JPWO2021122563A5 - - Google Patents

Description

The average pixel intensity for each target-specific nanoreactor was tracked throughout the entire cycle of PCR resulting in a real-time fluorescence curve for a single nanoreactor. These exhibit the normal logarithmic, linear and plateau phases of PCR, comparable to those observed in microliter scale reactions. Sigmoid and linear fits are performed on all nanoreactors using the Levenberg-Marquardt algorithm. Apply lift criteria (change in fluorescence after PCR vs. before PCR) to eliminate implausible curves. Create negative and positive real-time curves and determine the cycle threshold for positive cases. Identify false positives (evaporation nanoreactors, artifacts) and exclude them from analysis.
Literature:

The present invention provides, for example, the following items.
(Item 1)
A library of fabricated precursor microparticles for producing a library of fabricated microparticles, wherein the fabricated microparticles are configured for specific detection of one or more analytes of interest in a sample. and such specific detection is carried out within such microparticles by a suitable chemical or biochemical reaction, each of said fabricated precursor microparticles in said library comprising:
- a porous matrix, preferably a porous polymeric matrix, with a void volume for receiving the aqueous sample and providing a reaction space for said specific detection of the analyte; - analyte-specific detection of said precursor microparticles; a reagent binding component that allows attachment, preferably reversible attachment, of a reagent; said reagent binding component comprising:
(i) a polymer or polymer mixture forming or being said porous matrix;
(ii) reagent binding molecules attached to the porous matrix;
(iii) at least one ionizable group or a plurality of ionizable groups immobilized on the porous matrix, the ionizable group being capable of changing charge according to ambient conditions around the precursor microparticle; , ionizable group;
(iv) at least one charged group or groups immobilized on said porous matrix;
(v) Any combination of (i) to (iv)
a reagent binding component that is one of;
- when said analyte-specific reagent is attached to said precursor microparticle, a method attached to, contained within, or otherwise for identifying said analyte-specific reagent; the labeled component associated with it at
Library, including:
(Item 2)
There are at least two distinct subsets of fabricated precursor microparticles within said library, preferably three or more distinct subsets of fabricated precursor microparticles, each subset having the same characteristics as said precursor within said subset. the at least two or more distinct fabricated precursor microparticle subsets having different labeling components attached to, contained within, or otherwise associated with the body microparticles, each subset The library of item 1, wherein each label component attached to, contained within, or otherwise associated with is different.
(Item 3)
A library of fabricated microparticles for the specific detection of one or several analytes of interest in a sample, wherein such specific detection is achieved by a suitable chemical or biochemical reaction. each of the fabricated particles comprises a fabricated precursor particle according to any one of items 1 to 2, and the fabricated particle is attached, preferably reversibly attached, to the precursor particle. The library further comprises analyte-specific reagents.
(Item 4)
the analyte-specific reagent attached to each of the precursor microparticles,
a) direct binding of said analyte-specific reagent to said polymer or polymer mixture (i) forming or being part of said porous polymer matrix;
b) said analyte-specific reagent conjugated to a binding entity that binds to said reagent binding molecule (ii);
c) direct binding of said analyte-specific reagent to said ionizable group (iii) under conditions where said ionizable group has a suitable net charge;
d) Direct binding of said analyte-specific reagent to said charged group (iv) on said polymer, said analyte-specific reagent having at least one ionizable group or multiple ionizable groups. and the ionizable group is capable of changing charge according to the ambient conditions around the analyte-specific reagent; or
e) Any combination of (a) to (d)
The library of fabricated microparticles according to item 3, wherein the library of fabricated microparticles is attached via said reagent binding component, preferably reversibly attached, by said method.
(Item 5)
The polymer (i) is ia) a synthetic polymer, such as poly(methyl)(meth)acrylate, polyamide; ib) a silicone polymer, such as polydimethylsiloxane; ic) a polysaccharide, such as agarose, chitin, chitosan, dextran, alginate, carrageenan, cellulose, fucoidan, laminaran; gums selected from xanthan gum, gum arabic, gum gatchi, guar gum, locust bean gum, gum tragacanth, gum karaya and inulin; polypeptides, e.g. collagen, gelatin; polyamino acids, e.g. polylysine; a hydrogel-forming agent selected from the group comprising naturally occurring polymers selected from polynucleotides; said polymer mixture is a combination of any of the foregoing;
Said reagent binding molecule (ii) is avidin, in particular tetrameric avidin; streptavidin; monomeric avidin; avidin with a nitrated tyrosine in the biotin binding site; derived from or related to avidin; other proteins that retain the binding function of; biotin; desthiobiotin; iminobiotin; biotin with a cleavable spacer arm; selenobiotin; oxybiotin; homobiotin; norbiotin; iminobiotin; diaminobiotin; biotin sulfoxide ; biotin sulfone; epibiotin; 5-hydroxybiotin; 2-thiobiotin; azabiotin; carbobiotin; methylated derivative of biotin; ketone biotin; derived from or related to biotin and retains the binding function of biotin , selected from other molecules;
- the at least one ionizable group (iii) or the ionizable group according to item 4 d),
・N-2-acetamido-2-aminoethanesulfonic acid (ACES);
・N-2-acetamido-2-iminodiacetic acid (ADA);
・Aminomethylpropanediol (AMP);
・3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (AMPSO);
・N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES);
・N,N-bis-2-hydroxyethylglycine (BICINE);
・Bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris);
・1,3-Bis-Trishydroxymethylmethylaminopropane (Bis-Trispropane);
・4-cyclohexylamino-1-butanesulfonic acid (CABS);
・3-cyclohexylamino-1-propanesulfonic acid (CAPS);
・3-cyclohexylamino-2-hydroxy-1-propanesulfonic acid (CAPSO);
・2-N-cyclohexylaminoethanesulfonic acid (CHES);
・3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (DIPSO);
・N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS);
・N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS);
・N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES);
・N-2-hydroxyethylpiperazine-N-2-propanesulfonic acid (HEPPSO);
・2-N-morpholinoethanesulfonic acid (MES);
・4-N-morpholinobutanesulfonic acid (MOBS);
・3-N-morpholinopropanesulfonic acid (MOPS);
・3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO);
・Piperazine-N-N-bis-2-ethanesulfonic acid (PIPES);
・Piperazine-N-N-bis-2-hydroxypropanesulfonic acid (POPSO);
・N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS);
・N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS); ・3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid (TAPSO);
・N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES);
・N-Trishydroxymethylmethylglycine (TRICINE);
・Trishydroxymethylaminomethane (Tris);
・Polyhydroxylated amine;
- imidazole and its derivatives (i.e. imidazole), especially those containing a hydroxyl group;
- Dimers and polymers of triethanolamine; and
di/tri/oligo/polyamino acids such as Ala-Ala; Gly-Gly; Ser-Ser; Gly-Gly-Gly; or Ser-Gly. Oligo-His, Poly-His, Oligo-Lys, Poly-Lys
selected from
A library of manufactured precursor fine particles according to any one of items 1 to 2 or a library of manufactured fine particles according to any one of items 3 to 4.
(Item 6)
Said porous polymer matrix, or said polymer or polymer mixture forming said porous polymer matrix or being part of said porous polymer matrix, consists of a non-crosslinked polymer, preferably said porous polymer The polymer or polymer mixture forming the matrix or being part of the porous polymeric matrix consists of agarose or a combination of agarose and gelatin; more preferably, in said combination of agarose and gelatin, said The agarose is present in the range of 0.1% (w/v) to 4% (w/v) and the gelatin is present in the range of 0.1% (w/v) to 20% (w/v), preferably is present in the range of 0.5% (w/v) to 20% (w/v), or the library of prepared precursor particles according to any one of items 1 to 2, 5 or items 3 to 5. The library of manufactured fine particles according to any one of 5.
(Item 7)
The analyte-specific reagents include nucleic acids, including aptamers, spiegelmers; antibodies or antibody fragments; non-antibody proteins capable of specifically binding to the analyte or analyte complex, such as receptors, receptor fragments and affinities. Library of fabricated microparticles according to any of items 3 to 6, wherein said analyte-specific reagent is selected from proteins; preferably said analyte-specific reagent is selected from nucleic acids, in particular nucleic acid oligomers and nucleic acid primers.
