non-aqueous phase (e.g., PicoSurf oil) and purified using a method such as Loncarevic IF, et al., PLOS ONE 16(3):e0242529. https: / / doi.org / 10.1371 / journal.pone.0242529 The mixture is transferred to a reaction and detection chamber (RDC) and incubated at different temperatures as described in. The results are shown in Figure 9.

[0102] While not wishing to be bound by any theory, the inventors have demonstrated such behavior with cross-linked chitosan as the second polymer, and would expect similar behavior from other polymers (e.g., gelatin, poly(ethyleneimine), poly(2-dimethyl(aminoethyl)methacrylate), poly(l-lysine), poly(histidine), and similar polymers) when used in cross-linked form as the second polymer. Furthermore, it is also possible to use oligomers or monomers attached to an activated first polymer. Such oligomers or monomers can be oligo- or mono-histidine, oligo- or mono-lysine, oligo- or mono-arginine, and other chemically similar molecules. Similarly, the inventors have demonstrated such behavior with agarose as the first polymer, and would expect similar behavior to be achieved by other polymers (e.g., non-cross-linked gelatin, methylcellulose, hyaluronan, elastin-like polypeptides, and other derivatized polymers with similar gel-forming and thermoresponsive properties) when used in non-cross-linked form. Poly-N-isopropylacrylamide and its derivatives can also be used for this purpose.

[0103] Beads produced according to the listed protocol and treated with the appropriate buffer exhibit a predetermined response to temperature changes, as illustrated in Figure 10.

[0104] The images in Figure 10 clearly show the aggregation of the fluorescent particle labels with increasing temperature: upon cooling to room temperature, the first polymer, and thus the microspheres, solidify, but all embedded particles become concentrated within the core aggregate formed by the contracted second polymer.

[0105] E) Labeling / Coding of Microspheres (Beads) and Amplification Reactions Using Such Labeled / Coded Microspheres (Beads) Basics Multiplexing samples and analytes requires that each bead type be uniquely coded to distinguish between them. The bead coding strategy is based on labeling a secondary polymer or embedded microparticles with fluorescent dyes for four separate fluorescence channels (blue, green, red, and dark red). After heating, all labeled / coded particles are located in the inner compartment, clearly distinguishable from the fluorescent signal in the outer compartment of the nanoreactor bead. To adjust the fluorescence level, binary coding can be applied, meaning that a fluorochrome in a particular channel is either present or absent. The maximum number of bead types that can be coded using this strategy is 2. 4 = 16, where 2 is the number of resulting fluorescence intensity levels (0 and 1) and 4 is the number of fluorescence channels. The number of possible bead types can be further increased by allowing for various levels of fluorescent dye concentration, if not used to monitor / detect an enzymatic amplification reaction indicating the presence of an analyte / target, or by extending the coding of detectable agarose levels in the outer compartment of the bead.

[0106] Image analysis The image analysis software utilizes a segmentation algorithm that identifies the circular object contours of the outer and inner bead compartments. Based on this segmentation information, the mean fluorescence intensity is quantified. The number of independent fluorescence signal intensities assigned to each bead is 4*2=8, considering four channels and two compartments.

[0107] Bead Decoding Based on the fluorescence intensity level of the coding particles located in the internal compartment or the intensity level of the internal compartment itself, the bead decoding algorithm assigns each bead to one of the bead types identified by the underlying bead library. The embodiment illustrated in Figure 11 was derived from an experiment using two types of beads labeled with blue or red coding particles. Images were acquired at the end of PCR amplification. In addition to the coding particles in the blue and red channels, these channels also contain analyte detection information. Therefore, the outer bead compartment can be PCR-positive or PCR-negative. Image acquisition conditions for each channel were adjusted to ensure that pixels imaging coding particles with dye present were saturated. The absolute fluorescence level of coding particles in the absence of dye is attributed to the PCR results in the outer bead compartment. Consequently, fluorescence levels at or near saturation (e.g., 220-256) are classified as coding particle presence. Beads with uncertain fluorescence levels (e.g., 180-220) are invalid. Intermediate and low intensity (e.g., less than 180) are classified as no coding particle. Fusion beads with blue and red labels are invalid.

[0108] Data analysis Final data analysis for analyte detection and quantification is based on the fluorescence intensity measured in the outer bead compartment. Because the coding and detection information are spatially separated, it is possible to obtain analyte-specific information in the same fluorescence channel used for bead coding. Quantification can be performed using digital or real-time PCR analysis approaches. Digital PCR analysis relies on the fluorescence signal estimated from images taken at the end of PCR, while real-time analysis is based on the fluorescence signal estimated from images taken throughout the entire cycle of real-time PCR. Figure 12 shows the fluorescence signal intensity of the outer bead compartment in the blue and red detection channels for the embodiment of Figure 11. Four sample beads for each bead type cover four possible combinations of negative or positive PCR results in the blue and red channels, respectively. The intensities of the eight sample beads marked in the plot of Figure 11 are highlighted in the intensity plot of Figure 12.

[0109] Bead fusion control Fusion beads can be detected either by detecting the presence of unknown code combinations (eg, in the case of single-color codes) or by detecting various internal compartments (see Figure 11).

