WO2023241720A1

WO2023241720A1 - System for processing samples and method for preparing same

Info

Publication number: WO2023241720A1
Application number: PCT/CN2023/101043
Authority: WO
Inventors: 张经纬; 张川
Original assignee: 上海高探生物科技有限公司; 复旦大学
Priority date: 2022-06-17
Filing date: 2023-06-19
Publication date: 2023-12-21

Abstract

The present invention relates to a system and method for separating analytes and capturing biomolecules in a targeted manner in a reaction compartment composed of a permselective membrane. The system and method can be used for processing and packaging biomolecules in a series of reactions to give an analysis result.

Description

Sample processing system and preparation method thereof

Technical field

The present invention relates to a system for separating analytes (such as cells, bacteria, viruses, nucleic acids, biochemical compounds and/or other materials) and targeted capture of biomolecules in a reaction compartment composed of a selectively permeable membrane. methods and process encapsulated biomolecules through reactions/processes to perform multi-step reactions. Biomolecules derived from the same analyte are enriched in a reaction compartment composed of a selectively permeable membrane through a specific capture carrier. The remaining biomolecules can freely pass through the selective permeation membrane, thereby achieving various reaction substance exchanges required for purification, cleaning or multi-step reactions. After treatment with external stimuli, the captured enriched biomolecules and their reactive derivatives can be released from the reaction compartment consisting of a selectively permeable membrane at the desired step. The methods disclosed herein illustrate the use of reaction compartments composed of selectively permeable membranes for genotypic or phenotypic analysis of single cells.

Background technique

High-throughput processing and analysis of biological samples at single-cell or single-molecule resolution has important applications in many branches of the life sciences. Compartmentalization of cells, DNA, enzymes, or molecules with water-in-oil droplets or other forms of compartments enables massively parallel analysis with orders of magnitude higher analytical and processing throughput compared to 96-well microtiter plates. However, many molecular biology methods are based on sample processing based on serial multi-step biochemical reactions to trigger initiation, transformation or termination of reactions. Therefore, not all molecular biology sample processing workflows can be easily converted to droplet or other types of emulsion-based formats. Although some solutions such as droplet fusion, droplet reinjection, and droplet segmentation enable multi-step procedures (e.g., adding new reagents to preformed droplets), the required expertise and complexity of fluidic manipulations limits the wider use of such methods. Multi-step sample processing (such as most next-generation sequencing library construction pipelines) can become very challenging when the encapsulated cells or their genetic material must be processed through a series of consecutive and independent reactions. For example, for amplification and/or analysis of genetic material of encapsulated cells, cell lysis may be required, a step that may be inhibitory or deleterious to subsequent enzymatic steps. Therefore, it would be advantageous to have methods and/or systems capable of buffer/reagent exchange and/or removal of lysis reagents prior to the nucleic acid analysis/amplification step. On the other hand, purification-based operations in classic molecular biology methods are also difficult to implement in traditional droplets. Cells contain large amounts of impurities that sometimes have damaging effects on biomolecules. For example, RNase A, B, and C in cells can cause the degradation of mRNA.

Various attempts in the past to adapt reaction compartments consisting of selectively permeable membranes to suit tandem multi-step biochemical reactions have been neither successful nor practical. Although many studies have demonstrated reaction compartments composed of selectively permeable membranes composed of various polymers, no reaction compartment composed of selectively permeable membranes has been demonstrated or applied to multi-step reactions for processing or analysis. Encapsulated entities, such as cells, biomolecules, etc. The main reason is that: biomolecules are relatively similar in size and properties to each other, and the properties of biomolecules in the analytes and reaction reagents are similar. Simply using a semipermeable membrane to isolate based on physical methods cannot achieve the desired selectivity; and based on There is currently no efficient and low-cost method for mass production of bioselective semipermeable membranes (such as biological membrane proteins, transport proteins, etc.). At present, there is a lack of effective means to generate reaction compartments composed of selectively permeable membranes and to specifically capture and retain biomolecules in the compartments.

For example, Vijayakumar, K., Gulati, S., Demello, A.J. & Edel, J.B., in the paper "Rapid cell extraction in aqueous two-phase droplet systems" in "Chemical Science" (2010) 1,447-452, apply droplet microscopy fluidic system to generate an aqueous two-phase system (ATPS), in which aqueous droplets are composed of two phases: PEG-rich and dextran-rich, and they showed that T lymphoma cells have a preference between the two phases. However, the authors have not yet produced a reaction compartment composed of a selectively permeable membrane.

Tamminen, M.V. & Virta, M.P.J., in the article "Distinguishing a single microbial genome from a mixed microbial cell population based on a single gene" in "Frontiers of Microbiology" (2015) 6, 1-10, the method of producing a reaction compartment composed of a selectively permeable membrane The method differs from that described in the present invention and does not use capture vectors for targeted enrichment of genomic DNA. In this work, bacteria were encapsulated on acrylamide hydrogel beads, and then the hydrogel microspheres carrying embedded bacteria were resuspended in warmed agarose and emulsified again, forming a water-coated microsphere with an acrylamide composition. A capsule with a gel core and a hydrogel shell composed of agarose. The authors showed that the hydrogel core can be dissolved by DTT, resulting in the formation of liquid core capsules with agarose shells. The methods and systems disclosed here involve different steps and the generation of capsules relies on microfluidic systems.

Ma, S. and other authors in the article "Preparation of microgel particles with complex shapes through selective polymerization of aqueous two-phase systems" in "Small" (2012) 8, 2356-2360, although the authors used similar polymerization molecule, dextran and PEGDA to produce aqueous two-phase system (ATPS) droplets, but they proposed a method to create concave microparticles. The invention was used to produce semipermeable membrane particles, but biological samples cannot be encapsulated into open hydrogel particles.

Authors such as Watanabe, Motohiro and Ono "through dual Spontaneous cross-linking of microfluidics in droplets of aqueous phase systems to form hydrogel microcapsules with a single water core "In", we demonstrate the creation of aqueous cores and semipermeable hydrogels by forming droplets of aqueous two-phase systems (ATPS). Monodisperse four-arm polyethylene glycol (tetra-PEG) hydrogel microcapsules with a gel shell. A dextran (DEX)-rich core and a tetra-PEG macromonomer-rich shell, followed by tetra-PEG in the shell Spontaneous cross-end coupling reactions of macromonomers. The workflow generated by reaction compartments composed of selectively permeable membranes has similarities to the methods and systems reported here. However, the authors did not demonstrate any biological applications. For example: Analysis and processing of cells or biological samples, or the possibility of using reaction compartments composed of selectively permeable membranes for multi-step reactions; nor has any effective means of specific capture and retention of biomolecules within the compartment been demonstrated.

Leonaviciene, Leonavicius, et al. demonstrated a selective permeable membrane in the paper "Using semi-permeable capsules for multi-step processing of single cells" in "Lab on a Chip" (2020), and in the US20200400538A1 patent of Mazutis, Stonyte, Leonavicius, Zelvyte, etc. The resulting workflow for reaction compartment generation has similarities to the methods and systems reported here, but does not demonstrate any efficient means of specific capture and retention of biomolecules within the compartment. In addition, although the patent shows that genomic DNA, genomic Multiple Displacement Amplification (MDA) products, and large fragments of double-stranded DNA (>300kb molecular weight) can be selectively retained through the membrane, the shape factors of biomolecules are not considered: for example, biological The mRNA molecules in the body are single-stranded RNA molecules (generally between 75bp-3000bp, molecular weight about 25kDa-1.5MDa), which shrink into microspheres under biological conditions, with a radius of gyration of only about 16-21nm and a maximum diameter of about 50-70nm. , driven by the chemical potential energy inside and outside the reaction compartment composed of a selectively permeable membrane, it can easily and quickly penetrate a 1um-thick semipermeable membrane. In addition, the application of high-throughput sequencing often involves the fragmentation of large fragments of DNA material. Since the fragments required for sequencing are smaller than the pore size of the semipermeable membrane, this results in the preferential loss or overall leakage of the nucleic acid fragments to be analyzed.

This application demonstrates, but is not limited to, the production of a reaction compartment composed of a selectively permeable membrane, the encapsulation of analytes (such as cells), the specific capture of biomolecules, and the production of a reaction compartment composed of a selectively permeable membrane. Example of re-retention, serial multi-step biochemical reactions for encapsulated analytes. Perform genotypic and phenotypic analysis or other applications in massive parallelism.

Contents of the invention

The invention thus provides:

1. A sample processing system, comprising:

a) As a selectively permeable membrane on the outer layer of the reaction compartment, the selectively permeable membrane can selectively through analytical reagents;

b) Contents located inside the reaction compartment: including capture reagents and analytes;

in,

The analyte is directly or indirectly connected to the capture reagent to form a whole, or forms a complex or polymer through interaction, or the two undergo a biological or chemical reaction to generate a converted product;

The selectively permeable membrane can selectively retain the whole body, complex or polymer formed by connecting the capture reagent and the analyte, or the converted product;

The diameter of the whole or complex formed by connecting the capture reagent and the analyte, or the converted product, is greater than 1/2 of the pore size of the selective permeability membrane.

2. The sample processing system according to item 1, wherein,

The capture reagent, the analyte, and the integral, complex or polymer formed by connecting the capture reagent and the analyte located inside the reaction compartment, or the converted product is liquid, gelatinous or Semi-liquid state; the interior of the reaction compartment also includes a solution containing an osmotic pressure regulator;

Preferably the osmotic pressure regulator is dextran.

3. The sample processing system according to item 1 or 2, wherein the analyte is selected from one or both of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles, cells, etc. More than one kind, and the complex formed by them; preferably, the analyte is from the same cell; most preferably, the analyte is one or more of DNA, mRNA and protein from the same cell.

4. The sample processing system according to any one of items 1 to 3, wherein,

The capture reagent is composed of a targeting ligand and a carrier, which may or may not be specific to the analyte; the carrier can cause the capture reagent to remain in the reaction compartment through physical or chemical effects. ; Preferably, the carrier is one or more of organoids, cell clusters, cells, biological macromolecules and their analogs, high molecular polymers, nanoparticles, small molecules or their complexes;

The targeting ligand is a natural or artificial molecule and has specificity for the analyte;

Preferably, the targeting ligand is selected from one or more of the following group: locked nucleic acids and nucleic acids of XNA and their analogs, aptamers, small peptides, polypeptides, glycosylated peptides, polysaccharides, soluble receptors, Steroids, hormones, mitogens, antigens, superantigens, growth factors, cytokines, leptin, viral proteins, cell adhesion molecules, chemokines, streptavidin and its analogs, biotin and its analogs, Antibodies, antibody fragments, single-chain variable fragments (scFv), Nanobodies, T cell receptors, major histocompatibility complex (MHC) molecules, antigenic peptide-MHC molecule complexes (pMHC), DNA-binding proteins White, RNA-binding proteins, intracellular or cell surface receptor ligands, and their multiple ligands, complex ligands, and coupling ligands.