(Item 8)
For each of said microparticles, said analyte-specific reagent is transferred via said reagent-binding moiety by b) said analyte-specific reagent conjugated to a binding entity that binds said reagent-binding molecule (ii); reversibly attached to the fine particles,
- the binding entity is independently biotin, desthiobiotin, iminobiotin, biotin with a cleavable spacer arm, selenobiotin, oxybiotin, homobiotin, norbiotin, iminobiotin, diaminobiotin, biotin sulfoxide, biotin sulfone , epibiotin, 5-hydroxybiotin, 2-thiobiotin, azabiotin, carbobiotin, methylated derivatives of biotin, ketone biotin, other substances derived from or related to biotin and retaining the binding function of biotin. the reagent binding molecules are independently selected from avidin and streptavidin; or vice versa; or
- the binding entity is biotin, and the reagent binding molecule is derived from or related to monomeric avidin, avidin with a nitrated tyrosine in the biotin binding site, and avidin and which performs the binding function of avidin. selected from other proteins; or vice versa;
A library of manufactured fine particles according to any one of items 3 to 7.
(Item 9)
said library is a library for specific detection of a single analyte of interest in a number of samples, said library comprising at least two distinct fabricated microparticle subsets, preferably , there are three or more distinct fabricated microparticle subsets;
Each subset is
having different label components attached to, contained within, or otherwise associated with the microparticles of the subset;
All of said at least two, three or more distinct subsets,
having the same analyte-specific reagent attached to the porous matrix of the microparticles of the subset, the analyte-specific reagent being specific for one analyte of interest;
said at least two or more distinct fabricated microparticle subsets are identical with respect to attached analyte-specific reagent;
each label component attached to, contained within, or otherwise associated with the microparticles of each subset is different;
each subset is clearly defined and distinguishable by said respective label component;
A library of manufactured fine particles according to any one of items 3 to 8.
(Item 10)
said library is a library for specific detection of multiple analytes of interest in a single sample, said library comprising at least two distinct fabricated microparticle subsets, preferably: there are three or more distinct fabricated microparticle subsets;
Each subset is
having different label components attached to, contained within, or otherwise associated with the microparticles of the subset;
having different analyte-specific reagents attached to the porous matrix of the microparticles of the subset; each analyte-specific reagent being specific for one analyte of interest;
The at least two or more distinct subsets of fabricated microparticles include:
a respective labeling component attached to, contained within, or otherwise associated with said microparticles of each subset; and
said respective analyte-specific reagent attached to each subset.
are different;
each subset is clearly defined and distinguishable by said respective label moiety and said respective analyte-specific reagent;
A library of manufactured fine particles according to any one of items 3 to 8.
(Item 11)
the library is a library for specific detection of multiple analytes of interest in a number of samples, and within the library there are multiple different distinct fabricated microparticle subsets;
Each subset is
having different label components attached to, contained within, or otherwise associated with the microparticles of the subset;
Among the plurality of distinct fabricated microparticle subsets there are different classes of distinct fabricated microparticle subsets, each of said classes including a number of microparticle subsets, and each class having said plurality of microparticle subsets. all microparticle subsets within a class have the same analyte-specific reagent attached to the analyte-specific matrix; each analyte-specific reagent is attached to one analyte of interest. specific;
In the library, the plurality of different distinct fabricated microparticle subsets are:
each label component attached to, contained within, or otherwise associated with the microparticles of each subset is different;
each distinct particulate subset forms part of a class of particulate subsets;
each subset is clearly defined and distinguishable by said respective label moiety and said respective analyte-specific reagent;
The microparticle subsets of the different classes differ in their respective analyte-specific reagents attached to the porous matrix of the microparticles; each of the different classes includes several microparticle subsets, and each of the microparticle subsets within a class all of the subsets have the same analyte-specific reagent,
A library of manufactured fine particles according to any one of items 3 to 8.
(Item 12)
A kit for producing a library of manufactured microparticles according to any one of items 3 to 11, the kit comprising:
at least two containers, namely a first and a second container, and optionally further containers, each of which includes:
fabricated precursor particulate subsets as defined in any of items 1-2, 5 and 6, each subset being attached to, contained within, or contained in said precursor particulate within said subset; the two fabricated precursor microparticle subsets having different label components attached to, contained within, or otherwise associated with each subset; Each label component is different;
Further containers are
a) direct binding of said analyte-specific reagent to said polymer or polymer mixture (i) forming or being part of said porous polymer matrix;
b) said analyte-specific reagent conjugated to a binding entity that binds to said reagent binding molecule (ii);
c) direct binding of said analyte-specific reagent to said ionizable group (iii) under conditions where said ionizable group has a suitable net charge;
d) Direct binding of said analyte-specific reagent to said charged group (iv) on said polymer, said analyte-specific reagent having at least one ionizable group or multiple ionizable groups. and the ionizable group is capable of changing charge according to the ambient conditions around the analyte-specific reagent; or
e) Any combination of (a) to (d)
a conditioning solution that enables attachment, preferably reversible attachment, of an analyte-specific reagent to said subset of precursor microparticles via said reagent binding component; and the reagent binding molecule is as defined in any of items 1, 4 and 5, and the binding entity is as defined in any of items 4 to 5. said polymer or polymer mixture is as defined in any of items 1, 4 to 6; said ionizable group is as defined in any of items 1, 4 and 5; said analysis A kit, wherein the substance-specific reagent is as defined in any of items 3-4, 7 and 8.
(Item 13)
A method for producing a library of manufactured fine particles according to any one of items 3 to 11, the method comprising:
-
- a library of fabricated precursor particles as defined in any of items 1-2, 5 and 6;
- at least one analyte-specific reagent as defined in any of items 3-4, 7 and 8
providing in any order;
- said library of fabricated precursor microparticles under conditions that allow attachment, preferably reversible attachment, of said at least one analyte-specific reagent to some or all of said fabricated precursor microparticles; or mixing a selected subset thereof with said at least one analyte-specific reagent to generate a library of fabricated microparticles according to any of items 5 to 13;
- If necessary, washing the manufactured microparticles to remove unattached analyte-specific reagents.
including methods.
(Item 14)
A kit for detecting an analyte in a sample, the kit comprising:
a)
- a container containing a general detection composition comprising reagents for carrying out a chemical or biochemical detection reaction of an analyte, said chemical or biochemical detection reaction of an analyte comprising nucleic acid amplification; Yes, the general detection composition includes a buffer, a mononucleoside triphosphate, an amplification enzyme, e.g., a suitable nucleic acid polymerase, e.g., Taq polymerase, and an amplification product, e.g., a nucleic acid dye for detection of the amplified nucleic acid. containing, container, or
- a container comprising a first detection composition comprising a reagent for carrying out a chemical or biochemical detection reaction of an analyte; and a still further container comprising a second detection composition comprising a detection reagent; , the chemical or biochemical detection reaction of the analyte is an immunochemical detection reaction, and the first detection composition comprises a reagent, such as a buffer, and a reagent for carrying out the immunochemical detection reaction, and the immunochemical detection reaction. A secondary antibody or secondary antibody specific for the same analyte as the primary antibody, antibody fragment or non-antibody protein used as an analyte specific reagent (ASR) in a chemical detection reaction and conjugated to a suitable reporter enzyme. said second detection composition comprises as a detection reagent a suitable substrate for said suitable reporter enzyme, which substrate becomes detectable when reacted with said reporter enzyme; , a container and a still further container, which are optically detectable, more preferably fluorescently detectable, or - a first container comprising a reagent for carrying out a chemical or biochemical detection reaction of an analyte. and a second detection composition comprising a detection reagent, wherein said chemical or biochemical reaction is an immunochemical detection reaction; The detection composition includes reagents for carrying out the immunochemical detection reaction, such as a buffer, and a detection composition specific for the same analyte as the primary antibody used as the analyte-specific reagent (ASR) in said immunochemical detection reaction. a second antibody or a second antibody fragment attached to a suitable oligonucleotide tag; said second detection composition comprises a buffer, a mononucleoside triphosphate, an amplification enzyme, e.g. Detection reagents include a polymerase, such as Taq polymerase, and a nucleic acid dye for detection of the amplified nucleic acid, and a primer suitable for amplifying the oligonucleotide tag attached to the secondary antibody. , containers and still further containers;
and
b)
- a container containing a non-aqueous phase, for example an oil, for example a fluorocarbon oil, optionally supplemented with an emulsifier;
and
c)
- Mixing vessel for mixing ingredients
Preferably, the kit further comprises:
d)
- Container for carrying out the detection reaction
more preferably, the kit further comprises:
e)
- a kit as described in item 12, or
- A library of manufactured microparticles as described in any of items 3 to 11
Including the kit.