[0110] Example 3 Binding and quantification of bacterial gDNA with BLINK nanoreactor beads Bacterial gDNA from methicillin-resistant Staphylococcus aureus (MRSA) strain ST8 USA300, with a genome length of 2.8 Mb, was bound and quantified by digital PCR using BLINK nanoreactor beads. The culture was mechanically lysed using ceramic beads and a Minilys homogenizer (Bertin Technologies) at agitation level 3 for 2 min. The lysate was diluted 1:2000 in PBS and stored at -20°C. The nanoreactor beads used consist of an agarose hydrogel scaffold (A9793, Sigma), a cross-linked chitosan-HCl (Heppe Medical chitosan GmbH) capture matrix, fluorescent coding labeling with Lumogen Red F 300 nanoparticles (Kremer Pigmente GmbH & Co. KG), a fluorescent label (A9793-Cy3) for segmentation, and embedded 5 μm polystyrene magnetic microparticles (Sigma) for retention.

[0111] Bonding and washing The nanoreactor beads prepared in Example 1 are supplied as lyophilized pellets in 0.2 ml processing tubes containing a defined volume of 100 μm beads (n=56,000) for single-sample nucleic acid capture and subsequent digital amplification reactions. The nanoreactors are pre-washed by resuspending them in Component 1, which is necessary to ensure preconditioning of the capture matrix. To do this, a two-fold volume of Component 1 is added to the beads. The resulting bead slurry is placed in a magnet to retain the beads, and the supernatant is aspirated and discarded. This pre-wash is repeated twice to obtain equilibrated nanoreactor beads ready for the next nucleic acid binding reaction.

[0112] Component 1: Resuspension Buffer / Prewash Buffer 10 mM MES (2-(N-morpholino)ethanesulfonic acid), pH 5.0

[0113] To bind DNA to nanoreactor beads, as prepared in Example 1, 20 μL of the lysate diluted at three different concentrations was pipetted onto a 40 μL bead bed containing 56,000 beads, and 140 μL of binding buffer (component 2) was added. The binding mix was incubated for 5 min at 25°C and 1800 rpm in an Eppendorf thermomixer. After capture, the supernatant was gently removed using a magnetic tube rack and pipette, leaving the beads attached to the tube walls.

[0114] Component 2: Binding buffer 50mM malonic acid pH 3.5

[0115] A similar procedure was followed during washing, where the beads were washed three times with 300 μL of wash buffer (component 3) to remove unbound DNA and cellular debris.

[0116] Component 3: Wash Buffer Malonic acid 12.5mM (pH 3.5)

[0117] Loading of detection reagents into nanoreactor beads Analyte-specific and generic reagents for mecA target detection in nanoreactors were supplied as lyophilized pellets resuspended with Component 4. Primer and probe sequences targeted the mecA gene, which encodes the methicillin-resistant Staphylococcus aureus (MRSA) protein PBP2A (penicillin-binding protein 2A). The resulting Component 5 was combined with an equal volume of analyte-bound nanoreactor beads obtained from the upstream sample preparation process. The mixture was incubated at 25°C for 5 min with shaking at 800 rpm to allow the detection reagent to be incorporated into the porous nanoreactor beads. Excess liquid was removed by magnetically retaining the reagent-loaded nanoreactor beads.

[0118] Component 4 (general PCR buffer) 2xRT-PCR buffer: 40mM Tris HCl, 44mM KCl, 44mM NH4Cl, 6mM MgCl2

[0119] Component 5 (general and specific RT-PCR reagents) 2xRT-PCR buffer: 40mM Tris HCl, 44mM KCl, 44mM NH4Cl, 6mM MgCl2 0.4U / μl Hot Start Taq DNA polymerase (biotechrabbit GmbH) 0.8mM dNTPs (biotechrabbit GmbH) 0.8 μM sense primer (mecA_R: 5'-TGGCATGAGTAACGAAGAATATAA-3') (SEQ ID NO: 1) (Metabion International AG) 0.8 μM antisense primer (mecA_R: 5'-GAGTTGAACCTGGTGAAGTTG-3') (SEQ ID NO: 2) (Metabion International AG) 0.8 μM TaqMan probe (mecA_P: 5'-Atto647N-AAAGAACCTCTGCTCAACAAGTTCCAGA-BHQ2-3') (SEQ ID NO: 3) (Metabion International AG) 0.2% (w / v) low bioburden, protease-free, molecular biology grade BSA (Sigma)

[0120] Phase transfer in packed nanoreactor beads The reagent-filled nanoreactor beads were transferred to and dispersed in a non-aqueous phase (Component 6) to create individual nanoliter-sized reaction spaces. To do this, excess Component 6 (100 μl) was added to the beads, and the beads were emulsified using two stirring steps at agitation level 2 using a Minilys homogenizer (Bertin Technologies). The applied mechanical stress disrupted the attractive forces between the aqueous nanoreactor beads, creating surface tension that formed a suspension / emulsion. Both the hydrogel beads and the excess aqueous phase were emulsified in the oil phase. The submicron-scale droplets that formed as a by-product were removed by gentle suction with a pipette, as the microemulsion formed a separate layer below the emulsified nanoreactor beads. Repeated washing with the same emulsion / amplification oil (Component 6) essentially removed all unwanted droplets. An additional 100 μl of Component 6 was added.

