Method for detecting and / or quantifying a target analyte in multiple biological liquid samples

Abstract

The present invention relates to a method for detecting and / or quantifying analytes of interest in multiple biological fluid samples. Furthermore, the present invention relates to a kit and cartridge for carrying out the method for detecting and / or quantifying analytes of interest in multiple samples. The method includes two subprocesses: one sample-specific subprocess and one general subprocess. The former is a process in which each sample is separately subjected to a procedure or condition in which it is specifically labeled (sometimes referred to herein as "coded") so that it can be subsequently identified and distinguished from other samples. The latter is a process in which all samples undergo the same operation that allows for the detection of the analyte.

Description

Technical Field

[0001] The present invention relates to a method for detecting and / or quantifying a target analyte in a plurality of biological liquid samples.

Background Art

[0002] Background of the Invention In the field of diagnostics, sample pooling strategies and sample barcoding strategies are established and commonly used when processing a large number of biological samples.

[0003] Sample pooling strategies in the field of diagnostics are typically based on testing a pooled mixture of various samples of a defined volume. Usually, samples are obtained in pairs, one of the pair is maintained for storage, and the other of the pair is added to the pooled mixture. If such a pooled mixture is determined to be positive, the stored paired samples from the same pool are tested individually to finally identify the positive sample. The process of testing the individual paired samples from a positive resulting pooled mixture is sometimes also referred to as "re-flexing". In particular, in samples where a negative result is usually expected for each analyte being tested, such a pooling strategy provides a cost-effective approach. However, the degree of pooling directly affects the sensitivity of the test, and re-flexing requires obtaining at least paired samples from one origin, careful and controlled storage of such paired samples, as well as complex test logistics.

[0004] Sample barcoding, sometimes called sample indexing, is a widely used approach for labeling samples for multiplex sequencing and multiplex analysis. All nucleic acids in a sample are labeled with the same sequence tag, the resulting libraries are pooled with other libraries, and sequenced in parallel in a single run. A sample-specific index then allows software to separate the multiplexed sequence data within the sample-specific dataset.

[0005] Molecular barcoding differs from sample indexing in that each molecule in a sample is labeled with a different, unique sequence before PCR amplification. When each nucleic acid in the starting material (i.e., in the sample) is labeled with a unique sequence, or "molecular barcode" ("MBC"), sequence analysis software can report unique reads, filtering out duplicate reads and PCR errors. Both sample barcoding and molecular barcoding share the commonality of tagging nucleic acids with specific molecular sequence labels.

[0006] Another type of barcoding method is fluorescent cell barcoding (FCB), a cell-based multiplexing method for high-throughput flow cytometry. Because barcoded samples can be stained and acquired together, staining variability and antibody consumption are minimized, and the required sample volume is reduced (Krutzik et al., 2011 “Fluorescent cell barcoding for multiplex flowcytometry”, Curr. Protoc. Cytom., Chapter 6, Unit 6.31).

[0007] In particular, in cartridge-based testing scenarios, especially with a one-cartridge / one-sample approach, there are limitations in processing capacity due to the size and cost of the cartridges. Examples of such cartridges include Cepheid's GenExpert, Biocartis' Idylla, and Abbott's m-Pima. Higher processing capacity is usually achieved by adding the same instrument resources and running more cartridges at once. There are also cartridges that can process multiple samples on a single platform. However, such cartridges are usually designed to perform separate analytical processes for each individual sample, for example, when testing samples for various analytes, so such cartridges are merely components of a set of components, similar to individual analytical tools for each sample. Such setups are also useful when multiple samples need to be processed in parallel (e.g., when testing many samples for the same analyte), and such setups help improve the processing capacity of each instrument or simply reduce the time spent loading samples into analytical cartridges. An example of such a tool is the BDMAx System. A comprehensive review of existing cartridge-based diagnostic tools can be found in Relich et al. 2018; “Syndromic and Point-of-Care Molecular Testing”, Advances in Molecular Pathology 1(1):97-113. There is a continuing need in this field to increase processing capacity, make better use of equipment, while simultaneously reducing waste generation and lowering costs. Furthermore, there is a continuing need to process multiple samples in a single process within a single system to maximize the use of hardware and reagent resources. In addition, the field requires processes that facilitate the performance of a single detection reaction on a large number of individually identified samples by assigning individual identification to each sample being analyzed. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Krutzik et al.,2011 “Fluorescent cell barcoding for multiplex flowcytometry”,Curr.Protoc.Cytom.,Chapter 6,Unit 6.31 [Non-Patent Document 2] Relich et al.2018;“Syndromic and Point-of-Care Molecular Testing”,Advances in Molecular Pathology 1(1):97-113 [Overview of the project] [Means for solving the problem]

[0009] Summary of the Invention The present invention addresses these and related needs. In one embodiment, the present invention relates to a method for detecting and / or quantifying an analyte of interest in a plurality of biological liquid samples, the method comprising two subprocesses, namely one sample-specific subprocess and one general subprocess.

[0010] A “sample-specific subprocess,” which may be referred to herein as a “sample-specific process portion,” a “specific process portion,” or a “specific portion,” is a portion to which each sample is subjected separately to a procedure or condition that is specifically labeled (which may be referred to herein as “encoded”) so that it can be subsequently identified and distinguished from other samples.

[0011] A "general subprocess," "general process portion," or "general portion" is the part in which all samples undergo the same procedure that enables the detection of the analyte. Such a general portion does not involve any sample-specific procedures, handling, or manipulation and is typically performed on all samples under investigation, regardless of the nature of the sample.

[0012] In one embodiment, the sample-specific process portion consists of the following steps: - A step of separately providing, in any order, a plurality of different biological liquid samples suspected to contain the target analyte, and a plurality of differently labeled porous particulate subsets, wherein each of the plurality of porous particulate subsets is separated from the other subsets; - A step of specifically labeling each of the different biological samples by exposing each of the separate particulate subsets separately to one biological liquid sample, thereby enabling each sample to be absorbed by a specifically labeled porous particulate subset in an aqueous environment; - A process of separately transferring each subset of porous fine particles from the aqueous environment to a non-aqueous environment. Includes.

[0013] In one embodiment, the general process portion comprises the following steps: - A step of mixing together differently labeled porous microparticle subsets in the non-aqueous environment to produce a suspension of multiple differently labeled porous microparticle subsets; - A step of performing a detection reaction to detect the target analyte in the suspension of the plurality of differently labeled porous microparticle subsets; and - If the target analyte is present in any of the differently labeled subsets of suspended particulate matter, the step of detecting and / or quantifying the target analyte. Includes.

[0014] As used herein, the phrase "in the plurality of subsets, each of the porous particulate subsets is separated from the other subsets" and the term "distinct subset" mean a scenario in which such particulate subsets are physically separated from the other subsets. In one embodiment, two subsets that are "separated" from each other may be located in different locations and / or macroscopically identifiable and / or separated by a physical barrier so that mixing or cross-contamination between samples cannot occur. Thus, it is preferable that each "distinct subset" is provided in a form that allows for separate handling of such subset. Physical separation of different subsets can be achieved, for example, by a barrier around such subsets. In a simple embodiment, such distinct subsets may be placed in their own container or compartment. Typically, according to embodiments of the present invention, those subsets are separated from each other before the detection and / or quantification reaction (of the analyte) is carried out. During use, for example, during a detection reaction, or more specifically, during the general part of such a detection reaction, some or all subsets of a microparticle library may eventually be combined. Surprisingly, the inventors have found that in such a mixture, if the different microparticles are separated from each other by being transferred to a non-aqueous phase, then combining / mixing them does not actually result in overflow or “crosstalk” between those different microparticles. As a result, despite the fact that each microparticle is now mixed, there is also no cross-contamination between samples (tested for the presence of an analyte). The term “multiple differently labeled porous microparticle subsets” as used herein may also be referred to as “library of microparticles.”

[0015] As used herein, the term “biological liquid sample” refers to a liquid sample obtained from the body of an 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 a “biological liquid 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 biological liquid sample is plasma. Preferably, such a biological liquid 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 biological liquid sample is plasma, such plasma may be further dissolved and / or digested to remove components that may otherwise interfere with subsequent detection reactions. For example, if the analyte of interest is a specific nucleic acid, a biological liquid sample, such as plasma, may first be digested with a protease 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 undesirable components. Thus, as used herein, “biological liquid sample” may also refer to an extract obtained from any of the above-mentioned bodily fluids; this may be a nucleic acid extract obtained from any of the above-mentioned bodily fluids by a suitable extraction method, for example, for cell disruption (if necessary), removal of lipids, proteins and unwanted nucleic acids (if necessary), and purification of nucleic acids and / or concentration of a specific nucleic acid. In some embodiments, the nucleic acid extract may be produced using ethanol or another suitable alcohol.

[0016] As used herein, the term “library” (of microparticles) means a group of microparticles or a collection of microparticles, and such a library of microparticles or a collection 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 subsets of fabricated precursor microparticles, preferably three or more subsets of fabricated precursor microparticles, each subset having a different labeling component attached to, contained within, or otherwise associated with the microparticles within the subset, and the at least two or more microparticle subsets each have a different labeling component attached to, contained within, or otherwise associated with each subset. In such a library of different subsets, each subset is usually and preferably stored separately from one another, for example, in different containers. This means that the term “distinct subset” as used herein means a group of microparticles or microparticle subsets that are physically separated from other subsets. Detailed description of the invention

[0017] In one embodiment, the present invention relates to a method for detecting and / or quantifying a target analyte in a plurality of biological liquid samples, wherein the method comprises a sample-specific process portion followed by a general process portion; The aforementioned sample-specific process portion consists of the following steps: - A step of providing, separately in any order, a plurality of different biological liquid samples suspected to contain the target analyte, and a plurality of differently labeled porous particulate subsets, wherein each of the plurality of subsets is separated from the other subsets; - By separately exposing each of said separate subsets of microparticles to one biological liquid sample each, enabling each sample to be aspirated by one specifically labeled subset of porous microparticles in an aqueous environment, the step of specifically labeling each of said different biological samples; - The step of separately transferring each subset of porous microparticles from said aqueous environment to a non-aqueous environment comprising; Said general process part comprises the following steps: - By mixing together the differently labeled subsets of porous microparticles in said non-aqueous environment, the step of generating a suspension of a plurality of differently labeled subsets of porous microparticles; - Performing a detection reaction for detecting said target analyte on said suspension of a plurality of differently labeled subsets of porous microparticles; and The step of detecting and / or quantifying said target analyte if said target analyte is present in any of said differently labeled suspended microparticle subsets comprising.

[0018] Preferably, in said step of separately exposing each of said separate subsets of microparticles to one biological liquid sample each, each of said separate subsets of microparticles is exposed to one biological sample and a detection composition for performing a chemical or biochemical detection reaction in any order, and each microparticle subset aspirates the respective biological sample and detection composition to which it is exposed.

[0019] In one aspect, the present invention also relates to a method for detecting and / or quantifying a target analyte in a plurality of biological liquid samples, in particular according to the method as outlined in the previous paragraph, the sample-specific process of which is a. A step of separately providing, in any order, a plurality of distinct biological liquid samples and a plurality of porous microparticles suspected to contain a target analyte; each of the porous microparticles having a porous matrix and configured to receive a predetermined volume of liquid into the porous matrix, preferably to aspirate the analyte if one is present; the plurality of porous microparticles comprise different microparticle subsets provided, each microparticle subset characterized by a specific labeling component attached to, contained in, or otherwise associated with the respective subset; in the providing step, the number of different microparticle subsets provided is at least the same as the number of distinct biological liquid samples provided, and further, in the providing step, the different microparticle subsets are provided separately from one another; optionally, the different microparticle subsets comprise a detection composition comprising a reagent for carrying out a chemical or biochemical detection reaction of the analyte in their respective porous matrices; b. Exposing each distinct particulate subset to strictly one distinct biological liquid sample, thereby enabling each distinct particulate subset to incubate with a predetermined volume of strictly one distinct biological liquid sample and to aspirate such sample or a portion thereof, and, if necessary, to accumulate the analyte in or on the matrix of the particulates if the analyte is present in the sample; and, if further necessary, if the different distinct particulate subsets provided in step a) do not yet contain reagents for carrying out a chemical or biochemical detection reaction of the analyte, exposing each distinct particulate subset to a detection composition containing reagents for carrying out a chemical or biochemical detection reaction of the analyte, thereby enabling each distinct particulate subset to receive the reagents; c. A step of creating a plurality of separate, isolated reaction space subsets for detecting the analyte by transferring each particulate subset separately to a non-aqueous phase and removing part or all of the aqueous phase around the individual prepared particulates of the subset, wherein the reaction space comprises an aqueous phase containing the reagents for carrying out chemical or biochemical detection reactions of the sample and analyte, and the reaction space is limited to the void volume of the particulates. It is accompanied by.

[0020] In one embodiment, the general process is: d. Mixing separate and different particulate subsets in the non-aqueous phase so that all of the different particulate subsets form a suspension of different particulates in the non-aqueous phase; subjecting the mixed different particulate subsets to the conditions necessary for a chemical or biochemical detection reaction of the analyte; performing such a detection reaction of the analyte of interest; detecting and / or quantifying the analyte of interest using the signal generated in the detection reaction in each of the different particulate subsets if the analyte of interest is present in any member of each of the subsets. Includes.

[0021] In one embodiment, the method comprises the following steps e) A step to determine which of the multiple samples provided in step a) contains the target analyte by identifying the subset of fine particles in which the target analyte was detected in step d). It also includes.

[0022] In one embodiment, the identification of particulate subsets in step e) is performed using a specific labeling component attached to, contained in, or otherwise associated with each particulate subset.

[0023] In one embodiment, step b) further includes a substep of generating a first correlation record indicating which distinct particulate subsets are or have been exposed to which sample, and step d) includes a substep of generating a second correlation record indicating which particulate subsets produced a signal in the detection reaction.

[0024] In one embodiment, step e) is performed by referring to the records of the first and second correlations and concatenating the records, thereby enabling determination of which of the multiple samples provided in step a) contains the target analyte.

[0025] In one embodiment, each of the porous fine particles is (i) A polymer or polymer mixture that forms or is the porous polymer matrix; or (ii) 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; or (iii) at least one or more charged groups immobilized on the porous polymer matrix; or (iv) Any combination of (i) to (iii) This provides a porous matrix that allows for the accumulation of the target analyte.

[0026] In one embodiment, each of the porous microparticles has a porous matrix and comprises an analyte-specific reagent (ASR) attached to the porous matrix, preferably reversibly attached, such analyte-specific reagent enabling the concentration of the analyte of interest and / or a specific signal amplification or target amplification reaction involving the analyte; the analyte-specific reagent can specifically bind to the analyte of interest. In a preferred embodiment, the analyte-specific reagent enables a specific target amplification reaction, but preferably does not enable a specific signal amplification reaction. In particular, in some embodiments, the analyte-specific reagent does not enable and / or is not used in binding assays, such as bDNA assays.

[0027] 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.

[0028] In another embodiment, each of the porous microparticles has a porous matrix but does not contain an analyte-specific reagent (ASR) as defined in the preceding paragraph. In this embodiment, the concentration or accumulation of the analyte in the microparticles is achieved solely by the porous matrix.

