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JP-2026514412-A - Method for quantifying the concentration of an analyte in a sample

JP2026514412AJP 2026514412 AJP2026514412 AJP 2026514412AJP-2026514412-A

Abstract

The present invention relates to a method for quantifying the concentration cs of an analyte in a sample, and more particularly to a method for quantifying the concentration cs of a target nucleic acid in a sample based on the principle that the analyte/target provided in a sample of known volume is randomly distributed across a predetermined number of pre-fabricated reaction compartments. The present invention also relates to an aqueous suspension of a predetermined number N B of hydrogel microspheres for use in a method for quantifying the concentration cs of an analyte, particularly a target nucleic acid, in a sample. Furthermore, the present invention relates to macroparticles for use in such a method. Finally, the present invention also relates to a method for producing an aqueous suspension of a predetermined number N B of hydrogel microspheres. [Selection Diagram] None

Inventors

  • アーデルヘルム、カリン
  • エルマントラウト、オイゲン
  • エリンガー、トマス
  • フーボルト、シュテファン
  • レムート、オリヴァー
  • シアマン、ケルスティン
  • シュルツ、トルステン
  • シュタインメッツァー、カトリン
  • テプファー、スザンネ
  • トゥヒシェーラー、イェンス

Assignees

  • ブリンク アーゲー

Dates

Publication Date
20260511
Application Date
20240328
Priority Date
20230331

Claims (20)