[0121] Component 6 (Phase transfer oil) -HFE-7500 fluorocarbon oil (3M Deutschland GmbH) - Supplemented with 2–5% (v / v) PicoSurf (SphereFluidics) or 2–5% (v / v) FluorSurf (Emulseo)

[0122] Digital PCR amplification, detection, and gDNA quantification on nanoreactor beads The nanoreactor bead suspension was transferred to a polycarbonate reaction and detection chamber (RDC) and covered with a 250 μm transparent polycarbonate film to achieve a chamber height of approximately 100 μm, allowing the beads to disperse as a monolayer within the chamber. An example of a suitable reaction and detection chamber is described in Loncarevic IF, et al., PLOS ONE 16(3):e0242529. https: / / doi.org / 10.1371 / journal.pone.0242529. The RDC was then attached to a high-performance Peltier element mounted on a test fixture, with the membrane of the chamber in contact with the Peltier element. PCR was performed using the following cycling conditions: initial denaturation at 95°C for 120 s, followed by 45 cycles of 95°C for 5 s and 61°C for 15 s.

[0123] Upon completion of the PCR protocol, two images, shown in Figure 13B, were acquired at each chamber position, adding up to a total of 2 x 48 images. One channel was required for segmentation (green: λexc1 = 550 nm), and the second channel was required for mecA-specific amplification signal (red: λexc2 = 635 nm). Automated image acquisition was achieved using the BLINK toolbox software and a fluorescence microscope (Zeiss AxioObserver) equipped with a 5x objective (field of view 4.416 mm x 2.774 mm) and a pE-4000 (CoolLED Ltd.) light source. The microscope was further equipped with three fluorescence filter sets (Cy5 ET, Cy3 ET, AHF Analysentechnik) and an automated XY stage to which a thermocycler with reaction chambers could be attached.

[0124] DNA input per target was quantified by performing a poison assay based on the number of positive and negative beads detected per target. Diameter-normalized lambda values ​​were calculated for each lysate concentration level. The lambda value was multiplied by the initial number of beads used for binding to calculate gDNA copy output (cp output).

[0125] Nominal input was calculated by qPCR using the standard calibration curve shown in Figure 13A.

[0126] Example 4 Nucleic acid extraction from crude samples using nanoreactor beads In the following embodiments, the recombinant Accuplex TM(SeraCare Life Sciences, Inc.) EDTA plasma samples spiked with HCV high titer control material (Lot No. 10321228) are encapsulated in monodisperse emulsions using nanoreactor beads according to the present invention, which integrates RNA extraction and detection into a single workflow. Accuplex TM The control material was based on Sindbis virus, which mimics the viral complexity found in real patient samples, and contained HCV-specific RNA sequences. The nanoreactor beads used consisted of an agarose hydrogel scaffold (A9793, Sigma), a cross-linked chitosan-HCl (Heppe Medical chitosan GmbH) capture matrix, fluorescent coding labels with Lumogen Red F 300 nanoparticles (Kremer Pigmente GmbH & Co. KG), fluorescent labels for segmentation (A9793-Cy3), and embedded 5 μm polystyrene magnetic microparticles (Sigma) for retention. They were prepared essentially as described in Example 1.

[0127] Preparation of nanoreactors Nanoreactor beads are supplied as lyophilized pellets housed in 0.2 ml processing tubes containing a defined volume of 100 μm beads (n=53,000) for single-sample nucleic acid purification and subsequent digital amplification reactions. The nanoreactors are pre-washed by resuspending them in Component 1, which is necessary to ensure preconditioning of the capture matrix. To do this, two volumes of Component 1 are added to the beads. The resulting bead slurry is placed in a magnet to retain the beads, and the supernatant is aspirated and discarded. This pre-wash is repeated twice to obtain equilibrated nanoreactor beads ready for the next nucleic acid binding reaction.

[0128] Component 1: Resuspension Buffer / Wash Buffer 10 mM MES (2-(N-morpholino)ethanesulfonic acid), pH 6.5

[0129] Bead-based RNA extraction 50 μl of plasma was prepared by centrifugation of HCV-negative EDTA whole blood (provided by Universitatsklinikum Jena) and diluted Accuplex TM HCV control material was spiked and applied directly to the equilibrated nanoreactor beads. The original viral titer was 2.69 x 10 according to the manufacturer's information. 8 The control material had a final virus titer of 2.69 x 10 7 The solution was diluted with nuclease-free water to 100 cp / ml, of which 1 μl was spiked, corresponding to an analyte input size of 26,900 viral particles. Component 2, containing a chaotropic agent, was added to the processing tube in a volume of 133 μl and then incubated at 30°C with shaking at 1800 rpm for 5 min to efficiently homogenize the sample, lyse the viral particles, and stabilize the released fragile RNA. RNA binding was achieved by adding binding buffer (component 3) to a final binding volume of 300 μl and repeating the incubation at 25°C with shaking at 2000 rpm for an additional 5 min. Alternatively, lysis and binding can be combined into a single process step (incubation at 25°C for 10 min, shaking at 1800 rpm) using the same mixture containing equal volumes of components 2, 3, nanoreactor beads, and EDTA plasma. The magnetized microparticles / nanoparticles contained within the nanoreactor beads allow the nanoreactors to be transported through various sample preparation solutions, including wash buffer (Component 1). The processing tube is then transferred to a magnetic rack, retaining the beads. The binding supernatant is removed, and the nanoreactors are resuspended in 300 μl of Component 1. Effective repeated washing (5x) substantially removes most potential inhibitors that could interfere with downstream enzymatic reactions, making bead-based sample processing compatible with subsequent digital amplification reactions performed in the same compartment.