[0029] As used herein, the terms “signal amplification reaction” or “target amplification reaction” refer to a chemical or biochemical detection reaction in which a signal used for the detection of an analyte is amplified, or a target (i.e., analyte) to be detected and / or quantified is first amplified and then detected. A typical example of a target amplification reaction is a nucleic acid amplification reaction, e.g., PCR. A typical example of a signal amplification reaction is an immunochemical reaction, e.g., immunoassay. In preferred embodiments of the present invention, the chemical or biochemical detection reaction according to the present invention is a target amplification reaction and not a signal amplification reaction. In particular, the chemical or biochemical detection reaction according to the present invention is not a bDNA assay.

[0030] In one embodiment, 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, 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.

[0031] In one embodiment, each of the porous microparticles has the same analyte-specific reagent attached to the porous matrix, preferably reversibly attached. In one such embodiment, such same analyte-specific reagent is the only analyte-specific reagent attached to the microparticle.

[0032] As used herein, the term “same analyte-specific reagent” refers to a scenario in which various analyte-specific reagent molecules exist, all of which have the same specificity for a single particular analyte. For example, if an analyte-specific reagent is an antibody specific to a particular analyte, then all analyte-specific reagents that are “same” as such an analyte-specific reagent will have the same specificity for that analyte. Typically, in many cases, this means that such “same” analyte-specific reagents are identical in terms of their structure and / or sequence, or that their structure and sequence differ only to such an extent that their specificity for the same single particular analyte remains unaffected by these variations.

[0033] In one embodiment, the plurality of porous microparticles (or library of porous microparticles) contain different microparticle subsets. Each subset is, -Having different labeling components attached to, contained in, or otherwise associated with the fine particles of the subset; All of the aforementioned different subsets The subset of the fine particles has the same analyte-specific reagent attached to or contained therein, and the analyte-specific reagent is specific to one target analyte; The aforementioned different subsets of particulate matter are identical with respect to the analyte-specific reagents attached to or contained within them, Each subset of the fine particles has different labeling components attached to, contained therein, or otherwise associated with it; Each subset is clearly defined and identifiable by its respective labeling component.

[0034] In one embodiment, the method is a method for detecting and / or quantifying one target analyte in a plurality of biological liquid samples, wherein all of the different subsets have the same analyte-specific reagent attached to the microparticles of the subset, and the analyte-specific reagent is specific to one target analyte, and the number of different microparticle subsets provided, preferably in step a), is equal to the number of distinct biological liquid samples provided.

[0035] In another embodiment, the plurality of porous microparticles (or library of porous microparticles) contain several different analyte-specific reagents attached to or contained within the microparticles.

[0036] In such embodiments (i.e., embodiments in which several different analyte-specific reagents are attached to or contained in the plurality of porous microparticles), different microparticle subsets exist, Each subset is, -Having different labeling components attached to, contained in, or otherwise associated with the fine particles of the subset; Furthermore, among the multiple porous microparticles, there exist subsets of different classes of microparticles, and each class of the subset is: The microparticles have different analyte-specific reagents attached to or contained within the porous matrix of the microparticles; preferably, there are at least two different classes of microparticle subsets, more preferably at least three or more different classes of microparticle subsets; The aforementioned different particulate subsets differ in the respective labeling components attached to, contained in, or otherwise associated with the particulates of each subset; each particulate subset forms part of a class of particulate subsets; and 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, all of which have the same analyte-specific reagent attached to or included.

[0037] In such embodiments (i.e., embodiments in which several different analytes-specific reagents are attached to or contained in the plurality of porous microparticles), the method is a method for detecting and / or quantifying more than one of a target analytes in a plurality of biological liquid samples, wherein the number of different microparticle subsets provided, preferably in step a), is equal to the number of distinct biological liquid samples provided multiplied by the number of target analytes to be detected, and the same number of classes of microparticle subsets as the number of target analytes to be detected are provided, preferably in step a).

[0038] It should be noted that numerous types of microparticles can be used in accordance with the present invention, as long as the microparticles have a porous matrix and are configured to receive a predetermined volume of liquid into the porous matrix, and have a specific labeling component attached to, contained in, or otherwise associated with the microparticles, and such labeling component allows such microparticles to be classified as belonging to a particular subset of microparticles. Suitable microparticles may or may not have an analyte-specific reagent (ASR). Microparticles such as those disclosed in the EP application titled “A library of prefabricated microparticles and precursors thereof” (Agent reference number B33016EP), filed concurrently by the applicant Blink AG, have also proven to be particularly suitable for embodiments of the present invention, especially those in which more than one analyte is detected.

[0039] In one embodiment, the porous matrix is ​​a porous polymer matrix formed of a polymer or polymer mixture, preferably the porous polymer matrix is ​​composed of an uncrosslinked polymer or polymer mixture, more preferably the polymer or polymer mixture forming 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). In one particular 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). Embodiments in which the polymer or polymer mixture is not crosslinked are particularly excellent and versatile in switching between different states, for example, between a gel state and a sol state.

[0040] In one embodiment, 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 the 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 the 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 the precursor microparticles.

[0041] In one embodiment of a method for detecting and / or quantifying an analyte, the analyte of interest is a nucleic acid, the detection reaction is nucleic acid amplification, and the detection composition is a composition for performing nucleic acid amplification, comprising 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 detecting the amplified nucleic acid, and optionally one or more amplification primers, and further optionally, if such primers and / or probes are not yet provided as analyte-specific reagents (ASRs) attached to or contained in the microparticles, their respective molecular probes (e.g., TaqMan probes, molecular beacons, etc.).

[0042] In another embodiment of the method according to the present invention, the target analyte is a protein or other non-nucleic acid molecule, the detection reaction is an immunochemical detection reaction, and the detection composition is a composition for carrying out such an immunochemical detection reaction, provided in the method as two distinct components: the first component of the detection composition comprises a reagent necessary for carrying out the immunochemical detection reaction, e.g., a buffer, and a secondary antibody or secondary antibody fragment that is specific to the same analyte used as an analyte-specific reagent (ASR) in the immunochemical detection reaction and is conjugated to a suitable reporter enzyme; and optionally comprises a primary antibody, antibody fragment or non-antibody protein that can specifically bind to the protein analyte or other non-nucleic acid analyte if such a primary antibody, antibody fragment or non-antibody protein has not yet been provided as an analyte-specific reagent (ASR) attached to or contained in the microparticles; and the second component of the detection composition comprises a substrate suitable for the suitable reporter enzyme as a detection reagent, the substrate becoming detectable, preferably optically detectable, more preferably fluorescently detectable, when reacted with the reporter enzyme.

[0043] In such embodiments, i.e., embodiments in which the detection composition is provided as two distinct components, i.e., a first component and a second component, it is preferable that the particulate subset is exposed to the first component, and then to such second component, with the two exposures occurring sequentially. The two exposures may be further separated from each other by a suitable intermediate washing step, for example, a step of washing the particulate subset with a suitable washing buffer.

[0044] In another embodiment of the method according to the present invention, the target analyte is an enzyme or other clinical chemical analyte, the detection reaction is a chemical or biochemical (e.g., enzymatic) detection reaction, the detection composition is a composition for carrying out such a chemical or biochemical detection reaction, the sample is in contact with a detection reagent, both are in contact with fine particles according to the present invention, the detection composition contains as a detection reagent a substrate suitable for such a chemical or enzymatic reaction (creation), the substrate, upon reaction with the respective analyte, becomes detectable, preferably optically detectable, more preferably fluorescently detectable.

[0045] In one embodiment, 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, in particular digital immunochemical detection methods combined with nucleic acid amplification, e.g., immunopolymerase chain reaction; in particular digital immuno-PCR; and e) any combination of a) to d) 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).

[0046] As outlined above, the “biological liquid sample” as used herein may be a liquid sample obtained from the body of an 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 a “biological liquid 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 biological liquid sample is plasma. Preferably, such a biological liquid 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 biological liquid 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 a specific nucleic acid, a biological liquid sample, such as plasma, may first be digested with a protease 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 undesirable components. Thus, as used herein, “biological liquid sample” may also refer to an extract obtained from any of the above-mentioned bodily fluids; this may be a nucleic acid extract obtained from any of the above-mentioned bodily fluids by a suitable extraction method, for example, for cell disruption (if necessary), removal of lipids, proteins and unwanted nucleic acids (if necessary), and purification of nucleic acids and / or concentration of a specific nucleic acid. In some embodiments, the nucleic acid extract may be produced using ethanol or another suitable alcohol.

[0047] Cell disruption or breakdown can be achieved by physical and / or chemical methods, the primary objective of which is to disrupt the cell wall and / or cell membrane. Disruption methods are primarily based on the characteristics of the sample, and for this purpose, a wide range of tools and approaches are used individually or in combination to achieve tissue / cell disruption. Lysizing enzymes, chaotropic agents, and various types of washing agents are the main components of chemical lysis, while mechanical methods disrupt cells by grinding, shearing, bead beating, and shocking. It should be noted that even if one method does not yield good results, another may prove successful. In certain cases, osmotic shock has yielded better results than common nucleic acid purification protocols, e.g., phenol-chloroform extraction and bead beating. Another approach to cell disruption is to use a combination of different methods. A good example is the case with enzymatic lysis, where many protocols use proteases to release NA from the protective protein backbone. Also crucial is the inactivation of cellular nucleases released into solution to protect the new protein-free NA

[13] . A combination of a washing agent and a chaotropic salt may be used in a single solution to solubilize the cell wall and / or cell membrane, and to inactivate intracellular nucleases. On the other hand, mechanical disruption utilizes force to extract the cellular components. A classic example of grinding is the use of a mortar and pestle, which today is optimized by the use of liquid nitrogen (when the sample allows it). Cell walls can also be disrupted by shock waves produced by rapid pressure changes induced by sonication or cavitation. Other mechanical tools available for cell disruption are shearing, which uses tangential force to puncture the cell and bead beating, which uses various glass or steel beads to rupture the tough cell wall.

[0048] In a further embodiment, the present invention relates to a kit for detecting and / or quantifying a target analyte in a plurality of biological liquid samples, and in particular to a kit for carrying out a method according to the present invention, the kit is - A plurality of containers comprising a plurality of microparticles, each container comprising a plurality of porous microparticle subsets, each of the porous microparticles in each subset having a porous matrix and configured to receive a predetermined volume of liquid into the porous matrix; each microparticle subset is characterized by a specific labeling component attached to, contained therein, or otherwise associated with the respective subset, preferably the microparticles being as further defined above; and optionally comprising an aqueous cleaning reagent for cleaning the microparticles; - A container comprising a detection composition for detecting an analyte of interest; the detection composition comprising a reagent for carrying out a chemical or biochemical detection reaction of the analyte; the detection composition being a composition for carrying out nucleic acid amplification or an immunochemical detection reaction; - A container containing a non-aqueous phase for transferring each of the different particulate subsets to a non-aqueous phase after each of them has been exposed to a biological liquid sample, and for generating separate suspensions of the different particulate subsets in the non-aqueous phase; - A mixing vessel for mixing separate and different particulate subset suspensions together in the non-aqueous phase, wherein all of the different particulate subset suspensions form a single suspension of different particulates in the non-aqueous phase, which is then subjected to a detection reaction; - Container for conducting the detection reaction Includes.

[0049] In one embodiment, each of the porous microparticles has, or comprises, an analyte-specific reagent (ASR) attached, preferably reversibly attached, to its porous matrix, such analyte-specific reagent enabling the concentration of the analyte of interest and / or a specific signal amplification or target amplification reaction involving the analyte; the analyte-specific reagent can specifically bind to the analyte of interest. In preferred embodiments of the present invention, the analyte-specific reagent enables a specific target amplification reaction involving the analyte, but does not enable a signal amplification reaction. In particular, in some embodiments, the analyte-specific reagent does not enable and / or is not used in binding assays, such as bDNA assays.

[0050] In one embodiment, 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, 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.

[0051] In one embodiment, the target analyte is a nucleic acid, the detection reaction is nucleic acid amplification, and the detection composition is a composition for performing nucleic acid amplification, comprising 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 detecting the amplified nucleic acid, and optionally a pair of primers, if such primers have not already been provided as an analyte-specific reagent (ASR) attached to or contained in the microparticles.

[0052] In one embodiment, the target analyte is a protein or other non-nucleic acid molecule, the detection reaction is an immunochemical detection reaction, and the detection composition is a composition for carrying out such an immunochemical detection reaction, provided in the kit in two separate compartments or containers; the detection composition contains the reagents necessary to carry out the immunochemical detection reaction, e.g., a buffer, and a primary antibody, antibody fragment, or non-antibody protein used as an analyte-specific reagent (ASR) in the immunochemical reaction, and a secondary antibody or secondary antibody fragment conjugated to a suitable reporter enzyme, in the first compartment or The container contains; optionally, a primary antibody, antibody fragment, or non-antibody protein that can specifically bind to the protein analyte or other non-nucleic acid analyte, if such primary antibody, antibody fragment, or non-antibody protein has not yet been provided as an analyte-specific reagent (ASR) attached to or contained in the microparticles; the detection composition contains a substrate suitable for the preferred reporter enzyme as a detection reagent in a second compartment or container, the substrate becoming detectable, preferably optically detectable, and more preferably fluorescently detectable, upon reaction with the reporter enzyme.

[0053] In one embodiment, each of the porous microparticles has the same analyte-specific reagent attached to its porous matrix, or contains the same analyte-specific reagent.

[0054] In one embodiment, different subsets of microparticles exist among the plurality of porous microparticles. Each subset is, The subset of the fine particles has different labeling components attached to, contained therein, or otherwise associated therewith; All of the aforementioned different subsets The subset of the fine particles has the same analyte-specific reagent attached to or contained therein, and the analyte-specific reagent is specific to one target analyte; The aforementioned different subsets of particulate matter are identical with respect to the analyte-specific reagents attached to or contained within them, Each subset of the fine particles has different labeling components attached to, contained therein, or otherwise associated with it; Each subset is clearly defined and identifiable by its respective labeling component and is provided in a separate container.

[0055] In one embodiment, the kit is for detecting and / or quantifying one (i.e., at most one) target analyte in a plurality of biological liquid samples, wherein the number of different particulate subsets provided in the kit is equal to (preferably at least equal to) the number of separate biological liquid samples provided (or, in some embodiments, the number of different particulate subsets provided in the kit is greater than, and in any case not less than, the number of separate biological liquid samples provided).

[0056] In another embodiment, among the plurality of porous microparticles, there are several different analyte-specific reagents attached to or contained within the microparticles.

[0057] In such embodiments, different subsets of particulate matter exist, Each subset is, The subset of the fine particles has different labeling components attached to, contained therein, or otherwise associated therewith; Furthermore, among the multiple porous microparticles, there exist subsets of different classes of microparticles, and each class of the subset is: The microparticles have different analyte-specific reagents attached to or contained within the porous matrix of the microparticles; preferably, there are at least two different classes of microparticle subsets, more preferably at least three or more different classes of microparticle subsets; The aforementioned different particulate subsets differ in the respective labeling components attached to, contained in, or otherwise associated with the particulates of each subset; each particulate subset forms part of a class of particulate subsets; each subset is clearly defined and identifiable by its respective labeling component and its respective analyte-specific reagent, and is provided in separate containers; The aforementioned different classes of microparticle subsets differ in the analyte-specific reagents attached to or contained within the porous matrix of the microparticles; each of the aforementioned different classes comprises several microparticle subsets, all of which have the same analyte-specific reagents attached to or contained within them.

