A METHOD OF DETECTING AND/OR QUANTITATING AN ANALYTE OF INTEREST IN A PLURALITY OF BIOLOGICAL LIQUID SAMPLES

Abstract

The present invention relates to a method of detecting and/or quantitating an analyte of interest in a plurality of biological liquid samples. Furthermore, the present invention relates to a kit and a cartridge for performing a method for detecting and/or quantitating an analyte of interest in a plurality of samples.

Claims

1. A method of detecting and/or quantitating an analyte of interest in a plurality of biological liquid samples, said method comprising a sample-specific process part, followed by a generic process part; said sample-specific process part comprising the following steps: separately providing, in any order, a plurality of different biological liquid samples suspected of containing an analyte of interest, and a plurality of differently labelled subsets of porous microparticles, wherein, in said plurality of subsets, each of said subsets is separate from the other subsets; specifically labelling each of said different biological samples by separately exposing each of said separate subsets of microparticles to one biological liquid sample each, thus allowing each sample to be taken up by one specifically labelled subset of porous microparticles in an aqueous environment; separately transferring each subset of porous microparticles from said aqueous environment to a non-aqueous environment; said generic process part comprising the steps: mixing together the differently labelled subsets of porous microparticles in said non-aqueous environment and thus generating a suspension of a plurality of differently labelled subsets of porous microparticles therein; performing a detection reaction for detecting said analyte of interest, on said suspension of said plurality of differently labelled subsets of porous microparticles; and detecting and/or quantitating said analyte of interest, if present, in any of said differently labelled subsets of suspended microparticles.

2. The method according to claim 1, wherein, 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, in any order, one biological sample each and a detection composition for performing a chemical or biochemical detection reaction, such that each subset of microparticles takes up the respective biological sample and the detection composition to which it has been exposed.

3. A method of detecting and/or quantitating an analyte of interest in a plurality of biological liquid samples, according to claim 1, said method comprising the following steps: a) a step of separately providing, in any order, a plurality of separate biological liquid samples suspected of containing an analyte of interest, and a plurality of porous microparticles; each of said porous microparticles having a porous matrix and being configured to receive a volume of liquid in said porous matrix; wherein, in said plurality of porous microparticles, there are different subsets of microparticles provided, each subset of microparticles being characterized by a specific label component that is attached to, contained in or otherwise associated with the respective subset; wherein in said step of providing, the number of different subsets of microparticles provided is at least as big as the number of separate biological liquid samples provided, and wherein furthermore in said step of providing, the different subsets of microparticles are provided separate from each other; wherein, optionally, said different separate subsets of microparticles contain in their respective porous matrix a detection composition comprising reagents for performing a chemical or biochemical detection reaction of an analyte; b) a step of exposing each separate subset of microparticles to exactly one separate biological liquid sample, thereby allowing each separate subset of microparticles to incubate with a volume of exactly one separate biological liquid sample and to take up such sample or a portion thereof, and, optionally, to accumulate analyte, if present in said sample, in or on the matrix of the microparticle(s); and, further optionally, in case that said different separate subsets of microparticles, when provided in step a), do not yet comprise reagents for performing a chemical or biochemical detection reaction of an analyte, a step of exposing each separate subset of microparticles to a detection composition comprising reagents for performing a chemical or biochemical detection reaction of an analyte, thereby allowing each separate subset of microparticles to receive said reagents; c) transferring each subset of microparticles separately into a non-aqueous phase and removing some or all of the aqueous phase surrounding the individual prefabricated microparticle(s) of said subset(s), thereby creating a plurality of separate subsets of insulated reaction spaces for detecting said analyte, which reaction spaces comprise an aqueous phase including sample and said reagents for performing a chemical or biochemical detection reaction of an analyte, and which reaction spaces are confined to said void volume(s) of said microparticles; and d) mixing the separate different subsets of microparticles in said non-aqueous phase, such that all of said different subsets of microparticles form a suspension of different microparticles in said non-aqueous phase; subjecting said mixed different subsets of microparticles to conditions required for performing a chemical or biochemical detection reaction of an analyte; performing such detection reaction of said analyte of interest; and detecting and/or quantitating said analyte of interest, if present, in any of said different subsets of microparticles, by means of a signal generated in said detection reaction in a respective subset of microparticles, if said analyte of interest is present in said respective subset.

4. The method according to claim 3, said method further comprising the step e) determining which sample(s) of said plurality of samples provided in step a) does contain said analyte of interest by determining the identity of the subset(s) of microparticles in which said analyte of interest is detected in step d), wherein, the identity of the subset(s) of microparticles in step e) is determined by means of the specific label component that is attached to, contained in or otherwise associated with the respective subset of microparticle(s).

5. The method according to claim 3, wherein step b) further comprises a substep of generating a first record of correlation indicating which separate subset of microparticles is or has been exposed to which sample, and step d) comprises a substep of generating a second record of correlation indicating in which subset of microparticles a signal has been generated in said detection reaction.

6. The method according to claim 4, wherein step e) is performed by reference to said first and second records of correlation and by linking said records, thus allowing to determine which sample(s) of said plurality of samples provided in step a) does(do) contain said analyte of interest.

7. The method according to claim 1, wherein each of said porous microparticles has a porous matrix which allows to accumulate analyte of interest by binding to analyte through: (i) a polymer or polymer mixture that forms or is said porous matrix; or (ii) at least one ionisable group, or a plurality of ionisable groups, immobilized on said porous matrix, said ionisable group(s) being capable of changing its(their) charge(s) according to ambient conditions surrounding said microparticle(s); or (iii) at least one charged group, or a plurality of charged groups immobilized on said porous matrix; or (iv) a combination of any of (i)-(iii).

8. The method according to claim 1, wherein each of said porous microparticles has a porous matrix and comprises an analyte-specific reagent (ASR) that is attached to said porous matrix or contained by said microparticle, such analyte-specific reagent allowing an enrichment of an analyte of interest and/or allowing a specific signal or target amplification reaction involving said analyte; wherein said analyte-specific reagent is capable of specifically binding to an analyte of interest, wherein the analyte-specific reagent is selected from nucleic acids; antibodies or antibody fragments; and non-antibody proteins capable of specifically binding an analyte or analyte complex.

9. The method according to claim 8, wherein each of said porous microparticles contains or has the same analyte-specific reagent attached to its porous matrix.

10. The method according to claim 9, wherein, in said plurality of porous microparticles, there are different subsets of microparticles, with each subset having its distinct label component attached to, contained in or otherwise associated with said microparticles of said subset; and all of said different subsets having the same analyte-specific reagent attached to or contained in said microparticles of said subsets, said analyte-specific reagent being specific for one analyte of interest; such that said different subsets of microparticles are identical in terms of the analyte-specific reagent attached or contained, but differ by the respective label component attached to, contained in or otherwise associated with said microparticles of each subset; with each subset being unambiguously defined and identifiable by said respective label component.

11. The method according to claim 1, wherein said method is a method of detecting and/or quantitating one analyte of interest in a plurality of biological liquid samples, wherein the number of different subsets of microparticles provided equals the number of separate biological liquid samples provided.

12. The method according to claim 8, wherein, in said plurality of porous microparticles, there are several different analyte-specific reagents attached to or contained in said microparticles.

13. The method according to claim 12, wherein there are different subsets of microparticles, with each subset having its distinct label component attached to, contained in or otherwise associated with said microparticles of said subset; and wherein furthermore, in said plurality of porous microparticles, there are different classes of subsets of microparticles with each class of subsets having a different analyte-specific reagent attached to the porous matrix of said microparticles or contained in said microparticles; wherein there are at least two different classes of subsets of microparticles; such that said different subsets of microparticles differ by the respective label component attached to, contained in or otherwise associated with said microparticles of each subset; and each subset of microparticles forms part of one class of subsets of microparticles; with each subset being unambiguously defined and identifiable by the respective label component and the respective analyte-specific reagent; and such that said different classes of subsets of microparticles differ by the respective analyte-specific reagent attached or contained; and each of said different classes comprises several subsets of microparticles, all of which subsets have the same analyte-specific reagent attached or contained.