(Item 15)
A method for detecting and/or quantifying an analyte of interest in an aqueous sample, the method comprising the steps of:
-
- Aqueous samples known or suspected to contain the analyte of interest;
- a general detection composition comprising reagents for carrying out a chemical or biochemical detection reaction of said analyte;
- A library of manufactured microparticles according to any of items 3 to 11, wherein the analyte-specific reagent attached to the microparticle is characterized in that it specifically binds to the target analyte or A library of prefabricated microparticles selected to specifically react with the analytes of interest
providing in any order;
- optionally mixing said aqueous sample and said general detection composition;
- incubating said aqueous sample with said library of fabricated microparticles, whereby said library of microparticles absorbs the aqueous sample into the void volume of said microparticles and, optionally, said analyte of interest is absorbed into said sample; being capable of binding to said analyte of interest, if present in said analyte;
- a step of washing the fine particles as necessary;
- adding said generic detection composition to said microparticles, if said aqueous sample has not been previously mixed with said generic detection composition;
- transferring said library of fabricated microparticles to a non-aqueous phase and creating a plurality of isolated reaction spaces for detecting said analytes by removing the aqueous phase around individual fabricated microparticles; the reaction space comprises an aqueous phase and is limited to the void volume of the microparticle;
- optionally releasing the analyte-specific reagent attached to the microparticle by applying an external trigger;
- carrying out a detection reaction of the target analyte;
- detecting and/or quantifying said target analyte;
including methods.
(Item 16)
the target analyte is a nucleic acid,
- said analyte-specific reagent is a nucleic acid or a pair of nucleic acids sufficiently complementary to said analyte of interest to be able to hybridize to said analyte of interest under hybridizing conditions, preferably said analyte-specific reagent; The target reagent is a primer or primer pair suitable for amplifying the target analyte;
- said general detection composition comprises reagents other than primers for carrying out an amplification reaction of said nucleic acid analyte of interest, in particular said general detection composition comprises a buffer, a mononucleoside triphosphate, an amplification reaction; an enzyme, e.g., a suitable nucleic acid polymerase, e.g. Taq polymerase, and an amplification product, e.g., a nucleic acid dye for detection of the amplified nucleic acid;
- The transferring step is a step of transferring the manufactured fine particles to a non-aqueous phase, suspending them, and washing them repeatedly in the non-aqueous phase as necessary; filtration or mechanical stirring to ensure the formation of a monodisperse suspension of microparticles with no or substantially no remaining aqueous phase outside of the
- said optional step of releasing said analyte-specific reagent attached to said microparticle by applying an external trigger, temporarily increasing temperature, changing pH, or changing the conditions, preferably increasing the temperature, more preferably increasing the temperature from ambient temperature to >90°C;
- said step of performing a detection reaction is a step of performing an amplification reaction of said analyte when said analyte is present in said aqueous sample;
- the step of detecting and/or quantifying the analyte of interest is detecting and/or quantifying the amplified analyte;
The method described in item 15.
(Item 17)
A method for detecting and/or quantifying an analyte of interest in an aqueous sample, the method comprising the steps of:
-
- Aqueous samples known or suspected to contain the analyte of interest;
- a first detection composition comprising the reagents necessary to carry out a chemical or biochemical detection reaction of said analyte;
- a second detection composition comprising a detection reagent;
・Library of manufactured fine particles described in any of items 3 to 11
in any order, wherein the analyte-specific reagent attached to the microparticle is such that it specifically binds to or specifically reacts with the analyte of interest. The process is selected so that it can;
- optionally mixing said aqueous sample and said first detection composition;
- incubating said aqueous sample with said library of fabricated microparticles and, if said aqueous sample is not already mixed with said first detection composition, said microparticles; adsorbing the aqueous sample and the first detection composition into the void volume of the microparticle, and optionally binding to the analyte of interest if the analyte of interest is present in the sample; A process that makes it possible to;
- optionally washing the library of prepared microparticles to remove unabsorbed or unreacted first detection composition;
- incubating said library of fabricated microparticles containing an absorbed aqueous sample with said second detection composition, thereby enabling said library of microparticles to absorb said second detection composition; , process;
- optionally further washing the library of prepared microparticles to remove unabsorbed or unreacted second detection composition;
- transferring said library of fabricated microparticles to a non-aqueous phase and creating a plurality of isolated reaction spaces for detecting said analytes by removing the aqueous phase around individual fabricated microparticles; and the reaction space comprises an aqueous phase and is confined to the void volume of the microparticles; preferably said step of transferring to a non-aqueous phase transfers said library of fabricated microparticles to said second detection composition. a step carried out immediately after incubation with;
- optionally releasing the analyte-specific reagent attached to the microparticle by applying an external trigger;
- detecting and/or quantifying said analyte of interest by detecting and/or quantifying said detection reagent;
including methods.
(Item 18)
the analyte of interest is a protein or other non-nucleic acid molecule;
- said analyte-specific reagent is a primary antibody, antibody fragment or non-antibody protein capable of specifically binding to said protein analyte or other non-nucleic acid analyte;
- said first detection composition comprises the necessary reagents for carrying out an immunochemical detection reaction, such as a buffer, and a second antibody specific for said analyte and coupled to a suitable reporter enzyme; or comprising a secondary antibody fragment;
- said second detection composition comprises as a detection reagent a suitable substrate for said suitable reporter enzyme, which substrate becomes detectable when reacted by said reporter enzyme, preferably optically detectable; enabled, and more preferably detectable by fluorescence;
- said step of incubating said aqueous sample with said library of fabricated microparticles and said first detection composition comprises incubating said primary antibody, primary antibody fragment or non-antibody when said analyte is present in said aqueous sample; forming a complex between the analyte and the primary antibody, primary antibody fragment, or non-antibody protein by performing an immunochemical reaction involving binding of the analyte to a protein; further comprising binding said second antibody to a complex, forming a sandwich between the first antibody, antibody fragment or non-antibody protein, the analyte, and the second antibody;
- a first optional step of washing said library removes unbound secondary antibodies from said library;
- said step of incubating said library of fabricated microparticles containing absorbed aqueous samples with said second detection composition is a step of allowing said substrate to be reacted by said reporter enzyme;
- a further optional step of washing said library to remove unreacted substrate from said library;
- said transferring step is a step of transferring said fabricated microparticles to a non-aqueous phase, suspending, and optionally washing repeatedly in said non-aqueous phase; Also includes filtration or mechanical stirring to ensure the formation of a monodisperse suspension of microparticles with no or substantially no remaining aqueous phase;
- said optional step of releasing said analyte-specific reagent attached to said microparticle by applying an external trigger, temporarily increasing temperature, changing pH, or changing the conditions, preferably increasing the temperature, more preferably increasing the temperature from ambient temperature to >90°C;
- the step of detecting and/or quantifying the analyte of interest is detecting and/or quantifying the reacted substrate;
The method described in item 17.
(Item 19)
A method for detecting and/or quantifying an analyte of interest in an aqueous sample, the method comprising the steps of:
-
- Aqueous samples known or suspected to contain the analyte of interest;
- a first detection composition comprising the reagents necessary to carry out a chemical or biochemical detection reaction of said analyte;
- a second detection composition comprising a detection reagent;
・Library of manufactured fine particles described in any of items 3 to 11
in any order, wherein the analyte-specific reagent attached to the microparticle is such that it specifically binds to or specifically reacts with the analyte of interest. The process is selected so that it can;
- optionally mixing said aqueous sample and said first detection composition;
- incubating said aqueous sample with said library of fabricated microparticles and, if said aqueous sample is not already mixed with said first detection composition, said microparticles; adsorbing the aqueous sample and the first detection composition into the void volume of the microparticle, and optionally binding to the analyte of interest if the analyte of interest is present in the sample; A process that makes it possible to;
- optionally washing the library of prepared microparticles to remove unabsorbed or unreacted first detection composition;
- incubating said library of fabricated microparticles containing an absorbed aqueous sample with said second detection composition, thereby enabling said library of microparticles to absorb said second detection composition; , process;
- optionally further washing the library of prepared microparticles to remove unabsorbed or unreacted second detection composition;
- transferring said library of fabricated microparticles to a non-aqueous phase and creating a plurality of isolated reaction spaces for detecting said analytes by removing the aqueous phase around individual fabricated microparticles; the reaction space comprises an aqueous phase and is limited to the void volume of the microparticle;
- optionally releasing the analyte-specific reagent attached to the microparticle by applying an external trigger;
- carrying out a detection reaction of the target analyte; and
- detecting and/or quantifying said target analyte;
including methods.