5. The sample processing system according to any one of items 1 to 3, wherein,

The capture reagent is composed of a carrier, and the carrier has specificity for the analyte;

Preferably the carrier is a magnetic particle capable of binding DNA.

6. The sample processing system according to any one of items 1 to 5, wherein "located inside the reaction compartment" means located inside the reaction compartment and at the interface between the selectively permeable membrane and the inside of the reaction compartment. at or in the selectively permeable membrane.

7. The sample processing system according to any one of items 1 to 6, wherein,

The analyte is DNA, and the capture reagent is selected from one or more of proteins, nucleic acids, and their complexes;

Preferably, the capture reagent is a complex of protein and nucleic acid;

It is further preferred that the protein is DNA transposase and the nucleic acid is DNA;

Further preferably, the DNA transposase is Tn5, and the DNA forming a complex with the protein has a transposase recognition sequence at its end.

8. The sample processing system according to any one of items 1 to 6, wherein,

The analyte is DNA;

The capture reagent is a coupling couple composed of a polymer or nanoparticle and an oligonucleotide primer;

After the capture reagent and the DNA react in a premix reagent containing dNTPs, DNA polymerase, small molecule additives and PCR reaction buffer, the polymer or nanoparticle forms a complex with the analyte DNA. The diameter of the material is greater than 1/2 of the pore size of the selectively permeable membrane.

9. The sample processing system according to any one of items 1 to 6, wherein,

The analyte is DNA;

The capture reagent is an oligonucleotide primer;

After the capture reagent and the DNA react in a premix reagent containing dNTPs, DNA polymerase, small molecule additives and PCR reaction buffer, the diameter of the obtained product is greater than 1% of the pore size of the selective permeability membrane. /2.

10. The sample processing system according to any one of items 1 to 6, wherein,

The analyte is RNA;

The capture reagent is a coupling couple between a polymer or a nanoparticle and an oligonucleotide primer;

The capture reagent and the analyte RNA contain reverse transcriptase, dNTPs, and RNase inhibitors. After reacting in a premixed reagent composed of an agent, a small molecule additive and a PCT reaction buffer, the diameter of the obtained product is greater than 1/2 of the pore size of the selective permeability membrane.

11. The sample processing system according to any one of items 1 to 6, wherein,

The analyte is RNA;

The capture reagent is an oligonucleotide primer;

After the capture reagent reacts with the RNA to be analyzed in a premixed reagent containing reverse transcriptase, dNTPs, RNase inhibitors, small molecule additives and buffers, it is then reacted with oligonucleotide primers, dNTPs, DNA polymerase, small molecule After reacting in a premixed reagent composed of additives and PCR reaction buffer, the diameter of the obtained product is greater than 1/2 of the pore size of the selectively permeable membrane.

12. The sample processing system according to any one of items 1 to 6, wherein,

The analyte is protein;

The capture reagent is a coupling couple between a polymer or a nanoparticle and a targeting ligand;

The diameter of the complex formed by the capture reagent and the protein is greater than 1/2 of the pore size of the selectively permeable membrane.

13. The sample processing system according to any one of items 1 to 6, wherein,

The analyte is protein;

The capture reagent is a coupling couple between an initiating group and a long fragment of DNA;

The initiating group is an oligonucleotide primer;

After reacting in the premixed reagent composed of the initiating group, dNTP, DNA polymerase, small molecule additives, PCR reaction buffer and additional DNA template, a diameter larger than 1/2 of the pore size of the selective permeable membrane is formed. of DNA molecules.

14. The sample processing system according to any one of items 1 to 6, wherein,

The analyte is a T cell receptor;

The capture reagent is a coupling coupling between a polymer or a nanoparticle and an antigen peptide-MHC molecule complex (pMHC);

The diameter of the complex formed by the capture reagent and the T cell receptor is greater than 1/2 of the pore size of the selectively permeable membrane.

15. The sample processing system according to any one of items 1 to 6, wherein,

The analyte is a T cell receptor;

The capture reagent is a coupling couple between the initiating group and pMHC;

The initiating group is an oligonucleotide primer;

16. The sample processing system according to any one of items 1 to 15, wherein the connection between the analyte and the capture reagent is selected from the group consisting of covalent bonds, metal bonds, ionic bonds, van der Waals forces, and hydrogen bonds. , halogen bond, chalcogen bond, gold affinity, intercalation, overlap, cation-π bond, anion-π bond, salt bridge, secondary bond between non-metal atoms, secondary bond between metal atom and non-metal atom, gold affinity interaction, argyrophilic interaction, double hydrogen bond and secondary bond of gold bond; wherein, the interaction between the analyte and the capture reagent is selected from hydrogen bond interaction, ionic bond interaction, hydrophobic interaction and van der Waals force .

17. The sample processing system according to any one of items 1 to 16, wherein the selectively permeable membrane is one or more polymers selected from the following group: polyolefin, olefin copolymer, acrylic acid vinyl polymers, polyesters, polycarbonates, polyamides, polyimides, formaldehyde resins, polyurethanes, ether polymers, cellulose, thermoplastic elastomers and thermoplastic polyurethanes; preferably one or more of the above At least one of the polymers is a hydrogel.

18. The sample processing system according to any one of items 1 to 17, wherein the thickness of the selectively permeable membrane is less than 20 microns; preferably, the thickness of the selectively permeable membrane is 0.5-10 microns; most preferably, the thickness of the selectively permeable membrane is 0.5-10 microns. The thickness of the selectively permeable membrane is 1-5 microns.

19. The sample processing system according to item 18, wherein the selectively permeable membrane is:

A film formed from a hydrogel polymerized by PEGDA-containing monomers and then hardened by polymerization;

Or a film formed by hardening of polymerization initiated by light illumination or APS radical initiation.

20. The sample processing system according to any one of items 1 to 19, wherein,

The selectively permeable membrane can pass through molecules with a diameter of less than 70nm or a molecular weight of less than 2000kDa;

Preferably, the selectively permeable membrane can pass through molecules with a diameter of 50 nm or less or a molecular weight of 30 kDa or less;

It is further preferred that the selectively permeable membrane can transmit molecules with a diameter of 50 nm or more and 70 nm or less or a molecular weight of 30 kDa or more and 2000 kDa or less.

21. The sample processing system according to item 20, wherein the analysis reagent is selected from one or more of the following group or a complex composed of them: biological macromolecules and their analogs, high molecular polymers , nanoparticles, small molecules, inorganic solvents and organic solvents.

22. A method of manufacturing a sample processing system according to any one of items 1 to 21, comprising: Next steps:

preparing a first phase including an analyte, a capture reagent, an osmotic pressure regulator, and a first aqueous solvent;

preparing a second phase mixed with a selectively permeable membrane-forming material and a second aqueous solvent;

Mix the first phase and the second phase to form a mixed hydrophilic phase, and then mix the mixed hydrophilic phase with an oily solvent to prepare a water-in-oil emulsion to generate droplets; and

The above-mentioned water-in-oil emulsion is subjected to a curing or semi-curing reaction to form a selectively permeable membrane. In the curing or semi-curing reaction, the reaction conditions and the type of monomer or polymer in the selectively permeable membrane-forming material are adjusted or concentration to adjust the pore size of the selectively permeable membrane formed;

The water-in-oil emulsion that has been cured or semi-cured is demulsified to obtain a reaction compartment with a selectively permeable membrane on the outer layer and a content on the inside.

23. The manufacturing method according to item 22, wherein,

The first aqueous solvent includes one or more of the following group: ethanol, formaldehyde, polyvinyl alcohol, dextran, hydroxypropyl starch, Ficoll, methoxy polyethylene glycol, polyethylene glycol, dextran , Potassium phosphate, glucose, other inorganic salts (K ⁺ ,Na ⁺ ,Li ⁺ ,(NH ₄ ) ⁺ ,PO ₄ ^3– ,SO ₄ ^2- ), polyethylene glycol, polypropylene glycol, ethyl hydroxyethyl fiber ethylene oxide-propylene oxide, poly(N-isopropylacrylamide), poly(methyl methacrylate-co-methacrylic acid); or

The second aqueous solvent includes one or more of the following group: ethanol, formaldehyde, polyvinyl alcohol, dextran, hydroxypropyl starch, Ficoll, methoxy polyethylene glycol, polyethylene glycol, dextran , Potassium phosphate, glucose, other inorganic salts (K ⁺ ,Na ⁺ ,Li ⁺ ,(NH ₄ ) ⁺ ,PO ₄ ^3– ,SO ₄ ^2- ), polyethylene glycol, polypropylene glycol, ethyl hydroxyethyl fiber Element, ethylene oxide-propylene oxide, poly(N-isopropylacrylamide), poly(methyl methacrylate-co-methacrylic acid);

Preferably, both the first aqueous solvent and the second aqueous solvent are water;

It is further preferred that the osmotic pressure regulator is dextran and the concentration of dextran in the first aqueous solvent is 3%-10%; most preferably the osmotic pressure regulator is dextran and the dextran The concentration of sugar in the first aqueous solvent is 5.5%;

It is further preferred that the oil phase solvent is selected from one or more of the following groups: perfluoropolyether-polyethylene glycol-perfluoropolyether triblock copolymer, HFE-7500 fluorinated oil, Squalane oil, silicone oil and mineral oil;

Most preferably, the oil phase solvent is a perfluoropolyether-polyethylene glycol-perfluoropolyether triblock copolymer.

24. The manufacturing method according to item 22, wherein,

Using a surfactant to stabilize the droplets when generating the droplets, the surfactant is a non-ionic surfactant or an ionic surfactant;

Preferably, the nonionic surfactant is selected from one or more of the following group: PEG-PFPE ₂ , PFO, castor oil polyoxyester, polyoxyethylene 40 hydrogenated castor oil, and poloxamer;

Preferably, the ionic surfactant is selected from one or more of the following group: Krytox, sodium lauryl sulfate, and Janus nanoparticles; most preferably, the surfactant is PEG-PFPE ₂ .

25. The manufacturing method according to item 22, wherein,

The adjustment conditions for the curing or semi-curing reaction are the illumination intensity and illumination time of ultraviolet light;

Preferably, the curing or semi-curing reaction uses a catalyst and the catalyst is one or more TEMED initiators selected from the following group: 2-hydroxy-2-methyl-1-phenyl acetone, 1-hydroxycyclohexylbenzene Methyl ketone, 2-methyl-2-(4-morpholinyl)-1-[4-(methylthio)phenyl]-1-propanone, 2,4,6-trimethylbenzoyl- Diphenylphosphine oxide, ethyl 2,4,6-trimethylbenzoylphenylphosphonate, 2-dimethylamino-2-benzyl-1-[4-(4-morpholinyl)phenyl ]-1-Butanone, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone, MBF methyl benzoylformate, benzoin and derivatives Chemical substances (benzoin, benzoin dimethyl ether, benzoin ethyl ether, benzoin isopropyl ether, benzoin butyl ether), benzoyl compounds (diphenyl ethyl ketone, α, α-dimethoxy-α-phenylacetophenone) , Alkylphenones (α, α-diethoxyacetophenone, α-hydroxyalkylphenone, α-aminealkylphenone), acylphosphorus oxide (aroylphosphine oxide, bisbenzyl Acyl phenyl phosphine oxide), benzophenones (benzophenone, 2,4-dihydroxybenzophenone, Michinone), thioxanthone (thiopropoxythioxanthone, Isopropylthioanthrone); diaryliodonium salt, triaryliodonium salt, alkyl iodonium salt, cumene ferrocene hexafluorophosphate;

The selectively permeable membrane forming material is selected from one or more of the following group: polyolefin, olefin copolymer, acrylic, vinyl polymer, polyester, polycarbonate, polyamide, polyimide, Formaldehyde resin, polyurethane, ether polymer, cellulose, thermoplastic elastomer and thermoplastic polyurethane materials;

Preferably, the selectively permeable membrane-forming material is a hydrogel framework material; further preferably, the selectively permeable membrane-forming material is polyethylene glycol diacrylate (PEGDA), and further preferably, the concentration of PEGDA in the second aqueous solvent is 1%-10%,

Most preferably it is PEGDA and the concentration of PEGDA in the second aqueous solvent is 3%.

