WO2023093887A1 - Method for breaking poisson distribution to form reaction compartment group - Google Patents

Method for breaking poisson distribution to form reaction compartment group Download PDF

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WO2023093887A1
WO2023093887A1 PCT/CN2022/134738 CN2022134738W WO2023093887A1 WO 2023093887 A1 WO2023093887 A1 WO 2023093887A1 CN 2022134738 W CN2022134738 W CN 2022134738W WO 2023093887 A1 WO2023093887 A1 WO 2023093887A1
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carrier
microspheres
cells
reaction
cell
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Chinese (zh)
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张经纬
景祥益
梁雪
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复旦大学
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the application relates to the field of high-throughput chemical analysis, in particular to a targeted reaction complex and their use in breaking Poisson distribution to form reaction compartment groups.
  • the heterogeneity of cells is a ubiquitous life phenomenon. As an independent living entity, the properties and differences displayed by a single cell play a vital role in the development of the entire living system. Every tissue and organ in the human body contains a variety of cell types, and each type of cell will change with the life activity of the organism. If thousands of single cells are studied, it will be blurred Therefore, it is extremely important to understand the working principles of complex organisms, the life functions and immune responses of each cell type to reveal the working mechanism of human tissues and organs and the laws of gene regulation.
  • malignant tumors that cause human cancer are highly heterogeneous tissues, composed of tumor cells of various phenotypes, and the real malignant cells are mixed with normal cells, often accounting for only a small part of the entire tissue, so single-cell
  • the analysis can determine which cells are drug-resistant and which cells are easy to transfer, which plays an important role in guiding precise drug use, predicting the course of disease development, and clinical guidance.
  • expression may also vary between single cells.
  • the genome fundamentally determines cell behavior such as transcription or translation
  • gene expression is a random molecular process that is closely related to cellular Growth time and space are related, so that the analysis of the genome cannot accurately reflect the differences in the actual behavior of cells; as the main bearer of life activities, proteins have a direct impact on cell differences, dynamics and functions, but proteins in single cells Horizontal quantitative analysis, protein amplification, and efficient reading of protein sequences are always huge technical barriers; while RNA acts downstream of DNA and upstream of proteins, it has gradually become a powerful tool for indirectly judging gene expression and protein abundance. Analysis of the transcriptome can reveal the heterogeneity and randomness of genetic material and its expression at the single-cell level.
  • the second technical problem is the amplification of micro-contents in single cells, and how to label each cell during sample preparation in order to achieve high-throughput single-cell sequencing, that is, to introduce cell codes; There are deviations in the process. How to label the information of each transcript in a single cell, that is, to introduce molecular codes, and how to integrate cellular codes and molecular codes to achieve accurate quantification of cell contents have become the methodologies of scientific researchers in recent years. focus on innovation. In recent years, researchers have developed a technology to encode microspheres, which can be used for high-throughput labeling of single-cell content information.
  • the third technical challenge is to realize the high-throughput one-to-one pairing of single cells and single microspheres in microwell plates.
  • Fluorescence-activated flow cytometry which is widely used for microparticle sorting, is not suitable for the sorting of encoded microspheres.
  • the cost of encoding microspheres is expensive. Since fluorescence-activated flow cytometry sorting needs to consume a large amount of background microspheres, this method will cause a lot of waste of reagents.
  • the size of the microspheres often does not match the flow sorting consumables.
  • the sorting efficiency is low, and the number of cells and microspheres sorted per unit time is limited. It is impossible to achieve efficient high-throughput single cell and single microsphere one-to-one rapid pairing, which is far from the clinical requirements for the total number of cells. The selected single microspheres are easily broken.
  • the mRNA released by single cell cleavage is captured by the paired coding microspheres, and then reverse transcription and amplification are performed to encode the single cell mRNA information.
  • high-throughput sequencing and bioinformatics methods were used to analyze the expression of large cell mRNAs.
  • the capture of cells and microspheres in this method is based on the principle of Poisson distribution. Most of the droplets have no cells, and only ⁇ 1% of the droplets contain single cells. Combined with the Poisson distribution of microspheres, the effective analysis target is further reduced. Only a small number of cells in a large number of actual samples can be analyzed, which may ignore some important individual cells in the sample. In addition, this strategy is only suitable for samples with a large number of analysis objects.
  • Microfluidic chip is an emerging field that has developed rapidly and matured in recent years. It uses microchannels of various structures and various forms of external force fields to manipulate, process and control microfluids or samples on a microscopic scale, thereby It realizes the integration of some or even all functions of traditional laboratories on one chip.
  • the limitations of conventional microfluidic chips are also very obvious. It needs to design pumps and valves inside the chip to cooperate with external fluid control equipment with complex operations. The technical threshold is high, and it is difficult to reuse a chip.
  • reaction reagents need to consume a lot of chip production costs when performing multi-level and multi-scale analysis of the same sample (Macosko et al., 2015, Cell, 161, 1202-1214; Klein et al., 2015, Cell, 161, 1187- 1201; Han et al., 2018, Cell, 172, 1091–1107).
  • the fourth potential technical problem is that when the previous sequencing methods analyze actual samples, the currently reported sequencing methods based on encoded microspheres need to use fluorescence-activated flow cytometry to sort the target cells first, and then transfer them to each cell.
  • the information of the cell contents will change as the environment of the cells changes, which may lead to deviations between the sequencing information reflected in the final sequencing results and the information in the real environment of the cells at that time.
  • the fifth technical difficulty lies in the separation of rare cells.
  • the number of cells to be analyzed is very rare and the independent transcriptome information of each single cell needs to be analyzed, traditional techniques based on capillary picking, gradient dilution, or laser cutting require labor costs.
  • High, time-consuming, labor-intensive, and low throughput limit the high-throughput rapid separation and sequencing analysis of rare cells.
  • This application mainly focuses on the third type of technical problem above, that is, to solve the problem of one-to-one pairing of single cells and reaction reagents when realizing high-throughput analysis of multiple cells, and at the same time increase the number of droplet pairs between cells and microspheres.
  • the high co-capture rate solves the inefficiency caused by the "double Poisson distribution".
  • a method of forming a population of reaction compartments comprising:
  • the targeting reaction complex group is composed of more than two targeting reaction complexes, the targeting reaction complex includes a carrier, a targeting ligand corresponding to the substance to be detected connected to the carrier base, a reaction reagent corresponding to the substance to be detected connected to the carrier, optionally including a label molecule connected to the carrier corresponding to the substance to be detected;
  • a population of reaction compartments is formed by the conjugate itself or by surrounding the conjugate with a vehicle.
  • analyte is selected from one or more of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles, and cells, and by The complexes they form, most preferably the analytes are cells.
  • the shape of the carrier is selected from one or more of cube shape, tetrahedron shape, spherical shape, ellipsoid shape, octopus shape, bowl shape, red blood cell shape; most preferably bowl shape and/or red blood cells.
  • the carrier is made of a polymer or a small molecule, preferably a polymer carrier, more preferably a polystyrene carrier made of polystyrene or hydrogel , further the polymer carrier is made of polystyrene, most preferably the polymer is ferromagnetic or paramagnetic.
  • the targeting ligand can be natural or artificial, selected from nucleic acids comprising locked nucleic acids and XNA and their analogs, aptamers, small peptides, polypeptides, glycosyl peptides, polysaccharides, soluble receptors, steroids, hormones, mitogens, antigens, superantigens, growth factors, cytokines, leptin, viral proteins, cell adhesion molecules, chemokines, streptavidin, and the like Biotin and its analogues, antibodies, antibody fragments, single chain variable fragments (scFv), nanobodies, T cell receptors, major histocompatibility complex (MHC) molecules, antigenic peptide-MHC molecule complexes One or more of (pMHC), DNA binding protein, RNA binding protein, intracellular or cell surface receptor ligands, and multiple ligands, composite ligands, and coupling ligands formed by them.
  • nucleic acids comprising locked nucleic acids and XNA
  • the label molecule is selected from natural or artificial information molecules, including: oligonucleotide barcodes, oligopeptide or polypeptide barcodes, natural bases and LNA, PNA, XNA, etc. Nucleotides composed of unnatural bases, oligosaccharide or polysaccharide barcodes, chromophoric and auxochrome groups, metal atoms or ions, small molecules with distinguishable molecular weights, block polymers, polymeric One or more than two kinds of covalent linkages between substances and backbone molecules and the complex formed between them.
  • natural or artificial information molecules including: oligonucleotide barcodes, oligopeptide or polypeptide barcodes, natural bases and LNA, PNA, XNA, etc. Nucleotides composed of unnatural bases, oligosaccharide or polysaccharide barcodes, chromophoric and auxochrome groups, metal atoms or ions, small molecules with distinguishable molecular weights, block
  • reaction reagent is an oligonucleotide primer, an enzyme or a small molecule.
  • the surface of the carrier is coated with a mechanical buffer coating; further preferably coated with a hydrogel coating; most preferably the hydrogel coating has a ferromagnetic or paramagnetic particles.
  • connection is selected from covalent bonds, metallic bonds, ionic bonds, van der Waals forces, including hydrogen bonds, mechanical bonds, halogen bonds, chalcogen bonds, amophilic interactions, intercalation, Overlapping, cation– ⁇ bonds, anion– ⁇ bonds, salt bridges, secondary bonds between nonmetal atoms, secondary bonds between metal atoms and nonmetal atoms, aurophilic interactions, argentophilic interactions, double hydrogen bonds, and secondary bonds of gold bonds level key.
  • the targeting ligand is connected to the carrier through the connection of the reaction reagent, the connection of the label molecule or the connection of the linker; the reaction reagent and The connection of the carrier is through the connection of the targeting ligand, the connection of the label molecule or the connection of the linker; the connection of the label molecule and the carrier is through the connection of the targeting ligand, Ligation via the reagent or via a linker.
  • the medium is an oily medium, preferably a fluorinated oily medium, or a solid medium, preferably a microwell plate.
  • the two or more substances to be detected can be the same or not, and the two or more targeting reaction complexes can be the same or not, when the When the two or more substances to be detected are different and the two or more targeting reaction complexes are different, the targeting reaction complex includes label molecules corresponding to the substances to be detected connected to the carrier.
  • the one-to-one pairing problem of the analyte (single cell) and the carrier (single microsphere) is solved while realizing high-throughput analysis of multiple analytes (such as cells), At the same time, the co-capture rate of the medium (droplet) on the cells and microspheres is increased, which solves the problem of inefficiency caused by the "double Poisson distribution" phenomenon that occurs when the analyte is paired with the carrier.
  • erythrocyte-shaped and bowl-shaped carriers such as polystyrene microspheres
  • carriers with a large size and a large number relative to the object to be detected such as cells
  • the steric hindrance effect is used to further ensure that the "single cell and single cell
  • a layer of buffer layer hydrogel layer
  • the targeting complex group used in the present application has at least one reaction reagent, which realizes high-throughput analysis of the substance to be detected.
  • Figure 1 shows a schematic diagram of the composition of the targeting reaction complex according to the present application
  • the targeting reaction complex includes a carrier, a targeting ligand corresponding to the substance to be detected connected to the carrier, and a ligand corresponding to the substance to be detected connected to the carrier.
  • Reaction reagents optionally include label molecules corresponding to the substances to be detected connected to the carrier.
  • Figure 2A shows that the hard microspheres may crush the cells when they are combined with the cells
  • Figure 2B shows that the hydrogel coating on the surface of the hard microspheres has a protective effect on the cells; Hydrogel-coated targeting-response complexes (DNA-encoded microspheres coated with hydrogel coatings).
  • Figure 3A shows the hydrogel encapsulation by pipetting the microspheres
  • Figure 3B shows the image of the hydrogel after gel formation and demulsification.
  • Figure 4A shows that the diameter of the microsphere is larger than that of the cell, and the steric hindrance effect helps the combination of the microsphere and the cell at 1:1; it is difficult for a cell to combine with two or more microspheres;
  • Figure 4B shows that the diameter of the microsphere is smaller than that of the cell, and the formation of Multiple microspheres bound to one cell, or aggregates of cells and microspheres.
  • Figure 5A shows the preparation of hydrogel-coated magnetic microspheres
  • Figure 5B shows the magnetic purification after demulsification
  • Figure 5C shows the hydrogel-coated magnetic microspheres, and magnetic purification after demulsification
  • the antibody modified on the surface of the hydrogel layer after activation can be observed (the outer layer is bright white);
  • the white arrow refers to the formation of the antibody-modified hydrogel-coated microspheres through incubation with cells1: 1 combined.
  • Figure 6A-C shows the bowl-shaped carrier, and the steric hindrance effect helps the microspheres to combine with the cells at 1:1; it is difficult for a cell to combine with two or more bowl-shaped or red blood cell-shaped microspheres; The generation of bowl-shaped aqueous two-phase and ultraviolet crosslinking are carried out here;
  • Figure 6B shows the bowl-shaped particles;
  • Figure 6C shows the 1:1 combination of bowl-shaped particles and cells.
  • Figure 7A shows the finished hydrogel microspheres prepared by microfluidic equipment
  • Figure 7B shows that in the hydrogel microspheres, the label molecule and polyT are on the same reverse transcription primer
  • Figure 7C shows the hydrogel microspheres aggregate in droplets.
  • Figure 8A shows modified microspheres with anti-human-CD298, human ⁇ 2 microglobulin, mouse CD45, and mouse MHC class I antibodies, which can be used as universal binding microspheres to bind cells;
  • Figure 8B shows the water The gel microspheres bind to the cells to form a 1:1 complex.
  • Figure 9A is a flow chart of the entire high-throughput analysis process (targeted reaction complexes are pre-combined with analytes and then packaged into high-throughput compartments);
  • Figure 9B when the number of microspheres is 10 times or more than that of cells , can form a 1:1 combination, and there is a probability of 1/100 to observe that one cell binds to two microspheres; Poisson distribution improves the capture rate of cells;
  • Figure 9D shows that in some cases, the target reaction complex and the bound cells will be separated in the compartment;
  • Figure 9E shows that the analyte is a pair of cells interacting with each other.
  • Figure 10A-C shows the process of controlling the water/oil liquid in the microfluidics to form a water-in-oil compartment through a syringe pump; where Figure 10B shows the high-throughput formation of a microfluidic device; Figure 10C shows a microsphere The complex with cells enters the microfluidic intersection, and high-throughput droplets are generated; Figure 10D shows the results of single-cell analysis; shows the results of single-cell analysis, and human and mouse cell subpopulations can be separated.
  • Figure 11A shows the following steps: the first step of hydrogel encapsulation of microspheres, and cross-linked streptavidin; the second step of cell biotinylation; the third step, the biotinylated cells with streptavidin Incubation and binding of avidin; the fourth step, high-throughput packaging of the complexes of microspheres and cells to form compartments, release of reaction reagents in the compartments, and labeling of cells in the compartments;
  • Figure 11B shows the Gel-encapsulated target-tagged beads modified with fluorescent streptavidin;
  • Figure 11C shows biotinylated cells;
  • Figure 11D shows hydrogel beads bound to cells;
  • Figure 11E shows manual pipette tips
  • the compartments are formed outside the hydrogel-wrapped microspheres by pipetting, and the cells in the compartments are lysed to release the mRNA therein.
  • substantially free with respect to a particular component is used herein to mean that the particular component has not been purposefully formulated into the composition and/or is present only as a contaminant or in trace amounts.
  • the total amount of specific components resulting from any accidental contamination of the composition is below 0.05%, preferably below 0.01%.
  • Most preferred are compositions wherein the amount of a particular component is undetectable by standard analytical methods.
  • the present application provides methods of forming a population of reaction compartments.
  • a method of forming a population of reaction compartments comprising:
  • the targeting reaction complex group is composed of more than two targeting reaction complexes, the targeting reaction complex includes a carrier, a targeting ligand corresponding to the substance to be detected connected to the carrier base, a reaction reagent corresponding to the substance to be detected connected to the carrier, optionally including a label molecule connected to the carrier corresponding to the substance to be detected;
  • a population of reaction compartments is formed by the conjugate itself or by surrounding the conjugate with a vehicle.
  • compartment means providing a specific reaction space for a specific chemical reaction in which reactants can react with each other without reacting with reactants in adjacent compartments, however, Depending on the type, the product of the compartment may exchange substances with the product of the adjacent compartment (for example: dyes, surfactants, etc.).
  • targeting complies with the general definition in the field of biotechnology, typically antibody-antigen affinity reaction, affinity reaction of complementary nucleic acid sequences.
  • connection should be understood in a broad sense, at least including various connections through molecular bonds and without molecular bonds.
  • Figure 1 shows the components of the targeting reaction complex, where the structural relationship of targeting ligands, label molecules and reaction reagents respectively connected to the “carrier” is shown.
  • the "carrier” can be composed of the same molecules as the targeting ligand, label and reaction reagent, that is, it can be composed of small molecules. More usually, the carrier consists of a polymer, preferably polystyrene.
  • a method for forming a reaction compartment population wherein the analyte is selected from the group consisting of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles, and cells One or more than two, and complexes formed by them, most preferably the analytes are cells.
  • cell conforms to the general definition in the field of biology, which includes at least prokaryotic cells and eukaryotic cells.
  • prokaryotic cells include at least prokaryotic cells and eukaryotic cells.
  • eukaryotic cells include at least prokaryotic cells and eukaryotic cells.
  • immune cell populations include at least lymphoid B cells, lymphoid T cells, and NK cells.
  • organelle conforms to the general definition in the field of biology, and organelle is generally considered to be a microstructure or micro-organ scattered in the cytoplasm with a certain shape and function. They make up the basic structure of cells and enable cells to work and run normally.
  • the organelles in the cell mainly include: mitochondria, endoplasmic reticulum, centrosome, chloroplast, Golgi apparatus, ribosome, etc.
  • complex refers to two or more substances that interact with each other and have a certain binding strength. These substances can be composed of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria , viruses, organelles, and cells. For example: after incubation of HepG2 with T cells that recognize the cell line (genetically modified to carry the T cell receptor gene targeting the "major histocompatibility complex-AFP158 peptide"), cell-to-cell interactions will form; Specific examples are: complexes of bacteria and bacteriophages (viruses), complexes of cells and membrane proteins (polypeptides), and complexes of ribosomes and RNA (nucleic acids).
  • a method for forming a group of reaction compartments wherein the shape of the carrier is selected from one of cube shape, tetrahedron shape, sphere shape, ellipsoid shape, octopus shape, bowl shape, and red blood spheroid shape Or two or more; most preferably bowl-shaped and/or red blood cell-shaped.
  • “Bowl shape” specifically refers to a concave spherical shape, which is provided with a concave surface on the plane side of the "hemisphere".
  • the concave spherical shape specifically refers to a spherical circular plane with a concave surface.
  • the "red blood cell” carrier has two concave surfaces.
  • the substance to be detected is bound to the upper concave surface or the lower concave surface of the carrier, the substance to be detected will not bind to another carrier due to steric hindrance.
  • a method for forming a group of reaction compartments wherein the amount of the carrier is more than 10 times the amount of the substance to be detected, most preferably 10 times the amount of the substance to be detected times to 50 times.
  • the quantity of the carrier can be 10 times, 12 times, 14 times, 16 times, 18 times, 20 times, 22 times, 24 times, 26 times, 28 times, 30 times, 32x, 34x, 36x, 38x, 40x, 42x, 44x, 46x, 48x or 50x. It is desirable to control the amount of analyte interacting with the aforementioned targeted reaction complex or population of aforementioned reaction complexes and the assay process so that only one analyte per conjugate is analyzed. In the embodiment of the present application, specifically, when the number of microspheres carrying the reaction reagent is 10-20 times the number of analyzed cells, each microsphere has a high probability of binding to only one cell according to the Poisson distribution.
  • Poisson distribution is to describe the number of microspheres or cells randomly appearing in a unit space
  • double Poisson distribution is to describe the number of microspheres and cells randomly appearing in a unit space quantity.
  • breaking the Poisson distribution if there is a phenomenon that the number of microspheres and cells in a unit space is greater than the double Poisson distribution, it can be considered as "breaking the Poisson distribution".
  • a method for forming a group of reaction compartments wherein the maximum diameter of the carrier is more than 2 times that of the substance to be detected, and most preferably its maximum diameter is 3 times to 10 times that of the substance to be detected. times.
  • the maximum diameter of the carrier may be 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times that of the substance to be detected.
  • "diameter” specifically refers to the distance between the center of a plane figure or a solid (such as a circle, a conical section, a sphere, a cube) and two points on an edge).
  • the 1:1 combination of the carrier and the analyte is realized to the greatest extent.
  • the 1:1 combination of the carrier and the substance to be detected is realized to the greatest extent.
  • 10 times, 12 times, 14 times, 16 times, 18 times, 20 times, 22 times, 24 times, 26 times more than the substance to be detected can be used while using the "bowl-shaped" or "red blood cell”-shaped carrier.
  • Simultaneous use of 3 to 10 times the carrier size of the substance to be detected for example, 10 times, 12 times, 14 times, 16 times, 18 times, 20 times, 22 times, 24 times, 26 times, 28 times more than the substance to be detected , 30 times, 32 times, 34 times, 36 times, 38 times, 40 times, 42 times, 44 times, 46 times, 48 times or 50 times the number of carriers;
  • a method for forming a population of reaction compartments wherein the targeting ligand can be natural or artificial, selected from nucleic acids including locked nucleic acids and XNA and their analogs, adapters 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) Molecule, antigenic peptide-MHC molecular complex (pMHC), DNA-binding protein, RNA-binding protein, one or more than two of intracellular or cell surface receptor ligands, and multiple ligands and complex ligands
  • MHC major histocomp
  • an antibody conforms to the general definition in the field of biology. Specifically, an antibody refers to a protective protein produced by the body due to stimulation by an antigen. It is a large Y-shaped protein secreted by plasma cells (effector B cells) and used by the immune system to identify and neutralize foreign substances such as bacteria and viruses. It is only found in body fluids such as blood in vertebrates, and The cell membrane surface of its B cells. Antibodies recognize a unique feature of a particular foreign object, called an antigen. In the technical solution of the present application, for example, surface receptors of lymphocytes can be used as antigens to prepare corresponding antibodies, which are chemically modified on the surface of microspheres to separate lymphocyte populations.
  • aptamer conforms to the general definition in the field of biology, and in the technical solution of this application, it may specifically refer to nucleic acid aptamers.