14. The method according to claim 12, wherein said method is a method of detecting and/or quantitating more than one analyte of interest in a plurality of biological liquid samples, wherein, the number of different subsets of microparticles provided equals the number of separate biological liquid samples provided, multiplied by the number of analytes of interest to be detected, and wherein there are as many classes of subsets of microparticles provided as the number of analytes of interest to be detected.

15. The method according to claim 3, wherein said porous matrix is a porous polymer matrix formed by a polymer or polymer mixture, wherein said porous polymer matrix is composed of a polymer (or polymers) that is (are) not crosslinked, wherein said polymer or polymer mixture that forms said porous polymeric matrix, is composed of agarose or a combination of agarose and gelatin.

16. The method according to claim 3, wherein said analyte of interest is a nucleic acid, said detection reaction is a nucleic acid amplification, and said detection composition is a composition for performing a nucleic acid amplification which comprises a buffer, mono-nucleoside-triphosphates, an amplification enzyme, and a nucleic acid dye for the detection of an amplification product, and, optionally, one or more pairs of amplification primers and, further optionally, respective molecular probes, if such primers and/or probes are not already provided as analyte-specific reagent(s) (ASR) being attached to or contained in said microparticles; AND/OR wherein said analyte of interest is a protein or other non-nucleic acid molecule, said detection reaction is an immunochemistry detection reaction, and said detection composition is a composition for performing such immunochemistry detection reaction and is provided in said method as two separate components: wherein a first component of said detection composition comprises necessary reagents for performing an immunochemistry detection reaction, and a secondary antibody or secondary antibody fragment coupled to a suitable reporter enzyme and being specific for the same analyte as a primary antibody, antibody fragment, or non-antibody protein, used as analyte-specific reagent (ASR) in said immunochemistry detection reaction; and, optionally, a primary antibody, antibody fragment, or a non-antibody protein capable of specifically binding said protein analyte or other non-nucleic acid analyte, if such a primary antibody, antibody fragment, or non-antibody protein is not already provided as analyte-specific reagent(s) (ASR) being attached to or contained in said microparticles; and wherein a second component of said detection composition comprises, as a detection reagent, a suitable substrate for said suitable reporter enzyme which substrate upon having been reacted by said reporter enzyme, becomes detectable.

17. The method according to claim 1, wherein, in said step of detecting and quantitating said analyte of interest, quantitation of said analyte is performed by a method selected from: a) digital nucleic acid amplification; b) real-time quantitative nucleic acid amplification; c) immunochemistry detection methods; d) immunochemistry detection methods combined with nucleic acid amplification; and e) combination of any of a)-d); wherein quantitation is performed using any of methods a) orb), or a combination of a) and b), if the analyte of interest is a nucleic acid; and wherein quantitation is performed using any of methods c) or d), if the analyte is a protein, peptide or other non-nucleic acid analyte.

18. A kit for detecting an analyte of interest in a plurality of biological liquid samples, said kit comprising: a plurality of containers comprising a plurality of microparticles, each container comprising a subset of said plurality of porous microparticles, with each of said porous microparticles within each subset having a porous matrix and being configured to receive a volume of liquid in said porous matrix; each subset of microparticles being characterized by a specific label component that is attached to, contained in or otherwise associated with the respective subset, and, optionally, a container comprising an aqueous washing reagent for washing said microparticles; a container comprising a detection composition for detecting an analyte of interest; said detection composition comprising reagents for performing a chemical or biochemical detection reaction of an analyte; wherein said detection composition is either a composition for performing a nucleic acid amplification, or is a composition for performing an immunochemistry detection reaction; a container comprising a non-aqueous phase for transferring said different subsets of microparticles into a non-aqueous phase, once each subset has been exposed to a biological liquid sample, and for generating separate different suspensions of subsets of microparticles in a non-aqueous phase; a mixing container for mixing the separate different suspensions of subsets of microparticles in said non-aqueous phase together, such that all of said different suspensions of subsets of microparticles form a single suspension of different microparticles in said non-aqueous phase which is then subjected to a detection reaction; a container for performing a detection reaction.

19. The kit according to claim 18, wherein each of said porous microparticles has an analyte-specific reagent (ASR) attached to its porous matrix or contains an analyte-specific reagent (ASR), such analyte-specific reagent allowing an enrichment of an analyte of interest and/or allowing a specific signal or amplification reaction involving said analyte; wherein said analyte-specific reagent is capable of specifically binding to an analyte of interest, wherein said analyte-specific reagent is selected from nucleic acids; antibodies or antibody fragments; and non-antibody proteins capable of specifically binding an analyte or analyte complex.

20. The kit according to claim 18, wherein said analyte of interest is a nucleic acid, said detection reaction is a nucleic acid amplification, and said detection composition is a composition for performing a nucleic acid amplification which comprises a buffer, mono-nucleoside-triphosphates, an amplification enzyme, and a nucleic acid dye for the detection of an amplification product, and, optionally, a pair of primers, if such pair of primers are not already provided as analyte-specific reagent(s) (ASR) being attached to or contained in said microparticles; OR wherein said analyte of interest is a protein or other non-nucleic acid molecule, said detection reaction is an immunochemistry detection reaction, and said detection composition is a composition for performing such immunochemistry detection reaction and is provided in said kit in two separate compartments or containers; wherein said detection composition comprises, in a first compartment or container, necessary reagents for performing an immunochemistry detection reaction, and a secondary antibody or secondary antibody fragment coupled to a suitable reporter enzyme and being specific for the same analyte as a primary antibody, antibody fragment, or non-antibody protein, used as analyte-specific reagent (ASR) in said immunochemistry reaction; and, optionally, a primary antibody, antibody fragment, or a non-antibody protein capable of specifically binding said protein analyte or other non-nucleic acid analyte, if such a primary antibody, antibody fragment, or non-antibody protein is not already provided as analyte-specific reagent(s) (ASR) being attached to or contained in said microparticles; and wherein said detection composition comprises, in a second compartment or container, as a detection reagent, a suitable substrate for said suitable reporter enzyme which substrate upon having been reacted by said reporter enzyme, becomes detectable.

21. The kit according to claim 18, wherein each of said porous microparticles has the same analyte-specific reagent attached to its porous matrix or contains the same analyte-specific reagent.

22. The kit according to claim 21, wherein, in said plurality of porous microparticles, there are different subsets of microparticles, with each subset having its distinct label component attached to, contained in or otherwise associated with said microparticles of said subset; and all of said different subsets having the same analyte-specific reagent attached to or contained in said microparticles of said subsets, said analyte-specific reagent being specific for one analyte of interest; such that said different subsets of microparticles are identical in terms of the analyte-specific reagent attached or contained, but differ by the respective label component attached to, contained in or otherwise associated with said microparticles of each subset; with each subset being unambiguously defined and identifiable by said respective label component and being provided in a separate container.

23. The kit according to claim 21, wherein said kit is a kit for detecting one analyte of interest in a plurality of biological liquid samples, wherein the number of different subsets of microparticles provided in said kit equals the number of separate biological liquid samples provided.

24. The kit according to claim 18, wherein, in said plurality of porous microparticles, there are several different analyte-specific reagents attached to or contained in said microparticles.

25. The kit according to claim 24, wherein there are different subsets of microparticles, with each subset having its distinct label component attached to, contained in or otherwise associated with said microparticles of said subset; and wherein furthermore, in said plurality of porous microparticles, there are different classes of subsets of microparticles with each class of subsets having a different analyte-specific reagent attached to the porous matrix of said microparticles or contained in said microparticles; wherein there are at least two different classes of subsets of microparticles; such that said different subsets of microparticles differ by the respective label component attached to, contained in or otherwise associated with said microparticles of each subset; and each subset of microparticles forms part of one class of subsets of microparticles; with each subset being unambiguously defined and identifiable by the respective label component and the respective analyte-specific reagent and being provided in a separate container; and such that said different classes of subsets of microparticles differ by the respective analyte-specific reagent attached to the porous matrix of said microparticles or contained in said microparticles; and each of said different classes comprises several subsets of microparticles, all of which subsets have the same analyte-specific reagent attached or contained.

26. The kit according to claim 24, wherein said kit is a kit for detecting more than one analyte of interest in a plurality of biological liquid samples, wherein, the number of different subsets of microparticles provided in said kit equals the number of separate biological liquid samples provided, multiplied by the number of analytes of interest to be detected, and wherein, in said kit, there are as many classes of subsets of microparticles provided as the number of analytes of interest to be detected.