[0058] In such embodiments, the kit is for detecting more than one of several biological liquid samples, and the number of different particulate subsets provided in the kit is equal to (preferably at least equal to) the number of distinct biological liquid samples provided multiplied by the number of analytes to be detected, and the kit is provided with the same number (preferably at least equal to) classes of particulate subsets as the number of analytes to be detected; (or in some embodiments, the number of different particulate subsets provided in the kit may actually be greater than, and in any case not less than, the number of distinct biological liquid samples provided multiplied by the number of analytes to be detected; in embodiments of such kits, at least the same number of classes of particulate subsets as the number of analytes to be detected are provided, and in any case the number of classes of particulate subsets provided in such embodiments is not less than the number of analytes to be detected).

[0059] In a further embodiment, the present invention also relates to a cartridge for performing a method for detecting and / or quantifying a target analyte in a plurality of biological liquid samples, the method being as further defined above.

[0060] In one embodiment, the cartridge comprises multiple sample-specific modules, multiple storage chambers, at least one non-aqueous phase chamber for storing the non-aqueous phase, and an integrated single mixing and detection chamber or a combination of separate mixing chambers and separate detection chambers; Each sample-specific module includes a sample compartment having its own separate sample inlet, and each sample-specific module is configured to receive strictly one biological sample separately into its respective sample compartment; each sample-specific module is further configured to receive microparticles into the sample compartment, the microparticles being as further defined above; and each sample-specific module is further configured to facilitate the interphase transfer of the microparticles from an aqueous environment to a non-aqueous environment.

[0061] The term “sample-specific module” is meant to refer to a compartment within a cartridge that is explicitly designated to be used with a specific sample. Therefore, it typically includes a sample compartment and is configured to receive strictly one biological sample separately in each sample compartment. A “sample-specific module” may further include additional compartments and / or connection channels, each having its own specific sample inlet that allows for the addition of each sample to the sample-specific module. In one embodiment, the plurality of sample-specific modules each contain a plurality of porous microparticles in their respective sample compartments; each of the porous microparticles has a porous matrix and is configured to receive a predetermined volume of liquid into the porous matrix; each sample-specific module within its sample compartment contains a different subset of the plurality of microparticles, each microparticle subset characterized by a specific labeling component attached to, contained in, or otherwise associated with the respective subset; and / or the at least one non-aqueous phase chamber contains a non-aqueous phase, such as oil.

[0062] In one embodiment, each of the sample-specific modules further includes means for mechanically separating particulate matter from a liquid phase, preferably an aqueous liquid phase, such as a filter; the means are configured to facilitate interphase transfer of the particulate matter from an aqueous environment to a non-aqueous environment.

[0063] In one embodiment, the plurality of storage chambers include different groups of storage chambers, the first group of storage chambers containing reagents for performing chemical or biochemical detection reactions of the analyte, and separately, one or more further groups of storage chambers containing one or more of the following: lysis buffer, buffer for facilitating the binding of the analyte to an analyte-specific reagent, and washing buffer for washing fine particles.

[0064] In one embodiment, the cartridge includes a valve mechanism, which is configured to be attached to a pump, and which fluidly connects any of a) to c) to d) in succession or simultaneously; where a) is one or more of the storage chambers; b) is either an integrated single mixing and detection chamber or a combination of separate mixing chambers and separate detection chambers; c) is the at least one non-aqueous chamber; and d) is any of the sample-specific modules.

[0065] The inventors have provided a method for detecting and / or quantifying a target analyte in multiple biological liquid samples. While not wishing to be bound by any theory, the inventors believe that the novel method according to the present invention is based, in principle, on a novel assay comprising two fundamentally different parts: a sample-specific part and a general part (which may hereafter be referred to as the “specific process part” or “specific part” and “general process part,” respectively).

[0066] In the sample-specific portion of the above process, individual samples are coded, and if an analyte is present in an individual sample, the analyte is concentrated while unwanted material is removed from the sample. The coded and identifiable samples, then made identifiable by their individual codes / labels, are isolated as a type of aqueous droplet defined by fine particles in a non-aqueous environment. Such droplets act as reaction spaces for the detection of the analyte, if present.

[0067] Generally speaking, the above objectives are achieved by interrogating (and effectively “labeling” or “coding”) each sample using a specific type or subset of individually labeled microparticles, where “microparticles” may also be referred to herein as “beads,” and the specific type or subset of individually labeled microparticles has a defined specific label attached to, contained in, or otherwise associated with each microparticle. According to embodiments of the present invention, there may be multiple different types of microparticles (or “beads”) (such “types of microparticles” may also be referred to herein as “microparticle subsets”), where the microparticles are characterized by a specific label attached to, contained in, or otherwise associated with them. According to embodiments of the present invention, the microparticles can, by receiving a predetermined volume of liquid sample, draw up the analyte if it is present in the sample, bind to it as needed, and concentrate it. Binding of the analyte to the microparticles can be facilitated by exposing the microparticles to a sample suspected to contain the analyte of interest in the presence of a binding buffer. Such binding buffers are suitable for establishing conditions that promote the binding of analytes to the matrix of each microparticle and / or to any analyte-specific reagents (ASRs) attached to or contained within those microparticles.

[0068] Furthermore, according to embodiments of the present invention, each microparticle has an internal volume from which a sample can be drawn up, and reagents necessary to detect the analyte if it is present. The analyte in the sample is drawn up (or “absorbed”), and optionally, in the presence of a suitable binding buffer, is bound or concentrated (or “adsorbed”) by the microparticle, and after the respective washing steps as necessary, the microparticle is subsequently exposed to a suitable detection composition drawn up by the microparticle. As used herein, “detection composition” means a composition containing reagents for carrying out a chemical or biochemical detection reaction of an analyte. In a preferred embodiment, the “chemical or biochemical detection reaction” is a nucleic acid amplification or immunochemical detection reaction. Suitable “detection compositions” for carrying out such reactions are further outlined above. Once such a detection composition is drawn up by the microparticle, microparticles containing the biological liquid sample (or a portion of such a sample) and the suitable detection composition are obtained. Next, residual liquid located outside the microparticles but not contained within them is removed, for example, by filtration, centrifugation, shaking, or other mechanical stirring, or a combination thereof. Then, each microparticle is isolated by immersing it in a non-aqueous liquid. This non-aqueous liquid isolates each microparticle and prevents them from interacting with one another. Such a process is repeated for multiple samples, and each sample is interrogated by different specific microparticle subsets specifically labeled with a particular labeling component. The result of this process is distinct subset microparticle preparations, i.e., distinct subset preparations containing individual microparticles within each subset separated from one another, and each individual subset microparticle preparation is assigned its respective individual labeling component, so that such subset microparticle preparations can be clearly assigned to a particular sample.

[0069] At this stage, that is, after the microparticles have been isolated, preparations of different types of microparticles, i.e., different subsets corresponding to different samples, can be mixed and pooled together. Also at this stage, since the individual microparticles are isolated and no longer able to interact with each other, and have become individual aqueous droplets surrounded by a non-aqueous phase, preparations of different types of microparticles corresponding to different exposed samples can be pooled together and then subjected to the conditions necessary to carry out a detection reaction within the microparticles. Such a detection reaction could be, for example, nucleic acid amplification or an immunochemical reaction. Such a detection reaction corresponds to the general part of the method described above. Such a detection reaction is "general" because, insofar as such microparticles are interacted with the sample suspected to be the analyte, such a detection reaction is carried out for any microparticle, i.e., any type of microparticle. In other words, such a general part is carried out for all microparticles, regardless of the type of sample to which such microparticles are exposed, and preferably, it is carried out simultaneously as a single process.

[0070] The signals generated within each microparticle as a result of the detection reaction are assigned to the respective specific labeling components that are detected and attached to, contained in, or otherwise associated with each microparticle. Since it is known which labeling component corresponds to which sample, each generated signal can be effectively related to the respective sample, and as a result, it can be determined whether or not the target analyte is present in or was present in the sample being tested.

[0071] In effect, the aforementioned microparticles become individual reaction spaces or "reactors" in which trace amounts of each sample are confined and can be analyzed individually. Therefore, since a single molecule of the analyte can be distributed into a single microparticle, it is practically possible to carry out embodiments of the method according to the present invention in a manner that provides limiting dilution and allows the method according to the present invention to be carried out in a digital format. See also Sykes et al., 1992, Biotechniques 13(3): pp. 444-449. Such a form allows for exquisite sensitivity and good quantification of the analyte in each sample. The following are typical reagents used in embodiments of the present invention:

[0072] Firstly, there are the microparticles themselves, sometimes referred to herein as “beads.” These are highly porous, possessing a porous matrix and configured to receive a given volume of liquid within their porous matrix. Typically, they consist of a polymer or polymer mixture that provides mechanical stability and chemical integrity of the microparticles when a liquid sample is drawn up and contained within the porous matrix. Preferably, the matrix polymer may exhibit sol-gel characteristics, providing particles that are stable at room temperature and droplets at high temperatures. For example, when the microparticles are in a gel state, i.e., a gel phase or semi-solid phase, they form a suspension in the liquid phase. However, if the microparticles are then converted to a sol state, i.e., a soluble or semi-liquid state, they form an emulsion in the liquid phase, and the suspension changes to an emulsion. Embodiments in which the polymer or polymer mixture is not crosslinked are particularly good at switching between different states, for example, between a gel state and a sol state, and are versatile.

[0073] 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.

[0074] In a preferred embodiment, the microparticles according to the present invention are not only capable of aspirating such samples along with any analytes present in a liquid sample and accommodating them within a porous matrix, but also actually include binding members (e.g., analyte-specific binding molecules attached to the porous polymer matrix, e.g., analyte-specific reagents (ASRs); one or more ionizable groups immobilized on the porous polymer matrix, wherein the ionizable groups can change charge according to ambient conditions around the precursor microparticles; one or more charged groups immobilized on the porous polymer matrix; or any combination thereof) that facilitate or provide the necessary functions to bind to or concentrate the target analytes or analyte classes (e.g., nucleic acids) present in the sample, thereby increasing the local concentration of such analytes within the microparticles.

[0075] Furthermore, in a preferred embodiment of the present invention, the microparticles are further characterized by specific labels attached to, contained in, or otherwise associated with each (type) of microparticle.

[0076] Microparticles such as those disclosed in the EP application titled "A library of prefabricated microparticles and precursors thereof" (agent reference number B33016EP), filed concurrently by the present applicant Blink AG, have proven particularly suitable for embodiments of the present invention, especially those in which more than one analyte is detected in each sample.

[0077] In some embodiments, a lysis buffer may be necessary to release the analyte of interest from the sample or to make such analyte of interest available within the sample. Such a lysis buffer may contain an active agent that can cause the lysis of cells, particularly cell membranes, or organelles. As a result of such lysis, the analyte of interest may become accessible. In some embodiments, the lysis buffer may, alone or in combination with further buffers, promote the concentration or binding of the analyte of interest in particulate matter. Such further buffers that promote the concentration or binding of the analyte of interest in particulate matter may also be referred to herein as “binding buffers.” When such “binding buffers” are used to adjust the concentration of an analyte or other component, such “binding buffers” may also be referred to herein as “dilution buffers.”

[0078] As used herein, the terms “analyte” or “analyte” may also be referred to as “target” in this specification, particularly when nucleic acids are being analyzed.

[0079] In some embodiments, a washing buffer may also be required to remove unwanted components from the microparticles that could otherwise inhibit or prevent the subsequent detection reaction from functioning properly. For example, if the detection reaction is an immunochemical reaction, in some embodiments the microparticles may contain an analyte-specific reagent, which is a primary antibody specific to the analyte of interest. In such embodiments, the microparticles containing the primary antibody as the analyte-specific reagent are exposed to a sample suspected to contain the analyte of interest, and then the microparticles are exposed to a first detection composition containing a secondary antibody specific to the analyte of interest, which is conjugated to the primary antibody and is itself a suitable reporter, such as an enzyme. Once the microparticles have been exposed to the sample and the first detection composition, a washing step may be required to remove any unbound secondary antibodies that could interfere with the subsequent detection reaction. Thus, embodiments of the present invention assume the use of a washing step involving a washing buffer to remove unwanted components from the microparticles that could subsequently inhibit or prevent the proper detection reaction from occurring.

[0080] In a further step in one embodiment, the microparticles are exposed to a second detection composition containing a substrate suitable for a reporter enzyme attached to a secondary antibody as a detection reagent. When this substrate is reacted with the reporter enzyme, it becomes detectable, preferably optically detectable, and in particular fluorescently detectable.

[0081] In other embodiments of the present invention, the detection reaction may be a nucleic acid amplification reaction. In such embodiments, the microparticles may include oligonucleotide primers and probes for target-specific amplification and detection. Such analyte-specific reagents may already be provided reversibly bound to the microparticles or may be added together with other components of the general detection composition. Typically, such a general detection composition includes the reagents necessary to carry out the amplification reaction of the nucleic acid analyte of interest, and may or may not include primers and, optionally, suitable detection probes, depending on whether the primers and / or probes are already part of the microparticles. If such analyte-specific reagents are already part of the microparticles, they are not included in the general detection composition. However, such a general detection composition separately includes reagents other than primers necessary to carry out the amplification reaction of the nucleic acid analyte of interest, and in particular, the general detection composition includes a suitable buffer, a mononucleotide, an amplification enzyme (e.g., a suitable nucleic acid polymerase, e.g., Taq polymerase), and (optionally) a nucleic acid dye for detecting the amplification product (e.g., amplified nucleic acid).

[0082] According to the present invention, the above method also requires the transfer of the microparticles to a non-aqueous phase, which is used to isolate the individual microparticles and thereby effectively seal and confine their contents within the volume of the microparticles. Such a non-aqueous phase is typically a lipophilic compound, e.g., a lipophilic oil, e.g., a fluorocarbon oil, which preferably contains one or more suitable emulsifiers. Such a non-aqueous phase is required when the microparticles are transferred to the non-aqueous phase, which helps to replace any aqueous liquid that was not absorbed by the microparticles and to isolate such microparticles from one another, so that liquid exchange between different microparticles is no longer possible. As a result, after a stable microparticle suspension has been formed, there is no carryover of any sample from one microparticle to another, which could result in mixing of two different samples.

[0083] Furthermore, embodiments of the method according to the present invention enable 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 for microparticles, and PCR amplification can serve as an example illustrating the approach to quantifying targets by the present inventors' method. 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 digital PCR analysis 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.

[0084] The selection of the applicable quantification approach is based on the number / ratio of negative particles remaining after amplification. This information is obtained from digital PCR data. If the number / ratio of negative particles exceeds a predefined lower limit, endpoint Poisson analysis can be applied. Otherwise, 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 particles that enables robust quantification with Poisson is 0.5%. The average number of targets per corresponding particle is 5.3. To account for artifacts that may be present in fluorescence images, a further requirement may be a lower limit for the total number of negative particles, e.g., 50.

