A LIBRARY OF PREFABRICATED MICROPARTICLES AND PRECURSORS THEREOF
20230126528 · 2023-04-27
Inventors
- EUGEN ERMANTRAUT (JENA, DE)
- Ivan LONCAREVIC (Jena, DE)
- Katrin STEINMETZER (Jena, DE)
- Stephan HUBOLD (Jena, DE)
- Thomas ELLINGER (Jena, DE)
- Susanne KLINGER (Jena, DE)
- Oliver LEMUTH (Jena, DE)
- Lea KANITZ (Jena, DE)
Cpc classification
C12N15/1093
CHEMISTRY; METALLURGY
C12Q1/6806
CHEMISTRY; METALLURGY
International classification
C12N15/10
CHEMISTRY; METALLURGY
C12Q1/6806
CHEMISTRY; METALLURGY
Abstract
The present invention relates to prefabricated microparticles for performing a specific detection of one or several analytes in a sample, and to precursors of such microparticles, herein also sometimes referred to as “precursor-microparticles”. In particular, the present invention relates to libraries of such prefabricated microparticles and libraries of such precursor-microparticles. Furthermore, the present invention relates to kits for making such libraries and to kits of using such libraries for detecting an analyte of interest in a sample. Moreover, the present invention relates to methods of detecting and/or quantitating an analyte of interest in an aqueous sample, preferably using such kits.
Claims
1-25. (canceled)
26. A library of prefabricated precursor-microparticles for making a library of prefabricated microparticles, said prefabricated microparticles being configure for performing specific detection of one or several analytes of interest in a sample, such specific detection occurring within such microparticles by a suitable chemical or biochemical reaction, each of said prefabricated precursor-microparticles in said library comprising: a porous matrix having a void volume for receiving an aqueous sample and for providing a reaction space for the specific detection of an analyte; a reagent binding component allowing the attachment of an analyte-specific reagent to the precursor-microparticle; said reagent binding component being one of: (i) a polymer or polymer mixture that forms said porous matrix or is said porous matrix; (ii) a reagent binding molecule attached to said porous matrix; (iii) 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 precursor-microparticle; (iv) at least one charged group, or a plurality of charged groups immobilized on said porous matrix; (v) a combination of any of (i)-(iv); a label component attached to, contained within or otherwise associated with said precursor-microparticle for identifying said analyte-specific reagent, when attached to the precursor-microparticle.
27. The library according to claim 26, wherein, in said library, there are at least two separate subsets of prefabricated precursor-microparticles, with each subset having its distinct label component attached to, contained within or otherwise associated with said precursor-microparticles within said subset, such that said at least two or more separate subsets of prefabricated precursor-microparticles differ by the respective label component attached to, contained within or otherwise associated with each subset.
28. A library of prefabricated microparticles for performing specific detection of one or several analytes of interest in a sample, such specific detection occurring within such microparticles by a suitable chemical or biochemical reaction, each of said prefabricated microparticles comprising a prefabricated precursor-microparticle according to claim 26 and further comprising an analyte-specific reagent attached to said precursor-microparticle.
29. The library of prefabricated microparticles according to claim 28, wherein said analyte-specific reagent that is attached to each of said precursor-microparticles, is attached through said reagent binding component by a) direct binding of said analyte-specific reagent to said polymer or polymer mixture that forms said porous matrix or is part of said porous matrix (i); b) said analyte-specific reagent being conjugated to a binding entity which, in turn, binds to said reagent binding molecule (ii); c) direct binding of said analyte-specific reagent to said ionisable group(s) (iii) under conditions in which said ionisable group(s) has(have) a suitable net charge; d) direct binding of said analyte-specific reagent to said charged group(s) (iv) on said polymer, wherein said analyte-specific reagent has at least one ionisable group, or a plurality of ionisable groups, said ionisable group(s) being capable of changing its(their) charge(s) according to ambient conditions surrounding said analyte-specific reagent; or e) a combination of any of (a)-(d).
30. The library of prefabricated precursor-microparticles according to claim 26 or a library of prefabricated microparticles for performing specific detection of one or several analytes of interest in a sample, such specific detection occurring within such microparticles by a suitable chemical or biochemical reaction, each of said prefabricated microparticles comprising a prefabricated precursor-microparticle according to claim 1 and further comprising an analyte-specific reagent attached to said precursor-microparticle, wherein said polymer (i) is a hydrogel-forming agent selected from the group consisting of ia) synthetic polymers; ib) silicone-based polymers; ic) naturally occurring polymers selected from polysaccharides; gums selected from xanthan gum, arabic gum, ghatti gum, guar gum, locust bean gum, tragacanth gum, karaya gum, and inulin; polypeptides; poly-amino acids; polynucleotides; and mixture of any of the foregoing; said reagent binding molecule (ii) is selected from avidin; streptavidin; monomeric avidin; avidin having a nitrated tyrosine in its biotin binding site; other proteins, derived from or related to, avidin and retaining binding functionality of avidin; biotin; desthiobiotin; iminobiotin; biotin having a cleavable spacer arm; selenobiotin; oxybiotin; homobiotin; norbiotin; iminobiotin; diaminobiotin; biotin sulfoxide; biotin sulfone; epibiotin; 5-hydroxybiotin; 2-thiobiotin; azabiotin; carbobiotin; methylated derivatives of biotin; ketone biotin; other molecules, derived from or related to, biotin and retaining binding functionality of biotin; said at least one ionisable group (iii) is selected from N-2-acetamido-2-aminoethanesulfonic acid (ACES); N-2-acetamido-2-iminodiacetic acid (ADA); amino methyl propanediol (AMP); 3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxy propanesulfonic acid (AMPSO); N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES); N,N-bis-2-hydroxyethylglycine (BICINE); bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris); 1,3-bistrishydroxymethylmethylaminopropane (Bis-Tris Propane); 4-cyclohexylamino-1-butane sulfonic acid (CABS); 3-cyclohexylamino-1-propane sulfonic acid (CAPS); 3-cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO); 2-N-cyclohexylaminoethanesulfonic acid (CHES); 3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (DIPSO); N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS); N-2-hydroxyethylpiparazine-N-4-butanesulfonic acid (HEPBS); N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES); N-2-hydroxyethylpiperazine-N-2-propanesulfonic acid (HEPPSO); 2-N-morpholinoethanesulfonic acid (MES); 4-N-morpholinobutanesulfonic acid (MOBS); 3-N-morpholinopropanesulfonic acid (MOPS); 3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO); piperazine-N—N-bis-2-ethanesulfonic acid (PIPES); piperazine-N—N-bis-2-hydroxypropanesulfonic acid (POPSO); N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS); N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS); 3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid (TAPSO); N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES); N-trishydroxymethylmethylglycine (TRICINE); trishydroxymethylaminomethane (Tris); polyhydroxylated amines; imidazole, and derivatives thereof; triethanolamine dimers and polymers; and di/tri/oligo/poly amino acids.
31. The library of prefabricated precursor-microparticles according to claim 26; or a library of prefabricated microparticles for performing specific detection of one or several analytes of interest in a sample, such specific detection occurring within such microparticles by a suitable chemical or biochemical reaction, each of said prefabricated microparticles comprising a prefabricated precursor-microparticle according to claim 26, and further comprising an analyte-specific reagent attached to said precursor-microparticle; wherein said porous matrix, or said polymer or polymer mixture that forms or is part of said porous matrix, is composed of a polymer that is not crosslinked, wherein said polymer or polymer mixture that forms or is part of said porous polymeric matrix, is composed of agarose or a combination of agarose and gelatin.