27. A cartridge for performing a method of detecting and/or quantitating an analyte of interest in a plurality of biological liquid samples, wherein said 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 either a single combined mixing and detection chamber, or a combination of a separate mixing chamber and a separate detection chamber; wherein each sample-specific module comprises a sample compartment having its own separate sample inlet, each sample-specific module being configured to separately receive exactly one biological sample only, in the respective sample compartment; each sample-specific module being furthermore configured to receive microparticles in said sample compartment; each sample-specific module being further configured to facilitate a phase-transfer of said microparticles from an aqueous environment to a non-aqueous environment.

Description

[0154] Furthermore, reference is made to the figures which are given to illustrate, not to limit the present invention. More specifically,

[0155] FIG. 1A shows an embodiment of a basic scheme of an assay/method in accordance with embodiments of the present invention showing that the workflow of such method comprises a sample-specific part (referred to in the figure as “specific process part”) that is preferably performed for as many times as there are different samples to be tested, and a generic process part which is common to all microparticles, irrespective of which sample(s) they have been exposed to. The upper box depicts an embodiment of a basic process and possible components of a sample-specific part of the process. A sample is lysed using a lysis buffer, as a result of which the analyte(s) of interest, herein also sometimes referred to as “target(s)”, is (are) released. Optionally, a suitable dilution buffer (herein also sometimes referred to as “binding buffer”) may be used to adjust concentrations of certain reagents in the lysis buffer to an acceptable range, whereupon the sample including the suspected analyte of interest, is subjected to conditions in which the sample including the analyte(s) of interest, may be absorbed or adsorbed by microparticles that carry a certain label that is now assigned to the respective sample, such labelled microparticles herein also sometimes referred to as “encoded microparticles” or “encoded beads”. Such “encoded microparticles” that have been exposed to one sample, are herein also sometimes referred to as a “subset of microparticles”. Each such “subset” is characterized by a specific label component that is attached to, contained in or otherwise associated with the respective subset. In some embodiments, the respective analyte may also be bound to the microparticle(s) by affinity binding to the matrix forming the particle or to an analyte-specific reagent (ASR) that have been attached to or are contained in the particles for this purpose. Once the analyte(s) of interest has been incorporated/accommodated within the microparticle(s), the respective subset of microparticles is exposed to a suitable detection composition the nature and content of which depends on the detection reaction that is to be performed with the analyte of interest (which detection reaction, in turn, depends on the type of analyte to be detected). Such detection reaction comprises reagents necessary for performing a detection reaction of the analyte of interest. Preferred examples of such detection composition are further defined above. Subsequently, once the respective detection composition has been taken up in the microparticles, there may follow a step of removing any aqueous phase that is surrounding the respective microparticle(s). Such step may involve filtration, centrifugation, shaking or other mechanical agitation, and combinations thereof, that is performed with the respective microparticles. Thereafter, preferably after removal of any aqueous phase surrounding the respective microparticle(s), the respective microparticle(s) are transferred into a non-aqueous phase, such as an oil, optionally including an emulsifier. In preferred embodiments, the emulsifier helps in isolating the individual microparticle. As a result of such transfer into a non-aqueous phase, there will be a suspension of isolated microparticles with, possibly, analyte(s) of interest from this particular sample. The same process is repeated with a different type (“subset”) of microparticles, encoded differently, i. e. having a different label component attached to, contained in or otherwise associated therewith, with such differently encoded microparticles being exposed to a different sample. Once this process has been performed for as many different samples as necessary or desired, a first record of correlation may be generated indicating which separate subset of microparticles has been exposed to which sample. Such first record of correlation is herein also sometimes referred to as “sample/bead code list”. Once the process of exposure of different subsets of microparticles to different samples has been performed for as many different samples as desired or necessary, the respective isolated different subsets of microparticles may be mixed and subjected in the generic part of the process to a detection reaction which may be a nucleic acid amplification, or the performance of a immunochemistry reaction, or any other bio-chemical reaction suitable for detection an analyte of interest. Depending on the presence or absence of analyte in the respective sample (and subset of microparticles), there may be a signal generated in such detection reaction for a particular subset of microparticles. A list of for which beads a signal is detected (“Beads/signal code list” or “second record of correlation”) may thus be generated indicating in which subset of microparticles a signal has been generated in the detection reaction. Subsequently, the respective signal(s) generated in such detection reaction may subsequently be decoded (e.g. by aligning or comparing the second record of correlation (or the “Beads/signal code list”) with the first record of correlation (or the “sample/bead code list”), resulting in the finding that the analyte(s) of interest has been present (or absent) in one or several of the samples that had originally been used to fill/load the respective microparticle(s). Preferably, the determination of which sample contains the analyte of interest, i.e. the actual assignment of detected signal to is performed by linking the first record of correlation (i.e. the list indicating which subset has been exposed to which sample) with the second record of correlation (i.e. the list indicating in which subset a signal has been generated).

It should be noted that a major advantage of the method according to the present invention is the following: If samples were to be tested individually, a detection reaction would have to be performed individually and separately for each sample. The present invention allows for one part of the method (the “generic part”) which is identical and common to all the detection reactions that would otherwise be performed individually and separately for each sample, to be performed once for all samples being analyzed.

[0156] FIG. 1B shows an embodiment of a basic scheme of an assay/method in accordance with embodiments of the present invention showing that the workflow of such method can be also applied favorably for processing a single sample. The employed beads provide for a lean process that utilizes the porous material for target enrichment, clean-up and for performing a signal generation reaction such as PCR in individual nanoreactors, that provide for exquisite quantitation of target present in the sample across a broad measurement range.

[0157] FIG. 2 shows an embodiment of an exemplary microparticle in accordance with the present invention which has a porous matrix and is made up of a polymer which, in this particular case, is also capable of undergoing a phase transition upon an external trigger. For example, in a preferred embodiment, the microparticle having a porous matrix is composed of a porous polymeric matrix which polymer making up such porous polymeric matrix is capable of under undergoing a sol-gel transition, for example upon raising the temperature. Once the respective microparticles have been insulated in a non-aqueous phase, the temperature may be raised up to a level, where the microparticle undergoes such phase transition, resulting effectively in the formation of an aqueous droplet in which a detection reaction may take place. In accordance with this embodiment shown in FIG. 2, the microparticle(s) also has an increased binding affinity for a particular analyte of interest. This may be a feature of the polymer matrix itself or, for example, be achieved by an analyte-specific binding molecule (binder) reagent attached to the matrix. Such binder may, in some instances, for example, be an analyte-specific reagent (ASR), as defined further above. Analyte-specific binders may be selected from nucleic acids, including nucleic acid pairs, in particular primer pairs; aptamers, Spiegelmers; antibodies or antibody fragments; non-antibody proteins capable of specifically binding an analyte or analyte complex, such as receptors, receptor fragments, and affinity proteins. In a preferred embodiment, the analyte-specific reagent is selected from nucleic acid, in particular nucleic acid oligomers and nucleic acid primers. Furthermore, the microparticle(s) has (have) a label component attached to or contained within or otherwise associates therewith. Moreover such a microparticle may (optionally) comprise reversibly attached reagents for a signal generating detection reaction to be performed within the space provided the microparticle. It should be noted that, in accordance with embodiments of the present invention, a pair of primers that is suitable for amplifying a particular nucleic acid sequence (which is the “analyte” or “target”), may herein qualify and be regarded as a single analyte-specific reagent (ASR) despite the fact that such pair of primers comprises two different primers. They are, however, specific for a single nucleic acid sequence in that they flank the same region of such nucleic acid sequence, and thus are specific for the same nucleic acid analyte

In preferred embodiments, the label component is a fluorescent dye, more preferably a mixture of two different fluorescent dyes.
The microparticle(s) in accordance with embodiments of the present invention possess a capacity to accommodate, preferably bind or enrich, the analyte of interest in their porous matrix, and can be identified and distinguished against other microparticles by means of the respective label component that is attached to, contained in or otherwise associated with the respective microparticle(s). Because the microparticle(s) in accordance with the present invention is (are) of a porous nature, they provide for an accessible inner volume that is sufficient to take up (or “absorb”) sample, including any analyte of interest, if present, or to bind (to “adsorb”) the analyte to the matrix of the microparticle by e.g. an analyte specific binding molecule attached to said porous polymer matrix; ionizable groups, or a plurality of ionizable groups, immobilized on said porous polymer matrix, said ionizable group(s) being capable of changing its(their) charge(s) according to ambient conditions surrounding said precursor-microparticle; a charged group, or a plurality of charged groups immobilized on said porous polymer matrix; or a combination of any of those.
Preferably, such microparticles are furthermore capable of undergoing a phase transition upon the application of an external trigger, such that they may, for example upon raising the temperature above a certain threshold, transform from a gel state at room temperature into a soluble state at elevated temperature. When the particles at the same time are in a liquid, their suspension at room temperature will, thus, transform into an emulsion at elevated temperatures.
Preferred polymers that function in this manner are agarose polymers, alone or in conjunction with gelatin.