  • Aptamer is a DNA (deoxyribonucleic acid), RNA (ribonucleic acid) sequence, XNA (nucleic acid analog) or peptide.
  • XNA nucleic acid analog
  • Nucleic acid aptamers can bind to a variety of target substances with high specificity and high selectivity, so they are widely used in the field of biosensors.
  • the configuration of the nucleic acid aptamer itself will change accordingly.
  • the analyte is a nucleic acid fragment
  • its nucleic acid aptamer can be selected and chemically modified and fixed on the surface of the microsphere to separate cells.
  • a method for forming a group of reaction compartments wherein the tag molecules are selected from natural or artificial information molecules, including: oligonucleotide barcodes, oligopeptide or polypeptide barcodes, by Nucleotides, oligosaccharide or polysaccharide barcodes, chromophoric groups and auxochrome groups, metal atoms or ions, and molecular weights composed of natural bases and LNA, PNA, XNA and other unnatural bases can be distinguished One or more of the small molecules, block polymers, covalent links between polymers and backbone molecules, and complexes formed between them.
  • natural or artificial information molecules including: oligonucleotide barcodes, oligopeptide or polypeptide barcodes, by Nucleotides, oligosaccharide or polysaccharide barcodes, chromophoric groups and auxochrome groups, metal atoms or ions, and molecular weights composed of natural bases and LNA, PNA,
  • DNA barcode refers to a standard, sufficiently variable, easily amplified and relatively short DNA fragment that can represent the species in an organism.
  • DNA barcoding has become an important tool in ecological research, not only for species identification, but also to help biologists further understand the interactions that occur within ecosystems.
  • the researchers profile the DNA barcode of its tissue and compare it to other barcodes in an international database. A match with one of these would allow the researchers to confirm the species' identity.
  • DNA barcoding technology is an emerging technology for rapid and accurate identification of species by using a conserved fragment in the DNA of organisms.
  • the "analysis" is the transcriptome sequencing of immune cell populations
  • the use of DNA barcodes can deconvolute the analysis results after obtaining high-throughput analysis results of immune cell populations change.
  • a method of forming a population of reaction compartments wherein the reaction reagent is an oligonucleotide primer, an enzyme or a small molecule.
  • a reagent is a substance that reacts with an analyte and produces a signal that can be used for detection.
  • "reagent” may sometimes specifically refer to a fluorescent reagent capable of causing the analyte to emit fluorescence.
  • primer conforms to the general definition in the field of biotechnology, specifically, a primer refers to a macromolecule with a specific nucleotide sequence that stimulates synthesis at the initiation of nucleotide polymerization, A molecule that is hydrogen bonded to a reactant is called a primer.
  • Primers are usually two artificially synthesized oligonucleotide sequences, one primer is complementary to a DNA template strand at one end of the target region, and the other primer is complementary to the other DNA template strand at the other end of the target region, and its function is to act as a nucleotide
  • nucleic acid polymerase can synthesize a new nucleic acid chain from its 3-end.
  • primers are widely used in polymerase chain reaction, sequencing and probe synthesis, etc.
  • oligonucleotide primers can be used as "reaction reagents".
  • small molecules constituting reaction reagents can be various small molecules in the field of chemical technology.
  • the “analysis” is fluorescence detection, in particular, the “small molecules” can emit fluorescence Small molecules such as FAM, VIC, etc.
  • a method of forming a population of reaction compartments wherein said carrier diameter has a particle size distribution coefficient CV of less than 20%.
  • CV SD/average particle diameter, which can represent the width of the particle size distribution.
  • SD is the standard deviation (Standard Deviation), which is recorded as ⁇ in statistics
  • CV is also called relative standard deviation (Coefficient of variation), and is recorded as ⁇ in statistics.
  • the diameter of the microspheres used can be calculated by means and devices known to those skilled in the art, for example, it can be measured under a microscope and analyzed with image processing software, and can be measured by an instrument for measuring particle size (Bio-Rad T20 Cytometer) to determine, can also use the data provided by the microsphere supplier.
  • a method of forming a population of reaction compartments wherein the surface of the support is coated with a mechanical buffer coating; most preferably with a hydrogel coating.
  • the “mechanical buffer coating” should be regarded as a part of the “carrier”, that is, the “targeting ligand”, “reactive reagent” and “label molecule” described in this specification are all on the carrier.
  • the “mechanical buffer coating” is coated on the (non-coated mother core) before being connected to the carrier by, for example, chemical modification.
  • the tag molecule is first linked to the carrier, then coated with a mechanical buffer coating, and finally the reaction reagent and the targeting ligand are linked outside the mechanical buffer coating by, for example, chemical modification.
  • reaction reagent is first connected to the carrier, then coated with a mechanical buffer coating, and finally the label molecule and the targeting ligand are connected outside the mechanical buffer coating by, for example, chemical modification.
  • the targeting ligand is first linked to the carrier, then a mechanical buffer coating is applied, and finally the reaction reagent and the label molecule are linked outside the mechanical buffer coating by, for example, chemical modification.
  • the label molecule and the reaction reagent are first linked to the carrier, then coated with a mechanical buffer coating, and finally the targeting ligand is linked outside the mechanical buffer coating by, for example, chemical modification.
  • the label molecule and the targeting ligand are first linked to the carrier, then coated with a mechanical buffer coating, and finally the reaction reagent is linked outside the mechanical buffer coating through, for example, chemical modification.
  • reaction reagent and the targeting ligand are linked to the carrier first, then a mechanical buffer coating is applied, and finally the tag molecule is linked outside the mechanical buffer coating by, for example, chemical modification.
  • the label molecule, targeting ligand and label molecule are first linked to a carrier, and then a mechanical buffer coating is applied.
  • a method of forming a population of reaction compartments wherein the linkages are selected from covalent bonds, metallic bonds, ionic bonds, van der Waals forces, including hydrogen bonds, mechanical bonds, halogen bonds, sulfur Family bonds, aurophilic interactions, intercalation, overlapping, cation- ⁇ bonds, anion- ⁇ bonds, salt bridges, secondary bonds between non-metal atoms, secondary bonds between metal atoms and non-metal atoms, aurophilic interactions, argentophilic interactions , double hydrogen bonds and secondary bonds of gold bonds.
  • a method for forming a group of reaction compartments wherein the linking of the targeting ligand to the carrier is through the linking of the reaction reagent, the linking of the tag molecule or the linking of the labeling molecule.
  • a method for forming a group of reaction compartments wherein the small molecule is one or more of the targeting ligand, the reaction reagent, and the label molecule. That is, the targeting ligand, the reaction reagent, and the tag molecule themselves can form a part of the carrier.
  • a method for forming a group of reaction compartments wherein the medium is an oily medium, preferably a fluorinated oily medium, or a solid medium, preferably a microwell plate.
  • the oily medium can effectively block charged nucleic acid molecules from crossing different "compartments”. After marking, there will be no cross-contamination of products passing through the "compartment”.
  • the two or more substances to be detected can be the same or different,
  • the two or more targeting reaction complexes can be the same or not
  • the targeting reaction complex includes label molecules corresponding to the substances to be detected connected to the carrier.
  • the two or more substances to be detected are the same, and the two or more targeted reaction complexes are the same.
  • the two or more substances to be detected are the same, and the two or more target reaction complexes are different.
  • the two or more substances to be detected are different, and the two or more targeted reaction complexes are the same.
  • the two or more substances to be detected are different, and the two or more targeting reaction complexes are different; and the targeting The reaction complex includes a label molecule corresponding to the detection substance connected to the carrier.
  • Example 1 Preparation of a carrier (microsphere wrapped with a hydrogel layer) with a mechanical buffer coating
  • Polystyrene microspheres (Nanomicro Technology Uni-PS-30) can be customized by Chemgenes Corp to form microspheres with DNA coded (label) reverse transcription polyT primers (reaction reagents).
  • TEBST buffer (20mM Tris-HCl pH 8.0, 274mM NaCl, 5.4mM KCl, 20mM EDTA, 0.2% Triton X-100), mix well.
  • the targeting ligand, label or reaction reagent can be loaded in advance or afterwards.
  • EDC 1-ethyl-(3-dimethylaminopropyl) carbodiimide
  • NHS N-hydroxysuccinimide
  • Count cells 8.0*10 ⁇ 5 cells and incubate with antibody-modified (targeting ligand) hydrogel-coated microspheres in 200 ⁇ L PBS buffer and 2 mM EDTA, rotate the solution for 30 minutes
  • Embodiment 2 (two) The impact of carrier size on the combination of carrier and cell (as a comparative example)
  • This example is a comparative example, and the purpose of the experiment is to verify that if the size of the microsphere is too small, it will affect the combination with the cells. When the number is greater than the number of cells, a phenomenon in which multiple microspheres combine with one cell will be formed.
  • Example 3 Preparation of carrier (microsphere) with separation function (magnetic nanoparticle layer) and binding experiment with cells
  • Example 2 Prepare hydrogel-wrapped microspheres with a magnetic inner core by the method of Example 2 (Fig. 5A-C): the microsphere structure is a polystyrene inner core, coated with magnetic nanoparticles, and the outer layer is polypropylene with carboxyl groups Amide hydrogel layer (see Example 2 for PAA solution formulation and polymerization method)
  • Example 3 The influence of the shape of the carrier on the combination of the carrier and the cells
  • the purpose of this experiment is to prove that the bowl-shaped microspheres can bind to cells and break the Poisson distribution law by using the steric hindrance effect.
  • Step 1 Fabrication of bowl-shaped microspheres (carrier)
  • the dispersed phase will contain 6.3% w/v biotinylated polyethylene glycol, 4.5% w/v gelatin and 1.5% w/v lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)
  • a homogeneous precursor solution dissolved in DPBS was injected parallelized and emulsified step by step at a rate of 8 ⁇ L/min.
  • Novec 7500 containing 2% Pico-Surf was injected into the continuous phase at 16 ⁇ L/min.
  • Single-phase generated PEG/gelatin droplets flowed through Tygon tubes (0.03" ID, 0.0625" OD, Murdock) immersed in a 4°C water bath for temperature-induced phase separation of PEG and gelatin.
  • phase-separated droplet stream was introduced into a PDMS reservoir submerged in a water bath at 4 °C and exposed to UV light (200 mW/cm 2 ) for 1–3 seconds near the outlet region of the reservoir for polymerization.
  • UV light 200 mW/cm 2
  • the photocrosslinkable PEG component formed a polymer network, while the gelatin component remained unpolymerized (see Figure 6A for details).
  • the cross-linked particles are collected and oil and gelatin-rich droplets are removed in a series of washing steps. First remove the surfactant oil by pipetting.
  • Step 2 Binding of bowl-shaped microspheres to cells
  • Bowl-shaped microspheres were modified with AF350 streptavidin (Fisher Scientific) (targeting ligand) prior to use.
  • the bowl is washed with cell culture medium and concentrated in a conical tube.
  • the amount of concentrated bowl-shaped microspheres calculated to cover the entire pore area with a monolayer of bowl-shaped particles is 4.8 ⁇ L/cm 2 for 55 ⁇ m bowl-shaped microspheres, respectively. Transfer and disperse the calculated amount of bowl-shaped particles in the orifice plate.
  • the wells with a monolayer of bowl-shaped microspheres were seeded with Jurkat cells at a ratio of 1:1 between target cells and bowl-shaped microspheres.
  • the carrier (microsphere) carries a variety of targeting ligands: anti-human-CD298, human ⁇ 2 microglobulin, mouse CD45 and mouse MHC class I antibody.
  • microspheres contain DNA-encoded polyT capture oligonucleotides (reaction reagents and labels). It is coated with a carboxyl-containing hydrogel coating by droplet microfluidics, and the microspheres are counted in 500 ⁇ L MES buffer 8.0*10 ⁇ 5 microspheres
  • PAA polyacrylamide
  • TEBST buffer (20mM Tris-HCl pH 8.0, 274mM NaCl, 5.4mM KCl, 20mM EDTA, 0.2% Triton X-100), mix well.
  • Example 6 The reaction reagents and label molecules carried by the prepared microspheres can be combined into one substance (that is, the 5'-Acrydite-CTA CAC GAC GCT CTT CCG ATC T NNNNT 28 VN primer has dual functions of label molecules and reaction reagents) ;
  • V represents a mixture of G, C, and A;
  • N represents a mixture of A, G, C, and T.
  • one substance has two functions (label molecule and reaction reagent).
  • Example 7 Get the hydrogel microspheres prepared in Example 7 (the carboxyl group comes from the acrylic acid monomer), and count 8.0*10 ⁇ 5 microspheres in 500 ⁇ L of MES buffer
  • Count cells 8.0*10 ⁇ 5 PBMC cells Incubate with beads in 200 ⁇ L PBS buffer containing 2mM EDTA, swirl the solution for 30 minutes
  • Example 8 High-throughput formation of compartments and single-cell RNA sequencing (using the microspheres combined with human and mouse universal antibodies in Example 4)
  • the microspheres (Fig. 2C) and cell complexes were encapsulated with a vehicle (fluorine-containing oily medium) through a droplet microfluidic device (Fig. 10A).
  • a droplet microfluidic device Fig. 10A
  • the complex of single cells and microspheres from Example 4 was encapsulated into droplets using a custom-designed microfluidic device.
  • the device connects two aqueous phases before they separate into discrete droplets. Laminar flow prevents mixing of two aqueous input phases prior to droplet formation, one flow phase containing lysate and other buffers, and the other flow phase containing cells in complex with DNA-encoded microspheres suspended in buffer.
  • Droplet reaction compartments were generated using a microfluidic chip using a 90 ⁇ m height and 80 ⁇ m wide nozzle. Typical flow rates used are: ⁇ 200 ⁇ L/h for lysate-rich phase, ⁇ 100 ⁇ L/h for cell-enriched phase complexed with DNA-encoded microspheres and ⁇ 600 ⁇ L/h for droplet stabilization oil (2% PEG-PFPE 2 HFE7500). h (FIG. 10B).
  • the amplified library was sent to a third-party sequencing service company for sequencing.
  • Applicants use bioinformatics tools such as Seurat to process raw sequencing results, and cells with low overall expression or high mitochondrial ratio are removed.
  • bioinformatics tools such as Seurat to process raw sequencing results, and cells with low overall expression or high mitochondrial ratio are removed.
  • clustering principal component analysis was used, followed by k-means clustering to identify distinct cell states.
  • t-Distributed Stochastic Neighborhood Embedding (t-SNE) was used to visualize cell clusters.
  • t-SNE t-Distributed Stochastic Neighborhood Embedding
  • meta-clustering expression matrices from all hosts were pooled from four experiments using Seurat. Cell transcripts with fewer than 2000 cells were excluded from the analysis, and cells with fewer than 100 unique genes detected were excluded from the analysis (Fig. 10D).
  • Streptavidin is attached to the hydrogel coating of the microspheres to bind to the cells with surface-modified biotin.
  • the experimental process is shown in Figure 11A:
  • EDC (10mg) and NHS (5mg) were dissolved in 200 ⁇ l MES buffer respectively, and the stock solution was added to the washed microspheres to reach the final concentration
  • the particles were washed 4 times and stored in PBST at 4°C for long-term storage (Figure 11B).
  • the NeutrAvidin on the microspheres is a targeting ligand, which can be targetedly combined with downstream cells modified with biotin.
  • Step 2 Biotinylation of 293T cells (operate at 4°C throughout the process)
  • Mammalian cells were stained with Calcein AM (ThermoFisher #C3099) in 1 mL PBS containing 0.04% BSA.
  • Sulfo-NHS-SS-Biotin Put 1mg into a microcupule and add 164 ⁇ L of ultrapure water.
  • the reaction mixture was incubated at 4°C for 30 minutes.
  • Step 3 Streptavidinized microspheres capture 293T cells
  • the total solution volume is ⁇ 1mL. Prior to imaging, the solution was gently vortexed to remove non-adherent cells from the pellet.
  • Step 4 Encapsulate the hydrogel-encapsulated microspheres that have captured the cells.
  • the aqueous phase contained 29U/mL proteinase K (NEB #P8107S) and 70mM DTT (Sigma #D9779) and mixed 10 pipette strokes. Take care to avoid creating air bubbles when mixing the cell-bound microspheres with the solution. Add 280 ⁇ L of 0.5% ionic Krytox's HFE 7500 oil 65 to the cell-bead complex and vortex horizontally at 3000RPM for 15 s, then vertically for 2 min using a custom vortexer. Remove oil from beneath the emulsion such that less than 100 ⁇ L of oil remains.
  • the microsphere-cell emulsion was subsampled on a C-Chip disposable hemocytometer (Fisher Scientific #DHCN015), with each subsample containing 3.5 ⁇ L of emulsion per field. Chips were imaged in brightfield at 2X magnification. The remaining emulsion was enzymatically cleaved on a PCR thermocycler (Eppendorf Mastercycler Pro) at 65°C for 35 minutes with the lid temperature set at 105°C. After lysis was complete, fluorescence images were captured using a Nikon 2000 microscope (Thorlab M470L5) with 470 nm excitation light (FIG. 11E).
  • Applying the targeted reaction complex of the present application can realize single-cell analysis, and improve the efficiency of cell analysis while breaking the Poisson distribution.
  • the process involves two key steps: 1.
  • the analyte represented by cells is incubated with the target reaction complex, and is effectively combined with the target ligand; 2.
  • Tag molecules on the targeting reaction complex can be used to label the analytes during high-throughput reactions within the compartment.
  • Other common reactants can enter the reaction compartment through the mixed solution.
  • the embodiment demonstrates that the size of spherical microspheres must be larger than that of cells to break the probability of cell binding more efficiently (double Poisson distribution).
  • the number of microspheres is 10-20 times the number of cells, the 1 :1 combination (see Figure 8A and Figure 8B for details); and when bowl-shaped hydrogel microspheres are used, the steric hindrance effect is more significant, and the number of microspheres does not need to be too much in excess of the number of cells.
  • the high-throughput generation of the reaction compartment (the picture after generating the compartment should be the most important: 9A is the most important concept map; 5E, 8B, 6C, and 9B are the reaction compartments Before the formation; 9C is after the formation of the reaction compartment, as shown in 9C: no reaction compartment was found in the droplet, there were only cells, but no microspheres; it meant that all the cells were analyzed, which meant that the moorings were broken Loose distribution) of cells for various depth analysis including cell lysis, mRNA capture or single cell sequencing.

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Abstract

Provided is a method for breaking a Poisson distribution rule to form a reaction compartment group. The method comprises: preparing a to-be-detected object group, which consists of more than two to-be-detected objects; preparing a targeted reaction compound group, which consists of more than two targeted reaction compounds. The targeted reaction compounds comprise carriers, target ligands which are connected to the carriers and correspond to the to-be-detected objects and reaction reagents which are connected to the carriers and correspond to the to-be-detected objects, and optionally comprise tag molecules which are connected to the carriers and correspond to the to-be-detected objects.

Description

一种打破泊松分布形成反应隔室群的方法A Method of Breaking Poisson Distribution to Form Reacting Compartment Clusters 技术领域technical field
本申请涉及高通量化学分析领域，具体涉及一种靶向反应复合物以及它们在打破泊松分布形成反应隔室群的用途。The application relates to the field of high-throughput chemical analysis, in particular to a targeted reaction complex and their use in breaking Poisson distribution to form reaction compartment groups.
背景技术Background technique
细胞的异质性是普遍存在的生命现象，单细胞作为独立活动的生命个体，所展现出来的性质和差异性对于整个生命***的发展起到了至关重要的作用。人体内的每一个组织、器官都涵盖多种细胞类型，并且每一种类型的细胞随着生物体生命活动状态的不同而会发生改变，如果将成千上万个单细胞进行研究，就会模糊细胞之间的异质性信息，因此理解复杂的机体的工作原理、了解每一种细胞类型的生命功能和免疫应答对于揭示人体组织、器官工作的机理和基因调控的规律极为重要。例如导致人体患癌的恶性肿瘤为高度异质性的组织，由多种表型的肿瘤细胞组成，而真正的恶性细胞与正常细胞混杂，往往只占整个组织的一小部分，因此进行单细胞的分析，可以判断哪些细胞具有抗药性、哪些细胞易于转移，在指导精准用药、预测病程发展和临床指导等领域有重要的作用。The heterogeneity of cells is a ubiquitous life phenomenon. As an independent living entity, the properties and differences displayed by a single cell play a vital role in the development of the entire living system. Every tissue and organ in the human body contains a variety of cell types, and each type of cell will change with the life activity of the organism. If thousands of single cells are studied, it will be blurred Therefore, it is extremely important to understand the working principles of complex organisms, the life functions and immune responses of each cell type to reveal the working mechanism of human tissues and organs and the laws of gene regulation. For example, malignant tumors that cause human cancer are highly heterogeneous tissues, composed of tumor cells of various phenotypes, and the real malignant cells are mixed with normal cells, often accounting for only a small part of the entire tissue, so single-cell The analysis can determine which cells are drug-resistant and which cells are easy to transfer, which plays an important role in guiding precise drug use, predicting the course of disease development, and clinical guidance.
在看似同质的细胞亚群中，单细胞之间的表达也可能不尽相同，基因组虽从根本上决定了细胞的行为如转录或翻译，但基因表达是一个随机的分子进程，与细胞生长时间、空间均有关，使基因组的分析并不能准确反映细胞间实际行为的差异性；蛋白质作为生命活动的主要承担者，对细胞差异性、动力学以及功能直接产生影响，但蛋白质在单细胞水平的定量分析、蛋白质扩增以及蛋白质序列的高效读取始终是巨大的技术壁垒；而RNA作用于DNA的下游、蛋白质上游，已经逐渐成为间接判断基因表达情况及蛋白质丰度的有力工具，因此对转录组进行分析，可以揭示单细胞水平上遗传物质及其表达的异质性和随机性。In seemingly homogeneous cell subpopulations, expression may also vary between single cells. Although the genome fundamentally determines cell behavior such as transcription or translation, gene expression is a random molecular process that is closely related to cellular Growth time and space are related, so that the analysis of the genome cannot accurately reflect the differences in the actual behavior of cells; as the main bearer of life activities, proteins have a direct impact on cell differences, dynamics and functions, but proteins in single cells Horizontal quantitative analysis, protein amplification, and efficient reading of protein sequences are always huge technical barriers; while RNA acts downstream of DNA and upstream of proteins, it has gradually become a powerful tool for indirectly judging gene expression and protein abundance. Analysis of the transcriptome can reveal the heterogeneity and randomness of genetic material and its expression at the single-cell level.