(Item 20)
the analyte of interest is a protein or other non-nucleic acid molecule;
- said analyte-specific reagent is a primary antibody, antibody fragment or non-antibody protein capable of specifically binding to said protein analyte or other non-nucleic acid analyte;
- said first detection composition is coupled to a suitable oligonucleotide tag, specific for the same analyte as said first antibody, and the necessary reagents for carrying out an immunochemical detection reaction, such as a buffer; comprising a secondary antibody or secondary antibody fragment;
- said second detection composition comprises a buffer, a mononucleoside triphosphate, an amplification enzyme, e.g. a suitable nucleic acid polymerase, e.g. Taq polymerase, and an amplification product, e.g. a nucleic acid dye for detection of the amplified nucleic acid. , and a primer suitable for amplifying the oligonucleotide tag attached to the secondary antibody as a detection reagent;
- said step of incubating said aqueous sample with said library of fabricated microparticles and said first detection composition comprises incubating said primary antibody, primary antibody fragment or non-antibody when said analyte is present in said aqueous sample; forming a complex between the analyte and the primary antibody, primary antibody fragment, or non-antibody protein by performing an immunochemical reaction involving binding of the analyte to a protein; further comprising binding said second antibody to a complex, forming a sandwich between the first antibody, antibody fragment or non-antibody protein, the analyte, and the second antibody;
- a first optional step of washing said library removes unbound secondary antibodies from said library;
- said step of incubating said library of fabricated microparticles containing an absorbed aqueous sample with said second detection composition, wherein said primer in said second detection composition is attached to said secondary antibody; hybridizing to said oligonucleotide tag;
- a further optional step of washing said library to remove unhybridized primers from said library;
- said transferring step is a step of transferring said fabricated microparticles to a non-aqueous phase, suspending, and optionally washing repeatedly in said non-aqueous phase; Also includes filtration or mechanical stirring to ensure the formation of a monodisperse suspension of microparticles with no or substantially no remaining aqueous phase;
- said optional step of releasing said analyte-specific reagent attached to said microparticle by applying an external trigger, temporarily increasing temperature, changing pH, or changing the conditions, preferably increasing the temperature, more preferably increasing the temperature from ambient temperature to >90°C;
- the step of performing a detection reaction is a step of performing an amplification reaction of the oligonucleotide tag;
the step of detecting and/or quantifying the target analyte is detecting and/or quantifying the amplified oligonucleotide tag;
The method described in item 19.
(Item 21)
Any of items 15 to 20, wherein said step of releasing said analyte-specific reagent attached to said microparticle by applying an external trigger is carried out by increasing the temperature to which said microparticle is exposed. Method described.
(Item 22)
- the method detects and/or quantifies a single analyte in a plurality of aqueous samples, e.g. from different patients;
- the method provides a plurality of different aqueous samples known or suspected to contain a plurality of analytes of interest, e.g. samples from different patients;
- the library of fabricated microparticles provided in said method is a library according to item 9, in which said library contains a number equal to or equal to the number of different aqueous samples to be tested provided; at least the same number of distinct microparticle subsets are provided, all of said distinct subsets having the same analyte-specific reagent attached to said porous matrix of said microparticles of said subset, said analyte-specific reagent is specific for one analyte of interest;
- incubating said aqueous sample with said library of fabricated microparticles, incubating each of said different aqueous samples separately with a distinct subset of microparticles of said library;
- said step of incubating said library of fabricated microparticles containing an adsorbed aqueous sample with a second detection composition, wherein said step of incubating said library of fabricated microparticles containing an adsorbed aqueous sample with said second detection composition; Incubate;
- said step of transferring said library to a non-aqueous phase is carried out separately for each microparticle subset, i.e. each distinct microparticle subset is separately transferred to a non-aqueous phase, and the aqueous phase around said individual microparticles is By removing each subset separately, a plurality of isolated reaction spaces for detecting the analytes are created separately for each subset, the reaction spaces containing an aqueous phase and containing the void volume of the microparticles. limited to
The method continues, after the step of transferring the library to a non-aqueous phase, after the step of mixing together the plurality of isolated reaction spaces of all the distinct subsets in the non-aqueous phase. , further comprising the steps of performing a detection reaction for the target analyte, and then detecting and/or quantifying the target analyte.
The method described in any of items 15 to 21.
(Item 23)
- the method is a method for detecting and/or quantifying multiple analytes in a single aqueous sample;
- the method provides a single aqueous sample known or suspected to contain multiple analytes of interest;
- the library of fabricated microparticles provided in the method is a library according to item 10, and among such libraries there are at least the same number of different target analytes to be detected; distinct subsets of microparticles are provided, each of said distinct subsets having a different analyte-specific reagent attached to said porous matrix of said microparticles of said subset; each analyte-specific reagent comprising one specific for one target analyte;
- said step of incubating said aqueous sample with said library of fabricated microparticles, incubating said aqueous sample with all of said distinct microparticle subsets of said library;
- said step of incubating said library of fabricated microparticles containing an adsorbed aqueous sample with a second detection composition, wherein said step of incubating said library of fabricated microparticles containing an adsorbed aqueous sample includes all said distinct microparticle subsets of said library together with said second detection composition; Incubate;
- said step of transferring said library to a non-aqueous phase is performed for all microparticle subsets together, i.e. all microparticle subsets are transferred together to a non-aqueous phase and the aqueous phase around said individual microparticles is is removed to create a plurality of isolated reaction spaces for detecting and/or quantifying the plurality of analytes, the reaction spaces containing an aqueous phase and filling the void volume of the microparticle. limited,
The method described in any of items 15 to 21.
(Item 24)
- the method is a method for detecting and/or quantifying a plurality of analytes in a plurality of aqueous samples;
- the method provides a plurality of different aqueous samples known or suspected to contain a plurality of analytes of interest, e.g. samples from different patients;
- the library of fabricated microparticles provided in the method is a library according to item 11, in which there are distinct fabricated microparticle subsets of different classes; Each of the classes includes a number of microparticle subsets, each of said classes having a different analyte-specific reagent attached to said porous matrix of said microparticles, and for every microparticle subset within one class: the same analyte-specific reagents are attached; each analyte-specific reagent is specific for one analyte of interest; in said method, said step of providing said library comprises:
different classes of distinct particulate subsets are provided as many or at least as many as the number of different analytes to be detected;
- as many or at least as many different microparticle subsets are provided within each class as the number of aqueous samples to be tested that are provided;
- providing in said library as many or at least as many different microparticle subsets as the number of different aqueous samples to be tested multiplied by the number of different target analytes to be detected;
- said step of incubating said aqueous sample with said library of fabricated microparticles, in which exactly one aqueous sample is incubated with exactly one subset of microparticles from each class, each aqueous sample having a is incubated with a number of different microparticle subsets from different classes, equal to the number of microparticle classes in
- said step of incubating said library of fabricated microparticles containing adsorbed aqueous samples with a second detection composition, a combined subset of microparticles of different classes previously incubated with one respective aqueous sample; incubating with the second detection composition;
- said step of transferring said library to a non-aqueous phase is carried out separately for each aqueous sample, i.e. said combined microparticle subsets previously incubated with one respective aqueous sample are transferred together to a non-aqueous phase; By removing the aqueous phase around the individual microparticles separately for each aqueous sample, a plurality of isolated reaction spaces are created for detecting the plurality of analytes, the reaction spaces being separated from the aqueous phase. and limited to the void volume of the microparticle,
The method preferably continues after said step of transferring said library to a non-aqueous phase and after mixing together said plurality of isolated reaction spaces of said separate total samples in said non-aqueous phase. , further comprising the steps of performing a detection reaction for the target analyte, and then detecting and/or quantifying the target analyte.
The method described in any of items 15 to 21.