[0158] FIG. 3 shows the basic functionality of the microparticles (“Reactor beads”) in accordance with embodiments of the present invention. The figure shows three different types of microparticles, each being distinguished from each other by virtue of the respective label component associated with such respective type. In the present representation of FIG. 3, the different label components are symbolically represented by differently hatched areas within the circular representations of the microparticles/beads; such differently hatched areas may stand for any suitable label components as long as they allow to distinguish the different microparticles. E.g. they may represent different concentrations of a single dye, different ratios of two different dyes, different detectable physical labels, etc. In a first part of the process which is the “sample-specific part” of the process, the three different types of microparticles are separately exposed to three different samples 1-3, such that, effectively, each sample is encoded by the label component of the respective type (“subset”) of microparticle which is exposed to such sample. In a preferred embodiment, such correlation between label component and respective sample associated therewith, is achieved by generating a list of label components (of corresponding subsets of microparticles) with corresponding associated samples, herein also sometimes referred to as “a first record of correlation”. Such a list may herein also sometimes be referred to as a “sample/microparticle code list” or “sample/bead code list”, indicating which subset of microparticles has been exposed to which sample. In this embodiment, following a series of process steps, the microparticles are loaded with detection reagents and are collected in a non-aqueous environment that provides for an insulation of the microparticles and prevents any cross talk between the microparticles. In a second part of the process, which is the “generic part” of the process, the different suspensions of microparticles that encode and correspond to different samples in a non-aqueous phase are mixed and are subjected simultaneously (“in one go”, i.e. no longer separately from each other) to conditions that are necessary to perform the required detection reaction, for example a nucleic acid amplification, such as PCR involving a temperature cycling. Because such “generic part” allows the processing of a large number of samples, this facilitates the performance of a single detection process with samples of entire patient cohorts of a large size, e.g. in a clinical trial. The signal(s) produced in such detection reaction is(are) recorded, and the respective label components of the (subsets of) microparticles are determined/read. A second record of correlation is generated indicating in which subset of microparticles a signal has been generated. Such second record of correlation is herein also sometimes referred to as “Bead/signal code list”). The resultant detection signal/microparticle combinations in the “bead/signal code list” (i.e. the second record of correlation) are compared with the “sample/microparticle code list” (“i.e. the first record of correlation), as a result of which it can be determined which sample (of the plurality of tested samples) did or did not contain the respective analyte of interest.

Microparticles which are particularly suitable for embodiments, in accordance with the present invention are the microparticles as contained in a library of prefabricated microparticles for performing a specific detection of an analyte of interest in a sample, as described in co-pending European patent application entitled a library of prefabricated microparticles (Attorney docket B33016EP), filed concurrently herewith.

[0159] FIG. 4 shows a schematic diagram of an exemplary layout of a novel fluidic cartridge that may be suitable for performing embodiments of the method according to the present invention. Unique features of the cartridge are separate multiple inlets for the individual samples to be analyzed. Each inlet is separately connected with its own corresponding sample chamber or sample-specific module, and there are a number of different sample chambers or sample-specific modules provided in the cartridge each of which is intended and configured to be used for a separate sample. The terms “sample chamber”, “sample chamber module” and “sample-specific module” are used interchangeably herein and refer to a chamber that is specifically intended and configured to receive a specific sample. The actual space within the respective sample-specific module that is configured to contain the sample (and to contain the specific subset of microparticles for this particular sample) is herein also sometimes referred to as “sample compartment”. These specific chamber modules are provided to ensure a safe encoding process, i. e. a process in which each sample becomes encoded/labelled by exposing a specific separate type (“subset”) of microparticles to such sample, thus resulting in each of said different subsets of microparticles being “loaded” with a different sample, respectively. The different sample-specific chamber modules prevent any cross contamination between the different modules and thus different samples. The respective separate inlets are shown in the figure by triangular indentations in the sample chambers (or sample-specific modules) which are represented by rectangular boxes standing on their smaller side(s).

The exemplary cartridge layout in this figure comprises an exemplary valve and pump mechanism. Three separate groups of chambers (or modules) are provided on the cartridge layout: [0160] 1. modules facilitating the sample specific process and that among other parts contain the sample encoding microparticles [0161] 2. chambers for storing regents required to perform the specific (if not provided within the sample specific modules) and the generic processes; also shown is a specifically designated storage chamber for containing a non-aqueous phase (“non-aqueous phase chamber”) [0162] 3. chambers or modules facilitating the generic process (e.g. a mixing (optional) and a detection (compulsory) chamber, or a combined mixing and detection chamber, i.e. a chamber wherein both functions are combined (not shown))
All the reagents are delivered to the respective modules in a unidirectional fashion before the encoding process has been completed, and before the microparticles are insulated, i. e. secured in the non-aqueous phase. Eventually, a suspension of microparticles is collected from each sample module, and the different suspensions are then mixed together to form a single suspension. Such mixed microparticles are then transferred to a detection chamber and are subjected to the necessary conditions that are required for performing a detection reaction. A cartridge that may be adapted and, upon modification, be used in accordance with embodiments of the present invention is a cartridge as described in European patent application EP No. 19 187 064.1, filed on Jul. 18, 2019, which needs to be adapted however, insofar as it requires modification in that the cartridge according to the present invention has a plurality of sample chambers, and each of such sample chambers has its own sample inlet. This allows for the separate manipulation of a plurality of samples each of which becomes separately labelled in accordance with the present invention.

[0163] FIG. 5 shows an embodiment of a method according to the present invention. More specifically, different subsets of microparticles (referred to in the figure as “bead 1”, “bead 2”, “bead 3” and “bead 4”, respectively) have a different label component attached, but either contain no analyte-specific reagent at all, or they comprise, exactly, one type of analyte-specific reagent that is specific for one specific analyte of interest. The different subsets of microparticles are identifiable by their different label components and are provided separately, e.g in different containers, such as different sample chambers or sample-specific modules within a cartridge, as described further above, and each of them is exposed separately to a different sample. Effectively, there will be used as many subsets of microparticles as there are samples to be tested. Thereafter, the necessary (generic) detection reagent/reagent composition is added to each subset (with such detection composition optionally additionally also including an analyte-specific reagent, as defined further above, if such analyte-specific reagent had not been contained within the microparticles from the start). Then, the respective subsets of microparticles are, still separately, transferred into a non-aqueous phase (which may be an oil, optionally containing emulsifier), thereby becoming insulated from each other, as a result of which there will be no “cross talk”, i. e. mixing of samples between the spaces provided by the respective microparticles, anymore, and thereafter a (generic) detection reaction is performed (“one-pot detection reaction”). The results of such (generic) detection reaction allows for a conclusion to be drawn in respect of which sample the one analyte of interest has been present. Surprisingly, the present inventors have found that there is, indeed, no spilling over or “cross talk” between different microparticles once they have become isolated from each other by having been transferred into a non-aqueous phase. As a result there will also be no cross-contamination between samples (to be tested for the presence of an analyte), despite the fact that their respective microparticles have become mixed together.