发明内容Contents of the invention
目前高通量单细胞测序技术存在诸多技术难题：其一是高通量单细胞的分离，应用最广的高通量单细胞分离方法主要通过荧光激活细胞流式分选术来完成，荧光激活流式分选术可以同时通过多光谱通道扫描上千个细胞，通量高、速度快，可精确定位单细胞 分选的位置；可以进行特异与非特异性细胞的分选从而获得所需的细胞亚群，还可以实现多参数分析，在实际样品的分析中具有极大的优势，因此利用荧光激活流式分选对单细胞进行分离，使单细胞分隔在96或384微孔板中进行后续分析是目前在单细胞分析领域应用最广的技术之一(Jaitin,et al.,Science,343.776-779；Ba gnoli,et al.,Nature communications,9.2937.)At present, there are many technical problems in high-throughput single-cell sequencing technology: one is the separation of high-throughput single cells. The most widely used high-throughput single-cell separation method is mainly completed by fluorescence-activated cell flow sorting. Flow cytometry can scan thousands of cells through multispectral channels at the same time, with high throughput and fast speed, and can accurately locate the position of single cell sorting; it can sort specific and non-specific cells to obtain the desired cells Subgroups can also achieve multi-parameter analysis, which has great advantages in the analysis of actual samples. Therefore, fluorescence-activated flow sorting is used to separate single cells, so that single cells can be separated in 96 or 384 microwell plates for subsequent analysis. Analysis is currently one of the most widely used techniques in the field of single-cell analysis (Jaitin, et al., Science, 343.776-779; Bagnoli, et al., Nature communications, 9.2937.)
第二个技术难题是单细胞内微量内含物的扩增，以及为实现高通量的单细胞测序，如何在样品制备过程中对每个细胞进行标记，即引入细胞编码；并且由于扩增过程中存在偏差，如何对单细胞内每个转录本信息进行标记，即引入分子编码，如何将细胞编码与分子编码进行整合，实现细胞内含物的准确定量是科研工作者近几年来方法学上创新的重点。近年来，科研工作者发展了一种编码微球的技术，可用于高通量单细胞内含物信息的标记。The second technical problem is the amplification of micro-contents in single cells, and how to label each cell during sample preparation in order to achieve high-throughput single-cell sequencing, that is, to introduce cell codes; There are deviations in the process. How to label the information of each transcript in a single cell, that is, to introduce molecular codes, and how to integrate cellular codes and molecular codes to achieve accurate quantification of cell contents have become the methodologies of scientific researchers in recent years. focus on innovation. In recent years, researchers have developed a technology to encode microspheres, which can be used for high-throughput labeling of single-cell content information.
第三个技术难题在于实现微孔板内单细胞与单微球的高通量一对一配对，目前广泛用于微粒分选的荧光激活流式分选不适用于编码微球的分选，一是编码微球成本昂贵，由于荧光激活流式分选需要消耗大量的背景微球，这种方式会造成大量试剂浪费，二是微球大小往往与流式分选的耗材不匹配，三是分选效率低，单位时间内细胞与微球分选数量有限，无法实现高效的高通量单细胞与单微球一对一快速配对，距离临床对细胞总数的要求相去甚远，四是分选出的单微球容易破碎。The third technical challenge is to realize the high-throughput one-to-one pairing of single cells and single microspheres in microwell plates. Fluorescence-activated flow cytometry, which is widely used for microparticle sorting, is not suitable for the sorting of encoded microspheres. First, the cost of encoding microspheres is expensive. Since fluorescence-activated flow cytometry sorting needs to consume a large amount of background microspheres, this method will cause a lot of waste of reagents. Second, the size of the microspheres often does not match the flow sorting consumables. Third, The sorting efficiency is low, and the number of cells and microspheres sorted per unit time is limited. It is impossible to achieve efficient high-throughput single cell and single microsphere one-to-one rapid pairing, which is far from the clinical requirements for the total number of cells. The selected single microspheres are easily broken.
目前尚无能够将高通量捕获单细胞、无偏扩增微量单细胞内含物、全面分析单细胞内含物集成在一起的公开技术。但已有公开报道利用微流控技术高通量分析单细胞转录组。比如Cell文章(Macosko et al.，2015,Cell：161，1202-1214；Klein et al.,2015,Cell 161,1187-1201)报道的结合液滴微流控和编码微球的方法，基于泊松分布原理利用液滴微流控的方法配对捕获单细胞与单微球，单细胞裂解释放的mRNA被与之配对的编码微球捕获，再经过逆转录和扩增，将单细胞mRNA信息编码与放大，通过高通量测序与生物信息学方法分析大量大细胞mRNA的表达情况。该方法中细胞与微球的捕获是基于泊松分布原理，大部分的液滴没有细胞，只有～1％的液滴含有单个细胞，再结合微球的泊松分布，有效分析目标进一步减少，只能实现对大量实际样品中少部分细胞的分析，这样可能会忽略掉样品中一些重要的细胞个体。另外该策略只适合分析对象数目较多的样品，对于一些稀有细胞(比如循环肿瘤细胞)，由于其样品中细胞数量太少(10-100/mL血液)，无法用该方法来实现单细胞分析。这些技术都仅限于分析单细胞的mRNA，其他单细胞内含物无法进行分析，如基因组、miRNA、蛋白组、甲基化DNA、代谢产物、脂质体、磷脂等。目前公开的技术中，都没有涉及到高通量分析。Currently, there is no published technology that can integrate high-throughput capture of single cells, unbiased amplification of tiny single-cell contents, and comprehensive analysis of single-cell contents. However, there have been published reports of high-throughput analysis of single-cell transcriptomes using microfluidic technology. For example, the Cell article (Macosko et al., 2015, Cell: 161, 1202-1214; Klein et al., 2015, Cell 161, 1187-1201) reported the method of combining droplet microfluidics and encoding microspheres, based on Poor The principle of loose distribution uses the droplet microfluidic method to pair capture single cells and single microspheres. The mRNA released by single cell cleavage is captured by the paired coding microspheres, and then reverse transcription and amplification are performed to encode the single cell mRNA information. With amplification, high-throughput sequencing and bioinformatics methods were used to analyze the expression of large cell mRNAs. The capture of cells and microspheres in this method is based on the principle of Poisson distribution. Most of the droplets have no cells, and only ~1% of the droplets contain single cells. Combined with the Poisson distribution of microspheres, the effective analysis target is further reduced. Only a small number of cells in a large number of actual samples can be analyzed, which may ignore some important individual cells in the sample. In addition, this strategy is only suitable for samples with a large number of analysis objects. For some rare cells (such as circulating tumor cells), the number of cells in the sample is too small (10-100/mL blood), and this method cannot be used to achieve single-cell analysis. . These techniques are limited to the analysis of single-cell mRNA, and other single-cell contents cannot be analyzed, such as genome, miRNA, proteome, methylated DNA, metabolites, liposomes, phospholipids, etc. None of the currently disclosed technologies involves high-throughput analysis.
微流控芯片是近些年来新兴并且快速发展走向成熟的领域，它利用结构各异的微通道和形式多样的外加力场，对微量流体或样品在微观尺度上进行操纵、处理与控制，从而实现了传统实验室部分乃至全部功能在一块芯片上的集成。然而，常规微流控芯片的局限性也是非常明显的，其需要在芯片内部设计泵、阀与操作复杂的外部流体控制设备配合使用，技术门槛高，且一块芯片难以重复使用，当需要用不同的反应试剂对同一样本进行多层次、多尺度分析时需要消耗大量的芯片制作成本(Macosko et al.，2015,Cell,161，1202-1214；Klein et al.,2015,Cell,161,1187-1201；Han et al.,2018,Cell,172,1091–1107)。Microfluidic chip is an emerging field that has developed rapidly and matured in recent years. It uses microchannels of various structures and various forms of external force fields to manipulate, process and control microfluids or samples on a microscopic scale, thereby It realizes the integration of some or even all functions of traditional laboratories on one chip. However, the limitations of conventional microfluidic chips are also very obvious. It needs to design pumps and valves inside the chip to cooperate with external fluid control equipment with complex operations. The technical threshold is high, and it is difficult to reuse a chip. The reaction reagents need to consume a lot of chip production costs when performing multi-level and multi-scale analysis of the same sample (Macosko et al., 2015, Cell, 161, 1202-1214; Klein et al., 2015, Cell, 161, 1187- 1201; Han et al., 2018, Cell, 172, 1091–1107).
第四个潜在的技术问题在于以往的测序方法在对实际样品进行分析的时候，目前已报道的基于编码微球的测序方法都需要先用荧光激活流式分选出目标细胞，再转移至各个分析平台上，而细胞内含物信息会随着细胞所处环境的变化而发生改变，导致最后测序结果反映的测序信息对比细胞当时所处的真实环境下的信息可能会有所偏差。The fourth potential technical problem is that when the previous sequencing methods analyze actual samples, the currently reported sequencing methods based on encoded microspheres need to use fluorescence-activated flow cytometry to sort the target cells first, and then transfer them to each cell. On the analysis platform, the information of the cell contents will change as the environment of the cells changes, which may lead to deviations between the sequencing information reflected in the final sequencing results and the information in the real environment of the cells at that time.
第五个技术难题在于稀有细胞的分离，当待分析的细胞数量非常稀少又需要对每个单细胞独立的转录组信息进行分析时，传统基于毛细管挑取、梯度稀释或者激光切割等技术人力成本高、耗时耗力、通量低，限制了稀有细胞的高通量快速分离及测序分析。The fifth technical difficulty lies in the separation of rare cells. When the number of cells to be analyzed is very rare and the independent transcriptome information of each single cell needs to be analyzed, traditional techniques based on capillary picking, gradient dilution, or laser cutting require labor costs. High, time-consuming, labor-intensive, and low throughput limit the high-throughput rapid separation and sequencing analysis of rare cells.
本申请主要聚焦于上面的第三类技术难题，即，在实现对多个细胞进行高通量分析时解决了单细胞与反应试剂的一对一配对问题，同时增加液滴对细胞与微球的共捕获率，解决了“双泊松分布”造成的低效。This application mainly focuses on the third type of technical problem above, that is, to solve the problem of one-to-one pairing of single cells and reaction reagents when realizing high-throughput analysis of multiple cells, and at the same time increase the number of droplet pairs between cells and microspheres. The high co-capture rate solves the inefficiency caused by the "double Poisson distribution".
为了解决上述技术问题，本申请提供了：In order to solve the above technical problems, the application provides:
1.一种形成反应隔室群的方法，所述方法包括：1. A method of forming a population of reaction compartments, the method comprising:
准备待检测物群，其由两个以上待检测物构成；Prepare a group of substances to be detected, which is composed of more than two substances to be detected;
准备靶向反应复合物群，所述靶向反应复合物群由两个以上靶向反应复合物构成，所述靶向反应复合物包括载体、连接于载体上对应于待检测物的靶向配基、连接于载体上对应于待检测物的反应试剂、可选地包括连接于载体上对应于待检测物的标签分子；Prepare a targeting reaction complex group, the targeting reaction complex group is composed of more than two targeting reaction complexes, the targeting reaction complex includes a carrier, a targeting ligand corresponding to the substance to be detected connected to the carrier base, a reaction reagent corresponding to the substance to be detected connected to the carrier, optionally including a label molecule connected to the carrier corresponding to the substance to be detected;
使用所述待检测物群与所述靶向反应复合物群接触，形成结合物群；using the population of substances to be detected to be contacted with the population of targeting reaction complexes to form a population of conjugates;
由所述结合物自身或者由媒介物包裹所述结合物形成反应隔室群。A population of reaction compartments is formed by the conjugate itself or by surrounding the conjugate with a vehicle.
2.如项1所述的方法，其中，所述待分析物选自蛋白质、核酸、糖、脂、代谢物、多肽、细菌、病毒、细胞器以及细胞中的一种或两种以上，以及由它们形成的复合体，最优选所述待分析物为细胞。2. The method according to item 1, wherein the analyte is selected from one or more of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles, and cells, and by The complexes they form, most preferably the analytes are cells.
3.如项1所述的方法，其中所述载体的形状选自正方体形、四面体形，球形、椭球形、章鱼形、碗形、红血球形的一种或两种以上；最优选为碗形和/或红血球形。3. The method as described in item 1, wherein the shape of the carrier is selected from one or more of cube shape, tetrahedron shape, spherical shape, ellipsoid shape, octopus shape, bowl shape, red blood cell shape; most preferably bowl shape and/or red blood cells.
4.如项3所述的方法，其中所述载体的数量为所述待检测物数量的4倍及以上，优选为所述待检测物数量的10倍及以上，最优选为所述待检测物数量的10倍至30倍。4. The method according to item 3, wherein the quantity of the carrier is 4 times or more than the quantity of the substance to be detected, preferably 10 times or more than the quantity of the substance to be detected, most preferably the quantity of the substance to be detected 10 to 30 times the number of objects.
5.如项3或4所述的方法，其中所述载体为的最大直径为待检测物的1倍以上，优选其最大直径为待检测物的3倍及以上，最优选其最大直径为待检测物的4倍至10倍。5. The method as described in item 3 or 4, wherein the maximum diameter of the carrier is more than 1 time that of the substance to be detected, preferably 3 times or more than the maximum diameter of the substance to be detected, most preferably its maximum diameter is 4 times to 10 times that of the detected object.
6.如项3至5的任一项所述的方法，其中所述载体由聚合物或小分子构成，优选为聚合物载体，进一步优选为聚苯乙烯载体聚苯乙烯或水凝胶制成，进一步该聚合物载体由聚苯乙烯制成，最优选该聚合物带有铁磁性或顺磁性。6. The method according to any one of items 3 to 5, wherein the carrier is made of a polymer or a small molecule, preferably a polymer carrier, more preferably a polystyrene carrier made of polystyrene or hydrogel , further the polymer carrier is made of polystyrene, most preferably the polymer is ferromagnetic or paramagnetic.
7.如项1所述的方法，其中，所述靶向配基可以是天然或人造的，选自包括锁核酸和XNA的核酸及其类似物、适配体、小肽、多肽、糖基化肽、多糖、可溶性受体、类固醇、荷尔蒙、促***原、抗原、超级抗原、生长因子、细胞因子、瘦素、病毒蛋白、细胞黏附分子、趋化因子、链霉亲和素及其类似物、生物素及其类似物、抗体、抗体片段、单链可变片段(scFv)、纳米抗体、T细胞受体、主要组织相容性复合体(MHC)分子、抗原肽-MHC分子复合物(pMHC)、DNA结合蛋白、RNA结合蛋白、细胞内或细胞表面受体配基中的一种或两种以上以及它们共同形成的多重配基、复合配基、耦合配基。7. The method according to item 1, wherein the targeting ligand can be natural or artificial, selected from nucleic acids comprising locked nucleic acids and XNA and their analogs, aptamers, small peptides, polypeptides, glycosyl peptides, polysaccharides, soluble receptors, steroids, hormones, mitogens, antigens, superantigens, growth factors, cytokines, leptin, viral proteins, cell adhesion molecules, chemokines, streptavidin, and the like Biotin and its analogues, antibodies, antibody fragments, single chain variable fragments (scFv), nanobodies, T cell receptors, major histocompatibility complex (MHC) molecules, antigenic peptide-MHC molecule complexes One or more of (pMHC), DNA binding protein, RNA binding protein, intracellular or cell surface receptor ligands, and multiple ligands, composite ligands, and coupling ligands formed by them.
8.如项1所述的方法，其中，所述标签分子选自天然或人造的信息分子，包括：寡核苷酸条形码、寡肽或多肽条形码、由天然碱基与LNA、PNA、XNA等非天然碱基构成的核苷酸、寡糖或多糖条形码、生色团(chromophoric group)和助色团(auxochrome group)、金属原子或离子、分子量可区分的小分子、嵌段聚合物、聚合物与骨架分子共价连接物中的一种或两种以上以及它们之间形成的复合体。8. The method as described in item 1, wherein the label molecule is selected from natural or artificial information molecules, including: oligonucleotide barcodes, oligopeptide or polypeptide barcodes, natural bases and LNA, PNA, XNA, etc. Nucleotides composed of unnatural bases, oligosaccharide or polysaccharide barcodes, chromophoric and auxochrome groups, metal atoms or ions, small molecules with distinguishable molecular weights, block polymers, polymeric One or more than two kinds of covalent linkages between substances and backbone molecules and the complex formed between them.
9.如项1所述的方法，其中，所述反应试剂是寡聚核苷酸引物、酶或小分子。9. The method according to item 1, wherein the reaction reagent is an oligonucleotide primer, an enzyme or a small molecule.
10.如项1所述的方法，其中，所述载体直径粒径分布系数CV小于20％。10. The method according to item 1, wherein the carrier diameter particle size distribution coefficient CV is less than 20%.
11.如项10所述的方法，其中，所述载体表面涂覆有力学缓冲涂层；进一步优选涂覆有水凝胶涂层；最优选该水凝胶涂层带有铁磁性或顺磁性颗粒。11. The method as described in item 10, wherein, the surface of the carrier is coated with a mechanical buffer coating; further preferably coated with a hydrogel coating; most preferably the hydrogel coating has a ferromagnetic or paramagnetic particles.
12.如项1所述的方法，其中，所述连接选自共价键、金属键、离子键、范德华力、包括氢键、机械键、卤键、硫族键、亲金作用、嵌入、重叠、阳离子–π键、阴离子–π键、盐桥、非金属原子间次级键、金属原子与非金属原子间次级键、亲金作用、亲银作用、双氢键和金键的次级键。12. The method according to item 1, wherein the connection is selected from covalent bonds, metallic bonds, ionic bonds, van der Waals forces, including hydrogen bonds, mechanical bonds, halogen bonds, chalcogen bonds, amophilic interactions, intercalation, Overlapping, cation–π bonds, anion–π bonds, salt bridges, secondary bonds between nonmetal atoms, secondary bonds between metal atoms and nonmetal atoms, aurophilic interactions, argentophilic interactions, double hydrogen bonds, and secondary bonds of gold bonds level key.
13.如项1所述的方法，其中，所述靶向配基与所述载体连接是通过所述反应试剂的连接、通过所述标签分子的连接或通过接头的连接；所述反应试剂与所述载体的连接是通过所述靶向配基的连接、通过所述标签分子的连接或通过接头的连接；所述标签分子与所述载体的连接是通过所述靶向配基的连接、通过所述反应试剂的连接或通过接头 的连接。13. The method according to item 1, wherein the targeting ligand is connected to the carrier through the connection of the reaction reagent, the connection of the label molecule or the connection of the linker; the reaction reagent and The connection of the carrier is through the connection of the targeting ligand, the connection of the label molecule or the connection of the linker; the connection of the label molecule and the carrier is through the connection of the targeting ligand, Ligation via the reagent or via a linker.
14.如项6所述的方法，其中所述小分子为所述靶向配基、所述反应试剂、所述标签分子中的一项或两项以上。14. The method according to item 6, wherein the small molecule is one or more of the targeting ligand, the reaction reagent, and the label molecule.
15.如项1所述的方法，其中，所述媒介物是油性介质，优选含氟油性介质，或者固体介质，优选微孔板。15. The method according to item 1, wherein the medium is an oily medium, preferably a fluorinated oily medium, or a solid medium, preferably a microwell plate.
16.如项1所述的方法，其中所述两个以上待检测物是可以是相同或不相同的，且所述两个以上靶向反应复合物可以是相同的或者不相同的，当所述两个以上待检测物不相同且所述两个以上靶向反应复合物不相同时，所述靶向反应复合物包括连接于载体上对应于待检测物的标签分子。16. The method according to item 1, wherein the two or more substances to be detected can be the same or not, and the two or more targeting reaction complexes can be the same or not, when the When the two or more substances to be detected are different and the two or more targeting reaction complexes are different, the targeting reaction complex includes label molecules corresponding to the substances to be detected connected to the carrier.
本申请的技术方案实现的有益技术效果Beneficial technical effects realized by the technical solution of the present application
采用本申请的技术方案，在实现对多个的待分析物(例如细胞)进行高通量分析的同时解决了待分析物(单细胞)与载体(单微球)的一对一配对问题，同时增加媒介物(液滴)对细胞与微球的共捕获率，解决了待分析物与载体配对时出现的“双泊松分布”现象造成的低效问题。通过使用红细胞形和碗形的载体(例如聚苯乙烯微球)以及相对于待检测物(例如细胞)尺寸较大且数量较多的载体，利用空间位阻效应进一步确保了“单细胞与单微球的一对一配对”这一目的的实现。通过在载体(例如聚苯乙烯微球)的表面包覆一层缓冲层(水凝胶层)，避免了在细胞与微球结合过程中细胞被碾碎的危险，减少了待检测物损失。本申请所采用的靶向复合物群具有至少一种反应试剂，实现了对待检测物的高通量分析。By adopting the technical scheme of the present application, the one-to-one pairing problem of the analyte (single cell) and the carrier (single microsphere) is solved while realizing high-throughput analysis of multiple analytes (such as cells), At the same time, the co-capture rate of the medium (droplet) on the cells and microspheres is increased, which solves the problem of inefficiency caused by the "double Poisson distribution" phenomenon that occurs when the analyte is paired with the carrier. By using erythrocyte-shaped and bowl-shaped carriers (such as polystyrene microspheres) and carriers with a large size and a large number relative to the object to be detected (such as cells), the steric hindrance effect is used to further ensure that the "single cell and single cell The realization of the purpose of "one-to-one pairing of microspheres". By coating a layer of buffer layer (hydrogel layer) on the surface of the carrier (such as polystyrene microspheres), the danger of cells being crushed during the process of combining cells with microspheres is avoided, and the loss of the substance to be detected is reduced. The targeting complex group used in the present application has at least one reaction reagent, which realizes high-throughput analysis of the substance to be detected.
附图说明Description of drawings
图1显示了根据本申请的靶向反应复合物的构成示意图；靶向反应复合物包括载体、连接于载体上对应于待检测物的靶向配基、连接于载体上对应于待检测物的反应试剂、可选地包括连接于载体上对应于待检测物的标签分子。Figure 1 shows a schematic diagram of the composition of the targeting reaction complex according to the present application; the targeting reaction complex includes a carrier, a targeting ligand corresponding to the substance to be detected connected to the carrier, and a ligand corresponding to the substance to be detected connected to the carrier. Reaction reagents optionally include label molecules corresponding to the substances to be detected connected to the carrier.
