Library of fabricated microparticles and their precursors

Abstract

The present invention relates to prefabricated microparticles, and precursors of such microparticles (sometimes referred to herein as "precursor microparticles"), for the specific detection of one or several analytes in a sample. In particular, the present invention relates to libraries of such prefabricated microparticles and libraries of such precursor microparticles. Furthermore, the present invention relates to kits for producing such libraries and kits for using such libraries to detect analytes of interest in a sample. Furthermore, the present invention relates to methods of detecting and / or quantifying analytes of interest in an aqueous sample, preferably using such kits.

Description

Technical Field

[0001] Background of the Invention The present invention relates to fabricated microparticles for the specific detection of one or several analytes in a sample, and precursors of such microparticles (which may also be referred to herein as "precursor microparticles"). In particular, the present invention relates to libraries of such fabricated microparticles and libraries of such precursor microparticles. Furthermore, the present invention relates to kits for making such libraries and kits for using such libraries to detect a target analyte in a sample. Furthermore, the present invention relates to a method for detecting and / or quantifying a target analyte in an aqueous sample, preferably using such a kit.

Background Art

[0002] It is desirable to detect multiple analytes in a sample with high sensitivity. Various methods have been developed for simultaneously detecting analytes in solution. For example, in the case of highly sensitive detection of nucleic acids, methods that enable the detection of nucleic acid sequences in a sample by template-specific amplification are used. For this purpose, widely and well-reported methods such as polymerase chain reaction (PCR), recombinase polymerase reaction (RPA), transcription-mediated amplification (TMA), loop-mediated amplification reaction (LAMP), etc. are available and are being used. However, due to the cross-reactivity of the reagents used, the achievable level of multiplexing is limited, and the number of targets detected in the same assay is limited to a small number.

[0003] For highly sensitive detection of protein antigens, template-dependent amplification is not possible. However, nucleic acid amplification methods are combined with classical immunoassay formats. For example, immunoPCR (Adler, Wacker et al., 2003) uses a conjugated antibody immobilized on a solid phase and a detection antibody to which a nucleic acid sequence label is attached. After a sandwich consisting of the first antibody, antigen, and second antibody is formed, the nucleic acid sequence attached to the second antibody is amplified by an amplification reaction. In another assay format (Todd, Freese et al., 2007), a sandwich is formed between an antibody bound to a solid phase, the antigen, and a second antibody to which a fluorophore is provided. The fluorophore is selectively and quantitatively cleaved and counted one molecule at a time using a molecular flow counter. The number of detected fluorophores directly corresponds to the number of bound analytes.

[0004] The method known as SiMOA (Rissin, Kan et al., 2010) ("Single-molecule array") is also based on the digital measurement of single-binding events. First, the antigen is captured on the outside of a solid particle by a first antibody. A second antibody, labeled with an enzyme, binds to the antigen. These solid particles are brought into contact with a solution containing a substrate that is converted into a fluorescent product by the enzyme. The particles are placed in small cavities of an array so that only one particle is contained in each cavity and a small amount of the solution. The individual cavities are then isolated by covering them with a non-aqueous solution, which acts as a reaction space. By counting these individual cavities with a detectable signal and quantifying the signal within each cavity, the antigen concentration can be measured with ultra-high sensitivity.

[0005] Magnetic particles are used to facilitate improved handling of the microcarriers used (Tekin and Gijs 2013). (Leng, Zhang et al., 2010) describe hydrogel particles to which primer nucleotides are covalently attached, using a modified agarose polymer for primer binding. The primer-modified agarose is then mixed with an amplification reagent and a target-containing solution, and hydrogel microparticles are formed using a microfluidic droplet preparation apparatus. The agarose emulsion formed in oil is applied to amplification / detection.

[0006] Parallel detection of multiple analytes in a sample that meets the following requirements is needed in this field: a) No mutual interference of analyte-specific reactions. b) High sensitivity and high specificity for each analyte, c) It has a simple mechanism and consists of an analyte-specific reagent and minimal equipment.

[0007] The present invention aims to provide and satisfy these requirements. [Overview of the project] [Means for solving the problem]

[0008] Summary of the Invention The present invention will be described by reference to various aspects and embodiments:

[0009] In a first embodiment, the present invention relates to the following:

[0010] 1. A library of pre-fabricated precursor microparticles for preparing a library of pre-fabricated microparticles, wherein the pre-fabricated microparticles are configured to perform specific detection of one or more target analytes in a sample, and such specific detection is performed within such microparticles by a suitable chemical or biochemical reaction, and each of the pre-fabricated precursor microparticles in the library is - A porous matrix having void volume for receiving aqueous samples and providing a reaction space for specific detection of analytes; - A reagent-binding component that enables the attachment, preferably reversible attachment, of an analyte-specific reagent to precursor fine particles; the reagent-binding component is (i) A polymer or polymer mixture that forms or is the porous matrix; (ii) Reagent-binding molecules attached to the porous matrix; (iii) at least one or more ionizable groups immobilized on the porous matrix, wherein the ionizable groups are capable of changing their charge according to the ambient conditions around the precursor nanoparticles; (iv) at least one or more charged groups immobilized on the porous matrix; (v)(i)~(iv) Any combination One of them is a reagent-binding component; -When the analyte-specific reagent is attached to the precursor microparticles, a labeling component attached to, contained in, or otherwise associated with the precursor microparticles for identifying the analyte-specific reagent A library that includes this.

[0011] 2. The library according to Embodiment 1, wherein the library contains at least two separate fabricated precursor microparticle subsets, preferably three or more separate fabricated precursor microparticle subsets, each subset having a different labeling component attached to, contained in, or otherwise associated with the precursor microparticles within the subset, and each of the at least two or more separate fabricated precursor microparticle subsets has a different labeling component attached to, contained in, or otherwise associated with each subset.

[0012] 3. The library according to any one of Embodiments 1 to 2, wherein the labeling component is a mixture of at least two different dyes, preferably at least two fluorescent dyes, and the at least two different dyes, preferably the at least two fluorescent dyes, are present on each subset of the precursor microparticles in a predetermined ratio and / or predetermined amount of each of the at least two different dyes, and the different subsets of precursor microparticles differ from each other in terms of the respective ratio and / or amount of the at least two different dyes, and thus the different subsets of precursor microparticles can be distinguished by the respective ratio and / or amount of the at least two different dyes attached to or contained in each subset of precursor microparticles.

[0013] 4. The library according to any one of Embodiments 1 to 3, wherein the porous matrix is ​​a porous polymer matrix.

[0014] 5. In a further embodiment, the present invention also relates to a library of fabricated microparticles for specific detection of one or more target analytes in a sample, wherein such specific detection is carried out in such microparticles by a suitable chemical or biochemical reaction, and each of the fabricated microparticles comprises a fabricated precursor microparticle described in any of Embodiments 1 to 4, and further comprises an analyte-specific reagent attached, preferably reversibly attached, to the precursor microparticle.

[0015] 6. The analyte-specific reagent attached to each of the precursor microparticles a) Direct binding of the analyte-specific reagent to the polymer or polymer mixture (i) that forms or is part of the porous polymer matrix; b) The analyte-specific reagent conjugated to a binding entity that binds to the reagent-binding molecule (ii); preferably, the reagent-binding molecule and the binding entity are selected to interact with each other in a reversible manner, i.e., to bind to each other; c) Direct binding of the analyte-specific reagent to the ionizable group (iii) under conditions in which the ionizable group has a suitable net charge; d) Direct bonding of the analyte-specific reagent to the charged group(iv) on the polymer, wherein the analyte-specific reagent has at least one or more ionizable groups, and the ionizable groups are capable of changing charge according to ambient conditions around the analyte-specific reagent; or e) Any combination of (a) to (d) A library of fabricated microparticles according to Embodiment 5, which are attached via the reagent-binding component, preferably in a reversible manner.

[0016] 7. The polymer (i) is a hydrogel-forming agent selected from the group comprising naturally occurring polymers selected from: ia) synthetic polymers, e.g., poly(methyl)(meth)acrylate, polyamide; ib) silicone polymers, e.g., polydimethylsiloxane; ic) polysaccharides, e.g., agarose, chitin, chitosan, dextran, alginate, carrageenan, cellulose, fucoidan, laminaran; gums selected from xanthan gum, acacia gum, gatchigum, guar gum, locust bean gum, tragacanth gum, karaya gum, and inulin; polypeptides, e.g., collagen, gelatin; polyamino acids, e.g., polylysine; and polynucleotides; and the polymer mixture is any combination of the above; The reagent-binding molecule (ii) is selected from avidin, particularly tetrameric avidin; streptavidin; monomeric avidin; avidin having nitrated tyrosine in the biotin-binding site; other proteins derived from or related to avidin and retaining the binding function of avidin; biotin; desthiobiotin; iminobiotin; biotin having a cleavable spacer arm; selenobiotin; oxybiotin; homobiotin; norbiotin; iminobiotin; diaminobiotin; biotin sulfoxide; biotin sulfone; epibiotin; 5-hydroxybiotin; 2-thiobiotin; azabiotin; carbobiotin; methylated derivatives of biotin; ketone biotin; and other molecules derived from or related to biotin and retaining the binding function of biotin; in embodiments where the attachment of the analyte-specific reagent is reversible, the reagent-binding molecule and the binding entity are preferably selected to interact with each other in a reversible manner, i.e., to bind to each other; The at least one ionizable group (iii) or the ionizable group as described in d) of embodiment 6 is · N-2-acetamido-2-aminoethanesulfonic acid (ACES); · N-2-acetamido-2-imino diacetic acid (ADA); · Aminomethylpropanediol (AMP); · 3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (AMPSO); · N,N-bis 2-hydroxyethyl-2-aminoethanesulfonic acid (BES); · N,N-bis-2-hydroxyethylglycine (BICINE); · Bis-2-hydroxyethyliminotris hydroxymethylmethane (Bis-Tris); · 1,3-bis tris hydroxymethylmethylaminopropane (Bis-Tris propane); · 4-cyclohexylamino-1-butanesulfonic acid (CABS); · 3-cyclohexylamino-1-propanesulfonic acid (CAPS); · 3-Cyclohexylamino-2-hydroxy-1-propanesulfonic acid (CAPSO); · 2-N-Cyclohexylaminoethanesulfonic acid (CHES); · 3-N,N-Bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (DIPSO); · N-2-Hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS); · N-2-Hydroxyethylpiperazine (hydroxyethylpiparazine)-N-4-butanesulfonic acid (HEPBS); · N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES); · N-2-Hydroxyethylpiperazine-N-2-propanesulfonic acid (HEPPSO); · 2-N-Morpholinoethanesulfonic acid (MES); · 4-N-Morpholinobutanesulfonic acid (MOBS); · 3-N-Morpholinopropanesulfonic acid (MOPS); · 3-N-Morpholino-2-hydroxypropanesulfonic acid (MOPSO); · Piperazine-N-N-bis-2-ethanesulfonic acid (PIPES); · Piperazine-N-N-bis-2-hydroxypropanesulfonic acid (POPSO); · N-Tris hydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS); · N-Tris hydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS); · 3-N-Tris hydroxymethyl-methylamino-2-hydroxypropanesulfonic acid (TAPSO); · N-Tris hydroxymethyl-methyl-2-aminoethanesulfonic acid (TES); · N-Tris hydroxymethylmethylglycine (TRICINE); · Tris hydroxymethylaminomethane (Tris); · Polyhydroxylated amine; Imidazoles and their derivatives (i.e., imidazoles), particularly derivatives containing a hydroxyl group; Triethanolamine dimers and triethanolamine polymers; and Di / tri / oligo / polyamino acids, e.g., Ala-Ala; Gly-Gly; Ser-Ser; Gly-Gly-Gly; or Ser-Gly. Oligo-His, poly-His, oligo-Lys, poly-Lys Selected from, A library of fabricated precursor microparticles according to any one of Embodiments 1 to 4, or a library of fabricated microparticles according to any one of Embodiments 5 to 6.

[0017] 8. A library of fabricated precursor microparticles according to any one of Embodiments 1 to 4, 7 or a library of fabricated microparticles according to any one of Embodiments 5 to 7, wherein the porous polymer matrix, or the polymer or polymer mixture forming the porous polymer matrix or being part of the porous polymer matrix, is composed of an uncrosslinked polymer or polymer mixture, preferably the polymer or polymer mixture forming the porous polymer matrix or being part of the porous polymer matrix is ​​composed of agarose, or a combination of agarose and gelatin, more preferably the combination of agarose and gelatin, wherein the agarose is present in the range of 0.1% (w / v) to 4% (w / v), and the gelatin is present in the range of 0.1% (w / v) to 20% (w / v), preferably 0.5% (w / v) to 20% (w / v).

[0018] 9. A library of fabricated microparticles according to any one of Embodiments 5 to 8, wherein the analyte-specific reagent is selected from nucleic acids including aptamers and Spiegelmers; antibodies or antibody fragments; and non-antibody proteins capable of specifically binding to the analyte or analyte complex, such as receptors, receptor fragments, and affinity proteins; preferably, the analyte-specific reagent is selected from nucleic acids, particularly nucleic acid oligomers and nucleic acid primers.

[0019] 10. For each of the fine particles, the analyte-specific reagent is reversibly attached to the fine particles via the reagent-binding component by the analyte-specific reagent being conjugated to a binding entity that binds to the reagent-binding molecule (ii) (i). - The binding entity is independently selected from biotin, desthiobiotin, iminobiotin, biotin with a cleavable spacer arm, selenobiotin, oxybiotin, homobiotin, norbiotin, iminobiotin, diaminobiotin, biotin sulfoxide, biotin sulfone, epibiotin, 5-hydroxybiotin, 2-thiobiotin, azabiotin, carbobiotin, methylated derivatives of biotin, ketonebiotin, and other molecules derived from or related to biotin and retaining the biotin binding function; the reagent-binding molecule is independently selected from avidin and streptavidin; or vice versa; or - The binding entity is biotin, and the reagent-binding molecule is selected from monomeric avidin, avidin having nitrated tyrosine at the biotin-binding site, and other proteins derived from or related to avidin and retaining the avidin-binding function; or vice versa; in this preferred embodiment, the reagent-binding molecule and the binding entity are selected to interact with each other in a reversible manner, i.e., to bind to each other. A library of fabricated microparticles according to any of Embodiments 5 to 9.

[0020] 11. The library is a library for the specific detection of a single target analyte in several samples, and the library contains at least two distinct pre-fabricated microparticle subsets, preferably three or more distinct pre-fabricated microparticle subsets. Each subset is, The subset of the fine particles has different labeling components attached thereto, contained therein, or otherwise associated therewith; All of the aforementioned two, three or more distinct subsets The subset of the fine particles has the same analyte-specific reagent attached to its porous matrix, and the analyte-specific reagent is specific to one target analyte; The aforementioned two or more separate fabricated microparticle subsets are identical with respect to the attached analyte-specific reagent, Each subset of the fine particles has different labeling components attached to, contained within, or otherwise associated with them; Each subset is clearly defined and identifiable by its respective label component. A library of fabricated microparticles according to any of Embodiments 5 to 10.

[0021] 12. The library is a library for the specific detection of multiple target analytes in a single sample, and the library contains at least two distinct pre-fabricated microparticle subsets, preferably three or more distinct pre-fabricated microparticle subsets. Each subset is, The subset of the fine particles has different labeling components attached thereto, contained therein, or otherwise associated therewith; The subset of the fine particles has different analyte-specific reagents attached to its porous matrix; each analyte-specific reagent is specific to one target analyte; The aforementioned two or more separate fabricated microparticle subsets are Each of the labeling components attached to, contained within, or otherwise associated with the fine particles of each subset; and Each subset is fitted with its respective analyte-specific reagent. They are different; Each subset is clearly defined and identifiable by the respective labeling component and the respective analyte-specific reagent. A library of fabricated microparticles according to any of Embodiments 5 to 10.

[0022] 13. The library is a library for the specific detection of multiple target analytes in several samples, and the library contains multiple different and separate pre-fabricated microparticle subsets. Each subset is, The subset of the fine particles has different labeling components attached thereto, contained therein, or otherwise associated therewith; Among the aforementioned multiple separate pre-fabricated microparticle subsets, there exist separate pre-fabricated microparticle subsets of different classes, each of which comprises several microparticle subsets, and each class has different analyte-specific reagents attached to the porous matrix of the microparticles, with the same analyte-specific reagent attached to all microparticle subsets within a single class; each analyte-specific reagent is specific to one target analyte; In the aforementioned library, the multiple distinct and separate fabricated microparticle subsets are, Each subset of the fine particles has different labeling components attached to, contained within, or otherwise associated with them; Each separate microparticle subset forms part of a class of microparticle subsets; Each subset is clearly defined and identifiable by its respective labeling component and its respective analyte-specific reagent; The aforementioned different classes of microparticle subsets differ in the analyte-specific reagents attached to the porous matrix of the microparticles; each of the aforementioned different classes comprises several microparticle subsets, and all of those subsets within one class have the same analyte-specific reagents. A library of fabricated microparticles according to any of Embodiments 5 to 10.

[0023] 14. The aforementioned kit, It includes at least two containers, namely the first and second containers, and optionally further containers, each of which is The two fabricated precursor microparticle subsets include a subset of pre-fabricated precursor microparticles as defined in any of embodiments 1-4, 7, and 8, each subset having a different labeling component attached to, contained within, or otherwise associated with the precursor microparticles within the subset, and the two sets of pre-fabricated precursor microparticle subsets have different labeling components attached to, contained within, or otherwise associated with each subset; Further containers are a) Direct binding of the analyte-specific reagent to the polymer or polymer mixture (i) that forms or is part of the porous polymer matrix; b) The analyte-specific reagent, which has been conjugated to a binding entity that binds to the reagent-binding molecule (ii); c) Direct binding of the analyte-specific reagent to the ionizable group (iii) under conditions in which the ionizable group has a suitable net charge; d) Direct bonding of the analyte-specific reagent to the charged group(iv) on the polymer, wherein the analyte-specific reagent has at least one or more ionizable groups, and the ionizable groups are capable of changing charge according to ambient conditions around the analyte-specific reagent; or e) Any combination of (a) to (d) The conditioning solution comprises a reagent-binding component that enables the attachment of the analyte-specific reagent to the subset of precursor microparticles, preferably reversible attachment; A kit for preparing a library of pre-fabricated microparticles according to any of Embodiments 5 to 13, wherein the reagent-binding component is as defined in Embodiment 1, the reagent-binding molecule is as defined in any of Embodiments 1, 6, and 7, the binding entity is as defined in any of Embodiments 6 to 7; the polymer or polymer mixture is as defined in any of Embodiments 1, 6 to 8; the ionizable group is as defined in any of Embodiments 1, 6, and 7, and the analyte-specific reagent is as defined in any of Embodiments 5 to 6, 9, and 10.

[0024] 15. The kit according to Embodiment 14, further comprising a further container containing a washing buffer for removing free, i.e., unattached, analyte-specific reagents from the microparticles without removing any analyte-specific reagents attached to the microparticles.

[0025] 16. At least one mixing vessel for mixing the components, preferably several mixing vessels, more preferably the same number of mixing vessels as the number of vessels each containing a subset of precursor fine particles. A kit according to any one of embodiments 14 and 15, further comprising:

[0026] 17. In a further embodiment, the present invention also relates to a method for preparing a library of fabricated microparticles as described in any of Embodiments 5 to 13, the method being: - • A library of fabricated precursor microparticles as defined in any of embodiments 1-4, 7, and 8; • At least one analyte-specific reagent as defined in any of embodiments 5-6, 9, and 10 A process of providing in any order; - A step of producing a library of fabricated microparticles according to any of Embodiments 5 to 13 by mixing the library of fabricated precursor microparticles or a selected subset thereof with the at least one analyte-specific reagent, preferably under conditions that allow for the attachment of the at least one analyte-specific reagent to some or all of the fabricated precursor microparticles, preferably under conditions that allow for reversible attachment; - If necessary, wash the prepared microparticles to remove any unattached analyte-specific reagents. Methods that include...

[0027] 18. The method is carried out by using a kit as defined in any of embodiments 14 to 16, wherein the library is provided in the form of at least two containers of the kit, each container being The fabricated precursor microparticle subsets include those defined in any of Embodiments 1-4, 7, and 8, each subset having a different labeling component attached to, contained within, or otherwise associated with the precursor microparticles within the subset, and the at least two fabricated precursor microparticle subsets each have a different labeling component attached to, contained within, or otherwise associated with each subset; The method of Embodiment 17, wherein the conditions that enable the attachment of the at least one analyte-specific reagent to some or all of the fabricated precursor microparticles, preferably reversible attachment, are brought about by mixing the library of fabricated precursor microparticles or a selected subset thereof with the at least one analyte-specific reagent in the presence of a conditioning solution as defined in Embodiment 14, wherein the conditioning solution is preferably a buffer.

[0028] 19. A kit for detecting an analyte in a sample, wherein the kit comprises: a) - A container comprising a general detection composition comprising reagents for carrying out a chemical or biochemical detection reaction of an analyte, wherein the chemical or biochemical detection reaction of the analyte is nucleic acid amplification, and the general detection composition comprises a buffer, mononucleoside triphosphate, amplification enzyme, e.g., a suitable nucleic acid polymerase, e.g., Taq polymerase, and amplification product, e.g., a nucleic acid dye for detection of amplified nucleic acids, or - A container comprising a first detection composition comprising a reagent for carrying out a chemical or biochemical detection reaction of an analyte, and a further container comprising a second detection composition comprising a detection reagent, wherein the chemical or biochemical detection reaction of the analyte is an immunochemical detection reaction, and the first detection composition comprises a reagent for carrying out an immunochemical detection reaction, e.g., a buffer, and a secondary antibody or secondary antibody fragment that is specific to the same analyte as the primary antibody, antibody fragment or non-antibody protein used as an analyte-specific reagent (ASR) in the immunochemical detection reaction and is conjugated to a suitable reporter enzyme; the second detection composition comprises a substrate suitable for the reporter enzyme as a detection reagent, wherein the substrate becomes detectable, preferably optically detectable, more preferably fluorescently detectable, when reacted with the reporter enzyme, the container and further containers, or - A container comprising a first detection composition comprising a reagent for carrying out a chemical or biochemical detection reaction of an analyte, and a further container comprising a second detection composition comprising a detection reagent, wherein the chemical or biochemical reaction is an immunochemical detection reaction, and the first detection composition comprises a reagent for carrying out an immunochemical detection reaction, e.g., a buffer, and a secondary antibody or secondary antibody fragment that is specific to the same analyte as the primary antibody used as an analyte-specific reagent (ASR) in the immunochemical detection reaction and is conjugated to a suitable oligonucleotide tag; the second detection composition comprises a buffer, a mononucleoside triphosphate, an amplification enzyme, e.g., a suitable nucleic acid polymerase, e.g., Taq polymerase, and an amplification product, e.g., a nucleic acid dye for detection of amplified nucleic acids, and a primer suitable for amplifying the oligonucleotide tag attached to the secondary antibody, as a detection reagent; and b) - A container containing a non-aqueous phase, such as oil, such as fluorocarbon oil, supplemented with emulsifiers as needed; and c) - Mixing container for mixing ingredients A kit that includes this.

[0029] 20. d) - Container for conducting the detection reaction The kit according to embodiment 19, further comprising:

[0030] twenty one. e) - A kit as described in any of embodiments 14 to 16, or - Library of fabricated microparticles as described in any of Embodiments 5 to 13 A kit according to any one of embodiments 19 to 20, further comprising:

[0031] 22. In a further embodiment, the present invention also relates to a method for detecting and / or quantifying an analyte of interest in an aqueous sample, the method comprising the following steps: - • Aqueous samples known or suspected to contain the analyte of interest; • A general detection composition containing reagents for carrying out chemical or biochemical detection reactions of the analyte; A library of fabricated microparticles according to any one of Embodiments 5 to 13, wherein the analyte-specific reagent attached to the microparticles is selected to specifically bind to or react with the target analyte. A process of providing in any order; - If necessary, a step of mixing the aqueous sample with the general detection composition; -A step comprising incubating the aqueous sample with the library of fabricated microparticles, thereby allowing the library of microparticles to absorb the aqueous sample into the void volume of the microparticles, and, if necessary, enabling binding to the target analyte if present in the sample; - A step of washing the fine particles if necessary; - If the aqueous sample has not been previously mixed with the general detection composition, the step of adding the general detection composition to the fine particles; - A step of creating a plurality of isolated reaction spaces for detecting the analyte by transferring the library of fabricated microparticles to a non-aqueous phase and removing the aqueous phase around each individual fabricated microparticle, wherein the reaction spaces include an aqueous phase and are limited to the void volume of the microparticles; - A step of releasing the analyte-specific reagent attached to the fine particles by applying an external trigger, if necessary; - A step of performing a detection reaction for the target analyte; - Steps to detect and / or quantify the target analyte. Includes.

[0032] 23. The target analyte is a nucleic acid, - The analyte-specific reagent is a nucleic acid or nucleic acid pair that is sufficiently complementary to the target analyte to hybridize with the target analyte under hybridization conditions, and preferably the analyte-specific reagent is a primer or primer pair suitable for amplification of the target analyte; - The general detection composition comprises reagents other than primers for carrying out the amplification reaction of the nucleic acid analyte of the target, and in particular, the general detection composition comprises a buffer, a mononucleoside triphosphate, an amplification enzyme, such as a suitable nucleic acid polymerase, such as Taq polymerase, and an amplification product, such as a nucleic acid dye for the detection of amplified nucleic acids; - The transfer step comprises transferring the prepared fine particles to a non-aqueous phase, suspending them, and washing them repeatedly in the non-aqueous phase as necessary, and the step further includes, as necessary, filtration or mechanical stirring to ensure that no or substantially any aqueous phase remains on the outer surface of the fine particles to form a monodisperse suspension of the fine particles; - The optional step of applying an external trigger to release the analyte-specific reagent attached to the fine particles is a step of temporarily raising the temperature, changing the pH, or changing the salt conditions, preferably raising the temperature, more preferably raising the temperature to >90°C above ambient temperature. - The step of performing the detection reaction is a step of performing an amplification reaction of the analyte when the analyte is present in the aqueous sample; - The step of detecting and / or quantifying the target analyte is the step of detecting and / or quantifying the amplified analyte. The method according to Embodiment 22.