26. According to the manufacturing method described in item 22,

Wherein, the demulsification refers to using surfactant to shake and mix and then centrifuge at high speed,

Wherein, the surfactant is a nonionic surfactant or an ionic surfactant; the nonionic surfactant is selected from PEG-PFPE ₂ , PFO, castor oil polyoxyl ester, polyoxyethylene 40 hydrogenated One or more of castor oil and poloxamer;

The ionic surfactant is selected from Krytox, sodium lauryl sulfate and Janus nanoparticles One or more types of particles;

Most preferably the surfactant is PFO.

Beneficial technical effects achieved by the technical solution of this application

The sample processing system of the present application can be used to achieve the present invention. The present invention relates to a method for separating analytes (for example: cells, bacteria, viruses, nucleic acids, biochemical compounds and/or other materials) in a reaction compartment composed of a selectively permeable membrane. and systems and methods for targeted capture of biomolecules and processing of encapsulated biomolecules through reactions/processes to perform multi-step aqueous phase reactions. Biomolecules derived from the same analyte are enriched in a reaction compartment composed of a selectively permeable membrane through a specific capture carrier. The remaining biomolecules can freely pass through the selective permeation membrane, thereby achieving various reaction substance exchanges required for purification, cleaning or multi-step reactions. After treatment with external stimuli, the captured enriched biomolecules and their reactive derivatives can be released from the reaction compartment consisting of a selectively permeable membrane at the desired step. Using the sample processing system of the present application, target biomolecules with a volume smaller than the pore size can be specifically retained in a reaction compartment composed of a selectively permeable membrane, so that single-cell analysis of samples can be realized quickly and at low cost.

Description of the drawings

Figure 1A is a conceptual diagram of the functions implemented by the sample processing system; the sample preparation system achieves controlled substance exchange: analysis reaction reagents and impurities can freely enter and exit the reaction compartment; the analyte in the reaction compartment passes through the targeting ligand After specific binding to the capture reagent, it remains in the reaction compartment.

Figure 1B shows that the permselective reaction compartment achieves specific capture of the target analyte by retaining the capture reagent and the analyte as a whole or complex inside; elution purification can remove impurities and selectively retain the analyte to be analyzed. things.

Figure 2 shows the capture reagent forming a body or complex with the analyte.

Figure 3A shows an example in which the analyte is a DNA molecule, and the capture reagent is one or more of proteins, nucleic acids, and a complex formed by them together.

Figure 3B shows an example in which the analyte is a DNA molecule and the capture reagent is a coupling couple between a carrier (polymer or nanoparticle) and an oligonucleotide (primer).

Figure 3C shows an example in which the analyte is a DNA molecule and the capture reagent is an oligonucleotide (primer).

Figure 3D shows an example in which the analyte is an RNA molecule, and the capture reagent is a coupling couple between a carrier (polymer or nanoparticle) and an oligonucleotide (primer).

Figure 3E shows an example in which the analyte is an RNA molecule, and the capture reagent can be an oligonucleotide (primer).

Figure 3F shows an example in which the analyte is a protein molecule and the capture reagent is a coupling couple between a carrier (polymer or nanoparticle) and an antibody.

Figure 3G shows an example in which the analyte is a conjugate of a protein molecule and an antibody nucleic acid, and the capture reagent is a connection coupling between a carrier (polymer or nanoparticle) and a targeting ligand.

Figure 3H shows an example in which the analyte is a protein molecule and the capture reagent is a coupling couple between an initiating group and a targeting ligand.

Figure 3I shows an example in which the analyte is a T cell receptor and the capture reagent is a coupling couple between a carrier (polymer or nanoparticle) and an antigen peptide-MHC molecule complex (pMHC).

Figure 3J shows an example in which the analyte is a T cell receptor and the capture reagent is a coupling couple between the initiating group and pMHC.

Figure 4 shows a microfluidic device system that generates droplets containing an aqueous dual phase and a targeted capture carrier.

Figure 5 shows the following example: the first fluid (phase I solution, rich in dextran), the second fluid (phase II solution, rich in dextran-based) are injected into the microfluidic chip through the microfluidic device system. polymer of polyethylene glycol), continuous phase (the carrier oil is a fluorinated oil and contains a surfactant, such as PFPE-PEG-PFPE (perfluoropolyether-polyethylene glycol-perfluoropolyether) triblock copolymer substance); the targeted capture carrier enters the microfluidic system from the first fluid, the second fluid, the continuous phase (carrier oil), or any two or three.

Figure 6 shows an example of a two-step method to generate a sample preparation system: dissolving at least one layer of the inner polymer of the multi-layered polymer microspheres prepared in step 2 to obtain a selectively permeable membrane. reaction compartment.

Figure 7 shows an inner core of a dissolving band capture reagent forming an embodiment of a sample preparation system.

Figure 8 shows a sample preparation system composed of reaction compartments made of different materials.

Figure 9 shows an example of hardening the outer layer II phase by initiating polymerization.

Figure 10 shows a demulsification release reaction compartment and sample preparation system consisting of a selectively permeable membrane.

Figure 11 shows the sample preparation system of the present application, which is the state before the reaction compartment is gelled.

Figure 12 shows the sample preparation system of the present application, which is the state after the reaction compartment is gelled.

Figure 13 shows the sample preparation system of the present application in a state after demulsification of the reaction compartment.

Figure 14 shows that cells are wrapped in the sample preparation system of the present application. The cells are visible to the naked eye under bright field; after Syber Green staining, genomic DNA and nucleic acids emit strong signals under a fluorescence microscope.

Figure 15 shows the mRNA capture microspheres and cells in the reaction compartment; the size of the microspheres is ~2.7 microns; the outer wall pore size is approximately 50 nm.

Figure 16 shows a schematic representation of mRNA reverse transcription cDNA amplification.

Figure 17 shows a schematic representation of cDNA amplification library fragmentation.

Figure 18 shows a graphical representation of the genome gradually diffusing throughout the reaction compartment from the more compact state of Figure 14 after overnight incubation in the selectively permeable compartment.

Figure 19 shows a diagram of genome disruption by Tn5.

Figure 20 shows a diagram of genomic Tn5 fragmentation and amplification by P5 and P7 primers.

Figure 21 shows the experimental results of Example 3: that is, washing is performed after the amplification is completed: the DNA fragment of the amplification product remains in the membrane and is enriched on the magnetic beads.

Figure 22 shows a diagram of yeast co-encapsulated with Streptactin capture microspheres in a selectively permeable compartment before yeast growth and protein secretion.

Figure 23 shows an illustration of topological capture of fluorescent proteins on magnetic beads after yeast growth and protein secretion.

Detailed ways

Specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although specific embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to provide a thorough understanding of the invention, and to fully convey the scope of the invention to those skilled in the art.

It should be noted that certain words are used in the description and claims to refer to specific components. Those skilled in the art will understand that skilled persons may use different names to refer to the same component. This specification and the claims do not use difference in nouns as a way to distinguish components, but rather use differences in functions of the components as a criterion for distinction. If the words "include" or "include" mentioned throughout the description and claims are open-ended terms, they should be interpreted as "include but not limited to." The following descriptions of the description are preferred embodiments for implementing the present invention. However, the descriptions are for the purpose of general principles of the description and are not intended to limit the scope of the present invention. The protection scope of the present invention shall be determined by the appended claims.

As used herein, "substantially free" with respect to a particular component is used herein to mean that the particular component is not purposefully formulated into the composition and/or is present only as a contaminant or in trace amounts. Therefore, the total amount of a particular component resulting from any accidental contamination of the composition is less than 0.05%, preferably less than 0.01%. Most preferred are compositions in which the specific component is present in an amount undetectable by standard analytical methods.

As used in this specification, "a" or "an" may mean one or more. As used in the claims, the word "a" or "an" when used with the word "comprising" can mean one or more than one.

The term "or" is used in the claims to mean "and/or" unless it is expressly stated that only alternatives are to be referred to or the alternatives are mutually exclusive, although this disclosure supports reference to only alternatives and "and/or" Definition. As used herein, "another" may mean at least a second or more.

Throughout this application, the term "about" is used to indicate that a value includes the inherent variation in error of the device, the method used to determine the value, or the variation that exists between study subjects.

The ways to obtain various biological materials described in the examples are only to provide a way to obtain experimental materials to achieve the specific purpose of disclosure, and should not be used to limit the sources of biological materials of the present invention. In fact, the sources of biological materials used are wide, and any biological materials that can be obtained without violating laws and ethics can be replaced and used according to the tips in the embodiments.

The application relates in a first aspect to a sample processing system.

In a specific embodiment, a sample processing system is provided, which includes:

a) As a selectively permeable membrane on the outer layer of the reaction compartment, the selectively permeable membrane can selectively transmit analytical reagents;

in,

The selectively permeable membrane can selectively retain the capture reagent and the analyte to be connected to form a whole or a complex, or the converted product;

The diameter of the whole or complex formed by connecting the capture reagent and the analyte, or the converted product, is greater than 1/2 of the pore size of the selective permeability membrane. Advantageously, the diameter of the obtained whole or complex or converted product is greater than 0.5 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, 1.0 times, 1.5 times, 2 times the pore diameter of the selectively permeable membrane. times, 2.5 times, 3 times or greater.

In the context of this application, "reaction compartment" means a semi-enclosed reaction space surrounded by a semi-permeable membrane that allows the reaction of some substances (depending on their molecular size, hydrophobicity/hydrophilicity) The exchange of different, different charges, etc., specifically for this application, molecules with a smaller pore size relative to the pores of the semi-permeable membrane can freely enter or exit the reaction compartment, relative to the pores of the semi-permeable membrane Larger molecules can be trapped in the reaction compartment. In the context of this application, "selective permeable membrane" is sometimes also written as "semi-permeable membrane", specifically referring to the ability to carry charges on both sides of the membrane based on the difference in molecular size and hydrophobicity/hydrophilicity of the substance. A membrane structure that selectively allows substances to pass through different physical/chemical factors. In the context of this specification, unless otherwise stated, nucleic acid specifically refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In the context of this specification, "the pore size of a semipermeable membrane pore" may be understood as the average of the actual distances between the two furthest points on each membrane pore of the semipermeable membrane. In the context of this specification, the corresponding English word for "diameter" is diameter. More specifically, it can be understood as the two farthest points on a three-dimensional object. In yet another specific embodiment, the above-mentioned sample processing system is provided, wherein the capture reagent, the analyte, and the integral or complex or multiple combinations of the capture reagent and the analyte located inside the reaction compartment are provided. The polymer, or the converted product is in a liquid, colloidal or semi-liquid state; the interior of the reaction compartment further includes a solution containing an osmotic pressure regulator; preferably, the osmotic pressure regulator is dextran.