[0085] 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.

[0086] 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 individual 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. The offset term of the calibration curve may be a function of microparticle volume to explain the effect of variability in microparticle volume.

[0087] After performing a signal amplification reaction or a target amplification reaction in the reaction space created by the microparticles, by applying a simple rule based on the number of microparticles that remain negative (i.e., "dark"), it is possible to establish which approach is most suitable for obtaining quantitative results over a wide measurement range. The rule can be formulated as follows: a. In a given detection / quantification measurement using the library according to the present invention, when more than 0.5% of the microparticles specific to a given analyte remain negative (i.e., the probability of "dark" microparticles ("beads") ("PdarkBead" or "Pnegative") is >0.5%), Poisson analysis can be applied to measure the concentration of the target analyte in the sample. b. In a given detection / quantification measurement using the library according to the present invention, when less than 5% of the microparticles specific to a given analyte remain negative (i.e., the probability of "dark" microparticles ("beads") ("PdarkBead" or "Pnegative") is <5%), a real-time analysis algorithm can be applied to measure the concentration of the target analyte in the sample. c. In a given detection / quantification measurement using the library according to the present invention, when "negative" microparticles occur within the range of 0.5% < Pnegative < 5%, both approaches can be applied interchangeably.

[0088] This principle of quantification will also be illustrated in Figures 14 and 15 and Example 2.

[0089] Furthermore, reference is made to the drawings provided for purposes of illustration rather than limitation of the present invention. More specifically,

Brief Description of the Drawings

[0090] [Figure 1-1]Figure 1A shows an embodiment of the basic scheme of an assay / method according to an embodiment of the present invention, which shows that the workflow of such a method preferably includes a sample-specific portion (referred to in this drawing as the “specific process portion”) which is performed for each different sample being tested, and a general process portion which is common to all microparticles regardless of the sample being exposed. The rectangle above represents an embodiment of the basic process of the sample-specific portion of this process and any possible components present. The sample is dissolved using a lysis buffer. As a result, the analyte of interest, which may also be referred to herein as the “target,” is released. If necessary, a suitable dilution buffer (which may also be referred to herein as the “binding buffer”) may be used to adjust the concentration of a particular reagent in the lysis buffer to an acceptable range, and then the sample containing the analyte of interest is subjected to conditions under which the sample containing the analyte of interest can be absorbed or adsorbed by microparticles having a particular label assigned to each sample. Such labeled microparticles may also be referred to herein as “encoded microparticles” or “encoded beads.” Such “encoded microparticles” exposed to one sample may also be referred to herein as a “microparticle subset.” Each such “subset” is characterized by a specific labeling component attached to, contained within, or otherwise associated with that subset. In some embodiments, each analyte may be bound to the microparticles by affinity binding to the matrix forming the particles, or by affinity binding to an analyte-specific reagent (ASR) attached to or contained within the particles for this purpose. Once the analyte of interest is incorporated into / contained within the microparticles, each microparticle subset is exposed to a suitable detection composition. The nature and content of the detection composition depend on the detection reaction performed using the analyte of interest (and the detection reaction depends on the type of analyte being detected). Such a detection reaction includes the reagents necessary to perform the detection reaction of the analyte of interest. Preferred examples of such detection compositions are further defined above.Next, once each detection composition has been absorbed into the microparticles, a step may follow to remove the aqueous phase around each microparticle. Such a step may include filtration, centrifugation, shaking or other mechanical stirring, and combinations thereof, performed using each microparticle. Subsequently, preferably after removing the aqueous phase around each microparticle, each microparticle is transferred to a non-aqueous phase, such as oil, which may contain an emulsifier as needed. In a preferred embodiment, the emulsifier helps to isolate the individual microparticles. As a result of such transfer to the non-aqueous phase, a suspension of isolated microparticles is obtained, which may contain the analyte of interest derived from this particular sample. The same process is repeated with different types of microparticles ("subsets") that are differently coded, i.e., have different labeling components attached, contained, or otherwise associated. Such differently coded microparticles are exposed to different samples. Once this process has been performed for a required or desired number of different samples, a first record of correlations showing which distinct microparticle subsets were exposed to which samples may be generated. Such a first record of correlations may also be referred to herein as a "sample / bead code list". Once the process of exposing different particulate subsets to different samples has been carried out for a desired or required number of different samples, each isolated and distinct particulate subset can be mixed and subjected in a general part of the process to a detection reaction that may involve nucleic acid amplification, or to an immunochemical reaction, or to any other biochemical reaction suitable for the detection of the analyte of interest. Depending on the presence or absence of the analyte in each sample (and particulate subset), a signal may be generated in such a detection reaction for a particular particulate subset. In this way, a list of which beads produced a signal ("beads / signal code list" or "second record of correlation") may be generated, indicating which particulate subsets produced a signal in the detection reaction.Subsequently, by decoding each signal generated in such a detection reaction (for example, by aligning or comparing a second correlation record (or "beads / signal code list") with a first correlation record (or "sample / beads code list")), it may be found that the analyte of interest is present (or not present) in one or more of the samples originally used to fill / load each microparticle. Preferably, the determination of which sample contains the analyte of interest, i.e., the actual assignment of the detected signal, is made by linking a first correlation record (i.e., a list showing which subsets were exposed to which samples) with a second correlation record (i.e., a list showing which subsets generated the signal).

[0091] It should be noted that the main advantage of the method according to the present invention is as follows: When samples are tested individually, the detection reaction must be performed individually and separately for each sample. In the present invention, it is possible to perform the same and common part of the method ("general part") that can be performed individually and separately for all detection reactions that can be performed individually and separately for each sample, at once for all samples being analyzed.

[0092] [Figure 1-2] Figure 1B illustrates an embodiment of the basic scheme of an assay / method according to an embodiment of the present invention, showing that the workflow of such a method can also be advantageously applied to processing a single sample. The beads used provide a lean process that utilizes porous material for target enrichment, cleanup, and signal generation reactions, such as PCR, to be carried out in individual nanoreactors, providing precise quantification of targets present in the sample over a wide measurement range.

[0093] [Figure 2]Figure 2 shows an exemplary embodiment of microparticles according to the present invention, comprising a polymer having a porous matrix, which in this particular case can undergo a phase transition by an external trigger. For example, in a preferred embodiment, microparticles having a porous matrix are composed of a porous polymer matrix, and the polymer constituting such a porous polymer matrix can undergo a sol-gel transition, for example, when the temperature is increased. Once each microparticle is isolated in a non-aqueous phase, an aqueous droplet on which a detection reaction can be carried out can be effectively formed by raising the temperature to a level at which the microparticle undergoes such a phase transition. According to this embodiment shown in Figure 2, the microparticles also have a high binding affinity to a specific target analyte. This may be a feature of the polymer matrix itself, or it may be achieved, for example, by an analyte-specific binding molecule (binder) reagent attached to the matrix. Such a binder may, in some cases, be, for example, an analyte-specific reagent (ASR) as further defined above. Analyte-specific binders can be selected from nucleic acid pairs, particularly nucleic acid including primer pairs; aptamers, 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. In preferred embodiments, the analyte-specific reagent is selected from nucleic acids, particularly nucleic acid oligomers and nucleic acid primers. Furthermore, the microparticles have a labeled component attached to the microparticles, contained within the microparticles, or otherwise associated with the microparticles.

[0094] Furthermore, such microparticles may (optionally) contain reversibly attached reagents for a signal generation detection reaction to be carried out within the space provided to the microparticles. Note that, according to embodiments of the present invention, a primer pair suitable for amplifying a particular nucleic acid sequence (which is the “analyte” or “target”) may qualify as a single analyte-specific reagent (ASR) as herein, despite the fact that such a primer pair comprises two different primers. However, they are specific to a single nucleic acid sequence in that they are specific to the same nucleic acid analyte because they are adjacent to the same region of such nucleic acid sequence.

[0095] In a preferred embodiment, the labeling component is a fluorescent dye, more preferably a mixture of two different fluorescent dyes.

[0096] The microparticles according to embodiments of the present invention have the ability to contain, preferably bind to or concentrate, an analyte of interest in their porous matrix, and can be identified and distinguished from other microparticles by their respective labeling components attached to, contained therein, or otherwise associated with them. Because the microparticles according to the present invention are porous, they provide an internal volume sufficient and accessible to draw up (or "absorb") a sample containing any analyte of interest, or to bind ("adsorb") the analyte, if present, to the matrix of the microparticles, by, for example, an analyte-specific binding molecule attached to the porous polymer matrix; an ionizable group or a plurality of ionizable groups immobilized on the porous polymer matrix, wherein the ionizable group can change its charge according to the ambient conditions around the precursor microparticle; one or more charged groups immobilized on the porous polymer matrix; or any combination thereof.

[0097] Preferably, such fine particles can also undergo a phase transition when an external trigger is applied, for example, by changing from a gel state at room temperature to a soluble state at high temperatures when the temperature is raised above a certain threshold. Thus, when these particles are simultaneously present in a liquid, a suspension at room temperature will change to an emulsion at high temperatures.

[0098] A preferred polymer that functions in this way is an agarose polymer, either alone or in combination with gelatin.

[0099] [Figure 3]Figure 3 illustrates the basic function of microparticles ("reactor beads") according to an embodiment of the present invention. This figure shows three different types of microparticles, each distinguished from the others by a labeling component associated with that respective type. In the depiction of Figure 3, the different labeling components are symbolically represented by differently shaded regions within the circular depiction of the microparticles / beads; such differently shaded regions may represent any suitable labeling component, insofar as it is possible to distinguish the different microparticles. For example, they may represent different concentrations of a single dye, different ratios of two different dyes, different detectable physical labels, and so on. In the first part of this process, which is the "sample-specific part" of the process described above, the three different types of microparticles are separately exposed to three different samples 1-3, effectively encoding each sample by the labeling component of each type of microparticle ("subset") exposed to such sample. In a preferred embodiment, such correlations between labeling components and each sample associated with them are obtained by creating a list of labeling components (of the corresponding microparticle subset) including the corresponding associated samples, which may also be referred to herein as the "first record of correlations." Such lists may also be referred to herein as “sample / microparticle code lists” or “sample / bead code lists,” and they indicate which microparticle subsets were exposed to which samples. In this embodiment, after a series of process steps, detection reagents are loaded onto microparticles, and the microparticles are collected in a non-aqueous environment to isolate the microparticles and prevent crosstalk between them. In a second part of the process, which is the “general part” of the process described above, different samples are coded, and the corresponding different microparticle suspensions are mixed in a non-aqueous phase and subjected simultaneously (“at once,” i.e., no longer separately from one another) to the conditions necessary to perform the required detection reaction, e.g., nucleic acid amplification such as PCR, including temperature cycling. Since such a “general part” can process a large number of samples, this makes it easy to perform a single detection process using samples from an entire large patient cohort, for example, in a clinical trial.The signals generated in such a detection reaction are recorded, and the labeled components of each particulate (subset) are measured / read out. A second record of correlation is generated, showing which particulate subsets generated the signal. Such a second record of correlation may also be referred to herein as the “beads / signal code list.” The resulting “beads / signal code list” (i.e., the second record of correlation) is compared with the “sample / particulate code list” (i.e., the first record of correlation) to determine which of the samples (of which multiple samples were tested) contained or did not contain the respective analytes of interest.

[0100] Fine particles particularly suitable for embodiments of the present invention are fine particles that are included in a library of prefabricated microparticles for specific detection of an analyte in a sample, as described in a concurrently pending European patent application (agent reference number B33016EP) titled "a library of prefabricated microparticles," filed concurrently with this specification.

[0101] [Figure 4]Figure 4 shows a schematic diagram of an exemplary arrangement of a novel fluid cartridge that may be suitable for carrying out embodiments of the method according to the present invention. A unique feature of this cartridge is the presence of multiple separate inlets for individual samples to be analyzed. Each inlet is separately connected to its own corresponding sample chamber or sample-specific module, and several different sample chambers or sample-specific modules are provided in the cartridge, each of which is intended and configured for use with a distinct sample. The terms “sample chamber,” “sample chamber module,” and “sample-specific module” refer herein to a chamber that is interchangeable and explicitly intended and configured to receive a particular sample. The actual space within each sample-specific module configured to contain a sample (and a particular subset of particulate matter for this particular sample) may also be referred herein to as a “sample compartment.” These particular chamber modules are provided to ensure a secure coding process, i.e., a process in which each sample is coded / labeled by exposing such sample to a particular distinct type ("subset") of particulate matter, thereby “loading” a different sample into each of the different subsets of particulate matter. Different sample-specific chamber modules prevent cross-contamination between different modules and therefore between different samples. Each separate entrance is indicated in this diagram by a triangular indentation on the shorter side of the sample chamber (or sample-specific module), which is represented by a rectangle.

[0102] The exemplary cartridge arrangement in this figure includes exemplary valve and pump mechanisms. Three separate groups of chambers (or modules) are provided for the cartridge arrangement: 1. Facilitates sample-specific processing and includes sample-encoding microparticles among other components, modular 2. Chambers for storing reagents required for specific processes (if not provided within the sample-specific module) and general processes; a specially designated storage chamber for the non-aqueous phase ("non-aqueous phase chamber") is also indicated. 3. Chambers or modules to facilitate general processes (e.g., mixing (optional) and detection (mandatory) chambers, or integrated mixing and detection chambers, i.e., chambers combining both functions (not shown))

[0103] Before the coding process is completed and before the microparticles are isolated, i.e., before they are secured in the non-aqueous phase, all reagents are delivered unidirectionally to each module. Finally, suspensions of microparticles are collected from each sample module and then mixed together to form a single suspension. The thus mixed microparticles are then transferred to a detection chamber and subjected to the necessary conditions for carrying out the detection reaction. A cartridge that may be used when adapted and modified according to embodiments of the present invention is a cartridge such as that described in European Patent Application EP No. 19187064.1 filed on 18 July 2019, however, it needs to be adapted insofar as it requires modification in that the cartridge according to the present invention has multiple sample chambers, each of which has its own sample inlet. This makes it possible to handle multiple samples separately, each of which is labeled separately according to the present invention.

[0104] [Figure 5]Figure 5 shows an embodiment of the method according to the present invention. More specifically, different microparticle subsets (referred to in this figure as “Bead 1,” “Bead 2,” “Bead 3,” and “Bead 4,” respectively) are attached with different labeling components, but these subsets either contain no analyte-specific reagents at all, or contain strictly one type of analyte-specific reagent specific to one particular target analyte. These different microparticle subsets are distinguishable by their different labeling components and are supplied separately, for example, to different containers, e.g., different sample chambers or sample-specific modules in cartridges, as further described above, and each of them is exposed separately to a different sample. The same number of microparticle subsets as the sample to be tested are effectively used. The necessary (general) detection reagents / reagent compositions are then added to each subset (if such analyte-specific reagents were not present in the microparticles from the start, they are added along with such detection compositions, which may further include analyte-specific reagents as further defined above, as needed). Next, each microparticle subset is transferred separately to a non-aqueous phase (which may be an oil containing an emulsifier, if necessary), thereby isolating them from one another, and as a result, "crosstalk," i.e., mixing of the samples between the spaces provided by each microparticle, no longer occurs, after which a (general) detection reaction is performed ("one-pot detection reaction"). From the results of such a (general) detection reaction, it is possible to conclude which samples contained a single analyte of interest. Surprisingly, the inventors found that after the different microparticles were isolated from one another by being transferred to the non-aqueous phase, spillage or "crosstalk" between those different microparticles did not actually occur. As a result, despite the fact that each microparticle was mixed, there was also no cross-contamination between the samples (tested for the presence of the analyte).