[0033] 24. In a further embodiment, the present invention also relates to a method for detecting and / or quantifying an analyte of interest in an aqueous sample, the method comprising the following steps: - • Aqueous samples known or suspected to contain the analyte of interest; A first detection composition comprising reagents necessary for carrying out a chemical or biochemical detection reaction of the analyte; • A second detection composition containing a detection reagent; • Library of fabricated microparticles as described in any of Embodiments 5 to 13 A step of providing in any order, wherein the analyte-specific reagent attached to the fine particles is selected so as to specifically bind to or react with the target analyte; - If necessary, a step of mixing the aqueous sample with the first detection composition; -A step comprising incubating the aqueous sample with the library of fabricated microparticles, and if the aqueous sample has not yet been mixed with the first detection composition, also incubating it with the first detection composition, thereby allowing the library of microparticles to absorb the aqueous sample and the first detection composition into the void volume of the microparticles, and, if necessary, to bind to the target analyte if it is present in the sample; - If necessary, wash the library of the prepared microparticles to remove any unabsorbed or unreacted first detection composition; -A step comprising incubating the library of fabricated microparticles containing the absorbed aqueous sample with the second detection composition, thereby enabling the library of microparticles to absorb the second detection composition; - If necessary, further wash the library of the prepared microparticles to remove any unabsorbed or unreacted second detection composition; - A step of creating a plurality of isolated reaction spaces for detecting the analyte by transferring the library of fabricated microparticles to a non-aqueous phase and removing the aqueous phase around the individual fabricated microparticles, wherein the reaction spaces include an aqueous phase and are limited to the void volume of the microparticles; preferably, the step of transferring to the non-aqueous phase is performed immediately after incubating the library of fabricated microparticles with the second detection composition; - A step of releasing the analyte-specific reagent attached to the fine particles by applying an external trigger, if necessary; - A step of detecting and / or quantifying the target analyte by detecting and / or quantifying the detection reagent. Includes.

[0034] 25. The target analyte is a protein or other non-nucleic acid molecule. - The analyte-specific reagent is a primary antibody, antibody fragment, or non-antibody protein that can specifically bind to the protein analyte or other non-nucleic acid analyte; - The first detection composition comprises reagents necessary for carrying out an immunochemical detection reaction, such as a buffer, and a secondary antibody or secondary antibody fragment that is specific to the analyte and conjugated to a suitable reporter enzyme; - The second detection composition contains a substrate suitable for the preferred reporter enzyme as a detection reagent, wherein the substrate becomes detectable, preferably optically detectable, and more preferably fluorescently detectable, upon reaction with the reporter enzyme; - The step of incubating the aqueous sample with the library of fabricated microparticles and the first detection composition is, if the analyte is present in the aqueous sample, a step of forming a complex of the analyte and the primary antibody, primary antibody fragment or non-antibody protein by performing an immunochemical reaction including the binding of the analyte to the primary antibody, primary antibody fragment or non-antibody protein; the immunochemical reaction further includes the binding of the secondary antibody to the complex, thereby forming a sandwich between the primary antibody, antibody fragment or non-antibody protein, the analyte and the secondary antibody; -A first optional step for washing the library is a step of removing unbound secondary antibodies from the library; - The step of incubating the library of prepared microparticles containing the absorbed aqueous sample with the second detection composition is a step that allows the substrate to be reacted with the reporter enzyme; -A further optional step for washing the library is to remove unreacted substrates from the library; - The transfer step includes transferring the prepared fine particles to a non-aqueous phase, suspending them, and washing them repeatedly in the non-aqueous phase as necessary, and further including, as necessary, filtration or mechanical stirring to ensure that no or substantially any aqueous phase remains on the outside of the fine particles to form a monodisperse suspension of the fine particles; - The optional step of applying an external trigger to release the analyte-specific reagent attached to the fine particles is a step of temporarily increasing the temperature, changing the pH, or changing the salt conditions, preferably increasing the temperature, more preferably increasing the temperature to >90°C above ambient temperature; - The step of detecting and / or quantifying the target analyte is the step of detecting and / or quantifying the reacted substrate. The method according to Embodiment 24.

[0035] 26. In a further embodiment, the present invention also relates to a method for detecting and / or quantifying an analyte of interest in an aqueous sample, the method comprising the following steps: - • Aqueous samples known or suspected to contain the analyte of interest; A first detection composition comprising reagents necessary for carrying out a chemical or biochemical detection reaction of the analyte; • A second detection composition containing a detection reagent; • Library of fabricated microparticles as described in any of Embodiments 5 to 13 A step of providing in any order, wherein the analyte-specific reagent attached to the fine particles is selected so as to specifically bind to or react with the target analyte; - If necessary, a step of mixing the aqueous sample with the first detection composition; -A step comprising incubating the aqueous sample with the library of fabricated microparticles, and if the aqueous sample has not yet been mixed with the first detection composition, also incubating it with the first detection composition, thereby allowing the library of microparticles to absorb the aqueous sample and the first detection composition into the void volume of the microparticles, and, if necessary, to bind to the target analyte if it is present in the sample; - If necessary, wash the library of the prepared microparticles to remove any unabsorbed or unreacted first detection composition; -A step comprising incubating the library of fabricated microparticles containing the absorbed aqueous sample with the second detection composition, thereby enabling the library of microparticles to absorb the second detection composition; - If necessary, further wash the library of the prepared microparticles to remove any unabsorbed or unreacted second detection composition; - A step of creating a plurality of isolated reaction spaces for detecting the analyte by transferring the library of fabricated microparticles to a non-aqueous phase and removing the aqueous phase around each individual fabricated microparticle, wherein the reaction spaces include an aqueous phase and are limited to the void volume of the microparticles; - A step of releasing the analyte-specific reagent attached to the fine particles by applying an external trigger, if necessary; - A step of performing a detection reaction for the target analyte; and - Steps to detect and / or quantify the target analyte. Includes.

[0036] 27. The target analyte is a protein or other non-nucleic acid molecule. - The analyte-specific reagent is a primary antibody, antibody fragment, or non-antibody protein that can specifically bind to the protein analyte or other non-nucleic acid analyte; - The first detection composition comprises reagents necessary for carrying out an immunochemical detection reaction, such as a buffer, and a secondary antibody or secondary antibody fragment that is specific to the same analyte as the primary antibody and conjugated to a suitable oligonucleotide tag; -The second detection composition comprises a buffer, a mononucleoside triphosphate, an amplification enzyme, such as a suitable nucleic acid polymerase, such as Taq polymerase, and an amplification product, such as a nucleic acid dye for the detection of amplified nucleic acids, and a primer suitable for amplifying oligonucleotide tags attached to a secondary antibody, as a detection reagent; - The step of incubating the aqueous sample with the library of fabricated microparticles and the first detection composition is, if the analyte is present in the aqueous sample, a step of forming a complex of the analyte and the primary antibody, primary antibody fragment or non-antibody protein by performing an immunochemical reaction including the binding of the analyte to the primary antibody, primary antibody fragment or non-antibody protein; the immunochemical reaction further includes the binding of the secondary antibody to the complex, thereby forming a sandwich between the primary antibody, antibody fragment or non-antibody protein, the analyte and the secondary antibody; -A first optional step for washing the library is a step of removing unbound secondary antibodies from the library; - The step of incubating the library of fabricated microparticles containing the absorbed aqueous sample with the second detection composition is a step that allows the primers in the second detection composition to hybridize to oligonucleotide tags attached to the secondary antibody; -A further optional step for cleaning the library is to remove any unhybridized primers from the library; - The transfer step includes transferring the prepared fine particles to a non-aqueous phase, suspending them, and washing them repeatedly in the non-aqueous phase as necessary, and further including, as necessary, filtration or mechanical stirring to ensure that no or substantially any aqueous phase remains on the outside of the fine particles to form a monodisperse suspension of the fine particles; - The optional step of applying an external trigger to release the analyte-specific reagent attached to the fine particles is a step of temporarily increasing the temperature, changing the pH, or changing the salt conditions, preferably increasing the temperature, more preferably increasing the temperature to >90°C above ambient temperature; - The step of performing the detection reaction is a step of performing the amplification reaction of the oligonucleotide tag; The step of detecting and / or quantifying the target analyte is the step of detecting and / or quantifying the amplified oligonucleotide tag. The method according to Embodiment 26.

[0037] 28. The method according to any one of embodiments 22 to 27, wherein the step of releasing the analyte-specific reagent attached to the fine particles by applying an external trigger is performed by increasing the temperature to which the fine particles are exposed.

[0038] 29. - The method is, for example, a method for detecting and / or quantifying a single analyte in multiple aqueous samples, which may originate from different patients. -The method provides a number of different aqueous samples known or suspected to contain the analyte of interest, for example, samples from different patients. - The library of pre-fabricated microparticles provided in the above method is the library described in Embodiment 11, and such a library provides the same number or at least the same number of distinct microparticle subsets as the number of different aqueous samples to be tested, all of which have the same analyte-specific reagent attached to the porous matrix of the microparticles in the subset, and the analyte-specific reagent is specific to one target analyte; -In the step of incubating the aqueous sample with the library of fabricated microparticles, each of the different aqueous samples is incubated separately with a separate subset of microparticles in the library; -In the step of incubating the library of prepared microparticles containing the absorbed aqueous sample with a second detection composition, each of the distinct microparticle subsets of the library is incubated separately with the second detection composition; - The step of transferring the library to a non-aqueous phase is performed separately for each subset of fine particles, that is, each separate subset of fine particles is transferred separately to a non-aqueous phase, and the aqueous phase around each individual fine particle is removed separately for each subset, thereby creating a plurality of isolated reaction spaces for detecting the analyte separately for each subset, the reaction spaces comprising an aqueous phase and limited to the void volume of the fine particles, The method further comprises, after the step of transferring the library to a non-aqueous phase, the step of mixing together the plurality of isolated reaction spaces of all separate subsets in the non-aqueous phase, followed by a detection reaction of the target analyte, and then detecting and / or quantifying the target analyte, The method according to any one of embodiments 22 to 28.

[0039] 30. - The method described above is a method for detecting and / or quantifying multiple analytes in a single aqueous sample, -In the above method, a single aqueous sample known or suspected to contain multiple target analytes is provided. -The library of fabricated microparticles provided in the above method is the library described in Embodiment 12, and such a library provides as many or at least the same number of distinct microparticle subsets as the number of different target analytes to be detected, each of the distinct subsets having a different analyte-specific reagent attached to the porous matrix of the microparticles in the subset; each analyte-specific reagent is specific to one target analyte; -In the step of incubating the aqueous sample with the library of fabricated microparticles, the aqueous sample is incubated together with all of the separate microparticle subsets of the library; -In the step of incubating the library of prepared microparticles containing the absorbed aqueous sample with a second detection composition, all of the distinct microparticle subsets of the library are incubated together with the second detection composition; - The step of transferring the library to a non-aqueous phase is performed together for all particulate subsets, i.e., all particulate subsets are transferred together to a non-aqueous phase, and the aqueous phase around the individual particulates is removed, thereby creating multiple isolated reaction spaces for detecting and / or quantifying the multiple analytes, the reaction spaces comprising the aqueous phase and limited to the void volume of the particulates. The method according to any one of embodiments 22 to 28.

[0040] 31. - The method described above is a method for detecting and / or quantifying multiple analytes in multiple aqueous samples, -The method provides multiple different aqueous samples known or suspected to contain multiple target analytes, for example, samples from different patients. -The library of pre-fabricated microparticles provided in the method is the library described in Embodiment 13, and such a library contains separate subsets of pre-fabricated microparticles of different classes, each of which comprises several subsets of microparticles, each of which has different analyte-specific reagents attached to the porous matrix of the microparticles, the same analyte-specific reagent attached to all subsets of microparticles in one class; each analyte-specific reagent is specific to one target analyte; and in the method, the step of providing the library is, • A different class of distinct particulate subsets is provided, in the same number or at least the same number as the number of different analytes to be detected. • Each class is provided with the same number or at least the same number of different particulate subsets as the number of aqueous samples to be tested. The library contains a number of different particulate subsets equal to or at least the number obtained by multiplying the number of different aqueous samples to be tested by the number of different target analytes to be detected. -In the step of incubating the aqueous sample with the library of fabricated microparticles, strictly one aqueous sample is incubated with strictly one microparticle subset from each class, and each aqueous sample is incubated with the same number of different microparticle subsets from different classes as the number of classes of microparticles in the library. -In the step of incubating the library of fabricated microparticles containing the absorbed aqueous sample with a second detection composition, a subset of each aqueous sample combined with a different class of microparticles that have been previously incubated is incubated together with the second detection composition. - The step of transferring the library to a non-aqueous phase is performed separately for each aqueous sample, that is, each aqueous sample and the pre-incubated combined microparticle subset are transferred together to a non-aqueous phase, and the aqueous phase around the individual microparticles is removed separately for each aqueous sample, thereby creating multiple isolated reaction spaces for detecting the multiple analytes, the reaction spaces comprising the aqueous phase and limited to the void volume of the microparticles, Preferably, the method further includes, after the step of transferring the library to a non-aqueous phase, mixing together the multiple isolated reaction spaces of all the separate samples in the non-aqueous phase, then carrying out the detection reaction for the target analyte, and then detecting and / or quantifying the target analyte. The method according to any one of embodiments 22 to 28.

[0041] 32. In the step of detecting and quantifying the target analyte, the quantification of the analyte is a) Digital nucleic acid amplification, particularly digital polymerase chain reaction (PCR); b) Real-time quantitative nucleic acid amplification, particularly real-time polymerase chain reaction (PCR); c) Immunochemical detection methods, particularly digital immunochemical detection methods, such as digital immunoassays, such as digital enzyme-linked immunosorbent assays (ELISA); d) Immunochemical detection methods combined with nucleic acid amplification, e.g., immunopolymerase chain reaction; in particular, digital immunoPCR It is done by a method selected from; If the analyte is a nucleic acid, quantification is performed using either method a) or b) or a combination of a) and b); if the analyte is a protein, peptide, or other non-nucleic acid analyte, quantification is performed using either method c) or d). The method according to any one of embodiments 22 to 31. Detailed description of the invention

[0042] The inventors have invented a library of pre-fabricated precursor microparticles that serves as a starting point for creating a library of ready-to-use and / or "ready-to-fill" pre-fabricated microparticles. Such a library of pre-fabricated microparticles is intended and configured to perform specific detection of one or more (preferably more) target analytes in a sample, according to the user's needs. Such a library of pre-fabricated microparticles is highly versatile and can be prepared in a customized manner by the end user who selects the specific analytes to be detected in the sample. When used herein, the "library of pre-fabricated precursor microparticles" that serves as a starting point for creating a "library of pre-fabricated microparticles" differs from such a "library of pre-fabricated microparticles" in that each pre-fabricated precursor microparticle does not yet contain one or more attached analyte-specific reagents ("ASRs") (as sometimes abbreviated herein). However, such precursor microparticles can be readily converted into ready-to-use microparticles by attaching analyte-specific reagents to them. Since the precursor microparticles differ from each individual microparticle only in that they have analyte-specific reagents attached to them, the quality and details of each fabricated microparticle are the same as those described for the respective precursor microparticles from which they originate. Therefore, in a library of fabricated precursor microparticles, each of the fabricated precursor microparticles in the library contains a porous matrix, preferably a porous polymer matrix, having a void volume for receiving aqueous samples and providing a reaction space for the specific detection of the analyte. The same applies to each of the resulting microparticles. The specific detection of the analyte using such microparticles is carried out by chemical or biochemical reactions in the reaction space provided by the porous matrix of the microparticles. Typically, such chemical or biochemical reactions are target (analyte) amplification reactions or signal amplification reactions. Typical examples of target amplification reactions are nucleic acid amplification reactions, e.g., PCR. Typical examples of signal amplification reactions are immunochemical reactions, e.g., immunoassays.

[0043] Furthermore, each of the fabricated precursor microparticles in the library (and each of the fabricated microparticles in the library) contains a reagent-binding component that enables the attachment, preferably reversible attachment, of an analyte-specific reagent to the precursor microparticle. A preferred embodiment is one in which the analyte-specific reagent is reversibly attached to the precursor microparticle. As outlined above, such a reagent-binding component is (i) A polymer or polymer mixture that forms the porous matrix or is the polymer matrix; (ii) Reagent-binding molecules attached to the porous matrix; (iii) at least one or more ionizable groups immobilized on the porous polymer matrix, wherein the ionizable groups are capable of changing their charge according to the ambient conditions around the precursor nanoparticles; (iv) at least one or more charged groups immobilized on the porous matrix; (v)(i)~(iv) Any combination It is one of them.

[0044] Furthermore, each of the pre-fabricated precursor microparticles in the library contains a labeling component attached to, contained within, or otherwise associated with the precursor microparticle, and the same applies to each pre-fabricated microparticle resulting from such a pre-fabricated precursor microparticle. A labeling component attached to, contained within, or otherwise associated with a precursor microparticle or microparticle is suitable for identifying (meaning "uniquely labeling" or "uniquely marking") an analyte-specific reagent when the analyte-specific reagent is attached to the precursor microparticle. Examples of such labeling are described in (Mandecki, Ardelt et al., 2006, Wilson, Cossins et al., 2006, Birtwell and Morgan 2009), etc. A labeling component attached to, contained within, or otherwise associated with a precursor microparticle becomes particularly significant when it is a matter to consider in relation to the entire library of pre-fabricated precursor microparticles (or the entire library of pre-fabricated microparticles). As used herein, the term “library” means a plurality of microparticles or a group of microparticles, and such a plurality of microparticles or a group of microparticles or a library of microparticles contains at least two different types of such microparticles, which may also be referred to herein as “subsets.” In a preferred embodiment, there are at least two fabricated precursor microparticle subsets (or fabricated microparticle subsets), preferably three or more, each subset having a different labeling component attached to, contained in, or otherwise associated with the precursor microparticles (or microparticles) within the subset, and the at least two or more fabricated precursor microparticle subsets (or fabricated microparticle subsets) each have a different labeling component attached to, contained in, or otherwise associated with each subset.In such a library of different subsets, each subset is typically, and preferably, stored separately from one another, for example, in different containers. This means that the term “distinct subset” as used herein refers to a group or subset of precursor microparticles or microparticles that are physically separated from other subsets. Such physical separation can be achieved by a barrier around such subsets. In a simple embodiment, such distinct subsets may be placed in their own container or compartment. Typically, by combining multiple subsets, a library can be formed that targets different analytes / targets (with different subsets targeting different analytes), and that library can then be used to perform assays that distribute the analytes / targets to be detected / quantified in a sample to microparticles corresponding to multiple subsets within the library.

[0045] In a preferred embodiment, the labeling component that identifies each subset of precursor microparticles (and microparticles arising therefrom) is a mixture of at least two different dyes, preferably at least two fluorescent dyes, which are present preferably attached to each subset of the precursor microparticles (and microparticles arising therefrom) in a predetermined ratio and / or predetermined amount of the at least two different dyes. As a result, in such an embodiment, the different precursor microparticle subsets differ from one another in terms of the respective ratio and / or amount of the at least two different dyes, so that the different precursor microparticle subsets can be distinguished by the respective ratio and / or amount of the at least two different dyes attached to each precursor microparticle subset.

[0046] In another embodiment, the labeling component may be contained within the precursor microparticles. Furthermore, such a labeling component may be a mixture of at least two different dyes contained within the precursor microparticles, and each different precursor microparticle subset differs from one another in terms of the ratio and / or amount of each of the at least two different dyes contained within the microparticles, so that different precursor microparticle subsets can be distinguished by the ratio and / or amount of each of the at least two different dyes contained within each precursor microparticle subset.

[0047] Subset-specific labeling (or coding) can be achieved as previously established (Fulton, McDade et al., 1997).

[0048] In other embodiments, the labeling of each precursor microparticle (and corresponding microparticle) may also be achieved by the different shapes of each microparticle. This may be an example for embodiments of precursor microparticles (and corresponding microparticles) in which the labeling component is not attached to or contained within the precursor microparticle, but is "otherwise associated" with the precursor microparticle. Such embodiments assume the presence of at least two different labeling components simultaneously, i.e., in this case, each thus labeled precursor microparticle can be identified by the shape of the precursor microparticle. In this example, different shapes may mean different external shapes, different internal structures, different internal volumes within a porous matrix, and so on.

[0049] Preferably, the porous matrix of the precursor microparticles (and the microparticles) is a porous polymer matrix, which implicitly means that each precursor microparticle (and the microparticles arising therefrom) is made of or contains a polymer or a polymer mixture.

[0050] As outlined above, a library of fabricated precursor microparticles can be converted into a library of fabricated microparticles by attaching analyte-specific reagents to the precursor microparticles via reagent-binding components, preferably by reversibly attaching such reagents. Different microparticle subsets may have the same analyte-specific reagent attached, or they may have different analyte-specific reagents attached.

[0051] As used herein, the term “analyte-specific reagent” (“ASR”) means a reagent capable of specifically targeting or recognizing an analyte of interest. Such specific targeting or specific recognition manifests itself in the ability of such an analyte-specific reagent to specifically bind to or specifically react with such an analyte of interest. In one embodiment, the analyte-specific reagent is selected from nucleic acids, including aptamers, spiegelmers, nucleic acid oligomers, and nucleic acid primers; antibodies or antibody fragments; non-antibody proteins capable of specifically binding to an analyte or analyte complex; and affinity proteins. In a preferred embodiment, the analyte-specific reagent is selected from nucleic acids, particularly nucleic acid oligomers and nucleic acid primers. Nucleic acid primers are particularly suitable for performing nucleic acid amplification. In one embodiment, when the analyte-specific reagent is a nucleic acid primer, such an analyte-specific reagent is, in fact, a pair of primers adjacent to a region in the analyte of interest that is subsequently amplified (and detected). If necessary, in such embodiments (of a primer pair that is an analyte-specific reagent), such analyte-specific reagent may further include a detectable probe provided with the primers, which enables detection of each primer and the resulting amplification product.

[0052] As used herein, the term “analyte” means any molecular entity in a sample that is to be detected and / or quantified. Such term “analyte” may be used herein as a synonym for the term “target.” In one embodiment, the analyte may be a nucleic acid; in another embodiment, the analyte may be a protein, peptide, or other non-nucleic acid entity.

[0053] The libraries and their respective microparticles or precursor microparticles according to the present invention can be dried, for example, by freeze-drying. According to embodiments of the present invention, a gel-forming agent may be used to form the fabricated precursor microparticles (and their respective resulting microparticles), such a gel-forming agent is as further defined above. In one embodiment, such a gel-forming agent is used to form precursor microparticles (and their respective resulting microparticles), which can then be dried, preferably freeze-dried. After drying, the particles can be stored as a powder. In some embodiments involving a gel-forming agent, such a gel-forming agent further forms a gel that can undergo a transition to a sol state. In particular, such a transition may occur by applying an external trigger, for example, a change in temperature, pH, or salt conditions.

[0054] The inventors have found, surprisingly, that by providing an entire library of the fabricated microparticles or their precursors according to the present invention, it is possible to provide a miniaturized, versatile tool that can act as a defined reaction space that can be used in a highly versatile manner for detection reactions, for example, for digital detection of analytes in a sample, and that can be used in field environments. Since these microparticles act as or provide a closed reaction space, they may also be referred to herein as “nanoreactors.” Fabricated microparticles (or “nanoreactors”) according to embodiments of the present invention can be tailor-made by selecting appropriate analyte-specific reagents to be attached. Such tailor-made fabrication is particularly facilitated in preferred embodiments where the attachment of analyte-specific reagents is reversible. The same or different analyte-specific reagents may be attached to different subsets of microparticles in the library.

[0055] As used herein, the terms “same analyte-specific reagent” and “different analyte-specific reagent” mean the fact that when two analyte-specific reagents are the same, this means they target and / or recognize the same analyte of interest, and when they are different, they target and / or recognize different analytes of interest. Thus, in a library of fabricated microparticles where different microparticle subsets are different from each other with respect to the respective analyte-specific reagents attached to or contained therein, this means that these different subsets target and / or recognize different analytes of interest. On the other hand, when different subsets are attached to or contain the same analyte-specific reagent, this means that they recognize and / or target the same analyte of interest.

[0056] As used herein, the term “microparticles” means particles whose average dimensions are in the range of micrometers. In one embodiment, the microparticles according to the present invention have an average size or average dimension or average diameter of approximately 1 μm to 200 μm, preferably 5 μm to 150 μm, and more preferably 10 μm to 100 μm. In one embodiment, the microparticles according to the present invention are spherical, oval, or elliptical, preferably spherical, and the dimensions described above refer to the average diameter of such spherical, oval, or elliptical microparticles. In one embodiment, the microparticles have the shape of a (spherical) droplet. In another embodiment, the microparticles according to the present invention are spherical or quasi-spherical, i.e., have the shape of a sphere (or a shape nearly spherical), and such spheres have the average diameter of the dimensions described above. Typically, the microparticles according to the present invention are porous and have a porous polymer matrix having void volume for receiving aqueous samples and void volume for providing a reaction space for specifically detecting analytes.

[0057] When used herein, precursor microparticles are converted into microparticles by attaching analyte-specific reagents to them. Therefore, a library of fabricated precursor microparticles is suitable for the purpose of preparing a library of fabricated microparticles. Each resulting microparticle is suitable for the purpose of specific detection and / or quantification of one or more target analytes in a sample, depending on the number of different analyte-specific reagents attached to them (e.g., defined by the user). If only one analyte-specific reagent (or a single analyte-specific "reagent set" containing multiple components necessary to detect a single analyte, e.g., an amplification primer for the target nucleic acid and optionally a detection probe) is attached, such a library can only be used to detect a single target analyte, but can also be used in this way in multiple samples that can be distinguished thanks to the respective attached labeling components. If different analyte-specific reagents are attached to different microparticles in such a library of fabricated microparticles, such a library can be used to detect different target analytes in one or more samples.

[0058] In a preferred embodiment, the analyte-specific reagent is reversibly attached to each precursor microparticle. As used herein in the context of "reversibly attached" one entity to another, "reversibly attached" or "reversibly attached" preferably refers to a type of attachment where the attachment between the entities can "detach" under favorable conditions, but can also be reattached to each other under favorable conditions. Such reversible detachment can usually occur and be achieved in such a way that the entities remain intact, particularly without change in their structure and binding ability, and that the two entities can repeatedly attach (i.e., bind) to each other and be released from each other. A typical example of such reversible attachment is the hybridization of two complementary strands of nucleic acids under favorable conditions, for example, hybridization under hybridization conditions where the hybridization can be reversed if the resulting double strand is exposed to a different set of conditions, such as an increase in temperature that melts the double strand back into a single strand. The reversible nature of the adhesion is demonstrated because the two chains can be re-annealed once cooled. An example of adhesion that is not "reversible" in the sense described above is the binding between wild-type biotin and wild-type streptavidin. This binding is one of the strongest known non-covalent binding events and cannot be reversed unless one or both entities are structurally altered or perhaps destroyed, for example, by denaturation.