[0130] Component 2: Lysis buffer - 100 mM acetate buffer, pH 4 (Alfa Aesar) -3M guanidine sulfate (Sigma) -20mM ethylenediaminetetraacetic acid (Acros Organics) -10% (v / v) Tergitol (Sigma)

[0131] Component 3: Binding buffer 1M MES (2-(N-morpholino)ethanesulfonic acid), pH 6.5

[0132] Loading of detection reagents into nanoreactor beads Analyte-specific and generic reagents for HCV detection in nanoreactors were supplied as lyophilized pellets that were resuspended with component 4. The resulting component 5 was combined with an equal volume of the analyte-bound nanoreactor beads obtained in the upstream sample preparation process. The porous nanoreactors were allowed to incorporate the detection reagents by incubating at 30°C for 5 min with shaking at 1000 rpm. Excess liquid was removed by magnetically retaining the reagent-loaded nanoreactor beads.

[0133] Component 4 consisting of the following final concentrations 2xRT-PCR buffer: 40mM Tris HCl, 44mM KCl, 44mM NH4Cl, 6mM MgCl2

[0134] Component 5 consisting of the following final concentrations: -2xRT-PCR buffer: 40mM Tris HCl, 44mM KCl, 44mM NH4Cl, 6mM MgCl2 -0.4U / μl Hot Start Taq DNA polymerase (biotechrabbit GmbH) -0.8mM dNTPs (biotechrabbit GmbH) 0.8 μM sense primer (HCV_Brun_F: 5'-GTGGTCTGCGGAACCGGTGA-3') (SEQ ID NO: 4) (Metabion International AG) 0.8 μM antisense primer (HCV_Brun_R: 5'-CGCAAGCACCCTATCAGGCAGT-3') (SEQ ID NO: 5) (Metabion International AG) 0.8 μM TaqMan probe (HCV_Brun_P1: 5'-Atto488-CCGAGTAGCGTTGGGTTGCGAAAGG-BHQ1-3') (SEQ ID NO: 6) (Metabion International AG) -0.2% (w / v) low bioburden, protease-free, molecular biology grade BSA (Sigma)

[0135] Phase transfer in packed nanoreactors The reagent-filled nanoreactors were transferred to a non-aqueous phase (Component 6) and dispersed to create individual nanoliter-sized reaction spaces. To this end, excess Component 6 (100 μl) was added to the beads, and the beads were emulsified using two stirring steps at agitation level 2 using a Minilys homogenizer (Bertin Technologies). The applied mechanical stress disrupted the attractive forces between the aqueous beads, creating surface tension that formed a suspension / emulsion. Both the hydrogel beads and the excess aqueous phase were emulsified in the oil phase. The submicron-scale droplets that formed as a by-product were removed by gentle suction with a pipette, as the microemulsion formed a separate layer below the emulsified nanoreactor beads. Repeated washing with the same emulsion / amplification oil (Component 6) essentially removed all unwanted droplets. An additional 100 μl of Component 6 was added.

[0136] Component 6: Phase transition and signal amplification oil -HFE-7500 fluorocarbon oil (3M Deutschland GmbH) - Supplemented with 2–5% (v / v) PicoSurf (Dolomite Microfluidics) or 2–5% (v / v) FluoSurf (Emulseo)

[0137] Digital RT-PCR amplification in nanoreactors The nanoreactor bead suspension was transferred to a polycarbonate reaction and detection chamber (RDC) and covered with a 250 μm transparent polycarbonate film to create a chamber height of approximately 100 μm, allowing the beads to disperse as a monolayer within the chamber (1), forming a suspension array. The RDC was attached to a high-performance Peltier element, with the chamber's thin film in contact with the Peltier element. The nanoreactor was subjected to rapid temperature cycling to establish a chamber-specific PCR control mode. The applied temperature conditions were as follows: reverse transcription at 50 °C for 10 min, initial denaturation at 95 °C for 30 s, followed by 30–45 cycles of two-step PCR consisting of denaturation at 95 °C for 5 s and annealing / extension at 62 °C for 10 s. Due to the sol-gel switching ability, the suspension became an emulsion with individual liquid nanoreactor compartments.

[0138] Upon completion of the thermal protocol, three images were acquired at each chamber position, adding up to a total of 2 x 48 images, as shown in Figure 14. One channel was required for label identification (red: λexc1 = 650 nm), a second channel for HCV-specific amplification signal (blue: λexc2 = 470 nm), and a third channel for segmentation (green: λexc3 = 550 nm). Automated image acquisition was achieved using the BLINK toolbox software and a fluorescence microscope (Zeiss AxioObserver) equipped with a 5x objective (field of view 4.416 mm x 2.774 mm) and a pE-4000 (CoolLED Ltd.) light source. The microscope was further equipped with three fluorescence filter sets (Cy5 ET, Cy3 ET, FITC / FAM HC, AHF Analysentechnik) and an automated XY stage to which a thermocycler with reaction chambers could be attached.