[0105] [Figure 6]Figure 6 illustrates an embodiment of the method according to the present invention in which multiple samples are tested for multiple analytes. Essentially, this scheme is similar to that shown in Figure 5, except that it uses the same number of microparticle subsets as the number of samples tested multiplied by the number of different analytes to be detected. For example, the first sample in Figure 6 is tested / analyzed using two different microparticle subsets that differ not only in terms of the associated labeling component but also in terms of the analyte-specific reagent attached to such a type. The same applies to the second sample ("Sample 2"). Thus, in this example, there are a total of four different microparticle subsets, each of which has a unique combination of labeling component and analyte-specific reagent. The necessary (general) detection reagent / detection composition is then added to each sample individually, still to avoid cross-contamination between different samples. Each microparticle is then again transferred to a non-aqueous phase, one sample at a time, isolating each of these microparticles so that they cannot interfere with or cross-contaminate each other, before the necessary detection reaction is performed. The final result may be the finding that each sample tested contains no analytes to be detected, or one or two. As noted above, surprisingly, the inventors found that once the different microparticles were separated from each other by being transferred to the non-aqueous phase, no overflow or "crosstalk" between those different microparticles actually occurred. Consequently, despite the fact that each microparticle (each containing a small volume of the sample being tested) became mixed, no cross-contamination occurred between the samples (tested for the presence of one or more analytes).

[0106] [Figure 7] Figure 7 shows a micrograph of a microfluidic cross-junction setup used to produce microparticles according to the present invention. Microdroplets are formed in oil to form an aqueous hydrogel solution (left side). Subsequently, the droplets are cooled to form microparticles.

[0107] [Figure 8] Figure 8 shows fluorescence micrographs acquired using a Cy3 Filter Set on a Zeiss Axioscope equipped with a digital camera. Three different fluorescence levels are observed (bright, moderate, and low), which effectively correspond to three different concentrations of the labeling component (Cy3 dye), corresponding to three different labeling components.

[0108] [Figure 9] Figure 9 shows a stitched image of the detection chamber corresponding to the fluorescence channel encoding the bead label. In the magnified insert in the lower right, three different types of microparticles (or "beads"), identifiable (and therefore encoded) by their respective labeling components, are clearly recognizable (bright, moderate, and low signals). Because of the different labeling components, these beads can be used to label or "encode" different samples (hence, these beads may also be referred to herein as "sample-encoding beads"). These beads are spatially distributed in a hexagonal close-packed arrangement. The total number of sample-encoding beads (average diameter d=10⁵ μm) in this PCR chamber, as recognized by the software algorithm used, was found to be N=17.698.

[0109] [Figure 10]Figure 10A shows fluorescence images of color-labeled agarose-gelatin hybrid microparticles after PCR amplification. Three labels can be identified by their fluorescence intensity in channel 1. Channel 2 represents the fluorescence signal for the internal process control (MS2 phage), and channel 3 represents fluorescence specific to PCR amplification of the HCV target. Each label corresponds to a different sample that was processed and analyzed. Figure 10B (top graph) shows histograms of fluorescence intensities measured for different beads from channel 1 (top image in Figure 10A). Three distinct label species are visible. Violin plots showing the signal distribution observed across each bead species for channel 2 (MS2) and channel (3) are shown in the middle and bottom graphs. By applying Poisson analysis, the measured values ​​are converted to specific copy numbers per sample volume applied to the beads.

[0110] [Figure 11] Figure 11 summarizes the data from Example 3 regarding the binding efficiency of different microparticle formulations to RNA. This data indicates that the binding characteristics can be optimized by adjusting the composition of the microparticles.

[0111] [Figure 12] Figure 12 shows the time course of fluorescence images illustrating RNA binding to the prepared microparticles in Example 3.

[0112] [Figure 13] Figure 13 shows the concentration effect achieved by using microparticles. This graph shows data for microparticles of two different compositions. Black dots indicate the target concentration in the sample before incubation with the microparticles, as measured by digital RT-PCR. White bars represent the target concentration in the supernatant detected after incubation, and gray bars represent the target concentration on the microparticles. The clear concentration effect indicates that each supernatant was depleted.

[0113] [Figure 14] Figure 14 shows the accuracy of an analytical approach combining digital PCR and real-time quantitative PCR for quantifying targets in microparticles. Confidence intervals for both methods are shown. The vertical lines at λ = 5cp / microparticle (cp = copy) indicate approximations where the methods can be used interchangeably ("CI" = confidence interval).

[0114] [Figure 15-1] Figure 15 shows real-time fluorescence data obtained from a series of images collected on microparticles in oil during PCR amplification. The left side shows a stitched image of the detection chamber containing the endpoint fluorescence signal detected in a single fluorescence channel specific to amplification. The graph in the center exemplifies the fluorescence intensity over time 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 each microparticle detected in the fluorescence images. [Figure 15-2] Figure 15 shows real-time fluorescence data obtained from a series of images collected on microparticles in oil during PCR amplification. The left side shows a stitched image of the detection chamber containing the endpoint fluorescence signal detected in a single fluorescence channel specific to amplification. The graph in the center exemplifies the fluorescence intensity over time 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 each microparticle detected in the fluorescence images.

[0115] Furthermore, specific details, particularly the following embodiments, are provided not to limit the invention but to illustrate it. [Examples]

[0116] Example 1: Preparation of "sample-coding beads" for RNA detection assays Different labeled microparticles having a matrix containing gelatin and agarose were prepared, thereby allowing the gelatin fraction of the composition to function as a dye transporter in the matrix, resulting in target binding and concentration thanks to the composition. The following was performed to label gelatin with a fluorescent dye to create a particle code:

[0117] The acetone-insoluble fraction of gelatin derived from bovine skin type A is labeled with a fluorescent dye using NHS coupling chemistry. Cy(registered trademark)3 Mono NHS Ester (GE Healthcare) is dissolved in DMF to produce a 10% (w / v) final solution. Component 1 is dissolved in 70 mM sodium phosphate buffer (pH 8.0-8.3, filter-sterilized) to obtain a final concentration of 0.25% (w / v). 25 mL of any of the gelatin types is labeled using each dye with a molecular weight 10 times smaller than the free gelatin amino group. 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 and concentrated by repeated ammonium sulfate salting-out using a saturated (NH4)2SO4 solution. Alternatively, the gelatin can be in the form of gelled particle size for coupling and can be purified without ammonium sulfate salting-out by simply washing and centrifugation at ambient temperature. Gelatin can also be purified by ultrafiltration, solvent extraction using isopropanol, acetone, or methanol, gel filtration using a Sepharose column, or dialysis.

[0118] In either case, the purification is repeated until the eluate becomes clear and no longer fluoresces. Finally, after washing the pellet four times with dH2O, the purified fluorescently labeled gelatin sample is vacuum-dried to obtain component 3. Preparation of gelatin / agarose hybrid solution:

[0119] A hybrid hydrogel solution consisting of three components is prepared to fabricate a nanoreactor. Ingredient 1: Acetone-insoluble gelatin derived from bovine skin type A G1890 (Sigma). Ingredient 2: Low-gelling 2-hydroxyethyl agarose (A4018, Sigma) Ingredient 3: Cy3-labeled gelatin (Type A)

[0120] 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 labeled gelatin, dissolve the dry pellet of component 3 in each volume of nuclease-free water and incubate at 55°C until the gelatin melts. Mix all three components and fill with nuclease-free water to produce hybrid hydrogel solutions with final concentrations of 1.5% (w / v) gelatin and 0.5% (w / v) agarose A4018, respectively. Mix various volumes of component 3 and component 1 to obtain n sets of microparticles of different colors. In this embodiment, the resuspended components 3 and 1 were mixed in ratios of 1:1000, 1:500, and 1:250 to obtain three individual labeling components that enable the identification and analysis of three different samples. All solutions were maintained at 55°C until further use. Production of non-crosslinked gelatin / agarose microparticles

[0121] Color-coded monodisperse agarose-gelatin hybrid nanoparticles were fabricated using a microfluidic particle generation system (Dolomite, UK). The monodisperse hybrid nanoparticles were fabricated in a single-step emulsion formation process using a simple flow focus device. Specifically, a standard droplet junction tip (100 μm) with fluorophilic properties was used, along 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 delivered the hydrogel solution and carrier oil. This system was modified by incorporating 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, ensuring a constant temperature when the oil and gelatin / agarose hybrid solution contact at the tip junction. Both Picosurf 2 (Spherefluidics, UK) 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, respectively, within the heating device of the droplet system. The heating device temperature is set to 55°C. The fluid line is prepared for 1 minute at 2000 mbar using Flow Control Software. The flow rate is adjusted to 15-20 μl / min for stable droplet formation. Parameters are monitored using Dolomite Flow Control Advanced Software.

[0122] Figure 7 shows a micrograph of a microfluidic cross-junction containing microdroplets forming in the oil phase on the left. Subsequently, the hydrogel droplets solidify into fine particles.

[0123] 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 cooled to 4°C for at least 24 hours (preferably 48 hours) to form a stable hybrid framework. Recovery of hybrid microparticles from the continuous phase

[0124] The solidified hybrid beads 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 emulsion. 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.

[0125] Optional: This procedure can be repeated to remove residual fluorocarbon oil and surfactants. After washing the PFO, the recovered microparticles are washed twice with 1 mL of nuclease-free water. The quality and size of the microparticles are visually inspected using a microscope. The final result of the procedure described provides a set of nanoreactor microparticles (diameter d = 100 μm) having a porous polymer matrix and capable of encoding the sample.

[0126] Figure 8 shows fluorescence micrographs acquired using a Cy3 Filter Set on a Zeiss Axioscope equipped with a digital camera. Three different fluorescence levels (bright, moderate, and low) corresponding to three concentrations of the dye (Cy3 dye) are observed, making it possible to distinguish between each of the microparticles labeled with them by, in effect, three different labeling components. Freeze-drying of a set of fine particles

[0127] If necessary, the microsphere set can be freeze-dried to obtain microparticle pellets for long-term storage. Therefore, by supplementing the desired microsphere set and concentrate with an equal volume of 600 mg / mL trehalose solution, a microparticle library slurry containing 30% (w / v) trehalose is obtained. Subsequently, 100 μl library aliquots are prepared in RNase / DNase-free PCR strip tubes ready for freeze-drying. The type of excipient (e.g., trehalose) and its concentration in the freeze-dried formulation affect the degree of expansion of the freeze-dried microspheres when exposed to the eluate later in the process. The microsphere set was frozen on dry ice for 2 hours, and 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 stage involved gradually increasing the temperature from -25°C to 25°C while maintaining a pressure of 0.01 mbar for 3 hours. The final drying process was performed at 25°C and 0.05 mbar for 20 minutes. Example 2: Encoding of a sample using "sample coding beads" Sample preparation using hybrid beads Preparation of gel filtration resin

[0128] Dried P-2 Bio-Gel Media Extra fine <45 μm (BIO-RAD 150-4118) is hydrated with nuclease-free deionized H2O. Subsequently, the gel is placed on an empty spin column (i.e., SigmaPrep). TM Pack a spin column (Sigma-Aldrich SC1000-KT) to obtain a final bed volume of 400 μl. Remove the mobile phase by centrifugation at 1000 rcf for 1 minute. After adding 400 μl of 50 mM Tris HCl buffer pH 8.4, wash the column twice with the buffer by rotating it as described above. Dissolution of the sample

[0129] Three samples of 30 μl whole blood obtained from a healthy donor (Institute of Transfusion Medicine, University Hospital Jena) were combined with 300 cp / μL of MS2 virus (Samples 1, 2, and 3). Then, one of these three samples (Sample 1) was combined with AccuPlex HCV recombinant Sindbisvirus (SeraCare 0505-0036) diluted with Basematrix Negative Diluent (SeraCare 1805-0075), resulting in a nominal titer of 5,500 cp / μL.

[0130] All samples were separately mixed with 195 μl of lysis buffer containing 4.5 M guanidinium HCl, 9% Triton X100, 18 mM EDTA, and 100 mM Tris HCl pH 8.0. Each lysis mixture was incubated at 65°C for 5 minutes. Gel filtration for removing guanidinium HCl

[0131] Immediately after dissolution, each mixture was applied to a P-2 column. After centrifugation at 1,000 rcf for 1 minute, the flow-through / filtrate was collected in a new reaction tube containing 200 U of the RNase inhibitor (biotechrabbit, DE). Conjugation of analytes to sample coded beads

[0132] Assign the samples to fluorescently labeled microparticles as shown in the table below. Assigning samples to sample coding beads [Table 1]

[0133] The lyophilized particle pellet is resuspended in 70 μL of RNA-containing binding buffer (component 4). Each set consists of approximately 10,000 nanoreactors available for digital PCR analysis. By allowing these to absorb the buffer and swell immediately, a functional porous 3D gelatin-agarose matrix developed for efficient nonspecific binding of nucleic acids and digital PCR compatibility is provided. Nucleic acids are enriched in the nanoreactors by completely capturing HCV and MS2 RNA by incubating individual samples with the corresponding beads in separate tubes at 15°C for 5 minutes at 1000 rpm. Cleaning of fine particles

[0134] 200 μl of washing buffer (50 mM Tris HCl pH 8.4, 1 U / μl RNase inhibitor) was added to each sample. After a short 1-second vortexing cycle at 12,000–16,000 rpm, the microparticles were settled by centrifugation at 300 rcf for 30 seconds, and the supernatant was removed. This washing process was repeated three times. 1. Detection and quantification of molecular targets in different samples using "sample-coding beads". Loading reagents onto microparticles

[0135] The reagents for detecting the analytes in the nanoreactor are supplied as freeze-dried pellets, which are resuspended in 100 μL of 1× PCR buffer (component 7) to obtain a 2× reagent mixture (component 8). These reagents are carefully resuspended by a short vortex step. Equivolutes of the reagent mixture are distributed to individual containers containing sets of individual sample-coding beads that have captured RNA. The amplification and detection reagents are diffused into (and bound to) the hydrogel matrix by a brief incubation at 15°C for 5 minutes with shaking at 1000 rpm. Component 7: PCR buffer (20mM Tris HCl, 22mM KCl, 22mM NH4Cl, 3mM MgCl2, pH8.5) Component 8: Reagent mixture (2×) - 2× thermostable reverse transcriptase containing a ribonuclease inhibitor (biotechrabbit GmbH) -0.4U / μl Hot Start Taq DNA Polymerase (biotechrabbit GmbH) -0.8mM dNTP (biotechrabbit GmbH) -0.2% (w / v) low bioburden, protease-free BSA (Sigma) for molecular biology. Analyte-specific reagent (HCV) -0.8 μM HCV-Brun sense primer (5'-GTGGTCTGCGGAACCGGTGA-3') SEQ ID NO: 1 -0.8 μM HCV-Brun antisense primer (5'-CGCAAGCACCCTATCAGGCAGT-3') SEQ ID NO: 2 -0.8μM HCV-Brun TaqMan probe (5'-Atto647-CCGAGTAGYGTTGGGTYGCGAAAGG-BHQ-2-3') SEQ ID NO: 3 Internal process control specific reagent (MS2) -0.8 μM MS2-Nino sense primer (5'-CTCTGAGAGCGGCTCTATTGGT-3') SEQ ID NO: 4 -0.8 μM MS2-Nino sense primer (5'-GGTCCCTACAACGAGCCTAAATTC-3') SEQ ID NO: 5 -0.8μM MS2-Nino TaqMan probe (5'-FAM-TCAGACACGCGGTCCGCTATAACGA-BHQ-1-3') SEQ ID NO: 6 Emulsification of fine particles