(Item 25)
In the step of detecting and quantifying the analyte of interest, the quantification of the analyte comprises:
a) digital nucleic acid amplification, in particular digital polymerase chain reaction (PCR);
b) real-time quantitative nucleic acid amplification, in particular real-time polymerase chain reaction (PCR);
c) immunochemical detection methods, in particular digital immunochemical detection methods, e.g. digital immunoassays, e.g. digital enzyme-linked immunosorbent assay (ELISA);
d) Immunochemical detection methods combined with nucleic acid amplification, e.g. immunopolymerase chain reaction; in particular digital immuno-PCR
carried out by a method selected from;
When said analyte of interest is a nucleic acid, quantification is carried out using either method a) or b) or a combination of a) and b); when said analyte is a protein, peptide or other In the case of non-nucleic acid analytes, quantification is performed using either method c) or d).
The method described in any of items 15 to 24.

Claims (23)

A library of fabricated microparticles for the specific detection of one or several analytes of interest in a sample, wherein such specific detection is achieved by a suitable chemical or biochemical reaction. each of the fabricated particles includes a fabricated precursor particle, such fabricated precursor particles comprising:
- a porous polymer matrix with a void volume for receiving the aqueous sample and for providing a reaction space for said specific detection of the analyte;
- a reagent binding component that allows reversible attachment of an analyte-specific reagent to said precursor microparticle; said reagent binding component comprising:
(i) a polymer or polymer mixture forming or being said porous matrix;
(ii) reagent binding molecules attached to the porous matrix;
(iii) at least one ionizable group or a plurality of ionizable groups immobilized on the porous matrix, the ionizable group being capable of changing charge according to ambient conditions around the precursor microparticle; , ionizable group;
(iv) at least one charged group or groups immobilized on said porous matrix;
(v) Any combination of (i) to (iv)
a reagent binding component that is one of;
- when said analyte-specific reagent is attached to said precursor microparticle, a method attached to, contained within, or otherwise for identifying said analyte-specific reagent; the labeled component associated with it at
including;
Each of the fabricated microparticles further includes an analyte-specific reagent reversibly attached to the precursor microparticle ;
the analyte-specific reagent attached to each of the precursor microparticles,
a) direct binding of said analyte-specific reagent to said polymer or polymer mixture (i) forming or being part of said porous polymer matrix;
b) said analyte-specific reagent conjugated to a binding entity that binds said reagent binding molecule (ii); said reagent binding molecule and said binding entity interacting with each other in a reversible manner; said analyte-specific reagents selected to bind to each other;
c) direct binding of said analyte-specific reagent to said ionizable group (iii) under conditions where said ionizable group has a suitable net charge;
d) Direct binding of said analyte-specific reagent to said charged group (iv) on said polymer, said analyte-specific reagent having at least one ionizable group or multiple ionizable groups. and the ionizable group is capable of changing charge according to the ambient conditions around the analyte-specific reagent; or
e) Any combination of (a) to (d)
A library of fabricated microparticles, reversibly attached via said reagent-binding moiety, by . Within said library there are at least two distinct fabricated microparticle subsets, preferably three or more distinct fabricated microparticle subsets, each subset attached to said microparticle within said subset. , have different labeling components contained therein or otherwise associated therewith, such that said at least two or more distinct fabricated microparticle subsets have attached to each subset, 2. The library of fabricated microparticles of claim 1, wherein each label component contained therein or otherwise associated therewith is different. - said polymer (i) is ia) a synthetic polymer, such as poly(methyl)(meth)acrylate, polyamide; ib) a silicone-based polymer, such as polydimethylsiloxane; ic) a polysaccharide, such as agarose, chitin, chitosan; , dextran, alginate, carrageenan, cellulose, fucoidan, laminaran; gums selected from xanthan gum, gum arabic, gum gatchi, guar gum, locust bean gum, gum tragacanth, gum karaya and inulin; polypeptides, e.g. collagen, gelatin; polyamino acids, e.g. , polylysine; a hydrogel-forming agent selected from the group comprising naturally occurring polymers selected from polynucleotides; said polymer mixture is a combination of any of the foregoing;
- said reagent-binding molecule (ii) is derived from or related to avidin, in particular tetrameric avidin; streptavidin; monomeric avidin; avidin with a nitrated tyrosine in the biotin binding site; Other proteins that retain the binding function of avidin; biotin; desthiobiotin; iminobiotin; biotin with a cleavable spacer arm; selenobiotin; oxybiotin; homobiotin; norbiotin; iminobiotin; diaminobiotin; biotin sulfoxide; biotin sulfone; epibiotin; 5-hydroxybiotin; 2-thiobiotin; azabiotin; carbobiotin; methylated derivatives of biotin; ketone biotin; derived from or related to biotin and retaining the binding function of biotin the reagent-binding molecule and the binding entity are selected to interact with each other, i.e., bind to each other, in a reversible manner;
- the at least one ionizable group (iii) or the ionizable group according to claim 1 d),
・N-2-acetamido-2-aminoethanesulfonic acid (ACES);
・N-2-acetamido-2-iminodiacetic acid (ADA);
・Aminomethylpropanediol (AMP);
・3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (AMPSO);
・N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES);
・N,N-bis-2-hydroxyethylglycine (BICINE);
・Bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris);
・1,3-Bis-Trishydroxymethylmethylaminopropane (Bis-Trispropane);
・4-cyclohexylamino-1-butanesulfonic acid (CABS);
・3-cyclohexylamino-1-propanesulfonic acid (CAPS);
・3-cyclohexylamino-2-hydroxy-1-propanesulfonic acid (CAPSO);
・2-N-cyclohexylaminoethanesulfonic acid (CHES);
・3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (DIPSO);
・N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS);
・N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS);
・N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES);
・N-2-hydroxyethylpiperazine-N-2-propanesulfonic acid (HEPPSO);
・2-N-morpholinoethanesulfonic acid (MES);
・4-N-morpholinobutanesulfonic acid (MOBS);
・3-N-morpholinopropanesulfonic acid (MOPS);
・3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO);
・Piperazine-N-N-bis-2-ethanesulfonic acid (PIPES);
・Piperazine-N-N-bis-2-hydroxypropanesulfonic acid (POPSO);
・N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS);
・N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS);
・3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid (TAPSO);
・N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES);
・N-Trishydroxymethylmethylglycine (TRICINE);
・Trishydroxymethylaminomethane (Tris);
・Polyhydroxylated amine;
- imidazole and its derivatives (i.e. imidazole), especially those containing a hydroxyl group;
Dimers and polymers of triethanolamine; and di/tri/oligo/polyamino acids such as Ala-Ala; Gly-Gly; Ser-Ser; Gly-Gly-Gly; or Ser-Gly. Oligo-His, Poly-His, Oligo-Lys, Poly-Lys
selected from
A library of manufactured fine particles according to any one of claims 1 to 2 . Said porous polymer matrix, or said polymer or polymer mixture forming said porous polymer matrix or being part of said porous polymer matrix, consists of a non-crosslinked polymer, preferably said porous polymer The polymer or polymer mixture forming the matrix or being part of the porous polymeric matrix consists of agarose or a combination of agarose and gelatin; more preferably, in said combination of agarose and gelatin, said The agarose is present in the range of 0.1% (w/v) to 4% (w/v) and the gelatin is present in the range of 0.1% (w/v) to 20% (w/v), preferably A library of manufactured microparticles according to any one of claims 1 to 3 , wherein 0.5% (w/v) to 20% (w/v) is present. The analyte-specific reagents include nucleic acids, including aptamers, spiegelmers; antibodies or antibody fragments; non-antibody proteins capable of specifically binding to the analyte or analyte complex, such as receptors, receptor fragments and affinities. Library of fabricated microparticles according to any of claims 1 to 4 , wherein the analyte-specific reagent is selected from proteins; preferably, the analyte-specific reagent is selected from nucleic acids, in particular nucleic acid oligomers and nucleic acid primers. For each of said microparticles, said analyte-specific reagent is transferred via said reagent-binding moiety by b) said analyte-specific reagent conjugated to a binding entity that binds said reagent-binding molecule (ii); reversibly attached to the fine particles,
- the binding entity is independently biotin, desthiobiotin, iminobiotin, biotin with a cleavable spacer arm, selenobiotin, oxybiotin, homobiotin, norbiotin, iminobiotin, diaminobiotin, biotin sulfoxide, biotin sulfone , epibiotin, 5-hydroxybiotin, 2-thiobiotin, azabiotin, carbobiotin, methylated derivatives of biotin, ketone biotin, other substances derived from or related to biotin and retaining the binding function of biotin. molecule; 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 independently selected from avidin and streptavidin; avidin with a nitrated tyrosine in the biotin binding site, and other proteins derived from or related to avidin that retain the binding function of avidin; or vice versa. And;
- said reagent binding molecule and said binding entity are selected to interact with each other in a reversible manner, i.e. to bind to each other ;
A library of manufactured fine particles according to any one of claims 1 to 5 . said library is a library for specific detection of a single analyte of interest in a number of samples, said library comprising at least two distinct fabricated microparticle subsets, preferably , there are three or more distinct fabricated microparticle subsets;
Each subset is
- having different label components attached to, contained within or otherwise associated with said microparticles of said subset;
All of said at least two, three or more distinct subsets,
- having the same analyte-specific reagent attached to the porous matrix of the microparticles of the subset, said analyte-specific reagent being specific for one analyte of interest;