图2A显示硬质微球在与细胞结合时，可能将细胞碾碎；图2B显示在硬质微球表面包裹水凝胶涂层对细胞有保护作用；图2C显示了根据本申请的涂有水凝胶覆层的靶向反应复合物(包裹水凝胶镀层的DNA编码微球)。Figure 2A shows that the hard microspheres may crush the cells when they are combined with the cells; Figure 2B shows that the hydrogel coating on the surface of the hard microspheres has a protective effect on the cells; Hydrogel-coated targeting-response complexes (DNA-encoded microspheres coated with hydrogel coatings).
图3A显示了移液枪吹打微球实现水凝胶包裹；图3B显示水凝胶成胶破乳后的图像。Figure 3A shows the hydrogel encapsulation by pipetting the microspheres; Figure 3B shows the image of the hydrogel after gel formation and demulsification.
图4A显示微球直径大于细胞，位阻效应有助于微球与细胞1:1结合；一个细胞较难与两个及以上的微球结合；图4B显示微球比细胞尺径小时，形成多个微球结合一个细胞，或细胞与微球的聚团。Figure 4A shows that the diameter of the microsphere is larger than that of the cell, and the steric hindrance effect helps the combination of the microsphere and the cell at 1:1; it is difficult for a cell to combine with two or more microspheres; Figure 4B shows that the diameter of the microsphere is smaller than that of the cell, and the formation of Multiple microspheres bound to one cell, or aggregates of cells and microspheres.
图5A显示了水凝胶包裹磁性微球的制备；图5B显示了破乳后通过磁性纯化；图 5C显示了水凝胶包裹磁性微球，破乳后通过磁性纯化；图5D显示了在荧光显微镜下，可观察到活化后水凝胶层表面修饰的抗体(外层亮白色)；图5E中，白色箭头所指的是带抗体修饰的水凝胶包裹微球通过孵育与细胞形成1：1结合。Figure 5A shows the preparation of hydrogel-coated magnetic microspheres; Figure 5B shows the magnetic purification after demulsification; Figure 5C shows the hydrogel-coated magnetic microspheres, and magnetic purification after demulsification; Under the microscope, the antibody modified on the surface of the hydrogel layer after activation can be observed (the outer layer is bright white); in Figure 5E, the white arrow refers to the formation of the antibody-modified hydrogel-coated microspheres through incubation with cells1: 1 combined.
图6A-C显示了碗形载体，位阻效应有助于微球与细胞1:1结合；一个细胞较难与两个及以上的碗形或红细胞形微球结合；图6A为在液滴里进行碗形双水相的生成与紫外交联；图6B为碗形的颗粒；图6C为碗形的颗粒与细胞形成1：1结合。Figure 6A-C shows the bowl-shaped carrier, and the steric hindrance effect helps the microspheres to combine with the cells at 1:1; it is difficult for a cell to combine with two or more bowl-shaped or red blood cell-shaped microspheres; The generation of bowl-shaped aqueous two-phase and ultraviolet crosslinking are carried out here; Figure 6B shows the bowl-shaped particles; Figure 6C shows the 1:1 combination of bowl-shaped particles and cells.
图7A显示了用微流控设备制备的水凝胶微球成品；图7B显示了水凝胶微球中，标签分子与polyT在同一个逆转录引物上；图7C显示了水凝胶微球在液滴里聚合。Figure 7A shows the finished hydrogel microspheres prepared by microfluidic equipment; Figure 7B shows that in the hydrogel microspheres, the label molecule and polyT are on the same reverse transcription primer; Figure 7C shows the hydrogel microspheres aggregate in droplets.
图8A显示了经修饰的带有抗人-CD298、人β2微球蛋白、小鼠CD45以及小鼠MHC class I抗体的微球，可作为通用结合微球与细胞结合；图8B显示了该水凝胶微球与细胞结合，形成1：1复合物。Figure 8A shows modified microspheres with anti-human-CD298, human β2 microglobulin, mouse CD45, and mouse MHC class I antibodies, which can be used as universal binding microspheres to bind cells; Figure 8B shows the water The gel microspheres bind to the cells to form a 1:1 complex.
图9A为整个高通量分析过程的流程图(靶向反应复合物与待分析物预先结合，再被包裹进高通量隔室)；图9B，当微球数量为细胞10倍或以上时，可形成1：1结合，大概有1/100的概率观察到一个细胞结合两个微球；图9C为形成隔室后每个细胞都有靶向反应复合物微球与之结合，从而打破泊松分布，提高细胞的捕获率；图9D为部分情况下，靶向反应复合物与所结合的细胞会在隔室内分离；图9E显示待分析物为彼此相互作用的细胞对。Figure 9A is a flow chart of the entire high-throughput analysis process (targeted reaction complexes are pre-combined with analytes and then packaged into high-throughput compartments); Figure 9B, when the number of microspheres is 10 times or more than that of cells , can form a 1:1 combination, and there is a probability of 1/100 to observe that one cell binds to two microspheres; Poisson distribution improves the capture rate of cells; Figure 9D shows that in some cases, the target reaction complex and the bound cells will be separated in the compartment; Figure 9E shows that the analyte is a pair of cells interacting with each other.
图10A-C显示了通过注射泵控制微流控内的水/油液体形成油包水隔室的过程；其中图10B显示了微流控装置高通量形成隔室；图10C显示了微球与细胞复合物进入微流控十字交叉口，高通量液滴生成；图10D显示了单细胞分析结果；显示了单细胞分析结果，人和鼠的细胞亚群能够分离。Figure 10A-C shows the process of controlling the water/oil liquid in the microfluidics to form a water-in-oil compartment through a syringe pump; where Figure 10B shows the high-throughput formation of a microfluidic device; Figure 10C shows a microsphere The complex with cells enters the microfluidic intersection, and high-throughput droplets are generated; Figure 10D shows the results of single-cell analysis; shows the results of single-cell analysis, and human and mouse cell subpopulations can be separated.
图11A显示了下述步骤：第一步微球的水凝胶包裹，并交联链霉亲和素；第二步细胞生物素化；第三步，将生物素化的细胞与带链霉亲和素的孵育结合；第四步，高通量对微球与细胞的复合物进行包裹形成隔室，并在隔室里释放反应试剂、对隔室内细胞进行标签化；图11B显示了水凝胶包裹的靶向标签微球，经过带荧光的链霉亲和素修饰；图11C显示了生物素化的细胞；图11D显示了水凝胶微球结合细胞；图11E显示了手动枪头吹打在水凝胶包裹微球外形成隔室，对隔室中细胞进行细胞裂解释放其中的mRNA。Figure 11A shows the following steps: the first step of hydrogel encapsulation of microspheres, and cross-linked streptavidin; the second step of cell biotinylation; the third step, the biotinylated cells with streptavidin Incubation and binding of avidin; the fourth step, high-throughput packaging of the complexes of microspheres and cells to form compartments, release of reaction reagents in the compartments, and labeling of cells in the compartments; Figure 11B shows the Gel-encapsulated target-tagged beads modified with fluorescent streptavidin; Figure 11C shows biotinylated cells; Figure 11D shows hydrogel beads bound to cells; Figure 11E shows manual pipette tips The compartments are formed outside the hydrogel-wrapped microspheres by pipetting, and the cells in the compartments are lysed to release the mRNA therein.
具体实施方式Detailed ways
下面将参照附图更详细地描述本申请的具体实施例。虽然附图中显示了本申请的具 体实施例，然而应当理解，可以以各种形式实现本申请而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解本申请，并且能够将本申请的范围完整的传达给本领域的技术人员。Specific embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although specific embodiments of the present application are shown in the drawings, it should be understood that the present application may be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that the present application can be more thoroughly understood, and the scope of the present application can be fully conveyed to those skilled in the art.
需要说明的是，在说明书及权利要求当中使用了某些词汇来指称特定组件。本领域技术人员应可以理解，技术人员可能会用不同名词来称呼同一个组件。本说明书及权利要求并不以名词的差异来作为区分组件的方式，而是以组件在功能上的差异来作为区分的准则。如在通篇说明书及权利要求当中所提及的“包含”或“包括”为一开放式用语，故应解释成“包含但不限定于”。说明书后续描述为实施本申请的较佳实施方式，然所述描述乃以说明书的一般原则为目的，并非用以限定本申请的范围。本申请的保护范围当视所附权利要求所界定者为准。It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art should understand that they may use different terms to refer to the same component. The specification and claims do not use differences in nouns as a way of distinguishing components, but use differences in functions of components as a criterion for distinguishing. "Includes" or "comprises" mentioned throughout the specification and claims is an open term, so it should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred implementation mode for implementing the application, but the description is for the purpose of the general principles of the specification, and is not intended to limit the scope of the application. The scope of protection of the present application should be defined by the appended claims.
如本文所用，就特定组分而言“基本上不含”在本文中用于表示特定组分未被有目的地配制到组合物中和/或仅作为污染物或以痕量存在。因此，由组合物的任何意外污染导致的特定组分的总量低于0.05％，优选低于0.01％。最优选的是其中特定组分的量用标准分析方法检测不到的组合物。As used herein, "substantially free" with respect to a particular component is used herein to mean that the particular component has not been purposefully formulated into the composition and/or is present only as a contaminant or in trace amounts. Thus, the total amount of specific components resulting from any accidental contamination of the composition is below 0.05%, preferably below 0.01%. Most preferred are compositions wherein the amount of a particular component is undetectable by standard analytical methods.
如在本说明书中所使用的，“一”或“一个”可以表示一个或多个。如权利要求中所使用的，当与单词“包含”一起使用时，单词“一”或“一个”可以表示一个或多于一个。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" may mean one or more than one.
在权利要求中使用术语“或”用于表示“和/或”，除非明确指出仅指代替代方案或者替代方案是相互排斥的，尽管本公开内容支持仅指代替代方案和“和/或”的定义。如本文所用，“另一个”可以表示至少第二个或更多个。The term "or" is used in the claims to mean "and/or" unless it is expressly stated that only alternatives are referred to or that the alternatives are mutually exclusive, although this disclosure supports only references to alternatives and "and/or" Definition. As used herein, "another" may mean at least a second or more.
贯穿本申请，术语“约”用于指示值包括装置的误差的固有变化，该方法用于测定该值或存在于研究对象之间的变化。Throughout this application, the term "about" is used to indicate that a value includes inherent variation in error for the means used to determine the value or the variation that exists between study subjects.
实施例中描述到的各种生物材料的取得途径仅是提供一种实验获取的途径以达到具体公开的目的，不应成为对本申请生物材料来源的限制。事实上，所用到的生物材料的来源是广泛的，任何不违反法律和道德伦理能够获取的生物材料都可以按照实施例中的提示替换使用。The acquisition methods of various biological materials described in the examples are only to provide an experimental acquisition method to achieve the purpose of specific disclosure, and should not be a limitation on the source of biological materials in this application. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and ethics can be replaced according to the tips in the examples.
本申请提供了形成反应隔室群的方法。The present application provides methods of forming a population of reaction compartments.
在一个具体实施方式中，提供了一种形成反应隔室群的方法，所述方法包括：In a specific embodiment, a method of forming a population of reaction compartments is provided, the method comprising:
准备待检测物群，其由两个以上待检测物构成；Prepare a group of substances to be detected, which is composed of more than two substances to be detected;
准备靶向反应复合物群，所述靶向反应复合物群由两个以上靶向反应复合物构成，所述靶向反应复合物包括载体、连接于载体上对应于待检测物的靶向配基、连接于载体 上对应于待检测物的反应试剂、可选地包括连接于载体上对应于待检测物的标签分子；Prepare a targeting reaction complex group, the targeting reaction complex group is composed of more than two targeting reaction complexes, the targeting reaction complex includes a carrier, a targeting ligand corresponding to the substance to be detected connected to the carrier base, a reaction reagent corresponding to the substance to be detected connected to the carrier, optionally including a label molecule connected to the carrier corresponding to the substance to be detected;
使用所述待检测物群与所述靶向反应复合物群接触，形成结合物群；using the population of substances to be detected to be contacted with the population of targeting reaction complexes to form a population of conjugates;
由所述结合物自身或者由媒介物包裹所述结合物形成反应隔室群。A population of reaction compartments is formed by the conjugate itself or by surrounding the conjugate with a vehicle.
在此，“隔室”意指为特定的化学反应提供特定的反应空间，在该反应空间内，反应物相互之间可以发生反应，而不会与邻近隔室的反应物发生反应，但是，隔室的生成物依其类型有可能与邻近隔室的生成物发生物质交换(例如：染料、表面活性剂等)。Here, "compartment" means providing a specific reaction space for a specific chemical reaction in which reactants can react with each other without reacting with reactants in adjacent compartments, however, Depending on the type, the product of the compartment may exchange substances with the product of the adjacent compartment (for example: dyes, surfactants, etc.).
在本说明书的上下文中，“靶向”符合生物技术领域的一般定义，典型地为抗体-抗原亲和反应，互补的核酸序列的亲和反应。In the context of this specification, "targeting" complies with the general definition in the field of biotechnology, typically antibody-antigen affinity reaction, affinity reaction of complementary nucleic acid sequences.
在本说明书的上下文中，“连接”应作广义的理解，至少包括各种通过分子键的和不通过分子键的连接。In the context of this specification, "connection" should be understood in a broad sense, at least including various connections through molecular bonds and without molecular bonds.
图1给出了靶向反应复合物的组成成分，在此示出了靶向配基、标签分子和反应试剂分别连接于“载体”的结构关系。其中，“载体”可以由与靶向配基、标签和反应试剂同样的分子构成，即可以由小分子构成。更常见地，所述载体由聚合物，优选由聚苯乙烯构成。Figure 1 shows the components of the targeting reaction complex, where the structural relationship of targeting ligands, label molecules and reaction reagents respectively connected to the "carrier" is shown. Among them, the "carrier" can be composed of the same molecules as the targeting ligand, label and reaction reagent, that is, it can be composed of small molecules. More usually, the carrier consists of a polymer, preferably polystyrene.
在又一具体实施方式中，提供了一种形成反应隔室群的方法，其中，所述待分析物选自蛋白质、核酸、糖、脂、代谢物、多肽、细菌、病毒、细胞器以及细胞中的一种或两种以上，以及由它们形成的复合体，最优选所述待分析物为细胞。In yet another specific embodiment, a method for forming a reaction compartment population is provided, wherein the analyte is selected from the group consisting of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles, and cells One or more than two, and complexes formed by them, most preferably the analytes are cells.
在本说明书的上下文中，“细胞”符合生物学领域的一般定义，其至少包括原核细胞和真核细胞，针对本申请的技术的用途，有时可以特指多细胞生物的经过分化的细胞群，例如免疫细胞群，其至少包括淋巴B细胞、淋巴T细胞、NK细胞。In the context of this specification, "cell" conforms to the general definition in the field of biology, which includes at least prokaryotic cells and eukaryotic cells. For the application of the technology of this application, it can sometimes specifically refer to differentiated cell populations of multicellular organisms, For example, immune cell populations include at least lymphoid B cells, lymphoid T cells, and NK cells.
在本说明书的上下文中，“细胞器”符合生物学领域的一般定义，细胞器(organelle)一般认为是散布在细胞质内具有一定形态和功能的微结构或微器官。它们组成了细胞的基本结构，使细胞能正常的工作，运转。细胞中的细胞器主要有：线粒体、内质网、中心体、叶绿体，高尔基体、核糖体等。In the context of this specification, "organelle" conforms to the general definition in the field of biology, and organelle is generally considered to be a microstructure or micro-organ scattered in the cytoplasm with a certain shape and function. They make up the basic structure of cells and enable cells to work and run normally. The organelles in the cell mainly include: mitochondria, endoplasmic reticulum, centrosome, chloroplast, Golgi apparatus, ribosome, etc.
在本说明书的上下文中，“复合体”指的是彼此相互作用、具有一定结合强度的两种或两种以上的物质，这些物质可以由蛋白质、核酸、糖、脂、代谢物、多肽、细菌、病毒、细胞器以及细胞组成。例如：HepG2与识别该细胞系的T细胞(基因改造后，携带靶向“主要组织相容性复合体-AFP158肽段”的T细胞受体基因)孵育后会形成细胞与细胞的相互作用；具体的例子还有：细菌和噬菌体(病毒)的复合体、细胞和膜蛋白(多肽)的复合体、以及核糖体和RNA(核酸)的复合体。In the context of this specification, "complex" refers to two or more substances that interact with each other and have a certain binding strength. These substances can be composed of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria , viruses, organelles, and cells. For example: after incubation of HepG2 with T cells that recognize the cell line (genetically modified to carry the T cell receptor gene targeting the "major histocompatibility complex-AFP158 peptide"), cell-to-cell interactions will form; Specific examples are: complexes of bacteria and bacteriophages (viruses), complexes of cells and membrane proteins (polypeptides), and complexes of ribosomes and RNA (nucleic acids).
在一个具体实施方式中，提供了一种形成反应隔室群的方法，其中，所述载体的形 状选自正方体形、四面体形，球形、椭球形、章鱼形、碗形、红血球形的一种或两种以上；最优选为碗形和/或红血球形。“碗形”特指一种凹球形，其在“半球体”的平面一侧设置有凹面，当待检测物结合至所述载体的该凹面时，由于空间位阻效应，所述待检测物不会再结合至另一载体。凹球形特指一种球形的圆平面上有凹形面，当待检测物结合至所述载体的该凹面时，由于空间位阻效应，所述待检测物不会再结合至另一载体。而“红血球形”的载体具有两个凹面，当待检测物结合至所述载体的上凹面或下凹面时，由于空间位阻效应，所述待检测物不会再结合至另一载体。In a specific embodiment, a method for forming a group of reaction compartments is provided, wherein the shape of the carrier is selected from one of cube shape, tetrahedron shape, sphere shape, ellipsoid shape, octopus shape, bowl shape, and red blood spheroid shape Or two or more; most preferably bowl-shaped and/or red blood cell-shaped. "Bowl shape" specifically refers to a concave spherical shape, which is provided with a concave surface on the plane side of the "hemisphere". When the substance to be detected is bound to the concave surface of the carrier, due to the steric hindrance effect, the substance to be detected Will not bind to another carrier. The concave spherical shape specifically refers to a spherical circular plane with a concave surface. When the substance to be detected is bound to the concave surface of the carrier, the substance to be detected will not be bound to another carrier due to steric hindrance. The "red blood cell" carrier has two concave surfaces. When the substance to be detected is bound to the upper concave surface or the lower concave surface of the carrier, the substance to be detected will not bind to another carrier due to steric hindrance.
在又一具体实施方式中，提供了一种形成反应隔室群的方法，其中，所述载体的数量为所述待检测物数量的10倍以上，最优选为所述待检测物数量的10倍至50倍。具体地，所述载体的数量可以为所述待检测物数量的10倍、12倍、14倍、16倍、18倍、20倍、22倍、24倍、26倍、28倍、30倍、32倍、34倍、36倍、38倍、40倍、42倍、44倍、46倍、48倍或50倍。希望控制进行相互作用的待分析物与前述靶向反应复合物或前述反应复合物群的量以及分析过程，使得对每个结合物中仅含有一个待分析物的结合物进行分析。在本申请的实施例中，具体而言，当携带反应试剂的微球数量为被分析细胞数量10倍-20倍时，根据泊松分布每个微球大概率仅结合一个细胞。In yet another specific embodiment, a method for forming a group of reaction compartments is provided, wherein the amount of the carrier is more than 10 times the amount of the substance to be detected, most preferably 10 times the amount of the substance to be detected times to 50 times. Specifically, the quantity of the carrier can be 10 times, 12 times, 14 times, 16 times, 18 times, 20 times, 22 times, 24 times, 26 times, 28 times, 30 times, 32x, 34x, 36x, 38x, 40x, 42x, 44x, 46x, 48x or 50x. It is desirable to control the amount of analyte interacting with the aforementioned targeted reaction complex or population of aforementioned reaction complexes and the assay process so that only one analyte per conjugate is analyzed. In the embodiment of the present application, specifically, when the number of microspheres carrying the reaction reagent is 10-20 times the number of analyzed cells, each microsphere has a high probability of binding to only one cell according to the Poisson distribution.
在本说明书的上下文中，“泊松分布”的含义是描述单位空间内随机出现微球或细胞的数量；“双泊松分布”的含义是描述单位空间内随机出现同时有微球和细胞的数量。在实施例中，如果出现单位空间内出现同时有微球和细胞的数量大于双泊松分布的现象，则可以认为“打破泊松分布”。In the context of this specification, the meaning of "Poisson distribution" is to describe the number of microspheres or cells randomly appearing in a unit space; the meaning of "double Poisson distribution" is to describe the number of microspheres and cells randomly appearing in a unit space quantity. In an embodiment, if there is a phenomenon that the number of microspheres and cells in a unit space is greater than the double Poisson distribution, it can be considered as "breaking the Poisson distribution".
在一个具体实施方式中，提供了一种形成反应隔室群的方法，其中，所述载体的最大直径为待检测物的2倍以上，最优选其最大直径为待检测物的3倍至10倍。具体地，所述载体的最大直径可以为待检测物的2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍。在本说明书的上下文中，“直径”特指通过一平面图形或立体(如圆、圆锥截面、球、立方体)中心到边上两点间的距离)。在使用红细胞形和碗形的载体的情况下，由于空间位阻效应，最大程度地实现了载体和待检测物的1:1的结合。特别在使用红细胞形微球和免疫细胞，且红细胞形微球的直径为免疫细胞3倍至30倍的情况下，最大程度地实现了载体和待检测物的1:1的结合。In a specific embodiment, a method for forming a group of reaction compartments is provided, wherein the maximum diameter of the carrier is more than 2 times that of the substance to be detected, and most preferably its maximum diameter is 3 times to 10 times that of the substance to be detected. times. Specifically, the maximum diameter of the carrier may be 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times that of the substance to be detected. In the context of this specification, "diameter" specifically refers to the distance between the center of a plane figure or a solid (such as a circle, a conical section, a sphere, a cube) and two points on an edge). In the case of using erythrocyte-shaped and bowl-shaped carriers, due to the steric hindrance effect, the 1:1 combination of the carrier and the analyte is realized to the greatest extent. Especially when erythrocyte-shaped microspheres and immune cells are used, and the diameter of erythrocyte-shaped microspheres is 3 times to 30 times that of immune cells, the 1:1 combination of the carrier and the substance to be detected is realized to the greatest extent.