32. The library of prefabricated microparticles according to claim 28, 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.
33. The library of prefabricated microparticles according to claim 28, wherein, for each of said microparticles, said analyte-specific reagent is reversibly attached to said microparticle through said reagent binding component by b) said analyte-specific reagent being conjugated to a binding entity which, in turn, binds to said reagent binding molecule (ii), wherein said binding entity is independently selected from biotin, desthiobiotin, iminobiotin, biotin having a cleavable spacer arm, selenobiotin, oxybiotin, homobiotin, norbiotin, iminobiotin, diaminobiotin, biotin sulfoxide, biotin sulfone, epibiotin, 5-hydroxybiotin, 2-thiobiotin, azabiotin, carbobiotin, methylated derivatives of biotin, ketone biotin, other molecules, derived from or related to, biotin and retaining binding functionality of biotin; and said reagent binding molecule is independently selected from avidin and streptavidin; or vice versa; or said binding entity is biotin, and said reagent binding molecule is selected from monomeric avidin, avidin having a nitrated tyrosine in its biotin binding site, and other proteins, derived from or related to, avidin and retaining binding functionality of avidin; or vice versa.
34. The library of prefabricated microparticles according to claim 28, wherein said library is for performing a specific detection of a single analyte of interest in several samples, wherein, in said library, there are at least two separate subsets of prefabricated microparticles, with each subset having its distinct label component attached to, contained within or otherwise associated with said microparticles of said subset; and all of said at least two, three or more separate subsets having the same analyte-specific reagent attached to the porous matrix of said microparticles of said subsets, said analyte-specific reagent being specific for one analyte of interest; such that said at least two or more separate subsets of prefabricated microparticles are identical in terms of the analyte-specific reagent attached, but differ by the respective label component attached to, contained within or otherwise associated with said microparticles of each subset; with each subset being unambiguously defined and identifiable by said respective label component.
35. The library of prefabricated microparticles according to claim 28, wherein said library is for performing a specific detection of multiple analytes of interest in a single sample, wherein, in said library, there are at least two separate subsets of prefabricated microparticles, with each subset having its distinct label component attached to, contained within or otherwise associated with said microparticles of said subset; and having a different analyte-specific reagent attached to the porous matrix of said microparticles of said subset; each analyte-specific reagent being specific for one analyte of interest; such that said at least two or more separate subsets of prefabricated microparticles differ by the respective label component attached to, contained within or otherwise associated with said microparticles of each subset; and the respective analyte-specific reagent attached to each subset; with each subset being unambiguously defined and identifiable by said respective label component and said respective analyte-specific reagent.
36. The library of prefabricated microparticles according to claim 28, wherein said library is for performing a specific detection of multiple analytes of interest in several samples, wherein, in said library, there are a plurality of different separate subsets of prefabricated microparticles, with each subset having its distinct label component attached to, contained within or otherwise associated with said microparticles of said subset; and wherein, in said plurality of separate subsets of prefabricated microparticles, there are different classes of separate subsets of prefabricated microparticles, with each of said classes comprising several subsets of microparticles and each class having a different analyte-specific reagent attached to the porous matrix of said microparticles, and all subsets of microparticles within one class having the same analyte-specific reagent attached; each analyte-specific reagent being specific for one analyte of interest; such that, in said library, said plurality of different separate subsets of prefabricated microparticles differ by the respective label component attached to, contained within or otherwise associated with said microparticles of each subset; and each separate subset of microparticles forms part of one class of subsets of microparticles; with each subset being unambiguously defined and identifiable by said respective label component and said respective analyte-specific reagent; 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; and each of said different classes comprises several subsets of microparticles, all of which subsets within one class having the same analyte-specific reagent attached.
37. A kit for making a library of prefabricated microparticles according to claim 28, said kit comprising: at least two containers, namely a first and a second container, and, optionally, further containers, each containing: a subset of prefabricated precursor-microparticles each comprising: a porous matrix having a void volume for receiving an aqueous sample and for providing a reaction space for the specific detection of an analyte; and a reagent binding component allowing the attachment of an analyte-specific reagent to the precursor-microparticle; said reagent binding component being one of: (vi) a polymer or polymer mixture that forms said porous matrix or is said porous matrix; (vii) a reagent binding molecule attached to said porous matrix; (viii) 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 precursor-microparticle; (ix) at least one charged group, or a plurality of charged groups immobilized on said porous matrix; or a combination of any of (i)-(iv) with each subset having its distinct label component attached to, contained within or otherwise associated with said precursor-microparticles within said subset, such that said two subsets of prefabricated precursor-microparticles differ by the respective label component attached to, contained within or otherwise associated with each subset; a further container containing: a conditioning solution to enable the attachment of an analyte-specific reagent to said subset of said precursor-microparticles through said reagent binding component by a) direct binding of said analyte-specific reagent to said polymer or polymer mixture that forms said porous matrix or is part of said porous matrix (i); b) said analyte-specific reagent being conjugated to a binding entity which, in turn, binds to said reagent binding molecule (ii); c) direct binding of said analyte-specific reagent to said ionisable group(s) (iii) under conditions in which said ionisable group(s) has(have) a suitable net charge; d) direct binding of said analyte-specific reagent to said charged group(s) (iv) on said polymer, wherein said analyte-specific reagent has at least one ionisable group, or a plurality of ionisable groups, said ionisable group(s) being capable of changing its(their) charge(s) according to ambient conditions surrounding said analyte-specific reagent; or e) a combination of any of (a)-(d); said reagent binding component being one of: (i) a polymer or polymer mixture that forms said porous matrix or is said porous matrix; (ii) a reagent binding molecule attached to said porous matrix; (iii) 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 precursor-microparticle; (iv) at least one charged group, or a plurality of charged groups immobilized on said porous matrix; or (v) a combination of any of (i)-(iv); said polymer or polymer mixture having a void volume for receiving an aqueous sample and for providing a reaction space for the specific detection of an analyte; and said ionisable groups being capable of changing its(their) charge(s) according to ambient conditions surrounding said precursor-microparticle.
38. A method of making a library of prefabricated microparticles for performing specific detection of one or several analytes of interest in a sample, such specific detection occurring within such microparticles by a suitable chemical or biochemical reaction, each of said prefabricated microparticles comprising a prefabricated precursor-microparticle according to claim 26 and further comprising an analyte-specific reagent attached to said precursor-microparticle, said method comprising: providing, in any order: a library of prefabricated precursor-microparticles as defined in claim 26; at least one analyte-specific reagent; mixing said library of prefabricated precursor-microparticles, or selected subsets thereof, and the at least one analyte-specific reagent under conditions allowing the attachment, preferably the reversible attachment, of said at least one analyte-specific reagent to some or all of said prefabricated precursor-microparticles, thus generating a library of prefabricated microparticles; optionally, washing said prefabricated microparticles to remove any unattached analyte-specific reagent therefrom.