  1. A method for quantifying the concentration cs of an analyte in a sample: a) A step of providing an aqueous sample containing or presumed to contain an analyte, and a predetermined number of N B reaction compartments in any order, wherein the reaction compartments include a material capable of binding with the analyte; b) Exposing the predetermined number of N B reaction compartments to an aqueous sample in a manner that results in a random distribution of the analyte across the predetermined number of N B reaction compartments, thereby enabling the reaction compartments to take in the aqueous sample and, if present in the aqueous sample, to bind the analyte, preferably all of the analyte, thereby resulting in at least some of the predetermined number of N B reaction compartments that associate with or contain the analyte contained in the aqueous sample provided in step a); c) Optionally, if the flow and/or exchange of aqueous samples between different reaction compartments obtained from step b) is still possible and/or occurs after step b): a step of isolating the reaction compartments obtained from step b) so that the flow and/or exchange of aqueous samples between different reaction compartments is no longer possible and does not occur; d) A step of carrying out an enzyme amplification protocol in the reaction compartment, wherein the enzyme amplification protocol generates a first optically detectable signal in the reaction compartment associated with or containing the analyte; and the enzyme amplification protocol generates a second optically detectable signal in the reaction compartment not associated with or containing the analyte; e) A step of determining the number of reaction compartments having the first optically detectable signal (= "positive" reaction compartments) N pos and the number of reaction compartments having the second optically detectable signal (= "negative" reaction compartments) N neg by detecting the first and second optically detectable signals, respectively; the total number of reaction compartments detected here N = N pos + N neg ; f) The concentration c s of the analyte in the aqueous sample is given by the formula The calculation process is as follows (in the formula, c s = Concentration of the analyte in the aqueous sample N B = A predetermined number of reaction compartments exposed to the aqueous sample V S = Volume of the aqueous sample containing or suspected to contain the analyte N neg = Number of reaction compartments with a detected second optically detectable signal (= "negative" reaction compartments) N = Total number of detected reaction compartments = N neg + N pos (In the formula, N pos = Number of reaction compartments with the first optically detectable signal detected (= "positive" reaction compartments) A method that includes this.
  2. A method for quantifying the concentration cs of an analyte in a sample, particularly: a) A step of providing an aqueous sample containing or presumed to contain an analyte, and an aqueous suspension of a predetermined number N B of hydrogel microspheres in any order, wherein the hydrogel microspheres contain a material capable of binding to the analyte; b) Exposing the predetermined number N B hydrogel microspheres to the aqueous sample and mixing the aqueous sample with the suspension of the hydrogel microspheres, thereby enabling the hydrogel microspheres to take up the aqueous sample and, if present in the aqueous sample, bind to the analytes, preferably all of the analytes; thereby resulting in at least some of the predetermined number N B hydrogel microspheres that associate with or contain the analytes contained in the aqueous sample provided in step a); c) A step of transferring the hydrogel microspheres to a water- and aqueous-immiscible liquid phase, such as an oil phase, thereby generating a predetermined number N B of hydrogel microsphere suspensions in the water-immiscible phase, wherein the hydrogel microspheres are isolated from each other by the water-immiscible liquid phase so that the flow and/or exchange of aqueous samples between different hydrogel microspheres is no longer possible and does not occur; d) A step of performing an enzyme amplification protocol on the hydrogel microsphere, wherein the enzyme amplification protocol generates a first optically detectable signal in the hydrogel microsphere associated with or containing the analyte; and the enzyme amplification protocol generates a second optically detectable signal in the hydrogel microsphere not associated with or containing the analyte; e) A step of determining the number of hydrogel microspheres having the first optically detectable signal (= "positive" hydrogel microspheres) N pos and the number of hydrogel microspheres having the second optically detectable signal (= "negative" hydrogel microspheres) N neg by detecting the first and second optically detectable signals, respectively; the total number of hydrogel microspheres detected here N = N pos + N neg ; f) The concentration c s of the analyte in the sample is given by the formula The process of calculation according to (in the formula, c s = Concentration of the analyte in the aqueous sample N B = Default number of hydrogel microspheres exposed to the aqueous sample V S = Volume of the aqueous sample containing or suspected to contain the analyte N neg = Number of hydrogel microspheres with a detected second optically detectable signal (= "negative" hydrogel microspheres) N = Total number of detected hydrogel microspheres = N neg + N pos (In the formula, N pos = Number of hydrogel microspheres with the first optically detectable signal detected (= "positive" hydrogel microspheres) The method according to claim 1, including the method described in claim 1.
  3. The aforementioned analytes are: The method according to any one of claims 1 to 2, wherein a) a target nucleic acid; and the enzyme amplification protocol is a targeted amplification reaction; or b) a target protein; and the enzyme amplification protocol is a signal amplification reaction; or c) a biological cell, virus particle, or extracellular vesicle, each containing various types of nucleic acids within the cell, virus particle, or extracellular vesicle; and the enzyme amplification protocol is a targeted amplification reaction.