具体地，可以在使用“碗形”或“红血球”形载体的同时使用例如比待检测物多10倍、12倍、14倍、16倍、18倍、20倍、22倍、24倍、26倍、28倍、30倍、32倍、34倍、36倍、38倍、40倍、42倍、44倍、46倍、48倍或50倍的载体数量；具体地，可以在使用最大直径为待检测物的3倍至10倍的载体尺寸的同时使用例如比待检测 物多10倍、12倍、14倍、16倍、18倍、20倍、22倍、24倍、26倍、28倍、30倍、32倍、34倍、36倍、38倍、40倍、42倍、44倍、46倍、48倍或50倍的载体数量；具体地，可以在使用例如比待检测物多10倍、12倍、14倍、16倍、18倍、20倍、22倍、24倍、26倍、28倍、30倍、32倍、34倍、36倍、38倍、40倍、42倍、44倍、46倍、48倍或50倍的载体数量的同时使用“碗形”或“红血球”形载体；具体地，可以在使用例如比待检测物多10倍、12倍、14倍、16倍、18倍、20倍、22倍、24倍、26倍、28倍、30倍、32倍、34倍、36倍、38倍、40倍、42倍、44倍、46倍、48倍或50倍的载体数量的同时使用“碗形”或“红血球”形载体,且使用最大直径为待检测物的3倍至10倍的载体尺寸；以最终实现“根据泊松分布每个微球大概率仅结合一个细胞”的技术效果。Specifically, for example, 10 times, 12 times, 14 times, 16 times, 18 times, 20 times, 22 times, 24 times, 26 times more than the substance to be detected can be used while using the "bowl-shaped" or "red blood cell"-shaped carrier. times, 28 times, 30 times, 32 times, 34 times, 36 times, 38 times, 40 times, 42 times, 44 times, 46 times, 48 times or 50 times the number of carriers; Simultaneous use of 3 to 10 times the carrier size of the substance to be detected, for example, 10 times, 12 times, 14 times, 16 times, 18 times, 20 times, 22 times, 24 times, 26 times, 28 times more than the substance to be detected , 30 times, 32 times, 34 times, 36 times, 38 times, 40 times, 42 times, 44 times, 46 times, 48 times or 50 times the number of carriers; Times, 12 times, 14 times, 16 times, 18 times, 20 times, 22 times, 24 times, 26 times, 28 times, 30 times, 32 times, 34 times, 36 times, 38 times, 40 times, 42 times, 44 times, 46 times, 48 times or 50 times the number of carriers while using "bowl-shaped" or "red blood cell"-shaped carriers; times, 18 times, 20 times, 22 times, 24 times, 26 times, 28 times, 30 times, 32 times, 34 times, 36 times, 38 times, 40 times, 42 times, 44 times, 46 times, 48 times or 50 times the number of carriers while using "bowl-shaped" or "red blood cell"-shaped carriers, and using a carrier size with a maximum diameter of 3 to 10 times the size of the object to be detected; Probability of combining only one cell" technical effect.
在又一具体实施方式中，提供了一种形成反应隔室群的方法，其中，所述载体由聚苯乙烯或小分子制成，最优选由聚苯乙烯制成。In yet another embodiment, there is provided a method of forming a population of reaction compartments, wherein said carrier is made of polystyrene or small molecules, most preferably made of polystyrene.
在一个具体实施方式中，提供了一种形成反应隔室群的方法，其中，所述靶向配基可以是天然或人造的，选自包括锁核酸和XNA的核酸及其类似物、适配体、小肽、多肽、糖基化肽、多糖、可溶性受体、类固醇、荷尔蒙、促***原、抗原、超级抗原、生长因子、细胞因子、瘦素、病毒蛋白、细胞黏附分子、趋化因子、链霉亲和素及其类似物、生物素及其类似物、抗体、抗体片段、单链可变片段(scFv)、纳米抗体、T细胞受体、主要组织相容性复合体(MHC)分子、抗原肽-MHC分子复合物(pMHC)、DNA结合蛋白、RNA结合蛋白、细胞内或细胞表面受体配基中的一种或两种以上以及它们共同形成的多重配基、复合配基、耦合配基。在本说明书的上下文中，“抗体”符合生物学领域的一般定义，具体地，抗体(antibody)是指机体由于抗原的刺激而产生的具有保护作用的蛋白质。它是一种由浆细胞(效应B细胞)分泌，被免疫***用来鉴别与中和外来物质如细菌、病毒等的大型Y形蛋白质，仅被发现存在于脊椎动物的血液等体液中，及其B细胞的细胞膜表面。抗体能识别特定外来物的一个独特特征，该外来目标被称为抗原。在本申请的技术方案中，可以使用例如淋巴细胞的表面受体作为抗原，制备相应的抗体，化学修饰在微球表面，以起到分离淋巴细胞群的作用。在本说明书的上下文中，“适配体”符合生物学领域的一般定义，在本申请的技术方案中，可以特别指代核酸适配体。核酸适配体(Aptamer)是一段DNA(脱氧核糖核酸)，RNA(核糖核酸)序列，XNA(核酸类似物)或肽。通常是利用体外筛选技术——指数富集的配体***进化技术(Systematic evolution of ligands by exponential enrichment，SELEX)，从核酸分子文库中得到的寡核苷酸片段。核酸适配体能与多种目标物质高特异性、高选择性地结合，因此被广 泛应用于生物传感器领域。当核酸适配体与目标物质发生特异性结合时，核酸适配体自身的构型会随之发生变化。在本申请的技术方案中，当待分析物为核酸片段时，可以选用它的核酸适配体，经化学修饰固定在微球表面，以起到分离细胞的作用。In a specific embodiment, a method for forming a population of reaction compartments is provided, wherein the targeting ligand can be natural or artificial, selected from nucleic acids including locked nucleic acids and XNA and their analogs, adapters 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) Molecule, antigenic peptide-MHC molecular complex (pMHC), DNA-binding protein, RNA-binding protein, one or more than two of intracellular or cell surface receptor ligands, and multiple ligands and complex ligands formed by them , Coupling base. In the context of this specification, "antibody" conforms to the general definition in the field of biology. Specifically, an antibody refers to a protective protein produced by the body due to stimulation by an antigen. It is a large Y-shaped protein secreted by plasma cells (effector B cells) and used by the immune system to identify and neutralize foreign substances such as bacteria and viruses. It is only found in body fluids such as blood in vertebrates, and The cell membrane surface of its B cells. Antibodies recognize a unique feature of a particular foreign object, called an antigen. In the technical solution of the present application, for example, surface receptors of lymphocytes can be used as antigens to prepare corresponding antibodies, which are chemically modified on the surface of microspheres to separate lymphocyte populations. In the context of this specification, "aptamer" conforms to the general definition in the field of biology, and in the technical solution of this application, it may specifically refer to nucleic acid aptamers. Aptamer is a DNA (deoxyribonucleic acid), RNA (ribonucleic acid) sequence, XNA (nucleic acid analog) or peptide. Usually, it is an oligonucleotide fragment obtained from a nucleic acid molecule library by using an in vitro screening technique - Systematic evolution of ligands by exponential enrichment (SELEX). Nucleic acid aptamers can bind to a variety of target substances with high specificity and high selectivity, so they are widely used in the field of biosensors. When the nucleic acid aptamer specifically binds to the target substance, the configuration of the nucleic acid aptamer itself will change accordingly. In the technical solution of the present application, when the analyte is a nucleic acid fragment, its nucleic acid aptamer can be selected and chemically modified and fixed on the surface of the microsphere to separate cells.
在又一具体实施方式中，提供了一种形成反应隔室群的方法，其中，所述标签分子选自天然或人造的信息分子，包括：寡核苷酸条形码、寡肽或多肽条形码、由天然碱基与LNA、PNA、XNA等非天然碱基构成的核苷酸、寡糖或多糖条形码、生色团(chromophoric group)和助色团(auxochrome group)、金属原子或离子、分子量可区分的小分子、嵌段聚合物、聚合物与骨架分子共价连接物中的一种或两种以上以及它们之间形成的复合体。在本说明书的上下文中，DNA条形码(DNA barcode)是指生物体内能够代表该物种的、标准的、有足够变异的、易扩增且相对较短的DNA片段。DNA条形码已经成为生态学研究的重要工具，不仅用于物种鉴定，同时也帮助生物学家进一步了解生态***内发生的相互作用。在发现一种未知物种或者物种的一部分时，研究人员便描绘其组织的DNA条形码，而后与国际数据库内的其他条形码进行比对。如果与其中一个相匹配，研究人员便可确认这种物种的身份。DNA条形码技术是利用生物体DNA中一段保守片段对物种进行快速准确鉴定的新兴技术。具体到本申请的实施例，当所述“分析”是针对免疫细胞群的转录组测序时，使用DNA条形码可以在取得对免疫细胞群的高通量分析结果后，对这些分析结果去卷积化。In yet another specific embodiment, a method for forming a group of reaction compartments is provided, wherein the tag molecules are selected from natural or artificial information molecules, including: oligonucleotide barcodes, oligopeptide or polypeptide barcodes, by Nucleotides, oligosaccharide or polysaccharide barcodes, chromophoric groups and auxochrome groups, metal atoms or ions, and molecular weights composed of natural bases and LNA, PNA, XNA and other unnatural bases can be distinguished One or more of the small molecules, block polymers, covalent links between polymers and backbone molecules, and complexes formed between them. In the context of this specification, a DNA barcode (DNA barcode) refers to a standard, sufficiently variable, easily amplified and relatively short DNA fragment that can represent the species in an organism. DNA barcoding has become an important tool in ecological research, not only for species identification, but also to help biologists further understand the interactions that occur within ecosystems. When an unknown species or part of a species is discovered, the researchers profile the DNA barcode of its tissue and compare it to other barcodes in an international database. A match with one of these would allow the researchers to confirm the species' identity. DNA barcoding technology is an emerging technology for rapid and accurate identification of species by using a conserved fragment in the DNA of organisms. Specific to the embodiment of this application, when the "analysis" is the transcriptome sequencing of immune cell populations, the use of DNA barcodes can deconvolute the analysis results after obtaining high-throughput analysis results of immune cell populations change.
在一个具体实施方式中，提供了一种形成反应隔室群的方法，其中，所述反应试剂是寡聚核苷酸引物、酶或小分子。在本文中，反应试剂是用来与待分析物发生反应并产生能够用于检测信号的物质。在本说明书的上下文中，“反应试剂”有时可以特指能够促使待分析物发出荧光的荧光试剂。在本说明书的上下文中，“引物”符合生物技术领域的一般定义，具体地，引物是指在核苷酸聚合作用起始时，刺激合成的，一种具有特定核苷酸序列的大分子，与反应物以氢键形式连接，这样的分子称为引物。引物通常是人工合成的两段寡核苷酸序列，一个引物与靶区域一端的一条DNA模板链互补，另一个引物与靶区域另一端的另一条DNA模板链互补，其功能是作为核苷酸聚合作用的起始点，核酸聚合酶可由其3端开始合成新的核酸链。体外人工设计的引物被广泛用于聚合酶链反应、测序和探针合成等。具体到本申请的实施例，在所述“分析”是转录组测序时，可以采用寡聚核苷酸引物作为“反应试剂”。在本说明书的上下文中，“构成反应试剂的小分子”可以是化学技术领域的各种小分子，当所述“分析”是荧光检测时，特别地，所述“小分子”是可以发出荧光的小分子，例如FAM、VIC等。In a specific embodiment, a method of forming a population of reaction compartments is provided, wherein the reaction reagent is an oligonucleotide primer, an enzyme or a small molecule. In this context, a reagent is a substance that reacts with an analyte and produces a signal that can be used for detection. In the context of this specification, "reagent" may sometimes specifically refer to a fluorescent reagent capable of causing the analyte to emit fluorescence. In the context of this specification, "primer" conforms to the general definition in the field of biotechnology, specifically, a primer refers to a macromolecule with a specific nucleotide sequence that stimulates synthesis at the initiation of nucleotide polymerization, A molecule that is hydrogen bonded to a reactant is called a primer. Primers are usually two artificially synthesized oligonucleotide sequences, one primer is complementary to a DNA template strand at one end of the target region, and the other primer is complementary to the other DNA template strand at the other end of the target region, and its function is to act as a nucleotide At the starting point of polymerization, nucleic acid polymerase can synthesize a new nucleic acid chain from its 3-end. In vitro artificially designed primers are widely used in polymerase chain reaction, sequencing and probe synthesis, etc. Specific to the embodiment of the present application, when the "analysis" is transcriptome sequencing, oligonucleotide primers can be used as "reaction reagents". In the context of this specification, "small molecules constituting reaction reagents" can be various small molecules in the field of chemical technology. When the "analysis" is fluorescence detection, in particular, the "small molecules" can emit fluorescence Small molecules such as FAM, VIC, etc.
在一个具体实施方式中，提供了一种形成反应隔室群的方法，其中，所述载体直径 粒径分布系数CV小于20％。此处的英文缩写定义如下：CV＝SD/平均粒径，它可以表示粒度分布的宽窄。其中SD：是标准偏差(Standard Deviation)统计数学上记为σ；CV也称相对标准偏差(Coefficient of variation)统计数学上记为α。在本文中，采用的微球的直径可以采用本领域技术人员所公知的方式和装置来计算，例如可以在显微镜下进行测量并用图像处理软件分析，可以通过测定颗粒粒度的仪器(Bio-Rad T20细胞技术仪)来测定，也可以使用微球供应商提供的数据。In a specific embodiment, there is provided a method of forming a population of reaction compartments, wherein said carrier diameter has a particle size distribution coefficient CV of less than 20%. The English abbreviation here is defined as follows: CV=SD/average particle diameter, which can represent the width of the particle size distribution. Among them, SD: is the standard deviation (Standard Deviation), which is recorded as σ in statistics; CV is also called relative standard deviation (Coefficient of variation), and is recorded as α in statistics. Herein, the diameter of the microspheres used can be calculated by means and devices known to those skilled in the art, for example, it can be measured under a microscope and analyzed with image processing software, and can be measured by an instrument for measuring particle size (Bio-Rad T20 Cytometer) to determine, can also use the data provided by the microsphere supplier.
在一个具体实施方式中，提供了一种形成反应隔室群的方法，其中，所述载体表面涂覆有力学缓冲涂层；最优选涂覆有水凝胶涂层。在此，所述“力学缓冲涂层”应当视为“载体”的一部分，即，所述本说明书中所描述的“靶向配基”、“反应试剂”、“标签分子”都是在于载体(无涂层的母核)上涂覆完“力学缓冲涂层”之后才通过例如化学修饰连接在所述载体之上。In a specific embodiment, there is provided a method of forming a population of reaction compartments, wherein the surface of the support is coated with a mechanical buffer coating; most preferably with a hydrogel coating. Here, the "mechanical buffer coating" should be regarded as a part of the "carrier", that is, the "targeting ligand", "reactive reagent" and "label molecule" described in this specification are all on the carrier The "mechanical buffer coating" is coated on the (non-coated mother core) before being connected to the carrier by, for example, chemical modification.
作为替选，先将所述标签分子连接在载体上，随后涂覆力学缓冲涂层，最后在力学缓冲涂层之外通过例如化学修饰连接上所述反应试剂和所述靶向配基。Alternatively, the tag molecule is first linked to the carrier, then coated with a mechanical buffer coating, and finally the reaction reagent and the targeting ligand are linked outside the mechanical buffer coating by, for example, chemical modification.
作为替选，先将所述反应试剂连接在载体上，随后涂覆力学缓冲涂层，最后在力学缓冲涂层之外通过例如化学修饰连接上所述标签分子和所述靶向配基。As an alternative, the reaction reagent is first connected to the carrier, then coated with a mechanical buffer coating, and finally the label molecule and the targeting ligand are connected outside the mechanical buffer coating by, for example, chemical modification.
作为替选，先将所述靶向配基连接在载体上，随后涂覆力学缓冲涂层，最后在力学缓冲涂层之外通过例如化学修饰连接上所述反应试剂和所述标签分子。As an alternative, the targeting ligand is first linked to the carrier, then a mechanical buffer coating is applied, and finally the reaction reagent and the label molecule are linked outside the mechanical buffer coating by, for example, chemical modification.
作为替选，先将所述标签分子和反应试剂连接在载体上，随后涂覆力学缓冲涂层，最后在力学缓冲涂层之外通过例如化学修饰连接上所述靶向配基。Alternatively, the label molecule and the reaction reagent are first linked to the carrier, then coated with a mechanical buffer coating, and finally the targeting ligand is linked outside the mechanical buffer coating by, for example, chemical modification.
作为替选，先将所述标签分子和靶向配基连接在载体上，随后涂覆力学缓冲涂层，最后在力学缓冲涂层之外通过例如化学修饰连接上所述反应试剂。Alternatively, the label molecule and the targeting ligand are first linked to the carrier, then coated with a mechanical buffer coating, and finally the reaction reagent is linked outside the mechanical buffer coating through, for example, chemical modification.
作为替选，先将所述反应试剂和靶向配基连接在载体上，随后涂覆力学缓冲涂层，最后在力学缓冲涂层之外通过例如化学修饰连接上所述标签分子。As an alternative, the reaction reagent and the targeting ligand are linked to the carrier first, then a mechanical buffer coating is applied, and finally the tag molecule is linked outside the mechanical buffer coating by, for example, chemical modification.
作为替选，先将所述标签分子、靶向配基和标签分子连接在载体上，随后涂覆力学缓冲涂层。Alternatively, the label molecule, targeting ligand and label molecule are first linked to a carrier, and then a mechanical buffer coating is applied.
在一个具体实施方式中，提供了一种形成反应隔室群的方法，其中，所述连接选自共价键、金属键、离子键、范德华力、包括氢键、机械键、卤键、硫族键、亲金作用、嵌入、重叠、阳离子–π键、阴离子–π键、盐桥、非金属原子间次级键、金属原子与非金属原子间次级键、亲金作用、亲银作用、双氢键和金键的次级键。In a specific embodiment, there is provided a method of forming a population of reaction compartments, wherein the linkages are selected from covalent bonds, metallic bonds, ionic bonds, van der Waals forces, including hydrogen bonds, mechanical bonds, halogen bonds, sulfur Family bonds, aurophilic interactions, intercalation, overlapping, cation-π bonds, anion-π bonds, salt bridges, secondary bonds between non-metal atoms, secondary bonds between metal atoms and non-metal atoms, aurophilic interactions, argentophilic interactions , double hydrogen bonds and secondary bonds of gold bonds.
在一个具体实施方式中，提供了一种形成反应隔室群的方法，其中，所述靶向配基与所述载体连接是通过所述反应试剂的连接、通过所述标签分子的连接或通过接头的连 接；所述反应试剂与所述载体的连接是通过所述靶向配基的连接、通过所述标签分子的连接或通过接头的连接；所述标签分子与所述载体的连接是通过所述靶向配基的连接、通过所述反应试剂的连接或通过接头的连接。In a specific embodiment, a method for forming a group of reaction compartments is provided, wherein the linking of the targeting ligand to the carrier is through the linking of the reaction reagent, the linking of the tag molecule or the linking of the labeling molecule. The connection of the adapter; the connection of the reaction reagent and the carrier is through the connection of the targeting ligand, the connection of the label molecule or the connection of the linker; the connection of the label molecule and the carrier is through The linking of the targeting ligand, the linking through the reagent or the linking through the linker.
在一个具体实施方式中，提供了一种形成反应隔室群的方法，其中所述小分子为所述靶向配基、所述反应试剂、所述标签分子中的一项或两项以上。即，所述靶向配基、所述反应试剂、所述标签分子自身就可形成载体的一部分。In a specific embodiment, a method for forming a group of reaction compartments is provided, wherein the small molecule is one or more of the targeting ligand, the reaction reagent, and the label molecule. That is, the targeting ligand, the reaction reagent, and the tag molecule themselves can form a part of the carrier.
在一个具体实施方式中，提供了一种形成反应隔室群的方法，其中，所述媒介物是油性介质，优选含氟油性介质，或者固体介质，优选微孔板。使用油性介质作为“隔室”的“媒介物”的原因是油性介质可有效阻断带电荷的核酸分子跨越不同的“隔室“，好处是携带不同待分析物标签的反应试剂在对细胞内容进行标记后不会穿越”隔室“产物形成交叉污染。In a specific embodiment, a method for forming a group of reaction compartments is provided, wherein the medium is an oily medium, preferably a fluorinated oily medium, or a solid medium, preferably a microwell plate. The reason for using an oily medium as the "vehicle" of the "compartment" is that the oily medium can effectively block charged nucleic acid molecules from crossing different "compartments". After marking, there will be no cross-contamination of products passing through the "compartment".
在一个具体实施方式中，提供了一种形成反应隔室群的方法，其中，In a specific embodiment, a method of forming a population of reaction compartments is provided, wherein,
所述两个以上待检测物可以是相同或不相同的，The two or more substances to be detected can be the same or different,
且所述两个以上靶向反应复合物可以是相同的或者不相同的，And the two or more targeting reaction complexes can be the same or not,
当所述两个以上待检测物不相同且所述两个以上靶向反应复合物不相同时，所述靶向反应复合物包括连接于载体上对应于待检测物的标签分子。When the two or more substances to be detected are different and the two or more targeting reaction complexes are different, the targeting reaction complex includes label molecules corresponding to the substances to be detected connected to the carrier.
在一个具体实施方式中，在上述形成反应隔室群的方法中，所述两个以上待检测物是相同的，且所述两个以上靶向反应复合物是相同的。In a specific embodiment, in the above-mentioned method for forming a group of reaction compartments, the two or more substances to be detected are the same, and the two or more targeted reaction complexes are the same.
在一个具体实施方式中，在上述形成反应隔室群的方法中，所述两个以上待检测物是相同的，且所述两个以上靶向反应复合物是不同的。In a specific embodiment, in the above method for forming a group of reaction compartments, the two or more substances to be detected are the same, and the two or more target reaction complexes are different.
在一个具体实施方式中，在上述形成反应隔室群的方法中，所述两个以上待检测物是不同的，且所述两个以上靶向反应复合物是相同的。In a specific embodiment, in the above method for forming a group of reaction compartments, the two or more substances to be detected are different, and the two or more targeted reaction complexes are the same.