[0139] Bead segmentation and endpoint analysis All acquired images are subjected to an automated, multifaceted image processing algorithm developed by BLINK to detect and segment individual nanoreactor beads. Characteristics of all segmented nanoreactors, such as the fluorescence signal in each channel, their position, and their diameter / volume, are subsequently collected. Experimental data are processed using the open-source software Jupyter.

[0140] Accuplex HCV droplet digital PCR data are displayed in 1-D or 2-D plots. The software clustered the negative and positive fractions for each target and applied a Poisson algorithm to the fraction of positive events to determine the output copy number, expressed as a λ value (normalized to 100 μm bead diameter). The λ value was multiplied by the input number of nanoreactor beads to determine the output HCV copy number, respectively. The integrated sample preparation process and digital detection resulted in a mean recovery of 6697 copies with a standard deviation of 258 copies (n=2).

[0141] Example 5 Performing amplification and real-time detection using a primer bound to a first polymer Using biotinylated agarose as described in WO 2021 / 122563A1, beads containing biotinylated agarose as the first polymer were generated according to the protocol in Example 1. Analyte-specific beads were generated by conjugating target-specific primers and probes to precursor beads with defined fluorescent codes. All bead manipulations and washing steps were performed in a single laboratory tube (1.5 ml) with a magnetic rack. Precursor beads (25% volume bead bed in 100 mM Tris buffer, pH 8.9) were first derivatized with streptavidin (0.5 mg / ml in 100 mM Tris buffer, pH 8.9) at room temperature under constant shaking at 800 rpm for 30 min on a thermomixer. Excess streptavidin was removed by washing three times with 10 volumes of water. By adding 5' biotin to the primers and 3' biotin to the probe, bead-bound PCR reactions showed similar performance compared to PCR using unconjugated oligonucleotides. Primers and probes are immobilized on the beads in 100 mM Tris buffer (pH 8.9) at room temperature on a thermomixer for 30 min with constant shaking at 800 rpm. After incubation, unbound oligonucleotides are removed with 5 volumes of water. The biotin / streptavidin bond remains stable under acidic conditions during subsequent capture of genomic DNA (50 mM malonic acid, pH 2.5) onto the cross-linked chitosan polymer of the beads (room temperature on a thermomixer for 15 min with shaking at 800 rpm). After DNA capture, the beads are washed three times with 5 volumes of malonic acid (12.5 mM, pH 5.0) and then loaded with 2x concentrated PCR mix without oligonucleotides. 100 μl of PicoSurf oil (Spherefluidics) is then added to the suspension. The tube is closed, attached to a bead-beating device (Omnilyse, Bertin), and shaken three times for 5 seconds at 50% speed. The bead suspension is then transferred to an appropriate detection chamber for PCR incubation and image acquisition.

[0142] Detection of genomic targets using TaqMan-PCR probes in real-time PCR Beads containing Cy3-stained agarose, provided for background image segmentation, are pre-loaded with a desthiobiotinylated primer and a Cy5 / desthiobiotin-labeled probe (0.5 µM forward primer, 0.5 µM reverse primer, 0.4 µM probe) for detecting the gapA species marker of Staphylococcus aureus. As a negative control, use a population of beads labeled with FAM and lacking the primer and probe.

[0143] Capture of Staphylococcus aureus (S. aureus) genomic DNA isolates (input concentration set at 100 genome copies per bead) was performed by resuspending the beads in 100 μL of binding buffer (100 mM malonic acid, pH 2.5) and mixing the bead suspension with an equal volume of sample. After 15 min of incubation at room temperature, the beads were washed three times with 5x the volume of malonic acid (12.5 mM, pH 5.0) in a thermomixer at 800 rpm. Then, 2x PCR master mix was loaded onto the beads, resulting in the following PCR buffer composition on the beads: 100mM Tris / HCl pH 8.9 22mM ammonium chloride 22mM potassium chloride 3mM MgCl2 0.35mM dNTPs 0.1% BSA 0.2 units DNA polymerase

[0144] The primer / probe sequences used in this analysis were as follows:

[0145] [Table 2]

[0146] After resuspension in oil, amplification was performed (42 cycles of 2 min initial denaturation, 10 sec denaturation at 95°C, followed by 15 sec annealing / extension at 58°C). The PCR reaction was monitored in real time by fluorescent imaging on a Zeiss Axio Observer fluorescent microscope at every cycle during the annealing / extension step.

[0147] The image analysis algorithm segments beads on the image by recognizing bright, disc-shaped contours in the green channel, which correspond to the Cy3-generated signal. The resulting segmentation information allows us to estimate the average fluorescence level of all beads across all cycles (1-42) and all relevant channels (red / Cy5 for PCR probe signals, blue / Fam for control bead coding). All beads are assigned to a bead type using the signal intensity level measured in the blue channel (Fam) in the first cycle. Analyte-specific beads are characterized by low signal intensity, while negative control beads have high signal values.

[0148] In the field of view shown in the upper left panel of Figure 15, the total number of valid positive beads is 618, and the total number of valid control beads is 33. The real-time analysis algorithm fits a nonlinear function to the fluorescence signal course of each single bead, as shown in the lower left panel of Figure 15. The fitted nonlinear model combines a sigmoidal function and a linear function, where the sigmoidal component represents the amplification kinetics and the linear component represents the signal baseline. The cycle threshold (Ct) is calculated from the intersection of the tangent to the defined value of the sigmoidal function with the maximum quadratic derivative and the baseline, as shown in the right panel of Figure 15.