39. A kit for detecting an analyte in a sample, said kit comprising: a) a container containing a generic detection composition comprising reagents for performing a chemical or biochemical detection reaction of an analyte, wherein said chemical or biochemical detection reaction of an analyte is a nucleic acid amplification, said generic detection composition comprises a buffer, mono-nucleoside-triphosphates, an amplification enzyme and a nucleic acid dye for the detection of an amplification product, or a container containing a first detection composition comprising reagents for performing a chemical or biochemical detection reaction of an analyte, and yet a further container containing a second detection composition comprising a detection reagent, wherein said chemical or biochemical detection reaction of an analyte is an immunochemistry detection reaction, said first detection composition comprises 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 said second detection composition comprises, as a detection reagent, a suitable substrate for said reporter enzyme which substrate upon having been reacted by said reporter enzyme, becomes detectable, or a container containing a first detection composition comprising reagents for performing a chemical or biochemical detection reaction of an analyte, and yet a further container containing a second detection composition comprising a detection reagent, wherein the chemical or biochemical reaction is an immunochemistry detection reaction, said first detection composition comprises reagents for performing an immunochemistry detection reaction, and a secondary antibody or secondary antibody fragment coupled to a suitable oligonucleotide tag and being specific for the same analyte as a primary antibody, used as analyte-specific reagent (ASR) in said immunochemistry detection reaction; and said second detection composition comprises, as detection reagent(s), a buffer, mono-nucleoside-triphosphates, an amplification enzyme, and a nucleic acid dye for detection of an amplification product and primers suitable for amplifying the oligonucleotide tag attached to the secondary antibody; and b) a container containing a non-aqueous phase optionally supplemented with an emulsifier; and c) a mixing container for mixing components.
40. A method of detecting and/or quantitating an analyte of interest in an aqueous sample, said method comprising the steps: providing, in any order: an aqueous sample known or suspected to contain an analyte of interest; a generic detection composition comprising reagents for performing a chemical or biochemical detection reaction of the analyte; a library of prefabricated microparticles according to claim 28, wherein said analyte-specific reagent(s) attached to said microparticles, is(are) chosen such that it is (they are) capable of specifically binding to or reacting with said analyte of interest; optionally, mixing said aqueous sample and said generic detection composition; incubating said aqueous sample with said library of prefabricated microparticles, thereby allowing said library of microparticles to absorb aqueous sample in the void volumes of said microparticles and, optionally, to bind said analyte of interest, if present in said sample; optionally, washing said microparticles; adding said generic detection composition to the microparticles, if said aqueous sample has not been previously mixed with said generic detection composition; transferring said library of prefabricated microparticles into a non-aqueous phase and removing any aqueous phase surrounding the individual prefabricated microparticle(s), thereby creating a plurality of insulated reaction spaces for detecting said analyte, which reaction spaces comprise an aqueous phase and are confined to said void volume(s) of said microparticles; optionally, releasing the analyte-specific reagent(s) attached to said microparticles by applying an external trigger; performing a detection reaction of said analyte of interest; and detecting and/or quantitating said analyte of interest.
41. The method according to claim 40, wherein said analyte of interest is a nucleic acid, and said analyte-specific reagent is a nucleic acid or a pair of nucleic acids, sufficiently complementary to said analyte of interest to be able under hybridizing conditions to hybridize to said analyte of interest, wherein said analyte-specific reagent is a suitable primer or primer pair for amplification of said analyte of interest; said generic detection composition comprises reagents for performing an amplification reaction of said nucleic acid analyte of interest, except for primers, wherein said generic detection composition comprises a buffer, mono-nucleoside-triphosphates, an amplification enzyme and a nucleic acid dye for the detection of an amplification product; said step of transferring is a step wherein said prefabricated microparticles are transferred into a non-aqueous phase and suspended and, optionally, repeatedly washed with said non-aqueous phase, said step more optionally involving also a filtration or mechanical agitation to ensure the formation of a monodisperse suspension, of microparticles with no aqueous phase or no substantial aqueous phase remaining outside of any of the microparticles; said optional step of releasing the analyte-specific reagent(s) attached to said microparticles by applying an external trigger is a step of temporarily increasing the temperature, changing the pH, or changing the salt conditions, preferably of increasing the temperature, said step of performing a detection reaction is a step of performing an amplification reaction of said analyte if present in said aqueous sample; and said step of detecting and/or quantitating said analyte of interest is a step of detecting and/or quantitating said amplified analyte.
42. A method of detecting and/or quantitating an analyte of interest in an aqueous sample, said method comprising the steps: providing, in any order: an aqueous sample known or suspected to contain an analyte of interest; a first detection composition comprising necessary reagents for performing a chemical or biochemical detection reaction of the analyte; a second detection composition comprising a detection reagent; a library of prefabricated microparticles according to claim 28, wherein said analyte-specific reagent(s) attached to said microparticles, is(are) chosen such that it is (they are) capable of specifically binding to or reacting with said analyte of interest; optionally, mixing said aqueous sample and said first detection composition; incubating said aqueous sample with said library of prefabricated microparticles and, if said aqueous sample has not already been mixed with said first detection composition, also with said first detection composition, thereby allowing said library of microparticles to absorb aqueous sample and said first detection composition in the void volumes of said microparticles and, optionally, to bind said analyte of interest, if present in said sample; optionally, washing said library of prefabricated microparticles to remove any non-absorbed or non-reacted first detection composition; incubating said library of prefabricated microparticles including absorbed aqueous sample with said second detection composition, thereby allowing said library of microparticles to absorb said second detection composition; optionally, further washing said library of prefabricated microparticles to remove any non-absorbed or non-reacted second detection composition; transferring said library of prefabricated microparticles into a non-aqueous phase and removing any aqueous phase surrounding the individual prefabricated microparticle(s), thereby creating a plurality of insulated reaction spaces for detecting said analyte which reaction spaces comprise an aqueous phase and are confined to said void volume(s) of said microparticles; optionally, releasing the analyte-specific reagent(s) attached to said microparticles by applying an external trigger; and detecting and/or quantitating said analyte of interest by detecting and/or quantitating said detection reagent.
43. The method according to claim 42, wherein said analyte of interest is a protein or other non-nucleic acid molecule, and said analyte-specific reagent is a primary antibody, antibody fragment, or a non-antibody protein capable of specifically binding said protein analyte or other non-nucleic acid analyte; said first 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 said analyte; said second 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; said step of incubating said aqueous sample with said library of prefabricated microparticles and with said first detection composition is a step of performing an immunochemistry reaction involving the binding of said analyte, if present, in said aqueous sample, to said primary antibody, primary antibody fragment, or non-antibody protein, thus forming a complex of analyte and said primary antibody, primary antibody fragment, or non-antibody protein; said immunochemistry reaction further involving a binding of said secondary antibody to said complex, thus forming a sandwich between primary antibody, antibody fragment or non-antibody protein, analyte, and secondary antibody; said first optional step of washing said library is a step of removing any non-bound secondary antibody from said library; said step of incubating said library of prefabricated microparticles including absorbed aqueous sample with said second detection composition is a step of allowing said substrate to be reacted by said reporter enzyme; said further optional step of washing said library is a step of removing any non-reacted substrate from said library; said step of transferring is a step wherein said prefabricated microparticles are transferred into a non-aqueous phase and suspended and, optionally, repeatedly washed with said non-aqueous phase, more optionally involving also a filtration or mechanical agitation to ensure the formation of a monodisperse suspension of microparticles with no aqueous phase or no substantial aqueous phase remaining outside of any of the microparticles; said optional step of releasing the analyte-specific reagent(s) attached to said microparticles by applying an external trigger is a step of temporarily increasing the temperature, changing the pH, or changing the salt conditions; and said step of detecting and/or quantitating said analyte of interest is a step of detecting and/or quantitating said reacted substrate.