[0164] FIG. 6 shows an embodiment of a method according to the present invention in which multiple samples are tested for multiple analytes. In principle, the scheme is similar to what is shown in FIG. 5 except for that there will be used as many subsets of microparticles as there are samples to be tested, multiplied by the number of different analytes to be detected. As an example, the first sample in FIG. 6 is tested/analyzed using two different subsets of microparticles which not only differ in terms of the label component associated therewith but also in terms of the analyte-specific reagent attached to such type. The same is done for the second sample (“sample 2”). Hence, in this example there are altogether 4 different subsets of microparticles, with each of them having its unique combination of label component and analyte-specific reagent. Thereafter, the respective necessary (generic) detection reagent/detection composition is added, but still on a per-sample-basis, in order to avoid a cross-contamination between different samples. Thereafter, the respective microparticles are transferred, again, on a per-sample-basis into the non-aqueous phase, and only after these respective microparticles have been insulated in such a manner and can no longer interfere with and cross contaminate each other, the necessary detection reaction is performed. The final result will be a finding whether or not each sample tested had none, one or two of the analytes to be detected. Again, as pointed out above, surprisingly, the present inventors have found that there is, indeed, no spilling over or “cross talk” between different microparticles once they have become isolated from each other by having been transferred into a non-aqueous phase. As a result there will also be no cross-contamination between samples (to be tested for the presence of an analyte or several analytes), despite the fact that their respective microparticles (each of which comprises a small volume of the respective sample to be tested) have become mixed together.

[0165] FIG. 7 shows a micrograph of the microfluidic cross junction of the set up used to generate microparticles according to the invention. Microdroplets are formed in oil and form an aqueous hydrogel solution (at the left). Subsequently the droplets are cooled and microparticles formed.

[0166] FIG. 8 shows a fluorescence micrograph acquired with a Cy3 Filter Set on a Zeiss Axioscope equipped with a digital camera. Three different fluorescence levels can be seen (bright, medium, low) representing three concentrations of the label component (Cy3 dye), corresponding effectively to three different label components.

[0167] FIG. 9 shows a stitched image of the detection chamber representing the fluorescence channel encoding the label of the beads. Three different types of microparticles (or “beads”), distinguishable (and thus encoded) by their respective label components, can be clearly recognized (bright, medium and low signal) in the enlarged image insert on the lower right. Because of their different label components, these beads can be used to label or “encode” different samples (and are therefore also sometimes herein referred to as “sample encoding beads”). The beads are spatially distributed in hexagonal close packing. The total number of all Sample encoding beads (mean diameter d=105 μm) recognized by the employed software algorithm within this PCR chamber has been determined with N=17.698.

[0168] FIG. 10A shows fluorescence images of color-labeled agarose-gelatin hybrid microparticles post PCR amplification. Three labels can be recognized by their fluorescence intensity in channel 1. Channel 2 represents fluorescence signals for the internal process control (MS2 phage) and channel 3 represents fluorescence specific for PCR amplification of the HCV target. Each label corresponds to a different sample that has been processed and analyzed. FIG. 10B (upper graph) shows the histograms for the determined fluorescence intensity for the different beads from channel 1 (upper image in FIG. 10A). Three separate label species are found. Violin plots representing the signal distribution found across the respective bead species for channel 2 (MS2) and channels (3) are shown in the middle and lower graphs. By applying Poisson analysis the found numbers are translated into a specific copy number per volume sample applied to the beads.

[0169] FIG. 11 summarizes the data from example 3 on the binding efficiency of different microparticle formulation for RNA. The data shows that adjustments to the composition of the microparticles allow for optimization of binding characteristics.

[0170] FIG. 12 shows a time course fluorescence images showing RNA binding to the prepared microparticles in Example 3.

[0171] FIG. 13 illustrates the enrichment effect achieved by the use of the microparticles. The graph shows data for two different compositions of microparticles. Black dots indicate the target concentration in the sample before incubation with the microparticles determined by digital RT-PCR. White columns represent the target concentration detected after the incubation in the supernatant, grey columns target concentration on the microparticles. The enrichment effect is clearly visible, showing a depletion of the respective supernatant(s).

[0172] FIG. 14 shows the precision of the combined digital and real-time quantitative PCR analysis approach for target quantitation in microparticles. Confidence intervals are shown for both methods. The vertical line at λ=5 cp/microparticle (cp=copies) indicates the approximate value at which methods can be used interchangeably (“CI”=confidence interval).

[0173] FIG. 15 shows real time fluorescence data obtained from an image series collected on microparticles in oil during PCR amplification. A stitched image of a detection chamber with the endpoint fluorescence signal detected in one fluorescence channel specific for amplification is shown on the left. The graph in the center shows exemplarily fluorescence intensity for 12 representative individual microparticles selected from the fluorescence image on the left over time. A distribution of the calculated ct values for each microparticle detected in the fluorescence image is shown in the histogram on the right.

[0174] Moreover, reference is made to the specific description, in particular the following examples, which are given to illustrate not to limit the present invention.

EXAMPLES

Example 1: Generation of “Sample Encoding Beads” for a RNA Detection Assay

[0175] Differently labeled Microparticles with a matrix comprised of Gelatin and Agarose have been generated, whereby the Gelatin fraction of the composition served as the dye carrying part of the matrix and because of its composition provided for target binding and enrichment.

[0176] The Labeling of Gelatin with Fluorescent Dyes for Creating a Particle Code was Carried Out as Following:

[0177] The acetone insoluble fraction of gelatin from bovine skin type A is labelled with a fluorescent dye taking advantage of NHS coupling chemistry. Cy®3 Mono NHS Ester (GE Healthcare) were dissolved in DMF to make a final solution of 10% (w/v). Component 1 is dissolved in 70 mM sodium phosphate buffer (pH 8.0-8.3, sterile filtered) to make a final concentration of 0.25% (w/v). A 10-fold lower molecular amount of the respective dye over free gelatin amino groups is utilized to label 25 mL of either gelatine type. The label solutions are incubated at 4° C. overnight using the Multi-Rotator PTR-60 (Grant-bio) in the vertical mode. Fluorescently labelled gelatin purification and its concentration is accomplished by repeated ammonium sulfate precipitation using a saturated (NH.sub.4).sub.2SO.sub.4 solution. Alternatively, gelatin can be in a gelled particle-sized format for coupling and can be purified without ammonium sulfate precipitation by simply washing and centrifuging at ambient temperature. Also, ultrafiltration, solvent extraction with isopropanol, acetone or methanol, gel filtration using sepharose columns or dialysis can be performed to purify the gelatin.

[0178] In either case, purification is repeated until effluent appears clear and shows no fluorescence. Purified fluorescently labelled gelatin samples are finally vacuum-dried after washing the pellet four times with dH.sub.2O resulting in component 3.

[0179] Preparation of Gelatin/Agarose Hybrid Solution:

[0180] A hybrid hydrogel solution consisting of three components is prepared for fabricating nano-reactors. [0181] Component 1: Acetone-insoluble gelatin from bovine skin type A G1890 (Sigma) [0182] Component 2: Low-gelling 2-Hydroxyethyl agarose (A4018, Sigma) [0183] Component 3: Cy3-labeled gelatin (type A)

[0184] To generate a homogeneous 4% (w/v) solution of component 1, 40 mg of component 1 is dissolved in 1 mL nuclease-free water (Carl Roth) and incubated at 50° C. under gentle agitation (750 rpm). Likewise, 20 mg of component 2 is dissolved and melted in 1 mL nuclease-free water and incubated at 80° C. under gentle agitation to prepare a homogeneous 2% (w/v) agarose solution. To prepare a 4% (w/v) solutions of labelled gelatin, a dried pellet of component 3 is taken up in a respective volume of nuclease-free water and incubated at 55° C. until the gelatin is molten. All three components are mixed and filled up with nuclease-free water to generate a hybrid hydrogel solution with final concentrations of 1.5% (w/v) for gelatin and 0.5% (w/v) for agarose A4018, respectively. Various volumes of component 3 and component 1 are mixed yielding n distinctly colored microparticle sets. In this embodiment resuspended component 3 and component 1 are mixed in ratios 1:1000, 1:500, 1:250 to accomplish three individual label components to allow for identification and analysis of three different samples. All solutions were kept at 55° C. until further use.