在一个具体实施方式中，在上述形成反应隔室群的方法中，所述两个以上待检测物是不同的，且所述两个以上靶向反应复合物是不同的；且所述靶向反应复合物包括连接于所述载体上对应于所述待检测物的标签分子。In a specific embodiment, in the above-mentioned method for forming a group of reaction compartments, the two or more substances to be detected are different, and the two or more targeting reaction complexes are different; and the targeting The reaction complex includes a label molecule corresponding to the detection substance connected to the carrier.
实施例部分Example part
实施例一 带有力学缓冲涂层的载体(包裹有水凝胶层的微球)的制备Example 1 Preparation of a carrier (microsphere wrapped with a hydrogel layer) with a mechanical buffer coating
聚苯乙烯微球(纳微科技Uni-PS-30)经过Chemgenes Corp订制可形成带DNA编码(标签)的逆转录polyT引物(反应试剂)的微球。Polystyrene microspheres (Nanomicro Technology Uni-PS-30) can be customized by Chemgenes Corp to form microspheres with DNA coded (label) reverse transcription polyT primers (reaction reagents).
1、吸取1mL乙醇保存中的DNA编码polyT微球到2ml中离心管中；1. Pipette 1mL of DNA-encoded polyT microspheres stored in ethanol into a 2ml medium centrifuge tube;
2、移去上清，加入0.2ml PBS缓冲液(Teknova)冲洗微球，重复2遍；2. Remove the supernatant, add 0.2ml PBS buffer (Teknova) to wash the microspheres, repeat 2 times;
3、吸取10μl微球，在细胞计数板中计数；3. Take 10 μl of microspheres and count in the cell counting plate;
4、将剩余微球全部加入实施例一所述的聚丙烯酰胺(PAA)溶液中，同时充分混匀；4. Add all the remaining microspheres into the polyacrylamide (PAA) solution described in Example 1, and mix well at the same time;
5、吸取步骤4中全部溶液加入含氟油相中，使用200μl移液枪吹打3～4min(图3A)。5. Pipette the entire solution in step 4 and add it to the fluorine-containing oil phase, and pipette with a 200 μl pipette for 3-4 minutes (Figure 3A).
6、室温静置4h。6. Stand at room temperature for 4 hours.
7、用移液管除去下层的油。7. Remove the lower layer of oil with a pipette.
8、向2ml收集管中加入100uL HFE油中20％(v/v)全氟辛醇(PFO)作为化学破乳剂；8. Add 20% (v/v) perfluorooctyl alcohol (PFO) in 100uL HFE oil to a 2ml collection tube as a chemical demulsifier;
9、混合后，旋转2ml收集管2000g 2分钟。移液去除PFO/HFE上清液。重复1x；9. After mixing, spin the 2ml collection tube at 2000g for 2 minutes. Remove the PFO/HFE supernatant by pipetting. Repeat 1x;
10、移液去除上清，加入1ml含0.2％Tween20的PBS去除表面活性剂/溶液。重复2x；10. Remove the supernatant by pipetting, add 1ml of PBS containing 0.2% Tween20 to remove the surfactant/solution. Repeat 2x;
11、加入1mL TEBST缓冲液(20mM Tris-HCl pH 8.0,274mM NaCl,5.4mM KCl,20mM EDTA,0.2％Triton X-100)，混合均匀。11. Add 1mL TEBST buffer (20mM Tris-HCl pH 8.0, 274mM NaCl, 5.4mM KCl, 20mM EDTA, 0.2% Triton X-100), mix well.
12、3000x g旋转3分钟。移液去除上清液。重复3x。12. Spin at 3000x g for 3 minutes. Remove the supernatant by pipetting. Repeat 3x.
13、重悬于1ml TEBST中(图3B)。此微球在此溶液可在4℃下无限期保存。13. Resuspend in 1ml TEBST (Figure 3B). The microspheres can be stored indefinitely at 4°C in this solution.
在包裹有水凝胶层的微球上(载体)，可预先或之后装载靶向配基、标签或反应试剂。On the microsphere (carrier) coated with the hydrogel layer, the targeting ligand, label or reaction reagent can be loaded in advance or afterwards.
实施例二(一) 结合物的形成Example 2 (1) Formation of conjugates
称重10mg 1-乙基-(3-二甲基氨基丙基)碳二亚胺(缩写：EDC)和5mg N-羟基琥珀酰亚胺(NHS)，分别用200μL MES缓冲液(pH 6.5)溶解。Weigh 10 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (abbreviation: EDC) and 5 mg of N-hydroxysuccinimide (NHS), respectively with 200 μL of MES buffer (pH 6.5) dissolve.
将实施例一产生的带有羧基水凝胶涂层的微球和EDC/NHS溶液混合，在室温下旋转15分钟Mix the microspheres with carboxyl hydrogel coating and EDC/NHS solution produced in Example 1, and rotate at room temperature for 15 minutes
·以5rpm将微球旋转孵育5分钟Incubate the microspheres with rotation at 5 rpm for 5 minutes
·去除上层清液· Remove the supernatant
·在500μL PBS缓冲液中重悬微球Resuspend microspheres in 500 μL PBS buffer
·将200μL的500mg/ml Anti human CD20(Biolegend)添加到珠状溶液中Add 200 μL of 500mg/ml Anti human CD20 (Biolegend) to the bead solution
·在RT下旋转微球2小时· Spin the microspheres for 2 hours at RT
·以每分钟5转的速度向下旋转微球5分钟Spin the microspheres down at 5 rpm for 5 minutes
·计数细胞8.0*10^5细胞与带抗体修饰(靶向配基)的水凝胶包裹微球孵育在200μL PBS缓冲液与2mM EDTA，旋转溶液30分钟Count cells 8.0*10^5 cells and incubate with antibody-modified (targeting ligand) hydrogel-coated microspheres in 200 μL PBS buffer and 2 mM EDTA, rotate the solution for 30 minutes
·收集抗体修饰好的微球，去除所有上清计数上清中细胞，观察靶向配基修饰微球与细胞形成1：1结合物(图4A)Collect the antibody-modified microspheres, remove all the supernatant and count the cells in the supernatant, and observe the formation of a 1:1 conjugate between the targeting ligand-modified microspheres and the cells (Figure 4A)
由于细胞结合了微球，在单位体积中同时有微球和细胞的概率打破了泊松分布。Since cells bind microspheres, the probability of having both microspheres and cells per unit volume breaks the Poisson distribution.
实施例二(二) 载体尺寸对载体与细胞结合的影响(作为对比例)Embodiment 2 (two) The impact of carrier size on the combination of carrier and cell (as a comparative example)
之前的文献和商业化产品说明中有很多亲和力磁珠分选细胞的报导(例如：美天旎的
Figure PCTCN2022134738-appb-000001
MicroBead技术Technology、赛默飞的亲和力Dyna Bead产品等：https://www.miltenyibiotec.com/US-en/products/macs-cell-separation/cell-sep aration-reagents/realease-microbeads.html)，这些磁珠尺径小于细胞(即处于100nm～2.7微米)。 There are many reports of affinity magnetic bead sorting cells in previous literature and commercial product descriptions (for example: Miltenyi's
Figure PCTCN2022134738-appb-000001
MicroBead Technology, Thermo Fisher’s affinity Dyna Bead products, etc.: https://www.miltenyibiotec.com/US-en/products/macs-cell-separation/cell-sep aration-reagents/realease-microbeads.html), These magnetic beads are smaller in diameter than cells (ie, in the range of 100 nm to 2.7 microns).
本实施例为对比例，实验目的：验证微球尺寸如果太小，会对其与细胞结合的影响。其数量大于细胞数量时，会形成多个微球结合一个细胞的现象。This example is a comparative example, and the purpose of the experiment is to verify that if the size of the microsphere is too small, it will affect the combination with the cells. When the number is greater than the number of cells, a phenomenon in which multiple microspheres combine with one cell will be formed.
活化微球的步骤：Steps to activate microspheres:
1、在500ul MES缓冲液中计数带羧基的微球(Polysciences Inc.cat#18133)。该微球上的羧基活化后能够与细胞结合(靶向配基)：5.0*10^6个微球(90uL PBS缓冲液中的10uL微球(检测微球浓度：6.72x10^5/mL，转移～744uL微球→5.0x10^6)1. Count the carboxyl-containing microspheres (Polysciences Inc. cat#18133) in 500ul MES buffer. The carboxyl groups on the microspheres can be combined with cells after activation (targeting ligand): 5.0*10^6 microspheres (10uL microspheres in 90uL PBS buffer (detection microsphere concentration: 6.72x10^5/mL, Transfer ~744uL microspheres→5.0x10^6)
2、添加7.7mg(标准方案0.5mg/100uL)EDC和8.0mgNHS至744uL微球重悬MES缓冲液；2. Add 7.7mg (standard protocol 0.5mg/100uL) EDC and 8.0mg NHS to 744uL microsphere resuspension MES buffer;
3、混合微球和EDC/NHS溶液，在室温下旋转溶液20分钟，在10分钟间隔期间轻轻混合；3. Mix the microspheres and EDC/NHS solution, swirling the solution for 20 minutes at room temperature, mixing gently during 10-minute intervals;
4、以500rpm的速度将微球旋转5分钟(或使用磁性分离器)；4. Spin the microspheres at a speed of 500rpm for 5 minutes (or use a magnetic separator);
5、去除上清液(在此过程中丢失一些微球)；5. Remove the supernatant (lose some microspheres in the process);
6、用750uL DPBS缓冲液洗涤两次，pH＝7.06. Wash twice with 750uL DPBS buffer, pH=7.0
7、在300uL DPBS缓冲液中重悬；测量微球浓度(1.35x10^6/mL)，添加额外的99uL以在DPBS中达到1x10^6微球/mL7. Resuspend in 300uL DPBS buffer; measure the microsphere concentration (1.35x10^6/mL), add an additional 99uL to reach 1x10^6 microspheres/mL in DPBS
8、将100uL离心分装到4个试管中(对于各种微球：细胞比例进行测试，分别是1：1、1：5)8. Centrifuge and divide 100uL into 4 test tubes (test for various microsphere: cell ratios, respectively 1:1, 1:5)
细胞结合的步骤：Steps for cell binding:
1、将溶液置换到在DPBS+1mM EDTA buffer中进行细胞微球结合反应1. Replace the solution into DPBS+1mM EDTA buffer for cell microsphere binding reaction
2、在室温下旋转微球2小时；过程中t＝0min,30min,1h取样检查2. Rotate the microspheres at room temperature for 2 hours; during the process t=0min, 30min, 1h sampling inspection
3、显微镜下观察3. Observation under a microscope
本实验为对比例，推导出的结论是：微球尺寸大于细胞是非常重要的。This experiment is a comparative example, and the conclusion drawn is that it is very important that the size of the microsphere is larger than the cell.
因为如本对比例所示：当微球如果尺径小于细胞，则会出现多个微球结合一个细胞、甚至多个微球与多个微球形成聚团的现象(图4B)，无法打破细胞分析时细胞端的泊松分布；而当微球直径大于等于细胞直径时(在此实施例中是大于细胞直径的两倍)，位阻效应 导致微球不易与第二个细胞结合，从而打破泊松分布。Because as shown in this comparative example: if the diameter of the microsphere is smaller than the cell, multiple microspheres will combine with one cell, or even multiple microspheres and multiple microspheres will form aggregates (Figure 4B), which cannot be broken. Poisson distribution at the cell end during cell analysis; and when the microsphere diameter is greater than or equal to the cell diameter (in this example, it is greater than twice the cell diameter), the steric hindrance effect makes it difficult for the microsphere to combine with the second cell, thus breaking Poisson distribution.
实施例三(一) 带有分离功能(磁性纳米颗粒层)的载体(微球)的制备以及与细胞的结合实验Example 3 (1) Preparation of carrier (microsphere) with separation function (magnetic nanoparticle layer) and binding experiment with cells
1、用实施例2的方法制备带磁性内核的水凝胶包裹微球(图5A-C)：该微球结构为聚苯乙烯内核，涂有磁性纳米颗粒，外层为带羧基的聚丙烯酰胺水凝胶层(PAA溶液配方和聚合方式见实施例二)1. Prepare hydrogel-wrapped microspheres with a magnetic inner core by the method of Example 2 (Fig. 5A-C): the microsphere structure is a polystyrene inner core, coated with magnetic nanoparticles, and the outer layer is polypropylene with carboxyl groups Amide hydrogel layer (see Example 2 for PAA solution formulation and polymerization method)
2、加入5mg EDC和1.2mg NHS到100μL MES缓冲液中；2. Add 5mg EDC and 1.2mg NHS to 100μL MES buffer;
3、旋转15分钟，在RT旋转下降5点微球转5分钟去除上层清液；3. Rotate for 15 minutes, spin down the microsphere at RT for 5 minutes and spin for 5 minutes to remove the supernatant;
4、重悬微球500mg/μL PBS缓冲液；4. Resuspend the microspheres in 500mg/μL PBS buffer;
5、加入200抗-human CD2(Biolegend，靶向配基)旋转的微球；5. Add 200 microspheres of anti-human CD2 (Biolegend, targeting ligand) to rotate;
6、在室温下微球孵育2小时；6. Incubate the microspheres at room temperature for 2 hours;
7、微球旋转离心5分钟(图5D)；7. Spin and centrifuge the microspheres for 5 minutes (Figure 5D);
8、计数8.0*10^5个微球和不同数量的细胞在200μL PBS缓冲液，旋转30分钟；测量细胞数的步骤为：8. Count 8.0*10^5 microspheres and different numbers of cells in 200 μL PBS buffer and rotate for 30 minutes; the steps for measuring the number of cells are:
a、测量的Jurkat细胞浓度(7.62x10^5mL)*3.5mLa. Measured Jurkat cell concentration (7.62x10^5mL)*3.5mL
b、在100uL PBS+2mM EDTA中重悬细胞以达到1x10^6/100uL～1x10^7细胞/mLb. Resuspend cells in 100uL PBS+2mM EDTA to reach 1x10^6/100uL～1x10^7 cells/mL
c、对于各种微球：细胞比例的实验，将细胞等分到每个rxn管中(100uL Jurkat细胞,40uL Jurkat细胞+160uL DPBS,20uL Jurkat细胞+180uL DPBS)。c. For experiments with various microsphere:cell ratios, aliquot cells into each rxn tube (100uL Jurkat cells, 40uL Jurkat cells+160uL DPBS, 20uL Jurkat cells+180uL DPBS).
9、使用磁铁收集微球，可观察到细胞与微球1：1结合(图5E)，这说明，在细胞端打破了泊松分布规律。9. Use a magnet to collect the microspheres, and it can be observed that the cells and the microspheres are combined at a ratio of 1:1 (Figure 5E), which indicates that the Poisson distribution law is broken at the cell end.
实验结论：当微球与细胞预结合时，可提高细胞捕获效率，打破泊松分布。Experimental conclusion: When the microspheres are pre-combined with cells, the cell capture efficiency can be improved and the Poisson distribution can be broken.
实施例三(二) 载体形状对载体与细胞结合的影响Example 3 (2) The influence of the shape of the carrier on the combination of the carrier and the cells
本实验目的是证明碗形微球可与细胞结合，并利用位阻效应打破泊松分布规律。The purpose of this experiment is to prove that the bowl-shaped microspheres can bind to cells and break the Poisson distribution law by using the steric hindrance effect.
第一步：碗形微球的制造(载体)Step 1: Fabrication of bowl-shaped microspheres (carrier)
对于分散相，将含有6.3％w/v生物素化聚乙二醇、4.5％w/v明胶和1.5％w/v苯基-2,4,6-三甲基苯甲酰基次膦酸锂(LAP)溶解在DPBS中的均匀前体溶液注入平行化以8μL/min的速度分步乳化。将包含2％Pico-Surf的Novec 7500以16μL/min注入连续相。生成单相的PEG/明胶液滴流过浸入4℃水浴中的Tygon管(0.03"I.D.，0.0625"O.D，Murdock)，用于PEG和明胶的温度诱导相分离。调整管道的长度以确 保完全相分离(约60厘米，孵育10分钟)。将相分离的液滴流引入浸没在4℃水浴中的PDMS储液器中，并在储液器出口区域附近暴露于紫外线(200mW/cm 2)1-3秒以进行聚合。在紫外线照射下，可光交联的PEG组分形成聚合物网络，而明胶组分保持未聚合状态(详见图6A)。收集交联颗粒，并在一系列洗涤步骤中去除油和富含明胶的液滴。首先通过移液枪去除表面活性剂油。连续添加20％v/v全氟辛醇(PFO，Sigma)在Novec7500中的溶液和milli-q水以破乳并将颗粒转移至水相。样品以2500×g离心1分钟，去除油相。微球用Novec 7500洗涤两次以去除剩余的PFO和表面活性剂。用移液器去除油层后，残留的油用己烷(Sigma)洗涤三次。然后将样品用70％乙醇洗涤三次，然后用毫定量水洗涤两次以去除明胶。使用前通过在70％乙醇中孵育过夜对颗粒进行灭菌。碗形颗粒在显微镜下(如图6B所示)。 For the dispersed phase, will contain 6.3% w/v biotinylated polyethylene glycol, 4.5% w/v gelatin and 1.5% w/v lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) A homogeneous precursor solution dissolved in DPBS was injected parallelized and emulsified step by step at a rate of 8 μL/min. Novec 7500 containing 2% Pico-Surf was injected into the continuous phase at 16 μL/min. Single-phase generated PEG/gelatin droplets flowed through Tygon tubes (0.03" ID, 0.0625" OD, Murdock) immersed in a 4°C water bath for temperature-induced phase separation of PEG and gelatin. Adjust the length of the tubing to ensure complete phase separation (~60 cm, 10 min incubation). The phase-separated droplet stream was introduced into a PDMS reservoir submerged in a water bath at 4 °C and exposed to UV light (200 mW/cm 2 ) for 1–3 seconds near the outlet region of the reservoir for polymerization. Upon UV irradiation, the photocrosslinkable PEG component formed a polymer network, while the gelatin component remained unpolymerized (see Figure 6A for details). The cross-linked particles are collected and oil and gelatin-rich droplets are removed in a series of washing steps. First remove the surfactant oil by pipetting. A solution of 20% v/v perfluorooctyl alcohol (PFO, Sigma) in Novec 7500 and milli-q water was added sequentially to break the emulsion and transfer the particles to the aqueous phase. The samples were centrifuged at 2500 x g for 1 min to remove the oily phase. The microspheres were washed twice with Novec 7500 to remove remaining PFO and surfactant. After removing the oil layer with a pipette, the residual oil was washed three times with hexane (Sigma). The samples were then washed three times with 70% ethanol, followed by two milliliters of water to remove gelatin. Sterilize the pellets by incubating overnight in 70% ethanol before use. Bowl-shaped particles under the microscope (as shown in Figure 6B).
第二步：碗形微球与细胞结合Step 2: Binding of bowl-shaped microspheres to cells
碗形微球在使用前用AF350链霉亲和素(Fisher Scientific)修饰(靶向配基)。碗形微球用细胞培养基洗涤并浓缩在锥形管中。计算浓缩碗形微球的量以用碗形颗粒单层覆盖整个孔区域，对于55μm的碗形微球，分别为4.8μL/cm 2。将计算量的碗形颗粒转移并分散在孔板中。将用Jurkat细胞以目标细胞与碗形微球以1:1的比例接种到具有碗形微球单层的孔中。多次吸取孔中的溶液，使细胞和微球均匀分散在整个孔区域，并使细胞粘附在碗形微球上。为了去除未附着的细胞，用20μm可逆细胞过滤器(CellTrics)对样品进行过滤，并用由0.5％牛血清白蛋白(BSA)、1％P/S和含0.05％Pluronic F-127的DPBS组成的“洗涤缓冲液”洗涤。接下来，翻转细胞过滤器并用洗涤缓冲液洗涤，以在锥形管中回收碗形颗粒。将碗形颗粒离心以去除洗涤缓冲液。通过显微镜观察碗形颗粒与细胞结合(详见图6C)。 Bowl-shaped microspheres were modified with AF350 streptavidin (Fisher Scientific) (targeting ligand) prior to use. The bowl is washed with cell culture medium and concentrated in a conical tube. The amount of concentrated bowl-shaped microspheres calculated to cover the entire pore area with a monolayer of bowl-shaped particles is 4.8 μL/cm 2 for 55 μm bowl-shaped microspheres, respectively. Transfer and disperse the calculated amount of bowl-shaped particles in the orifice plate. The wells with a monolayer of bowl-shaped microspheres were seeded with Jurkat cells at a ratio of 1:1 between target cells and bowl-shaped microspheres. Pipette the solution in the well several times to evenly disperse the cells and microspheres throughout the well area and make the cells adhere to the bowl-shaped microspheres. To remove non-attached cells, samples were filtered through a 20 μm reversible cell strainer (CellTrics) and washed with a solution consisting of 0.5% bovine serum albumin (BSA), 1% P/S, and DPBS containing 0.05% Pluronic F-127. "Wash Buffer" washes. Next, invert the cell strainer and wash with wash buffer to recover the bowl-shaped particles in the conical tube. Centrifuge the bowl-shaped pellet to remove wash buffer. The association of bowl-shaped particles with cells was observed by microscopy (see Figure 6C for details).
实验结论：当微球为碗形时，可更好地实现微球与细胞1：1结合(详见图6C)。相较于实施例二(二)，位阻效应导致微球不易与第二个细胞结合，从而打破泊松分布。Experimental conclusion: When the microspheres are bowl-shaped, the 1:1 combination of microspheres and cells can be better achieved (see Figure 6C for details). Compared with Example 2 (2), the steric hindrance effect makes it difficult for the microspheres to combine with the second cell, thereby breaking the Poisson distribution.
实施例四 通用性载体的制备：载体(微球)带有多种靶向配基：抗人-CD298、人β2微球蛋白、小鼠CD45以及小鼠MHC class I抗体。Example 4 Preparation of a universal carrier: the carrier (microsphere) carries a variety of targeting ligands: anti-human-CD298, human β2 microglobulin, mouse CD45 and mouse MHC class I antibody.