[0059] The reversible nature of the attachment of analyte-specific reagents to each precursor microparticle according to the embodiments of the present invention facilitates the customization of microparticle production according to user needs and allows each microparticle library to be adapted to a wide variety of different methods. Therefore, individualized and / or customized libraries can be easily produced according to each field environment and its needs. Furthermore, in situations where the removal of analyte-specific reagents may be desirable, for example, when such analyte-specific reagents should be available (and freely diffusible) for subsequent reactions in method protocols that require, for example, freely available (and freely diffusible) analyte-specific reagents, the reversible attachment of such analyte-specific reagents makes this possible. Preferably, such reversible attachment occurs because the precursor microparticles have a reagent-binding component that enables the reversible attachment of analyte-specific reagents to the precursor microparticles. In a preferred embodiment, such a reagent-binding component is a reagent-binding molecule attached to a porous polymer matrix of the precursor microparticles. Such a reagent-binding molecule is designed and intended to bind to a binding entity conjugated to the analyte-specific reagent. Therefore, in this preferred embodiment, an interaction occurs between a reagent-binding molecule attached to the porous polymer matrix of one precursor microparticle and a binding entity conjugated to the other analyte-specific reagent. In this preferred embodiment, the reagent-binding molecule and the binding entity are selected to interact with each other in a reversible manner, i.e., to bind to each other. For example, as long as the interaction between the two entities is reversible and the binding can be reversed again, the reagent-binding molecule attached to the porous polymer matrix may be streptavidin or a derivative thereof, and the binding entity conjugated to the analyte-specific reagent may be biotin or a biotin derivative, e.g., desthiobiotin, or vice versa (i.e., the reagent-binding molecule is biotin or a biotin derivative, e.g., desthiobiotin, and the binding entity is streptavidin or a derivative thereof).For example, if the analyte-specific reagent is a nucleic acid primer (or a pair of nucleic acid primers, and optionally a detection probe), such primers can be readily conjugated to, for example, desthiobiotin, or are even commercially available in such desthiotinated form. On the other hand, a reagent-binding molecule, which may be streptavidin, can be attached to a porous polymer matrix. Under ambient conditions, selecting a pair of desthiobiotin-primers specific to the analyte of interest and reversibly attaching them to precursor microparticles having streptavidin attached to a porous polymer matrix is ​​extremely simple and easy for the end user. The binding event between the biotin derivative and streptavidin under ambient conditions is easily achieved by simply exposing precursor microparticles to a solution of primers labeled with such biotin derivatives under favorable conditions. A “conditioning solution” can be used to provide conditions that facilitate binding. Such a conditioning solution may be, for example, a buffer that provides a suitable pH and salt environment. On the other hand, if the temperature is subsequently increased, for example during nucleic acid amplification, the analyte-specific reagent can be easily removed again from the microparticles due to the reversible nature of the bond between the reagent-binding molecule and the binding entity. This is because, under high-temperature conditions, further binding between the reagent-binding molecule (e.g., streptavidin) attached to the porous polymer matrix of one of the precursor microparticles and the binding entity on the analyte-specific reagent (e.g., desthiobiotin on the primer) is impossible. Therefore, after such removal, the analyte-specific reagent becomes fully available for any resulting amplification reaction.

[0060] As used herein, the term "protein derived from or related to avidin" means any protein that is structurally or sequentially similar to avidin and possesses or retains the ability to bind to avidin.

[0061] As used herein, the term "molecule derived from or related to biotin" means any molecule that is structurally similar to biotin and possesses or retains the function of binding to biotin.

[0062] In one embodiment of each library, each of the fabricated precursor microparticles or microparticles in that library comprises a porous polymer matrix having void volume for receiving aqueous samples and providing a reaction space for specific detection of analytes. Such a porous polymer matrix is ​​formed by a polymer or polymer mixture, more preferably by a hydrogel-forming agent or a mixture of such hydrogel-forming agents, or is a hydrogel-forming agent or a mixture of such hydrogel-forming agents. In a preferred embodiment, the polymer or polymer mixture forming the porous polymer matrix has the ability to switch between a gel state and a sol state. In one embodiment, the polymer or polymer mixture forming the porous polymer matrix or being part of the porous polymer matrix is ​​not a crosslinked polymer. Embodiments in which the polymer or polymer mixture is not crosslinked are particularly excellent in switching between different states, for example, between a gel state and a sol state, and are versatile. In a particularly preferred embodiment, the polymer is agarose, more preferably a combination of agarose and gelatin. When a combination of agarose and gelatin is used, the agarose is preferably present in the range of 0.1%(w / v) to 4%(w / v), and the gelatin is preferably present in the range of 0.1%(w / v) to 20%(w / v), more preferably 0.5%(w / v) to 20%(w / v). In one identification embodiment, the concentration of agarose in the porous polymer matrix is ​​0.5%(w / v), and the concentration of gelatin is in the range of 1%(w / v) to 2%(w / v).

[0063] The technology invented by the present inventors is highly versatile in that it enables the detection of multiple analytes in a sample by using a library of pre-fabricated microparticles to which the same number of different analyte-specific reagents as the number of analytes to be detected in the sample are attached. Thus, according to one embodiment, the library of pre-fabricated microparticles contains at least two, preferably three or more, subsets of pre-fabricated microparticles, each subset having a different labeling component attached to, contained within, or otherwise associated with the microparticles of that subset; each subset having a different analyte-specific reagent attached to the microparticles of that subset; the at least two or more subsets of pre-fabricated microparticles differ in the respective labeling components attached or contained within, and the respective analyte-specific reagents attached to each subset; thus, each subset is clearly defined and identifiable by its respective labeling component and its respective analyte-specific reagent. Such a library provides as many or at least the same number of distinct microparticle subsets as there are different target analytes to be detected, each of which has a different analyte-specific reagent attached to the porous matrix of the microparticles in the subset; each analyte-specific reagent is specific to one target analyte. Such a library is described in particular in Embodiment 12, claimed in Claim 10, and its use is described in particular in Embodiment 30, claimed in Claim 23, examples of which are shown in Figures 10B), 10C), and 10E), and will be further explained with reference thereto.

[0064] Even when there is only one target analyte to be detected, but it may be present in different samples, the present invention, in one embodiment, provides a library of fabricated microparticles, the library containing at least two, preferably three or more, fabricated microparticle subsets, each subset having a different labeling component attached to, contained in, or otherwise associated with the microparticles of the subset; all of the at least two, three or more subsets having the same analyte-specific reagent attached to the microparticles of the subset; the at least two or more fabricated microparticle subsets are identical with respect to the attached analyte-specific reagent, but each subset has a different labeling component attached to, contained in, or otherwise associated with the microparticles of the subset. This library of identification is particularly useful for detecting a single analyte in a very large number of samples, i.e., preferably the same number of samples as the number of subsets in the library. In other words, such a library provides as many or at least the same number of distinct particulate subsets as the number of different aqueous samples to be tested, all of which have the same analyte-specific reagent attached to the porous matrix of the particulates in the subset, and the analyte-specific reagent is specific to one target analyte. Such libraries are described in particular in Embodiment 11, claimed in claim 9, and their use is described in particular in Embodiment 29, claimed in claim 22, examples of which are shown in Figure 10A) and further described with reference thereto.

[0065] The technology invented by the present inventors further enables the detection of multiple analytes in several samples by using a library of pre-fabricated microparticles to which the same number of different analytes-specific reagents as the number of analytes to be detected in the sample, wherein the library contains multiple distinct microparticle subsets, the total number of which is equal to the number of samples tested multiplied by the number of analytes to be detected and / or quantified. Thus, according to one such embodiment, the library of such pre-fabricated microparticles contains distinct microparticle subsets, each subset having a different labeling component attached, contained in, or otherwise associated with it. Furthermore, the library contains distinct pre-fabricated microparticle subsets of different classes, each of which contains several microparticle subsets to which the same analytes-specific reagent is attached within that class; the microparticle subsets belonging to different classes have different analytes-specific reagents attached to the porous matrix of the microparticles. In other words, all particulate subsets within a class are coated with the same analyte-specific reagent (and therefore intended to be used to detect and quantify the same analyte), but different subsets within a class are intended to be used with different samples. When such a library is used in a method for detecting and / or quantifying multiple analytes in multiple samples, such a library contains: • A different class of distinct particulate subsets is provided, in the same number or at least the same number as the number of different analytes to be detected. • Each class is provided with the same number or at least the same number of different particulate subsets as the number of aqueous samples to be tested. Overall, the library will contain a number of different particulate subsets equal to or at least equal to the number obtained by multiplying the number of different aqueous samples to be tested by the number of different target analytes to be detected. Such libraries are described in particular in Embodiment 13 and claimed in Claim 11, and their use is described in particular in Embodiment 31 and claimed in Claim 24, examples of which are shown in Figures 10D), 11A), and 11B) and further described with reference thereto.

[0066] The versatility of the present invention is also reflected in the fact that libraries according to the present invention can be prepared very easily by an appropriate kit that can be used by an end user to determine which analytes to focus on, and therefore which analytes should be detected, and how many samples to examine.

[0067] Therefore, in a further embodiment, the present invention relates to a kit for preparing a library of fabricated microparticles as defined above, the kit being: The system includes at least two containers, namely the first and second containers, and additional containers as needed, each container being: • comprising a subset of fabricated precursor microparticles as further defined above, each subset having a different labeling component attached to, contained within, or otherwise associated with the precursor microparticles within the subset, and the two subsets of fabricated precursor microparticles contained in the at least two containers having different labeling components attached to, contained within, or otherwise associated with them; Further containers are · a) Direct binding of the analyte-specific reagent to the polymer mixture (i) which forms or is part of the porous polymer matrix; b) The analyte-specific reagent conjugated to a binding entity that binds to the reagent-binding molecule (ii) as further defined above; c) Direct binding of the analyte-specific reagent to the ionizable group (iii) as defined above, under conditions in which the ionizable group has a suitable net charge; d) Direct bonding of the analyte-specific reagent to the charged group(iv) on the polymer as defined above, wherein the analyte-specific reagent has at least one or more ionizable groups, and the ionizable groups are capable of changing charge according to ambient conditions around the analyte-specific reagent; or e) any combination of a) to d) The solution comprises a conditioning solution that enables the reversible attachment of an analyte-specific reagent to the subset of precursor microparticles via the reagent-binding component of the precursor microparticles, as further defined above; the reagent-binding component, reagent-binding molecule, binding entity, polymer or polymer mixture, ionizable group and analyte-specific reagent are as further defined above.

[0068] Using such embodiments, end users can generate a library of their own pre-fabricated microparticles that are specific to a selected number and type of target analytes and can be used with a selected number of samples. The attachment of each desired analyte-specific reagent, such as a primer or antibody, is simply carried out by exposing the pre-fabricated precursor microparticles to the analyte-specific reagent under appropriate conditions, preferably those established or brought about by the above-described conditioning solution, such as a buffer.

[0069] In one embodiment, when a very large number of target analytes are detected in a single sample, a library of corresponding pre-fabricated microparticles is prepared using the kit according to the present invention, the library containing different microparticle subsets, each subset being specific to the target analyte for identification, and each subset being different with respect to the respective labeling components attached to, contained within, or otherwise associated with each subset. Such libraries are described in particular in Embodiment 12, claimed in claim 10, and their use is described in particular in Embodiment 30, claimed in claim 23, examples of which are shown in Figures 10B), 10C), and 10E) and further described with reference thereto.

[0070] On the other hand, if, for example, detection of a single analyte in multiple samples from different patients is desired and intended, a corresponding library is generated in which different pre-fabricated microparticle subsets exist, all of which are coated with the same analyte-specific reagent, but still differ with respect to the respective labeling components coated, contained within, or otherwise associated with each subset. Such libraries are described in particular in Embodiment 11, claimed in claim 9, and their use is described in particular in Embodiment 29, claimed in claim 22, examples of which are shown in Figure 10A) and further described with reference thereto. Note that in the conventional “pooling” approach, different samples from different patients are pooled together, and then such a pool is examined for the presence of an analyte. In such a conventional approach, if the pool is found to show a positive signal indicating the presence of an analyte in at least one of the samples originally used to create the pool, all the samples must be retested individually to finally determine which individual sample was positive. In contrast, the library according to the present invention does not require such repeated testing of individual samples if a positive signal is detected. This is because the library according to the present invention makes it possible to determine at the level of individual microparticles (showing such a positive signal) which sample a positive microparticle belongs to. This is possible even if such individual (positive) microparticles are mixed with other microparticles used to search for different (patient) samples (i.e., they exist in a "pool" of those microparticles), and such a "mixture" (or "pool") can usually be generated when a detection reaction is performed on all the microparticles in the library.

[0071] As a third possibility, a library useful for detecting more than one target analytes in multiple samples, for example, two target analytes, may also be provided. In such a library, each particulate subset has two, three, four, or n subsets attached to each subset, each with a different labeling component, but the same analyte-specific reagent attached (when two, three, four, or n samples are tested for the target analyte of identification). Furthermore, within such a library, there may be two, three, four, or n particulate subsets, and each subset within such two, ..., n subsets has a different analyte-specific reagent attached (when two, three, four, or n different target analytes are detected). Such a library is described in particular in Embodiment 13 and claimed in Claim 11, and its use is described in particular in Embodiment 31 and claimed in Claim 24, examples of which are shown in Figures 10D), 11A), and 11B), and will be further explained with reference thereto. Furthermore, for the reasons outlined above, this differs from traditional pooling approaches, which require repeated testing of the entire pool and individual samples if the pool is found to be positive.

[0072] In one embodiment, a kit for preparing a library of pre-fabricated microparticles may further include a further container containing a washing buffer or washing solution for removing free, i.e., unattached, analyte-specific reagents from the microparticles without removing any analyte-specific reagents attached to the microparticles. In a further embodiment, a kit for preparing pre-fabricated microparticles may further include at least one mixing container for mixing components, preferably several mixing containers, more preferably the same number of mixing containers as the number of containers containing the precursor microparticle subsets.

[0073] In one embodiment, the mixing of components may be carried out in the same container containing each of the prepared precursor microparticle subsets. However, in other embodiments, it may be prudent to provide one or more of such clearly designated mixing containers separately. This may be particularly useful when the microparticle subsets are pooled after being provided separately with different analyte-specific reagents.

[0074] In a further embodiment, the present invention also relates to a method for preparing a library of fabricated microparticles, which provides a library of fabricated precursor microparticles and at least one analyte-specific reagent (both as defined above) in any order. In such a method, the library of fabricated microparticles according to the present invention is produced by mixing the library of fabricated precursor microparticles or a selected subset thereof with at least one analyte-specific reagent under conditions that allow for the attachment, preferably reversible attachment, of the at least one analyte-specific reagent to some or all of the fabricated precursor microparticles.

[0075] If necessary, the above method further includes a step of washing the fabricated microparticles to remove any unadhered analyte-specific reagents.

[0076] In one embodiment, such a method may be carried out by using a kit for preparing a library as defined above, wherein the library is provided in the form of the at least two containers of the kit, each container containing a prepared precursor microparticle subset as further defined above, each subset having a different labeling component attached to, contained in, or otherwise associated with the precursor microparticles within the subset, and the at least two prepared precursor microparticle subsets each have a different labeling component attached to, contained in, or otherwise associated with the respective subset; The conditions that enable the attachment of the at least one analyte-specific reagent to some or all of the fabricated precursor microparticles, preferably reversible attachment, are brought about by mixing the library of the fabricated precursor microparticles or a selected subset thereof with the at least one analyte-specific reagent in the presence of a conditioning solution as defined above, preferably a buffer solution. In a preferred embodiment of a method for preparing a library of fabricated microparticles, as further defined above, the method is: - A library of fabricated precursor microparticles, where each subset is provided in a separate container, as further defined above, or where several subsets of fabricated precursor microparticles are provided together in separate containers; • At least one analyte-specific reagent as further defined above A process of providing in any order; The step of generating a library of fabricated microparticles as further defined above by separately mixing a subset of the library of fabricated precursor microparticles with one or more analyte-specific reagents, or by mixing a selected mixed subset of the library of fabricated precursor microparticles with at least one analyte-specific reagent, under conditions that allow for the attachment of at least one analyte-specific reagent to some or all of the fabricated precursor microparticles, preferably under conditions that allow for reversible attachment; - If necessary, the prepared microparticles are washed to remove any unattached analyte-specific reagents. • (If necessary) The process of combining individual or mixed subsets into a single microparticle library containing different subsets having analyte-specific reagents selectively attached to a specified subset of the library. Includes.

[0077] In a further embodiment, a kit for preparing a library of pre-fabricated microparticles, as defined above, which may also be referred to herein as a “fabrication kit,” may serve as a platform for providing a kit for detecting an analyte. The latter kit, unlike the “fabrication kit” which is a kit for preparing a library of pre-fabricated microparticles, may also be referred to herein as a “detection kit.”

[0078] In a further embodiment, the present invention also relates to a kit for detecting an analyte in a sample ("detection kit"), the detection kit is: a) - A container comprising a general detection composition for nucleic acid amplification, wherein the general detection composition comprises reagents for carrying out a chemical or biochemical detection reaction of an analyte, the chemical or biochemical detection reaction of the analyte being nucleic acid amplification, and the general detection composition comprising a buffer, mononucleoside triphosphate, amplification enzyme, for example, a suitable nucleic acid polymerase, for example, Taq polymerase, and amplification product, for example, a nucleic acid dye for detection of amplified nucleic acids, or - A container comprising a first detection composition comprising a reagent for performing a chemical or biochemical detection reaction of an analyte, wherein the chemical or biochemical detection reaction of the analyte is an immunochemical detection reaction; and a further container comprising a second detection composition comprising a detection reagent, wherein the first detection composition comprises a reagent for performing an immunochemical detection reaction, e.g., a buffer, and a secondary antibody or secondary antibody fragment that is specific to the same analyte as the primary antibody, antibody fragment or non-antibody protein used as an analyte-specific reagent (ASR) in the immunochemical detection reaction and is conjugated to a suitable reporter enzyme; the second detection composition comprises a substrate suitable for the reporter enzyme as a detection reagent, wherein the substrate becomes detectable, preferably optically detectable, more preferably fluorescently detectable, when reacted with the reporter enzyme; and the further container. or - A container comprising a first detection composition comprising a reagent for carrying out a chemical or biochemical detection reaction of an analyte, wherein the chemical or biochemical reaction is an immunochemical detection reaction; and a further container comprising a second detection composition comprising a detection reagent, wherein the first detection composition comprises a reagent for carrying out an immunochemical detection reaction, e.g., a buffer, and a secondary antibody or secondary antibody fragment that is specific to the same analyte as the primary antibody used as an analyte-specific reagent (ASR) in the immunochemical detection reaction and is conjugated to a suitable oligonucleotide tag; the second detection composition comprises a buffer, mononucleoside triphosphate, an amplification enzyme, e.g., a suitable nucleic acid polymerase, e.g., Taq polymerase, and an amplification product, e.g., a nucleic acid dye for detection of amplified nucleic acids, and a primer suitable for amplifying the oligonucleotide tag attached to the secondary antibody, as the detection reagent; and a further container; and b) - A container containing a non-aqueous phase, such as oil, such as fluorocarbon oil, supplemented with emulsifiers as needed; and c) - Mixing container for mixing ingredients Includes.

[0079] In one embodiment, the detection kit may include d) a further container for carrying out the detection reaction.

[0080] According to the present invention, the detection kit is used in conjunction with a library of particulate matter as further defined herein and above.

[0081] Therefore, in some embodiments, the detection kit may also include a library of fabricated microparticles as further defined above. In such a library of microparticles according to the present invention, the microparticles include analyte-specific reagents as defined above. These microparticles contain analyte-specific reagents that act as a defined reaction space for detection by chemical or biochemical reactions. Each analyte-specific reagent specifically targets or recognizes the analyte of interest.

[0082] In other embodiments, a library of pre-fabricated microparticles as defined above is provided separately from the kit and does not form part of it.

[0083] In the detection kit according to the embodiment of the present invention, component a) is, in effect, a determining component of the type of detection used. Therefore, component a) has various possibilities. Depending on what kind of detection is to be performed, and therefore depending on the analyte to be detected, such component a) may be configured to perform detection in a nucleic acid amplification context, or in an immunochemical detection context, or in an immunoamplification reaction context, such as immunoPCR.

[0084] Therefore, such component a) may be a container containing a general detection composition used in nucleic acid amplification situations. Such a general detection composition used in nucleic acid amplification situations comprises a buffer, a mononucleoside triphosphate, an amplification enzyme, for example, a suitable nucleic acid polymerase, for example, Taq polymerase, and an amplification product, for example, a nucleic acid dye for detection of amplified nucleic acids.

[0085] Alternatively, in the context of immunochemical detection, component a) may be a combination of two separate containers, the first container of such a combination comprising a reagent for performing immunochemical detection, e.g., a buffer, and a first detection composition comprising a secondary antibody or secondary antibody fragment that is specific to the same analyte used as the analyte-specific reagent in the immunochemical reaction and is conjugated to a suitable reporter enzyme. In this context, the term “analyte-specific,” when used in the context of a secondary antibody, means that the secondary antibody can specifically recognize or target the same analyte used as the analyte-specific reagent, regardless of whether such analyte is free or already conjugated to the primary antibody, antibody fragment, etc., used as the analyte-specific reagent in the immunochemical detection reaction. The primary antibody, antibody fragment, or non-antibody protein used as the analyte-specific reagent is a part of the microparticles as defined above, and a library of these microparticles as defined above may form part of a detection kit or may be provided separately from such a kit.

[0086] A third option of component a) is an option for detection in the context of an immunoamplification reaction, e.g., immunoPCR. In this embodiment, component a) is a combination of two separate containers, the first container comprising a reagent for performing an immunochemical detection reaction, e.g., a buffer, and a first detection composition comprising a primary antibody, antibody fragment, or non-antibody protein used as an analyte-specific reagent, a secondary antibody or secondary antibody fragment that is specific to the same analyte and conjugated to a suitable oligonucleotide tag; the second container comprising a second detection composition, such a second detection composition comprising a buffer, mononucleoside triphosphate, an amplification enzyme, e.g., a suitable nucleic acid polymerase, e.g., Taq polymerase, and an amplification product, e.g., a nucleic acid dye for detection of amplified nucleic acids, and a primer suitable for amplifying the oligonucleotide tag attached to the secondary antibody as a detection reagent.

[0087] The library according to the present invention is extremely easy to prepare and can be used very versatility for a wide variety of analytical or diagnostic applications:

[0088] For example, as already outlined above, a library according to one embodiment of the present invention may be used in a method for detecting a single analyte in a plurality of different samples. Each subset is typically provided in a separate container, thereby allowing interrogation of the subset by each sample. In such embodiments, the number of distinct particulate subsets present in the library is at least equal to (or may be greater than) the number of samples in the plurality of different samples. If the number of distinct particulate subsets present in the library is greater than the number of samples in the plurality of different samples, it is not necessary to use the entire library (including all subsets). Such a library is described in Embodiment 11.

[0089] As another example, as already outlined above, a library according to one embodiment of the present invention may be used in a method for detecting multiple analytes in a single sample. A subset of the library may be provided together in a single container, or may be initially provided in separate containers and then combined into a single container and simultaneously interrogated with the sample. In such embodiments, the number of separate particulate subsets present in the library is at least equal to (or may be greater than) the number of analytes to be detected in the single sample. If the number of separate particulate subsets present in the library is greater than the number of analytes to be detected in the single sample, it is not necessary to use the entire library (including all subsets). Such a library is described in Embodiment 12.

[0090] As another example, a library according to one embodiment of the present invention may be used in a method for detecting multiple analytes in multiple different samples. In this case, the library comprises sublibraries of different subsets, each subset corresponding to a different class of analyte-specific reagents, and each sublibrary is used to be interrogated with different samples. Such a library is described in Embodiment 13. As used herein, the terms “sublibrary” and “class” mean the following:

[0091] As used herein, the term “class” refers to the entire set of all particulate subsets in a library that have the same analyte-specific reagent (ASR). All subsets within a class are specific to a single analyte of interest. Within a class, different subsets have different labeling components from one another. Within a class, each of the different subsets is used to interrogate a different sample; therefore, these different subsets are maintained in separate containers.

[0092] As used herein in the context of a method for detecting multiple analytes in multiple different samples, the term “sublibrary” means the entire set of all microparticle subsets in a library used to interrogate a sample of one identification at a time. Since multiple different samples are present, the library includes different sublibraries of separate prepared microparticle subsets, each of which is used to interrogate a different sample. Within each sublibrary, each subset of prepared microparticles in a sublibrary is contaminated with a different analyte-specific reagent; each analyte-specific reagent is specific to one target analyte.

[0093] Therefore, in effect, each sublibrary contains one microparticle subset derived from each class, and within each sublibrary, each microparticle subset is coated with a different analyte-specific reagent.

[0094] As used herein, the term “interrogate a sample” is used in the context of a subset used to “interrogate a sample” to mean a scenario in which such a subset is exposed to or incubated with the identification sample, thereby enabling the particulate subset to draw the sample into the void volume of the particulates.

[0095] When the terms “incubate,” “interrogate,” and “expose” are used in the context of a sample being incubated or interrogated with a library of particulate matter, or a library of particulate matter being exposed to a sample, all of these terms mean a scenario in which the sample and the library are in contact with each other for a sufficient amount of time for the particulate matter to draw the sample into its void volume.

[0096] As used herein, the terms “sample” or “aqueous sample” mean any suitable fluid (e.g., body fluid) or its components isolated from an environment to be analyzed for the presence and / or amount of one or more analytes. For example, a sample may be an aqueous solution to be analyzed, obtained from, for example, an environment of identification, e.g., a patient, particularly patient tissue or patient body fluid. Such a sample may be a body fluid or its components. Examples include blood, plasma, serum, saliva, urine, tears, sweat, lymph, semen, and cerebrospinal fluid. The term may also mean a sample that has undergone several processing steps, e.g., nucleic acid extraction, cell depletion, analyte concentration, etc., and corresponds to a liquid containing the analyte that is subjected to the reagents and processes outlined herein. In one embodiment, as used herein, the terms “sample” or “aqueous sample” mean a liquid sample obtained from the body of a living organism, which may or may not be further processed, for example, to make the analyte of interest available for further analysis or to be subject to further analysis. Preferably, such “sample” or “aqueous sample” is selected from blood, plasma, serum, urine, sweat, tears, sputum, lymph, semen, ascites, amniotic fluid, bile, breast milk, synovial fluid, ascites, pericardial fluid, cerebrospinal fluid, chyle, and urine, and more preferably, the sample is plasma. Preferably, such aqueous sample may be further processed, for example, dissolved or digested before being subjected to the method according to the present invention, or otherwise processed. For example, if the aqueous sample is plasma, such plasma may be further dissolved and / or digested to remove components that may otherwise interfere with the subsequent detection reaction. For example, if the analyte of interest is nucleic acid of identification, the aqueous sample, e.g., plasma, may first be digested with proteases and other suitable enzymes before being subjected to the method according to the present invention to remove any unwanted proteins or peptides, as well as lipids or other unwanted components.Therefore, as used herein, “aqueous sample” may also refer to an extract obtained from any of the aforementioned body fluids; for example, it may be a nucleic acid extract obtained from any of the aforementioned body fluids by a suitable extraction method, for example, by disrupting cells (if necessary), removing lipids, proteins and unwanted nucleic acids (if necessary), and concentrating nucleic acids for the purification and / or identification of nucleic acids. In some embodiments, the nucleic acid extract may be produced using ethanol or another suitable alcohol.