[0149] More specifically, Figure 15 shows the monitoring of a bead digital PCR reaction with the detection of S. aureus genomic DNA.

[0150] Figure 15, upper left panel: Shows the field of view of the Cy5 channel after amplification (PCR signals, bright and dark beads are clearly visible). The Cy3 image taken before PCR shows the analyte-specific beads, and the FAM image shows the control beads. The corresponding signals in the Cy5 image indicate that all analyte-specific beads show positive PCR signals, while the control beads remain dark. In this example, the target concentration was set at a very high level (approximately 100 cp / bead), so all analyte-specific beads show bright Cy5 signals. The graph in the lower left panel shows the real-time curves of Cy5 fluorescence for individual beads shown in the inset, as well as the average intensity of PCR-positive and negative beads. Three beads belong to the positive bead type and show strong amplification kinetics, with a Ct value of approximately 21.6 and a lift ratio of approximately 2.5. The density distribution of Ct values ​​estimated from all 618 valid beads of the positive bead type is shown on the right side of Figure 15. The fourth bead is a control bead with no increase in fluorescence intensity. Instead of analyzing the real-time curves for all beads individually and then summarizing the Ct values, this method also allows for the calculation of a single mean fluorescence intensity for each bead type. As shown in Figure 15 (left side) of this embodiment, there are two mean real-time curves, one for the positive bead type and one for the negative control bead type.

[0151] The features of the invention disclosed in this specification, in the claims and / or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

Claims

1. A microsphere comprising a first material and a second material, wherein the first material is capable of forming a porous hydrogel when exposed to or containing an aqueous solution and has a melting temperature ranging from 40°C to 90°C; the second material forms a network within the porous hydrogel, or the second material is attached to the first material and both materials together form a network within the porous hydrogel; the second material has a pKa value and is capable of coagulating in an aqueous environment where the pH is above the pKa value; the network is capable of shrinking when exposed to a temperature above the melting temperature of the first material; the microsphere further comprises at least one of a) a fluorescent label and b) a magnetic particle.

2. the first material is a first polymer, and the second material is a second polymer, oligomer, or monomer; When the second material is a second polymer, the second polymer is a polymer crosslinked with itself or with the first polymer; When the second material is an oligomer or a monomer, the oligomer or the monomer is attached to the first polymer. The microsphere of claim 1.

3. said second material, preferably said second polymer or said oligomer or said monomer, is further capable of binding nucleic acids at a pH below said pKa value, preferably said second material, preferably said second polymer or said oligomer or said monomer, has a pKa value above 6; The microsphere according to any one of claims 1 to 2.

4. 3. Microspheres according to any of claims 1 to 2, wherein the fluorescent label is selected from: a) fluorescent particles with a size ranging from 50 nm to 10 μm, preferably from 100 nm to 5 μm, more preferably from 1 μm to 5 μm, even more preferably from 1 μm to 3 μm; b) non-particulate fluorescent dyes attached to the first and / or second material, preferably the first and / or second polymer / oligomer / monomer; and c) a combination of a) and b).

5. Microspheres according to any of claims 1 to 2, wherein the size of the magnetic particles ranges from 50 nm to 10 μm, preferably from 100 nm to 5 μm, more preferably from 1 μm to 5 μm, even more preferably from 1 μm to 3 μm.

6. The first material, preferably the first polymer, comprises a binding member that allows reversible and pH-independent immobilization of one or several amplification primers to the first material, preferably the first polymer, and preferably, for such reversible and pH-independent immobilization, the binding member specifically interacts with a conjugate attached to the one or several amplification primers; more preferably, the microsphere is a binding member that is attached to and immobilized on the first material, preferably the first polymer, via the specific interaction between the binding member on the first material, preferably the first polymer, and the conjugate on the one or several amplification primers.

3. Microspheres according to any of claims 1 to 2, further comprising one or several amplification primers carrying a conjugate; even more preferably, said binding member is selected from avidin, streptavidin and derivatives thereof, and said conjugate is selected from desthiobiotin, iminobiotin, biotin with a cleavable spacer arm, selenobiotin, oxybiotin, homobiotin, norbiotin, diaminobiotin, biotin sulfoxide, biotin sulfone, epibiotin, 5-hydroxybiotin, 2-thiobiotin, azabiotin, carbobiotin, methylated derivatives of biotin, and ketone biotin, or vice versa.