44. A method of detecting and/or quantitating an analyte of interest in an aqueous sample, said method comprising the steps: providing, in any order: an aqueous sample known or suspected to contain an analyte of interest; a first detection composition comprising necessary reagents for performing a chemical or biochemical detection reaction of the analyte; a second detection composition comprising a detection reagent; a library of prefabricated microparticles according to claim 28, wherein said analyte-specific reagent(s) attached to said microparticles, is(are) chosen such that itis (they are) capable of specifically binding to or reacting with said analyte of interest; optionally, mixing said aqueous sample and said first detection composition; incubating said aqueous sample with said library of prefabricated microparticles and, if said aqueous sample has not already been mixed with said first detection composition, also with said first detection composition, thereby allowing said library of microparticles to absorb aqueous sample and said first detection composition in the void volumes of said microparticles and, optionally, to bind said analyte of interest, if present in said sample; optionally, washing said library of prefabricated microparticles to remove any non-absorbed or non-reacted first detection composition; incubating said library of prefabricated microparticles including absorbed aqueous sample with said second detection composition, thereby allowing said library of microparticles to absorb said second detection composition; optionally, further washing said library of prefabricated microparticles to remove any non-absorbed or non-reacted second detection composition; transferring said library of prefabricated microparticles into a non-aqueous phase and removing any aqueous phase surrounding the individual prefabricated microparticle(s), thereby creating a plurality of insulated reaction spaces for detecting said analyte which reaction spaces comprise an aqueous phase and are confined to said void volume(s) of said microparticles; optionally, releasing the analyte-specific reagent(s) attached to said microparticles by applying an external trigger; performing a detection reaction of said analyte of interest; and detecting and/or quantitating said analyte of interest.
45. The method according to claim 44, wherein said analyte of interest is a protein or other non-nucleic acid molecule, and said analyte-specific reagent is a primary antibody, antibody fragment, or a non-antibody protein capable of specifically binding said protein analyte or other non-nucleic acid analyte; said first detection composition comprises necessary reagents for performing an immunochemistry detection reaction, and a secondary antibody or secondary antibody fragment coupled to a suitable oligonucleotide tag and being specific for the same analyte as said primary antibody; said second detection composition comprises, as detection reagent(s), a buffer, mono-nucleoside-triphosphates, an amplification enzyme, and a nucleic acid dye for detection of an amplification product, and primers suitable for amplifying the oligonucleotide tag attached to the secondary antibody; said step of incubating said aqueous sample with said library of prefabricated microparticles and with said first detection composition is a step of performing an immunochemistry reaction involving the binding of said analyte, if present, in said aqueous sample, to said primary antibody, primary antibody fragment, or non-antibody protein, thus forming a complex of analyte and said primary antibody, primary antibody fragment, or non-antibody protein; said immunochemistry reaction further involving a binding of said secondary antibody to said complex, thus forming a sandwich between primary antibody, antibody fragment or non-antibody protein, analyte, and secondary antibody; said first optional step of washing said library is a step of removing any non-bound secondary antibody from said library; said step of incubating said library of prefabricated microparticles including absorbed aqueous sample with said second detection composition is a step of allowing said primers in said second detection composition hybridize to the oligonucleotide tag attached to the secondary antibody; said further optional step of washing said library is a step of removing any non-hybridized primers from said library; said step of transferring is a step wherein said prefabricated microparticles are transferred into a non-aqueous phase and suspended and, optionally, repeatedly washed with said non-aqueous phase, more optionally involving also a filtration or mechanical agitation to ensure the formation of a monodisperse suspension, of microparticles with no aqueous phase or no substantial aqueous phase remaining outside of any of the microparticles; said optional step of releasing the analyte-specific reagent(s) attached to said microparticles by applying an external trigger is a step of temporarily increasing the temperature, changing the pH, or changing the salt conditions; said step of performing a detection reaction is a step of performing an amplification reaction of said oligonucleotide tag; and said step of detecting and/or quantitating said analyte(s) of interest is a step of detecting and/or quantitating said amplified oligonucleotide tag(s).
46. The method according to claim 40, wherein said step of releasing the analyte-specific reagent(s) attached to said microparticles by applying an external trigger is performed by raising the temperature to which said microparticles are exposed.
47. A method of detecting and/or quantitating an analyte of interest in an aqueous sample, said method comprising the steps: providing, in any order: an aqueous sample known or suspected to contain an analyte of interest; a generic detection composition comprising reagents for performing a chemical or biochemical detection reaction of the analyte; a library of prefabricated microparticles for performing specific detection of one or several analytes of interest in a sample, such specific detection occurring within such microparticles by a suitable chemical or biochemical reaction, each of said prefabricated microparticles comprising a prefabricated precursor-microparticle according to claim 1 and further comprising an analyte-specific reagent attached to said precursor-microparticle, wherein said analyte-specific reagent(s) attached to said microparticles, is(are) chosen such that it is (they are) capable of specifically binding to or reacting with said analyte of interest; optionally, mixing said aqueous sample and said generic detection composition; incubating said aqueous sample with said library of prefabricated microparticles, thereby allowing said library of microparticles to absorb aqueous sample in the void volumes of said microparticles and, optionally, to bind said analyte of interest, if present in said sample; optionally, washing said microparticles; adding said generic detection composition to the microparticles, if said aqueous sample has not been previously mixed with said generic detection composition; transferring said library of prefabricated microparticles into a non-aqueous phase and removing any aqueous phase surrounding the individual prefabricated microparticle(s), thereby creating a plurality of insulated reaction spaces for detecting said analyte, which reaction spaces comprise an aqueous phase and are confined to said void volume(s) of said microparticles; optionally, releasing the analyte-specific reagent(s) attached to said microparticles by applying an external trigger; performing a detection reaction of said analyte of interest; and detecting and/or quantitating said analyte of interest, wherein, the method is a method of detecting and/or quantitating a single analyte in a plurality of aqueous samples, in said method, there are provided a plurality of different aqueous samples known or suspected to contain an analyte of interest, the library of prefabricated microparticles that is provided in said method, is a library according to claim 9, wherein, in such library, there are as many or at least as many separate subsets of microparticles provided, as there are different aqueous samples provided and to be tested, wherein all of said separate substeps have the same analyte-specific reagent attached to the porous matrix of said microparticles of said subsets, said analyte-specific reagent being specific for one analyte of interest; in said step of incubating said aqueous samples with said library of prefabricated microparticles, each of said different aqueous samples is incubated separately with a separate subset of microparticles of said library; in said step of incubating said library of prefabricated microparticles including absorbed aqueous sample with a second detection composition, each of said separate subsets of microparticles of said library is incubated separately with said second detection composition; said step of transferring said library into a non-aqueous phase is performed for each subset of microparticles separately, i.e. each separate subset of microparticles is separately transferred into a non-aqueous phase, and the aqueous phase surrounding the individual microparticles is removed for each subset separately, thereby creating, for each subset separately, a plurality of insulated reaction spaces for detecting said analyte which reaction spaces comprise an aqueous phase and are confined to said void volume(s) of said microparticles, wherein said method further comprises, after said step of transferring said library into a non-aqueous phase, a step of mixing said plurality of insulated reaction spaces of all the separate subsets in said non-aqueous phase together, before subsequently a detection reaction of said analyte of interest is performed, and before detecting and/or quantitating said analyte of interest.