[0097] According to embodiments of the present invention, the library used in a method for detecting multiple analytes in multiple different samples contains multiple different distinct pre-fabricated microparticle subsets, Each subset is, The subset of the fine particles has different labeling components attached thereto, contained therein, or otherwise associated therewith; Among the aforementioned multiple separate pre-fabricated microparticle subsets, there exist separate pre-fabricated microparticle subsets of different classes, each of which comprises several microparticle subsets, and each class has different analyte-specific reagents attached to the porous matrix of the microparticles, with the same analyte-specific reagent attached to all microparticle subsets within a single class; each analyte-specific reagent is specific to one target analyte; In the aforementioned library, the multiple distinct and separate fabricated microparticle subsets are, Each subset of the fine particles has different labeling components attached to, contained within, or otherwise associated with them; Each separate microparticle subset forms part of a class of microparticle subsets; Each subset is clearly defined and identifiable by its respective labeling component and its respective analyte-specific reagent; The aforementioned subsets of different classes of microparticles each have different analyte-specific reagents attached to the porous matrix of the microparticles; each of the aforementioned different classes comprises several subsets of microparticles, all of which within a class have the same analyte-specific reagent attached.

[0098] When such a library is actually used in a method for detecting multiple analytes in multiple different samples, it should be noted that such a library contains multiple distinct pre-fabricated microparticle subsets, and each of these distinct pre-fabricated microparticle subsets contains a different sublibrary, each of which is used to interrogate different samples. Each of these sublibraries contains several pre-fabricated microparticle subsets, and each subset within the sublibrary is coated with a different analyte-specific reagent.

[0099] Such a library is described in Embodiment 13.

[0100] One particularly useful feature of the fine particles according to some embodiments of the present invention is, (i) Analyte-specific reagents bound to fine particles; (ii) A polymer or polymer mixture that forms or is the porous polymer matrix; (iii) One or more ionizable groups immobilized on the porous matrix, wherein the ionizable groups are capable of changing their charge according to the ambient conditions around the precursor nanoparticles or nanoparticles; (iv) One or more charged groups immobilized on the porous polymer matrix; (v)(i)~(iv) Any combination By binding the analyte to the sample, the concentration of the analyte from the sample can be promoted, thus making it a means of concentrating, cleaning up, and detecting / quantifying the analyte / target in the sample.

[0101] Typical examples of analyte-specific reagents conjugated to microparticles (i.e., option (i) above) that can be used to facilitate the concentration of analytes from a sample include antibodies or antibody fragments; and non-antibody proteins that can specifically bind to the analyte or analyte complex, such as receptors, receptor fragments, and affinity proteins.

[0102] In a further embodiment, the present invention relates to a method for detecting and / or quantifying a target analyte in an aqueous sample, the method comprising the following steps: - • Aqueous samples known or suspected to contain the analyte of interest; • A general detection composition containing reagents for carrying out chemical or biochemical detection reactions of the analyte; A library of fabricated microparticles according to any of the embodiments defined above, wherein the analyte-specific reagent attached to the microparticles is selected to specifically bind to or react with the target analyte. A process of providing in any order; - If necessary, a step of mixing the aqueous sample with the general detection composition; -A step comprising incubating the aqueous sample with the library of fabricated microparticles, thereby allowing the library of microparticles to absorb the aqueous sample into the void volume of the microparticles, and, if necessary, enabling binding to the target analyte if present in the sample; - A step of washing the fine particles if necessary; - If the aqueous sample has not been previously mixed with the general detection composition, the step of adding the general detection composition to the fine particles; - A step of creating a plurality of isolated reaction spaces for detecting the analyte by transferring the library of fabricated microparticles to a non-aqueous phase and removing the aqueous phase around each individual fabricated microparticle, wherein the reaction spaces include an aqueous phase and are limited to the void volume of the microparticles; - A step of releasing the analyte-specific reagent attached to the fine particles by applying an external trigger, if necessary; - A step of performing a detection reaction for the target analyte; - Steps to detect and / or quantify the target analyte. Includes.

[0103] Such methods are particularly useful when the target analyte is nucleic acid and the detection reaction is a nucleic acid amplification reaction.

[0104] In a further embodiment, the present invention relates to a method for detecting and / or quantifying a target analyte in an aqueous sample, the method comprising the following steps: - • Aqueous samples known or suspected to contain the analyte of interest; A first detection composition comprising reagents necessary for carrying out a chemical or biochemical detection reaction of the analyte; • A second detection composition containing a detection reagent; A library of fabricated microparticles according to any of the embodiments defined above, wherein the analyte-specific reagent attached to the microparticles is selected to specifically bind to or react with the target analyte. A process of providing in any order; - If necessary, a step of mixing the aqueous sample with the first detection composition; -A step comprising incubating the aqueous sample with the library of fabricated microparticles, and if the aqueous sample has not yet been mixed with the first detection composition, also incubating it with the first detection composition, thereby allowing the library of microparticles to absorb the aqueous sample and the first detection composition into the void volume of the microparticles, and, if necessary, to bind to the target analyte if it is present in the sample; - If necessary, wash the library of the prepared microparticles to remove any unabsorbed or unreacted first detection composition; -A step comprising incubating the library of fabricated microparticles containing the absorbed aqueous sample with the second detection composition, thereby enabling the library of microparticles to absorb the second detection composition; - If necessary, further wash the library of the prepared microparticles to remove any unabsorbed or unreacted second detection composition; - A step of transferring the library of fabricated microparticles to a non-aqueous phase and removing the aqueous phase around each individual fabricated microparticle to create a plurality of isolated reaction spaces for detecting the analyte, wherein the reaction spaces include an aqueous phase and are limited to the void volume of the microparticles; preferably, the step of transferring to the non-aqueous phase is performed immediately after incubating the library of fabricated microparticles with the second detection composition; - A step of releasing the analyte-specific reagent attached to the fine particles by applying an external trigger, if necessary; A step of detecting and / or quantifying the target analyte by detecting and / or quantifying the detection reagent. Includes.

[0105] Such methods are particularly useful when the analyte of interest is a protein or other non-nucleic acid, and the detection reaction is immunochemical detection.

[0106] In a further embodiment, the present invention also relates to a method for detecting and / or quantifying a target analyte in an aqueous sample, the method comprising the following steps: - • Aqueous samples known or suspected to contain the analyte of interest; A first detection composition comprising reagents necessary for carrying out a chemical or biochemical detection reaction of the analyte; • A second detection composition containing a detection reagent; A library of fabricated microparticles according to any of the embodiments defined above, wherein the analyte-specific reagent attached to the microparticles is selected to specifically bind to or react with the target analyte. A process of providing in any order; - If necessary, a step of mixing the aqueous sample with the first detection composition; -A step comprising incubating the aqueous sample with the library of fabricated microparticles, and if the aqueous sample has not yet been mixed with the first detection composition, also incubating it with the first detection composition, thereby allowing the library of microparticles to absorb the aqueous sample and the first detection composition into the void volume of the microparticles, and, if necessary, to bind to the target analyte if it is present in the sample; - If necessary, wash the library of the prepared microparticles to remove any unabsorbed or unreacted first detection composition; -A step comprising incubating the library of fabricated microparticles containing the absorbed aqueous sample with the second detection composition, thereby enabling the library of microparticles to absorb the second detection composition; - If necessary, further wash the library of the prepared microparticles to remove any unabsorbed or unreacted second detection composition; - A step of creating a plurality of isolated reaction spaces for detecting the analyte by transferring the library of fabricated microparticles to a non-aqueous phase and removing the aqueous phase around each individual fabricated microparticle, wherein the reaction spaces include an aqueous phase and are limited to the void volume of the microparticles; - A step of releasing the analyte-specific reagent attached to the fine particles by applying an external trigger, if necessary; - A step of performing a detection reaction for the target analyte; and - Steps to detect and / or quantify the target analyte. Includes.

[0107] Such methods are particularly useful when the analyte of interest is a protein or other non-nucleic acid, and the detection of the analyte is performed by immunochemical detection coupled with an amplification reaction. A typical example of such a reaction is immunoPCR.

[0108] The microparticle library according to the present invention can be tailor-made according to the user's needs and can be used in a wide variety of different ways:

[0109] Therefore, in one embodiment, -The above method is, for example, a method for detecting and / or quantifying a single analyte in multiple aqueous samples, which may originate from different patients. -The method provides a number of different aqueous samples known or suspected to contain the analyte of interest, for example, samples from different patients. - The library of pre-fabricated microparticles provided in the above method is the library described in Embodiment 11, and such a library provides the same number or at least the same number of distinct microparticle subsets as the number of different aqueous samples to be tested, all of which have the same analyte-specific reagent attached to the porous matrix of the microparticles in the subset, and the analyte-specific reagent is specific to one target analyte; -In the step of incubating the aqueous sample with the library of fabricated microparticles, each of the different aqueous samples is incubated separately with a separate subset of microparticles in the library; -In the step of incubating the library of prepared microparticles containing the absorbed aqueous sample with a second detection composition, each of the distinct microparticle subsets of the library is incubated separately with the second detection composition; - The step of transferring the library to a non-aqueous phase is performed separately for each particulate subset, that is, each separate particulate subset is transferred separately to a non-aqueous phase, and the aqueous phase around the individual particulates is removed separately for each subset, thereby creating a plurality of isolated reaction spaces for detecting the analyte separately for each subset, the reaction spaces comprising the aqueous phase and limited to the void volume of the particulates, The method further comprises, after the step of transferring the library to a non-aqueous phase, the step of mixing together the plurality of isolated reaction spaces of all separate subsets in the non-aqueous phase, followed by a detection reaction for the target analyte, and then detecting and / or quantifying the target analyte.

[0110] In another embodiment, -The above method is a method for detecting and / or quantifying multiple analytes in a single aqueous sample. -In the above method, a single aqueous sample known or suspected to contain multiple target analytes is provided. - The library of pre-fabricated microparticles provided in the above method is the library described in Embodiment 12, and such a library provides as many or at least the same number of distinct microparticle subsets as the number of different target analytes to be detected, each of the distinct subsets having a different analyte-specific reagent attached to the porous matrix of the microparticles in the subset; each analyte-specific reagent is specific to one target analyte; -In the step of incubating the aqueous sample with the library of fabricated microparticles, the aqueous sample is incubated together with all of the separate microparticle subsets of the library; -In the step of incubating the library of prepared microparticles containing the absorbed aqueous sample with a second detection composition, all of the distinct microparticle subsets of the library are incubated together with the second detection composition; - The step of transferring the library to a non-aqueous phase is performed together for all particulate subsets, i.e., all particulate subsets are transferred together to a non-aqueous phase, and the aqueous phase around the individual particulates is removed, thereby creating multiple isolated reaction spaces for detecting and / or quantifying the multiple analytes, the reaction spaces comprising the aqueous phase and limited to the void volume of the particulates.

[0111] In another embodiment, -The above method is a method for detecting and / or quantifying multiple analytes in multiple aqueous samples. -The method provides multiple different aqueous samples known or suspected to contain multiple target analytes, for example, samples from different patients. -The library of pre-fabricated microparticles provided in the method is the library described in Embodiment 13, in which there are separate subsets of pre-fabricated microparticles of different classes, each of which comprises several subsets of microparticles, each of which has different analyte-specific reagents attached to the porous matrix of the microparticles, the same analyte-specific reagent attached to all subsets of microparticles in one class; each analyte-specific reagent is specific to one target analyte; in the method, the step of providing the library, • A different class of distinct particulate subsets is provided, in the same number or at least the same number as the number of different analytes to be detected. • Each class is provided with the same number or at least the same number of different particulate subsets as the number of aqueous samples to be tested. The library contains a number of different particulate subsets equal to or at least the number obtained by multiplying the number of different aqueous samples to be tested by the number of different target analytes to be detected. -In the step of incubating the aqueous sample with the library of fabricated microparticles, strictly one aqueous sample is incubated with strictly one microparticle subset from each class, and each aqueous sample is incubated with the same number of different microparticle subsets from different classes as the number of classes of microparticles in the library. -In the step of incubating the library of fabricated microparticles containing the absorbed aqueous sample with a second detection composition, a subset of each aqueous sample combined with a different class of microparticles that have been previously incubated is incubated together with the second detection composition. - The step of transferring the library to a non-aqueous phase is performed separately for each aqueous sample, that is, each aqueous sample and the pre-incubated combined microparticle subset are transferred together to a non-aqueous phase, and the aqueous phase around the individual microparticles is removed separately for each aqueous sample, thereby creating multiple isolated reaction spaces for detecting the multiple analytes, the reaction spaces comprising the aqueous phase and limited to the void volume of the microparticles, The method preferably further includes, after the step of transferring the library to a non-aqueous phase, mixing together the multiple isolated reaction spaces of the separate samples in the non-aqueous phase, then carrying out the detection reaction for the target analyte, and then detecting and / or quantifying the target analyte.

[0112] Regarding quantification in the above-described method, in some embodiments, in the step of detecting and quantifying the target analyte, the quantification of the analyte is performed as follows: a) Digital nucleic acid amplification, particularly digital polymerase chain reaction (PCR); b) Real-time quantitative nucleic acid amplification, particularly real-time polymerase chain reaction (PCR); c) Immunochemical detection methods, particularly digital immunochemical detection methods, such as digital immunoassays, such as digital enzyme-linked immunosorbent assays (ELISA); d) Immunochemical detection methods combined with nucleic acid amplification, e.g., immunopolymerase chain reaction; in particular, digital immunoPCR It is done by a method selected from; If the analyte is a nucleic acid, quantification shall be performed using either method a) or b) or a combination of a) and b); if the analyte is a protein, peptide, or other non-nucleic acid analyte, quantification shall be performed using either method c) or d).

[0113] The versatility of the present invention, particularly with respect to analyte quantification, is also reflected in the fact that embodiments of the microparticle library according to the present invention enable parallel (multiplexed) ultra-high-sensitivity detection of multiple analytes in a sample. In particular, this works in the library described in any of embodiments 12 to 13. More specifically, this works in particular in the library described in embodiment 12 (a library for detecting and / or quantifying multiple analytes in a sample). Each microparticle corresponds to a discontinuous signal amplification space or target amplification space that enables the detection of a single molecule similar to established digital detection methods, while these microparticles can be used to simultaneously detect and even quantify multiple different analytes (i.e., multiple analyte species) (>1) in a sample, thereby the achievable theoretical detection limit for each different analyte can be determined by the following formula:

number

[0114] The above formula is due to the binomial distribution of analyte molecules across all interrogated particles in the sample. The detection limit is represented by N, thereby setting P to 95%. For clarity, the above formula does not take into account the distribution of the analyte in the body fluid sample being analyzed regarding the presence and amount of the analyte.

[0115] Furthermore, the microparticle library embodiment according to the present invention enables a remarkable approach to quantifying analytes by bridging the gap between a quantitative digital analysis approach and a quantitative real-time analysis approach. This claim applies to any type of signal amplification assay or target amplification assay used on microparticles, and PCR amplification can serve as an example illustrating the approach to quantifying targets by embodiments of the method according to the present invention. Quantification of target molecules is preferably performed by digital PCR (dPCR) because this method allows for more sensitive and accurate quantification than real-time quantitative PCR (qPCR). A drawback of digital PCR is that its inherent measurement range limitations depend on the number of microparticles specific to a single sample / analyte. To overcome this limitation, the present invention complements the analysis of digital PCR with real-time quantitative PCR for target concentrations exceeding the measurement range of digital PCR. Implementing this approach requires the acquisition of fluorescence images at the end of PCR and throughout the entire cycle of real-time PCR. All acquired images are analyzed by an image analysis algorithm to quantify the fluorescence level of each microparticle in the reactor chamber. A segmentation algorithm separates bright, disc-shaped microparticle objects from a dark background. Based on this segmentation information, the average fluorescence and average volume of the microparticles can finally be estimated by fitting circles to the contours of the microparticle objects. Real-time PCR image analysis involves tracking the position of microparticles in sequential images, thereby enabling monitoring of fluorescence during the reaction process at each individual microparticle.

[0116] The selection of the applicable quantification approach is based on the number / ratio of microparticles that remain negative after the amplification reaction, i.e., do not show amplification. This information is obtained from the digital PCR data. If the number / ratio of negative microparticles exceeds a predefined lower limit (i.e., the number / ratio of negative microparticles is higher than such a predefined lower limit), endpoint Poisson analysis can be applied. Otherwise, i.e., if the number / ratio of negative microparticles is lower than such a predefined lower limit, real-time analysis is performed using real-time quantitative PCR data acquired throughout the entire cycle. A reasonable lower limit for the ratio of negative microparticles that enables robust quantification with Poisson is 0.5%. The average number of targets per corresponding microparticle is 5.3. A further requirement may be a lower limit for the total number of negative microparticles, e.g., 50, to account for artifacts that may be present in the fluorescence images.

[0117] Endpoint Poisson analysis is performed by measuring the proportion of negative particles and applying a Poisson correction to account for the fact that positive particles may contain more than one target molecule. The threshold for distinguishing between positive and negative particles is directly estimated from the fluorescence signal intensity of particles known to be negative. Possible variability in particle volume is incorporated into the quantification by performing a Poisson correction specific to particle volume. Variation in the total volume of particles that may occur between measurements is also corrected by the algorithm described above.

[0118] If the target concentration exceeds the measurement range of digital PCR, real-time analysis fits a nonlinear function to the progression of the fluorescence signal of each single microparticle. This fitted nonlinear model is a combination of a sigmoid function and a linear function, where the sigmoid component reveals the amplification dynamics and the linear component represents the signal baseline. The cycle threshold (Ct) is calculated from the intersection of the baseline and the tangent line at the defined value of the sigmoid function where the second derivative is maximized. The number of targets per microparticle is calculated using a calibration dataset.

[0119] This quantification principle is illustrated in Figures 12 and 13 and in Example 3. See also the drawings. [Brief explanation of the drawing]

[0120] [Figure 1] Figure 1 shows embodiments of the process for generating a library of fabricated precursor microparticles (left portion of the figure) and for generating a library of fabricated microparticles thereafter (right portion of the figure). In the left portion of the figure, fabricated precursor microparticles are generated, each having a different labeling component attached to, contained within, or otherwise associated with the precursor microparticle; different subsets of fabricated precursor microparticles are generated by repeating this process in the left portion of the figure for different fabricated precursor microparticles with different labeling components. The result is a library of fabricated precursor microparticles, which may contain at least two distinct subsets of fabricated microparticles, or possibly three or more distinct subsets, each having a different labeling component attached to, contained within, or otherwise associated with the precursor microparticles in the subset, and the at least two or more distinct subsets of fabricated precursor microparticles each have a different labeling component attached to or contained within each subset. In the right portion of the figure, an analyte-specific reagent (ASR) is loaded into each of the fabricated precursor microparticle subsets. When using a library of microparticles to detect different analytes, the analyte-specific reagents attached to each microparticle subset will be different. When using the library to detect the same analyte using multiple samples, the analyte-specific reagents attached to different microparticle subsets will be the same. The resulting pre-fabricated microparticle library is highly versatile and therefore can be prepared and used according to different needs. It may be used to detect multiple analytes in a single sample, or to detect a single analyte in multiple samples.

[0121] In the embodiment shown in Figure 1, binding of the analyte-specific reagent (ASR) is carried out via a reagent-binding component which may be one of the possibilities (i) to (v) further enumerated above. For example, in its simplest form, the reagent-binding component may be a polymer or polymer mixture which forms a porous polymer matrix or a polymer matrix of fine particles. In another embodiment, it may be a reagent-binding molecule of identification attached to a porous polymer matrix. In yet another embodiment, it may be one or more ionizable groups, or one or more charged groups immobilized on a porous polymer matrix, or any combination of the foregoing. In a preferred embodiment, the reagent-binding component is a reagent-binding molecule attached to a porous polymer matrix, and the porous polymer matrix interacts with the binding entity conjugated to the analyte-specific reagent. An example of this embodiment is further shown below, for example, the reagent-binding molecule attached to the porous polymer matrix is ​​a streptavidin molecule or a streptavidin-related molecule. The binding entity on the analyte-specific reagent, i.e., the binding entity that binds to the reagent-binding molecule, is desthiobiotin, or a similar molecule that enables reversible attachment to streptavidin or avidin. Reversibility is achieved in such a way that the binding between the binding entity (on the analyte-specific reagent (ASR)) and the reagent-binding molecule (on the porous polymer matrix) can be released by applying an external trigger, for example, by changing the temperature to which the particulate matter is exposed. It will be apparent to those skilled in the art that many different variations of such embodiments are possible and conceivable.

[0122] [Figure 2]Figure 2 shows an embodiment of a scheme outlining the relationship between precursor microparticles and the resulting fabricated microparticles when analyte-specific reagents are bound to them. Fabricated precursor microparticles having a porous polymer matrix are provided. The porous polymer matrix has void volumes for receiving aqueous samples and providing a reaction space for the specific detection of analytes. The fabricated precursor microparticles further include a reagent-binding component that enables the attachment, preferably reversible, of analyte-specific reagents to the precursor microparticles. Furthermore, the precursor microparticles are coated with a labeling component that enables the identification of the precursor microparticles (and the subsequently resulting fabricated precursor microparticles, as well as the analyte-specific reagents that will be coated to the precursor microparticles). Analyte-specific reagents (ASRs) that will be coated to the fabricated precursor microparticles are also shown. Note that, as an example, such analyte-specific reagents may be nucleic acid primer pairs and, optionally, probes that are specific to the (nucleic acid) analyte of identification and enable amplification and detection of such analytes if such analytes are present in the sample to which each microparticle is subsequently exposed. In this figure, all ASRs are shown similarly, but such primer pairs and, if applicable, probes are assumed to be eligible as a single analyte-specific reagent. Another example of an analyte-specific reagent could be a (primary) antibody specific to the analyte of identification.

[0123] [Figure 3-1]Figure 3A shows an exemplary scheme embodiment for a method for detecting an analyte of interest in an aqueous sample, in which an embodiment is provided of a library of microparticles according to the present invention and exposed to such an aqueous sample. The library is exposed (or "incubated") to a detection composition containing the sample and amplification / detection reagents. As a result of such exposure, the library of microparticles can absorb the aqueous sample and detection composition into the void volume of the microparticles and, if necessary, bind to or concentrate the analyte of interest if it is present in the sample. Depending on the type and number of different analyte-specific reagents attached to the microparticles in the library, one or more different analytes may be detected. After the library has been exposed to the sample and detection composition, the library is transferred to a non-aqueous phase, and the aqueous phase around the individual prepared microparticles is removed, for example, by applying mechanical force to the microparticles. However, each microparticle still has an aqueous phase inside its respective void volume. As a result of such migration to the non-aqueous phase and removal of the surrounding aqueous phase, multiple isolated reaction spaces are created, which act as "reactors" enabling the detection of the analyte. These reaction spaces contain the aqueous phase and are limited to the void volume of each microparticle. Depending on the precise properties of each microparticle, it may be preferable to induce a phase transition in such microparticles, e.g., a sol-gel transition (i.e., a transition from a solid or semi-solid gel state to a liquid soluble state), which triggers the release of the respective analyte-specific reagents effectively attached to the microparticles. This can be achieved by applying an external trigger, such as a change in temperature, a change in pH, or the addition of an identifying chemical agent. In a preferred embodiment, such an external trigger is an increase in temperature. The sol-gel transition also typically results in a transition from a suspension of microparticles (solid in liquid) to a suitable emulsion of microdroplets (formerly (formlerly) solid microparticles) in the liquid phase (liquid in liquid). Each microparticle is transferred to the non-aqueous phase and, if necessary, released with the analyte-specific reagents before undergoing the detection reaction for the target analyte.Since each of these microparticles is isolated in the non-aqueous phase, there is no crosstalk between different microparticles / between the reaction spaces provided by such microparticles. Depending on the type of analyte and analyte-specific reagent, such a detection reaction may be an amplification reaction (if the analyte is a nucleic acid) or an immunochemical reaction (e.g., if the analyte is a protein). Subsequently, the analyte can be detected in the individual microparticles. Alternatively, the detection reaction may also be an immunoamplification reaction, where a primary antibody conjugated to the analyte is used in the first step, and a sandwich is formed using a secondary antibody attached to an oligonucleotide tag that can be amplified using suitable primers in the second step. Since each microparticle has a specific labeling component, it is possible to assign the presence of the analyte of identification and the associated (or generated) signal to the labeling component of each microparticle in which the signal is detected.

[0124] [Figure 3-2]Figure 3B shows an exemplary scheme embodiment for a method of detecting and / or quantifying an analyte of interest in an aqueous sample, in which a library of microparticles according to the embodiment of the present invention is provided and the library is exposed to such an aqueous sample under conditions favorable for binding the analyte to the microparticles. To remove unwanted material, the microparticles having bound analytes may be washed with a suitable buffer. Subsequently, the library is exposed to a detection composition containing an amplification / detection reagent. After exposure to the detection composition containing the required amplification / detection reagent, the library is transferred to a non-aqueous phase, thereby creating a virtually isolated reaction space within each microparticle, and thus multiple isolated reaction spaces are effectively created. Each microparticle contains an aqueous phase containing the sample and the required amplification / detection reagent, and is isolated from other microparticles by the surrounding non-aqueous phase. Subsequently, an amplification / detection reaction is performed, and the analyte of interest may be detected in each microparticle. Since each microparticle has its own labeling component, each analyte-specific signal generated when the analyte is present in each microparticle, such signals, and the presence of the corresponding analyte can be assigned to the respective labeling component of the individual microparticle and therefore to the individual microparticle.

[0125] [Figure 4] Figure 4 shows an embodiment of the synthesis of encoded precursor microparticles, as described in detail in Example 1. Using differently labeled gelatin and agarose, agarose / gelatin microparticles with different labeling components are generated. Each differently labeled type of gelatin is shown on the left. The center photograph shows images of several differently labeled microparticles thus generated, and on the right are scatter plots of the fluorescence signals obtained for individual microparticles in two separate fluorescence channels, and pseudo-color images of different microprecursor microparticles that can be detected by their respective fluorescence. Nine different types of precursor microparticles can be clearly distinguished as distinct particle populations according to their fluorescence.