In this application, the "reaction compartment" forms a "mesh-shaped" semi-permeable membrane structure as its boundary, which is suitable for molecules with large diameters (large diameters generally correspond to molecules with large molecular weights, but are also related to the shape of the molecules). Relevant) has a clear interception effect, which can be adjusted through the pore size of the membrane pores of the "mesh-shaped semipermeable membrane".

In yet another specific embodiment, the above sample processing system is provided, wherein the analyte is selected from one or both of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles, cells, etc. More than one kind, and the complex formed by them; preferably, the analyte is from the same cell; most preferably, the analyte is one or more of DNA, mRNA and protein from the same cell.

In the context of this specification, a reaction compartment consisting of a selectively permeable membrane and a liquid-like core has dimensions ranging from approximately 10 μm to 150 μm. The analytes targeted by the reaction compartment can be small molecules, macromolecules, and cell-scale structures.

In a specific embodiment, the above sample processing system is provided, wherein,

The capture reagent consists of a targeting ligand and a carrier;

The carrier is specific to the presence or absence of the analyte; the function of the carrier is to allow the capture reagent to remain in the reaction compartment as a whole through physical or chemical effects; preferably, the carrier is organoids, cell clusters, cells, One or more of biological macromolecules and their analogs, high molecular polymers, nanoparticles, small molecules or their complexes;

Preferably, the targeting ligand is selected from one or more of the following group: locked nucleic acids and nucleic acids of XNA and their analogs, aptamers, small peptides, polypeptides, glycosylated peptides, polysaccharides, soluble receptors, Steroids, hormones, mitogens, antigens, superantigens, growth factors, cytokines, leptin, viral proteins, cell adhesion molecules, chemokines, streptavidin and its analogs, biotin and its analogs, Antibodies, antibody fragments, single-chain variable fragments (scFv), Nanobodies, T cell receptors, major histocompatibility complex (MHC) molecules, antigenic peptide-MHC molecule complexes (pMHC), DNA-binding proteins, RNA Binding proteins, intracellular or cell surface receptor ligands, and their multiple ligands, complex ligands, and coupling ligands.

Overall, the role of capture reagents is to enrich specific biomolecules within the reaction compartment.

In yet another specific embodiment, the above-mentioned sample processing system is provided, wherein the capture reagent is composed of a carrier, and the carrier has specificity for the analyte; preferably, the carrier is a magnetic particle capable of binding DNA. .

In the context of this specification, "magnetic particles" are also called magnetic beads, which refer to magnetic particles with sizes ranging from nanometers to micrometers. Magnetic particles are mainly composed of magnetite (Magnetite, Fe ₃ O ₄ ) or maghemite (maghemite, γ-Fe ₂ O ₃ ). For example, the magnetic particles used in this application are magnetic particles with the trade name BeaverBeads Oligo(dT) magnetic beads and model number Oligo(dT) (2.8 μm). The principle of the capture reagent is oligo-dT and oligo-A of mRNA. Tail complementary combination. In yet another specific embodiment, the above-mentioned sample processing system is provided, wherein being located inside the reaction compartment refers to being located inside the reaction compartment, at the interface between the selectively permeable membrane and the inside of the reaction compartment, or at the selectively permeable membrane.

Preferably, the capture reagent is a complex of protein and nucleic acid;

Further preferably, the DNA transposase is Tn5, and the DNA forming a complex with the protein has a transposase recognition sequence at its end, especially a 19 bp transposase recognition sequence.

Here, the mechanism by which Tn5 transposase can serve as a capture reagent (to form a larger diameter molecule to be intercepted) is that the protein interaction affinity between Tn5 dimers allows short DNA fragments to remain physically separated even though they are broken. The complex form exists stably. In yet another specific embodiment, the above sample processing system is provided, wherein,

The analyte is DNA;

In yet another specific embodiment, the above sample processing system is provided, wherein,

The analyte is DNA;

The capture reagent is an oligonucleotide primer;

In the context of this specification, dNTP is the English abbreviation of deoxy-ribonucleoside triphosphate. It includes dATP, dGTP, dTTP, dCTP, N refers to the nitrogenous base, and the variable represented by N refers to any one of A, T, G, and C; further, the dNTP as the reaction substrate can also be other Substrates that can be integrated by the polymerase: such as dUTP or dNTP analogs, such as biotinylated dNTPs. In biological DNA synthesis, as well as various PCR

(RT-PCR (reverse transcription PCR), Real-time PCR) plays the role of raw material.

The analyte is RNA;

After the capture reagent reacts with the analyte RNA in a premixed reagent containing reverse transcriptase, dNTPs, RNase inhibitors, small molecule additives and PCT reaction buffer, the diameter of the obtained product is larger than the 1/2 of the pore size of the selectively permeable membrane.

The analyte is RNA;

The capture reagent is an oligonucleotide primer;

The analyte is protein;

The initiating group is an oligonucleotide primer;

In the context of this specification, "priming group" refers to an oligonucleotide primer that can react with a specific sequence on a protein, and the long fragment of DNA coupled thereto retains the nucleic acid fragment on the protein and is used to analyze protein information.

The analyte is a T cell receptor;

In yet another specific embodiment, the above-mentioned sample processing system is provided, wherein the connection between the analyte and the capture reagent is selected from covalent bonds, metal bonds, ionic bonds, van der Waals forces, including hydrogen bonds, halogen bonds, Chalcogen bond, gold affinity, intercalation, overlap, cation-π bond, anion-π bond, salt bridge, secondary bond between non-metal atoms, secondary bond between metal atom and non-metal atom, gold affinity, silver affinity interaction, double hydrogen bond and secondary bond of gold bond; wherein, the interaction between the analyte and the capture reagent is selected from the group consisting of hydrogen bond interaction, ionic bond interaction, hydrophobic interaction and van der Waals force.

In a specific embodiment there is provided the above sample processing system, wherein the selectively permeable membrane is one or more polymers selected from the group consisting of polyolefins, olefin copolymers, acrylics, vinyl polymers polymers, polyesters, polycarbonates, polyamides, polyimides, formaldehyde resins, polyurethanes, ether polymers, cellulose, thermoplastic elastomers and thermoplastic polyurethanes; preferably at least one of the one or more polymers is One is hydrogel.

In yet another specific embodiment, the above-mentioned sample processing system is provided, wherein the thickness of the selectively permeable membrane is less than 20 microns; preferably, the thickness of the selectively permeable membrane is 0.5-10 microns; most preferably, the thickness of the selectively permeable membrane is 0.5-10 microns. The film thickness is 1-5 microns.

In particular, the thickness of the selectively permeable membrane can be 0.5 microns, 1.0 microns, 1.5 microns, 2.0 microns, 2.5 microns, 3.0 microns, 3.5 microns, 4.0 microns, 4.5 microns, 5.0 microns, 5.5 microns, 6.0 microns, 6.5 micron, 7.0 micron, 7.5 micron, 8.0 micron, 8.5 micron, 9.0 micron, 9.5 micron, 10.0 micron.

In yet another specific embodiment, the above-mentioned sample processing system is provided, wherein the selectively permeable membrane is: a membrane formed from a hydrogel polymerized by a PEGDA-containing monomer and then hardened by polymerization;

In particular, the selectively permeable membrane can pass through molecules with diameters of 50nm, 52nm, 54nm, 56nm, 58nm, 60nm, 62nm, 64nm, 66nm, 68nm, 70nm, or can pass through molecules with molecular weights of 30kDa, 65kDa, 100kDa, 200kDa, Molecules of 300kDa, 400kDa, 500kDa, 600kDa, 700kDa, 800kDa, 900kDa, 1000kDa, 1100kDa, 1200kDa, 1300kDa, 1400kDa, 1500kDa, 1600kDa, 1700kDa, 1800kDa, 1900kDa, 2000kDa.

In yet another specific embodiment, the above-mentioned sample processing system is provided, wherein the analysis reagent is selected from one or more of the following groups or a complex composed of them: biological macromolecules and their analogs, polymers Polymers, nanoparticles, small molecules, inorganic and organic solvents.

In a second aspect, the present application relates to a method for preparing a sample processing system.

In a specific embodiment, the above sample processing system preparation method is provided, wherein the method includes the following steps:

The first phase and the second phase are mixed into a mixed hydrophilic phase, and then the mixed hydrophilic phase and the oily phase are mixed The solvents are mixed to produce a water-in-oil emulsion; and

As used herein, the term "Phase I solution" also becomes "first phase" in the context of this application. refers to a solution that is miscible with a phase II solution (also referred to as "second phase" in the context of this application) but that can form a separate phase during a so-called liquid-liquid phase separation process, which occurs passively or is subject to external forces (such as gravity or surface tension). Similarly, as used herein, the term "Phase II solution" refers to a solution that is miscible with a Phase I solution but that can form a separate phase during a liquid-liquid phase separation process.

On the one hand, the phase I solution is rich in dextran.

Phase II solutions, on the other hand, are rich in polyethylene glycol-based polymers.

In an exemplary embodiment, droplet generation occurs at the intersection of the nozzles (constrictions) where breakup of the fluid stream into monodisperse droplets occurs.

In the context of this description, "generated droplets" or "droplets" and "reaction compartment" are to be understood as synonyms; and "carrier oil" and "oil solvent" are to be understood as synonyms.

In an exemplary embodiment, the present invention includes a microfluidic device system for generating droplets containing an aqueous dual phase and a targeted capture carrier. The device system includes:

(i) The inlet of the continuous phase (carrier oil);

(ii) the inlet of the first fluid;

(iii) Inlet for the second fluid;

(iv) The nozzle or flow focusing joint where droplet formation occurs;

(v) Droplet collection outlet.

(vi) Channel connecting the nozzle to the collection outlet.

(vii) The analyte enters the microfluidic system through the first fluid, the second fluid, or both.

(viii) The targeted capture carrier enters the microfluidic system from the first fluid, the second fluid, the continuous phase (carrier oil), or any two or three.

In another aspect, the invention includes methods for forming microfluidic systems containing droplets containing an aqueous dual phase and a targeted capture carrier:

(i) The target capture carrier is combined with the first fluid, the second fluid, the continuous phase (carrier oil), or any two or a mixture of the three, ready to enter the microfluidic system.