As such, said at least two or more distinct fabricated microparticle subsets are identical with respect to the attached analyte-specific reagent;
- each label component attached to, contained within, or otherwise associated with said microparticles of each subset is different;
each subset is clearly defined and distinguishable by said respective label component;
A library of manufactured fine particles according to any one of claims 1 to 6 . said library is a library for specific detection of multiple analytes of interest in a single sample, said library comprising at least two distinct fabricated microparticle subsets, preferably: there are three or more distinct fabricated microparticle subsets;
Each subset is
- having different label components attached to, contained within or otherwise associated with said microparticles of said subset;
- having different analyte-specific reagents attached to the porous matrix of the microparticles of the subset; each analyte-specific reagent being specific for one analyte of interest;
As such, said at least two or more distinct subsets of fabricated microparticles include:
- a respective label component attached to, contained within, or otherwise associated with said microparticles of each subset; and
- said respective analyte-specific reagents attached to each subset are different;
each subset is clearly defined and distinguishable by said respective label moiety and said respective analyte-specific reagent;
A library of manufactured fine particles according to any one of claims 1 to 6 . the library is a library for specific detection of multiple analytes of interest in a number of samples, and within the library there are multiple different distinct fabricated microparticle subsets;
Each subset is
- having different label components attached to, contained within or otherwise associated with said microparticles of said subset;
Among the plurality of distinct fabricated microparticle subsets there are different classes of distinct fabricated microparticle subsets, each of said classes including a number of microparticle subsets, and each class having said plurality of microparticle subsets. all microparticle subsets within a class have the same analyte-specific reagent attached to the analyte-specific matrix; each analyte-specific reagent is attached to one analyte of interest. specific;
Therefore, in said library, said plurality of different distinct fabricated microparticle subsets are:
- each label component attached to, contained within, or otherwise associated with said microparticles of each subset is different;
each distinct particulate subset forms part of a class of particulate subsets;
each subset is clearly defined and distinguishable by said respective label moiety and said respective analyte-specific reagent;
As such, the microparticle subsets of the different classes differ in their respective analyte-specific reagents attached to the porous matrix of the microparticles; each of the different classes includes several microparticle subsets, and each of the microparticle subsets within a class all of those subsets have the same analyte-specific reagent,
A library of manufactured fine particles according to any one of claims 1 to 6 . A kit for producing a library of manufactured microparticles according to any one of claims 1 to 9 , the kit comprising:
- at least two containers, namely a first and a second container, and optionally further containers, each of which contains:
- comprising fabricated precursor particulate subsets as defined in any of claims 1 to 4 , each subset comprising: a prefabricated precursor particulate subset in said subset; The two fabricated precursor microparticle subsets have different label components associated therewith in a manner such that the two fabricated precursor microparticle subsets have different label components attached to, contained within, or otherwise associated with each subset. Each label component is different;
-Further containers include :
- A conditioning solution,
a) direct binding of said analyte-specific reagent to said polymer or polymer mixture (i) forming or being part of said porous polymer matrix;
b) said analyte-specific reagent conjugated to a binding entity that binds to said reagent binding molecule (ii);
c) direct binding of said analyte-specific reagent to said ionizable group (iii) under conditions where said ionizable group has a suitable net charge;
d) Direct binding of said analyte-specific reagent to said charged group (iv) on said polymer, said analyte-specific reagent having at least one ionizable group or multiple ionizable groups. and e) the ionizable group is attached to the reagent-binding moiety by a direct bond, the charge of which can be changed according to the ambient conditions around the analyte-specific reagent; or e) by any combination of (a)-(d). a conditioning solution that enables reversible attachment of an analyte-specific reagent to the subset of precursor microparticles via;
The reagent binding component is as defined in claim 1, the reagent binding molecule is as defined in any of claims 1 and 3 , and the binding entity is as defined in any of claims 1 and 3. the polymer or polymer mixture is as defined in any of claims 1, 3 to 4 ; the ionizable group is as defined in any of claims 1 and 3 ; A kit as defined in any of claims 1, 5 and 6, wherein said analyte-specific reagent is as defined in any of claims 1, 5 and 6 . A method for producing a library of manufactured fine particles according to any one of claims 1 to 9 , the method comprising:
-
- a library of fabricated precursor particles as defined in any of claims 1 to 4 ;
- providing at least one analyte-specific reagent as defined in any of claims 1, 5 and 6 in any order;
- said library of fabricated precursor microparticles or a selection thereof under conditions that allow reversible attachment of said at least one analyte-specific reagent to some or all of said fabricated precursor microparticles; producing a library of fabricated microparticles according to any of claims 1 to 9 by mixing the subset of microparticles with the at least one analyte-specific reagent;
- A method, optionally comprising the step of washing said fabricated microparticles to remove unattached analyte-specific reagents. A kit for detecting an analyte in a sample, the kit comprising:
a)
- a container containing a general detection composition comprising reagents for carrying out a chemical or biochemical detection reaction of an analyte, said chemical or biochemical detection reaction of an analyte comprising nucleic acid amplification; Yes, the general detection composition includes a buffer, a mononucleoside triphosphate, an amplification enzyme, e.g., a suitable nucleic acid polymerase, e.g., Taq polymerase, and an amplification product, e.g., a nucleic acid dye for detection of the amplified nucleic acid. or - a container containing a first detection composition containing a reagent for carrying out a chemical or biochemical detection reaction of an analyte, and a second detection composition containing a detection reagent. is also a further container, wherein the chemical or biochemical detection reaction of an analyte is an immunochemical detection reaction, and the first detection composition comprises a reagent for carrying out the immunochemical detection reaction, e.g. buffer, and specific for the same analyte as the primary antibody, antibody fragment or non-antibody protein used as the analyte-specific reagent (ASR) in the immunochemical detection reaction and conjugated to a suitable reporter enzyme. , a second antibody or a second antibody fragment; said second detection composition comprises as a detection reagent a substrate suitable for said suitable reporter enzyme, which substrate, when reacted with said reporter enzyme, causes detection. containers and still further containers that enable, preferably become optically detectable, more preferably become detectable by fluorescence, or - for carrying out a chemical or biochemical detection reaction of an analyte. and a second detection composition comprising a detection reagent, wherein the chemical or biochemical reaction is an immunochemical detection reaction. and said first detection composition is the same as a reagent for carrying out an immunochemical detection reaction, e.g. a buffer, and a primary antibody used as an analyte specific reagent (ASR) in said immunochemical detection reaction. a second antibody or a second antibody fragment specific for the analyte and conjugated to a suitable oligonucleotide tag; said second detection composition comprising a buffer, a mononucleoside triphosphate, an amplification enzyme; , a suitable nucleic acid polymerase, e.g. Taq polymerase, and a nucleic acid dye for detection of the amplified nucleic acid, e.g. a nucleic acid dye suitable for amplifying the oligonucleotide tag attached to the secondary antibody. containers and still further containers containing primers as detection reagents;
and b)
- a container containing a non-aqueous phase, for example an oil, for example a fluorocarbon oil, optionally supplemented with an emulsifier;
and c)
- a mixing container for mixing the components , said kit further comprising:
d)
- a container for carrying out a detection reaction ; said kit further comprises:
e)
- a kit according to claim 10 , or - a kit comprising a library of manufactured microparticles according to any of claims 1 to 9 . A method for detecting and/or quantifying an analyte of interest in an aqueous sample, the method comprising the steps of:
-
- Aqueous samples known or suspected to contain the analyte of interest;
- a general detection composition comprising reagents for carrying out a chemical or biochemical detection reaction of said analyte;
- A library of fabricated microparticles according to any one of claims 1 to 9 , wherein the analyte-specific reagent attached to the microparticle is characterized in that it specifically binds to the analyte of interest or providing in any order a library of fabricated microparticles that are selected to be capable of specifically reacting with said analyte of interest;
- optionally mixing said aqueous sample and said general detection composition;
- incubating said aqueous sample with said library of fabricated microparticles, whereby said library of microparticles absorbs the aqueous sample into the void volume of said microparticles and, optionally, said analyte of interest is absorbed into said sample; being capable of binding to said analyte of interest, if present in said analyte;
- a step of washing the fine particles as necessary;