[0126] [Figure 5]Figure 5 shows that by selecting an appropriate binding entity for a primer oligonucleotide (which acts as an analyte-specific reagent), these can be reversibly attached to streptavidin-coated microparticles prepared according to embodiments of the present invention. In this example, the reagent-binding molecule on the microparticles is streptavidin, and the binding entity on the analyte-specific reagent is biotin or desthiobiotin. Desthiobiotin allows for reversible attachment, while biotin does not. To demonstrate the reversible nature of attachment in the case of desthiobiotin, crosslinked microparticles were incubated with biotin-tagged fluorescently labeled oligonucleotides (Panel A) or desthiobitin-tagged fluorescently labeled oligonucleotides (Panel C) ("loading" stage). The incubation ("loading") was performed at room temperature. The microparticles were washed and fluorescence images were taken. In both images (upper left and upper center images, Panels A and C), the microparticles are clearly visible. Subsequently, the temperature was raised to 95°C ("denaturation" stage), and the microparticles were washed again. The biotin-labeled oligonucleotides remained bound to the microparticles (as can be seen from the remaining fluorescence in panel B), while the desthiobiotin-labeled oligonucleotides (and their associated fluorescence) were clearly removed by this treatment (no fluorescence is seen in panel D). The bar graph on the right (panel E) is a quantitative representation of the signals obtained from the microparticles before and after heating.

[0127] Therefore, it has been shown that by using such reversibly bound entities (e.g., desthiobiotin), primer oligonucleotides can be released from the microparticle matrix. This is a desirable feature for highly efficient amplification reactions in droplet spaces created by microparticles in a non-aqueous environment. While some amplification may be possible even with primers attached, for optimal amplification reaction conditions, it is ideal for the primer oligonucleotides to be unbound to any matrix and therefore intentionally released before the amplification reaction.

[0128] [Figure 6] Figure 6 shows a microscopic image containing microparticles prepared according to embodiments of the present invention. These microparticles were coated with an anti-CD45 antibody and incubated with whole blood stained with the fluorescent dye acridine orange. After carefully washing the sample with PBS buffer, the microparticles were imaged. The microparticles, having a variable diameter of 35–50 μm, prepared according to embodiments of the present invention, are distinguishable from the background, and some of these microparticles hold single cells attached to them. Since CD45 is a surface antigen characteristic of leukocytes, the bound cells are likely to be leukocytes. This embodiment clearly demonstrates that such microparticles can also be used to selectively bind to cell populations and subsequently to select cells on individual particles that can be processed according to the present invention in accordance with the process outlined in Figures 3A and 3B.

[0129] [Figure 7]Figure 7 illustrates an embodiment of a method for detecting several analytes in a single sample using a library of four differently labeled microparticles ("nanoractors") according to the present invention ("analyte multiplexing"). This Figure 7 illustrates the experiment performed in Example 3. Four different types of microparticles (sometimes referred to as synonyms for "nanoractors" in this figure and elsewhere in this specification) that can be distinguished according to their respective labeling components are used (specific to four different molecular targets rpoB, IS6110, IS1081, and atpD by attaching target-specific primers and probes (as analyte-specific reagents) to each microparticle). Figure 7A shows three grayscale images representing three fluorescence channels used to microimage the color-labeled agarose-gelatin hybrid microparticles. The four different labels can be assigned to the detected microparticles according to the fluorescence signals obtained in channels 1 and 2, as illustrated by the scatter plot. A further channel, channel 3, is used to monitor the nucleic acid amplification signal (e.g., PCR signal). Each labeling component corresponds to the target of identification (analyte) when present. The 1D plots for each type (or "subset") of microparticles in Figure 7B show positive and negative signals, which translate to the number of detected copies per sample volume. Figure 7B shows the signal intensity for different nanoreactor (or microparticle) types (i.e., different analytes). After amplification, it can be seen that microparticle type ("subset") 0 and microparticle type ("subset") 3, which are specific to rpoB and atpD, respectively, show positive signals in the tested sample, indicating the presence of such analytes in the original sample. These graphs also show the presence of positive and negative microparticles. By counting the positive and negative microparticles and applying Poisson analysis, the number of targets in the sample can be determined with high accuracy. Thus, this also enables high-accuracy quantification.In contrast, particulate type ("subset") 1 and particulate type ("subset") 2, which are specific to analytes IS6110 and IS1081, respectively, do not yield a positive signal, indicating that such analytes are not present from the original sample.

[0130] [Figure 8] Figure 8 shows an embodiment of a kit for preparing a library of pre-fabricated microparticles ("fabrication kit"). Such an embodiment of such a kit comprises at least two or more containers, each containing a subset of pre-fabricated precursor microparticles as further defined above. Each subset of pre-fabricated precursor microparticles has a different labeling component attached to, contained within, or otherwise associated with the precursor microparticles, and different subsets of pre-fabricated precursor microparticles have different respective labeling components attached to, contained within, or otherwise associated with them. The kit also comprises a further container containing a conditioning solution (e.g., a buffer, e.g., PBS buffer, or water) which enables the attachment, preferably reversible, of analyte-specific reagents to the subsets of precursor microparticles. In a preferred embodiment, the kit further comprises a further container containing a washing buffer which removes free, i.e., unattached, analyte-specific reagents from the microparticles, but does not remove analyte-specific reagents attached to the microparticles. Furthermore, in a preferred embodiment, such a kit also includes one or more mixing vessels for mixing the components together. Different analyte-specific reagents that users of such a kit can select and provide are also shown, which enable such users to generate a library of microfabricated microparticles according to their needs. The adhesion of each analyte-specific reagent is facilitated by using the conditioning solution included in the kit.

[0131] [Figure 9]Figure 9 shows an embodiment of a kit for detecting an analyte in a sample ("detection kit") according to an embodiment of the present invention. In this exemplary figure, the kit includes a container containing a general detection composition (a composition containing reagents necessary to carry out the detection reaction but not containing any analyte-specific reagents (ASRs)), a container containing a non-aqueous phase, such as oil, along with a suitable emulsifier as needed. Furthermore, such a kit may optionally include one or more mixing containers. Furthermore, as needed, such a kit may optionally include a further container that functions as a reactor. In this embodiment, a library of fabricated microparticles may be provided separately, as with the sample, and such a library is exposed to the sample and the detection reagents in the mixing container of the kit. Phase transfer is then carried out by using the "reactor" container in which the next detection reaction takes place.

[0132] [Figure 10-1]Figures 10A-D show examples of different libraries according to embodiments of the present invention. Figure 10A shows an example of a library for detecting a single analyte in several samples. The library shown in Figure 10A is the simplest library for detecting a single analyte in two different samples. As can be seen, such a library contains two subsets of particulate matter, each with the same analyte-specific reagent attached, but with different labeling components ("1" and "2"). Each subset in such a library is used to detect the same analyte in different samples. Figure 10B shows a simple example of a library for detecting several analytes in a single sample. In this case, since the library contains two different subsets (each subset with a different analyte-specific reagent attached), the analytes that can be detected are two different analytes. Furthermore, these subsets have different labeling components ("1" and "2"). These two different subsets may initially be stored in separate containers, but may ultimately be combined into a single container when exposed to a sample. Figure 10C shows another example of a library for detecting several analytes in a single sample. This exemplary library provides four different microparticle subsets, each of which has its own different analyte-specific reagent attached. Furthermore, each subset has a different labeling component ("1", "2", "3", and "4") from one another. Initially, these different subsets may be stored in separate containers but may eventually be combined when exposed to a sample, and it is suspected that four different analytes are present in those samples. Figure 10D shows an example of a library for detecting several analytes in several samples. As can be seen, there are eight different microparticle subsets. Each microparticle subset has a different labeling component ("1" through "8"), but there are four pairs of microparticle subsets, each pair having a different analyte-specific reagent attached, and two subsets within each pair having the same analyte-specific reagent attached.In this application, a subset of microparticles that are occasionally coated with the same analyte-specific reagent but have different labeling components may also be referred to herein as part of a “class”. An example of a library shown in Figure 10D may be used to detect the presence of four analytes in two samples. Thus, each sample is explored with, interrogated with, or exposed to four microparticle subsets, each of which is coated with a different analyte-specific reagent. Such subsets of different microparticles used to interrogate the same single sample (of several samples) may also be referred herein as a “sublibrary” of microparticle subsets. The concepts of “class” and “sublibrary” are further outlined and explained in Figure 11.

[0133] [Figure 10-2] Figure 10E shows an exemplary schematic diagram of a method for detecting several analytes in a single sample, illustrating the preparation of a library that allows for the detection of four different analytes using four different analyte-specific reagents (ASRs) in such a library. Each microparticle is distinguishable by being differently labeled with a different labeling component ("1" to "4"). The resulting library is the exemplary library shown in Figure 10C. The single sample is exposed to the library and the required detection compositions, and the microparticles are transferred to a non-aqueous phase. The detection reaction is then carried out, and the resulting signal is detected and analyzed.

[0134] [Figure 11-1]Figures 11A and 11B show exemplary libraries for detecting several analytes in several samples. In this identification case shown here, there are three different samples to be tested for the presence of four different analytes. As seen in Figure 11A, there are four different particulate subsets for each sample tested. However, at the same time, there are also three particulate subsets to which the same analyte-specific reagent is attached, so that three (or N-threads) particulate subsets to which the same analyte-specific reagent is attached are formed. Such an N-thread (to which the same analyte-specific reagent is attached) has as many different subsets as there are samples tested (if not all subsets of that N-thread are ultimately used in that experiment, there may be "at least the same number" of different subsets). Thus, in effect, N indicates the number of samples tested. Such an N-thread may also be referred to herein as a "class," which refers to the entirety of all particulate subsets in the library to which the same analyte-specific reagent is attached. All subsets within a class are specific to one target analyte. In contrast, the term “sublibrary” refers to the entire set of all microparticle subsets in a library used to interrogate a sample of one identification at a time. Within each sublibrary, each subset of prepared microparticles is contaminated with a different analyte-specific reagent; each analyte-specific reagent is specific to one target analyte. In Figure 11A, sublibraries 1, 2, and 3 each contain four microparticle subsets, and these four microparticle subsets within each sublibrary may initially be stored in separate containers. However, when each sublibrary is brought into contact with its respective sample, the corresponding microparticle subsets within each sublibrary may be combined. The microparticles exposed to each sample may then be washed as necessary and subsequently exposed to a suitable detection composition containing the reagents required to carry out the amplification / detection reaction. Subsequently, phase transfer is performed, and each microparticle is placed into a non-aqueous phase, thereby creating multiple isolated reaction spaces for each sample, effectively as a suspension.After each particle is transferred to a non-aqueous phase, they can be pooled in a single container, where suitable amplification / detection reactions can be carried out. [Figure 11-2] Same as above.

[0135] [Figure 12]Figure 12 shows a combination of digital PCR and real-time quantitative PCR for quantifying targets in microparticles. The quantification approach shown here utilizes digital PCR when the target concentration does not exceed the upper limit of the measurement range. Digital PCR is fairly accurate and robust to PCR inhibitors. Confidence intervals ("CI") for digital PCR, resulting from statistical effects, are shown. These are determined by the Poisson distribution of sample collection at the lower limit of the measurement range and the binomial distribution of the target on the microparticle at the upper limit of the measurement range. The Poisson distribution of sample collection also contributes to the inaccuracy of real-time PCR. However, real-time PCR is also highly susceptible to the variability of the PCR process. Quantitative fluorescence readings during amplification are considerably more likely to be affected by variability in reaction efficiency than binary fluorescence readings at the completion of PCR. Typical reproducibility for commercial test assays based on real-time quantitative PCR ranges from approximately 0.30 log cp / mL (copies / ml) at very low target concentrations to 0.10 log cp / mL at high target concentrations. The advantage of the proposed quantification approach is that real-time quantitative PCR is preferably applied only to high target concentrations, in which case the method can yield more accurate results than at low concentrations. Generally, this approach makes it possible to utilize the large dynamic range of quantitative PCR (qPCR), as well as the exquisite sensitivity and quantification accuracy of digital PCR at the lower limit of the measurement range. In one embodiment, after the detection method according to the present invention has been performed, quantification may be achieved using the following illustrative guidelines: If the number of negative (dark) particles is >0.5% of the total particles available in the test assay, Poisson analysis (digital) is applied. For lower values, real-time analysis is applied. This corresponds to an approximate average concentration (lambda) of 5.3 targets / particle. Artifacts that may be present in the fluorescence image can be accounted for by introducing a minimum requirement for the total number of negative particles, e.g., 50 per analysis.

[0136] More generally, after performing the detection method according to the present invention, quantification can be achieved using the following illustrative guidelines: If the number of negative particles (i.e., particles that do not produce a signal) exceeds a percentage within the range of 0.1 to 1.0%, preferably 0.5 to 1.0%, and more preferably 0.5 to 0.8%, Poisson analysis is applied. If the number of negative particles (i.e., particles that do not produce a signal) is less than a percentage within the range of 0.1 to 1.0%, preferably less than a percentage within the range of 0.5 to 1.0%, and more preferably less than a percentage within the range of 0.5 to 0.8%, then, for example, a method including measurement of the cycle threshold (e.g., comparative C, also known as the 2-ΔΔCT method) is applied. T Quantitative real-time analysis is applied using methods (see, for example, Schmittgen et al., 2008, Nature Protocols, 3, pp. 1101-1108).

[0137] Further details are described in Example 3.

[0138] [Figure 13-1] Figure 13 shows real-time fluorescence data obtained from a series of images collected in microparticles in oil during PCR amplification. The left side shows an image of the detection chamber containing the endpoint fluorescence signal in a single fluorescence channel specific to the amplification. The central graph shows the fluorescence intensity for 12 representative individual microparticles selected from the fluorescence images on the left. The histogram on the right shows the distribution of calculated ct values ​​for all microparticles detected in the fluorescence images. [Figure 13-2] Same as above.

[0139] Furthermore, the following embodiments are provided not to limit the invention, but to illustrate it. [Examples]

[0140] Example 1 Synthesis of coded precursor microparticles Labeling of gelatin with fluorescent dyes for identification of analyte-specific reagents. Acetone-insoluble fractions of gelatin derived from bovine skin type A or porcine skin type B are labeled with different mixtures of two fluorescent dyes using NHS coupling chemistry. Cy®3 Mono NHS Ester and Cy®5 Mono NHS Ester (GE Healthcare) are dissolved in DMSO to produce a 1% (w / v) final solution in potassium phosphate buffer (pH 8.0, filter-sterilized). 25 mL of 0.25% (w / v) of either gelatin type is labeled using an 8-fold molar excess of each dye compared to the free gelatin amino groups. These labeled solutions are incubated overnight at 4°C using a Multi-Rotator PTR-60 (Grant-bio) in vertical mode. The fluorescently labeled gelatin is purified by repeated ammonium sulfate salting-out using a saturated (NH4)2SO4 solution. Alternatively, ultracentrifugation, solvent extraction with isopropanol, acetone, or methanol, gel filtration using a Sepharose column, or dialysis can be performed. In either case, the purification process is repeated until the eluate becomes clear and no longer fluoresces. Finally, the purified fluorescently labeled gelatin sample is vacuum-dried. Preparation of gelatin / agarose hybrid solution

[0141] A hybrid hydrogel solution consisting of four components is prepared to fabricate a nanoreactor. Ingredient 1: Acetone-insoluble gelatin derived from bovine skin type A G1890 (Sigma) or porcine skin type B G9391 (Sigma). Ingredient 2: Low-gelling 2-hydroxyethyl agarose (A4018, Sigma) Ingredient 3: Cy3-labeled gelatin (Type A or Type B) Ingredient 4: Cy5-labeled gelatin (Type A or Type B)

[0142] To produce a homogeneous 4% (w / v) solution of component 1, dissolve 40 mg of component 1 in 1 mL of nuclease-free water (Carl Roth) and incubate at 50°C with gentle stirring (750 rpm). Similarly, dissolve and melt 20 mg of component 2 in 1 mL of nuclease-free water and incubate at 80°C with gentle stirring to prepare a homogeneous 2% (w / v) agarose solution. To prepare a 4% (w / v) solution of each labeled gelatin, dissolve the dry pellets of components 3 and 4 in their respective volumes of nuclease-free water and incubate at 55°C until the gelatin is melted. Mix all four components and fill with nuclease-free water to produce hybrid hydrogel solutions with final concentrations of 1% (w / v) gelatin and 0.5% (w / v) agarose A4018, respectively. Mixing components 3 and 4 in varying volumes yields n sets of microspheres of different colors. In this embodiment, the resuspended components 3 and 4 are mixed in ratios of 1:0, 3:1, 1:3, and 0:1 at a maximum of 3% (v / v) of the total gelatin fraction to obtain four individual labeled components (labeled components 1-4, respectively) for identifying analyte-specific reagents and enabling a quadruple reaction assay. Maintain all solutions at 55°C until further use. Production of non-crosslinked gelatin / agarose microparticles

[0143] Color-coded monodisperse agarose-gelatin hybrid microparticles are subjected to QX100 / QX200 Droplet Digital (ddPCR) TMThe system was prepared using a part of the BioRad system or a modified μEncapsulator system (Dolomite microfluidics). In the case of the BioRad system, the DG8 cartridge is maintained on a Thermomixer to maintain the solution at 55°C. After loading 50 μL of gelatin / agarose hybrid solution, 100 μL of emulsion reagent HFE-7500 containing 2-5% Picosurf 2 (Sphere Fluidics) is applied to the bottom well of the cartridge. Vacuum is applied to the collection well by gently pulling the syringe connected to the collection well. Alternatively, QX200 TM / QX100 TM A Droplet Generator can be used for droplet production. Approximately 80,000 hydrogel droplets with a diameter of 100 μm can be produced per well.

[0144] In the case of the Dolomite microfluidics system, monodisperse hybrid microparticles can be fabricated in a single-step suspension formation process using a simple flow focus device. Specifically, a standard droplet junction tip (100 μm) with fluorophilic properties is used with a 4-way linear connector and tip interface H to mediate the fluid connection between the tube material and the tip. Two Mitos P-Pumps deliver the hydrogel solution and carrier oil. By modifying this system to incorporate a heating device placed on a hot plate, which allows for maintaining the gelatin / agarose hybrid solution in a liquid state and heating the driving fluid, a constant temperature is ensured when the oil and gelatin / agarose hybrid solution come into contact at the tip junction. Both HFE-7500 / Picosurf 2 and the hybrid hydrogel solution are pre-filtered through a 0.22 μm filter and then placed into the P-Pump (Mitos) and hydrogel reservoir in the heating device of the droplet system, respectively. The heating device temperature is set to 55°C. Prepare the fluid line at 2000 mbar for 1 minute. Adjust the flow rate to 15-17 μl / min for stable droplet formation. Monitor the parameters using Dolomite Flow Control Advanced Software.

[0145] In both cases, the color-coded agarose-gelatin hybrid microparticles are collected in a 2 mL microcentrifuge tube or 15 mL Falcon tube on ice to initiate the solidification of the hybrid hydrogel. To prevent the loss of the aqueous phase of the microparticles at the oil-air boundary, 500 μL of emulsion oil containing the microparticles is covered with 500 μL of nuclease-free water. The microparticles are then stored at 4°C for at least 1 hour (preferably overnight) to form a stable hybrid skeleton. Recovery of hybrid microparticles from the continuous phase

[0146] The solidified hybrid microparticles accumulate on top of the emulsion oil. Carefully remove the emulsion oil with a pipette, taking care not to remove the particles. Then, add 500 μL of 1H,1H,2H,2H-perfluorooctanol (PFO; Sigma) to the tube to break up the suspension. Vortex the tube for 5 seconds and centrifuge at 2,500 × g for 5 seconds to transfer the hybrid hydrogel particles to the oil phase. Transfer the hybrid hydrogel microparticles to a new 1.5 mL microcentrifuge tube. Optional: This procedure can be repeated to remove residual fluorocarbon oil and surfactant. After washing with PFO, wash the recovered microparticles once with 1 mL of nuclease-free water. Visually inspect the quality and size of the microparticles using a microscope. Nine exemplary microparticle preparations are shown in Figure 4 (colored precipitates of individual microparticle preparations in microtubes on the left, and a mixed set of nine different microparticles in the center image). Functionalization of hybrid precursor nanoparticles

[0147] To enable the attachment of analyte-specific components to hybrid precursor nanoparticles, a mobile binding chemistry is established. Streptavidin is covalently attached to the amine-containing fraction of the hybrid matrix of the nanoparticles using the following three-step protocol. First, a sulfhydryl group is added to streptavidin using the amine-reactive portion of the SPDP reagent (NHS) ester, followed by a reduction step. In the second step, the amino group fraction of the precursor nanoparticles is activated with maleimide using a sulfo-SMCC crosslinking reagent. Finally, the activated streptavidin and the maleimide-activated hybrid nanoparticles are combined to obtain a nanoreactor precursor having a porous polymer matrix and reagent-binding components. Protocol 1: SPDP Crosslinking

[0148] To prevent condensation, the vial of the crosslinking agent 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide (N-succinimidyl 3-(2-pyridyldithio)propionate; SPDP; P3415, Sigma) is equilibrated to ambient temperature before opening. For SPDP modification of streptavidin, a 2-fold molar excess of SPDP is used compared to streptavidin. SPDP is dissolved in 50 μL of DMF to obtain a 0.23 molar SPDP solution. Additionally, 300 mg of 15.8 U / mg streptavidin (SA10; Prozyme) is dissolved in 10 mL of 100 mM potassium phosphate buffer containing 20 mM NaCl (pH 7.5) to produce a 30 mg / mL solution. This solution is centrifuged at 3000 rpm, and the supernatant is kept on ice for further experiments. The entire volume (50 μL) of the SPDP solution is added to 30 mL of streptavidin solution, and the reaction is allowed to proceed overnight at 4°C. The reaction is quenched by adding Tris to a final concentration of 100 mM, and the mixture is incubated further at room temperature for 30 minutes. In the next step, unreacted SPDP is removed from the streptavidin solution by centrifugation at 8000 rpm for 15 minutes using a Vivacon 500 ultrafiltration column (100 kDa MWCO) (Sartorius Stedim Biotech). The flow-through is discarded, and the mixture is washed five times with 100 mM potassium phosphate buffer containing 20 mM NaCl. The activated streptavidin is reduced by incubation with 2 mM DTT at ambient temperature for 30 minutes. In this way, the pyridine-2-thione group is removed from the modified streptavidin. Protocol 2: Maleimide activation of the gelatin fraction (sulfo-SMCC coupling)

[0149] Open a new vial of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide sodium salt (sulfo-SMCC; M6035, Sigma) after it has been fully equilibrated to ambient temperature. Wash the amine-containing hybrid microparticles three times with amine-free conjugation buffer (potassium phosphate, 20 mM NaCl pH 7.2). Prepare a 10 mg / mL sulfo-SMCC stock solution for conjugation immediately before use. Add a sufficient amount of sulfo-SMCC stock solution to the microparticle solution to obtain a crosslinking reagent in excess of 10 molars from the available amino groups (for gelatin type B, it is expected that 10% of the gelatin amino acids will have free amino groups). Incubate the 50% (v / v) hydrogel microparticle slurry with 10 mM sulfo-SMCC and immediately place on ice. Allow the reaction to proceed overnight at 4°C at 100 rpm using a Multi-Rotator PTR-60 (Grant-bio) in vertical mode. The reaction is quenched by adding Tris to a final concentration of 100 mM, and then incubated at room temperature for 30 minutes. Maleimide-activated particles are purified by repeated washing with conjugation buffer and centrifugation at 1000 rcf. Protocol 3: Conjugation of activated proteins

[0150] Maleimide-activated microparticles are combined with sulfhydryl-modified streptavidin. The maleimide group reacts with the sulfhydryl group at pH 6.5-7.5 to form a stable, cleavable thioether bond.

[0151] Quenching is performed by adding 2-mercaptoethanol (Sigma) to a final concentration of 2 mM and incubating at RT (Restoration Time) for 30 minutes at 1000 rpm in a shaking incubator. A second quenching step is performed using N-(2-hydroxyethyl)maleimide (Sigma) to a final concentration of 6 mM, and incubation at RT while mixing. Binding ability is measured using biotinylated dye conjugates. Example 2 Library preparation Reversible Attachment of Target-Specific Primers as Analyte-Specific Reagents to Precursor Particles

[0152] Each hybrid hydrogel particle labeled with a fluorescent color can specifically detect different target targets. To equip each class of particles with a primer set compatible with its target, a functionalized gelatin / agarose hybrid particle aliquot is incubated with desthiobiotinylated primers in individual 2.0 mL microcentrifuge tubes. When the temperature is raised and an emulsion is formed in the subsequent signal amplification step, the desthiobiotin moiety of the oligonucleotide promotes the release of the oligonucleotide from the particles. Thereby, the oligonucleotide becomes immediately available for the detection reaction.

[0153] Therefore, after washing the precursor particles once with nuclease-free water, a 50% particle slurry is obtained by resuspending the particle pellet in an equal volume of nuclease-free water. Different labeled slurry aliquots are prepared in that an aliquot of the 50% particle slurry is pelleted. A 50% bead slurry with a final concentration of 200 nM for each primer is obtained by adding an equal volume of each target-specific desthiobiotin-labeled primer pair (components 5-8) to one microsphere pellet, thereby assigning specific analyte-specific reagents. To ensure efficient binding of the primers to the hybrid hydrogel matrix, both components are incubated at 20 °C for 15 minutes while shaking at 1000 rpm. The microspheres are washed three times with a five-fold larger volume of nuclease-free water to remove unbound primers. Component 5: rpoB Primer Pair (Analyte-Specific Reagent 1) -0.2 μM desthiobiotin-labeled rpoB sense primer (5’-ATCAACATCCGGCCGGTGGTCGCC-3’) SEQ ID NO: 1 (Metabion International AG) -0.2 μM desthiobiotin-labeled rpoB antisense primer (5’-TCACGTGACAGACCGCCGGGC-3’), SEQ ID NO: 2 (Metabion International AG) Component 6: IS6110 primer pair (Analyte-specific reagent 2) -0.2 μM desthiobiotin-labeled IS6110 sense primer (5’-CGCCGCTTCGGACCACCAGCAC-3’), SEQ ID NO: 3 (Metabion International AG) -0.2 μM desthiobiotin-labeled IS6110 antisense primer (5’-GTGACAAAGGCCACGTAGGCGAACC-3’), SEQ ID NO: 4 (Metabion International AG) Component 7: IS1081 primer pair (Analyte-specific reagent 3) -0.2 μM desthiobiotin-labeled IS1081 sense primer (5’-GCGCGGCAAGATCATCAATGTGGAG-3’), SEQ ID NO: 5 (Metabion International AG) -0.2 μM desthiobiotin-labeled IS1081 antisense primer (5’-GCCACCGCGGGGAGTTTGTCG-3’), SEQ ID NO: 6 (Metabion International AG) Component 8: atpD (Internal standard) primer pair (Analyte-specific reagent 4) -0.2 μM desthiobiotin-labeled internal standard (Bac. globigii) sense primer (5’-GCGCGGCAAGATCATCAATGTGGAG-3’), SEQ ID NO: 7 (Metabion International AG) -0.2 μM desthiobiotin-labeled internal standard (Bac. globigii) antisense primer (5’-GCCACCGCGGGGAGTTTGTCG-3’), SEQ ID NO: 8 (Metabion International AG) Lyophilization of the library of microparticles

[0154] If necessary, the microsphere library is lyophilized to obtain a dry analyte / target-specific microparticle pellet for long-term storage. Therefore, equal volumes of the desired target-specific microparticles are combined using a pipette and thoroughly mixed using a vortexer. An equal volume of 600 mg / mL trehalose solution is added to the microparticle library to produce a slurry of 30% (w / v) trehalose-containing microparticle library. Subsequently, 100 μl library aliquots are prepared in RNase / DNase-free PCR strip tubes ready for lyophilization. The type of excipient (e.g., trehalose) and its concentration in the lyophilized formulation affect the degree of expansion of the freeze-dried microparticles when exposed to the eluate later in the process.