48. A method of detecting and/or quantitating an analyte of interest in an aqueous sample, said method comprising the steps: providing, in any order: an aqueous sample known or suspected to contain an analyte of interest; a generic detection composition comprising reagents for performing a chemical or biochemical detection reaction of the analyte; a library of prefabricated microparticles for performing specific detection of one or several analytes of interest in a sample, such specific detection occurring within such microparticles by a suitable chemical or biochemical reaction, each of said prefabricated microparticles comprising a prefabricated precursor-microparticle comprising: a porous matrix having a void volume for receiving an aqueous sample and for providing a reaction space for the specific detection of an analyte; a reagent binding component allowing the attachment of an analyte-specific reagent to the precursor-microparticle; said reagent binding component being one of: (x) a polymer or polymer mixture that forms said porous matrix or is said porous matrix; (xi) a reagent binding molecule attached to said porous matrix; (xii) 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 precursor-microparticle; (xiii) at least one charged group, or a plurality of charged groups immobilized on said porous matrix; (xiv) a combination of any of (i)-(iv); and further comprising an analyte-specific reagent attached to said precursor-microparticle, wherein said analyte-specific reagent(s) attached to said microparticles, is(are) chosen such that it is (they are) capable of specifically binding to or reacting with said analyte of interest; optionally, mixing said aqueous sample and said generic detection composition; incubating said aqueous sample with said library of prefabricated microparticles, thereby allowing said library of microparticles to absorb aqueous sample in the void volumes of said microparticles and, optionally, to bind said analyte of interest, if present in said sample; optionally, washing said microparticles; adding said generic detection composition to the microparticles, if said aqueous sample has not been previously mixed with said generic detection composition; transferring said library of prefabricated microparticles into a non-aqueous phase and removing any aqueous phase surrounding the individual prefabricated microparticle(s), thereby creating a plurality of insulated reaction spaces for detecting said analyte, which reaction spaces comprise an aqueous phase and are confined to said void volume(s) of said microparticles; optionally, releasing the analyte-specific reagent(s) attached to said microparticles by applying an external trigger; performing a detection reaction of said analyte of interest; and detecting and/or quantitating said analyte of interest, wherein, the method is a method of detecting and/or quantitating multiple analytes in a single aqueous sample, in said method, there is provided a single aqueous sample known or suspected to contain multiple analytes of interest, the library of prefabricated microparticles that is provided in said method, is a library according to claim 35, wherein, in such library, there are as many or at least as many separate subsets of microparticles provided, as there are different analytes of interest to be detected, with each of said separate subsets having a different analyte-specific reagent attached to the porous matrix of said microparticles of said subset; each analyte-specific reagent being specific for one analyte of interest; in said step of incubating said aqueous sample with said library of prefabricated microparticles, said aqueous sample is incubated with all of said separate subsets of microparticles of said library together; in said step of incubating said library of prefabricated microparticles including absorbed aqueous sample with a second detection composition, all of said separate subsets of microparticles of said library are incubated together with said second detection composition; and said step of transferring said library into a non-aqueous phase is performed for all subsets of microparticles together, and the aqueous phase surrounding the individual microparticles is removed, thereby creating a plurality of insulated reaction spaces for detecting and/or quantitating said multiple analytes which reaction spaces comprise an aqueous phase and are confined to said void volume(s) of said microparticles.
49. A method of detecting and/or quantitating an analyte of interest in an aqueous sample, said method comprising the steps: providing, in any order: an aqueous sample known or suspected to contain an analyte of interest; a generic detection composition comprising reagents for performing a chemical or biochemical detection reaction of the analyte; a library of prefabricated microparticles for performing specific detection of one or several analytes of interest in a sample, such specific detection occurring within such microparticles by a suitable chemical or biochemical reaction, each of said prefabricated microparticles comprising a prefabricated precursor-microparticle comprising: a porous matrix having a void volume for receiving an aqueous sample and for providing a reaction space for the specific detection of an analyte; a reagent binding component allowing the attachment of an analyte-specific reagent to the precursor-microparticle; said reagent binding component being one of: (xv) a polymer or polymer mixture that forms said porous matrix or is said porous matrix; (xvi) a reagent binding molecule attached to said porous matrix; (xvii) 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 precursor-microparticle; (xviii) at least one charged group, or a plurality of charged groups immobilized on said porous matrix; (xix) a combination of any of (i)-(iv); and further comprising an analyte-specific reagent attached to said precursor-microparticle, wherein said analyte-specific reagent(s) attached to said microparticles, is(are) chosen such that it is (they are) capable of specifically binding to or reacting with said analyte of interest; optionally, mixing said aqueous sample and said generic detection composition; incubating said aqueous sample with said library of prefabricated microparticles, thereby allowing said library of microparticles to absorb aqueous sample in the void volumes of said microparticles and, optionally, to bind said analyte of interest, if present in said sample; optionally, washing said microparticles; adding said generic detection composition to the microparticles, if said aqueous sample has not been previously mixed with said generic detection composition; transferring said library of prefabricated microparticles into a non-aqueous phase and removing any aqueous phase surrounding the individual prefabricated microparticle(s), thereby creating a plurality of insulated reaction spaces for detecting said analyte, which reaction spaces comprise an aqueous phase and are confined to said void volume(s) of said microparticles; optionally, releasing the analyte-specific reagent(s) attached to said microparticles by applying an external trigger; performing a detection reaction of said analyte of interest; and detecting and/or quantitating said analyte of interest, wherein, the method is a method of detecting and/or quantitating multiple analytes in a plurality of aqueous samples, in said method, there are provided a plurality of different aqueous samples known or suspected to contain multiple analytes of interest, the library of prefabricated microparticles that is provided in said method, is a library according to claim 36, wherein, in such library, there are different classes of separate subsets of prefabricated microparticles, with each of said classes comprising several subsets of microparticles and each of said classes having a different analyte-specific reagent attached to the porous matrix of said microparticles, and all subsets of microparticles within one class having the same analyte-specific reagent attached; each analyte-specific reagent being specific for one analyte of interest; wherein, in said method, in said step of providing said library, there are as many or at least as many different classes of separate subsets of microparticles provided, as there are different analytes to be detected, as many or at least as many different subsets of microparticles provided within each class, as there are aqueous samples provided and to be tested, as many or at least as many different subsets of microparticles provided within the library as there are different aqueous samples to be tested multiplied by the number of different analytes of interest to be detected, in said step of incubating said aqueous sample with said library of prefabricated microparticles, exactly one aqueous sample is incubated with exactly one subset of microparticles from each class, with each aqueous sample being incubated with as many different subsets of microparticles from different classes together as there are classes of microparticles in said library, in said step of incubating said library of prefabricated microparticles including absorbed aqueous sample with a second detection composition, the combined subsets from different classes of microparticles with which subsets one respective aqueous sample had been previously incubated, are incubated together with said second detection composition, said step of transferring said library into a non-aqueous phase is performed for each aqueous sample separately, namely the combined subsets of microparticles with which one respective aqueous sample had been previously incubated, are transferred together into a non-aqueous phase, and the aqueous phase surrounding the individual microparticles is removed, thereby creating, for each aqueous sample separately, a plurality of insulated reaction spaces for detecting said multiple analytes which reaction spaces comprise an aqueous phase and are confined to said void volume(s) of said microparticles, wherein said method further comprises, after said step of transferring said library into a non-aqueous phase, a step of mixing said pluralities of insulated reaction spaces of all the separate samples in said non-aqueous phase together, before subsequently a detection reaction of said analytes of interest is performed, and before detecting and/or quantitating said analytes of interest.