  4. The aforementioned analytes are: a) A target nucleic acid; and the enzyme amplification protocol is a nucleic acid amplification protocol that results in specific amplification of the target nucleic acid if the target nucleic acid is associated with or contained by any of the hydrogel microspheres, and results in the generation of a first optically detectable signal in the hydrogel microsphere if the hydrogel microsphere contains or is associated with the amplified target nucleic acid; or b) A target protein; and the enzyme amplification protocol is a signal amplification reaction that involves the formation of an analyte-specific immune complex on or in the hydrogel microsphere if the hydrogel microsphere is associated with or contains the analyte; where a chromogenic enzyme is attached to the analyte-specific immune complex, and the enzyme amplification reaction results in the generation of a first optically detectable signal in the hydrogel microsphere if the hydrogel microsphere contains or is associated with the analyte; or c) A target protein; and the enzyme amplification protocol is a signal amplification reaction that involves the formation of an analyte-specific immune complex on or in the hydrogel microsphere if the hydrogel microsphere is associated with or contains the analyte If the analyte is included, the signal amplification reaction is characterized by the formation of an analyte-specific immune complex on or within the hydrogel microsphere; where the nucleic acid label is attached to the analyte-specific immune complex, and the enzyme amplification reaction further involves specific nucleic acid amplification of the nucleic acid label, resulting in the accumulation of the amplified nucleic acid label and the generation of a first optically detectable signal within the hydrogel microsphere if the hydrogel microsphere contains or associates with the analyte; or d) biological cells, virus particles, or extracellular vesicles, each containing various types of nucleic acids within the cell, virus particle, or extracellular vesicle; and the enzyme amplification protocol is characterized by the resulting specific amplification of one or more types of nucleic acids contained within the biological cell, virus particle, or extracellular vesicle if the biological cell, virus particle, or extracellular vesicle is associated with or included by any of the hydrogel microspheres, and the nucleic acid amplification protocol is characterized by the resulting generation of a first optically detectable signal within the hydrogel microsphere if the hydrogel microsphere contains or associates with the biological cell, virus particle, or extracellular vesicle. The method according to any one of claims 1, 2 to 3.
  5. Materials that can be bonded to the aforementioned analytes include: a) When the analyte is a nucleic acid, and the material capable of binding to the analyte binds nonspecifically to the nucleic acid, cationic polymers, cationic oligomers, cationic monomers, and silica; b) If the analyte is a nucleic acid, and the material capable of binding to the analyte specifically binds to a particular nucleic acid, such as a target nucleic acid, then an oligonucleotide; where the oligonucleotide is complementary to a given nucleic acid analyte; c) If the analyte is a protein, biological cell, viral particle, or extracellular vesicle, and the material capable of binding to the analyte specifically binds to a particular protein, such as a target protein, or specifically binds to a label, tag, prosthetic group, or other component associated with the analyte, or to an antibody, antibody fragment, or other component associated with an antibody that specifically binds to the analyte, the antibody, antibody fragment, and protein receptor, A method selected from any of claims 1, 2 to 4.
  6. The analyte is nucleic acid, and a material capable of binding to the analyte binds nonspecifically to the nucleic acid; the material capable of binding nonspecifically to the nucleic acid is: A polymer skeleton to which chitosan and its derivatives, gelatin and its derivatives, poly(ethyleneimine), poly(2-dimethyl(aminoethyl) methacrylate), poly(lysine), poly(histidine), poly(arginine), and basic amino acids are attached or incorporated as parts of such skeleton; an oligopeptide comprising or consisting of basic amino acids such as histidine, lysine, and arginine; and a monomer selected from basic amino acids such as histidine, lysine, and arginine, according to any one of claims 1, 2 to 5, particularly the method according to claim 5.
  7. The method according to any one of claims 1, 2 to 6, wherein the predetermined number N B of the hydrogel microspheres is in the range of 1,000 to 1,000,000, preferably in the range of 5,000 to 500,000, more preferably in the range of 5,000 to 100,000, even more preferably in the range of 5,000 to 50,000, and even more preferably in the range of 5,000 to 20,000.
  8. The method according to any one of claims 1, 2 to 7, wherein the hydrogel microspheres include magnetic particles that enable mechanical handling and migration of the hydrogel microspheres.
  9. The method according to any one of claims 1, 2 to 8, wherein the method comprises an additional step b * ) performed after step b) and before step c): b * ) is a step of washing the predetermined number N B hydrogel microspheres by exposing them to a washing buffer.
  10. The method includes an additional step b ** ), and step b ** is performed after step b) and before step c), and if step b * ) is performed in addition according to claim 9, it is performed after step b * ) and before step c): b ** ) is a step of treating the predetermined number N B hydrogel microspheres by exposing them to a solution for carrying out the enzyme amplification reaction, The solution comprises (i) a buffer, a mono-nucleoside triphot, an amplification enzyme, a nucleic acid dye for detecting the amplified nucleic acid, optionally one or more amplification primers or one or more sets of amplification primers if not already present in the hydrogel microsphere, and optionally a molecular probe such as a TaqMan probe or molecular beacon; or (ii) a buffer, an analyte-specific antibody or antibody fragment to which a label has been attached, and a detection reagent for detecting the label; or (iii) a buffer, an analyte-specific antibody or antibody fragment, an analyte detection antibody bound to the analyte-specific antibody or antibody fragment, an analyte detection antibody to which the label has been attached, and a detection reagent for detecting the label; wherein in (ii) and (iii), the label is either a nucleic acid tag or a chromogenic enzyme; - If the label is a nucleic acid tag, the detection reagent is a solution containing a mono-nucleoside-triphosphate, an amplification enzyme, a nucleic acid dye for detecting the amplified nucleic acid, optionally one or more amplification primers or one or more sets of amplification primers if they are not already present in the hydrogel microsphere, and optionally a molecular probe such as a TaqMan probe or molecular beacon; and, - If the label is a chromogenic enzyme, the detection reagent is a solution containing the substrate for the chromogenic enzyme. The method according to any one of claims 1, 2 to 9.