[0155] Library aliquots were frozen on dry ice for 2 hours, then freeze-dried under vacuum (-25°C and 0.1 mbar) using an Alpha 2-4 LSCplus freeze-dryer (Christ). The samples were left in the freeze-dryer for a total of 200 minutes. The main drying step involved holding at 0.01 mbar for 3 hours while gradually increasing the temperature from -25°C to 25°C. The final drying step was performed at 25°C and 0.05 mbar for 20 minutes. The final product is as follows:

[0156] Ingredient 9: - Target-specific reagents required for parallel detection of rpoB, IS6110, IS1081, and internal standards in a single sample Freeze-dried nanoreactor library containing Example 3 Multiplexing of analytes using nanoreactors

[0157] In the following embodiment, a sample (eluate) is encapsulated in a monodisperse suspension using target-specific microparticles from a nanoreactor library. Loading analytes and common detection reagents into a nanoreactor library

[0158] Typical reagents for analyte detection within the nanoreactor are supplied as freeze-dried pellets, which are then resuspended in analyte-containing eluates obtained from the upstream sample preparation process. In this example, actual samples were simulated by using purified PCR products at specified concentrations for rpoB, IS6110, IS1081, and atpD, containing the target sequences to be amplified (originally derived from H37Rv DNA), in nuclease-free water.

[0159] The entire volume (100 μl) of a sample that does not contain the template molecule or contains a specified amount of the target molecule is diluted with sputum eluate from an MTB (= Mycobacterium tuberculosis)-negative patient and added to a lyophilized general reagent pellet (component 10). The reagent is carefully resuspended by a short vortex step. Similarly, the entire volume of the general reagent mixture containing the template molecule is added to the nanoreactor library pellet (component 9). The entire liquid containing all detection reagents and template molecules is absorbed into porous microparticles for 15 minutes at ambient temperature with shaking at 1000 rpm. In this process, four templates are randomly distributed to four types of reagent-specific nanoreactors. In the subsequent digital amplification step, only templates that match the specific reagent set of the nanoreactor can be amplified and detected. As a result, the number of positive events in digital amplification is reduced by factor n, where n corresponds to the number of nanoreactor types in the nanoreactor library.

[0160] Component 10 consists of the following final concentrations. -PCR buffer: 20mM Tris HCl, 22mM KCl, 22mM NH4Cl, 3mM MgCl2 -0.2U / μl Hot Start Taq DNA Polymerase (biotechrabbit GmbH) -0.4mM dNTP (biotechrabbit GmbH) -1 μM EvaGreen® Fluorescent DNA Stain (JenaBioscience GmbH) or 0.4 μM TaqMan probe -0.1% (w / v) low bioburden, protease-free BSA (Sigma) for molecular biology. Phase transfer of loaded particles

[0161] To prevent crosstalk between target-specific reactions, the nanoreactor library is transferred to a non-aqueous phase by dispersing the microparticles in component 11. Using a 1.5 mL microcentrifuge tube, all of the aqueous phase (100 μL) is brought into contact with an excess amount of component 11 (500 μL). High shear force is applied to deagglomerate the aqueous microparticles and emulsify them in fluorocarbon oil to form a single nanoreactor. Sonifier TM S-450 and Ultrasonics Sonifier TM The mixture is agitated by applying ultrasound using a Cup Horn (Branson) or by simply sliding the tube over the holes of a microcentrifuge rack 20 times at a frequency of approximately 20 times / second while pressing the tube against the rack surface. Applying mechanical stress to this breaks the attractive forces between aqueous microparticles and creates surface tension, thereby forming a suspension. Both the hydrogel microparticles and the excess aqueous phase are emulsified in the oil phase. Submicron-scale droplets formed as byproducts are removed by washing the suspension three times by gentle centrifugation (400 rcf). All undesirable liquid droplets are essentially removed by repeated washing with the same oil (component 11). Component 11 also provides efficient thermal stability of the suspension for subsequent digital emulsion / suspension PCR.

[0162] Component 11: Oil for phase transfer and signal amplification -HFE-7500 fluorocarbon oil (3M Deutschland GmbH) - Supplemented with 2-5% (v / v) PicoSurf (Dolomite Microfluidics) or 2-5% (v / v) FluoSurf (Emulseo) Parallel digital PCR amplification reaction in a reaction space using microparticles as a template

[0163] Place the monodisperse suspension containing the encapsulated sample in approximately 2.5 cm. 2 The nanoreactors are transferred to a detection chamber having a certain area and a layer thickness of 100 μm. The detection window of the chamber is made of 0.8 mm polycarbonate (Makrolon 6555; Covestro AG), and the opposite side of the chamber consists of a polished, unmodified, transparent 125-micron polycarbonate (Lexan 8010) film (Koenig Kunststoffe GmbH) that facilitates efficient heat transfer required for individual nanoliter reactions. The nanoreactors suspended in fluorocarbon oil are forced to form a single layer due to the dimensions of the reaction chamber. Thus, the microcapsules provide an array of approximately 20,000 to 30,000 nanoreactors (5,000 to 7,500 per target) arranged at equal intervals for subsequent parallel signal amplification reactions.

[0164] Using a modified 30×30×4.7mm, 19.3W Peltier element (Quick-Ohm, Kupper & Co. GmbH, #QC-71-1.4-3.7M) and an established chamber-specific PCR control mode, microparticles are subjected to ultrafast temperature cycling. The applied temperature conditions are 30-45 cycles of a two-step PCR consisting of an initial denaturation at 95°C for 30 seconds, followed by denaturation at 95°C for 1 second and annealing / extension at 64°C for 4 seconds. Due to its sol-gel switching ability, the suspension becomes an emulsion containing individual liquid nanoliter droplets. Furthermore, upon initial heating to 95°C to make it available for PCR, the microparticles release target-specific oligonucleotides bound to desthiobiotin from the gelatin matrix. Multiplexed amplification of individual targets occurs in the resulting nanoreaction compartment.

[0165] Automated image acquisition was initiated by BLINK toolbox software and performed using a fluorescence microscope (Zeiss AxioObserver) equipped with a 5× objective lens (field of view 4.416 mm × 2.774 mm) and a pE-4000 (CoolLED Ltd.) light source. This microscope was further equipped with an automated x-y stage with three fluorescence filter sets (Cy5 ET, Cy3 ET, FITC / FAM HC, AHF Analysentechnik), and a thermocycler with a reaction chamber.

[0166] The settings for image acquisition were as follows: 100 - 1000 ms and 1 - 10× gain. Two images were required for label identification (λexc1 = 550 nm, λexc2 = 650 nm), and one image was required for specific PCR signals (λexc3 = 470 nm). When performing free-choice nanoreactor-specific real-time analysis, three images corresponding to three fluorescence channels can be taken at the end of each annealing step of the temperature protocol at one position in the chamber.

[0167] Once the temperature protocol is complete, scan the entire detection chamber using the same instrument with the settings described above. For the settings outlined earlier, a total of 30 images are required to cover the dimensions of the amplification / detection chamber. Figure 7A shows images of the same region for three fluorescence channels, where channels 1 and 2 represent the ratio of labels encoding the respective analyte-specific reagents provided to the microparticles, and channel 3 shows microparticles with negative (dark) and positive (bright) amplification signals. The graph on the right shows a scatter plot of the fluorescence signals obtained for each microparticle in channels 1 and 2. Four different microparticle species are clearly recognizable. If necessary, analyze the melting behavior of the amplified product's DNA by subjecting the nanoreactor to stepwise increases (2°C / step) from 50°C to 90°C to measure the degree of specificity or to identify SNPs, etc. By acquiring fluorescence images at each temperature step, monitor the denaturation of the DNA strain and the resulting attenuation of the fluorescence signal. Decoding and multiplexed analysis of microparticles

[0168] All acquired images are subjected to an automated multi-faceted image processing algorithm. This method uses Maximally Stable Extremal Regions (MSER) ​​image segmentation to detect adjacent droplets from the MSER-based image segmentation results. Specifically, the images are first subjected to preprocessing including median filtering. Next, the MSER algorithm is applied to the image background to determine convex turning points in the background contour, and Delaunay triangulation is applied to identify appropriate cuts between droplets / microparticles. Furthermore, the droplets / microparticles are segmented using the MSER algorithm. Finally, the plausibility of the droplet / microparticle contours is verified (contrast, shape, convexity). Subsequently, features including fluorescence signal, location, and diameter / volume in each channel are collected for all segmented droplets / microparticles. The experimental data is applied to a Jupyter script that identifies individual labels (combinations of fluorescent dyes Cy3 and Cy5) and their respective specific amplification and melting curve signals.

[0169] The following data readings are possible: a) Endpoint analysis (digital readings)

[0170] This PCR is amplified to the endpoint, so the total number of fluorescence-positive and fluorescence-negative droplets is measured for each individual label. Positive droplets contain at least one copy of the identification target and therefore show an increase in fluorescence signal above a specified intensity threshold. This threshold is derived from a previous amplification reaction performed without a template. Digital PCR data for droplets against each labeled target is displayed in a 1D or 2D plot. The software first clusters the fluorescence-negative and fluorescence-positive fractions for each nanoreactor volume, and then applies the fluorescence-positive droplet fraction to a Poisson algorithm to determine the starting concentration of the target DNA molecule in units of copies / mL input. Since the assay described above is a quadruple reaction, the droplets are clustered into different groups depending on the concentration of the template added: a) Labeled component 1 (Cy3 and Cy5 in a 1:0 ratio), detection signal (EvaGreen / probe) negative. b) Labeled component 1 (Cy3 and Cy5 in a 1:0 ratio), detection signal (EvaGreen / probe) positive. c) Labeled component 2 (Cy3 and Cy5 in a 3:1 ratio), detection signal (EvaGreen / probe) negative. d) Labeled component 2 (Cy3 and Cy5 in a 3:1 ratio), detection signal (EvaGreen / probe) positive. e) Labeled component 3 (Cy3 and Cy5 in a 1:3 ratio), detection signal (EvaGreen / probe) negative. f) Labeled component 3 (Cy3 and Cy5 in a 1:3 ratio), detection signal (EvaGreen / probe) positive. g) Labeled component 4 (Cy3 and Cy5 in a 0:1 ratio), detection signal (EvaGreen / probe) negative. h) Labeled component 4 (Cy3 and Cy5 in a 0:1 ratio), detection signal (EvaGreen / probe) positive.

[0171] Such 1D plots are shown in Figure 7B for each microparticle with its respective labeled component. Positive microparticles are clearly distinguishable from negative microparticles for types 0 and 3, while types 2 and 3 only represent negative microparticles. The calculated target concentrations in the sample are shown in the table in Figure 7B. b) Real-time analysis