7. the first material is a first polymer selected from agarose, gelatin, hyaluronan, elastin, elastin-like polypeptides, thermoresponsive polymers having an upper critical solution temperature (UCST), such as poly(N-acryloylglycinamide) (PNAGA), poly(allylamine)-co-poly(allylurea) and its derivatives, poly(methacrylamide), poly(N-acryloylaspartamide), poly(N-methacryloylglutamine amide), poly(acrylamide)-co-(acrylonitrile), poly(sulfobetaine), poly(phosphorylcholine); and other polymers capable of forming a porous hydrogel when exposed to or containing an aqueous solution and having a melting temperature ranging from 40° C. to 90° C.; and the second material is a second polymer selected from chitosan and its derivatives, gelatin and its derivatives, methylcellulose, poly(N-isopropylacrylamide) (pNIPAM), poly(ethyleneimine), poly(2-dimethyl(aminoethyl)methacrylate), poly(lysine), poly(histidine), poly(arginine), and a polymer backbone having a basic amino acid attached or incorporated as part of such backbone; or said second material is an oligomer, preferably an oligopeptide, selected from said oligopeptides comprising or consisting of basic amino acids such as histidine, lysine, and arginine; or said second material is a monomer selected from basic amino acids such as histidine, lysine, and arginine; with the proviso that when said first and second materials are first and second polymers, respectively, said polymers are different; Preferably, the first material is a non-crosslinked first polymer and the second material is a crosslinked second polymer; or equally preferably, the first material is a first polymer and the second material is a second polymer crosslinked to the first polymer; more preferably, the first polymer is agarose and the second polymer is crosslinked chitosan.

8. 10. The microsphere of claim 1, wherein the first material, preferably the first polymer, comprises an aqueous solution such that the first material is in the form of a porous hydrogel.

9. It also includes nucleic acids that are not oligonucleotide primers or oligonucleotide primers, - bound to said second material, preferably said second polymer, when said aqueous solution has a pH below said pKa value of said second material, preferably said second polymer; and - when the aqueous solution has a pH equal to or greater than the pKa value of the second material, preferably the second polymer, the first material, preferably the first polymer, is still held within microspheres beside or within the porous hydrogel, but is not necessarily bound or is no longer bound to the second material, preferably the second polymer, The microsphere of claim 8.

10. The aqueous solution is one of the following: a) a first composition for washing and / or promoting binding of nucleic acids to said second material, e.g., said second polymer, said first composition having a pH value below the pKa value of said second material, e.g., said second polymer, or more preferably said first composition comprising a buffer that buffers to a pH range below the pKa value of said second material, e.g., said second polymer; or b) a second composition for promoting and performing nucleic acid amplification, said second composition comprising a buffer for buffering to a pH range suitable for performing nucleic acid amplification, mono-nucleoside-triphosphates, an amplification enzyme such as a suitable nucleic acid polymerase, e.g., Taq polymerase, optionally one or more amplification primers or one or more sets of amplification primers if not already immobilized on said first material, preferably said first polymer; and optionally a nucleic acid dye for detection of an amplification product, e.g., an amplified nucleic acid; and / or optionally a respective molecular probe, e.g., a TaqMan probe or a molecular beacon; said pH range suitable for performing nucleic acid amplification is higher than the pKa value of said second material (e.g., said second polymer), The microsphere according to any one of claims 8 to 9.

11. 2. The microsphere of claim 1, wherein the second material, preferably the second polymer, is in a non-aggregated form.

12. 2. The microsphere of claim 1, wherein the second material, preferably the second polymer, is in an aggregated form.

13. A liquid phase immiscible with water and aqueous solutions, such as an oil phase, comprising a plurality of microspheres according to claim 11 or a plurality of microspheres according to claim 12.

14. 1. A method for capturing and / or concentrating a nucleic acid or nucleic acids from a sample, said method comprising any of the following: a) providing a plurality of microspheres as defined in claim 1 in or exposing the microspheres to a first composition for promoting binding of nucleic acids to the second material, preferably the second polymer; the first composition has a pH value less than the pKa value of the second material, e.g., the second polymer, or preferably, the first composition comprises a buffer that buffers the first composition to a pH range less than the pKa value of the second material, e.g., the second polymer; thereby allowing the microspheres to equilibrate with and incorporate the first composition; preferably, the pKa value of the second material, e.g., the second polymer, is greater than 6; and b) exposing the microspheres from step a) to a sample containing or suspected of containing nucleic acid, whereby the nucleic acid, if present, can be bound to the second material, e.g., the second polymer, the sample preferably also containing a buffer that buffers the pH range to a range below the pKa value of the second material, e.g., the second polymer, thereby capturing nucleic acid in the sample, if present; or a') exposing a plurality of microspheres as defined in claim 1 to a sample containing or suspected of containing nucleic acid, whereby said nucleic acid, if present, can be bound to said second material, e.g., said second polymer, said sample having a pH below the pKa value of said second material, e.g., said second polymer, preferably said sample comprising a buffer that buffers the pH range to below the pKa value of said second material, e.g., said second polymer; thereby capturing nucleic acid in said sample, if present, as a result of such exposure.