(ii) Inject the first fluid (Phase I solution);

(iii) Inject the second fluid (Phase II solution);

(iv) Inject carrier oil;

(v) bringing the carrier oil, phase I solution and phase II solution to the fluid intersection;

(vi) wrapping the Phase I solution and the Phase II solution in droplets suspended in a carrier oil;

(vii) Off-chip droplet collection.

(viii) The analyte enters the microfluidic system through the first fluid, the second fluid, or both.

In another aspect, the present invention includes Phase I and Phase II solutions that form internal and external phases, respectively, in the droplets. In the method of the present invention, the phase I and phase II solutions can be selected from a range of available polymer systems, as described in J.M.S. Cabral, Cell Partitioning in Aqueous Two-Phase Polymer Systems, Adv Biochem Engin/Biotechnol (2007).

In one embodiment, droplets are generated on a microfluidic chip that includes fluid junctions that allow the generation of droplets of different sizes. The droplet size can be controlled by adjusting the flow rate of phase I, the composition of the phase solution and the carrier oil and/or the cross-section of the nozzle and/or the cross-section of the microfluidic channel.

In one embodiment, droplets are produced at a frequency ranging from 0.01 Hz to 10 kHz, preferably from 0.1 kHz to 5 kHz, more preferably from 0.5 kHz to 2.5 kHz. A frequency of 1kHz means delivering droplets at a rate of 1000 droplets per second. In particular, the droplet generation frequency may be 0.1kHz, 0.2kHz, 0.3kHz, 0.4kHz, 0.5kHz, 0.6kHz, 0.7kHz, 0.8kHz, 0.9kHz, 1.0kHz, 1.5kHz, 2.0kHz, 2.5kHz, 3.0 kHz, 3.5kHz, 4.0kHz, 4.5kHz, 5.0kHz.

In yet another specific embodiment, the above preparation method is provided, wherein,

In a specific embodiment, the first aqueous solvent includes one or more of the following group: ethanol, formaldehyde, polyvinyl alcohol, dextran, hydroxypropyl starch, Ficoll, methoxypolyethylene glycol alcohol, polyethylene glycol, dextran, potassium phosphate, glucose, other inorganic salts (K ⁺ ,Na ⁺ ,Li ⁺ ,(NH ₄ ) ⁺ ,PO ₄ ^3– ,SO ₄ ^2- ), polyethylene glycol, poly Propylene glycol, ethylhydroxyethylcellulose, ethylene oxide-propylene oxide, poly(N-isopropylacrylamide), poly(methyl methacrylate-co-methacrylic acid).

In a specific embodiment, the second aqueous solvent includes one or more of the following group: ethanol, formaldehyde, polyvinyl alcohol, dextran, hydroxypropyl starch, Ficoll, methoxypolyethylene glycol alcohol, polyethylene glycol, dextran, potassium phosphate, glucose, other inorganic salts (K ⁺ ,Na ⁺ ,Li ⁺ ,(NH ₄ ) ⁺ ,PO ₄ ^3– ,SO ₄ ^2- ), polyethylene glycol, poly Propylene glycol, ethylhydroxyethylcellulose, ethylene oxide-propylene oxide, poly(N-isopropylacrylamide), poly(methyl methacrylate-co-methacrylic acid).

In a specific embodiment, the first aqueous solvent includes one or more of the following group: ethanol, formaldehyde, polyvinyl alcohol, dextran, hydroxypropyl starch, Ficoll, methoxypolyethylene glycol alcohol, polyethylene glycol, dextran, potassium phosphate, glucose, other inorganic salts (K ⁺ ,Na ⁺ ,Li ⁺ ,(NH ₄ ) ⁺ ,PO ₄ ^3– ,SO ₄ ^2- ), polyethylene glycol, poly Propylene glycol, ethyl hydroxyethyl cellulose, ethylene oxide-propylene oxide, poly(N-isopropylacrylamide), poly(methyl methacrylate-co-methacrylic acid); the second water-based The solvent contains one or more of the following group: ethanol, formaldehyde, polyvinyl alcohol, dextran, hydroxypropyl starch, Ficoll, methoxypolyethylene glycol, polyethylene glycol, dextran, potassium phosphate, glucose , other inorganic salts (K ⁺ ,Na ⁺ ,Li ⁺ ,(NH ₄ ) ⁺ ,PO ₄ ^3– ,SO ₄ ^2- ), polyethylene glycol, polypropylene glycol, ethyl hydroxyethyl cellulose, ethylene oxide Alkane-propylene oxide, poly(N-isopropylacrylamide), poly(methyl methacrylate-co-methacrylic acid).

In a specific embodiment, both the first aqueous solvent and the second aqueous solvent are water;

Preferably, the osmotic pressure regulator is dextran and the concentration of dextran in the first aqueous solvent is 3%-10%; most preferably, the osmotic pressure regulator is dextran and the dextran The concentration in the first aqueous solvent is 5.5%;

Preferably, the oil phase solvent is selected from one or more of perfluoropolyether-polyethylene glycol-perfluoropolyether triblock copolymer, HFE-7500 fluorinated oil, Squalane oil, silicone oil and mineral oil; Most preferably, the oil phase solvent is a perfluoropolyether-polyethylene glycol-perfluoropolyether triblock copolymer.

In yet another specific embodiment, the above preparation method is provided, wherein a surfactant is used to stabilize the droplets when the droplets are generated, and the surfactant is a nonionic surfactant or an ionic surfactant; preferably PFO ; The nonionic surfactant is selected from one or more of PEG-PFPE ₂ , castor oil polyoxyester, polyoxyethylene 40 hydrogenated castor oil, and poloxamer; the ionic surfactant Select one or more from Krytox, sodium lauryl sulfate and Janus nanoparticles.

The catalyst used in the curing or semi-curing reaction is one or more TEMED initiators selected from the following group: 2-hydroxy-2-methyl-1-phenyl acetone, 1-hydroxycyclohexyl phenyl ketone , 2-methyl-2-(4-morpholinyl)-1-[4-(methylthio)phenyl]-1-propanone, 2,4,6-trimethylbenzoyl-diphenyl Phosphine oxide, ethyl 2,4,6-trimethylbenzoylphenylphosphonate, 2-dimethylamino-2-benzyl-1-[4-(4-morpholinyl)phenyl]-1 -Butanone, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone, MBF methyl benzoylformate, benzoin and derivatives (benzoin , Benzoin Dimethyl Ether, Benzoin Ethyl Ether, Benzoin isopropyl ether, benzoin butyl ether), benzils (diphenyl ethyl ketone, α, α-dimethoxy-α-phenyl acetophenone), alkylbenzophenones (α, α-di Ethoxyacetophenone, α-hydroxyalkylphenone, α-aminoalkylphenone), acylphosphine oxide (aroylphosphine oxide, bisbenzoylphenylphosphine oxide), benzophenones (Benzophenone, 2,4-dihydroxybenzophenone, Michler's ketone), thioxanthone (thiopropoxythioxanthone, isopropylthioxanthone); diaryl Ionium salt, triaryliodonium salt, alkyl iodonium salt, cumene ferrocene hexafluorophosphate;

The selectively permeable membrane forming material is selected from one or more of the following group: polyolefin, olefin copolymer, acrylic, vinyl polymer, polyester, polycarbonate, polyamide, polyimide, Formaldehyde resin, polyurethane, ether polymer, cellulose, thermoplastic elastomer and thermoplastic polyurethane material; preferably it is a hydrogel skeleton material, further preferably it is polyethylene glycol diacrylate (PEGDA) and the PEGDA is in the second The concentration in the aqueous solvent is 1%-10%, most preferably it is PEGDA and the concentration of PEGDA in the second aqueous solvent is 3%.

In particular, the concentration of the PEGDA in the second aqueous solvent may be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%.

In yet another specific embodiment, the above preparation method is provided, wherein the demulsification refers to using a surfactant to shake and mix and then centrifuge at high speed, wherein the surfactant is a nonionic surfactant or an ionic surfactant. Surfactant; the nonionic surfactant is selected from one or more of PEG-PFPE ₂ , castor oil polyoxyester, polyoxyethylene 40 hydrogenated castor oil, and poloxamer; the ionic surfactant The surfactant is selected from one or more of Krytox, sodium lauryl sulfate and Janus nanoparticles; the most preferred surfactant is PFO. The term "about" used in this specification refers to a value within ±10% of the specified value. For example, "about 20" includes ±10% of 20, or from 18 to 22. Preferably, the term "about" refers to a value range of ±5% of the specified value.

In one embodiment, the volume of the droplets ranges from 0.01 pL to 1000 nL, preferably from 100 pL to 500 pL, more preferably from 10 pL to 100 μL. Specifically, the volume of the droplets can be 10pL, 20pL, 50pL, 100pL, 200pL, 500pL, 1000pL, 2nL, 5pL, 10nL, 20nL, 50nL, 100nL, 200nL, 500nL, 1000nL, 2μL, 5μL, 10μL, 20μL, 50μL ,100μL.

In a specific embodiment, phase I, phase II, or both solutions may contain, for example, various compounds, such as buffers, salts, carbohydrates, lipids, polymers, proteins, nucleic acids, polypeptides, organelles, cells, One or more types of bacteria, viruses or microorganisms, and complexes formed by them.

In a specific embodiment, the carrier oil used to generate the droplets is a fluorinated oil and contains the surfactant, PFPE-PEG-PFPE (perfluoropolyether-polyethylene glycol-perfluoropolyether) triblock copolymer . The surfactant is present in the carrier oil at a concentration of 0.05% to 5% (w/w), preferably 0.1% to 5% (w/w), more preferably 1% to 5% (w/w).

The method of the present invention is not limited by the type of surfactant or carrier oil used. One of ordinary skill in the art will be able to select appropriate surfactants, dispersed phases and carrier oils based on the desired characteristics of the droplets used and the reaction conditions. Surfactants, also called emulsifiers, act at the water/oil interface to prevent (or partially reduce) phase separation.

In one embodiment, the carrier oil (oil solvent) is selected from fluorinated oils such as FC40 oil FC43 FC77 oil FC72 FC84 FC70 HFE-7500 HFE-7100 Perfluorohexane, perfluorooctane, perfluorodecane, Galden-HT135 oil (SolvaySolexis), Galden-HT170 oil (SolvaySolexis), Galden-HT110 oil (SolvaySolexis), Galden-HT90 oil (SolvaySolexis), Galden-HT70 Oil (SolvaySolexis), GaldenPFPE liquid, SVFluids or ZVFluids; and hydrocarbon oils such as mineral oil, light mineral oil, Adepsine oil, Albolene, cable oil, baby oil, Drakeol, electrical insulating oil, heat treatment oil, hydraulic oil, lignite oil, liquid paraffin, mineral seal oil, paraffin oil, Petroleum, industrial oil, white oil, silicone oil or vegetable oil. In a specific embodiment, the carrier oil is a fluorinated oil. In a more specific embodiment, the carrier oil is HFE-7500 oil.

In a preferred embodiment, the depth of all channels on the microfluidic chip is the same and is in the range of 1 μm to 1000 μm, preferably in the range of 50-500 μm and more preferably in the range of 20-300 μm, and even more preferably In the range of 10-100μm.