50. The method according to claim 40, 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, such as immuno-polymerase chain reaction; in particular digital immune-PCR; wherein quantitation is performed using any of methods a) or b), 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.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0428]
[0429] In the embodiment shown in
[0430]
[0431]
[0432]
[0433]
[0434]
[0435] Thus it has been shown that by using such reversibly binding entities (e.g. desthiobiotin) the primer oligonucleotide can be released from the microparticle matrix. This is a preferable feature for highly efficient amplification reactions in the droplet space created by the microparticles in a non-aqueous environment. Whilst some degree of amplification may be possible even with attached primers, for optimum amplification reaction conditions, primer oligonucleotides should ideally not be bound to any matrix and, thus, will be deliberately released prior to any amplification reaction.
[0436]
[0437]
[0438]
[0439]
[0440]
[0441]
[0442]
[0443]
[0444] More generally speaking, once a method of detection in accordance with the present invention has been performed, a quantitation can be achieved using the following exemplary guideline: If the number of negative microparticles (i.e. microparticles in which no signal can be detected), exceeds a percentage in the range of from 0.1-1.0%, preferably 0.5-1.0%, more preferably 0.5-0.8%, then Poisson analysis is to be applied. If the number of negative microparticles (i.e. microparticles in which no signal can be detected) is below a percentage in the range of from 0.1-1.0%, preferably below a percentage in the range of from 0.5-1.0%, more preferably below a percentage in the range of from 0.5-0.8%, then quantitative real time analysis is to be applied, e.g. involving determination of cycle threshold values (e.g. using a comparative C.sub.T method also referred to as 2-Δ Δ CT method (see for example Schmittgen et al., 2008, Nature Protocols, 3, pp. 1101-1108)
[0445] Further details are explained in Example 3.
[0446]
[0447] Furthermore, reference is made to the following examples, which are given to illustrate, not to limit the present invention.
EXAMPLES
Example 1
[0448] Synthesis of Encoded Precursor Microparticles
[0449] Labeling of Gelatin with Fluorescent Dyes for Identification of Analyte-Specific Reagents
[0450] The acetone insoluble fraction of gelatin from bovine skin type A or porcine skin type B is labelled with a distinct mixture of two fluorescent dyes taking advantage of NHS coupling chemistry. Cy®3 Mono NHS Ester and Cy®5 Mono NHS Ester (GE Healthcare) were dissolved in DMSO to make a final solution of 1% (w/v) in potassium phosphate buffer (pH 8.0, sterile filtered). An 8-fold molar excess of the respective dye over free gelatin amino groups is utilized to label 25 mL of 0.25% (w/v) 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. Purification of the fluorescently labelled gelatin is accomplished by repeated ammonium sulfate precipitation using a saturated (NH.sub.4).sub.2SO.sub.4 solution. Alternatively, ultracentrifugation, solvent extraction with isopropanol, acetone or methanol, gel filtration using sepharose columns or dialysis can be performed. In either case, purification is repeated until effluent appears clear and shows no fluorescence. Purified fluorescently labelled gelatin samples are finally vacuum-dried.
[0451] Preparation of Gelatin/Agarose Hybrid Solution
[0452] A hybrid hydrogel solution consisting of four components is prepared for fabricating nano-reactors. [0453] Component 1: Acetone-insoluble gelatin from bovine skin type A G1890 (Sigma) or porcine skin type B G9391 (Sigma) [0454] Component 2: Low-gelling 2-Hydroxyethyl agarose (A4018, Sigma) [0455] Component 3: Cy3-labeled gelatin (type A or type B) [0456] Component 4: Cy5-labeled gelatin (type A or type B)
[0457] 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 each labelled gelatin, the dried pellets of component 3 and 4 are taken up in a respective volume of nuclease-free water and incubated at 55° C. until the gelatin is molten. All four components are mixed and filled up with nuclease-free water to generate a hybrid hydrogel solution with final concentrations of 1% (w/v) total for gelatin and 0.5% (w/v) for agarose A4018, respectively. Various volumes of component 3 and component 4 are mixed yielding n distinctly coloured microsphere sets. In this embodiment resuspended component 3 and component 4 are mixed in ratios 1:0, 3:1, 1:3, 0:1 at a maximum of 3% (v/v) of the total gelatine fraction to accomplish four individual label components (label components 1-4, respectively) for identifying analyte specific reagents and allowing a 4-plex reaction assay. All solutions are kept at 55° C. until further use.
[0458] Generation of Non-Crosslinked Gelatin/Agarose Microparticles
[0459] Monodisperse color coded agarose-gelatin hybrid microparticles are fabricated using either parts of the QX100/QX200 Droplet Digital (ddPCR™) system (BioRad) or a modified Encapsulator system (Dolomite microfluidics). In the case of the BioRad system, a DG8 Cartridge is kept on a Thermomixer to keep solutions at 55° C. After loading 50 μL of the gelatin/agarose hybrid solution, 100 μL of the emulsion reagent HFE-7500 containing 2-5% Picosurf 2 (Sphere Fluidics) 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. Alternatively, the QX200™/QX100™ Droplet Generator can be used for droplet generation. Approximately 80,000 hydrogel droplets of 100 μm in diameter are produced per well.
[0460] In the case of the Dolomite microfluidics system, monodisperse hybrid microparticles can be fabricated in a one-step process of suspension 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 by 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 get in contact at the chip junction. HFE-7500/Picosurf 2 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. A flow rate of 15-17 μl/min is adjusted for stable droplet formation. Parameters are monitored with the Dolomite Flow Control Advanced Software.
[0461] 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. Subsequently, microparticles are stored at 4° C. for at least 1h (preferentially overnight) to form stable hybrid scaffolds.
[0462] Recovery of Hybrid Microparticles from Continuous Phase
[0463] Solidified hybrid microparticles 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 suspension. To transfer the hybrid hydrogel particles into the oil phase, the tube is vortexed for 5s and centrifuged at 2,500×g for 5s. The hybrid hydrogel microparticles are transferred to a fresh 1.5 mL microcentrifuge tube. Optional: This procedure can be repeated to remove residual fluorocarbon oil and surfactant. Following the PFO wash, recovered microparticles are washed once with 1 mL nuclease-free water. Microparticle quality and sizes are visually examined using a microscope. Exemplary nine microparticle preparations are shown in
[0464] Functionalisation of Hybrid Precursor Microparticles
[0465] To be able to equip hybrid precursor microparticles with analyte-specific components, a flexible binding chemistry is established. Streptavidin is covalently attached to the amine-containing fraction of the hybrid matrix of the microparticles using the following 3-step protocol. First, sulfhydryl groups are added to Streptavidin using the amine-reactive portion of SPDP reagent (NHS) ester and conducting a subsequent reduction step. In a second step, a fraction of the amino groups of precursor microparticles is maleimide-activated using a Sulfo-SMCC crosslinking reagent. Finally, activated streptavidin and the maleimide-activated hybrid microparticles are conjugated resulting in nanoreactor precursors with a porous polymeric matrix and a reagent binding component.