- adding said generic detection composition to said microparticles, if said aqueous sample has not been previously mixed with said generic detection composition;
- transferring said library of fabricated microparticles to a non-aqueous phase and creating a plurality of isolated reaction spaces for detecting said analytes by removing the aqueous phase around individual fabricated microparticles; the reaction space comprises an aqueous phase and is limited to the void volume of the microparticle;
- optionally releasing the analyte-specific reagent attached to the microparticle by applying an external trigger;
- carrying out a detection reaction of the target analyte;
- A method comprising detecting and/or quantifying said analyte of interest. the target analyte is a nucleic acid,
- said analyte-specific reagent is a nucleic acid or a pair of nucleic acids sufficiently complementary to said analyte of interest to be able to hybridize to said analyte of interest under hybridizing conditions, preferably said analyte-specific reagent; The target reagent is a primer or primer pair suitable for amplifying the target analyte;
- said general detection composition comprises reagents other than primers for carrying out an amplification reaction of said nucleic acid analyte of interest, in particular said general detection composition comprises a buffer, a mononucleoside triphosphate, an amplification reaction; an enzyme, e.g., a suitable nucleic acid polymerase, e.g. Taq polymerase, and an amplification product, e.g., a nucleic acid dye for detection of the amplified nucleic acid;
- The transferring step is a step of transferring the manufactured fine particles to a non-aqueous phase, suspending them, and washing them repeatedly in the non-aqueous phase as necessary; filtration or mechanical stirring to ensure the formation of a monodisperse suspension of microparticles with no or substantially no remaining aqueous phase outside of the
- said optional step of releasing said analyte-specific reagent attached to said microparticle by applying an external trigger, temporarily increasing temperature, changing pH, or changing the conditions, preferably increasing the temperature, more preferably increasing the temperature from ambient temperature to >90°C;
- said step of performing a detection reaction is a step of performing an amplification reaction of said analyte when said analyte is present in said aqueous sample;
- the step of detecting and/or quantifying the analyte of interest is detecting and/or quantifying the amplified analyte;
14. The method according to claim 13 . A method for detecting and/or quantifying an analyte of interest in an aqueous sample, the method comprising the steps of:
-
- Aqueous samples known or suspected to contain the analyte of interest;
- a first detection composition comprising the reagents necessary to carry out a chemical or biochemical detection reaction of said analyte;
- a second detection composition comprising a detection reagent;
- providing a library of fabricated microparticles according to any one of claims 1 to 9 in any order, wherein the analyte-specific reagent attached to the microparticle is a step selected to be capable of specifically binding to or specifically reacting with said analyte of interest;
- optionally mixing said aqueous sample and said first detection composition;
- incubating said aqueous sample with said library of fabricated microparticles and, if said aqueous sample is not already mixed with said first detection composition, said microparticles; adsorbing the aqueous sample and the first detection composition into the void volume of the microparticle, and optionally binding to the analyte of interest if the analyte of interest is present in the sample; A process that makes it possible to;
- optionally washing the library of prepared microparticles to remove unabsorbed or unreacted first detection composition;
- incubating said library of fabricated microparticles containing an absorbed aqueous sample with said second detection composition, thereby enabling said library of microparticles to absorb said second detection composition; , process;
- optionally further washing the library of prepared microparticles to remove unabsorbed or unreacted second detection composition;
- transferring said library of fabricated microparticles to a non-aqueous phase and creating a plurality of isolated reaction spaces for detecting said analytes by removing the aqueous phase around individual fabricated microparticles; and the reaction space includes an aqueous phase and is limited to the void volume of the microparticles; preferably, said step of transferring to a non-aqueous phase transfers said library of fabricated microparticles to said second detection composition. a step carried out immediately after incubation with;
- optionally releasing the analyte-specific reagent attached to the microparticle by applying an external trigger;
- a method comprising detecting and/or quantifying said analyte of interest by detecting and/or quantifying said detection reagent. the analyte of interest is a protein or other non-nucleic acid molecule;
- said analyte-specific reagent is a primary antibody, antibody fragment or non-antibody protein capable of specifically binding to said protein analyte or other non-nucleic acid analyte;
- said first detection composition comprises the necessary reagents for carrying out an immunochemical detection reaction, such as a buffer, and a second antibody specific for said analyte and coupled to a suitable reporter enzyme; or comprising a secondary antibody fragment;
- said second detection composition comprises as a detection reagent a suitable substrate for said suitable reporter enzyme, which substrate becomes detectable when reacted by said reporter enzyme, preferably optically detectable; enabled, and more preferably detectable by fluorescence;
- said step of incubating said aqueous sample with said library of fabricated microparticles and said first detection composition comprises incubating said primary antibody, primary antibody fragment or non-antibody when said analyte is present in said aqueous sample; forming a complex between the analyte and the primary antibody, primary antibody fragment, or non-antibody protein by performing an immunochemical reaction involving binding of the analyte to a protein; further comprising binding said second antibody to a complex, forming a sandwich between the first antibody, antibody fragment or non-antibody protein, the analyte, and the second antibody;
- a first optional step of washing said library removes unbound secondary antibodies from said library;
- said step of incubating said library of fabricated microparticles containing absorbed aqueous samples with said second detection composition is a step of allowing said substrate to be reacted by said reporter enzyme;
- a further optional step of washing said library to remove unreacted substrate from said library;
- said transferring step is a step of transferring said fabricated microparticles to a non-aqueous phase, suspending, and optionally washing repeatedly in said non-aqueous phase; Also includes filtration or mechanical stirring to ensure the formation of a monodisperse suspension of microparticles with no or substantially no remaining aqueous phase;
- said optional step of releasing said analyte-specific reagent attached to said microparticle by applying an external trigger, temporarily increasing temperature, changing pH, or changing the conditions, preferably increasing the temperature, more preferably increasing the temperature from ambient temperature to >90°C;
- the step of detecting and/or quantifying the analyte of interest is detecting and/or quantifying the reacted substrate;
16. The method according to claim 15 . A method for detecting and/or quantifying an analyte of interest in an aqueous sample, the method comprising the steps of:
-
- Aqueous samples known or suspected to contain the analyte of interest;
- a first detection composition comprising the reagents necessary to carry out a chemical or biochemical detection reaction of said analyte;
- a second detection composition comprising a detection reagent;
- providing a library of fabricated microparticles according to any one of claims 1 to 9 in any order, wherein the analyte-specific reagent attached to the microparticle is a step selected to be capable of specifically binding to or specifically reacting with said analyte of interest;
- optionally mixing said aqueous sample and said first detection composition;
- incubating said aqueous sample with said library of fabricated microparticles and, if said aqueous sample is not already mixed with said first detection composition, said microparticles; adsorbing the aqueous sample and the first detection composition into the void volume of the microparticle, and optionally binding to the analyte of interest if the analyte of interest is present in the sample; A process that makes it possible to;
- optionally washing the library of prepared microparticles to remove unabsorbed or unreacted first detection composition;
- incubating said library of fabricated microparticles containing an absorbed aqueous sample with said second detection composition, thereby enabling said library of microparticles to absorb said second detection composition; , process;
- optionally further washing the library of prepared microparticles to remove unabsorbed or unreacted second detection composition;
- transferring said library of fabricated microparticles to a non-aqueous phase and creating a plurality of isolated reaction spaces for detecting said analytes by removing the aqueous phase around individual fabricated microparticles; the reaction space comprises an aqueous phase and is limited to the void volume of the microparticle;
- optionally releasing the analyte-specific reagent attached to the microparticle by applying an external trigger;
A method comprising: - performing a detection reaction for the target analyte; and - detecting and/or quantifying the target analyte. the analyte of interest is a protein or other non-nucleic acid molecule;
- said analyte-specific reagent is a primary antibody, antibody fragment or non-antibody protein capable of specifically binding to said protein analyte or other non-nucleic acid analyte;
- said first detection composition is coupled to a suitable oligonucleotide tag, specific for the same analyte as said first antibody, and the necessary reagents for carrying out an immunochemical detection reaction, such as a buffer; comprising a secondary antibody or secondary antibody fragment;