[0136] To prevent crosstalk between sample-encoded beads, individual nanoreactor sets are transferred separately to the non-aqueous phase by dispersing the microparticles in component 9. The beads are centrifuged at 500 rcf for 30 seconds, and the supernatant is discarded. The bead bed is brought into contact with an excess volume of component 9 (200 μL) using a 1.5 mL microcentrifuge tube. High shear force is required 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 tube rack 20 times at a frequency of approximately 20 / 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 / emulsion. 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 emulsion three times by gentle centrifugation (500 rcf). All undesirable liquid droplets are essentially removed by repeated washing with the same oil (component 12). Component 12 also provides efficient thermal stability of the emulsion for subsequent digital emulsion PCR. Here, three emulsified sample-coding bead sets can simply be combined in a single container (tube) prepared for parallel digital signal amplification and detection of the encoded samples. Component 9: Oil for phase transfer and signal amplification -HFE-7500 fluorocarbon oil (3M Deutschland GmbH) - Supplemented with 2-5% (v / v) PicoSurf (Dolomite Microfluidics), 2-5% (v / v) FluoSurf (Emulseo), or 2-5% (v / v) 008-fluorinated surfactant (RAN Biotechnologies) Parallel digital PCR amplification reactions in sample-encoded beads

[0137] The monodisperse emulsion containing encapsulated samples 1, 2, and 3 was placed approximately 2.5 cm apart. 2 The samples 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 microspheres provide an array of approximately 20,000 to 30,000 nanoreactors (approximately 7,000 to 10,000 per sample) arranged at equal intervals for subsequent parallel signal amplification reactions.

[0138] Microparticles are subjected to ultrafast temperature cycling 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. The applied RT-PCR temperature conditions are 30-45 cycles of a two-step PCR consisting of reverse transcription at 50°C for 10 minutes, initial denaturation at 95°C for 30 seconds, followed by denaturation at 95°C for 2 seconds and annealing / extension at 65°C for 5 seconds. The suspension becomes an emulsion containing individual liquid nanoliter droplets due to its sol-gel switching ability. Multiple dual amplifications of HCV and MS2 (control) from different samples occur in the labeled nano-reaction compartment.

[0139] 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 4.416mm × 2.774mm) and a pE-4000 (CoolLED Ltd.) light source. This microscope was further equipped with an automated XY stage with a thermocycler containing three sets of fluorescence filters (Cy5 ET, Cy3 ET, FITC / FAM HC, AHF Analysentechnik) and a reaction chamber.

[0140] The image acquisition settings are as follows: 100-1000 ms and gain of 1-10×. One image is required for label identification (λexc1=580 nm), one image is required for the internal standard PCR signal (λexc2=470 nm), and one image is required for the specific PCR signal (λexc3=635 nm). For freely selected nanoreactor-specific real-time analysis, three images corresponding to the three fluorescence channels can be acquired in each cycle at a suitable position in the chamber.

[0141] Once the temperature protocol is complete, scan the entire detection chamber area using the same equipment with the settings described above. A total of 48-56 images are required to cover the dimensions of the amplification / detection chamber.

[0142] Figure 9 shows a stitched image of the detection chamber corresponding to the fluorescent channel encoding the bead labels. In the magnified insert in the lower right, three different sample-coding beads are clearly recognizable (bright, moderate, and low signal). These beads are spatially distributed in a hexagonal close-packed pattern. The total number of sample-coding beads (average diameter d=105 μm) in this PCR chamber, as recognized by the software algorithm used, was found to be N=17.698. Sample decoding and endpoint analysis

[0143] 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 of all segmented droplets / microparticles, including fluorescence signal, location, and diameter / volume in each channel, are collected. The experimental data is applied to a Jupyter script that identifies individual labels (graduation of bound Cy3 dye) and their respective specific amplifications.

[0144] 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 a specific target and therefore show an increase in fluorescence signal above a defined intensity threshold. This threshold is derived from previously performed template-free amplification reactions or determined statistically in each experiment. The negative and positive fractions for each nanoreactor type are clustered, and the positive droplet fraction is applied to a Poisson algorithm to determine the starting concentration of the target RNA molecule in units of copies / μL (copies / mL). Since the assay described above results in the simultaneous detection of HCV in three different samples, their reactors are clustered into six groups:

[0145] Digital PCR endpoint data cluster [Table 2]

[0146] Fluorescence images of color-labeled agarose-gelatin hybrid microparticles after PCR amplification are shown in Figure 10A. Three labels are recognizable by their fluorescence intensity in channel 1. Channel 2 represents the fluorescence signal for the internal process control (MS2 phage), and channel 3 represents fluorescence specific to PCR amplification of the HCV target. Each label corresponds to a different sample that was processed and analyzed. Figure 10B (top graph) shows histograms of fluorescence intensities measured for different beads. Three distinct label types are visible. Violin plots showing the signal distribution observed across each bead type for channel 2 (MS2) and channel (3) are shown in the middle and bottom graphs. By applying Poisson analysis, the measured values ​​are converted to specific copy numbers (cp / μl) per sample volume applied to the beads. Example 3: RNA binding ability of agarose-gelatin hybrid beads

[0147] RNA binding was investigated for various hybrid beads consisting of gelatin and agarose A4018 at different concentrations. Binding RNA on microparticles was qualitatively measured using an RNA dye assay or quantitatively using the digital PCR method described above.

[0148] Bead preparation The following modifications were made to create the microparticles as described above: Unlabeled microparticles were obtained by removing the labeled fraction of gelatin. • Hybrid hydrogel solutions for producing microparticles are made from different concentrations of agarose and gelatin, i.e.: a) 1.3% gelatin, 1.0% agarose b) 0.5% gelatin, 0.5% agarose c) 1.3% gelatin, 0.5% agarose d) 2.0% gelatin, 0.5% agarose e) 4.0% gelatin, 0.5% agarose It was • Two different gelatin types from each of the two manufacturers, namely: GELITA Imagel AP (Type A) ·GELITA Imagel SI (Type B) • Acetone-insoluble fraction of G1890 (Sigma, Type A) • Acetone-insoluble fraction of G9391 (Sigma type B) This was used for the production of microparticles. RiboGreen RNA Binding Assay

[0149] Fill the wells of a UV-Star microtiter plate (Greiner) with 3 μL of 50% bead slurry in 1× binding buffer (component 4). Incubate RNA at a final concentration of 1 ng / μL from different preparations—i.e., RNA spacer (Metabion), brewer's yeast-derived tRNA (Roche Diagnostic), and total RNA extracted from blood—at 15°C for 5–10 minutes to permeate and bind to the porous hydrogel matrix. Because binding occurs rapidly, mixing immediately after adding the RNA to the beads is crucial for uniform distribution of RNA throughout the beads.

[0150] To visualize RNA bound to hybrid microparticles, 0.5 μL of Quant-iT 1:2 diluted solution was used. TM RiboGreen® RNA reagent aliquots were pipetteed into microtiter wells and mixed. The beads were washed twice with 200 μL of TE buffer, and then the fluorescence signal was measured using a fluorescence microscope (Zeiss AxioObserver) and a pE-4000 (CoolLED Ltd.) light source.

[0151] Figure 11 shows that binding characteristics can be optimized by adjusting the composition of the microparticles. When combined with A4018, GELITA Imagel A gelatin yields a relatively weak RiboGreen signal on the beads compared to the acetone-insoluble fraction of Sigma's G1890 type A gelatin. Hybrid beads made from >1.3% gelatin combined with 0.5% agarose can bind more total RNA, tRNA, and short RNA fragments. Combinations with 1% agarose also yield better signals for the RNAs tested (results for total RNA samples are missing). Gelatin type B behaves differently from type A in terms of RNA enrichment on the beads. For any of the above RNAs, GELITA SI gelatin beads show almost no signal (meaning enrichment is not achieved by diffusion into the particles), while beads made from the acetone-insoluble fraction of G9391 gelatin type B show a weak RiboGreen signal for RNA spacer 1 and tRNA. Overall, total RNA, tRNA, and short RNA fragments can be bound more effectively to hybrid beads made with a combination of >1.3% gelatin and 0.5% agarose.

[0152] Figure 12 shows Quant-iT TM This shows the binding of total yeast RNA to microparticles visualized with the RiboGreen® RNA reagent over time. This assay format also allows for direct monitoring of RNA binding to microparticles. Images are acquired every 6 seconds, and the increase in the RiboGreen signal can be directly measured. Quantification of RNA binding by digital PCR

[0153] To quantitatively evaluate the RNA binding ability of the microparticles according to the present invention, digital PCR in a nanoreactor was performed as described above. Since these microparticles can be transferred from solidified particles to liquid droplets, they can provide both binding and a digital PCR detection matrix. Therefore, the bound HCV RNA molecules can be measured directly on the beads. Alternatively, the supernatant can be measured digitally using a DG8 cartridge (Bio-Rad) to generate droplets.

[0154] Add 40 μL of binding buffer (component 4) containing purified (QIAamp Viral RNA Mini Kit) Accuplex HCV RNA (Seracare Life Sciences Inc.) to a 40 μL bead bed and incubate at 15°C for 5 minutes at 1000 rpm to bind the RNA. Then, centrifuge the beads at 300 × g for 30 seconds and keep the supernatant on ice for further analysis. Add 40 μL of 2 × reagent mixture (component X) to the bead bed (40 μL) and allow the amplification and detection reagents to diffuse into the hydrogel matrix with a short incubation of 5 minutes at 15°C while shaking at 1000 rpm. Component X: Reagent mixture (2×) - 2× thermostable reverse transcriptase containing a ribonuclease inhibitor (biotechrabbit GmbH) -0.4U / μl Hot Start Taq DNA Polymerase (biotechrabbit GmbH) -0.8mM dNTP (biotechrabbit GmbH) -0.2% (w / v) low bioburden, protease-free BSA (Sigma) for molecular biology. Analyte-specific reagent (HCV) -0.8 μM HCV-Brun sense primer (5'-GTGGTCTGCGGAACCGGTGA-3') SEQ ID NO: 1 -0.8 μM HCV-Brun antisense primer (5'-CGCAAGCACCCTATCAGGCAGT-3') SEQ ID NO: 2 -0.8μM HCV-Brun TaqMan probe (5'-Atto647-CCGAGTAGYGTTGGGTYGCGAAAGG-BHQ-2-3') SEQ ID NO: 3

[0155] The phase transfer, amplification reaction, and analysis of the beads are performed as previously described. The supernatant (40 μL) is supplemented with PCR reagent (40 μL of 2× reagent mixture), and then emulsified using a DG8 cartridge (Bio-Rad). 100 μL of emulsion reagent HFE-7500 containing 2-5% Picosurf2 (Sphere Fluidics) and 40 μL are 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. The droplets are collected and transferred to a separate detection chamber for digital analysis. The amplification reaction and analysis settings for the droplets are the same as those for the beads. A reference sample consisting of equal volumes of 2× reagent mixture and PCR buffer is also emulsified and analyzed in the same manner.

[0156] Figure 13 shows summary data from these experiments designed to evaluate the enrichment ability of microparticles of two different compositions. Black dots indicate the target concentration in the sample before incubation with the microparticles. White bars represent the target concentration in the supernatant detected after incubation, and gray bars represent the target concentration on the microparticles. A clear enrichment effect is observed.

[0157] Figure 14 shows the accuracy of the combined analytical approach of digital PCR and real-time quantitative PCR, further outlined above, for quantifying targets in microparticles. Confidence intervals for both methods are shown. The vertical lines at λ = 5cp / microparticle (cp = copy) indicate approximations where the methods can be used interchangeably ("CI" = confidence interval).