In another embodiment, the method of the present invention further includes collecting the droplets off-chip.

In another embodiment, the collected droplets are broken up, thereby releasing the reaction compartment consisting of a selectively permeable membrane into the surrounding medium. This can be accomplished by destabilizing the droplet water-oil interface using chemical means or using electrofusion, temperature, dilution, etc. In this particular embodiment, the droplet water-oil interface is destabilized by mixing the emulsion with chemicals such as fluorinated octanol.

In an exemplary embodiment, the reaction compartment consists of a membrane-like selectively permeable shell and a liquid-like core.

In a preferred embodiment, the selectively permeable membrane is a hydrogel polymerized with PEGDA, and the liquid core is rich in dextran.

In an exemplary embodiment, cellular, biochemical and biological compounds are introduced into a reaction compartment consisting of droplets/selectively permeable membranes by providing said entities with:

(i) In the "first solution";

(ii) in the "second solution";

(iii) In both solutions.

In the method of the present invention, by suspending the reaction compartment composed of the selectively permeable membrane in a required aqueous solution or polar solvent, the analytes wrapped in the reaction compartment composed of the selectively permeable membrane are substances are displaced to different biochemical environments.

In another aspect of the invention, substances enclosed in reaction compartments composed of selectively permeable membranes react with chemical, biochemical or biological compounds present in an aqueous solution.

In another exemplary embodiment, the encapsulated cells are lysed within a reaction compartment consisting of a selectively permeable membrane.

In another example, the cell-lysing material remains completely or partially within the reaction compartment consisting of a selectively permeable membrane.

In yet another example, a reaction compartment composed of a selectively permeable membrane is suspended in another solution to displace reagents that lyse the surrounding cells.

In an exemplary embodiment, cell-lysing mRNA is captured by polyT-rich nanoparticles. In a specific embodiment, cells enclosed in reaction compartments composed of selectively permeable membranes are lysed and their mRNA is captured by polyT-rich nanoparticles. In an exemplary embodiment, the mRNA from lysed cells is converted to cDNA via a reverse transcription reaction. In a specific embodiment, cells enclosed in a reaction compartment composed of a selectively permeable membrane are lysed and their mRNA is converted into cDNA using reverse transcriptase.

In the methods of the present invention, the DNA and/or RNA of the lysed cells may be modified/processed using chemical or biochemical means. For example, add poly(A) tails to nucleic acids, add nucleic acid barcodes, add indexes, connect adapters, digest, fragment, etc.

In the method of the invention, the encapsulated cDNA of a single cell can be labeled (barcoded) with a barcoded poly(T) primer.

In this approach, barcoded poly(T) primers can carry cellular barcodes, molecular barcodes (unique molecular identifiers), sequencing adapters, poly-dT moieties, and/or other components required for the barcoding reaction.

In another exemplary embodiment, cDNA from lysed cells is enzymatically amplified by PCR inside or outside a reaction compartment consisting of a selectively permeable membrane.

In the method of the present invention, lysis and nucleic acid amplification are performed by performing sequential multi-step reactions on material from the same encapsulated cell.

In a preferred embodiment, a reaction compartment composed of a selectively permeable membrane is used for genotypic analysis of single cells.

In an exemplary embodiment, the reaction trapped in the selectively permeable membrane is released by displacing the selectively permeable membrane-composed reaction compartment into another fluid, such as a biological buffer, water, and/or other aqueous solutions. Biochemical and biological molecules inside compartments.

In exemplary embodiments, performing the methods described above includes, but is not limited to, the use of microfluidic systems.

Example part

Example 1: Single-cell mRNA capture

Materials and reagents

Equipment manufacturing and operation. Polydimethylsiloxane (PDMS) microfluidic devices were fabricated and operated using standard procedures as described. Preparation of ATPS (aqueous two-phase system) and target capture reagents. All chemicals were ordered from Sigma-Aldrich and Fisher Scientific. Use APS (ammonium persulfate), 10% (w/v) dextran (MW 500K), 10mg/mL polyT magnetic beads, 5% (w/v) PEGDA (MW 8K), 5% (v/v) PEGDA (MW 575), 0.5% (w/v) to prepare ATPS droplets. Other concentrations of PEGDA (MW 8K) and PEGDA (MW 575) as well as other polymers can be used. Solutions containing the above ingredients were mixed and liquid-liquid phase separation was induced in a tabletop centrifuge.

emulsification. For the generation of larger-sized reaction compartments composed of selectively permeable membranes, as shown in Figure 1: The reaction compartment composed of droplets and selectively permeable membranes was generated using a microfluidic chip with a 50μm height and a 40μm wide nozzle. of. Typical flow rates used are: PEGD(M)A-rich phase - flow rate 200 μL/h, dextran-rich phase containing 293T cells - flow rate 100 μL/h and droplet stabilizing oil

(2% PEG-PFPE ₂ HFE7500) - flow rate 600 μL/h. As the viscosity of the biphasic system increases, droplet breakup by the jetting mechanism can be observed, which can shift the droplet generation mode by adjusting the flow rate of the system.

Cross-linking. The emulsion was collected in a 1.5 ml tube and immediately cross-linked using a high-intensity ultraviolet inspection lamp UVP (UVP, 95-0127-01) exposed at 365 nm wavelength for 2.5 minutes. After the PEGDA shell has hardened, a demulsifier (surfactant: PFO) is used to recover the reaction compartment consisting of a selectively permeable membrane from the emulsion.

Cell lysis, reverse transcription and amplification of mRNA. Lysis of the encapsulated cells was performed by suspending the reaction compartment consisting of a selectively permeable membrane in a lysis buffer containing: 200 μg/mL proteinase K (Invitrogen, AM2546), 0.1% (v/v) TritonX- 100 (Sigma-Aldrich, T8787-100ML), 10mM Tris-HCl [pH 7.5] and 1mM EDTA. The reaction compartment consisting of a selectively permeable membrane suspended in lysis buffer was incubated at 37°C for 30 min and then at 50°C for an additional 30 min.

During this process, RNA is captured by the capture reagent (polyT magnetic beads) and remains in the reaction compartment.

After lysis, the reaction compartment consisting of a selectively permeable membrane was washed in buffer (10 mM Tris-HCl [pH 7.5] and 0.05% (v/v) TritonX-100). Then follow the manufacturer's recommendations by suspending the reaction compartment consisting of a selectively permeable membrane in reverse transcriptase containing 0.5 U/μL SMARTScribe II reverse transcriptase, 1X First StrandBuffer, and incubating for an additional 30 minutes at 50°C. . PCR, which is similar to a macro reaction, is used to amplify fragments of specific regions of cDNA or specific genes. Use Takara single cells according to manufacturer's recommendations cDNA PCR reagent (Takara ) for 10 cycles of amplification. In all enzymatic reactions, the reaction compartment composed of selectively permeable membranes occupies approximately 40-50% of the final reaction volume.

Table 1 cDNA amplification reaction system

Interruption and capture of amplification products. The amplified products were fragmented using the DNA fragmentation kit (Nextera XT DNA Library Preparation Kit (24 samples), FC-131-1024) according to the manufacturer's recommendations. Using Tn5 dimers as capture carriers can maintain short fragments of DNA in a polymeric state in a reaction compartment composed of a selectively permeable membrane.

The results of this example are shown in Figures 9-17. It can be seen from these figures that the two-phase aqueous system, the carrier and the capture reagent can be packaged through the microfluidic device to form selective transmission under light curing conditions. reaction compartment. This reaction compartment retains RNA molecules (fluorescence is achieved through subsequent cDNA amplification) Color development verified that RNA molecules and cDNA molecules were retained through topological capture). After being interrupted by Tn5, the Tn5 dimer allows the broken short DNA fragments to remain in a complex with a larger diameter through protein interactions, thereby being retained in a compartment surrounded by a selectively permeable membrane.

Example 2: Capture of single cell fragmented genomes

Materials and reagents

Equipment manufacturing and operation. Polydimethylsiloxane (PDMS) microfluidic devices were fabricated and operated using standard procedures as described.

Preparation of ATPS and target capture reagents. All chemicals were ordered from Sigma-Aldrich and Fisher Scientific. Use APS (ammonium persulfate), 10% (w/v) dextran (MW 500K), 10mg/mL polyT magnetic beads, 5% (w/v) PEGDA (MW 8K), 5% (v/v) PEGDA (MW575), 0.5% (w/v) was used to prepare ATPS droplets. Other concentrations of PEGDA (MW 8K) and PEGDA (MW 575) as well as other polymers can be used. Solutions containing the above ingredients were mixed and liquid-liquid phase separation was induced in a tabletop centrifuge.

emulsification. For the generation of a reaction compartment surrounded by a larger size selectively permeable membrane, as shown in Figure 1: the reaction compartment composed of droplets and selectively permeable membrane is a micro-tube using a 50μm height and 40μm wide nozzle. Produced by fluidic chips. Typical flow rates used were: PEGD(M)A-rich phase - flow rate 200 μL/h, dextran-rich phase containing 293T cells - flow rate 100 μL/h and droplet stabilizing oil (2% PEG-PFPE ₂ HFE7500) - flow rate is 600μL/h. As the viscosity of the biphasic system increases, droplet breakup by the jetting mechanism can be observed, which can shift the droplet generation mode by adjusting the flow rate of the system.

Cross-linking. The emulsion was collected in a 1.5 ml tube and immediately cross-linked using a high-intensity ultraviolet inspection lamp UVP (UVP, 95-0127-01) exposed at 365 nm wavelength for 2.5 minutes. After the PEGDA shell hardens, a demulsifier (surfactant PFO) is used to recover the reaction compartment consisting of a selectively permeable membrane from the emulsion.

Cell lysis, genomic DNA fragmentation and amplification. Lysis of the encapsulated cells was performed by suspending the reaction compartment consisting of a selectively permeable membrane in a lysis buffer containing: 200 μg/mL Proteinase K (Invitrogen, AM2546), 0.1% (v/v) Triton X- 100 (Sigma-Aldrich, T8787-100ML), 10mM Tris-HCl [pH 7.5] and 1mM EDTA. The reaction compartment consisting of a selectively permeable membrane suspended in lysis buffer was incubated overnight at room temperature (approximately 25°C) so that the genome fully filled the entire reaction compartment.

After lysis, place the reaction compartment consisting of a selectively permeable membrane in wash buffer (10mM Tris-HCl [pH 7.5] and 0.05% (v/v) Triton X-100). The amplified products were then prepared by suspending the reaction compartment consisting of a selectively permeable membrane in the DNA fragmentation kit (Nextera XT DNA Library Preparation Kit (24 samples), FC-131-1024) according to the manufacturer's recommendations. break. Using Tn5 dimers as capture carriers can maintain short fragments of DNA in a polymeric state in a reaction compartment composed of a selectively permeable membrane.

Interruption and capture of amplification products. Amplification was performed for 35 cycles using the KAPA PCR kit (KAPA Biosystems, KK2602) according to the manufacturer's recommendations.