[0185] Generation of Un-Crosslinked Gelatin/Agarose Microparticles

[0186] Monodisperse color coded agarose-gelatin hybrid microparticles were fabricated using a microfluidic particle generator system (Dolomite, UK). Monodisperse hybrid microparticles are fabricated in a one-step process of emulsion formation using a simple flow-focus device. In detail, a standard droplet junction chip (100 μm) of fluorophilic nature is used with a 4-way linear connector and a chip interface H to interface the fluidic connection between tubing and chip. Two Mitos P-Pumps deliver the hydrogel solution and the carrier oil. The system is modified with the integration of a heating rig which is placed on top of a hot plate and allows for maintaining the gelatin/agarose hybrid solution in liquid state and heating up the driving fluid ensuring consistent temperature when oil and gelatin/agarose hybrid solution contact at the chip junction. Picosurf 2 (Spherefluidics, UK) and the hybrid hydrogel solution are both pre-filtered with a 0.22 μm filter before placing them into the P-Pump (Mitos) and the hydrogel reservoir within the heating rig of the droplet system, respectively. Temperature of the heating rig is set to 55° C. The fluid lines are primed at 2000 mbar for 1 min using the Flow Control Software. A flow rate of 15-20 μl/min is adjusted for stable droplet formation. Parameters are monitored with the Dolomite Flow Control Advanced Software.

[0187] FIG. 7 shows a micrograph of the microfluidic cross junction with microdroplets forming on the left in the oil phase. Subsequently the hydrogel droplets solidify ad become microparticles.

[0188] In both cases, the color-coded agarose-gelatin hybrid microparticles are collected on ice in either 2 mL microcentrifuge tubes or 15 mL falcon tubes to initiate solidification of the hybrid hydrogel. To prevent loss of aqueous phase of the microparticles at the oil-air boundary, 500 μL of the emulsion oil containing the microparticles is overlaid with 500 μL of nuclease-free water. Microparticles are subsequently cooled down to 4° C. for at least 24 hours (preferentially 48 h) to form stable hybrid scaffolds.

[0189] Recovery of Hybrid Microparticles from Continuous Phase

[0190] Solidified hybrid beads accumulate on top of the emulsion oil. The emulsion oil is removed carefully with a pipette taking care not to remove the particles. Afterwards, 500 μL 1H,1H,2H,2H-perfluorooctanol (PFO; Sigma) is added to the tube to break the emulsion. To transfer the hybrid hydrogel particles into the oil phase, the tube is vortexed for 5 s and centrifuged at 2,500×g for 5 s. The hybrid hydrogel microparticles are transferred to a fresh 1.5 mL microcentrifuge tube.

[0191] Optional: This procedure can be repeated to remove residual fluorocarbon oil and surfactant. Following the PFO wash, recovered microparticles are washed twice with 1 mL nuclease-free water. Microparticle quality and sizes are visually examined using a microscope. The final result of the described procedure yields nanoreactor microparticles (diameter d=100 μm) sets with a porous polymeric matrix and a sample encoding capacity.

[0192] FIG. 8 shows a fluorescence micrograph acquired with a Cy3 Filter Set on a Zeiss Axioscope equipped with a digital camera. Three different fluorescence levels can be seen (bright, medium, low) representing three concentrations of the dye (Cy3 dye), and thus effectively three different label components allowing a distinction between the respective microparticles labelled therewith.

[0193] Lyophilisation of Microparticle Sets

[0194] Optionally, the microsphere sets can be lyophilized to give microparticle pellets for long-term storage. Therefore, desired microsphere sets, and concentrations are supplemented with an equal volume of a 600 mg/mL trehalose solution to generate a 30% (w/v) trehalose containing microparticle library slurry. Subsequently, library aliquots of 100 μl are prepared in RNase/DNase-free PCR strip tubes ready for lyophilisation. The type of excipient (e.g. trehalose) and its concentration in the lyophilisation formulation affects the degree of swelling of the freeze-dried microspheres when exposed to an eluate later in the process. The microsphere sets were freeze-dried under vacuum (−25° C. and 0.1 mbar) using the Alpha 2-4 LSCplus freeze-drier (Christ) after freezing on dry ice for 2 h. The samples were left in the freeze dryer for a total time of 200 min. The main drying stage was held at 0.01 mbar for 3 h with a stepwise increase in temperature, from −25° C. to 25° C. The final drying step was conducted at 25° C. and 0.05 mbar for 20 min.

Example 2: Encoding of Samples with “Sample Encoding Beads”

[0195] Sample Preparation Using Hybrid Beads

[0196] Preparation of Gelfiltration Resin

[0197] Dry P-2 Bio-Gel Media Extra fine <45 μm (BIO-RAD 150-4118) is allowed to hydrate in nuclease-free deionized H.sub.2O. Subsequently the gel is filled into an empty spin column (i.e. SigmaPrep™ spin columns, Sigma-Aldrich SC1000-KT) to yield a final bed volume of 400 μl. The column is subjected to a short centrifugation step of 1 min at woo rcf to remove the mobile phase. The column is washed two times with 400 μl of 50 mM Tris HCl buffer pH 8.4 by adding the buffer and subsequently spin the columns as described above.

[0198] Sample Lysis

[0199] Three Samples of 30 μl of whole blood obtained from a healthy donor (Institute of Transfusion Medicine, University Hospital Jena) have been spiked with 300 cp/μL of MS2 virus (Sample 1, 2 and 3) and one of the three samples (Sample 1) has been spiked with AccuPlex HCV Recombinant Sindbis Virus (SeraCare 0505-0036) diluted in Basematrix Negative Diluent (SeraCare 1805-0075), resulting in a nominal titer of 5.500 cp/μL sample.

[0200] All sample have been separately mixed with 195 μl of lysis buffer each, containing 4.5 M Guanidinium HCl, 9% Triton X100, 18 mM EDTA and 100 mM Tris HCl pH 8.0. Each lysis mix has incubated at 65° C. for 5 minutes.

[0201] Gelfiltration to Remove Guanidinium HCl

[0202] Immediately after lysis each mix has been applied to the P-2 column. The flow through/filtrat has been recovered after 1 minute of centrifugation at 1.000 rcf into a fresh reaction tube containing 200 U of RNase Inhibitor (biotechrabbit, DE).

[0203] Binding of the Analyte to Sample Encoding Beads

[0204] Samples are assigned to the fluorescently labelled microparticles as shown in the following Table.

TABLE-US-00001 Assignment of samples to Sample Encoding Beads Sample Encoding Subtype HCV MS2 Sample 1 0 + + Sample 2 1 − + Sample 3 2 − + + = positive; − = negative

[0205] Pellets of lyophilized microparticles are resuspended in 70 μL RNA-containing binding buffer (component 4), with each set consisting of roughly 10.000 nanoreactors available for digital PCR analysis. These are allowed to absorb the buffer and to instantly swell providing for a functional porous 3D gelatin-agarose matrix developed for efficient and unspecific binding of nucleic acids and digital PCR compatibility. Individual samples are incubated with their corresponding beads at 15° C. for 5 min at 1000 rpm in separate tubes to fully capture HCV and MS2 RNA resulting in an enrichment of nucleic acids in the nanoreactors.

[0206] Washing the Microparticles

[0207] 200 μl of washing buffer (50 mM Tris HCl pH 8.4, 1 U/μ1 RNaseInhibitor) were added to each sample. After a short vortexing step at 12.000 to 16.000 rpm for 1 second, the microparticles are sedimented by centrifugation at 300 rcf for 30 seconds and the supernatant is removed. The washing step is repeated 3×.

[0208] 1. Detection and Quantitation of a Molecular Target in Different Samples with “Sample Encoding Beads”

[0209] Loading the Reagents onto the Microparticles

[0210] The reagents for the detection of an analyte within the nano-reactors are supplied as a freeze-dried pellet that is resuspended with 100 μL of 1× PCR buffer (component 7) making a 2× reagent mixture (component 8). The reagents are carefully resuspended by a short vortexing step. An equal volume of the reagent mix with regard to the bead slurry is distributed into the individual containers with individual sample encoding bead sets that have captured the RNAs. The amplification and detection reagents are allowed to diffuse into (and bind to) the hydrogel matrix by a short incubation of 5 min at 15° C. while shaking at 1000 rpm.