[0172] The average pixel intensity for each target-specific nanoreactor was tracked throughout the entire PCR cycle, yielding a real-time fluorescence curve for a single nanoreactor. These exhibit the typical logarithmic, linear, and plateau phases of PCR, comparable to those observed in microliter-scale reactions. Sigmoid and linear fitting were performed for all nanoreactors using the Levenberg-Marquardt algorithm. Lift criteria (change in fluorescence after PCR vs. before PCR) were applied to eliminate unsuitable curves. Real-time negative and positive curves were created, and the cycle threshold for positive cases was determined. False positives (evaporated nanoreactors, artifacts) were identified and excluded from the analysis. Literature: [ka] The present invention provides, for example, the following items: (Item 1) A library of pre-fabricated precursor microparticles for preparing a library of pre-fabricated microparticles, wherein the pre-fabricated microparticles are configured to perform specific detection of one or more target analytes in a sample, and such specific detection is performed within such microparticles by a suitable chemical or biochemical reaction, and each of the pre-fabricated precursor microparticles in the library is - A porous matrix having void volume for receiving aqueous samples and providing a reaction space for the specific detection of the analyte, preferably a porous polymer matrix; - A reagent-binding component that enables the attachment of the analyte-specific reagent to the precursor fine particles, preferably reversible attachment; the reagent-binding component is (i) A polymer or polymer mixture that forms or is the porous matrix; (ii) Reagent-binding molecules attached to the porous matrix; (iii) at least one or more ionizable groups immobilized on the porous matrix, wherein the ionizable groups are capable of changing their charge according to the ambient conditions around the precursor nanoparticles; (iv) at least one or more charged groups immobilized on the porous matrix; (v)(i)~(iv) Any combination One of them is a reagent-binding component; -When the analyte-specific reagent is attached to the precursor microparticles, a labeling component attached to, contained in, or otherwise associated with the precursor microparticles for identifying the analyte-specific reagent A library that includes this. (Item 2) The library according to item 1, wherein the library contains at least two separate fabricated precursor microparticle subsets, preferably three or more separate fabricated precursor microparticle subsets, each subset having a different labeling component attached to, contained in, or otherwise associated with the precursor microparticles within the subset, and each of the at least two or more separate fabricated precursor microparticle subsets has a different labeling component attached to, contained in, or otherwise associated with each subset. (Item 3) A library of fabricated microparticles for specific detection of one or more target analytes in a sample, wherein such specific detection is carried out in such microparticles by a suitable chemical or biochemical reaction, and each of the fabricated microparticles comprises a fabricated precursor microparticle as described in any of items 1-2, and further comprises an analyte-specific reagent attached, preferably reversibly attached, to the precursor microparticle. (Item 4) The analyte-specific reagent attached to each of the precursor microparticles is a) Direct binding of the analyte-specific reagent to the polymer or polymer mixture (i) that forms or is part of the porous polymer matrix; b) The analyte-specific reagent, which has been conjugated to a binding entity that binds to the reagent-binding molecule (ii); c) Direct binding of the analyte-specific reagent to the ionizable group (iii) under conditions in which the ionizable group has a suitable net charge; d) Direct bonding of the analyte-specific reagent to the charged group(iv) on the polymer, wherein the analyte-specific reagent has at least one or more ionizable groups, and the ionizable groups are capable of changing charge according to ambient conditions around the analyte-specific reagent; or e) Any combination of (a) to (d) A library of pre-fabricated microparticles according to item 3, which are attached, preferably reversibly, via the reagent-binding component. (Item 5) The polymer (i) is a hydrogel-forming agent selected from the group comprising naturally occurring polymers selected from: ia) synthetic polymers, e.g., poly(methyl)(meth)acrylate, polyamide; ib) silicone polymers, e.g., polydimethylsiloxane; ic) polysaccharides, e.g., agarose, chitin, chitosan, dextran, alginate, carrageenan, cellulose, fucoidan, laminaran; gums selected from xanthan gum, acacia gum, gatchigum, guar gum, locust bean gum, tragacanth gum, karaya gum, and inulin; polypeptides, e.g., collagen, gelatin; polyamino acids, e.g., polylysine; and polynucleotides; and the polymer mixture is any combination of the above; The reagent-binding molecule (ii) is selected from avidin, particularly tetrameric avidin; streptavidin; monomeric avidin; avidin having nitrated tyrosine at the biotin-binding site; other proteins derived from or related to avidin and retaining the avidin-binding function; biotin; desthiobiotin; iminobiotin; biotin having a cleavable spacer arm; selenobiotin; oxybiotin; homobiotin; norbiotin; iminobiotin; diaminobiotin; biotin sulfoxide; biotin sulfone; epibiotin; 5-hydroxybiotin; 2-thiobiotin; azabiotin; carbobiotin; methylated derivatives of biotin; ketonebiotin; and other molecules derived from or related to biotin and retaining the biotin-binding function; - The at least one ionizable group (iii) or the ionizable group described in item 4(d) is N-2-acetamido-2-aminoethanesulfonic acid (ACES); • N-2-acetamido-2-iminodiacetic acid (ADA); • Aminomethylpropanediol (AMP); 3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (AMPSO); • N,N-bis-2-hydroxyethyl-2-aminoethanesulfonic acid (BES); ·N,N-bis-2-hydroxyethylglycine (BICINE); • Bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris); • 1,3-Bis-Trispropane (Bis-Trispropane); 4-Cyclohexylamino-1-butanesulfonic acid (CABS); 3-Cyclohexylamino-1-propanesulfonic acid (CAPS); 3-Cyclohexylamino-2-hydroxy-1-propanesulfonic acid (CAPSO); 2-N-cyclohexylaminoethanesulfonic acid (CHES); • 3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (DIPSO); • N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS); · N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS); • N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES); • N-2-hydroxyethylpiperazine-N-2-propanesulfonic acid (HEPPSO); 2-N-morpholinoethanesulfonic acid (MES); · 4-N-morpholinobutanesulfonic acid (MOBS); • 3-N-morpholinopropanesulfonic acid (MOPS); · 3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO); • Piperazine-NN-bis-2-ethanesulfonic acid (PIPES); • Piperazine-NN-bis-2-hydroxypropanesulfonic acid (POPSO); N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS); • N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS); • 3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid (TAPSO); N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES); N-Trishydroxymethylmethylglycine (TRICINE); Trishydroxymethylaminomethane (Tris); • Polyhydroxylated amines; Imidazole and its derivatives (i.e., imidazole), in particular derivatives containing a hydroxyl group; • Dimers and polymers of triethanolamine; and Di / tri / oligo / polyamino acids, e.g., Ala-Ala; Gly-Gly; Ser-Ser; Gly-Gly-Gly; or Ser-Gly. Oligo-His, poly-His, oligo-Lys, poly-Lys Selected from, A library of pre-fabricated precursor microparticles as described in either item 1 or 2, or a library of pre-fabricated microparticles as described in either item 3 or 4. (Item 6) A library of fabricated precursor microparticles according to any one of items 1 to 2, 5, or a library of fabricated microparticles according to any one of items 3 to 5, wherein the porous polymer matrix, or the polymer or polymer mixture forming the porous polymer matrix or being part of the porous polymer matrix, is composed of an uncrosslinked polymer, preferably the polymer or polymer mixture forming the porous polymer matrix or being part of the porous polymer matrix is ​​composed of agarose, or a combination of agarose and gelatin, more preferably the combination of agarose and gelatin, wherein the agarose is present in the range of 0.1% (w / v) to 4% (w / v), and the gelatin is present in the range of 0.1% (w / v) to 20% (w / v), preferably 0.5% (w / v) to 20% (w / v). (Item 7) The analyte-specific reagent is selected from nucleic acids including aptamers and Spiegelmers; antibodies or antibody fragments; and non-antibody proteins capable of specifically binding to the analyte or analyte complex, such as receptors, receptor fragments, and affinity proteins; preferably, the analyte-specific reagent is selected from nucleic acids, particularly nucleic acid oligomers and nucleic acid primers, in a library of pre-fabricated microparticles according to any one of items 3 to 6. (Item 8) For each of the aforementioned fine particles, the analyte-specific reagent is reversibly attached to the fine particles via the reagent-binding component by the analyte-specific reagent being conjugated to a binding entity that binds to the reagent-binding molecule (ii), - The binding entity is independently selected from biotin, desthiobiotin, iminobiotin, biotin with a cleavable spacer arm, selenobiotin, oxybiotin, homobiotin, norbiotin, iminobiotin, diaminobiotin, biotin sulfoxide, biotin sulfone, epibiotin, 5-hydroxybiotin, 2-thiobiotin, azabiotin, carbobiotin, methylated derivatives of biotin, ketonebiotin, and other molecules derived from or related to biotin and retaining the biotin binding function; the reagent-binding molecule is independently selected from avidin and streptavidin; or vice versa; or - The binding entity is biotin, and the reagent-binding molecule is selected from monomeric avidin, avidin having nitrated tyrosine at the biotin-binding site, and other proteins derived from or related to avidin and retaining the avidin-binding function; or vice versa. A library of fabricated microparticles as described in any of items 3-7. (Item 9) The library is a library for the specific detection of a single target analyte in several samples, and the library contains at least two distinct pre-fabricated microparticle subsets, preferably three or more distinct pre-fabricated microparticle subsets. Each subset is, The subset of the fine particles has different labeling components attached thereto, contained therein, or otherwise associated therewith; All of the aforementioned two, three or more distinct subsets The subset of the fine particles has the same analyte-specific reagent attached to the porous matrix, and the analyte-specific reagent is specific to one target analyte; The aforementioned two or more separate fabricated microparticle subsets are identical with respect to the attached analyte-specific reagent, Each subset of the fine particles has different labeling components attached to, contained within, or otherwise associated with them; Each subset is clearly defined and identifiable by its respective label component. A library of fabricated microparticles as described in any of items 3-8. (Item 10) The library is a library for the specific detection of multiple target analytes in a single sample, and the library contains at least two distinct pre-fabricated microparticle subsets, preferably three or more distinct pre-fabricated microparticle subsets. Each subset is, The subset of the fine particles has different labeling components attached thereto, contained therein, or otherwise associated therewith; The subset of the fine particles has different analyte-specific reagents attached to the porous matrix; each analyte-specific reagent is specific to one target analyte; The aforementioned two or more separate fabricated microparticle subsets are Each of the labeling components attached to, contained within, or otherwise associated with the fine particles of each subset; and Each subset is fitted with the respective analyte-specific reagent They are different; Each subset is clearly defined and identifiable by the respective labeling component and the respective analyte-specific reagent. A library of fabricated microparticles as described in any of items 3-8. (Item 11) The library is a library for the specific detection of multiple target analytes in several samples, and the library contains multiple different and separate pre-fabricated microparticle subsets. Each subset is, The subset of the fine particles has different labeling components attached thereto, contained therein, or otherwise associated therewith; Among the aforementioned multiple separate fabricated microparticle subsets, there exist separate fabricated microparticle subsets of different classes, each of which comprises several microparticle subsets, each class having different analyte-specific reagents attached to the porous matrix of the microparticles, all microparticle subsets within a single class having the same analyte-specific reagent; each analyte-specific reagent is specific to one target analyte; In the aforementioned library, the multiple distinct and separate fabricated microparticle subsets are, Each subset of the fine particles has different labeling components attached to, contained within, or otherwise associated with them; Each separate microparticle subset forms part of a class of microparticle subsets; Each subset is clearly defined and identifiable by its respective labeling component and its respective analyte-specific reagent; The aforementioned different classes of microparticle subsets differ in the analyte-specific reagents attached to the porous matrix of the microparticles; each of the aforementioned different classes comprises several microparticle subsets, and all of those subsets within one class have the same analyte-specific reagent. A library of fabricated microparticles as described in any of items 3-8. (Item 12) A kit for preparing a library of pre-fabricated microparticles as described in any of items 3 to 11, wherein the kit is It includes at least two containers, namely the first and second containers, and optionally further containers, each of which is The fabricated precursor microparticle subsets include those defined in any of items 1-2, 5, and 6, each subset having a different labeling component attached to, contained within, or otherwise associated with the precursor microparticles within the subset, and the two fabricated precursor microparticle subsets have different labeling components attached to, contained within, or otherwise associated with each subset; Further containers are a) Direct binding of the analyte-specific reagent to the polymer or polymer mixture (i) that forms or is part of the porous polymer matrix; b) The analyte-specific reagent, which has been conjugated to a binding entity that binds to the reagent-binding molecule (ii); c) Direct binding of the analyte-specific reagent to the ionizable group (iii) under conditions in which the ionizable group has a suitable net charge; d) Direct bonding of the analyte-specific reagent to the charged group(iv) on the polymer, wherein the analyte-specific reagent has at least one or more ionizable groups, and the ionizable groups are capable of changing charge according to ambient conditions around the analyte-specific reagent; or e) Any combination of (a) to (d) The kit comprises a conditioning solution that enables the attachment, preferably reversible, of an analyte-specific reagent to the subset of precursor microparticles via the reagent-binding component; the reagent-binding component is as defined in item 1; the reagent-binding molecule is as defined in any of items 1, 4, and 5; the binding entity is as defined in any of items 4-5; the polymer or polymer mixture is as defined in any of items 1, 4-6; the ionizable group is as defined in any of items 1, 4, and 5; and the analyte-specific reagent is as defined in any of items 3-4, 7, and 8. (Item 13) A method for preparing a library of fabricated microparticles as described in any of items 3 to 11, wherein the method is: - • A library of fabricated precursor microparticles as defined in any of items 1-2, 5, and 6; • At least one analyte-specific reagent as defined in any of items 3-4, 7, and 8. A process of providing in any order; - A step of producing a library of fabricated microparticles according to any one of items 5 to 13 by mixing the library of fabricated precursor microparticles or a selected subset thereof with the at least one analyte-specific reagent, preferably under conditions that allow for the attachment of the at least one analyte-specific reagent to some or all of the fabricated precursor microparticles, preferably under conditions that allow for reversible attachment; - If necessary, wash the prepared microparticles to remove any unattached analyte-specific reagents. Methods that include... (Item 14) A kit for detecting analytes in a sample, wherein the kit is a) - A container comprising a general detection composition comprising reagents for carrying out a chemical or biochemical detection reaction of an analyte, wherein the chemical or biochemical detection reaction of the analyte is nucleic acid amplification, and the general detection composition comprises a buffer, mononucleoside triphosphate, amplification enzyme, e.g., a suitable nucleic acid polymerase, e.g., Taq polymerase, and amplification product, e.g., a nucleic acid dye for detection of amplified nucleic acids, or - A container comprising a first detection composition comprising a reagent for carrying out a chemical or biochemical detection reaction of an analyte, and a further container comprising a second detection composition comprising a detection reagent, wherein the chemical or biochemical detection reaction of the analyte is an immunochemical detection reaction, and the first detection composition comprises a reagent for carrying out an immunochemical detection reaction, e.g., a buffer, and a secondary antibody or secondary antibody fragment that is specific to the same analyte as the primary antibody, antibody fragment or non-antibody protein used as an analyte-specific reagent (ASR) in the immunochemical detection reaction and is conjugated to a suitable reporter enzyme; the second detection composition comprises a substrate suitable for the suitable reporter enzyme as a detection reagent, wherein the substrate becomes detectable, preferably optically detectable, more preferably fluorescently detectable, when reacted with the reporter enzyme, the container and further containers, or - Analysis A container comprising a first detection composition comprising a reagent for carrying out a chemical or biochemical detection reaction of a substance, and a further container comprising a second detection composition comprising a detection reagent, wherein the chemical or biochemical reaction is an immunochemical detection reaction, and the first detection composition comprises a reagent for carrying out an immunochemical detection reaction, e.g., a buffer, and a secondary antibody or secondary antibody fragment that is specific to the same analyte as the primary antibody used as an analyte-specific reagent (ASR) in the immunochemical detection reaction and is conjugated to a suitable oligonucleotide tag; the second detection composition comprises a buffer, a mononucleoside triphosphate, an amplification enzyme, e.g., a suitable nucleic acid polymerase, e.g., Taq polymerase, and an amplification product, e.g., a nucleic acid dye for detection of amplified nucleic acids, and a primer suitable for amplifying the oligonucleotide tag attached to the secondary antibody, as a detection reagent; and a further container; and b) - A container containing a non-aqueous phase, such as oil, such as fluorocarbon oil, supplemented with emulsifiers as needed; and c) - Mixing container for mixing ingredients The kit includes, preferably, d) - Container for conducting the detection reaction Includes; more preferably, the kit further, e) - The kit described in item 12, or - Library of fabricated microparticles as described in any of items 3-11 A kit that includes this. (Item 15) A method for detecting and / or quantifying a target analyte in an aqueous sample, the method comprising the following steps: - • Aqueous samples known or suspected to contain the analyte of interest; A general detection composition comprising reagents for carrying out chemical or biochemical detection reactions of the analyte; A library of pre-fabricated microparticles as described in any of items 3 to 11, wherein the analyte-specific reagent attached to the microparticles is selected to specifically bind to or react with the target analyte. A process of providing in any order; - If necessary, a step of mixing the aqueous sample with the general detection composition; -A step comprising incubating the aqueous sample with the library of fabricated microparticles, thereby allowing the library of microparticles to absorb the aqueous sample into the void volume of the microparticles, and, if necessary, enabling binding to the target analyte if present in the sample; - A step of washing the fine particles if necessary; - If the aqueous sample has not been previously mixed with the general detection composition, the step of adding the general detection composition to the fine particles; - A step of creating a plurality of isolated reaction spaces for detecting the analyte by transferring the library of fabricated microparticles to a non-aqueous phase and removing the aqueous phase around each individual fabricated microparticle, wherein the reaction spaces include an aqueous phase and are limited to the void volume of the microparticles; - A step of releasing the analyte-specific reagent attached to the fine particles by applying an external trigger as necessary; - A step of performing a detection reaction for the target analyte; - Steps to detect and / or quantify the target analyte. Methods that include... (Item 16) The target analyte is nucleic acid, - The analyte-specific reagent is a nucleic acid or nucleic acid pair that is sufficiently complementary to the target analyte to hybridize with the target analyte under hybridization conditions, and preferably the analyte-specific reagent is a primer or primer pair suitable for amplification of the target analyte; - The general detection composition comprises reagents other than primers for carrying out the amplification reaction of the nucleic acid analyte of the target, and in particular, the general detection composition comprises a buffer, a mononucleoside triphosphate, an amplification enzyme, such as a suitable nucleic acid polymerase, such as Taq polymerase, and an amplification product, such as a nucleic acid dye for the detection of amplified nucleic acids; - The transfer step comprises transferring the prepared fine particles to a non-aqueous phase, suspending them, and washing them repeatedly in the non-aqueous phase as necessary, and the step further includes, as necessary, filtration or mechanical stirring to ensure that no or substantially any aqueous phase remains on the outside of the fine particles to form a monodisperse suspension of the fine particles; - The optional step of applying an external trigger to release the analyte-specific reagent attached to the fine particles is a step of temporarily increasing the temperature, changing the pH, or changing the salt conditions, preferably increasing the temperature, more preferably increasing the temperature to >90°C above ambient temperature. - The step of performing the detection reaction is a step of performing an amplification reaction of the analyte when the analyte is present in the aqueous sample; - The step of detecting and / or quantifying the target analyte is the step of detecting and / or quantifying the amplified analyte. The method described in item 15. (Item 17) A method for detecting and / or quantifying a target analyte in an aqueous sample, the method comprising the following steps: - • Aqueous samples known or suspected to contain the analyte of interest; A first detection composition comprising reagents necessary for carrying out a chemical or biochemical detection reaction of the analyte; • A second detection composition containing a detection reagent; • Library of fabricated microparticles as described in any of items 3-11 A step of providing in any order, wherein the analyte-specific reagent attached to the fine particles is selected so as to specifically bind to or react with the target analyte; - If necessary, a step of mixing the aqueous sample with the first detection composition; -A step comprising incubating the aqueous sample with the library of fabricated microparticles, and if the aqueous sample has not yet been mixed with the first detection composition, also incubating it with the first detection composition, thereby allowing the library of microparticles to absorb the aqueous sample and the first detection composition into the void volume of the microparticles, and, if necessary, to bind to the target analyte if it is present in the sample; - If necessary, wash the library of the prepared microparticles to remove any unabsorbed or unreacted first detection composition; -A step comprising incubating the library of fabricated microparticles containing the absorbed aqueous sample with the second detection composition, thereby enabling the library of microparticles to absorb the second detection composition; - If necessary, further wash the library of the prepared microparticles to remove any unabsorbed or unreacted second detection composition; - A step of creating a plurality of isolated reaction spaces for detecting the analyte by transferring the library of fabricated microparticles to a non-aqueous phase and removing the aqueous phase around the individual fabricated microparticles, wherein the reaction spaces include an aqueous phase and are limited to the void volume of the microparticles; preferably, the step of transferring to the non-aqueous phase is performed immediately after incubating the library of fabricated microparticles with the second detection composition; - A step of releasing the analyte-specific reagent attached to the fine particles by applying an external trigger as necessary; - A step of detecting and / or quantifying the target analyte by detecting and / or quantifying the detection reagent. Methods that include... (Item 18) The target analyte is a protein or other non-nucleic acid molecule. - The analyte-specific reagent is a primary antibody, antibody fragment, or non-antibody protein that can specifically bind to the protein analyte or other non-nucleic acid analyte; - The first detection composition comprises reagents necessary for carrying out an immunochemical detection reaction, such as a buffer, and a secondary antibody or secondary antibody fragment that is specific to the analyte and conjugated to a suitable reporter enzyme; - The second detection composition contains a substrate suitable for the preferred reporter enzyme as a detection reagent, wherein the substrate becomes detectable, preferably optically detectable, and more preferably fluorescently detectable, upon reaction with the reporter enzyme; - The step of incubating the aqueous sample with the library of fabricated microparticles and the first detection composition is, if the analyte is present in the aqueous sample, a step of forming a complex of the analyte and the primary antibody, primary antibody fragment or non-antibody protein by performing an immunochemical reaction including the binding of the analyte to the primary antibody, primary antibody fragment or non-antibody protein; the immunochemical reaction further includes the binding of the secondary antibody to the complex, thereby forming a sandwich between the primary antibody, antibody fragment or non-antibody protein, the analyte and the secondary antibody; -A first optional step for washing the library is a step of removing unbound secondary antibodies from the library; - The step of incubating the library of prepared microparticles containing the absorbed aqueous sample with the second detection composition is a step that allows the substrate to be reacted with the reporter enzyme; -A further optional step for washing the library is to remove unreacted substrates from the library; - The transfer step includes transferring the prepared fine particles to a non-aqueous phase, suspending them, and washing them repeatedly in the non-aqueous phase as necessary, and further including, as necessary, filtration or mechanical stirring to ensure that no or substantially any aqueous phase remains on the outside of the fine particles to form a monodisperse suspension of the fine particles; - The optional step of applying an external trigger to release the analyte-specific reagent attached to the fine particles is a step of temporarily increasing the temperature, changing the pH, or changing the salt conditions, preferably increasing the temperature, more preferably increasing the temperature to >90°C above ambient temperature; - The step of detecting and / or quantifying the target analyte is the step of detecting and / or quantifying the reacted substrate. The method described in item 17. (Item 19) A method for detecting and / or quantifying a target analyte in an aqueous sample, the method comprising the following steps: - • Aqueous samples known or suspected to contain the analyte of interest; A first detection composition comprising reagents necessary for carrying out a chemical or biochemical detection reaction of the analyte; • A second detection composition containing a detection reagent; • Library of fabricated microparticles as described in any of items 3-11 A step of providing in any order, wherein the analyte-specific reagent attached to the fine particles is selected so as to specifically bind to or react with the target analyte; - If necessary, a step of mixing the aqueous sample with the first detection composition; -A step comprising incubating the aqueous sample with the library of fabricated microparticles, and if the aqueous sample has not yet been mixed with the first detection composition, also incubating it with the first detection composition, thereby allowing the library of microparticles to absorb the aqueous sample and the first detection composition into the void volume of the microparticles, and, if necessary, to bind to the target analyte if it is present in the sample; - If necessary, wash the library of the prepared microparticles to remove any unabsorbed or unreacted first detection composition; -A step comprising incubating the library of fabricated microparticles containing the absorbed aqueous sample with the second detection composition, thereby enabling the library of microparticles to absorb the second detection composition; - If necessary, further wash the library of the prepared microparticles to remove any unabsorbed or unreacted second detection composition; - A step of creating a plurality of isolated reaction spaces for detecting the analyte by transferring the library of fabricated microparticles to a non-aqueous phase and removing the aqueous phase around each individual fabricated microparticle, wherein the reaction spaces include an aqueous phase and are limited to the void volume of the microparticles; - A step of releasing the analyte-specific reagent attached to the fine particles by applying an external trigger as necessary; - A step of performing a detection reaction for the target analyte; and - Steps to detect and / or quantify the target analyte. Methods that include... (Item 20) The target analyte is a protein or other non-nucleic acid molecule. - The analyte-specific reagent is a primary antibody, antibody fragment, or non-antibody protein that can specifically bind to the protein analyte or other non-nucleic acid analyte; - The first detection composition comprises reagents necessary for carrying out an immunochemical detection reaction, such as a buffer, and a secondary antibody or secondary antibody fragment that is specific to the same analyte as the primary antibody and conjugated to a suitable oligonucleotide tag; - The second detection composition comprises a buffer, a mononucleoside triphosphate, an amplification enzyme, such as a suitable nucleic acid polymerase, such as Taq polymerase, and an amplification product, such as a nucleic acid dye for the detection of amplified nucleic acids, and a primer suitable for amplifying the oligonucleotide tag attached to the secondary antibody, as a detection reagent; - The step of incubating the aqueous sample with the library of fabricated microparticles and the first detection composition is, if the analyte is present in the aqueous sample, a step of forming a complex of the analyte and the primary antibody, primary antibody fragment or non-antibody protein by performing an immunochemical reaction including the binding of the analyte to the primary antibody, primary antibody fragment or non-antibody protein; the immunochemical reaction further includes the binding of the secondary antibody to the complex, thereby forming a sandwich between the primary antibody, antibody fragment or non-antibody protein, the analyte and the secondary antibody; -A first optional step for washing the library is a step of removing unbound secondary antibodies from the library; - The step of incubating the library of fabricated microparticles containing the absorbed aqueous sample with the second detection composition is a step that allows the primers in the second detection composition to hybridize to the oligonucleotide tags attached to the secondary antibody; -A further optional step for cleaning the library is to remove any unhybridized primers from the library; - The transfer step includes transferring the prepared fine particles to a non-aqueous phase, suspending them, and washing them repeatedly in the non-aqueous phase as necessary, and further including, as necessary, filtration or mechanical stirring to ensure that no or substantially any aqueous phase remains on the outside of the fine particles to form a monodisperse suspension of the fine particles; - The optional step of applying an external trigger to release the analyte-specific reagent attached to the fine particles is a step of temporarily increasing the temperature, changing the pH, or changing the salt conditions, preferably increasing the temperature, more preferably increasing the temperature to >90°C above ambient temperature; - The step of performing the detection reaction is a step of performing the amplification reaction of the oligonucleotide tag; The step of detecting and / or quantifying the target analyte is the step of detecting and / or quantifying the amplified oligonucleotide tag. The method described in item 19. (Item 21) The method according to any one of items 15 to 20, wherein the step of releasing the analyte-specific reagent attached to the fine particles by applying an external trigger is performed by increasing the temperature to which the fine particles are exposed. (Item 22) - The method is, for example, a method for detecting and / or quantifying a single analyte in multiple aqueous samples, which may originate from different patients. -The method provides multiple different aqueous samples known or suspected to contain multiple target analytes, for example, samples from different patients. - The library of pre-fabricated microparticles provided in the above method is the library described in item 9, and such a library provides the same number or at least the same number of distinct microparticle subsets as the number of different aqueous samples to be tested, all of which have the same analyte-specific reagent attached to the porous matrix of the microparticles in the subset, and the analyte-specific reagent is specific to one target analyte; -In the step of incubating the aqueous sample with the library of fabricated microparticles, each of the different aqueous samples is incubated separately with a separate subset of microparticles in the library; -In the step of incubating the library of prepared microparticles containing the absorbed aqueous sample with a second detection composition, each of the distinct microparticle subsets of the library is incubated separately with the second detection composition; - The step of transferring the library to a non-aqueous phase is performed separately for each subset of particulate matter, that is, each separate subset of particulate matter is transferred separately to a non-aqueous phase, and the aqueous phase around each individual particulate matter is removed separately for each subset, thereby creating a plurality of isolated reaction spaces for detecting the analyte separately for each subset, the reaction spaces comprising an aqueous phase and limited to the void volume of the particulate matter, The method further comprises, after the step of transferring the library to a non-aqueous phase, mixing together the plurality of isolated reaction spaces of all the separate subsets in the non-aqueous phase, then carrying out a detection reaction for the target analyte, and then detecting and / or quantifying the target analyte, The method described in any of items 15-21. (Item 23) - The method described above is a method for detecting and / or quantifying multiple analytes in a single aqueous sample, -In the above method, a single aqueous sample known or suspected to contain multiple target analytes is provided. - The library of pre-fabricated microparticles provided in the method is the library described in item 10, and such a library provides as many or at least as the number of different target analytes to be detected, each of the distinct subsets having a different analyte-specific reagent attached to the porous matrix of the microparticles in the subset; each analyte-specific reagent is specific to one target analyte; -In the step of incubating the aqueous sample with the library of fabricated microparticles, the aqueous sample is incubated together with all of the separate microparticle subsets of the library; -In the step of incubating the library of prepared microparticles containing the absorbed aqueous sample with a second detection composition, all of the distinct microparticle subsets of the library are incubated together with the second detection composition; - The step of transferring the library to a non-aqueous phase is performed together for all particulate subsets, i.e., all particulate subsets are transferred together to a non-aqueous phase, and the aqueous phase around the individual particulates is removed, thereby creating multiple isolated reaction spaces for detecting and / or quantifying the multiple analytes, the reaction spaces comprising the aqueous phase and limited to the void volume of the particulates. The method described in any of items 15-21. (Item 24) - The method described above is a method for detecting and / or quantifying multiple analytes in multiple aqueous samples, -The method provides multiple different aqueous samples known or suspected to contain multiple target analytes, for example, samples from different patients. -The library of pre-fabricated microparticles provided in the method is the library described in item 11, and such a library contains separate subsets of pre-fabricated microparticles of different classes, each of which comprises several subsets of microparticles, each of which has different analyte-specific reagents attached to the porous matrix of the microparticles, and all subsets of microparticles in one class have the same analyte-specific reagent attached; each analyte-specific reagent is specific to one target analyte; and in the step of providing the library in the method, • A different class of distinct particulate subsets is provided, in the same number or at least the same number as the number of different analytes to be detected. • Each class is provided with the same number or at least the same number of different particulate subsets as the number of aqueous samples to be tested. The library contains a number of different particulate subsets equal to or at least the number obtained by multiplying the number of different aqueous samples to be tested by the number of different target analytes to be detected. -In the step of incubating the aqueous sample with the library of fabricated microparticles, strictly one aqueous sample is incubated with strictly one microparticle subset from each class, and each aqueous sample is incubated with the same number of different microparticle subsets from different classes as the number of classes of microparticles in the library. -In the step of incubating the library of fabricated microparticles containing the absorbed aqueous sample with a second detection composition, a subset of each aqueous sample combined with a different class of microparticles that have been previously incubated is incubated together with the second detection composition. - The step of transferring the library to a non-aqueous phase is performed separately for each aqueous sample, that is, each aqueous sample and the pre-incubated combined microparticle subset are transferred together to a non-aqueous phase, and the aqueous phase around the individual microparticles is removed separately for each aqueous sample, thereby creating a plurality of isolated reaction spaces for detecting the plurality of analytes, the reaction spaces comprising the aqueous phase and limited to the void volume of the microparticles, Preferably, the method further includes, after the step of transferring the library to a non-aqueous phase, mixing together the multiple isolated reaction spaces of the separate samples in the non-aqueous phase, then carrying out the detection reaction for the target analyte, and then detecting and / or quantifying the target analyte. The method described in any of items 15-21. (Item 25) In the step of detecting and quantifying the target analyte, the quantification of the analyte is a) Digital nucleic acid amplification, particularly digital polymerase chain reaction (PCR); b) Real-time quantitative nucleic acid amplification, particularly real-time polymerase chain reaction (PCR); c) Immunochemical detection methods, particularly digital immunochemical detection methods, such as digital immunoassays, such as digital enzyme-linked immunosorbent assays (ELISA); d) Immunochemical detection methods combined with nucleic acid amplification, e.g., immunopolymerase chain reaction; in particular, digital immunoPCR It is done by a method selected from; If the target analyte is a nucleic acid, quantification is performed using either method a) or b) or a combination of a) and b); if the analyte is a protein, peptide, or other non-nucleic acid analyte, quantification is performed using either method c) or d). The method described in any of items 15-24.

Claims

1. A library of fabricated microparticles for the specific detection of one or more target analytes in a sample, wherein such specific detection is carried out in such microparticles by a suitable chemical or biochemical reaction, and each of the fabricated microparticles comprises a fabricated precursor microparticle, - A porous polymer matrix having void volume for receiving aqueous samples and providing a reaction space for the specific detection of the analyte; - A reagent-binding component that enables the reversible attachment of an analyte-specific reagent to the precursor microparticles; the reagent-binding component is (i) A polymer or polymer mixture that forms or is the porous polymer matrix; (ii) Reagent-binding molecules attached to the porous polymer matrix; (iii) at least one or more ionizable groups immobilized on the porous polymer matrix, wherein the ionizable groups can change their charge according to the ambient conditions around the precursor nanoparticles; (iv) at least one or more charged groups immobilized on the porous polymer matrix; (v)(i) to (iv) any combination One of them is a reagent-binding component; - When the analyte-specific reagent is attached to the precursor microparticles, a labeling component attached to, contained in, or otherwise associated with the precursor microparticles for identifying the analyte-specific reagent Includes, Each of the fabricated microparticles further comprises an analyte-specific reagent reversibly attached to the precursor microparticles. The analyte-specific reagent attached to each of the precursor microparticles is a) Direct binding of the analyte-specific reagent to the polymer or polymer mixture (i) that forms or is part of the porous polymer matrix; b) The analyte-specific reagent conjugated to a binding entity that binds to the reagent-binding molecule (ii); the analyte-specific reagent wherein the reagent-binding molecule and the binding entity are selected to interact with each other in a reversible manner, i.e., to bind to each other; c) Direct binding of the analyte-specific reagent to the ionizable group (iii) under conditions in which the ionizable group has a suitable net charge; d) Direct bonding of the analyte-specific reagent to the charged group (iv) on the polymer, wherein the analyte-specific reagent has at least one or more ionizable groups, and the ionizable groups can change charge according to ambient conditions around the analyte-specific reagent; or e) Any combination of (a) to (d) A library of fabricated microparticles reversibly attached via the reagent-binding component.

2. The library of fabricated microparticles according to claim 1, wherein the library contains at least two distinct fabricated microparticle subsets, or at least three or more distinct fabricated microparticle subsets, each subset having a different labeling component attached to, contained in, or otherwise associated with the microparticles within the subset, so that the at least two or more distinct fabricated microparticle subsets each have a different labeling component attached to, contained in, or otherwise associated with each subset.

3. - The polymer (i) is a hydrogel-forming agent selected from the group comprising ia) synthetic polymers; ib) silicone polymers; ic) polysaccharides; rubbers selected from xanthan gum, gum arabic, gum gulch, gum guar, locust bean gum, gum tragacanth, gum karaya, and inulin; polypeptides; polyamino acids; and naturally occurring polymers selected from polynucleotides; the polymer mixture is any combination of the above; - The reagent-binding molecule (ii) is selected from avidin; streptavidin; avidin having nitrated tyrosine at the biotin-binding site; other proteins derived from or related to avidin and retaining the avidin-binding function; biotin; desthiobiotin; iminobiotin; biotin having a cleavable spacer arm; selenobiotin; oxybiotin; homobiotin; norbiotin; iminobiotin; diaminobiotin; biotin sulfoxide; biotin sulfone; epibiotin; 5-hydroxybiotin; 2-thiobiotin; azabiotin; carbobiotin; methylated derivatives of biotin; ketonebiotin; other molecules derived from or related to biotin and retaining the biotin-binding function; the reagent-binding molecule and the binding entity are selected to interact with each other in a reversible manner, i.e., to bind to each other; - The at least one ionizable group (iii) or The ionizable group described in claim 1(d) is • N-2-acetamido-2-aminoethanesulfonic acid (ACES); • N-2-acetamido-2-iminodiacetic acid (ADA); • Aminomethylpropanediol (AMP); 3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (AMPSO); • N,N-bis-2-hydroxyethyl-2-aminoethanesulfonic acid (BES); • N,N-bis-2-hydroxyethylglycine (BICINE); • Bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris); 1,3-Bistrishydroxymethylmethylaminopropane (Bis-Trispropane); 4-Cyclohexylamino-1-butanesulfonic acid (CABS); 3-Cyclohexylamino-1-propanesulfonic acid (CAPS); 3-Cyclohexylamino-2-hydroxy-1-propanesulfonic acid (CAPSO); 2-N-cyclohexylaminoethanesulfonic acid (CHES); 3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (DIPSO); • N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS); • N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS); • N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES); • N-2-hydroxyethylpiperazine-N-2-propanesulfonic acid (HEPPSO); 2-N-morpholinoethanesulfonic acid (MES); 4-N-morpholinobutanesulfonic acid (MOBS); 3-N-morpholinopropanesulfonic acid (MOPS); 3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO); • Piperazine-N-N-bis-2-ethanesulfonic acid (PIPES); • Piperazine-N-N-bis-2-hydroxypropanesulfonic acid (POPSO); N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS); • N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS); • 3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid (TAPSO); N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES); N-trishydroxymethylmethylglycine (TRICINE); Trishydroxymethylaminomethane (Tris); • Polyhydroxylated amines; Imidazoles and their derivatives (i.e., imidazoles), especially derivatives containing a hydroxyl group; • Dimers and polymers of triethanolamine; and Di / Tri / Oligo / Polyamino Acids Selected from, A library of fabricated microparticles according to claim 1 or 2.

4. - The synthetic polymer is selected from poly(methyl)(meth)acrylate and polyamide; - The polysaccharide is selected from agarose, chitin, chitosan, dextran, alginate, carrageenan, cellulose, fucoidan, and laminaran; - The polypeptide is selected from collagen and gelatin; - The polyamino acid is selected from polylysine; - The avidin is selected from tetrameric avidin and monomeric avidin; and - The di / tri / oligo / polyamino acids are selected from Ala-Ala, Gly-Gly; Ser-Ser; Gly-Gly-Gly; Ser-Gly; oligo-His; poly-His; oligo-Lys; and poly-Lys. A library of fabricated microparticles as described in claim 3.

5. A library of fabricated microparticles according to any one of claims 1 to 4, wherein the porous polymer matrix, or the polymer or polymer mixture forming the porous polymer matrix or being part of the porous polymer matrix, is composed of an uncrosslinked polymer.

6. The library of fabricated microparticles according to claim 5, wherein the polymer or polymer mixture forming the porous polymer matrix or being part of the porous polymer matrix is ​​composed of agarose or a combination of agarose and gelatin.

7. The library of fabricated microparticles according to claim 6, wherein in the above combination of agarose and gelatin, the agarose is present in the range of 0.1% (w / v) to 4% (w / v) and the gelatin is present in the range of 0.1% (w / v) to 20% (w / v).

8. The library of fabricated microparticles according to claim 7, wherein in the above combination of agarose and gelatin, the gelatin is present in the range of 0.5% (w / v) to 20% (w / v).

9. A library of fabricated microparticles according to any one of claims 1 to 8, wherein the analyte-specific reagent is selected from nucleic acids including aptamers and Spiegelmers; antibodies or antibody fragments; and non-antibody proteins that can specifically bind to the analyte or analyte complex.

10. The library of fabricated microparticles according to claim 9, wherein the non-antibody protein capable of specifically binding to the analyte or analyte complex is selected from receptors, receptor fragments, and affinity proteins.

11. The library of fabricated microparticles according to claim 9, wherein the analyte-specific reagent is selected from nucleic acid oligomers and nucleic acid primers.

12. For each of the fine particles, the analyte-specific reagent is reversibly attached to the fine particles via the reagent-binding component by the analyte-specific reagent being conjugated to a binding entity that binds to the reagent-binding molecule (ii). - The binding entity is independently selected from biotin, desthiobiotin, iminobiotin, biotin with a cleavable spacer arm, selenobiotin, oxybiotin, homobiotin, norbiotin, iminobiotin, diaminobiotin, biotin sulfoxide, biotin sulfone, epibiotin, 5-hydroxybiotin, 2-thiobiotin, azabiotin, carbobiotin, methylated derivatives of biotin, ketonebiotin, and other molecules derived from or related to biotin and retaining the biotin binding function; the reagent-binding molecule is independently selected from avidin and streptavidin; or vice versa; or - The binding entity is biotin, and the reagent-binding molecule is selected from monomeric avidin, avidin having nitrated tyrosine at the biotin-binding site, and other proteins derived from or related to avidin and retaining the avidin-binding function; or vice versa; - The reagent-binding molecule and the binding entity interact with each other in a reversible manner, that is, they are selected to bind to each other. A library of fabricated microparticles according to any one of claims 1 to 11.