Table 2 Library construction reaction system

Table 3 PCR program settings:

The results of this example are shown in Figures 18-20. It can be seen from these figures that using Tn5 dimers as capture carriers can maintain short fragments of DNA in a polymer state and remain in a membrane composed of a selective permeable membrane. in the reaction compartment.

Example 3: Topological capture of DNA short fragments based on magnetic beads

Materials and reagents

Preparation of primer-containing magnetic beads.

1. Shake and resuspend the magnetic beads (beaver bio). Take 30ul magnetic beads (0.3mg) into a new centrifuge tube. Place the centrifuge tube on the magnetic stand, let it stand for 1 minute, absorb the supernatant, and remove the centrifuge tube from the magnetic stand. (It is recommended that the amount of biotinylated molecules added is 1-2 times the loading capacity of the magnetic beads to saturate the magnetic beads)

2. Add 1mL buffer I (Beaver Bio) into the centrifuge tube, shake thoroughly to resuspend the magnetic beads, perform magnetic separation, and remove the supernatant.

3. Repeat step 2 once

Prepare 500uL buffer I diluted biotinylated nucleic acid

Table 4 PCR reaction solution

4. Add 500uL of biotinylated nucleic acid diluted with buffer I (so that the magnetic bead concentration is 2 mg/mL), and shake thoroughly to resuspend the magnetic beads. Place the centrifuge tube on a rotating mixer and rotate and mix at room temperature for 30 minutes.

5. Magnetic separation, transfer the supernatant to a new centrifuge tube.

Preparation of ATPS and target capture reagents. All chemicals were ordered from Sigma-Aldrich and Fisher Scientific. Primers are ordered from the manufacturer. Use APS (ammonium persulfate), 10% (w/v) dextran (MW 500K), 10mg/mL polyT magnetic beads, 5% (w/v) PEGDA (MW 8K), 5% (v/v) PEGDA (MW 575), 0.5% (w/v) was used to prepare ATPS droplets. Other concentrations of PEGDA (MW 8K) and PEGDA (MW 575) as well as other polymers can be used. The solution containing all components was mixed and liquid-liquid phase separation was induced in a tabletop centrifuge.

emulsification. For the generation of larger-sized reaction compartments composed of selectively permeable membranes, as shown in Figure 1: The reaction compartment composed of droplets and selectively permeable membranes was generated using a microfluidic chip with a 50μm height and a 40μm wide nozzle. of. Typical flow rates used are: PEGDA-rich phase - flow rate is 200 μL/h, dextran-rich phase (in which added: amplification template plasmid (pBbSLactamC-mCherry), biotinylated 150bp-F-primer ( 41bp) magnetic beads 20mg, 400nM 5'biotin-MID42 (41bp), 400nM 150bp-R-Primer-Fam) - flow rate 100μL/h and droplet stabilizing oil (2% PEG-PFPE ₂ HFE7500) - flow rate 600μL/h. As the viscosity of the biphasic system increases, droplet breakup by the jetting mechanism can be observed, which can shift the droplet generation mode by adjusting the flow rate of the system.

Cross-linking. The emulsion was collected in a 1.5 ml tube and immediately cross-linked using a high-intensity ultraviolet inspection lamp UVP (UVP, 95-0127-01) exposed at 365 nm wavelength for 2.5 minutes. After the PEGDA shell hardens, a demulsifier (PFO) is used to recover the reaction compartment consisting of a selectively permeable membrane from the emulsion.

Table 5 PCR reaction solution

Table 6 PCR amplification conditions

Amplification was performed for 40 cycles, and the annealing temperature was adjusted to 65°C.

Figure 21 shows the experimental results of this embodiment: that is, after the amplification is completed, washing is performed: the amplified product DNA fragments remain in the membrane and are enriched on the magnetic beads.

Example 4: Topological capture of proteins based on modified antibody magnetic beads

Materials and reagents

Preparation of antibody-containing vectors.

1. Shake and resuspend the vector (GE Healthcare). Take 10ul magnetic beads (0.2mg) into a new centrifuge tube. Place the centrifuge tube on the magnetic stand, let it stand for 1 minute, absorb the supernatant, and remove the centrifuge tube from the magnetic stand. (It is recommended that the amount of biotinylated molecules added is 1-2 times the loading capacity of the magnetic beads to saturate the magnetic beads)

2. Add 1mL buffer W (GE Healthcare) to the centrifuge tube, shake thoroughly to resuspend the magnetic beads, perform magnetic separation, and remove the supernatant.

3. Repeat step 2 once

Preparation of ATPS and target capture reagents. All chemicals were ordered from Sigma-Aldrich and Fisher Scientific. The Strep-tactin vector and the yeast secreting strep-tag-II-GFP were both constructed independently in the laboratory. Use APS (ammonium persulfate), 10% (w/v) dextran (MW 500K), 10mg/mL polyT magnetic beads, 5% (w/v) PEGDA (MW 8K), 5% (v/v) PEGDA (MW575), 0.5% (w/v) was used to prepare ATPS droplets. Other concentrations of PEGDA (MW 8K) and PEGDA (MW 575) as well as other polymers can be used. The solution containing all components was mixed and liquid-liquid phase separation was induced in a tabletop centrifuge.

emulsification. For the generation of larger-sized reaction compartments composed of selectively permeable membranes, as shown in Figure 1: The reaction compartment composed of droplets and selectively permeable membranes was generated using a microfluidic chip with a 50μm height and a 40μm wide nozzle. of. Typical flow rates used are: PEGDA-rich phase - flow rate 200 μL/h, dextran-rich phase (in which yeast secreting strep-tag-II-GFP protein and Strep-Tactin microspheres are added), flow rate was 100 μL/h and droplet stabilizing oil (2% PEG-PFPE ₂ HFE7500) - flow rate was 600 μL/h. As the viscosity of the biphasic system increases, droplet breakup by the jetting mechanism can be observed, which can shift the droplet generation mode by adjusting the flow rate of the system.

The experimental results of this step are shown in Figure 22. As can be seen from Figure 22, the green fluorescence field is that the strep-tactin microspheres do not emit significant light initially (they do not contain fluorescent proteins, and there is no topological capture of the proteins).

The final experimental results are shown in Figure 23. It can be seen from Figure 23 that the semipermeable membrane wrapped with yeast and strep-Tactin microspheres was placed in YPD culture medium and grown for 48 hours. After the yeast grew, the GPF protein secreted with strep-tag-II was blocked by strep-Tactin microspheres. The balls are captured and retained within the membrane. GFP has its own green fluorescence and develops color after being enriched on the surface of the microspheres. Compared with the original microspheres, the fluorescence intensity of the microspheres is very high.

Although the embodiments of the present invention have been described above in conjunction with the accompanying drawings, the present invention is not limited to the above-mentioned specific embodiments and application fields. The above-mentioned specific embodiments are only illustrative and instructive, rather than restrictive. . Under the inspiration of this description and without departing from the scope of protection of the claims of the present invention, those of ordinary skill in the art can also make many forms, which are all included in the protection of the present invention.

Claims

A sample processing system including:

a) As a selectively permeable membrane on the outer layer of the reaction compartment, the selectively permeable membrane can selectively transmit analytical reagents;

b) Contents located inside the reaction compartment: including capture reagents and analytes;

in,

The analyte is directly or indirectly connected to the capture reagent to form a whole, or forms a complex or polymer through interaction, or the two undergo a biological or chemical reaction to generate a converted product;

The selectively permeable membrane can selectively retain the whole body, complex or polymer formed by connecting the capture reagent and the analyte, or the converted product;

The diameter of the whole or complex formed by connecting the capture reagent and the analyte, or the converted product, is greater than 1/2 of the pore size of the selective permeability membrane.
The sample processing system according to claim 1, wherein

The capture reagent, the analyte, and the integral, complex or polymer formed by connecting the capture reagent and the analyte located inside the reaction compartment, or the converted product is liquid, gelatinous or Semi-liquid state; the interior of the reaction compartment also includes a solution containing an osmotic pressure regulator;

Preferably the osmotic pressure regulator is dextran.
The sample processing system according to claim 1 or 2, wherein the analyte is selected from one or two types of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles, cells, etc. The above, and the complex formed by them; preferably, the analyte is from the same cell; most preferably, the analyte is one or more of DNA, mRNA and protein from the same cell.
The sample processing system according to any one of claims 1 to 3, wherein

The capture reagent is composed of a targeting ligand and a carrier, which may or may not be specific to the analyte; the carrier can cause the capture reagent to remain in the reaction compartment through physical or chemical effects. ; Preferably, the carrier is one or more of organoids, cell clusters, cells, biological macromolecules and their analogs, high molecular polymers, nanoparticles, small molecules or their complexes;

The targeting ligand is a natural or artificial molecule and has specificity for the analyte;

Preferably, the targeting ligand is selected from one or more of the following group: locked nucleic acids and nucleic acids of XNA and their analogs, aptamers, small peptides, polypeptides, glycosylated peptides, polysaccharides, soluble receptors, Steroids, hormones, mitogens, antigens, superantigens, growth factors, cytokines, leptin, viral proteins, cell adhesion molecules, chemokines, streptavidin and its analogs, biotin and its analogs, Antibodies, antibody fragments, single-chain variable fragments (scFv), Nanobodies, T cell receptors, major histocompatibility complex (MHC) molecules, antigenic peptide-MHC molecule complexes (pMHC), DNA-binding proteins, RNA Binding proteins, intracellular or cell surface receptor ligands, and their multiple ligands, complex ligands, and coupling ligands.
The sample processing system according to any one of claims 1 to 3, wherein

The capture reagent is composed of a carrier, and the carrier has specificity for the analyte;

Preferably the carrier is a magnetic particle capable of binding DNA.
The sample processing system according to any one of claims 1 to 5, wherein "located inside the reaction compartment" means located inside the reaction compartment and at the interface between the selectively permeable membrane and the inside of the reaction compartment. Or in the selective permeability membrane.
The sample processing system according to any one of claims 1 to 6, wherein

The analyte is DNA, and the capture reagent is selected from one or more of proteins, nucleic acids, and their complexes;

Preferably, the capture reagent is a complex of protein and nucleic acid;

It is further preferred that the protein is DNA transposase and the nucleic acid is DNA;

Further preferably, the DNA transposase is Tn5, and the DNA forming a complex with the protein has a transposase recognition sequence at its end.
The sample processing system according to any one of claims 1 to 6, wherein

The analyte is DNA;

The capture reagent is a coupling couple composed of a polymer or nanoparticle and an oligonucleotide primer;

After the capture reagent and the DNA are reacted in a premix reagent containing dNTPs, DNA polymerase, small molecule additives and PCR reaction buffer, the polymer or nanoparticle is reacted with the to-be- The diameter of the complex formed by the analyte DNA is greater than 1/2 of the pore size of the selectively permeable membrane.
The sample processing system according to any one of claims 1 to 6, wherein

The analyte is DNA;

The capture reagent is an oligonucleotide primer;

After the capture reagent and the DNA react in a premix reagent containing dNTPs, DNA polymerase, small molecule additives and PCR reaction buffer, the diameter of the obtained product is greater than 1% of the pore size of the selective permeability membrane. /2.
The sample processing system according to any one of claims 1 to 6, wherein