[0158] Figure 15 shows real-time fluorescence data obtained from a series of images collected on microparticles in oil during PCR amplification. The stitched image of the detection chamber, including the endpoint fluorescence signal detected in a single fluorescence channel specific to amplification, is shown on the left. The graph in the center exemplifies the fluorescence intensity over time 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 each microparticle detected in the fluorescence images. The present invention provides, for example, the following items: (Item 1) A method for detecting and / or quantifying a target analyte in multiple biological liquid samples, wherein the method comprises a sample-specific process portion followed by a general process portion; The aforementioned sample-specific process portion consists of the following steps: - A step of providing, separately in any order, a plurality of different biological liquid samples suspected to contain the target analyte, and a plurality of differently labeled porous particulate subsets, wherein each of the plurality of subsets is separated from the other subsets; - A step of specifically labeling each of the different biological samples by exposing each of the separate subsets of particulate matter separately to one biological liquid sample, thereby enabling each sample to be drawn up by a specifically labeled subset of porous particulate matter in an aqueous environment; - A process of separately transferring each subset of porous fine particles from the aqueous environment to a non-aqueous environment. Includes; The aforementioned general process portion consists of the following steps: - A step of mixing the differently labeled porous microparticle subsets together in the non-aqueous environment to produce a suspension of multiple differently labeled porous microparticle subsets; - A step of performing a detection reaction on the suspension of the multiple differently labeled porous microparticle subsets to detect the target analyte; and - If the target analyte is present in any of the differently labeled subsets of suspended particulate matter, the step of detecting and / or quantifying the target analyte. Methods that include... (Item 2) The method according to item 1, wherein each of the separate particulate subsets is separately exposed to a biological liquid sample, each of the separate particulate subsets is exposed to a biological sample and a detection composition for performing a chemical or biochemical detection reaction in any order, and each particulate subset absorbs the respective exposed biological sample and the detection composition. (Item 3) The above method comprises the following steps: a. A step of separately providing, in any order, a plurality of distinct biological liquid samples and a plurality of porous microparticles suspected to contain a target analyte; each of the porous microparticles having a porous matrix and configured to receive a predetermined volume of liquid into the porous matrix; the plurality of porous microparticles having different microparticle subsets to be provided, each microparticle subset characterized by a specific labeling component attached to, contained therein, or otherwise associated with therein; in the providing step, the number of different microparticle subsets to be provided is at least the same as the number of distinct biological liquid samples to be provided, and further, in the providing step, the different microparticle subsets are provided separately from one another; optionally, the different microparticle subsets include a detection composition containing a reagent for carrying out a chemical or biochemical detection reaction of the analyte in their respective porous matrices; b. Exposing each distinct particulate subset to exactly one distinct biological liquid sample, thereby enabling each distinct particulate subset to incubate with a predetermined volume of exactly one distinct biological liquid sample and to aspirate such sample or a portion thereof, and, if necessary, to accumulate the analyte in or on the matrix of the particulates if the analyte is present in the sample; and, if further necessary, if the different distinct particulate subsets provided in step a) do not yet contain reagents for carrying out a chemical or biochemical detection reaction of the analyte, exposing each distinct particulate subset to a detection composition containing reagents for carrying out a chemical or biochemical detection reaction of the analyte, thereby enabling each distinct particulate subset to receive the reagents; c. A step of creating a plurality of separate, isolated reaction space subsets for detecting the analyte by transferring each particulate subset separately to a non-aqueous phase and removing part or all of the aqueous phase around the individual prepared particulates of the subset, wherein the reaction space comprises an aqueous phase containing the reagents for carrying out chemical or biochemical detection reactions of the sample and analyte, and the reaction space is limited to the void volume of the particulates; d. Mixing the separate and different particulate subsets in the non-aqueous phase so that all of the different particulate subsets form a suspension of different particulates in the non-aqueous phase; subjecting the mixed different particulate subsets to the conditions necessary for a chemical or biochemical detection reaction of the analyte; performing such a detection reaction of the target analyte; detecting and / or quantifying the target analyte if it is present in any of the different particulate subsets, using the signal generated in the detection reaction in each of the respective subsets. A method for detecting and / or quantifying a target analyte in multiple biological liquid samples, particularly those described in either item 1 or 2. (Item 4) The method of item 3, further comprising step e) which determines which of the plurality of samples provided in step a) contains the target analyte by identifying a subset of particulate matter in which the target analyte was detected in step d), preferably, the identification of the subset of particulate matter in step e) is performed using the specific labeling component attached to, contained in, or otherwise associated with each of the subsets of particulate matter. (Item 5) The method according to any one of items 3 to 4, wherein step b) further comprises a substep of generating a first correlation record indicating which distinct particulate subsets are or have been exposed to which sample, and step d) comprises a substep of generating a second correlation record indicating which particulate subsets produced a signal in the detection reaction. (Item 6) The method according to items 4-5, wherein step e) is performed by referring to and concatenating the records of the first and second correlations, thereby enabling determination of which of the multiple samples provided in step a) contains the target analyte. (Item 7) Each of the porous fine particles is (i) A polymer or polymer mixture that forms or is the porous matrix; or (ii) 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 fine particles; or (iii) at least one or more charged groups immobilized on the porous matrix; or (iv) Any combination of (i) to (iii) The method according to any of the above items, having a porous matrix that allows for the accumulation of the target analyte by binding to the analyte. (Item 8) Each of the porous microparticles has a porous matrix and comprises an analyte-specific reagent (ASR) attached to or incorporated by the microparticle, such analyte-specific reagent enables the concentration of the analyte of interest and / or a specific signal amplification or target amplification reaction involving the analyte; the analyte-specific reagent can specifically bind to the analyte of interest, and preferably 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, 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, according to any one of items 1 to 6 or 7. (Item 9) The method according to item 8, wherein each of the porous microparticles contains or has the same analyte-specific reagent attached to its porous matrix. (Item 10) Among the aforementioned multiple porous microparticles, there exist different subsets of microparticles. Each subset is, The subset of the fine particles has different labeling components attached to, contained therein, or otherwise associated therewith; All of the aforementioned different subsets The subset of the fine particles has the same analyte-specific reagent attached to or contained therein, and the analyte-specific reagent is specific to one target analyte; The aforementioned different subsets of particulate matter are identical with respect to the analyte-specific reagents attached to or contained within them, Each subset of the fine particles has different labeling components attached to, contained therein, or otherwise associated with it; Each subset is clearly defined and identifiable by its respective label component. The method described in item 9. (Item 11) The method described above is a method for detecting and / or quantifying one target analyte in a plurality of biological liquid samples, provided, preferably in step a), the number of different particulate subsets provided is equal to the number of separate biological liquid samples provided, according to any of items 1-7 or any of items 8-10. (Item 12) The method according to item 8, wherein several different analyte-specific reagents are attached to or contained within the plurality of porous microparticles. (Item 13) Different subsets of particles exist, and each subset is, The subset of the fine particles has different labeling components attached to, contained therein, or otherwise associated therewith; Furthermore, among the multiple porous microparticles, there exist subsets of different classes of microparticles, and each class of the subset is: The microparticles have different analyte-specific reagents attached to or contained within the porous matrix of the microparticles; preferably, there are at least two different classes of microparticle subsets, more preferably at least three or more different classes of microparticle subsets; The different microparticle subsets differ in the respective labeling components attached to, contained in, or otherwise associated with the microparticles of each subset; each 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 different classes of microparticle subsets differ in the respective analyte-specific reagents attached to or contained within them; each of the different classes comprises several microparticle subsets, all of which have the same analyte-specific reagent attached to or contained within them. The method described in item 12. (Item 14) The method described above is a method for detecting and / or quantifying one or more target analytes in a plurality of biological liquid samples, wherein the number of different particulate subsets provided, preferably in step a), is equal to the number of distinct biological liquid samples provided multiplied by the number of target analytes to be detected, and the same number of classes of particulate subsets as the number of target analytes to be detected are provided, preferably in step a), according to any one of items 12 to 13. (Item 15) The method according to any one of items 3 to 14, wherein the porous matrix is ​​a porous polymer matrix formed of a polymer or polymer mixture, preferably the porous polymer matrix is ​​composed of an uncrosslinked polymer, more preferably the polymer or polymer mixture forming 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 16) The target analyte is a nucleic acid, the detection reaction is nucleic acid amplification, and the detection composition is a composition for performing nucleic acid amplification comprising 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 detecting the amplified nucleic acid, and optionally one or more amplification primers, and further optionally, if such primers and / or probes are not yet provided as analyte-specific reagents (ASRs) attached to or contained in the microparticles, their respective molecular probes (e.g., TaqMan probes, molecular beacons, etc.); and / or The method according to any one of items 3 to 15, wherein the target analyte is a protein or other non-nucleic acid molecule, the detection reaction is an immunochemical detection reaction, and the detection composition is a composition for carrying out such an immunochemical detection reaction, provided in the method as two distinct components: the first component of the detection composition comprises a reagent necessary for carrying out the 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; and optionally comprises a primary antibody, antibody fragment or non-antibody protein that can specifically bind to the protein analyte or other non-nucleic acid analyte if such a primary antibody, antibody fragment or non-antibody protein has not yet been provided as an analyte-specific reagent (ASR) attached to or contained in the microparticles; and the second component of the detection composition comprises a substrate suitable for the suitable reporter enzyme as a detection reagent, the substrate becoming detectable, preferably optically detectable, more preferably fluorescently detectable, when reacted with the reporter enzyme. (Item 17) 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, in particular digital immunochemical detection methods combined with nucleic acid amplification, e.g., immunopolymerase chain reaction; in particular digital immuno-PCR; and e) any combination of a) to d) 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). A method using any of the items mentioned above. (Item 18) A kit for detecting a target analyte in multiple biological liquid samples, particularly for performing the method described in any of items 1 to 17, wherein the kit is - A plurality of containers comprising a plurality of microparticles, each container comprising a plurality of porous microparticle subsets, each of the porous microparticles in each subset having a porous matrix and configured to receive a predetermined volume of liquid into the porous matrix; each microparticle subset is characterized by a specific labeling component attached to, contained therein, or otherwise associated with the respective subset, preferably the microparticles being as defined in any of items 1 to 17; and optionally comprising an aqueous cleaning reagent for cleaning the microparticles; - A container comprising a detection composition for detecting an analyte of interest; the detection composition comprising a reagent for carrying out a chemical or biochemical detection reaction of the analyte; the detection composition being a composition for carrying out nucleic acid amplification or an immunochemical detection reaction; - A container containing a non-aqueous phase for transferring each of the different particulate subsets to a non-aqueous phase after each of them has been exposed to a biological liquid sample, and for generating separate suspensions of the different particulate subsets in the non-aqueous phase; - A mixing vessel for mixing the separate and different particulate subset suspensions together in the non-aqueous phase, wherein all of the different particulate subset suspensions form a single suspension of different particulates in the non-aqueous phase, which is then subjected to a detection reaction; - Container for conducting the detection reaction A kit that includes this. (Item 19) Each of the porous microparticles has or comprises an analyte-specific reagent (ASR) attached to its porous matrix, the analyte-specific reagent enabling the concentration of the analyte of interest and / or a specific signal amplification or amplification reaction involving the analyte; the analyte-specific reagent is capable of specifically binding to the analyte of interest, and preferably 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, as described in item 18. (Item 20) The target analyte is a nucleic acid, the detection reaction is nucleic acid amplification, and the detection composition is a composition for performing nucleic acid amplification comprising 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 detecting the amplified nucleic acid, and optionally a pair of primers, if such primers have not yet been provided as an analyte-specific reagent (ASR) attached to or contained in the microparticles; or The target analyte is a protein or other non-nucleic acid molecule, the detection reaction is an immunochemical detection reaction, and the detection composition is a composition for carrying out such an immunochemical detection reaction, provided in the kit in two separate compartments or containers; the detection composition contains in the first compartment or container the reagents necessary for carrying out the immunochemical detection reaction, such as a buffer, and a primary antibody, antibody fragment, or non-antibody protein used as an analyte-specific reagent (ASR) in the immunochemical reaction, and a secondary antibody or secondary antibody fragment that is specific to the same analyte and conjugated to a suitable reporter enzyme; if necessary, If a primary antibody, antibody fragment, or non-antibody protein capable of specifically binding to a protein analyte or other non-nucleic acid analyte has not yet been provided as an analyte-specific reagent (ASR) attached to or contained in the microparticles, the kit comprises such a primary antibody, antibody fragment, or non-antibody protein; the detection composition comprises a substrate suitable for the preferred reporter enzyme as a detection reagent in a second compartment or container, the substrate becoming detectable, preferably optically detectable, more preferably fluorescently detectable, when reacted with the reporter enzyme, according to any one of items 18-19. (Item 21) A kit according to any one of items 18 to 20, wherein each of the porous microparticles has the same analyte-specific reagent attached to its porous matrix, or contains the same analyte-specific reagent. (Item 22) Among the aforementioned multiple porous microparticles, there exist different subsets of microparticles. Each subset is, The subset of the fine particles has different labeling components attached to, contained therein, or otherwise associated therewith; All of the aforementioned different subsets The subset of the fine particles has the same analyte-specific reagent attached to or contained therein, and the analyte-specific reagent is specific to one target analyte; The aforementioned different subsets of particulate matter are identical with respect to the analyte-specific reagents attached to or contained within them, Each subset of the fine particles has different labeling components attached to, contained therein, or otherwise associated with it; Each subset is clearly defined and identifiable by its respective labeling component and is provided in a separate container. The kit described in item 21. (Item 23) The kit is for detecting one target analyte in multiple biological liquid samples, and the number of different particulate subsets provided in the kit is equal to the number of separate biological liquid samples provided, as described in any of items 21-22. (Item 24) A kit according to any one of items 18-20, wherein several different analyte-specific reagents are attached to or contained within the plurality of porous microparticles. (Item 25) Different subsets of particles exist, Each subset is, The subset of the fine particles has different labeling components attached to, contained therein, or otherwise associated therewith; Furthermore, among the multiple porous microparticles, there exist subsets of different classes of microparticles, and each class of the subset is: The microparticles have different analyte-specific reagents attached to or contained within the porous matrix of the microparticles; preferably, there are at least two different classes of microparticle subsets, more preferably at least three or more different classes of microparticle subsets; The aforementioned different particulate subsets differ in the respective labeling components attached to, contained in, or otherwise associated with the particulates of each subset; each particulate subset forms part of a class of particulate subsets; each subset is clearly defined and identifiable by its respective labeling component and its respective analyte-specific reagent, and is provided in separate containers; The aforementioned different classes of microparticle subsets differ in the analyte-specific reagents attached to or contained within the porous matrix of the microparticles; each of the aforementioned different classes comprises several microparticle subsets, all of which have the same analyte-specific reagent attached to or contained within them. The kit described in item 24. (Item 26) The kit is for detecting more than one of several biological liquid samples, the number of different particulate subsets provided in the kit is equal to the number of distinct biological liquid samples provided multiplied by the number of target analytes to be detected, and the kit provides the same number of classes of particulate subsets as the number of target analytes to be detected, as described in any of items 24-25. (Item 27) A cartridge for performing a method for detecting and / or quantifying a target analyte in multiple biological liquid samples as described in any of items 1 to 17, wherein the cartridge preferably comprises multiple sample-specific modules, multiple storage chambers, at least one non-aqueous phase chamber for storing a non-aqueous phase, and an integrated single mixing and detection chamber or a combination of separate mixing chambers and separate detection chambers; Each sample-specific module comprises a sample compartment having its own separate sample inlet, and each sample-specific module is configured to receive strictly one biological sample separately into its respective sample compartment; each sample-specific module is further configured to receive microparticles into the sample compartment, the microparticles being as defined in any of items 1 to 17; and each sample-specific module is further configured to facilitate the interphase transfer of the microparticles from an aqueous environment to a non-aqueous environment, in a cartridge.