15. 15. A method for enriching nucleic acids from a sample, said method comprising carrying out the method of claim 14; and: Either (Option A): c) exposing the microspheres from step b) or step a') to a second composition for promoting and performing nucleic acid amplification, the second composition comprising a second buffer buffering to a pH range suitable for performing nucleic acid amplification, mono-nucleoside-triphosphates, an amplification enzyme such as a suitable nucleic acid polymerase, e.g., Taq polymerase, and, if the microsphere does not already comprise one or several amplification primers immobilized to the first material, preferably the first polymer, via specific interactions between binding members on the first material, preferably the first polymer, and conjugates on the one or several amplification primers, further comprising one or more amplification primers or one or more sets of amplification primers; thereby allowing the microspheres to equilibrate with and take up the second composition for promoting and performing nucleic acid amplification; optionally, the second composition further comprising a nucleic acid dye and / or a respective molecular probe, e.g., TaqMan probe or molecular beacon, for detection of amplification products, e.g., amplified nucleic acids; preferably, the pH range suitable for performing nucleic acid amplification is higher than the pKa value of the second material, e.g., the second polymer; d) subjecting the microspheres of step c) to an amplification protocol within the composition to promote and effect nucleic acid amplification, such protocol comprising, or preceded by, or followed by, at least one step of heating the suspension to a temperature above the melting temperature of the first material, preferably the first polymer, and preferably comprising repeatedly and cyclically raising and lowering the temperature to which the suspension of microspheres is exposed, if nucleic acids are present in the microspheres, to a level that allows amplification of nucleic acids that match the sequences of the one or more amplification primers or one or more sets of amplification primers; Or (option B): e) exposing the microspheres from step b) or step a') to a second composition for promoting and carrying out nucleic acid amplification, the second composition comprising a second buffer buffering to a suitable pH range for carrying out nucleic acid amplification, mono-nucleoside-triphosphates, an amplification enzyme such as a suitable nucleic acid polymerase, e.g., Taq polymerase, and wherein the microspheres bind to one or more binding members immobilized on the first material, preferably the first polymer, via specific interactions between binding members on the first material, preferably the first polymer, and conjugates on the one or several amplification primers. further comprises one or more amplification primers or one or more sets of amplification primers if it does not already comprise some amplification primers; thereby allowing the microspheres to equilibrate with and incorporate the second composition for facilitating and performing nucleic acid amplification; the second composition optionally further comprises a nucleic acid dye and / or a respective molecular probe, e.g., TaqMan probe or molecular beacon, for detection of amplification products, e.g., amplified nucleic acids; preferably, the pH range suitable for performing nucleic acid amplification is higher than the pKa value of the second material, e.g., the second polymer; f) transferring the microspheres from step e) to a liquid phase immiscible with water and aqueous solutions, such as an oil phase, thereby producing a suspension of the microspheres separated from each other by the water-immiscible liquid phase, such as an oil phase; and g) subjecting the microspheres of step f) to an amplification protocol, which protocol comprises or is preceded or followed by at least one step of heating the suspension to a temperature above the melting temperature of the first material, preferably the first polymer, and preferably comprises repeatedly and cyclically raising and lowering the temperature to which the suspension of microspheres is exposed to a level that allows amplification of nucleic acids that match the sequences of the one or more amplification primers or one or more sets of amplification primers, if nucleic acids are present in the microspheres, and Upon heating to a temperature above the melting temperature of the microsphere, the second material in the microsphere, preferably the second polymer, shrinks, thereby producing a shrunken core within each microsphere, and fluorescent labels and / or magnetic particles, if present or contained within each microsphere, accumulate within the shrunken core, thereby facilitating detection and / or further manipulation of each microsphere; preferably, i) the fluorescent labels and / or ii) the magnetic particles accumulated within the shrunken core of the microsphere are used to locate and / or identify an individual microsphere or multiple microspheres.

16. 16. The method of claim 15, further comprising the steps of: h) detecting the amplified nucleic acid, if present, in any of the following: in said compositions for promoting and performing nucleic acid amplification (Option A); or in said microspheres (option B); wherein the amplification protocol results in an optically detectable signal, and an optically detectable signal is generated. in said compositions for promoting and performing nucleic acid amplification (Option A); or in the space surrounding the contracted core of each microsphere (option B); wherein detection of said optically detectable signal indicates the presence of amplified nucleic acid.

17. 17. The method of any of claims 15-16, wherein an individual microsphere or a plurality of microspheres are located and / or identified using i) the fluorescent label and / or ii) the magnetic particles accumulated within the contracted core of the microsphere.

18. 17. The method of any of claims 15-16, wherein fused microspheres are distinguished from non-fused microspheres using i) the fluorescent label and / or ii) the magnetic particles accumulated within the shrunken core of the microsphere; and / or iii) the presence of more than one shrunken core in fused microspheres; and / or iv) the presence of only one shrunken core in non-fused microspheres.

19. 17. Use of a plurality of microspheres as defined in any of claims 1-2 in an assay for the detection of multiple nucleic acid analytes, said use comprising carrying out a method according to any of claims 15-16, said use comprising different microspheres, each microsphere comprising a different fluorescent label and specific for the detection of a particular nucleic acid analyte by comprising a specific amplification primer or a specific set of such amplification primers for the amplification of said particular nucleic acid analyte, wherein said different microspheres are detectably distinguishable from one another by means of said fluorescent labels, and wherein each microsphere is specific for a different nucleic acid analyte.

20. 17. Use of a plurality of microspheres as defined in any of claims 1-2 in an assay for the detection of a single nucleic acid analyte in multiple samples, said use comprising carrying out a method according to any of claims 15-16, said use comprising different microspheres, each microsphere comprising a different fluorescent label, and further comprising an amplification primer or a set of such amplification primers for amplification of said single nucleic acid analyte, each microsphere being specific for a particular sample by being separately exposed to said particular sample in step b) or a'), said different microspheres being detectably distinguishable from one another by means of said fluorescent labels, and each microsphere being specific for the same nucleic acid analyte but for different samples.

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