The analyte is RNA;

The capture reagent is a coupling couple between a polymer or a nanoparticle and an oligonucleotide primer;

After the capture reagent reacts with the analyte RNA in a premixed reagent containing reverse transcriptase, dNTPs, RNase inhibitors, small molecule additives and PCT reaction buffer, the diameter of the obtained product is larger than the 1/2 of the pore size of the selectively permeable membrane.
The sample processing system according to any one of claims 1 to 6, wherein

The analyte is RNA;

The capture reagent is an oligonucleotide primer;

After the capture reagent reacts with the RNA to be analyzed in a premixed reagent containing reverse transcriptase, dNTPs, RNase inhibitors, small molecule additives and buffers, it is then reacted with oligonucleotide primers, dNTPs, DNA polymerase, small molecule After reacting in a premixed reagent composed of additives and PCR reaction buffer, the diameter of the obtained product is greater than 1/2 of the pore size of the selectively permeable membrane.
The sample processing system according to any one of claims 1 to 6, wherein

The analyte is protein;

The capture reagent is a coupling couple between a polymer or a nanoparticle and a targeting ligand;

The diameter of the complex formed by the capture reagent and the protein is greater than 1/2 of the pore size of the selectively permeable membrane.
The sample processing system according to any one of claims 1 to 6, wherein

The analyte is protein;

The capture reagent is a coupling couple between an initiating group and a long fragment of DNA;

The initiating group is an oligonucleotide primer;

After reacting in the premixed reagent composed of the initiating group, dNTP, DNA polymerase, small molecule additives, PCR reaction buffer and additional DNA template, a diameter larger than 1/2 of the pore size of the selective permeable membrane is formed. of DNA molecules.
The sample processing system according to any one of claims 1 to 6, wherein

The analyte is a T cell receptor;

The capture reagent is a coupling coupling between a polymer or a nanoparticle and an antigen peptide-MHC molecule complex (pMHC);

The diameter of the complex formed by the capture reagent and the T cell receptor is greater than 1/2 of the pore size of the selectively permeable membrane.
The sample processing system according to any one of claims 1 to 6, wherein

The analyte is a T cell receptor;

The capture reagent is a coupling couple between the initiating group and pMHC;

The initiating group is an oligonucleotide primer;

After reacting in the premixed reagent composed of the initiating group, dNTP, DNA polymerase, small molecule additives, PCR reaction buffer and additional DNA template, a diameter larger than 1/2 of the pore size of the selective permeable membrane is formed. of DNA molecules.
The sample processing system according to any one of claims 1 to 15, wherein the connection between the analyte and the capture reagent is selected from the group consisting of covalent bonds, metal bonds, ionic bonds, van der Waals forces, including hydrogen bonds, Halogen bond, chalcogen bond, gold affinity, intercalation, overlap, cation-π bond, anion-π bond, salt bridge, secondary bond between non-metal atoms, secondary bond between metal atom and non-metal atom, gold affinity , argyrophilic interaction, double hydrogen bond and secondary bond of gold bond; wherein, the interaction between the analyte and the capture reagent is selected from hydrogen bond interaction, ionic bond interaction, hydrophobic interaction and van der Waals force.
The sample processing system according to any one of claims 1 to 16, wherein the selectively permeable membrane is one or more polymers selected from the group consisting of: polyolefin, olefin copolymer, acrylic acid vinyl polymers, polyesters, polycarbonates, polyamides, polyimides, formaldehyde resins, polyurethanes, ether polymers, cellulose, thermoplastic elastomers and thermoplastic polyurethanes; preferably one or more of the above At least one of the polymers is a hydrogel.
The sample processing system according to any one of claims 1 to 17, wherein the thickness of the selectively permeable membrane is less than 20 microns; preferably the thickness of the selectively permeable membrane is 0.5-10 microns; most preferably the selection The thickness of the permeable membrane is 1-5 microns.
The sample processing system according to claim 18, wherein the selectively permeable membrane is:

A film formed from a hydrogel polymerized by PEGDA-containing monomers and then hardened by polymerization;

Or a film formed by hardening of polymerization initiated by light illumination or APS radical initiation.
The sample processing system according to any one of claims 1 to 19, wherein

The selectively permeable membrane can pass through molecules with a diameter of less than 70nm or a molecular weight of less than 2000kDa;

Preferably, the selectively permeable membrane can pass through molecules with a diameter of 50 nm or less or a molecular weight of 30 kDa or less;

It is further preferred that the selectively permeable membrane can transmit molecules with a diameter of 50 nm or more and 70 nm or less or a molecular weight of 30 kDa or more and 2000 kDa or less.
The sample processing system according to claim 20, wherein the analysis reagent is selected from one or more of the following group or a complex composed of them: biological macromolecules and their analogs, high molecular polymers, Nanoparticles, small molecules, inorganic solvents and organic solvents.
A method of manufacturing a sample processing system according to any one of claims 1 to 21, comprising the following steps:

preparing a first phase including an analyte, a capture reagent, an osmotic pressure regulator, and a first aqueous solvent;

preparing a second phase mixed with a selectively permeable membrane-forming material and a second aqueous solvent;

Mix the first phase and the second phase to form a mixed hydrophilic phase, and then mix the mixed hydrophilic phase with an oily solvent to prepare a water-in-oil emulsion to generate droplets; and

The water-in-oil emulsion is cured or semi-cured to form a selectively permeable membrane. In the curing or semi-curing reaction, the pore size of the selectively permeable membrane formed is adjusted by adjusting the reaction conditions and the type or concentration of the monomer or polymer in the selectively permeable membrane-forming material;

The water-in-oil emulsion that has been cured or semi-cured is demulsified to obtain a reaction compartment with a selectively permeable membrane on the outer layer and a content on the inside.
The manufacturing method according to claim 22, wherein

The first aqueous solvent includes one or more of the following group: ethanol, formaldehyde, polyvinyl alcohol, dextran, hydroxypropyl starch, Ficoll, methoxy polyethylene glycol, polyethylene glycol, dextran , Potassium phosphate, glucose, other inorganic salts (K + ,Na + ,Li + ,(NH 4 ) + ,PO 4 3– ,SO 4 2- ), polyethylene glycol, polypropylene glycol, ethyl hydroxyethyl fiber ethylene oxide-propylene oxide, poly(N-isopropylacrylamide), poly(methyl methacrylate-co-methacrylic acid); or

The second aqueous solvent includes one or more of the following group: ethanol, formaldehyde, polyvinyl alcohol, dextran, hydroxypropyl starch, Ficoll, methoxy polyethylene glycol, polyethylene glycol, dextran , Potassium phosphate, glucose, other inorganic salts (K + ,Na + ,Li + ,(NH 4 ) + ,PO 4 3– ,SO 4 2- ), polyethylene glycol, polypropylene glycol, ethyl hydroxyethyl fiber Element, ethylene oxide-propylene oxide, poly(N-isopropylacrylamide), poly(methyl methacrylate-co-methacrylic acid);

Preferably, both the first aqueous solvent and the second aqueous solvent are water;

It is further preferred that the osmotic pressure regulator is dextran and the concentration of dextran in the first aqueous solvent is 3%-10%; most preferably the osmotic pressure regulator is dextran and the dextran The concentration of sugar in the first aqueous solvent is 5.5%;

It is further preferred that the oil phase solvent is selected from one or more of the following groups: perfluoropolyether-polyethylene glycol-perfluoropolyether triblock copolymer, HFE-7500 fluorinated oil, Squalane oil, silicone oil and mineral oil;

Most preferably, the oil phase solvent is a perfluoropolyether-polyethylene glycol-perfluoropolyether triblock copolymer.
The manufacturing method according to claim 22, wherein

Using a surfactant to stabilize the droplets when generating the droplets, the surfactant is a non-ionic surfactant or an ionic surfactant;

Preferably, the nonionic surfactant is selected from one or more of the following group: PEG-PFPE 2 , PFO, castor oil polyoxyester, polyoxyethylene 40 hydrogenated castor oil, and poloxamer;

Preferably, the ionic surfactant is selected from one or more of the following group: Krytox, sodium lauryl sulfate, and Janus nanoparticles; most preferably, the surfactant is PEG-PFPE 2 .
The manufacturing method according to claim 22, wherein

The adjustment conditions for the curing or semi-curing reaction are the illumination intensity and illumination time of ultraviolet light;

Preferably, the curing or semi-curing reaction uses a catalyst and the catalyst is one or more TEMED initiators selected from the following group: 2-hydroxy-2-methyl-1-phenyl acetone, 1-hydroxycyclohexylbenzene Methyl ketone, 2-methyl-2-(4-morpholinyl)-1-[4-(methylthio)phenyl]-1-propanone, 2,4,6-trimethylbenzoyl- Diphenylphosphine oxide, ethyl 2,4,6-trimethylbenzoylphenylphosphonate, 2-dimethylamino-2-benzyl-1-[4-(4-morpholinyl)phenyl ]-1-Butanone, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone, MBF methyl benzoylformate, benzoin and derivatives Chemical substances (benzoin, benzoin dimethyl ether, benzoin ethyl ether, benzoin isopropyl ether, benzoin butyl ether), benzoyl compounds (diphenyl ethyl ketone, α, α-dimethoxy-α-phenylacetophenone) , Alkylphenones (α, α-diethoxyacetophenone, α-hydroxyalkylphenone, α-aminealkylphenone), acylphosphorus oxide (aroylphosphine oxide, bisbenzyl Acyl phenyl phosphine oxide), benzophenones (benzophenone, 2,4-dihydroxybenzophenone, Michinone), thioxanthone (thiopropoxythioxanthone, Isopropylthioanthrone); diaryliodonium salt, triaryliodonium salt, alkyl iodonium salt, cumene ferrocene hexafluorophosphate;

The selectively permeable membrane forming material is selected from one or more of the following group: polyolefin, olefin copolymer, acrylic, vinyl polymer, polyester, polycarbonate, polyamide, polyimide, Formaldehyde resin, polyurethane, ether polymer, cellulose, thermoplastic elastomer and thermoplastic polyurethane materials;

Preferably, the selectively permeable membrane-forming material is a hydrogel framework material; further preferably, the selectively permeable membrane-forming material is polyethylene glycol diacrylate (PEGDA), and further preferably, the concentration of PEGDA in the second aqueous solvent is 1%-10%,

Most preferably it is PEGDA and the concentration of PEGDA in the second aqueous solvent is 3%.
The manufacturing method according to claim 22,

Wherein, the demulsification refers to using surfactant to shake and mix and then centrifuge at high speed,

Wherein, the surfactant is a nonionic surfactant or an ionic surfactant; the nonionic surfactant is selected from PEG-PFPE 2 , PFO, castor oil polyoxyl ester, polyoxyethylene 40 hydrogenated One or more of castor oil and poloxamer;

The ionic surfactant is selected from one or more of Krytox, sodium lauryl sulfate and Janus nanoparticles;

Most preferably the surfactant is PFO.