(这些都是抗体吧？加上个“抗”字比较好？对)(These are antibodies, right? Would it be better to add the word "anti"? Yes)
1、商业化微球(Chemgenes Corp.Cat#Macosko-2011-10)含有带DNA编码的polyT捕获寡核苷酸(反应试剂和标签)。通过液滴微流控对其包覆带羧基的水凝胶涂层，在500μL MES缓冲液中对微球进行计数8.0*10^5个微球1. Commercial microspheres (Chemgenes Corp. Cat#Macosko-2011-10) contain DNA-encoded polyT capture oligonucleotides (reaction reagents and labels). It is coated with a carboxyl-containing hydrogel coating by droplet microfluidics, and the microspheres are counted in 500 μL MES buffer 8.0*10^5 microspheres
2、将5mg EDC和1.2mg NHS添加到100μL MES缓冲液中2. Add 5mg EDC and 1.2mg NHS to 100μL MES buffer
3、混合微球和EDC/NHS溶液，在室温下旋转溶液15分钟3. Mix the microspheres and EDC/NHS solution, and rotate the solution for 15 minutes at room temperature
4、以5rpm的速度离心微球5分钟4. Centrifuge the microspheres at 5 rpm for 5 minutes
5、去除上清液5. Remove the supernatant
6、在500μL PBS缓冲液中重悬微球6. Resuspend the microspheres in 500 μL PBS buffer
7、将200μL的500mg/mL人-CD298,人β2微球蛋白,小鼠CD45以及小鼠MHC class I添加到微球溶液中(靶向配基)7. Add 200 μL of 500 mg/mL human-CD298, human β2 microglobulin, mouse CD45 and mouse MHC class I to the microsphere solution (targeting ligand)
8、在室温下旋转微球2小时8. Spin the microspheres at room temperature for 2 hours
9、以5rpm的速度离心微球5分钟9. Centrifuge the microspheres at 5 rpm for 5 minutes
10、计数细胞8.0*10^5个细胞在200μL含2mM EDTA的PBS缓冲液中与珠一起孵育，旋转溶液30分钟10. Count cells 8.0*10^5 cells and incubate with beads in 200 μL PBS buffer containing 2mM EDTA, swirl the solution for 30 minutes
11、使用磁铁收集微球，去除所有上清液(图2C)。11. Collect the microspheres using a magnet and remove all supernatant (Figure 2C).
实验结论：通过给含DNA编码(标签)的逆转录引物(反应试剂)的水凝胶包裹微球(载体)装载多种抗体(靶向配基)，可进一步扩大微球的细胞结合的范围。Experimental conclusion: By loading various antibodies (targeting ligands) on the hydrogel-coated microspheres (carriers) containing DNA-encoded (labeled) reverse transcription primers (reaction reagents), the range of cell binding of the microspheres can be further expanded .
实施例五 水凝胶微球载体的制备(载体为不含包裹层的实心水凝胶微球)Example 5 Preparation of hydrogel microsphere carrier (the carrier is a solid hydrogel microsphere without a coating layer)
如无特别说明，下述实施例中化学试剂均采购自Sigma Aldrich。Unless otherwise specified, the chemical reagents in the following examples were purchased from Sigma Aldrich.
1、制备由6.2％丙烯酰胺，0.18％N,N'-亚甲基双(丙烯酰胺)，0.3％过硫酸铵，0.6％丙烯酸钠组成的聚丙烯酰胺(PAA)溶液。将此溶液装入带有28G针头的1ml注射器(BD公司)中。1. Prepare a polyacrylamide (PAA) solution consisting of 6.2% acrylamide, 0.18% N,N'-methylenebis(acrylamide), 0.3% ammonium persulfate, and 0.6% sodium acrylate. This solution was filled into a 1 ml syringe (BD) with a 28G needle.
2、在氢氟醚(HFE)油中制备5％(w/w)含氟表面活性剂和1％N,N,nn-四甲基乙基二胺(TEMED)组成的不溶连续相，用于液滴的生成和稳定。将溶液装入新的1ml注射器。2. Prepare an insoluble continuous phase composed of 5% (w/w) fluorosurfactant and 1% N,N,nn-tetramethylethyldiamine (TEMED) in hydrofluoroether (HFE) oil, and use for droplet formation and stabilization. Fill the solution into a new 1ml syringe.
3、在15ml的收集管中收集1ml的液滴，在室温下培养3小时用于聚合。孵育后，用移液管除去下层的油。3. Collect 1ml droplets in a 15ml collection tube and incubate at room temperature for 3 hours for polymerization. After incubation, remove the underlying oil with a pipette.
4、向15ml收集管中加入1ml HFE油中20％(v/v)全氟辛醇(PFO)作为化学破乳剂4. Add 20% (v/v) perfluorooctyl alcohol (PFO) in 1ml HFE oil to a 15ml collection tube as a chemical demulsifier
5、混合后，旋转15ml收集管2000x g 2分钟。移液去除PFO/HFE上清液。重复1x。5. After mixing, spin the 15ml collection tube at 2000x g for 2 minutes. Remove the PFO/HFE supernatant by pipetting. Repeat 1x.
6、移液去除上清，加入1ml含0.2％Tween20的PBS去除表面活性剂/溶液。重复2x。6. Remove the supernatant by pipetting, add 1ml of PBS containing 0.2% Tween20 to remove the surfactant/solution. Repeat 2x.
7、加入1mL TEBST缓冲液(20mM Tris-HCl pH 8.0,274mM NaCl,5.4mM KCl,20mM EDTA,0.2％Triton X-100)，混合均匀。7. Add 1mL TEBST buffer (20mM Tris-HCl pH 8.0, 274mM NaCl, 5.4mM KCl, 20mM EDTA, 0.2% Triton X-100), mix well.
8、3000x g旋转3分钟。移液去除上清液。重复3x。8. Spin at 3000x g for 3 minutes. Remove the supernatant by pipetting. Repeat 3x.
9、重悬于1ml TEBST中(图7A)。此微球在此溶液可在4℃下无限期保存。9. Resuspend in 1ml TEBST (Figure 7A). The microspheres can be stored indefinitely at 4°C in this solution.
实施例六 所制备的微球所携带的反应试剂和标签分子可合并为同一物质(即5’-Acrydite-CTA CAC GAC GCT CTT CCG ATC T NNNNT 28VN引物兼有标签分子与反应试剂双重功能)；序列中，V代表G、C、A的混合；N代表A、G、C、T四种碱基的混合。 Example 6 The reaction reagents and label molecules carried by the prepared microspheres can be combined into one substance (that is, the 5'-Acrydite-CTA CAC GAC GCT CTT CCG ATC T NNNNT 28 VN primer has dual functions of label molecules and reaction reagents) ; In the sequence, V represents a mixture of G, C, and A; N represents a mixture of A, G, C, and T.
应用实施例六中的微流控***制备由40％丙烯酸-N,N'-亚甲基双(丙烯酰 胺)(37.5:1)，10％过硫酸铵，10X Gel buffer(100uL 1M的Tris HCl pH＝7.5；20uL pH＝8的0.5M EDTA；150uL 1M NaCl；730uL ddH 2O)，2X Oligo solution(100uM)，的聚丙烯酰胺(PAA)溶液形成的水凝胶微球(配方如图7B)。其中Oligo序列为：5’-Acrydite-CTA CAC GAC GCT CTT CCG ATC T NNNNT 28VN(SEQ ID NO：1)。 Using the microfluidic system in Example 6 to prepare 40% acrylic acid-N,N'-methylenebis(acrylamide) (37.5:1), 10% ammonium persulfate, 10X Gel buffer (100uL 1M Tris HCl pH=7.5; 20uL pH=8 0.5M EDTA; 150uL 1M NaCl; 730uL ddH 2 O), 2X Oligo solution (100uM), polyacrylamide (PAA) solution formed hydrogel microspheres (formula shown in Figure 7B ). Wherein the Oligo sequence is: 5'-Acrydite-CTA CAC GAC GCT CTT CCG ATC T NNNNT 28 VN (SEQ ID NO: 1).
装载含低聚物的溶液，用于生成液滴。当液滴不稳定时，拍摄液滴照片，确定液滴大小。运行后，取出油，加入TEMED，油中含有0.4％TEMED，为将油倒回珠状乳剂中，放置在37度使微球凝固(图7C)。Load the oligomer-containing solution for droplet generation. When the droplet is unstable, take a picture of the droplet to determine the droplet size. After the run, the oil was removed and TEMED was added. The oil contained 0.4% TEMED. To pour the oil back into the bead emulsion, the microspheres were solidified at 37 degrees (FIG. 7C).
在此，一个物质(引物)兼有两种功能(标签分子和反应试剂)。Here, one substance (primer) has two functions (label molecule and reaction reagent).
实施例七 靶向细胞的水凝胶微球的制备，并与细胞结合形成复合物Example 7 Preparation of hydrogel microspheres targeting cells and combining with cells to form complexes
取实施例七制备的水凝胶微球(羧基来自丙烯酸单体)，在500μL MES缓冲液中对微球进行计数8.0*10^5个微球Get the hydrogel microspheres prepared in Example 7 (the carboxyl group comes from the acrylic acid monomer), and count 8.0*10^5 microspheres in 500 μL of MES buffer
·将5mg EDC和1.2mg NHS添加到100μL MES缓冲液中Add 5 mg EDC and 1.2 mg NHS to 100 μL MES buffer
·混合微球和EDC/NHS溶液，在室温下旋转溶液15分钟· Mix the microspheres and EDC/NHS solution, swirling the solution for 15 minutes at room temperature
·以5rpm的速度离心微球5分钟Centrifuge the microspheres at 5 rpm for 5 minutes
·去除上清液· Remove the supernatant
·在500μL PBS缓冲液中重悬微球Resuspend microspheres in 500 μL PBS buffer
·将200μL含各种反应试剂的微球分别与500mg/mL抗CD45抗体(BioLegend，靶向配基)混合Mix 200 μL microspheres containing various reaction reagents with 500 mg/mL anti-CD45 antibody (BioLegend, targeting ligand)
·在室温下旋转微球2小时(图8A)Spin the microspheres for 2 hours at room temperature (Figure 8A)
·以5rpm的速度离心微球5分钟Centrifuge the microspheres at 5 rpm for 5 minutes
·计数细胞8.0*10^5个PBMC细胞在200μL含2mM EDTA的PBS缓冲液中与珠一起孵育，旋转溶液30分钟Count cells 8.0*10^5 PBMC cells Incubate with beads in 200 μL PBS buffer containing 2mM EDTA, swirl the solution for 30 minutes
·使用重力离心收集微球，去除所有上清液(图8B)Collect the microspheres by gravity centrifugation and remove all supernatant (Figure 8B)
此实施例证明，当反应试剂与标签分子属于同一种物质时，携带了靶向配基的微球也能与细胞结合。This example proves that when the reaction reagent and the label molecule belong to the same substance, the microspheres carrying the targeting ligand can also bind to cells.
实施例八 隔室的高通量形成及单细胞RNA测序(使用实施例四的同时结合人与鼠通用抗体的微球)Example 8 High-throughput formation of compartments and single-cell RNA sequencing (using the microspheres combined with human and mouse universal antibodies in Example 4)
通过液滴微流控设备(图10A)用媒介物(含氟油性介质)来包裹微球(图2C)与细胞复合物。如图10A所示，使用定制设计的微流体装置将单个细胞与微球的复合物(来自于实施例四)一起包裹到液滴中。该装置在它们分成离散的液滴之前连接两路水相。层流防止在液滴形成之前混合两种水相输入，一路流相包含裂解液和其他缓冲液，另一路流相包含悬浮在缓冲液中的细胞与DNA编码微球复合物。液滴反应隔室是使用使用90μ m高度和80μm宽喷嘴的微流控芯片产生的。使用的典型流速为：富含裂解液的相～200μL/h，富含细胞与DNA编码微球复合物的相～100μL/h和液滴稳定油(2％PEG-PFPE 2HFE7500)～600μL/h(图10B)。 The microspheres (Fig. 2C) and cell complexes were encapsulated with a vehicle (fluorine-containing oily medium) through a droplet microfluidic device (Fig. 10A). As shown in FIG. 10A , the complex of single cells and microspheres (from Example 4) was encapsulated into droplets using a custom-designed microfluidic device. The device connects two aqueous phases before they separate into discrete droplets. Laminar flow prevents mixing of two aqueous input phases prior to droplet formation, one flow phase containing lysate and other buffers, and the other flow phase containing cells in complex with DNA-encoded microspheres suspended in buffer. Droplet reaction compartments were generated using a microfluidic chip using a 90 μm height and 80 μm wide nozzle. Typical flow rates used are: ~200 μL/h for lysate-rich phase, ~100 μL/h for cell-enriched phase complexed with DNA-encoded microspheres and ~600 μL/h for droplet stabilization oil (2% PEG-PFPE 2 HFE7500). h (FIG. 10B).
微流体装置组件列表List of Microfluidic Device Components
(1)倒置显微镜(1) Inverted Microscope
(2)压力控制器+3流量传感器/三个注射泵(2) Pressure controller + 3 flow sensors / three syringe pumps
(3)3个falcon管/3mL注射器(3) 3 falcon tubes/3mL syringes
(4)磁力搅拌***(4) Magnetic stirring system
(5)用于实验装置元件连接的微流体导管(5) Microfluidic conduits for connection of experimental device components
(6)微流体配件和连接器(6) Microfluidic accessories and connectors
(7)PDMS共流微流体液滴生成装置(7) PDMS co-flow microfluidic droplet generation device
(8)用于beads的100微米细胞过滤器(8) 100 micron cell strainer for beads
(9)用于细胞的40微米细胞过滤器(9) 40 micron cell strainer for cells
(10)计数室(10) counting room
物料清单：Bill of Materials:
Figure PCTCN2022134738-appb-000002
Figure PCTCN2022134738-appb-000002
Figure PCTCN2022134738-appb-000003
Figure PCTCN2022134738-appb-000003
表1单细胞转录组测序的反应物料清单Table 1 List of reaction materials for single-cell transcriptome sequencing
1.用30mL 100％EtOH X2清洗微球(来自于实施例8)1. Wash the microspheres with 30mL 100% EtOH X2 (from Example 8)
2.用30mL TE/TW buffer X2清洗微球2. Wash the microspheres with 30mL TE/TW buffer X2
3.将微球重悬于10ml TE/TW缓冲液中3. Resuspend the microspheres in 10ml TE/TW buffer
4.通过100um过滤器(@4℃长期保存)4. Pass through a 100um filter (@4°C for long-term storage)
5.对于单细胞转录组分析，用生理盐水冲液清洗微球与细胞形成的复合物5. For single-cell transcriptome analysis, wash the complexes formed by microspheres and cells with saline flush
6.在生理盐水中重悬清洗微球与细胞形成的复合物，浓度约120个复合物/μL6. Resuspend the complex formed by washing microspheres and cells in physiological saline, the concentration is about 120 complexes/μL
7.用6.4mm磁力搅拌器将细胞-微球复合物的悬浮液装入3ml塑料注射器中；裂解液从另一个针管中装入(10％Sarkosyl的生理盐水)7. Use a 6.4mm magnetic stirrer to fill the suspension of the cell-microsphere complex into a 3ml plastic syringe; the lysate is loaded from another needle tube (10% Sarkosyl normal saline)
8.将液滴生成油装入10ml塑料注射器8. Fill the droplet generating oil into a 10ml plastic syringe
9.将注射器连接到具有0.38mm内径管的125um同流装置9. Connect the syringe to a 125um co-flow device with 0.38mm inner diameter tubing
10.通过连续磁力搅拌使微球-细胞复合物保持悬浮状态10. Keeping Microsphere-Cell Complexes in Suspension by Continuous Magnetic Stirring
将收集的液滴放置在显微镜下观察(图10C)Place the collected droplets under a microscope for observation (Figure 10C)
11.添加30ml 6X SSC@室温11. Add 30ml 6X SSC @ room temperature
12.用P1000移液器从每个等分液滴底部除去油12. Remove oil from the bottom of each aliquot drop with a P1000 pipette
13.加入600ul全氟-1-辛醇13. Add 600ul perfluoro-1-octanol
14.用手大力震荡管子～20s14. Shake the tube vigorously by hand for ~20s
15.旋转1,000g～1min15. Rotate 1,000g～1min
16.将样品放在冰上以减少退火的mRNA解离16. Keep samples on ice to reduce dissociation of annealed mRNA
17.除去油水层上方约5ml的上清液17. Remove about 5ml of supernatant above the oil-water layer
18.用另外30ml的6X SSC缓冲液在室温清洗微球18. Wash the microspheres with another 30ml of 6X SSC buffer at room temperature
19.将水层转移到新管中19. Transfer the aqueous layer to a new tube
20.旋转1,000g～1min20. Rotate 1,000g～1min
21.将微球转移到不粘的1.5ml管中21. Transfer the microspheres to a non-stick 1.5ml tube
22.用1ml 6X SSC缓冲液洗涤两次22. Wash twice with 1ml 6X SSC buffer
23.用300ul 5x Maxima H-RT缓冲液清洗一次23. Wash once with 300ul 5x Maxima H-RT buffer
24.对于90,000颗微球，添加200ul RT混合液：24. For 90,000 microspheres, add 200ul RT mixture:
i.1X Maxima RT缓冲液i.1X Maxima RT Buffer
ii.4％Ficoll PM-400ii. 4% Ficoll PM-400
iii.1mM dNTPiii. 1mM dNTP
iv.1U/ul核糖核酸酶抑制剂iv.1U/ul ribonuclease inhibitor
v.2.5uM的TSOv.2.5uM TSO
vi.10U/ul Maxima H-RTvi.10U/ul Maxima H-RT
25.室温孵育～30min25. Incubate at room temperature for ~30min
26. 42℃孵育～90min26. Incubate at 42°C for ~90min
27.用1ml 1X TE清洗一次：含0.5％十二烷基硫酸钠27. Wash once with 1ml 1X TE: with 0.5% sodium lauryl sulfate
28.用1ml TE/TW缓冲液清洗微球两次28. Wash the microspheres twice with 1ml TE/TW buffer
29.用10mM Tris pH＝7.5清洗微球一次29. Wash the microsphere once with 10mM Tris pH=7.5
30.将微球重悬于200ul核酸外切酶I混合液中30. Resuspend the microspheres in 200ul exonuclease I mixture
1X核酸外切酶I缓冲液(NEB)1X Exonuclease I Buffer (NEB)
1U/ul核酸外切酶I(NEB)1U/ul Exonuclease I (NEB)
31.孵育@37℃～45min31. Incubate @37℃～45min
32.用1mL TE/SDS清洗一次32. Wash once with 1mL TE/SDS
33.用1mL TE/TW洗涤两次33. Wash twice with 1 mL TE/TW
34.用ddH 2O清洗一次 34. Wash once with ddH 2 O
35.重悬于ddH 2O 35. Resuspend in ddH 2 O
36.将1000个微球的等分试样通过PCR扩增，每份体积为50uL36. PCR amplified aliquots of 1000 microspheres with a volume of 50uL each
1x Hifi HotStart Readymix(Roche)1x Hifi HotStart Readymix(Roche)
0.8uM TSO引物0.8uM TSO primer
37.PCR程序如下：37. The PCR program is as follows:
温度temperature 时间time 循环cycle 循环数number of cycles
95℃95°C 3min3min  the  the
 the  the  the  the
98℃98°C 20s20s 11 44
65℃65°C 45s45s 11  the
72℃72°C 3min3min 11  the
 the  the  the  the
98℃98°C 20s20s 22 Xx
67℃67°C 20s20s 22  the
72℃72°C 3min3min 22  the
 the  the  the  the
72℃72°C 5min5min  the  the
表2 cDNA扩增的PCR反应程序Table 2 PCR reaction program for cDNA amplification
38.制备3'-末端cDNA片段进行测序38. Preparation of 3'-end cDNA fragments for sequencing
a.将四等分的600pg cDNA放到到四个标准Nextera XT标记反应中a. Place quartered 600pg cDNA into four standard Nextera XT labeling reactions
b.使用P5-TSO-hybrid和Nextera_N701代替试剂盒提供的寡核苷酸b. Use P5-TSO-hybrid and Nextera_N701 instead of the oligonucleotides provided in the kit
Figure PCTCN2022134738-appb-000004
Figure PCTCN2022134738-appb-000004
Figure PCTCN2022134738-appb-000005
Figure PCTCN2022134738-appb-000005
表3文库构建的PCR反应程序Table 3 PCR reaction program for library construction
该扩增文库送至第三方测序服务公司进行测序。The amplified library was sent to a third-party sequencing service company for sequencing.
实施例九 分析实施例八的实验结果Embodiment nine Analyzing the experimental result of embodiment eight
申请人使用Seurat等生物信息学工具处理原始测序结果，整体表达低或线粒体比例高的细胞被去除。对于聚类，使用了主成分分析，然后进行k均值聚类以识别不同的细胞状态。t-分布式随机邻域嵌入(t-SNE)用于可视化细胞聚类。对于元聚类，来自所有宿主的表达矩阵使用Seurat合并了四个实验。少于2000个细胞转录本被排除在分析之外，少于100个的细胞检测到的独特基因被排除在分析之外(图10D)。Applicants use bioinformatics tools such as Seurat to process raw sequencing results, and cells with low overall expression or high mitochondrial ratio are removed. For clustering, principal component analysis was used, followed by k-means clustering to identify distinct cell states. t-Distributed Stochastic Neighborhood Embedding (t-SNE) was used to visualize cell clusters. For meta-clustering, expression matrices from all hosts were pooled from four experiments using Seurat. Cell transcripts with fewer than 2000 cells were excluded from the analysis, and cells with fewer than 100 unique genes detected were excluded from the analysis (Fig. 10D).
实施例十 微球带有另一种靶向配基(链霉亲和素)Example 10 Microspheres with another targeting ligand (streptavidin)
本实验目的：微球的水凝胶涂层上连接链霉亲和素，与表面修饰生物素的细胞进行结合。实验流程如图11A所示：The purpose of this experiment: Streptavidin is attached to the hydrogel coating of the microspheres to bind to the cells with surface-modified biotin. The experimental process is shown in Figure 11A:
第一步：微球的水凝胶珠功能化Step 1: Hydrogel Bead Functionalization of Microspheres
1、__1.12_μL 1×PBST中的微球(__8.90x10^3_微球/μL)～5000-10k微球(载体)1. Microspheres in __1.12_μL 1×PBST (__8.90x10^3_microspheres/μL)～5000-10k microspheres (carrier)
使用600μl MES缓冲液(pH＝4.7,0.1M)重悬水凝胶珠去除缓冲液。Use 600 μl of MES buffer (pH=4.7, 0.1 M) to resuspend the hydrogel beads to remove the buffer.