[0211] Component 7: PCR buffer (20 mM Tris HCl, 22 mM KCl, 22 mM NH4Cl, 3 mM MgCl2, pH 8.5)

[0212] Component 8: Reagent mixture (2×) [0213] 2× thermostable Reverse Transcriptase with Ribonuclease Inhibitor (biotechrabbit GmbH) [0214] 0.4 U/μl Hot Start Taq DNA Polymerase (biotechrabbit GmbH) [0215] 0.8 mM dNTPs (biotechrabbit GmbH) [0216] 0.2% (w/v) low bioburden, protease free, for molecular biology BSA (Sigma)

[0217] Analyte-specific reagents (HCV)

TABLE-US-00002   0.8 μM HCV-Brun sense primer SEQ ID NO: 1 (5′-GTGGTCTGCGGAACCGGTGA-3′) 0.8 μM HCV-Brun antisense primer SEQ ID NO: 2 (5′-CGCAAGCACCCTATCAGGCAGT-3′) 0.8 μM HCV-Brun TaqMan probe SEQ ID NO: 3 (5′-Atto647-CCGAGTAGYGTTGGGTYGCGAAAGG-BHQ-2-3′)

[0218] Internal Process Control-specific reagents (MS2)

TABLE-US-00003   0.8 μM MSs-Nino sense Primer SEQ ID NO: 4 (5′-CTCTGAGAGCGGCTCTATTGGT-3′) 0.8 μM MS2-Nino sense Primer SEQ ID NO: 5 (5′-GGTCCCTACAACGAGCCTAAATTC-3′) 0.8 μM MS2-Nino TaqMan probe SEQ ID NO: 6 (5′-FAM-TCAGACACGCGGTCCGCTATAACGA-BHQ-1-3′)

[0219] Emulsification of Microparticles

[0220] Individual nano-reactor sets are transferred separately into a non-aqueous phase by dispersing microparticles in component 9 to prevent crosstalk between the sample-encoded beads. The beads are centrifuged at 500 rcf for 30 s and the supernatant is discarded. The bead bed is brought in contact with an excess of component 9 (200 μL) using a 1.5 mL microcentrifuge tube. High shear forces need to be applied to deagglomerate and emulsify aqueous microparticles in the fluorocarbon oil into single nano-reactors. The mixtures are agitated by either application of ultrasound using the Sonifier™ S-450 and the Ultrasonics Sonifier™ 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/s while pressing the tube against the rack surface. This applies mechanical stress and breaks attracting forces between the aqueous microparticles and creates surface tension forming a suspension/emulsion. Both the hydrogel microparticles and excess aqueous phase are emulsified in the oil phase. The submicron-scaled droplets that are produced as a byproduct are eliminated by washing the emulsion three times by mild centrifugation (500 rcf). Repeated washing with the same oil (component 12) removes essentially all undesirable liquid droplets. Component 12 also yields efficient thermostability of the emulsion for subsequent digital emulsion PCR. The three emulsified Sample Encoding Bead sets can now be simply combined into one container (tube) prepared for parallel digital signal amplification and detection of encoded samples.

[0221] Component 9: phase transfer & signal amplification oil [0222] HFE-7500 fluorocarbon oil (3M Deutschland GmbH) supplemented with [0223] 2-5% (v/v) PicoSurf (Dolomite Microfluidics) OR 2-5% (v/v) FluoSurf (Emulseo) OR 2-5% (v/v) 008-FluoroSurfactant (RAN Biotechnologies)

[0224] Parallel digital PCR amplification reactions in Sample Encoding Beads

[0225] The monodispersed emulsion with encapsulated sample 1, sample 2 and sample 3 is transferred into a detection chamber with an area of approximately 2.5 cm.sup.2 and a layer thickness of 100 μm. The chamber detection window is made of a 0.8 mm Polycarbonate (Makrolon 6555; Covestro AG) while the opposite side of the chamber is composed of polished and unmodified transparent 125-micron Polycarbonate (Lexan 8010) film (Koenig Kunststoffe GmbH) facilitating efficient heat transfer necessary for individual nanoliter reactions. Nanoreactors suspended in the fluorocarbon oil are forced to form a monolayer owing to the dimension of the reaction chamber. Thus, microspheres provide an evenly spaced array of approximately 20.000-30.000 nano-reactors (≈70.000-10.000/sample) for subsequent parallel signal amplification reactions.

[0226] Microparticles are subjected to ultra-rapid temperature cycling using a modified PELTIER element 30×30×4.7 mm, 19.3 W (Quick-Ohm, Küpper & Co. GmbH, #QC-71-1.4-3.7M) and an established chamber-specific PCR control mode. The thermal RT-PCR conditions applied are: Reverse Transcription for at 50° C. for 10 min, Initial denaturation at 95° C. for 30 s followed by 30-45 cycles of a two-step PCR consisting of Denaturation at 95° C. for 2 s and Annealing/Elongation at 65° C. for 5 s. Due to their sol-gel switching capability, the suspension becomes an emulsion with individual liquid nanoliter droplets. The multiple duplexed amplifications of HCV and MS2 (control) from different samples takes place in the labelled nano reaction compartments.

[0227] Automated Image acquisition is triggered by the BLINK toolbox software and is done with a Fluorescence microscope (Zeiss AxioObserver) equipped with a 5× objective (field of view 4.416 mm×2.774 mm) and a pE-4000 (CoolLED Ltd.) light source. The microscope was further equipped with three fluorescence filter sets (Cy5 ET, Cy3 ET, FITC/FAM HC, AHF Analysentechnik) and an automated x-y stage to which the thermocycler with the reaction chamber was mounted.

[0228] Image acquisition settings are as following: 100-1000 ms and gain of 1-10×. One image is required for label identification (λexc 1=580 nm), one image for the internal control PCR signals (λexc 2=470 nm) and one image for specific PCR signals (λexc 3=635 nm). For optional nanoreactor specific real-time analysis, three images corresponding to three fluorescence channels can be taken at each cycle at a suitable position of the chamber.

[0229] Upon completion of the thermal protocol, the whole detection chamber field is scanned using the same equipment at the settings mentioned above. In total 48-56 images are required to cover the dimension of the amplification/detection chamber.

[0230] A stitched image of the detection chamber representing the fluorescence channel encoding the label of the beads is shown in FIG. 9. Three different Sample Encoding Beads can be clearly recognized (bright, medium and low signal) in the enlarged image insert on the lower right. The beads are spatially distributed in hexagonal close packing. The total number of all Sample encoding beads (mean diameter d=105 μm) recognized by the employed software algorithm within this PCR chamber has been determined with N=17.698.

[0231] Decoding of Samples and Endpoint Analysis

[0232] All images acquired are subjected to an automated multifaceted image processing algorithm. The method employs image segmentation exploiting the Maximally Stable Extremal Regions (MSER) to detect neighboring droplets from the result of MSER-based image segmentation. In detail, images are first subjected to preprocessing involving a median filter. Secondly, the MSER algorithm is applied to the image background to determined convex turning points of background outlines and their Delaunay triangulation to identify appropriate cuts between the droplets/microparticles. Furthermore, droplets/microparticles are segmented using the MSER algorithm. Finally, a plausibility check for droplet/microparticle outlines is performed (contrast, form, convexity). Features including fluorescence signals in each channel, position, diameter/volume, etc. of all segmented droplets/microparticles are subsequently collected. Experiment data is applied to a Jupyter script identifying individual labels (graduation of coupled Cy3 dye) and their respective specific amplification.

[0233] The PCR is amplified to an endpoint and thus the total number of fluorescent positive and negative droplets is determined for each individual label. Positive droplets contain at least 1 copy of the specific target and thus show an increase fluorescence signal above a defined intensity threshold. The threshold value is derived from previously performed amplification reactions without template or is determined in each experiment using statistics. Negative and positive fractions for each nanoreactor type are clustered and the fraction of positive droplets is fitted to a Poisson algorithm to determine the initial concentration of the target RNA molecules in units of copies/μL (copies/mL) input.