Claims

1. A method for detecting and / or quantifying a target analyte in multiple biological liquid samples, wherein the method comprises a sample-specific process portion followed by a general process portion; The aforementioned sample-specific process portion consists of the following steps: - A step of providing, separately in any order, a plurality of different biological liquid samples suspected to contain the target analyte, and a plurality of differently labeled porous particulate subsets, wherein each of the subsets is separated from the other subsets; - A step of specifically labeling each of the different biological samples by exposing each of the separate particulate subsets separately to one biological liquid sample, thereby enabling each sample to be drawn up by a specifically labeled porous particulate subset in an aqueous environment; - A process of separately transferring each subset of porous fine particles from the aqueous environment to a non-aqueous environment. Including; The aforementioned general process portion consists of the following steps: - A step of mixing the differently labeled porous microparticle subsets in the non-aqueous environment to produce a suspension of multiple differently labeled porous microparticle subsets; - A step of performing a detection reaction on the suspension of the multiple differently labeled porous microparticle subsets for the target analyte to be detected; and - If the target analyte is present in any of the differently labeled suspended particulate subsets, the step of detecting and / or quantifying the target analyte. Includes, Each of the aforementioned porous microparticles is a porous matrix, (i) a polymer or polymer mixture forming the porous matrix, or a polymer or polymer mixture that is the porous matrix; or (ii) at least one or more ionizable groups immobilized on the porous matrix, wherein the ionizable groups can change their charge according to the ambient conditions around the fine particles; or (iii) at least one or more charged groups immobilized on the porous matrix; or (iv)(i) to (iii) any combination It has a porous matrix that allows for the accumulation of the target analyte by binding to the analyte; The above method comprises the following steps: a. A step of separately providing, in any order, a plurality of separate biological liquid samples and a plurality of porous microparticles suspected to contain a target analyte; each of the porous microparticles having a porous matrix and configured to receive a predetermined volume of liquid into the porous matrix; the plurality of porous microparticles having different microparticle subsets to be provided, each microparticle subset characterized by a specific labeling component attached to, contained therein, or otherwise associated with the respective subset; in the providing step, the number of different microparticle subsets to be provided is at least the same as the number of separate biological liquid samples to be provided, and further, in the providing step, the different microparticle subsets are provided separately from one another; b. Exposing each separate particulate subset separately to strictly one separate biological liquid sample, thereby enabling each separate particulate subset to incubate with a predetermined volume of strictly one separate biological liquid sample and to aspirate such sample or a portion thereof, and, if an analyte is present in the sample, to accumulate the analyte in or on the matrix of the particulates; and, if the different separate particulate subsets provided in step a. do not yet contain reagents for performing a chemical or biochemical detection reaction of the analyte, exposing each separate particulate subset to a detection composition containing reagents for performing a chemical or biochemical detection reaction of the analyte, thereby enabling each separate particulate subset to receive the reagents; c. A step of creating a plurality of separate, isolated reaction space subsets for detecting the analyte by transferring each particulate subset separately to a non-aqueous phase and removing part or all of the aqueous phase around the individual prepared particulates of the subset, wherein the reaction space comprises an aqueous phase containing the reagents for carrying out chemical or biochemical detection reactions of the sample and analyte, and the reaction space is limited to the void volume of the particulates; d. Mixing the separate different particulate subsets in the non-aqueous phase so that all of the different particulate subsets form a suspension of different particulates in the non-aqueous phase; subjecting the mixed different particulate subsets to the conditions necessary for a chemical or biochemical detection reaction of the analyte; performing such a detection reaction of the target analyte; detecting and / or quantifying the target analyte if it is present in any of the different particulate subsets, using the signal generated in the detection reaction in each of the respective subsets. Includes, The target analyte is a nucleic acid, the detection reaction is nucleic acid amplification, and the detection composition is a composition for performing nucleic acid amplification, comprising a buffer, a mononucleoside triphosphate, an amplification enzyme, and a nucleic acid dye for detecting the amplification product; and / or The method wherein the target analyte is a protein or other non-nucleic acid molecule, the detection reaction is an immunochemical detection reaction, and the detection composition is a composition for carrying out such an immunochemical detection reaction, provided in the method as two separate components: the first component of the detection composition comprises a reagent comprising a buffer necessary for carrying out the immunochemical detection reaction, 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; and the second component of the detection composition comprises a substrate suitable for the suitable reporter enzyme as a detection reagent, the substrate becoming optically detectable when reacted with the reporter enzyme.

2. The method according to claim 1, wherein, in the step of separately exposing each of the separate particulate subsets to a biological liquid sample, each of the separate particulate subsets is exposed in any order to a biological sample and a detection composition for carrying out a chemical or biochemical detection reaction, and each particulate subset absorbs the respective exposed biological sample and the detection composition.

3. The method according to any one of claims 1 to 2, wherein the different microparticle subsets each include a detection composition comprising a reagent for carrying out a chemical or biochemical detection reaction of an analyte in their respective porous matrices.

4. The method according to any one of claims 1 to 3, further comprising step e., which determines which of the plurality of samples provided in step a. contains the target analyte by identifying a subset of particulate matter in which the target analyte was detected in step d., wherein the identification of the subset of particulate matter in step e. is performed using the specific labeling component attached to, contained in, or otherwise associated with each of the subsets of particulate matter.

5. The method according to any one of claims 1 to 4, wherein step b. further comprises a substep of generating a first correlation record indicating which separate particulate subsets are or have been exposed to which sample, and step d. comprises a substep of generating a second correlation record indicating which particulate subsets produced a signal in the detection reaction.

6. The method according to claim 5, wherein step e is performed by referring to and concatenating the records of the first and second correlations, thereby enabling determination of which of the plurality of samples provided in step a contains the target analyte.

7. The method according to any one of claims 1 to 6, wherein each of the porous microparticles comprises an analyte-specific reagent (ASR) attached to or incorporated by the microparticles, the ASR enabling a specific signal amplification reaction or target amplification reaction involving the analyte; the ASR can specifically bind to the analyte of interest, and the ASR is selected from nucleic acids including aptamers and Spiegelmers; antibodies or antibody fragments; and non-antibody proteins selected from receptors, receptor fragments, and affinity proteins that can specifically bind to the analyte or analyte complex.

8. The method according to claim 7, wherein the analyte-specific reagent is selected from nucleic acids.

9. The method according to claim 8, wherein the analyte-specific reagent is selected from nucleic acid oligomers and nucleic acid primers.

10. The method according to any one of claims 7 to 9, wherein each of the porous microparticles contains or has the same analyte-specific reagent attached to its porous matrix.

11. Among the aforementioned plurality of porous microparticles, there exists a subset of microparticles that are labeled differently. Each subset is, - Having different labeling components attached to, contained in, or otherwise associated with the fine particles of the subset; and All of the aforementioned different subsets - The subset of the fine particles has the same analyte-specific reagent attached to or contained therein, wherein the analyte-specific reagent is specific to one target analyte; Therefore, the aforementioned different subsets of particulate matter are identical with respect to the analyte-specific reagents attached to or contained within them, - Each subset of the fine particles has different labeling components attached to, contained therein, or otherwise associated with them; and Each subset is clearly defined and identifiable by its respective label component. The method according to claim 10.

12. The method according to any one of claims 1 to 6 or any one of claims 7 to 11, wherein the method is a method for detecting and / or quantifying one target analyte in a plurality of biological liquid samples, and the number of different particulate subsets provided is equal to the number of separate biological liquid samples provided.

13. The method according to any one of claims 7 to 9, wherein several different analyte-specific reagents are attached to or contained in the plurality of porous microparticles.

14. Different subsets of particles exist, and each subset is, - Having different labeling components attached to, contained in, or otherwise associated with the fine particles of the subset; Furthermore, among the multiple porous microparticles, there exist subsets of different classes of microparticles, and each class of the subset is: - Having different analyte-specific reagents attached to or contained in the porous matrix of the fine particles; Therefore, the different particulate subsets differ in the labeling components attached to, contained in, or otherwise associated with the particulates of each subset; each particulate subset forms part of a class of particulate subsets; and each subset is clearly defined and identifiable by its respective labeling component and its respective analyte-specific reagent; Therefore, the different classes of particulate subsets each have different analyte-specific reagents attached to or contained within them; each of the different classes comprises several particulate subsets, all of which have the same analyte-specific reagent attached to or contained within them. The method according to claim 13.

15. The method according to claim 14, wherein at least two different classes of microparticle subsets are present among the plurality of porous microparticles.

16. The method according to any one of claims 14 to 15, wherein at least three or more subsets of different classes of fine particles are present among the plurality of porous fine particles.

17. The method according to any one of claims 14 to 16, wherein the method is a method for detecting and / or quantifying one or more target analytes in a plurality of biological liquid samples, the number of different particulate subsets provided is equal to the number of separate biological liquid samples provided multiplied by the number of target analytes to be detected, and the same number of classes of particulate subsets as the number of target analytes to be detected are provided.

18. The method according to any one of claims 1 to 17, wherein the porous matrix is ​​a porous polymer matrix formed of a polymer or a polymer mixture, and the porous polymer matrix is ​​composed of an uncrosslinked polymer.

19. The method according to claim 18, wherein the polymer or polymer mixture forming the porous polymer matrix is ​​composed of agarose or a combination of agarose and gelatin.

20. The method according to claim 19, 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).

21. The method according to claim 20, wherein the gelatin is present in a range of 0.5% (w / v) to 20% (w / v).

22. The method according to any one of claims 1 to 21, wherein the detection composition in the nucleic acid detection reaction comprises one or more amplification primers, and, if such primers and probes have not yet been provided as analyte-specific reagents (ASRs) attached to or contained in the fine particles, the respective molecular probes.

23. The first component of the detection composition in the immunochemical detection reaction is If a primary antibody, antibody fragment, or non-antibody protein capable of specifically binding to the protein analyte or other non-nucleic acid analyte has not yet been provided as an analyte-specific reagent (ASR) attached to or contained in the microparticles, the method according to any one of claims 1 to 22, comprising such a primary antibody, antibody fragment, or non-antibody protein.

24. The method according to any one of claims 1 to 23, wherein the substrate becomes detectable by fluorescence when reacted with the reporter enzyme.

25. In the step of detecting and quantifying the target analyte, the quantification of the analyte is performed as follows: a) Digital nucleic acid amplification; b) Real-time quantitative nucleic acid amplification; c) Immunochemical detection methods; d) Immunochemical detection methods combined with nucleic acid amplification; and e) any combination of a) to d) 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 1 to 24.

26. A kit for carrying out the method according to any one of claims 1 to 25, the kit comprising: a plurality of containers comprising a plurality of microparticles, each container comprising a plurality of porous microparticle subsets, each of the porous microparticles in each subset having a porous matrix and configured to receive a predetermined volume of liquid into the porous matrix; each microparticle subset characterized by a specific labeling component attached to, contained therein, or otherwise associated with the respective subset, the microparticles being as defined in any one of claims 1 to 25; - A container comprising a detection composition for detecting an analyte of interest; the detection composition comprising a reagent for carrying out a chemical or biochemical detection reaction of the analyte; the detection composition being a composition for carrying out nucleic acid amplification or an immunochemical detection reaction; - A container containing a non-aqueous phase for transferring each of the different particulate subsets to a non-aqueous phase after each of the different particulate subsets has been exposed to a biological liquid sample, and for generating separate suspensions of the different particulate subsets in the non-aqueous phase; - A mixing vessel for mixing the separate and different particulate subset suspensions together in the non-aqueous phase, wherein all of the different particulate subset suspensions form a single suspension of different particulates in the non-aqueous phase, which is then subjected to a detection reaction; - Container for conducting the detection reaction Including; The target analyte is a nucleic acid, the detection reaction is nucleic acid amplification, and the detection composition is a composition for performing nucleic acid amplification, comprising a buffer, a mononucleoside triphosphate, an amplification enzyme, and a nucleic acid dye for detecting the amplification product; or The kit comprises the following: the target analyte is a protein or other non-nucleic acid molecule, the detection reaction is an immunochemical detection reaction, and the detection composition is a composition for carrying out such an immunochemical detection reaction, provided in two separate compartments or containers in the kit; the detection composition comprises in a first compartment or container a reagent comprising a buffer necessary for carrying out the immunochemical detection reaction, 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 reaction, and conjugated to a suitable reporter enzyme; and the detection composition comprises in a second compartment or container a substrate suitable for the suitable reporter enzyme as a detection reagent, the substrate becoming optically detectable when reacted with the reporter enzyme.

27. The kit according to claim 26, further comprising a container containing an aqueous cleaning reagent for cleaning the fine particles.

28. The kit according to any one of claims 26 to 27, wherein each of the porous microparticles has an analyte-specific reagent (ASR) attached to its porous matrix, or comprises an analyte-specific reagent (ASR) that enables a specific signal amplification reaction or amplification reaction involving the analyte; the analyte-specific reagent can specifically bind to the target analyte, and the analyte-specific reagent is selected from nucleic acids including aptamers and Spiegelmers; antibodies or antibody fragments; and non-antibody proteins selected from receptors, receptor fragments, and affinity proteins that can specifically bind to the analyte or analyte complex.

29. The kit according to claim 28, wherein the analyte-specific reagent is selected from nucleic acid oligomers and nucleic acid primers.

30. The kit according to any one of claims 26 to 29, wherein the detection composition in the nucleic acid detection reaction comprises a pair of primers if such primers have not yet been provided as an analyte-specific reagent (ASR) attached to or contained in the fine particles.

31. The first component of the detection composition in the immunochemical detection reaction is If a primary antibody, antibody fragment, or non-antibody protein capable of specifically binding to the protein analyte or other non-nucleic acid analyte has not yet been provided as an analyte-specific reagent (ASR) attached to or contained in the microparticles, the kit according to any one of claims 26 to 30, comprising such primary antibody, antibody fragment, or non-antibody protein.

32. The kit according to any one of claims 26 to 31, wherein the substrate becomes detectable by fluorescence when reacted with the reporter enzyme.

33. The kit according to any one of claims 26 to 32, wherein each of the porous microparticles has the same analyte-specific reagent attached to its porous matrix, or contains the same analyte-specific reagent.

34. Among the aforementioned multiple porous microparticles, there exist different subsets of microparticles. Each subset is, - Having different labeling components attached to, contained in, or otherwise associated with the fine particles of the subset; All of the aforementioned different subsets - The subset of the fine particles has the same analyte-specific reagent attached to or contained therein, wherein the analyte-specific reagent is specific to one target analyte; Therefore, the aforementioned different subsets of particulate matter are identical with respect to the analyte-specific reagents attached to or contained within them, - Each subset of the fine particles has different labeling components attached to, contained in, or otherwise associated with them; Each subset is clearly defined and identifiable by its respective labeling component and is provided in separate containers. The kit according to claim 33.

35. The kit according to any one of claims 33 to 34, wherein the kit is for detecting one target analyte in a plurality of biological liquid samples, and the number of different particulate subsets provided in the kit is equal to the number of separate biological liquid samples provided.

36. The kit according to any one of claims 26 to 32, wherein several different analyte-specific reagents are attached to or contained in the plurality of porous microparticles.

37. Different subsets of particles exist, Each subset is, - Having different labeling components attached to, contained in, or otherwise associated with the fine particles of the subset; Furthermore, among the multiple porous microparticles, there exist subsets of different classes of microparticles, and each class of the subset is: - Having different analyte-specific reagents attached to or contained in the porous matrix of the fine particles; Therefore, the different particulate subsets differ in the respective labeling components attached to, contained in, or otherwise associated with the particulates of each subset; each particulate subset forms part of a class of particulate subsets; each subset is clearly defined and identifiable by its respective labeling component and its respective analyte-specific reagent, and is provided in separate containers; Therefore, the different classes of microparticle subsets differ in the analyte-specific reagents attached to or contained within the microparticles; each of the different classes comprises several microparticle subsets, all of which have the same analyte-specific reagents attached to or contained within them. The kit according to claim 36.

38. The kit according to claim 37, wherein at least two different classes of microparticle subsets are present among the plurality of porous microparticles.

39. The kit according to any one of claims 37 to 38, wherein the plurality of porous microparticles include at least three or more subsets of microparticles of different classes.

40. The kit according to any one of claims 36 to 39, wherein the kit is for detecting more than one of a target analytes in a plurality of biological liquid samples, the number of different particulate subsets provided in the kit is equal to the number of separate biological liquid samples provided multiplied by the number of target analytes to be detected, and the kit provides the same number of classes of particulate subsets as the number of target analytes to be detected.

41. A cartridge for performing a method for detecting and / or quantifying a target analyte in a plurality of biological liquid samples according to any one of claims 1 to 25, wherein the cartridge comprises a plurality of sample-specific modules, a plurality of storage chambers, at least one non-aqueous phase chamber for storing a non-aqueous phase, and an integrated single mixing and detection chamber or a combination of separate mixing chambers and separate detection chambers; Each sample-specific module comprises a sample compartment having its own separate sample inlet, and each sample-specific module is configured to receive strictly one biological sample separately into its respective sample compartment; each sample-specific module is further configured to receive microparticles into the sample compartment, the microparticles being as defined in any of claims 1 to 25; and each sample-specific module is further configured to facilitate the interphase transfer of the microparticles from an aqueous environment to a non-aqueous environment, in a cartridge.

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