13. The library is a library for the specific detection of a single target analyte in several samples, and the library contains at least two distinct pre-fabricated microparticle subsets, or at least three or more distinct pre-fabricated microparticle subsets. Each subset is, - Having different labeling components attached to, contained within, or otherwise associated with the fine particles of the subset; All of the aforementioned two, three or more distinct subsets - The subset of the fine particles has the same analyte-specific reagent attached to the porous polymer matrix, wherein the analyte-specific reagent is specific to one target analyte; Therefore, the aforementioned two or more separate pre-fabricated microparticle subsets are identical with respect to the attached analyte-specific reagent, - Each subset of the fine particles has different labeling components attached to, contained within, or otherwise associated with them; Each subset is clearly defined and identifiable by its respective label component. A library of fabricated microparticles according to any one of claims 1 to 12.

14. The library is a library for the specific detection of multiple target analytes in a single sample, and the library contains at least two separate pre-fabricated microparticle subsets, or at least three or more separate pre-fabricated microparticle subsets. Each subset is, - Having different labeling components attached to, contained within, or otherwise associated with the fine particles of the subset; - The subset of the fine particles has different analyte-specific reagents attached to the porous polymer matrix; each analyte-specific reagent is specific to one target analyte; Therefore, the at least two or more separate fabricated microparticle subsets are: the respective labeling components attached to, contained within, or otherwise associated with the microparticles of each subset; and - The respective analyte-specific reagents attached to each subset They differ; Each subset is clearly defined and identifiable by the respective labeling component and the respective analyte-specific reagent. A library of fabricated microparticles according to any one of claims 1 to 13.

15. The library is a library for the specific detection of multiple target analytes in several samples, and the library contains multiple different and separate pre-fabricated microparticle subsets. Each subset is, - Having different labeling components attached to, contained within, or otherwise associated with the fine particles of the subset; Among the aforementioned multiple separate pre-fabricated microparticle subsets, there exist separate pre-fabricated microparticle subsets of different classes, each of which comprises several microparticle subsets, and each class has different analyte-specific reagents attached to the porous polymer matrix of the microparticles, and all microparticle subsets within a class have the same analyte-specific reagent; each analyte-specific reagent is specific to one target analyte; Therefore, in the library, the multiple distinct fabricated microparticle subsets are - Each subset of the fine particles has different labeling components attached to, contained within, or otherwise associated with them; Each separate subset of particulate matter forms part of a class of particulate matter subsets; Each subset is clearly defined and identifiable by its respective labeling component and its respective analyte-specific reagent; Therefore, the different classes of microparticle subsets have different analyte-specific reagents attached to the porous polymer matrix of the microparticles; each of the different classes comprises several microparticle subsets, and all of those subsets within one class have the same analyte-specific reagent. A library of fabricated microparticles according to any one of claims 1 to 12.

16. A kit for preparing a library of fabricated microparticles according to any one of claims 1 to 15, wherein the kit comprises: - Including at least two containers, namely a first and a second container, each of which is, - comprising a fabricated precursor microparticle subset as defined in any of claims 1 to 8, each subset having a different labeling component attached to, contained within, or otherwise associated with the precursor microparticles within the subset, so that the two fabricated precursor microparticle subsets have different labeling components attached to, contained within, or otherwise associated with each subset; - Further containers are, - A conditioning solution, a) Direct binding of the analyte-specific reagent to the polymer or polymer mixture (i) that forms or is part of the porous polymer matrix; b) The analyte-specific reagent conjugated to a binding entity that binds to the reagent-binding molecule (ii); c) Direct binding of the analyte-specific reagent to the ionizable group (iii) under conditions in which the ionizable group has a suitable net charge; d) Direct bonding of the analyte-specific reagent to the charged group (iv) on the polymer, wherein the analyte-specific reagent has at least one or more ionizable groups, and the ionizable groups can change charge according to ambient conditions around the analyte-specific reagent; or e) Any combination of (a) to (d) The conditioning solution comprises the reagent-binding component, thereby enabling the reversible attachment of the analyte-specific reagent to the subset of precursor microparticles; A kit comprising: the reagent-binding component as defined in claim 1; the reagent-binding molecule as defined in any of claims 1, 3 to 4; the binding entity as defined in any of claims 1, 3 to 4; the polymer or polymer mixture as defined in any of claims 1, 3 to 8; the ionizable group as defined in any of claims 1, 3 to 4; and the analyte-specific reagent as defined in any of claims 1, 9 to 12.

17. The kit according to claim 16, further comprising further containers, each containing a subset of pre-fabricated precursor microparticles as defined in claim 16.

18. A method for preparing a library of fabricated microparticles according to any one of claims 1 to 15, wherein the method is: － A library of fabricated precursor microparticles as defined in any one of claims 1 to 8; - At least one analyte-specific reagent as defined in any of claims 1, 9 to 12 A process of providing in any order; A method comprising the step of producing a library of pre-fabricated microparticles according to any one of claims 1 to 15, by mixing the library of pre-fabricated precursor microparticles or a selected subset thereof with the at least one analyte-specific reagent under conditions that allow for the reversible attachment of the at least one analyte-specific reagent to some or all of the pre-fabricated precursor microparticles.

19. The method according to claim 18, further comprising the step of washing the prepared fine particles after the mixing to remove any unadhered analyte-specific reagents.

20. A kit for detecting analytes in a sample, wherein the kit is a) - A container comprising a general detection composition comprising reagents for carrying out a chemical or biochemical detection reaction of an analyte, wherein the chemical or biochemical detection reaction of the analyte is nucleic acid amplification, and the general detection composition comprises a buffer, mononucleoside triphosphate, amplification enzyme, and nucleic acid dye for detection of the amplification product, or - A container and further containers comprising a first detection composition comprising a reagent for carrying out a chemical or biochemical detection reaction of an analyte, and a second detection composition comprising a detection reagent, wherein the chemical or biochemical detection reaction of the analyte is an immunochemical detection reaction, and the first detection composition comprises a reagent for carrying out an immunochemical detection reaction; the second detection composition comprises a suitable substrate suitable for a suitable reporter enzyme as a detection reagent, the substrate becoming detectable when reacted with the reporter enzyme; or - A container and further containers comprising a first detection composition comprising a reagent for carrying out a chemical or biochemical detection reaction of an analyte, and a second detection composition comprising a detection reagent, wherein the chemical or biochemical reaction is an immunochemical detection reaction, and the first detection composition comprises a reagent for carrying out an immunochemical detection reaction; the second detection composition comprises a buffer, a mononucleoside triphosphate, an amplification enzyme, a nucleic acid dye for detecting the amplification product, and a primer suitable for amplifying an oligonucleotide tag attached to a secondary antibody as a detection reagent; and b) - A container containing a non-aqueous phase; and c) - Mixing container for mixing ingredients The kit includes, d) - Container for conducting the detection reaction The kit includes; the kit further includes, e) - The kit according to claim 16 or 17, or - Library of fabricated microparticles according to any one of claims 1 to 15 A kit that includes this.

21. The kit according to claim 20, wherein the amplification enzyme is a suitable nucleic acid polymerase, and the amplification product is an amplified nucleic acid.

22. The kit according to claim 21, wherein the preferred nucleic acid polymerase is Taq polymerase.

23. The reagent for performing the immunochemical detection reaction is a buffer, and a secondary antibody or secondary antibody fragment that is specific to the same analyte as the primary antibody, antibody fragment, or non-antibody protein used as an analyte-specific reagent (ASR) in the immunochemical detection reaction, and is conjugated to a suitable reporter enzyme, or The kit according to any one of claims 20 to 22, wherein the reagent for performing the immunochemical detection reaction is a buffer and a secondary antibody or secondary antibody fragment that is specific to the same analyte as the primary antibody used as an analyte-specific reagent (ASR) in the immunochemical detection reaction and is conjugated to a suitable oligonucleotide tag.

24. The kit according to any one of claims 20 to 23, wherein the suitable substrate is a substrate that becomes detectable by fluorescence when reacted with the reporter enzyme.

25. The kit according to any one of claims 20 to 24, wherein the non-aqueous phase is oil.

26. The kit according to any one of claims 20 to 25, wherein the non-aqueous phase is supplemented with an emulsifier.

27. The kit according to claim 25 or 26, wherein the non-aqueous phase is a fluorocarbon oil.

28. A method for detecting and / or quantifying a target analyte in an aqueous sample, the method comprising the following steps: － • Aqueous samples known or suspected to contain the analyte of interest; A general detection composition comprising reagents for carrying out chemical or biochemical detection reactions of the analyte; A library of pre-fabricated microparticles according to any one of claims 1 to 9, wherein the analyte-specific reagent attached to the microparticles is selected to specifically bind to or react specifically with the target analyte. A process of providing in any order; - A step comprising incubating the aqueous sample with the library of fabricated microparticles, thereby allowing the library of microparticles to absorb the aqueous sample into the void volume of the microparticles, and enabling binding to the target analyte if present in the sample; - If the aqueous sample has not been previously mixed with the general detection composition, the step of adding the general detection composition to the fine particles; - A step of creating a plurality of isolated reaction spaces for detecting the analyte by transferring the library of fabricated microparticles to a non-aqueous phase and removing the aqueous phase around each individual fabricated microparticle, wherein the reaction spaces include an aqueous phase and are limited to the void volume of the microparticles; - A step of performing the detection reaction for the target analyte; - Steps to detect and / or quantify the target analyte. Methods that include...

29. The method of claim 28, further comprising the step of mixing the aqueous sample with the general detection composition before incubating the aqueous sample with the library of prepared microparticles.

30. The method according to claim 28 or 29, further comprising the step of washing the microparticles after incubating the aqueous sample with the library of fabricated microparticles, but before adding the general detection composition.

31. The method according to any one of claims 28 to 30, further comprising the step of applying an external trigger to release the analyte-specific reagent attached to the fine particles after transferring the library of fine particles to a non-aqueous phase and before performing the detection reaction of the target analyte.

32. The target analyte is nucleic acid, - The analyte-specific reagent is a nucleic acid or nucleic acid pair that is sufficiently complementary to the target analyte to be able to hybridize to the target analyte under hybridization conditions; - The general detection composition comprises reagents other than primers for carrying out the amplification reaction of the target nucleic acid analyte, and in particular, the general detection composition comprises a buffer, a mononucleoside triphosphate, an amplification enzyme, and a nucleic acid dye for detecting the amplification product; - The transfer step is a step of transferring the manufactured fine particles to a non-aqueous phase and suspending them; - The step of releasing the analyte-specific reagent attached to the fine particles by applying an external trigger is a step of temporarily increasing the temperature, changing the pH, or changing the salt conditions. - The step of performing the detection reaction is a step of performing an amplification reaction of the analyte when the analyte is present in the aqueous sample; - The step of detecting and / or quantifying the target analyte is the step of detecting and / or quantifying the amplified analyte. The method according to any one of claims 28 to 31.

33. The method according to any one of claims 28 to 32, wherein the analyte-specific reagent is a primer or primer pair suitable for amplification of the target analyte; the amplification enzyme is a suitable nucleic acid polymerase; and the amplification product is an amplified nucleic acid.

34. The method according to claim 33, wherein the preferred nucleic acid polymerase is Taq polymerase.

35. The method according to any one of claims 28 to 34, wherein, in the transfer step, the fine particles are transferred to the non-aqueous phase and then repeatedly washed in the non-aqueous phase, and the step also includes filtration or mechanical stirring to ensure that no or substantially any aqueous phase remains on the outside of the fine particles to form a monodisperse suspension of the fine particles.

36. The method according to any one of claims 32 to 35, wherein the step of releasing the analyte-specific reagent attached to the fine particles by applying an external trigger is a step of raising the temperature from the ambient temperature to a temperature of >90°C.

37. A method for detecting and / or quantifying a target analyte in an aqueous sample, the method comprising the following steps: － • Aqueous samples known or suspected to contain the analyte of interest; A first detection composition comprising reagents necessary for carrying out a chemical or biochemical detection reaction of the analyte; • A second detection composition containing a detection reagent; Library of fabricated microparticles according to any one of claims 1 to 15 A step of providing in any order, wherein the analyte-specific reagent attached to the fine particles is selected so as to specifically bind to or react specifically with the target analyte; - A step comprising incubating the aqueous sample with the library of fabricated microparticles, and if the aqueous sample has not yet been mixed with the first detection composition, also incubating it with the first detection composition, thereby allowing the library of microparticles to absorb the aqueous sample and the first detection composition into the void volume of the microparticles, and enabling binding to the target analyte if the target analyte is present in the sample; - A step comprising incubating the library of fabricated microparticles containing the absorbed aqueous sample with the second detection composition, thereby enabling the library of microparticles to absorb the second detection composition; - A step of creating a plurality of isolated reaction spaces for detecting the analyte by transferring the library of fabricated microparticles to a non-aqueous phase and removing the aqueous phase around each individual fabricated microparticle, wherein the reaction spaces include an aqueous phase and are limited to the void volume of the microparticles; - A step of detecting and / or quantifying the target analyte by detecting and / or quantifying the detection reagent. Methods that include...

38. - A step of mixing the aqueous sample with the first detection composition before incubating the aqueous sample with the library of fabricated microparticles; - Before incubating the library of fabricated microparticles containing the absorbed aqueous sample with the second detection composition, the library of fabricated microparticles is washed to remove any unabsorbed or unreacted first detection composition; - A step of further washing the library of fabricated microparticles to remove any unabsorbed or unreacted second detection composition before transferring it to a non-aqueous phase; and - A step of applying an external trigger to release the analyte-specific reagent attached to the fine particles before detecting and / or quantifying the target analyte by detecting and / or quantifying the detection reagent. The method according to claim 37, further comprising:

39. The method according to any one of claims 37 to 38, wherein the step of transferring to a non-aqueous phase is performed immediately after incubating the library of fabricated microparticles with the second detection composition.

40. The target analyte is a protein or other non-nucleic acid molecule. - The analyte-specific reagent is a primary antibody, antibody fragment, or non-antibody protein that can specifically bind to the protein analyte of interest or other non-nucleic acid analytes; - The first detection composition comprises reagents necessary for carrying out an immunochemical detection reaction; - The second detection composition contains a suitable substrate as a detection reagent for a suitable reporter enzyme, and the substrate becomes detectable when reacted with the reporter enzyme; - The step of incubating the aqueous sample with the library of fabricated microparticles and the first detection composition is, if the analyte is present in the aqueous sample, a step of forming a complex of the analyte with the primary antibody, primary antibody fragment or non-antibody protein by performing an immunochemical reaction including the binding of the analyte to the primary antibody, primary antibody fragment or non-antibody protein; the immunochemical reaction further includes the binding of a secondary antibody to the complex, thereby forming a sandwich between the primary antibody, antibody fragment or non-antibody protein, the analyte and the secondary antibody; - The first step of washing the library as defined in claim 38 is the step of removing unbound secondary antibodies from the library; - The step of incubating the library of prepared microparticles containing the absorbed aqueous sample with the second detection composition is a step that enables the substrate to be reacted with the reporter enzyme; - A further step of washing the library as defined in claim 38 is a step of removing unreacted substrates from the library; - The transfer step is a step of transferring the manufactured fine particles to a non-aqueous phase and suspending them; - The step of releasing the analyte-specific reagent attached to the fine particles by applying an external trigger is a step of temporarily increasing the temperature, changing the pH, or changing the salt conditions; - The step of detecting and / or quantifying the target analyte is the step of detecting and / or quantifying the reacted substrate. The method according to claim 38 or 39.

41. The method according to claim 40, wherein the reagent for performing an immunochemical detection reaction is a buffer and a secondary antibody or secondary antibody fragment that is specific to the analyte and conjugated to a suitable reporter enzyme.

42. The method according to claim 40 or 41, wherein the suitable substrate is a substrate that becomes detectable by fluorescence when reacted with the reporter enzyme.

43. The method according to any one of claims 37 to 42, wherein the transfer step is a step of transferring the manufactured fine particles to a non-aqueous phase, suspending them, and repeatedly washing them in the non-aqueous phase, and further includes filtration or mechanical stirring to ensure that no aqueous phase remains on the outside of the fine particles to form a monodisperse suspension of fine particles.

44. The method according to any one of claims 40 to 43, wherein the step of releasing the analyte-specific reagent attached to the fine particles by applying an external trigger is a step of raising the temperature from the ambient temperature to a temperature of >90°C.

45. A method for detecting and / or quantifying a target analyte in an aqueous sample, the method comprising the following steps: － • Aqueous samples known or suspected to contain the analyte of interest; A first detection composition comprising reagents necessary for carrying out a chemical or biochemical detection reaction of the analyte; • A second detection composition containing a detection reagent; Library of fabricated microparticles according to any one of claims 1 to 15 A step of providing in any order, wherein the analyte-specific reagent attached to the fine particles is selected so as to specifically bind to or react specifically with the target analyte; - A step comprising incubating the aqueous sample with the library of fabricated microparticles, and if the aqueous sample has not yet been mixed with the first detection composition, also incubating it with the first detection composition, thereby allowing the library of microparticles to absorb the aqueous sample and the first detection composition into the void volume of the microparticles, and enabling binding to the target analyte if the target analyte is present in the sample; - A step comprising incubating the library of fabricated microparticles containing the absorbed aqueous sample with the second detection composition, thereby enabling the library of microparticles to absorb the second detection composition; - A step of creating a plurality of isolated reaction spaces for detecting the analyte by transferring the library of fabricated microparticles to a non-aqueous phase and removing the aqueous phase around each individual fabricated microparticle, wherein the reaction spaces include an aqueous phase and are limited to the void volume of the microparticles; - A step of performing the detection reaction for the target analyte; and - Steps to detect and / or quantify the target analyte. Methods that include...

46. - A step of mixing the aqueous sample with the first detection composition before incubating the aqueous sample with the library of fabricated microparticles; - Before incubating the library of fabricated microparticles containing the absorbed aqueous sample with the second detection composition, the library of fabricated microparticles is washed to remove any unabsorbed or unreacted first detection composition; - A step of further washing the library of fabricated microparticles to remove any unabsorbed or unreacted second detection composition before transferring it to a non-aqueous phase; and - A step of applying an external trigger to release the analyte-specific reagent attached to the fine particles before performing the detection reaction of the target analyte. The method according to claim 45, further comprising:

47. The target analyte is a protein or other non-nucleic acid molecule. - The analyte-specific reagent is a primary antibody, antibody fragment, or non-antibody protein that can specifically bind to the protein analyte of interest or other non-nucleic acid analytes; - The first detection composition comprises reagents necessary for carrying out an immunochemical detection reaction; - The second detection composition comprises a buffer, a mononucleoside triphosphate, an amplification enzyme, a nucleic acid dye for detecting the amplification product, and a primer suitable for amplifying an oligonucleotide tag attached to a secondary antibody as a detection reagent; - The step of incubating the aqueous sample with the library of fabricated microparticles and the first detection composition is, if the analyte is present in the aqueous sample, a step of forming a complex of the analyte and the primary antibody, primary antibody fragment or non-antibody protein by performing an immunochemical reaction including the binding of the analyte to the primary antibody, primary antibody fragment or non-antibody protein; the immunochemical reaction further includes the binding of the secondary antibody to the complex, thereby forming a sandwich between the primary antibody, antibody fragment or non-antibody protein, the analyte and the secondary antibody; - The first step of washing the library is the step of removing unbound secondary antibodies from the library; - The step of incubating the library of fabricated microparticles containing the absorbed aqueous sample with the second detection composition is a step that allows the primer in the second detection composition to hybridize to the oligonucleotide tag attached to the secondary antibody; - A further step of washing the library is to remove primers that did not hybridize from the library; - The transfer step is a step of transferring the manufactured fine particles to a non-aqueous phase and suspending them; - The step of releasing the analyte-specific reagent attached to the fine particles by applying an external trigger is a step of temporarily increasing the temperature, changing the pH, or changing the salt conditions; - The step of performing the detection reaction is a step of performing the amplification reaction of the oligonucleotide tag; The step of detecting and / or quantifying the target analyte is the step of detecting and / or quantifying the amplified oligonucleotide tag. The method according to claim 46.

48. The method according to claim 47, wherein the reagent for performing an immunochemical detection reaction is a buffer and a secondary antibody or secondary antibody fragment that is specific to the same analyte as the primary antibody and conjugated to a suitable oligonucleotide tag; the amplification enzyme is a suitable nucleic acid polymerase; and the amplification product is an amplified nucleic acid.

49. The method according to claim 48, wherein the preferred nucleic acid polymerase is Taq polymerase.

50. The method according to any one of claims 47 to 49, wherein the transfer step is a step of transferring the manufactured fine particles to a non-aqueous phase, suspending them, and repeatedly washing them in the non-aqueous phase, and further includes filtration or mechanical stirring to ensure that no aqueous phase remains on the outside of the fine particles to form a monodisperse suspension of fine particles.

51. The method according to any one of claims 47 to 50, wherein the step of releasing the analyte-specific reagent attached to the fine particles by applying an external trigger is a step of raising the temperature from the ambient temperature to a temperature of >90°C.

52. The method according to any one of claims 28 to 51, wherein the step of releasing the analyte-specific reagent attached to the fine particles by applying an external trigger is performed by increasing the temperature to which the fine particles are exposed.

53. - The method described above is a method for detecting and / or quantifying a single analyte in multiple aqueous samples, - The above method provides several different aqueous samples that are known or suspected to contain several target analytes. - The library of pre-fabricated microparticles provided in the method is the library according to claim 13, wherein such a library provides the same number or at least the same number of distinct microparticle subsets as the number of different aqueous samples to be tested, all of which have the same analyte-specific reagent attached to the porous polymer matrix of the microparticles in the subset, and the analyte-specific reagent is specific to one target analyte; - In the step of incubating the aqueous sample with the library of fabricated microparticles, each of the different aqueous samples is incubated separately with a separate subset of microparticles of the library; - In the step of incubating the library of prepared microparticles containing the absorbed aqueous sample with a second detection composition, each of the distinct microparticle subsets of the library is incubated separately with the second detection composition; - The step of transferring the library to a non-aqueous phase is performed separately for each subset of fine particles, that is, each separate subset of fine particles is transferred separately to a non-aqueous phase, and the aqueous phase around each individual fine particle is removed separately for each subset, thereby creating a plurality of isolated reaction spaces for detecting the analyte separately for each subset, the reaction spaces comprising an aqueous phase and limited to the void volume of the fine particles, The method further comprises, after the step of transferring the library to a non-aqueous phase, mixing together the plurality of isolated reaction spaces of all the separate subsets in the non-aqueous phase, then carrying out a detection reaction for the target analyte, and then detecting and / or quantifying the target analyte, The method according to any one of claims 28 to 52.

54. - The method described above is a method for detecting and / or quantifying multiple analytes in a single aqueous sample, - The above method provides a single aqueous sample that is known or suspected to contain multiple target analytes, - The library of fabricated microparticles provided in the method is the library according to claim 14, wherein such a library provides as many or at least the same number of distinct microparticle subsets as the number of different target analytes to be detected, each of the distinct subsets having a different analyte-specific reagent attached to the porous polymer matrix of the microparticles in the subset; each analyte-specific reagent is specific to one target analyte; -In the step of incubating the aqueous sample with the library of fabricated microparticles, the aqueous sample is incubated together with all of the separate microparticle subsets of the library; - In the step of incubating the library of prepared microparticles containing the absorbed aqueous sample with a second detection composition, all of the distinct microparticle subsets of the library are incubated together with the second detection composition; - The step of transferring the library to a non-aqueous phase is performed together for all particulate subsets, i.e., all particulate subsets are transferred together to a non-aqueous phase, and the aqueous phase around the individual particulates is removed, thereby creating multiple isolated reaction spaces for detecting and / or quantifying the multiple analytes, the reaction spaces comprising the aqueous phase and limited to the void volume of the particulates. The method according to any one of claims 28 to 52.

55. - The method described above is a method for detecting and / or quantifying multiple analytes in multiple aqueous samples, - The above method provides several different aqueous samples that are known or suspected to contain several target analytes. - The library of pre-fabricated microparticles provided in the method is the library according to claim 15, wherein such a library contains separate subsets of pre-fabricated microparticles of different classes, each of which comprises several subsets of microparticles, each of which has different analyte-specific reagents attached to the porous polymer matrix of the microparticles, the same analyte-specific reagent attached to all subsets of microparticles in one class; each analyte-specific reagent is specific to one target analyte; and in the step of providing the library in the method, - A different class of distinct particulate subsets is provided, in the same number or at least the same number as the number of different analytes to be detected. • Each class will provide the same number or at least the same number of different particulate subsets as the number of aqueous samples to be tested provided. The library contains a number of different particulate subsets equal to or at least the number obtained by multiplying the number of different aqueous samples to be tested by the number of different target analytes to be detected. - In the step of incubating the aqueous sample with the library of fabricated microparticles, strictly one aqueous sample is incubated with strictly one microparticle subset derived from each class, and each aqueous sample is incubated with the same number of different microparticle subsets derived from different classes as there are classes of microparticles in the library. -In the step of incubating the library of fabricated microparticles containing the absorbed aqueous sample with a second detection composition, a subset of each aqueous sample combined with pre-incubated microparticles of different classes is incubated together with the second detection composition. - The step of transferring the library to a non-aqueous phase is performed separately for each aqueous sample, that is, each aqueous sample and the pre-incubated combined microparticle subset are transferred together to a non-aqueous phase, and the aqueous phase around the individual microparticles is removed separately for each aqueous sample, thereby creating multiple isolated reaction spaces for detecting the multiple analytes, the reaction spaces containing the aqueous phase and limited to the void volume of the microparticles. The method according to any one of claims 28 to 52.

56. The method according to claim 55, wherein the method further comprises, after the step of transferring the library to a non-aqueous phase, mixing together the plurality of isolated reaction spaces of the separate samples in the non-aqueous phase, then carrying out a detection reaction for the target analyte, and then detecting and / or quantifying the target analyte.

57. In the step of detecting and quantifying the target analyte, the quantification of the analyte is a) Digital nucleic acid amplification, particularly digital polymerase chain reaction (PCR); b) Real-time quantitative nucleic acid amplification, in particular, real-time polymerase chain reaction (PCR); c) Immunochemical detection methods; d) Immunochemical detection methods combined with nucleic acid amplification It is done by a method selected from; If the target analyte is a nucleic acid, quantification is performed using either method a) or b) or a combination of a) and b); if the analyte is a protein, peptide or other non-nucleic acid analyte, quantification is performed using either method c) or d). The method according to any one of claims 28 to 56.

58. The method according to claim 57, wherein the immunochemical detection method is a digital immunoassay.

59. The method according to claim 58, wherein the immunochemical detection method is digital enzyme-linked immunosorbent assay (ELISA).

60. The method according to any one of claims 57 to 59, wherein the immunochemical detection method combined with nucleic acid amplification is digital immunoPCR.

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