  11. The method according to any one of claims 1, 2 to 10, wherein in step e), the first optically detectable signal generated in the hydrogel microspheres, and optionally the second optically detectable signal, are also detected via the first channel, preferably the fluorescence channel, and optionally, the total number N of detected microspheres is determined by optical imaging via an additional second channel.
  12. The method according to any one of claims 1, 2 to 11, wherein the hydrogel microspheres have an average diameter in the range of 10 μm to 500 μm, preferably 20 μm to 500 μm, more preferably 20 μm to 200 μm, more preferably 20 μm to 100 μm, and even more preferably 40 μm to 100 μm.
  13. A method for quantifying the concentration cs of a target nucleic acid in a sample: a) A step of providing an aqueous sample containing or presumed to contain a target nucleic acid, and an aqueous suspension of a predetermined number N B of hydrogel microspheres in any order, wherein the hydrogel microspheres contain a material that can nonspecifically bind to the nucleic acid, such as the target nucleic acid, or specifically bind to the target nucleic acid; b) Exposing the predetermined number N B hydrogel microspheres to an aqueous sample and mixing the aqueous sample with the suspension of the hydrogel microspheres, thereby enabling the hydrogel microspheres to take up the aqueous sample and bind the target nucleic acid, preferably all of the target nucleic acid, if present in the aqueous sample; thereby resulting in at least some of the predetermined number N B hydrogel microspheres that associate with or contain the target nucleic acid contained in the aqueous sample provided in step a); c) A step of transferring the hydrogel microspheres to a water- and aqueous-immiscible liquid phase, such as an oil phase, thereby generating a predetermined number N B of hydrogel microsphere suspensions in the water-immiscible phase, wherein the hydrogel microspheres are isolated from each other by the water-immiscible liquid phase so that the flow and/or exchange of aqueous samples between different hydrogel microspheres is no longer possible and does not occur; d) A step of performing a nucleic acid amplification protocol on the hydrogel microspheres specific to the target nucleic acid; wherein the nucleic acid amplification protocol results in specific amplification of the target nucleic acid if the target acetate is associated with or included in any of the hydrogel microspheres, and results in the generation of a first optically detectable signal in the hydrogel microspheres if the hydrogel microspheres include or associate with the amplified target nucleic acid; wherein the nucleic acid amplification protocol does not result in amplification of the target nucleic acid and results in the generation of a second optically detectable signal in the hydrogel microspheres if the hydrogel microspheres are not associated with and do not include the amplified target nucleic acid; e) A step of determining the number of hydrogel microspheres having the first optically detectable signal (= "positive" hydrogel microspheres) N pos and the number of hydrogel microspheres having the second optically detectable signal (= "negative" hydrogel microspheres) N neg by detecting the first and second optically detectable signals, respectively; the total number of microspheres detected here N = N pos + N neg ; f) The concentration c s of the target nucleic acid in the aqueous sample is given by the formula The process of calculating according to (in the formula, cs = Concentration of target nucleic acid in aqueous sample NB = Default number of hydrogel microspheres exposed to aqueous sample Vs = Volume of aqueous sample containing or suspected to contain target nucleic acid Nneg = Number of hydrogel microspheres with a detected second optically detectable signal (= "negative" hydrogel microspheres) N = Total number of detected hydrogel microspheres = Nneg + Npos (In the formula, N pos = Number of hydrogel microspheres with the first optically detectable signal detected (= "positive" hydrogel microspheres) The method of any of the prior claims, including the method of any of the prior claims.
  14. An aqueous suspension of a predetermined number of N B hydrogel microspheres for use in a method for quantifying the concentration cs of an analyte, preferably a target nucleic acid, in a sample according to any one of claims 2 to 13, wherein the aqueous suspension comprises an aqueous solvent and a predetermined number of N B hydrogel microspheres, wherein the hydrogel microspheres comprise a material capable of binding to an analyte, preferably a material capable of binding to nucleic acids.
  15. The aqueous suspension according to claim 14, wherein the predetermined number N B of hydrogel microspheres is in the range of 1,000 to 1,000,000, preferably in the range of 5,000 to 500,000, more preferably in the range of 5,000 to 100,000, even more preferably in the range of 5,000 to 50,000, and even more preferably in the range of 5,000 to 20,000.
  16. Macroparticles for use in a method for quantifying the concentration cs of an analyte, preferably a target nucleic acid, in a sample according to any one of claims 1 to 13, wherein the macroparticles comprise a matrix and a predetermined number of N B hydrogel microspheres embedded in the matrix of the macroparticles, wherein the macroparticles are dried, preferably freeze-dried.
  17. The macroparticle according to claim 16, wherein the predetermined number N B of the hydrogel microspheres is in the range of 1,000 to 1,000,000, preferably in the range of 5,000 to 500,000, more preferably in the range of 5,000 to 100,000, even more preferably in the range of 5,000 to 50,000, and even more preferably in the range of 5,000 to 20,000.
  18. The macroparticle according to any one of claims 16 and 17, wherein the matrix comprises or consists of a material that is an excipient for a drying process, particularly freeze-drying.
  19. The macroparticles according to claim 18, wherein the excipient for drying, particularly for freeze-drying, is selected from saccharides such as trehalose, sucrose, mannitol, glucose, fructose, lactose, mannitol, inositol, hydroxypropyl-β-cyclodextrin and combinations thereof; polymers such as polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), dextran, and gelatin; amino acids such as arginine, histidine, and glycine; and any combination thereof.
  20. The macroparticles are substantially spherical, spherical, droplet-shaped, ellipsoidal, or other circular in shape, and have an average diameter ranging from 1 mm to 50 mm, preferably 2 mm to 20 mm, more preferably 2 mm to 15 mm, even more preferably 2 mm to 10 mm, and even more preferably 2 mm to 5 mm, according to any one of claims 16 to 19.