[0466] Protocol 1: SPDP Crosslinking
[0467] A vial of 3-(2-Pyridyldithio)propionic acid N-hydroxysuccinimide ester (N-Succinimidyl 3-(2-pyridyldithio)propionate; SPDP; P3415, Sigma) crosslinker is allowed to equilibrate to ambient temperatures before opening to prevent condensation. For the SPDP modification of Streptavidin, a 2-fold molar excess of SPDP over streptavidin is used. SPDP is dissolved in 50 μL DMF to give a 0.23 molar SPDP solution. Also, 300 mg of 15.8 U/mg Streptavidin (SA10; Prozyme) is dissolved in 10 mL 100 mM potassium phosphate buffer containing 20 mM NaCl (pH 7.5) to make a 30 mg/mL solution. The solution is centrifuged at 3000 rpm and the supernatant is kept on ice for further experiments. The full volume (50 μL) of the SPDP solution is added to 30 mL of the streptavidin solution and the reaction is allowed to proceed overnight at 4° C. The reaction is quenched by adding Tris to a final concentration of 100 mM and further incubated at room temperature for 30 min. In a subsequent step, unreacted SPDP is removed from the streptavidin solution by centrifugation at 8000 rpm for 15 min using Vivacon 500 Ultrafiltration columns (100 kDa MWCO) (Sartorius Stedim Biotech). The flow-through is discarded and washing with 100 mM potassium phosphate buffer containing 20 mM NaCl repeated 5 times. The activated streptavidin is reduced by incubation with 2 mM DTT at ambient temperatures for 30 min. Thus, the pyridine-2-thione groups are removed from the modified streptavidin.
[0468] Protocol 2: Maleimid-Activation of Gelatin Fraction (Coupling of Sulfo-SMCC)
[0469] A fresh vial of 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC; M6035, Sigma) is allowed to fully equilibrate to ambient temperature before opening. Amine-containing hybrid microparticles are washed three times in non-amine containing conjugation buffer (potassium phosphate, 20 mM NaCl pH 7.2). Immediately before use, a 10 mg/mL of Sulfo-SMCC stock solution is prepared for conjugation. Sufficient Sulfo-SMCC stock solution is added to the microparticle solution to obtain 10 molar excess of crosslinking reagent over available amino groups (in the case of gelatine typeB-10% of amino acids of gelatine are expected to carry free amino groups. A 50% (v/v) hydrogel microparticle slurry is incubated with 10 mM Sulfo-SMCC and immediately put on ice. The reaction is allowed to proceed overnight at 4° C. at 100 rpm using the Multi-Rotator PTR-60 (Grant-bio) in the vertical mode. The reaction is quenched by adding Tris to a final concentration of 100 mM followed by an incubation at room temperature for 30 min. Maleimide activated particles are purified by repeated washing in conjugation buffer and centrifugation at 1000 rcf.
[0470] Protocol 3: Conjugation of Activated Proteins
[0471] The maleimide-activated microparticles and the sulfhydryl-modified Streptavidin are conjugated. The Maleimide groups react with sulfhydryl groups at pH 6.5-7.5 to form stable non-cleavable thioether bonds.
[0472] Quenching is performed by adding 2-mercaptoethanol (Sigma) to a final concentration of 2 mM and incubation at RT for 30 min at 1000 rpm in a shaker incubator. A second quenching step is conducted with N-(2-hydroxyethyl)maleimide (Sigma) to give final concentration of 6 mM and Incubation at RT while mixing. Binding capacity was determined using a biotinylated dye-conjugate.
Example 2
[0473] Library Preparation
[0474] Reversible Attachment of Target-Specific Primers as Analyte-Specific Reagent(s) to Precursor Microparticles
[0475] Each fluorescently colour-labelled hybrid hydrogel microparticle is capable of specifically detecting a different target of interest. To equip each class of microparticle with a primer set that is compatible with its target, functionalized gelatin/agarose hybrid microparticle aliquots are incubated with desthio-biotinylated primers in individual 2.0 mL microcentrifuge tubes. The desthiobiotin moiety of the oligonucleotide facilitates release of the oligonucleotides from the microparticle when temperature is raised, and an emulsion formed in the subsequent signal amplification step. This makes the oligonucleotides immediately available for the detection reaction.
[0476] Thus, precursor microparticles are washed once with nuclease-free water followed by resuspension of the microparticle pellet in equal amounts of nuclease-free water to make a 50% microparticle slurry. Differently labelled slurry aliquots are prepared in that aliquots of the 50% microparticle slurry are pelleted. An equal volume of each target-specific desthiobiotin-labelled primer pair (component 5-8) is added to one microsphere pellet to obtain a 50% bead slurry at a final concentration of 200 nM of each primer thereby assigning a specific analyte-specific reagent. To ensure efficient binding of primers to the hybrid hydrogel matrix, both components are incubated at 20° C. for 15 min while shaking at 1000 rpm. Microspheres are washed three times with a five times higher volume of nuclease-free water to remove unattached primers. [0477] Component 5: rpoB primer pair (analyte-specific reagent 1)
TABLE-US-00001 0.2 μM desthiobiotin-labeled rpoB sense primer SEQ ID NO: 1 (5′-ATCAACATCCGGCCGGTGGTCGCC-3′) (Metabion International AG) 0.2 μM desthiobiotin-labeled rpoB antisense primer SEQ ID NO: 2 (5′-TCACGTGACAGACCGCCGGGC-3′) (Metabion International AG) [0478] Component 6: IS6110 primer pair (analyte-specific reagent 2)
TABLE-US-00002 0.2 μM desthiobiotin-labeled IS6110 sense primer SEQ ID NO: 3 (5′-CGCCGCTTCGGACCACCAGCAC-3′) (Metabion International AG) 0.2 μM desthiobiotin-labeled IS6110 antisense primer SEQ ID NO: 4 (5′-GTGACAAAGGCCACGTAGGCGAACC-3′) (Metabion International AG) [0479] Component 7: IS1081 primer pair (analyte-specific reagent 3)
TABLE-US-00003 0.2 μM desthiobiotin-labeled IS1081 sense primer SEQ ID NO: 5 (5′-GCGCGGCAAGATCATCAATGTGGAG-3′) (Metabion International AG) 0.2 μM desthiobiotin-labeled IS1081 antisense primer SEQ ID NO: 6 (5′-GCCACCGCGGGGAGTTTGTCG-3′) (Metabion International AG) [0480] Component 8: atpD (internal control) primer pair (analyte-specific reagent 4)
TABLE-US-00004 0.2 μM desthiobiotin-labeled internal control (Bac. globigii) sense primer SEQ ID NO: 7 (5′-GCGCGGCAAGATCATCAATGTGGAG-3′) (Metabion International AG) 0.2 μM desthiobiotin-labeled internal control (Bac. globigii) antisense primer SEQ ID NO: 8 (5′-GCCACCGCGGGGAGTTTGTCG-3′) (Metabion International AG)
[0481] Lyophilisation of Microparticle Library
[0482] Optionally, the microsphere library is lyophilized to give dry analyte/target-specific microparticle pellets for long-term storage. Therefore, equal amounts of desired target-specific microparticle are pipetted together and sufficiently mixed using a vortexer. The microparticle library is 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 microparticle when exposed to an eluate later in the process.