[0234] Since the assay described above provides simultaneous detection of HCV in 3 different samples, the reactors cluster into 6 groups:

TABLE-US-00004 Digital PCR endpoint data cluster. Sample Encoding HCV MS2 phage Cluster Sample Bead type (label) RNA RNA 1 Sample 1 0 Positive Pass 2 Negative 3 Sample 2 1 Positive Pass 4 Negative 5 Sample 3 2 Positive Pass 6 Negative

[0235] Fluorescence images of color-labeled agarose-gelatin hybrid microparticles are shown in FIG. 10A post PCR amplification. Three labels can be recognized by the their fluorescence intensity in channel 1. Channel 2 represents fluorescence signals for the internal process control (MS2 phage) and channel 3 represents fluorescence specific for PCR amplification of the HCV target. Each label corresponds to a different sample that has been processed and analyzed. FIG. 10B (upper graph) shows the histograms for the determined fluorescence intensity for the different beads. Three separate label species are found. Violin plots representing the signal distribution found across the respective bead species for channel 2 (MS2) and channels (3) are shown in the middle and lower graphs. By applying Poisson analysis the found numbers are translated into a specific copy number per volume sample (cp/μl) applied to the beads.

Example 3: RNA Binding Capability of Agarose-Gelatin Hybridbeads

[0236] Binding of RNA is examined for various hybrid beads consisting of various concentrations of different gelatins and agarose A4018. Bound RNA on microparticles was either measured qualitatively by using an RNA dye assay or quantitatively using the digital PCR format described above.

[0237] Preparation of Beads

[0238] Microparticles were generated as described before with the following deviations: [0239] Labelled fraction of gelatin was omitted thereby producing unlabelled microparticles [0240] The hybrid hydrogel solutions for generation of microparticles consisted of different concentrations of agarose and gelatin, namely: [0241] a) 1.3% gelatin, 1.0% agarose [0242] b) 0.5% gelatin, 0.5% agarose [0243] c) 1.3% gelatin, 0.5% agarose [0244] d) 2.0% gelatin, 0.5% agarose [0245] e) 40.0% gelatin, 0.5% agarose [0246] Two different gelatin types each of two producers were used for microparticle fabrication, namely: [0247] GELITA Image1 AP (typeA) [0248] GELITA Image1 SI (typeB) [0249] acetone-insoluble fraction of G1890 (Sigma, type A) [0250] acetone-insoluble fraction of G9391 (Sigma type B)

[0251] RiboGreen RNA Binding Assay

[0252] Wells of UV-Star microtiter plates (Greiner) are filled with 3 μL of a 50% bead slurry in a 1× binding buffer (component 4). Final concentrations of 1 ng/μL of RNA from different preparations, namely an RNA-spacer (Metabion), a tRNA from brewer's yeast (Roche Diagnostic) and a total RNA extracted from blood are incubated at 15° C. for 5-10 min allowing the RNA to penetrate into/onto the porous hydrogel matrix and bind. As binding occurs quickly, mixing directly after addition of RNA to the beads is crucial for homogenous distribution of RNA across the beads.

[0253] To visualize RNA that is bound to the hybrid microparticles 0.5 μL of a 1:2 diluted Quant-iT™ RiboGreen® RNA reagent aliquot is pipetted to the microtiter wells and mixed. The beads were washed twice with 200 μL of TE buffer and their fluorescence signal subsequently measured using a Fluorescence microscope (Zeiss AxioObserver) and a pE-4000 (CoolLED Ltd.) light source.

[0254] FIG. 11 illustrates that adjustments to the composition of the microparticles allow for optimization of binding characteristics. GELITA Image1 A gelatine in combination with A4018 leads to relatively low RiboGreen signals on the beads in comparison to the aceton-insoluble fraction of G1890 type A gelatine from Sigma. Total RNA, tRNA and the short RNA fragment can be bound to a greater extent with hybrid beads made of >1.3% gelatine in combination with 0.5% agarose. In combination with 1% agarose, better signals are also obtained for the RNAs tested (total RNA sample results are missing). Gelatine type B behaves differently in comparison to type A with regard to RNA enrichment on the beads. While almost no signals are visible for any of the GELITA SI gelatine beads for any of the RNAs (meaning only diffusion into particle but no enrichment), the beads made of the acetone-insoluble fraction of G9391 gelatine typeB show RiboGreen signals although those are weak for RNA spacer 1 and tRNA. Overall total RNA, tRNA and the short RNA fragment can be bound to a greater extent with hybridbeads made of >1.3% gelatine in combination with 0.5% agarose.

[0255] FIG. 12 shows binding of total RNA from yeast over the time to microparticles visualized with Quant-iT™ RiboGreen® RNA reagent. The assay format also allows for direct monitoring of RNA binding to the microparticles. Images have been acquired every 6 s and the increase in RiboGreen signal can be directly measured.

[0256] Quantifying RNA Binding by Digital PCR

[0257] For quantitative assessment of RNA binding capacity of the microparticles according to the invention digital PCR within the nanoreactors was performed as described above. The ability of the microparticles to transition from a solidified particle to a liquid droplet allows for providing both the binding and digital PCR detection matrix. Thus, bound HCV RNA molecules can be measured directly on the beads. Alternatively, the supernatant can be measured in a digital format using the DG8 Cartridge (Bio-Rad) for droplet generation.

[0258] 40 μL of binding buffer (component 4) containing purified (QIAamp Viral RNA Mini Kit) Accuplex HCV RNA (Seracare Life Sciences Inc.) were added to 40 μL of a bead bed and incubated at 15° C. for 5 min at 1000 rpm to bind the RNA. Beads were subsequently centrifuged at 300 xg for 30 s and the supernatant is kept on ice for subsequent analysis. 40 μL of the 2× reagent mixture (component X) is added to the bead bed (40 μL) and the amplification and detection reagents diffuse into the hydrogel matrix by a short incubation of 5 min at 15° C. while shaking at 1000 rpm.

[0259] Component X: Reagent Mixture (2×) [0260] 2× thermostable Reverse Transcriptase with Ribonuclease Inhibitor (biotechrabbit GmbH) [0261] 0.4 U/μl Hot Start Taq DNA Polymerase (biotechrabbit GmbH) [0262] 0.8 mM dNTPs (biotechrabbit GmbH) [0263] 0.2% (w/v) low bioburden, protease free, for molecular biology BSA (Sigma) Analyte-specific reagents (HCV) [0264] 0.8 μM HCV-Brun sense primer (5′-GTGGTCTGCGGAACCGGTGA-3′) SEQ ID NO: 1 [0265] 0.8 μM HCV-Brun antisense primer (5′-CGCAAGCACCCTATCAGGCAGT-3′) SEQ ID NO: 2 [0266] 0.8 μM HCV-Brun TaqMan probe (5′-Atto647-CCGAGTAGYGTTGGGTYGCGAAAGG-BHQ-2-3′) SEQ ID NO:3

[0267] Bead phase transfer, amplification reaction and analysis are conducted as described before. The supernatant (40 μL) is supplemented with PCR reagents (40 μL of 2× reagent mix) and subsequently emulsified using the DG8 Cartridge (Bio-Rad). 100 μL of the emulsion reagent HFE-7500 containing 2-5% Picosurf 2 (Sphere Fluidics) and 40 μL is applied into the bottom wells of the cartridge. Vacuum is applied in the collection well by pulling gently on a syringe connected to the well. Droplets are collected and transferred to a separate detection chamber for digital analysis. The settings for the amplification reaction and analysis of the droplets are identical to the settings for the beads. Also, a reference sample consisting of equal volumes of 2× reagent mix and the PCR buffer is emulsified and analysed likewise.

[0268] FIG. 13 shows summary data on these experiments that were designed to assess the enrichment capacity of two different compositions of microparticles. Black dots indicate the target concentration in the sample before incubation with the microparticles. White columns represent the target concentration detected after the incubation in the supernatant, grey columns target concentration on the microparticles. The enrichment effect is clearly visible.

[0269] FIG. 14 shows the precision of the combined digital and real-time quantitative PCR analysis approach, outlined further above, for target quantitation in microparticles. Confidence intervals are shown for both methods. The vertical line at λ=5 cp/microparticle (cp=copies) indicates the approximate value at which methods can be used interchangeably (“CI”=confidence interval).

[0270] FIG. 15 shows real time fluorescence data obtained from an image series collected on microparticles in oil during PCR amplification. A stitched image of a detection chamber with the endpoint fluorescence signal detected in one fluorescence channel specific for amplification is shown on the left. The graph in the center shows exemplarily fluorescence intensity for 12 representative individual microparticles selected from the fluorescence image on the left over time. A distribution of the calculated ct values for each microparticle detected in the fluorescence image is shown in the histogram on the right.