Description

Field of Invention The present invention relates to a method for quantifying the concentration cs of an analyte in a sample, and more particularly to a method for quantifying the concentration cs of a target nucleic acid in a sample. The present invention also relates to a principle by which, by binding the analyte/target, the analyte/target in a sample of known volume is randomly distributed across a predetermined number of pre-fabricated reaction compartments. The present invention also relates to aqueous suspensions of hydrogel microspheres of a predetermined number N B for use in a method for quantifying the concentration cs of an analyte, particularly a target nucleic acid, in a sample. Furthermore, the present invention also relates to macroparticles for use in such a method. Finally, the present invention also relates to a method for producing aqueous suspensions of hydrogel microspheres of a predetermined number N B. Background of the Invention Digital detection assays, such as digital PCR (dPCR) assays, offer specificity, sensitivity, and accuracy in targeted analysis, such as nucleic acid analysis. Compared to bulk methods, digital assays such as dPCR offer the advantage of absolute quantification without the use of calibration curves. For this purpose, the assay mix, for example, an amplification mix containing the reagents and analytes/targets necessary for target detection, is divided into thousands of compartments. For example, one method is to dispense a liquid across multiple wells on a solid substrate. Another approach is the preparation of aqueous microdroplets in an immiscible liquid, as disclosed, for example, in Hindson et al., 2011, Anal. Chem., 83, 22, pp. 8604-8610, in relation to dPCR. In such conventional dPCR, the quantification of the target is based on the detection of PCR-positive compartments with known volumes. Hydrogels have been used to encapsulate cells in dPCR (Geng et al., 2014, Anal. Chem. 86, 703-712) and to generate beads immobilized with amplification products for post-PCR analysis (Leng et al., 2010, Lab Chip. 10, 2481-2843). To overcome the inherent need for complex, expensive, and error-prone microfluidics, hydrogel beads have also been successfully used in particle template emulsification, where they function as mechanical templates for droplet formation in fluorocarbon oil (Loncarevic et al., 2021, Plos One, 16, e0242529). In the approach used by Loncarevic et al. (2021, ibid.), hydrogel beads are used to compartmentalize the sample, but binding of the analyte to/on the hydrogel beads, or binding or concentration therein, is virtually nonexistent. In the method described by Loncarevic et al. (2021), the hydrogel beads merely divide the sample, resulting in random distribution of the analyte between the beads, leaving a large amount of sample "unused," and preventing quantitative incorporation of the analyte into the hydrogel beads. Furthermore, Loncarevic et al.'s (2021) methodology requires knowledge of the hydrogel bead volume and its volume variability, as well as correction for such volume variability. Therefore, there remains a need in this field to promote and provide digital assays that are compartment volume independent for quantification and do not require microfluidic devices such as dPCR. There is also a need in this field for digital assay methodologies, such as dPCR methodologies, that are sample volume independent and provide target quantification without accurately measuring the volume of the reaction/amplification compartment. Additionally, there is a need for digital assay methodologies, such as dPCR methodologies, that are easy to implement and capable of handling a wide variety of sample volumes. All these objectives are addressed by a method for quantifying the concentration cs of the analyte in a sample, and the method is: a) A step of providing an aqueous sample containing or presumed to contain an analyte, and a predetermined number of N B reaction compartments in any order, wherein the reaction compartments contain a material capable of binding with the analyte; b) Exposing the predetermined number of N B reaction compartments to an aqueous sample in a manner that results in a random distribution of the analyte across the predetermined number of N B reaction compartments, thereby enabling the reaction compartments to take up the aqueous sample and bind to the analyte, preferably all of the analyte, present in the aqueous sample, thereby resulting in at least some of the predetermined number of N B reaction compartments that associate with or contain the analyte contained in the aqueous sample provided in step a); c) Optionally, if the flow and/or exchange of aqueous samples between different reaction compartments obtained from step b) is still possible and/or occurs after step b): a step of isolating the reaction compartments obtained from step b) so that the flow and/or exchange of aqueous samples between different reaction compartments