2、EDC(10mg)和NHS(5mg)分别溶解于200μl MES缓冲液中，并在洗涤后的微球中加入储备液以达到最终浓度2. EDC (10mg) and NHS (5mg) were dissolved in 200μl MES buffer respectively, and the stock solution was added to the washed microspheres to reach the final concentration
物质substance 重量(g)Weight (g)
EDCEDC 0.010.01
磺基-NHSSulfo-NHS 0.0050.005
表4微球活化试剂Table 4 Microsphere Activation Reagents
样本sample
200uL含10mg/ml EDC的MES200uL MES containing 10mg/ml EDC
200uL含5mg/ml Sulfo-NHS的MES200uL MES containing 5mg/ml Sulfo-NHS
3、溶液涡旋30s，置于水平振荡器(650rpm)室温下振荡30min。3. The solution was vortexed for 30s, placed on a horizontal shaker (650rpm) and shaken at room temperature for 30min.
4、离心在PBST中的颗粒，来回漂洗四次4. Centrifuge the pellet in PBST, rinse back and forth four times
5、向颗粒中加入NeutrAvidin原液(5mg/ml)使蛋白终浓度为1mg/mL，水平摇床孵育2.5h(650rpm，室温)。5. Add NeutrAvidin stock solution (5mg/ml) to the granules to make the final protein concentration 1mg/mL, and incubate on a horizontal shaker for 2.5h (650rpm, room temperature).
 the 样本sample 对照组control group
NeutrAvidin_μlNeutrAvidin_μl 100100 //
荧光链霉亲和素(2μg/ml)_μLFluorescent streptavidin (2μg/ml)_μL // 22
表5链霉亲和素的修饰条件Modification conditions of table 5 streptavidin
6、颗粒漂洗4次后保存在PBST中4℃可长期保存(图11B)，该微球上的NeutrAvidin为靶向配基，可与下游修饰了生物素的细胞靶向结合。6. The particles were washed 4 times and stored in PBST at 4°C for long-term storage (Figure 11B). The NeutrAvidin on the microspheres is a targeting ligand, which can be targetedly combined with downstream cells modified with biotin.
第二步：293T细胞生物素化(全程4℃操作)Step 2: Biotinylation of 293T cells (operate at 4°C throughout the process)
1、哺乳动物细胞在含有0.04％BSA的1mL PBS中用钙黄绿素AM(ThermoFisher#C3099)染色。1. Mammalian cells were stained with Calcein AM (ThermoFisher #C3099) in 1 mL PBS containing 0.04% BSA.
2、准备90-95％融合细胞的6x6cm 2培养皿 2. Prepare a 6x6cm 2 Petri dish with 90-95% confluent cells
3、用冰冷的PBS(pH 8.0)洗涤细胞3次，去除细胞中含胺的培养基和蛋白质。快速除去PBS(注意：如果细胞在悬浮液中生长，每毫升步骤3中制备的生物素溶液使用1×10^6个细胞。每个标记反应的细胞总数不要超过4×10^7个)3. Wash the cells 3 times with ice-cold PBS (pH 8.0) to remove the amine-containing medium and proteins in the cells. Quickly remove PBS (Note: If cells are growing in suspension, use 1 x 10 cells per mL of biotin solution prepared in step 3. Do not exceed 4 x 10 total cells per labeling reaction)
注意：不要让PBS与细胞保持接触超过5秒，以防止细胞变圆和脱落。Note: Do not keep the PBS in contact with the cells for more than 5 s to prevent the cells from rounding and falling off.
4、将细胞轻轻刮入溶液中，将细胞悬浮在1ml PBS(pH 8.0)中，浓度约为25×10^6个细胞/mL。4. Gently scrape the cells into the solution, suspend the cells in 1ml PBS (pH 8.0), the concentration is about 25×10^6 cells/mL.
5、1000_μL 293T cell in 1×PBS(__1.90x106_cell per mL)～10^6cell5. 1000_μL 293T cell in 1×PBS(__1.90x106_cell per mL)～10^6cell
6、每毫升细胞悬液加入1.0mg Sulfo-NHS-SS-Biotin(不称量)试剂(产生～2mM生物素试剂)。6. Add 1.0 mg Sulfo-NHS-SS-Biotin (no weighing) reagent per ml of cell suspension (yielding ~2mM biotin reagent).
注意：在使用前立即制备10mM的Sulfo-NHS-SS-Biotin溶液，如下所示：Note: Prepare a 10 mM solution of Sulfo-NHS-SS-Biotin immediately before use as follows:
Sulfo-NHS-SS-Biotin：将1mg放入微量管中，加入164μL超纯水。Sulfo-NHS-SS-Biotin: Put 1mg into a microcupule and add 164μL of ultrapure water.
将反应混合物在4℃温度下孵育30分钟。The reaction mixture was incubated at 4°C for 30 minutes.
注意：在4℃下进行孵育可能会降低生物素试剂的活性内化。Note: Incubation at 4 °C may reduce the active internalization of the biotin reagent.
7、将PBS加入细胞沉淀中，用血清移液管轻轻上下吸取细胞3次。以500×g离心1分钟并弃去上清液。7. Add PBS to the cell pellet, and gently pipette the cells up and down 3 times with a serological pipette. Centrifuge at 500 x g for 1 min and discard the supernatant.
注意：淬灭反应并去除多余的生物素试剂和副产物(图11C)。NOTE: Quench the reaction and remove excess biotin reagent and by-products (Figure 11C).
第三步：链霉亲和素化的微球捕获293T细胞Step 3: Streptavidinized microspheres capture 293T cells
1、所有实验中的微球和细胞均重悬在100％PBS。1. Microspheres and cells in all experiments were resuspended in 100% PBS.
水凝胶包裹微球的浓度(/mL)Concentration of hydrogel-coated microspheres (/mL) 细胞浓度(/mL)Cell concentration (/mL)
1000010000 10001000
表6细胞与微球孵育时的浓度Concentration when table 6 cells were incubated with microspheres
2、4℃温育30min，轻轻搅动。2. Incubate at 4°C for 30 minutes with gentle agitation.
3、总溶液体积≈1mL。在成像之前，将溶液轻轻涡旋以去除颗粒中的非贴壁细胞。3. The total solution volume is ≈1mL. Prior to imaging, the solution was gently vortexed to remove non-adherent cells from the pellet.
细胞结合后形成微球与细胞1:1捕获(图11D)Cells combine to form microspheres and capture cells 1:1 (Figure 11D)
第四步：对捕获了细胞的水凝胶包裹微球进行包裹。Step 4: Encapsulate the hydrogel-encapsulated microspheres that have captured the cells.
对水凝胶包裹的微球进行吹打，形成液滴隔室Pipetting hydrogel-encapsulated microspheres to form droplet compartments
 the 油相oil phase 水相water box 总体积total capacity 吹打时间blow time
样本sample 60ul60ul 30ul30ul 90ul90ul 3min3min
表7液滴生成的条件Table 7 Conditions for droplet generation
水相中含有29U/mL蛋白酶K(NEB#P8107S)和70mM DTT(Sigma#D9779)，并混合10次移液器冲程。在将结合了细胞的微球与溶液混合时要小心避免产生气泡。将280μL 0.5％离子型Krytox的HFE 7500油65添加到细胞-珠复合物中，并以3000RPM的速度水平涡旋15秒，然后使用定制涡旋仪垂直涡旋2分钟。从乳液下方去除油，使得剩余的油少于100μL。在裂解之前，微球-细胞的乳液在C-Chip一次性血细胞计数器(Fisher Scientific#DHCN015)上进行二次采样，每个子样本每个视野包含3.5μL乳液。芯片以2倍放大倍数在明场中成像。剩余的乳液在PCR热循环仪(Eppendorf Mastercycler Pro)上在65℃下进行酶裂解35分钟，盖子温度设置为105℃。裂解完成后，使用具有470nm激发光的Nikon 2000显微镜(Thorlab M470L5)捕获荧光图像(图11E)。The aqueous phase contained 29U/mL proteinase K (NEB #P8107S) and 70mM DTT (Sigma #D9779) and mixed 10 pipette strokes. Take care to avoid creating air bubbles when mixing the cell-bound microspheres with the solution. Add 280 µL of 0.5% ionic Krytox's HFE 7500 oil 65 to the cell-bead complex and vortex horizontally at 3000RPM for 15 s, then vertically for 2 min using a custom vortexer. Remove oil from beneath the emulsion such that less than 100 µL of oil remains. Prior to lysis, the microsphere-cell emulsion was subsampled on a C-Chip disposable hemocytometer (Fisher Scientific #DHCN015), with each subsample containing 3.5 μL of emulsion per field. Chips were imaged in brightfield at 2X magnification. The remaining emulsion was enzymatically cleaved on a PCR thermocycler (Eppendorf Mastercycler Pro) at 65°C for 35 minutes with the lid temperature set at 105°C. After lysis was complete, fluorescence images were captured using a Nikon 2000 microscope (Thorlab M470L5) with 470 nm excitation light (FIG. 11E).
实施例结果总结：Example result summary:
本申请列举了三种靶向反应复合物的生产工艺。这些靶向反应复合物都可以与待检测物(细胞)结合：A)包裹水凝胶涂层的聚合物微球；B)一般球形水凝胶微球；C)碗形水凝胶微球。The application lists three production techniques of targeting reaction complexes. These targeted reaction complexes can be combined with the analytes (cells): A) polymer microspheres coated with hydrogel; B) generally spherical hydrogel microspheres; C) bowl-shaped hydrogel microspheres .
应用本申请的靶向反应复合物，可实现单细胞分析，在打破泊松分布的同时提高细胞分析的效率。该流程涉及两个关键步骤：1.以细胞为代表的待分析物与靶向反应复合物进行孵育，并通过靶向配基进行有效结合；2.通过液滴微流控或枪头吹打，可高通量形成反应隔室，打破细胞分析的双泊松分布；Applying the targeted reaction complex of the present application can realize single-cell analysis, and improve the efficiency of cell analysis while breaking the Poisson distribution. The process involves two key steps: 1. The analyte represented by cells is incubated with the target reaction complex, and is effectively combined with the target ligand; 2. Through droplet microfluidics or tip pipetting, High-throughput reaction compartments can be formed to break the double Poisson distribution of cell analysis;
在隔室内进行高通量反应时，靶向反应复合物上的标签分子可对待分析物进行标记。其他公共反应物可通过混合溶液进入反应隔室。Tag molecules on the targeting reaction complex can be used to label the analytes during high-throughput reactions within the compartment. Other common reactants can enter the reaction compartment through the mixed solution.
实施例论证了球形微球尺寸必须大于细胞才能更高效地打破细胞结合的概率(双泊松分布)，当微球数量为细胞数量的10-20倍时，可进一步实现微球与细胞的1:1结合 (具体见图8A和图8B)；而当使用碗形的水凝胶微球时，位阻效应更为显著，微球数量无需比细胞数量过量太多。The embodiment demonstrates that the size of spherical microspheres must be larger than that of cells to break the probability of cell binding more efficiently (double Poisson distribution). When the number of microspheres is 10-20 times the number of cells, the 1 :1 combination (see Figure 8A and Figure 8B for details); and when bowl-shaped hydrogel microspheres are used, the steric hindrance effect is more significant, and the number of microspheres does not need to be too much in excess of the number of cells.
一旦靶向反应复合物与细胞结合完成，高通量生成反应隔室(生成隔室后的图片应该是最重要的：9A为最重要的概念图；5E、8B、6C、9B为反应隔室形成前；9C为反应隔室形成后，如9C所示：液滴中没有找到一个反应隔室，仅有细胞、但没有微球的；意味着所有的细胞都被分析了，意味着打破泊松分布)可对细胞进行各种深度分析(包括细胞裂解、mRNA捕获或单细胞测序)。Once the target reaction complex is combined with the cells, the high-throughput generation of the reaction compartment (the picture after generating the compartment should be the most important: 9A is the most important concept map; 5E, 8B, 6C, and 9B are the reaction compartments Before the formation; 9C is after the formation of the reaction compartment, as shown in 9C: no reaction compartment was found in the droplet, there were only cells, but no microspheres; it meant that all the cells were analyzed, which meant that the moorings were broken Loose distribution) of cells for various depth analysis including cell lysis, mRNA capture or single cell sequencing.
尽管以上结合附图对本申请的实施方案进行了描述，但本申请并不局限于上述的具体实施方案和应用领域，上述的具体实施方案仅仅是示意性的、指导性的，而不是限制性的。本领域的普通技术人员在本说明书的启示下和在不脱离本申请权利要求所保护的范围的情况下，还可以做出很多种的形式，这些均属于本申请保护之列。Although the embodiments of the present application have been described above in conjunction with the accompanying drawings, the present application is not limited to the above-mentioned specific embodiments and application fields, and the above-mentioned specific embodiments are only illustrative, instructive, and not restrictive . Those skilled in the art can also make many forms under the enlightenment of this description and without departing from the protection scope of the claims of the application, and these all belong to the protection list of the application.

Claims (16)

  1. 一种形成反应隔室群的方法，所述方法包括：A method of forming a population of reaction compartments, the method comprising:
    准备待检测物群，其由两个以上待检测物构成；Prepare a group of substances to be detected, which is composed of more than two substances to be detected;
    准备靶向反应复合物群，所述靶向反应复合物群由两个以上靶向反应复合物构成，所述靶向反应复合物包括载体、连接于载体上对应于待检测物的靶向配基、连接于载体上对应于待检测物的反应试剂、可选地包括连接于载体上对应于待检测物的标签分子；Prepare a targeting reaction complex group, the targeting reaction complex group is composed of more than two targeting reaction complexes, the targeting reaction complex includes a carrier, a targeting ligand corresponding to the substance to be detected connected to the carrier base, a reaction reagent corresponding to the substance to be detected connected to the carrier, optionally including a label molecule connected to the carrier corresponding to the substance to be detected;
    使用所述待检测物群与所述靶向反应复合物群接触，形成结合物群；using the population of substances to be detected to be contacted with the population of targeting reaction complexes to form a population of conjugates;
    由所述结合物自身或者由媒介物包裹所述结合物形成反应隔室群。A population of reaction compartments is formed by the conjugate itself or by surrounding the conjugate with a vehicle.
  2. 如权利要求1所述的方法，其中，所述待分析物选自蛋白质、核酸、糖、脂、代谢物、多肽、细菌、病毒、细胞器以及细胞中的一种或两种以上，以及由它们形成的复合体，最优选所述待分析物为细胞。The method according to claim 1, wherein the analyte is selected from one or more of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles and cells, and by them The complex formed, most preferably the analyte is a cell.
  3. 如权利要求1所述的方法，其中所述载体的形状选自正方体形、四面体形，球形、椭球形、章鱼形、碗形、红血球形的一种或两种以上；最优选为碗形和/或红血球形。The method according to claim 1, wherein the shape of the carrier is selected from one or more of cube shape, tetrahedron shape, sphere, ellipsoid, octopus shape, bowl shape, red blood cell shape; most preferably bowl shape and / or red blood cells.
  4. 如权利要求3所述的方法，其中所述载体的数量为所述待检测物数量的4倍及以上，优选为所述待检测物数量的10倍及以上，最优选为所述待检测物数量的10倍至30倍。The method according to claim 3, wherein the quantity of the carrier is 4 times or more than the quantity of the substance to be detected, preferably 10 times or more than the quantity of the substance to be detected, most preferably the quantity of the substance to be detected 10 to 30 times the quantity.
  5. 如权利要求3或4所述的方法，其中所述载体为的最大直径为待检测物的1倍以上，优选其最大直径为待检测物的3倍及以上，最优选其最大直径为待检测物的4倍至10倍。The method according to claim 3 or 4, wherein the maximum diameter of the carrier is more than 1 time of the substance to be detected, preferably 3 times and above the maximum diameter of the substance to be detected, and most preferably its maximum diameter is 3 times that of the substance to be detected. 4 to 10 times that of objects.
  6. 如权利要求3至5的任一项所述的方法，其中所述载体由聚合物或小分子构成，优选为聚合物载体，进一步优选为聚苯乙烯载体聚苯乙烯或水凝胶制成，进一步该聚合物载体由聚苯乙烯制成，最优选该聚合物带有铁磁性或顺磁性。The method according to any one of claims 3 to 5, wherein the carrier is made of a polymer or a small molecule, preferably a polymer carrier, more preferably a polystyrene carrier made of polystyrene or hydrogel, Further the polymer carrier is made of polystyrene, most preferably the polymer is ferromagnetic or paramagnetic.
  7. 如权利要求1所述的方法，其中，所述靶向配基可以是天然或人造的，选自包括锁核酸和XNA的核酸及其类似物、适配体、小肽、多肽、糖基化肽、多糖、可溶性受体、 类固醇、荷尔蒙、促***原、抗原、超级抗原、生长因子、细胞因子、瘦素、病毒蛋白、细胞黏附分子、趋化因子、链霉亲和素及其类似物、生物素及其类似物、抗体、抗体片段、单链可变片段(scFv)、纳米抗体、T细胞受体、主要组织相容性复合体(MHC)分子、抗原肽-MHC分子复合物(pMHC)、DNA结合蛋白、RNA结合蛋白、细胞内或细胞表面受体配基中的一种或两种以上以及它们共同形成的多重配基、复合配基、耦合配基。The method according to claim 1, wherein the targeting ligand can be natural or artificial, selected from nucleic acids including locked nucleic acids and 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 their 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 ( One or more of pMHC), DNA binding protein, RNA binding protein, intracellular or cell surface receptor ligands, and multiple ligands, composite ligands, and coupling ligands formed by them.
  8. 如权利要求1所述的方法，其中，所述标签分子选自天然或人造的信息分子，包括：寡核苷酸条形码、寡肽或多肽条形码、由天然碱基与LNA、PNA、XNA等非天然碱基构成的核苷酸、寡糖或多糖条形码、生色团(chromophoric group)和助色团(auxochrome group)、金属原子或离子、分子量可区分的小分子、嵌段聚合物、聚合物与骨架分子共价连接物中的一种或两种以上以及它们之间形成的复合体。The method according to claim 1, wherein the label molecule is selected from natural or artificial information molecules, including: oligonucleotide barcodes, oligopeptide or polypeptide barcodes, natural bases and LNA, PNA, XNA, etc. Nucleotides composed of natural bases, oligosaccharide or polysaccharide barcodes, chromophoric groups and auxochrome groups, metal atoms or ions, small molecules with distinguishable molecular weights, block polymers, polymers One or more than two covalently linked substances with the backbone molecules and the complex formed between them.
  9. 如权利要求1所述的方法，其中，所述反应试剂是寡聚核苷酸引物、酶或小分子。The method of claim 1, wherein the reaction reagent is an oligonucleotide primer, an enzyme or a small molecule.
  10. 如权利要求1所述的方法，其中，所述载体直径粒径分布系数CV小于20％。The method according to claim 1, wherein the carrier diameter particle size distribution coefficient CV is less than 20%.
  11. 如权利要求10所述的方法，其中，所述载体表面涂覆有力学缓冲涂层；进一步优选涂覆有水凝胶涂层；最优选该水凝胶涂层带有铁磁性或顺磁性颗粒。The method according to claim 10, wherein the surface of the carrier is coated with a mechanical buffer coating; further preferably coated with a hydrogel coating; most preferably the hydrogel coating has ferromagnetic or paramagnetic particles .
  12. 如权利要求1所述的方法，其中，所述连接选自共价键、金属键、离子键、范德华力、包括氢键、机械键、卤键、硫族键、亲金作用、嵌入、重叠、阳离子-π键、阴离子-π键、盐桥、非金属原子间次级键、金属原子与非金属原子间次级键、亲金作用、亲银作用、双氢键和金键的次级键。The method of claim 1, wherein the linkage is selected from the group consisting of covalent bonds, metallic bonds, ionic bonds, van der Waals forces, including hydrogen bonds, mechanical bonds, halogen bonds, chalcogen bonds, alkophilic interactions, intercalation, overlapping , cation-π bond, anion-π bond, salt bridge, secondary bond between non-metal atoms, secondary bond between metal atom and non-metal atom, auphilic interaction, argentophilic interaction, secondary hydrogen bond and gold bond key.
  13. 如权利要求1所述的方法，其中，所述靶向配基与所述载体连接是通过所述反应试剂的连接、通过所述标签分子的连接或通过接头的连接；所述反应试剂与所述载体的连接是通过所述靶向配基的连接、通过所述标签分子的连接或通过接头的连接；所述标签分子与所述载体的连接是通过所述靶向配基的连接、通过所述反应试剂的连接或通过接头的连接。The method according to claim 1, wherein the targeting ligand is connected to the carrier through the connection of the reaction reagent, the connection of the label molecule or the connection of the linker; the connection of the reaction reagent and the The connection of the carrier is through the connection of the targeting ligand, the connection of the label molecule or the connection of the linker; the connection of the label molecule and the carrier is through the connection of the targeting ligand, through The coupling of the reagents or via a linker.
  14. 如权利要求6所述的方法，其中所述小分子为所述靶向配基、所述反应试剂、 所述标签分子中的一项或两项以上。The method according to claim 6, wherein the small molecule is one or more of the targeting ligand, the reaction reagent, and the label molecule.
  15. 如权利要求1所述的方法，其中，所述媒介物是油性介质，优选含氟油性介质，或者固体介质，优选微孔板。The method according to claim 1, wherein the medium is an oily medium, preferably a fluorinated oily medium, or a solid medium, preferably a microwell plate.
  16. 如权利要求1所述的方法，其中所述两个以上待检测物可以是相同或不相同的，且所述两个以上靶向反应复合物可以是相同的或者不相同的，当所述两个以上待检测物不相同且所述两个以上靶向反应复合物不相同时，所述靶向反应复合物包括连接于所述载体上对应于所述待检测物的标签分子。The method according to claim 1, wherein the two or more substances to be detected can be the same or not, and the two or more targeting reaction complexes can be the same or not, when the two When the more than one detection substance is different and the two or more targeting reaction complexes are different, the targeting reaction complex includes a label molecule corresponding to the detection substance connected to the carrier.
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