[0483] The library aliquots 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 2h. 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 3h 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. The final product is: [0484] Component 9: lyophilized nanoreactor library [0485] comprising target-specific reagents required for parallel detection of rpoB, IS6110, IS1081 and internal control in one sample
Example 3
[0486] Analyte Multiplexing Using Nanoreactors
[0487] In the following embodiment, a sample (eluate) is encapsulated in a monodispersed suspension using the target-specific microparticles of the nano-reactor library.
[0488] Loading of the Analyte and the Generic Detection Reagents to the Nanoreactor Library
[0489] The generic reagents for analyte detection within the nano-reactors are supplied as a freeze-dried pellet that is resuspended with an analyte-containing eluate derived from an upstream sample preparation process. In this example a real sample mimicked by using a defined concentration of purified PCR products for rpoB, IS6110, IS1081 and atpD with target sequences to be amplified (originally derived from H37Rv DNA) in nuclease-free water.
[0490] The entire volume of the sample (100 μl) containing either no template molecules or defined amounts of target molecules are diluted in a MTB (=Mycobacterium tuberculosis) negative patient sputum eluate and added to a lyophilised generic reagent pellet (component 10). The reagents are carefully resuspended by a short vortexing step. Likewise, the entire volume of the generic reagent mix including the template molecules is added to the nano-reactor library pellet (component 9). The porous microparticles are allowed to absorb the entire liquid including all detection reagents and template molecules for 15 min at ambient temperatures while shaking at 1000 rpm. In this process, the four templates are distributed randomly among the four types of reagent specific nanoreactors. In the subsequent digital amplification step only templates matching the specific reagent set of the nanoreactors can be amplified and detected. Consequently, the number of positive events in the digital amplification is reduced by a factor n, where n corresponds to the number of nanoreactor types in the nanoreactor library. [0491] Component 10 consists of the following final concentrations [0492] PCR Buffer: 20 mM Tris HCl, 22 mM KCl, 22 mM NH.sub.4Cl, 3 mM MgCl.sub.2 [0493] 0.2 U/μl Hot Start Taq DNA Polymerase (biotechrabbit GmbH) [0494] 0.4 mM dNTPs (biotechrabbit GmbH) [0495] 1 μM EvaGreen® Fluorescent DNA Stain (JenaBioscience GmbH) or 0.4 μM TaqMan probes [0496] 0.1% (w/v) low bioburden, protease free, for molecular biology BSA (Sigma)
[0497] Phase Transfer of Loaded Microparticles
[0498] The nano-reactor library is transferred into a non-aqueous phase by dispersing microparticles in component 11 to prevent crosstalk between target-specific reactions. The complete aqueous phase (100 μL) is brought in contact with an excess of component 11 (500 μL) using a 1.5 mL microcentrifuge tube. High shear forces are applied to deagglomerate and emulsify aqueous microparticles in the fluorocarbon oil into single nano-reactors. The mixture is 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/suspension. 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 suspension three times the mild centrifugation (400 rcf). Repeated washing with the same oil (component 11) removes essentially all undesirable liquid droplets. Component 11 also yields efficient thermostability of the suspension for subsequent digital emulsion/suspension PCR. [0499] Component 11: phase transfer & signal amplification oil [0500] HFE-7500 fluorocarbon oil (3M Deutschland GmbH) supplemented with [0501] 2-5% (v/v) PicoSurf (Dolomite Microfluidics) OR 2-5% (v/v) FluoSurf (Emulseo)
[0502] Parallel Digital PCR Amplification Reactions in Microparticle-Templated Reaction Spaces
[0503] The monodispersed suspension with encapsulated sample 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, microcapsules provide an evenly spaced array of approximately 20000-30000 nano-reactors (5000-7500/target) for subsequent parallel signal amplification reactions.
[0504] 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 conditions applied are: Initial denaturation for 30s at 95° C. followed by 30-45 cycles of a two-step PCR consisting of Denaturation at 95° C. for is and Annealing/Elongation at 64° C. for 4s. Due to their sol-gel switching capability, the suspension becomes an emulsion with individual liquid nanoliter droplets. Furthermore, microparticles release desthiobiotin bound target-specific oligonucleotides from the gelatin matrix when initially heated to 95° C. making it available for the PCR. The multiplexed amplification of individual targets takes place in the resulting nano reaction compartments.
[0505] 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.
[0506] Image acquisition settings are as following: 100-1000 ms and gain of 1-10×. Two images are required for label identification (λexc 1=550 nm, λexc 2=650 nm) and one image for specific PCR signals (λexc 3=470 nm). For optional nanoreactor specific real time analysis, three images corresponding to three fluorescence channels can be taken at the end of each annealing step of the thermal protocol at one position of the chamber.
[0507] Upon completion of the thermal protocol, the whole detection chamber is scanned using the same equipment at the settings mentioned above. With the set up outlined before in total 30 images are required to cover the dimension of the amplification/detection chamber.
[0508] Decoding of Microparticles & Multiplexed Analysis
[0509] 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 (combination of fluorescent dyes Cy3 & Cy5) and their respective specific amplification and melting curve signals.
[0510] The following data readouts are possible:
[0511] a) Endpoint Analysis (Digital Readout)
[0512] 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. Droplet digital PCR data for each labelled target is viewed in a 1-D or 2-D plot. The software first clusters the negative and positive fractions for each nanoreactor volume and then fits the fraction of positive droplets to a Poisson algorithm to determine the initial concentration of the target DNA molecules in units of copies/mL input. Since the assay described above is a 4-plex reaction the droplets cluster into various groups depending on the concentration of templates added: [0513] a) Label component 1 (1:0 ratio Cy3 & Cy5), detection signal (EvaGreen/probe) negative [0514] b) Label component 1 (1:0 ratio Cy3 & Cy5), detection signal (EvaGreen/probe) positive [0515] c) Label component 2 (3:1 ratio Cy3 & Cy5), detection signal (EvaGreen/probe) negative [0516] d) Label component 2 (3:1 ratio Cy3 & Cy5), detection signal (EvaGreen/probe) positive [0517] e) Label component 3 (1:3 ratio Cy3 & Cy5), detection signal (EvaGreen/probe) negative [0518] f) Label component 3 (1:3 ratio Cy3 & Cy5), detection signal (EvaGreen/probe) positive [0519] g) Label component 4 (0:1 ratio Cy3 & Cy5), detection signal (EvaGreen/probe) negative [0520] h) Label component 4 (0:1 ratio Cy3 & Cy5), detection signal (EvaGreen/probe) positive
[0521] Such 1-D plots are shown for each microparticle with its respective label component in
[0522] b) Real-Time Analysis
[0523] Average pixel intensities for each target-specific nanoreactor were tracked through all cycles of the PCR generating real-time fluorescence curves for single nanoreactors. These exhibit the typical exponential, linear, and plateau phases of the PCR, comparable to those observed in microliter-scale reactions. Sigmoid and linear fitting is performed on all nanoreactors using the Levenberg-Marquardt algorithm. Lift (change in fluorescence post PCR vs. prePCR) criteria are applied to eliminate implausible curves. Negative and positive real-time curves are generated, and a cycle threshold value is determined if positive. False positives (evaporating nanoreactors, artefacts) are identified and excluded from the analysis.
LITERATURE
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