- said second detection composition comprises a buffer, a mononucleoside triphosphate, an amplification enzyme, e.g. a suitable nucleic acid polymerase, e.g. Taq polymerase, and an amplification product, e.g. a nucleic acid dye for detection of the amplified nucleic acid. , and a primer suitable for amplifying the oligonucleotide tag attached to the secondary antibody as a detection reagent;
- said step of incubating said aqueous sample with said library of fabricated microparticles and said first detection composition comprises incubating said primary antibody, primary antibody fragment or non-antibody when said analyte is present in said aqueous sample; forming a complex between the analyte and the primary antibody, primary antibody fragment, or non-antibody protein by performing an immunochemical reaction involving binding of the analyte to a protein; further comprising binding said second antibody to a complex, forming a sandwich between the first antibody, antibody fragment or non-antibody protein, the analyte, and the second antibody;
- a first optional step of washing said library removes unbound secondary antibodies from said library;
- said step of incubating said library of fabricated microparticles containing an absorbed aqueous sample with said second detection composition, wherein said primer in said second detection composition is attached to said secondary antibody; hybridizing to said oligonucleotide tag;
- a further optional step of washing said library to remove unhybridized primers from said library;
- said transferring step is a step of transferring said fabricated microparticles to a non-aqueous phase, suspending and, if necessary, washing repeatedly in said non-aqueous phase; Also includes filtration or mechanical stirring to ensure the formation of a monodisperse suspension of microparticles with no or substantially no remaining aqueous phase;
- said optional step of releasing said analyte-specific reagent attached to said microparticle by applying an external trigger, temporarily increasing temperature, changing pH, or changing the conditions, preferably increasing the temperature, more preferably increasing the temperature from ambient temperature to >90°C;
- the step of performing a detection reaction is a step of performing an amplification reaction of the oligonucleotide tag;
the step of detecting and/or quantifying the target analyte is detecting and/or quantifying the amplified oligonucleotide tag;
18. The method according to claim 17 . Any of claims 13 to 18 , wherein the step of releasing the analyte-specific reagent attached to the microparticle by applying an external trigger is performed by increasing the temperature to which the microparticle is exposed. The method described in. - the method detects and/or quantifies a single analyte in a plurality of aqueous samples, e.g. from different patients;
- the method provides a plurality of different aqueous samples known or suspected to contain a plurality of analytes of interest, e.g. samples from different patients;
- the library of fabricated microparticles provided in the method is a library according to claim 7 , in which there are as many different aqueous samples provided as the number of different aqueous samples to be tested; or at least the same number of distinct microparticle subsets are provided, all of said distinct subsets having the same analyte-specific reagent attached to said porous matrix of said microparticles of said subset, said analyte-specific The reagent is specific for one analyte of interest;
- incubating said aqueous sample with said library of fabricated microparticles, incubating each of said different aqueous samples separately with a distinct subset of microparticles of said library;
- said step of incubating said library of fabricated microparticles containing an adsorbed aqueous sample with a second detection composition, wherein said step of incubating said library of fabricated microparticles containing an adsorbed aqueous sample with said second detection composition; incubate;
- said step of transferring said library to a non-aqueous phase is carried out separately for each microparticle subset, i.e. each distinct microparticle subset is separately transferred to a non-aqueous phase, and the aqueous phase around said individual microparticles is By removing each subset separately, a plurality of isolated reaction spaces for detecting the analytes are created separately for each subset, the reaction spaces containing an aqueous phase and containing the void volume of the microparticles. limited to
The method continues, after the step of transferring the library to a non-aqueous phase, after the step of mixing together the plurality of isolated reaction spaces of all the distinct subsets in the non-aqueous phase. , further comprising the steps of performing a detection reaction for the target analyte, and then detecting and/or quantifying the target analyte.
The method according to any one of claims 13 to 19 . - the method is a method for detecting and/or quantifying multiple analytes in a single aqueous sample;
- the method provides a single aqueous sample known or suspected to contain multiple analytes of interest;
- the library of fabricated microparticles provided in the method is a library according to claim 8 , and in such a library there are at least the same number of different target analytes to be detected. a number of distinct microparticle subsets are provided, each of said distinct subsets having a different analyte-specific reagent attached to said porous matrix of said microparticles of said subset; each analyte-specific reagent comprising: specific for one analyte of interest;
- said step of incubating said aqueous sample with said library of fabricated microparticles, incubating said aqueous sample with all of said distinct microparticle subsets of said library;
- said step of incubating said library of fabricated microparticles containing an adsorbed aqueous sample with a second detection composition, wherein said step of incubating said library of fabricated microparticles containing an adsorbed aqueous sample includes all said distinct microparticle subsets of said library together with said second detection composition; incubate;
- said step of transferring said library to a non-aqueous phase is performed for all microparticle subsets together, i.e. all microparticle subsets are transferred together to a non-aqueous phase and the aqueous phase around said individual microparticles is is removed to create a plurality of isolated reaction spaces for detecting and/or quantifying the plurality of analytes, the reaction spaces containing an aqueous phase and filling the void volume of the microparticle. limited,
The method according to any one of claims 13 to 19 . - the method is a method for detecting and/or quantifying a plurality of analytes in a plurality of aqueous samples;
- the method provides a plurality of different aqueous samples known or suspected to contain a plurality of analytes of interest, e.g. samples from different patients;
- the library of fabricated microparticles provided in the method is a library according to claim 9 , in which there are distinct fabricated microparticle subsets of different classes; Each of the classes includes several subsets of microparticles, each of the classes having a different analyte-specific reagent attached to the porous matrix of the microparticles, and each of the classes having a different analyte-specific reagent attached to the porous matrix of the microparticles; , the same analyte-specific reagents are attached; each analyte-specific reagent is specific for one analyte of interest; in said method, said step of providing said library comprises:
different classes of distinct particulate subsets are provided as many or at least as many as the number of different analytes to be detected;
- as many or at least as many different microparticle subsets are provided within each class as the number of aqueous samples to be tested that are provided;
- providing in said library as many or at least as many different microparticle subsets as the number of different aqueous samples to be tested multiplied by the number of different target analytes to be detected;
- said step of incubating said aqueous sample with said library of fabricated microparticles, in which exactly one aqueous sample is incubated with exactly one subset of microparticles from each class, each aqueous sample having a is incubated with a number of different microparticle subsets from different classes, equal to the number of microparticle classes in
- said step of incubating said library of fabricated microparticles containing adsorbed aqueous samples with a second detection composition, a combined subset of microparticles of different classes previously incubated with one respective aqueous sample; incubating with the second detection composition;
- said step of transferring said library to a non-aqueous phase is carried out separately for each aqueous sample, i.e. said combined microparticle subsets previously incubated with one respective aqueous sample are transferred together to a non-aqueous phase; By removing the aqueous phase around the individual microparticles separately for each aqueous sample, a plurality of isolated reaction spaces are created for detecting the plurality of analytes, the reaction spaces being separated from the aqueous phase. and limited to the void volume of the microparticle,
The method preferably continues after said step of transferring said library to a non-aqueous phase and after mixing together said plurality of isolated reaction spaces of said separate total samples in said non-aqueous phase. , further comprising the steps of performing a detection reaction for the target analyte, and then detecting and/or quantifying the target analyte.
The method according to any one of claims 13 to 19 . In the step of detecting and quantifying the analyte of interest, the quantification of the analyte comprises:
a) digital nucleic acid amplification, in particular digital polymerase chain reaction (PCR);
b) real-time quantitative nucleic acid amplification, in particular real-time polymerase chain reaction (PCR);
c) immunochemical detection methods, in particular digital immunochemical detection methods, such as digital immunoassays, such as digital enzyme-linked immunosorbent assays (ELISA);
d) Immunochemical detection methods combined with nucleic acid amplification, e.g. immunopolymerase chain reaction; in particular digital immuno-PCR
carried out by a method selected from;
When said analyte of interest is a nucleic acid, quantification is carried out using either method a) or b) or a combination of a) and b); when said analyte is a protein, peptide or other In the case of non-nucleic acid analytes, quantification is performed using either method c) or d).
The method according to any one of claims 13 to 22 .