WO2023093886A1 - 靶向反应复合物及其在靶向多重检测中的用途 - Google Patents

靶向反应复合物及其在靶向多重检测中的用途 Download PDF

Info

Publication number
WO2023093886A1
WO2023093886A1 PCT/CN2022/134736 CN2022134736W WO2023093886A1 WO 2023093886 A1 WO2023093886 A1 WO 2023093886A1 CN 2022134736 W CN2022134736 W CN 2022134736W WO 2023093886 A1 WO2023093886 A1 WO 2023093886A1
Authority
WO
WIPO (PCT)
Prior art keywords
reaction
targeting
analyte
carrier
cell
Prior art date
Application number
PCT/CN2022/134736
Other languages
English (en)
French (fr)
Inventor
张经纬
景祥益
梁雪
Original Assignee
复旦大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 复旦大学 filed Critical 复旦大学
Publication of WO2023093886A1 publication Critical patent/WO2023093886A1/zh

Links

Images

Classifications

    • 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
    • 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
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • 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
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the application relates to the field of high-throughput chemical analysis, in particular to a targeted reaction complex and its use in targeted multiplex detection.
  • 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 types of cells; Compared with the traditional method of flow cytometry and single-cell analysis, this method simplifies the process of high-throughput analysis of multiple types of cells.
  • a target and analyze the target reaction complex of analyte it comprises:
  • a targeting ligand for the analyte linked to the carrier
  • reaction reagent linked to the carrier for detecting the analyte
  • a tag molecule corresponding to the analyte linked to the carrier is A tag molecule corresponding to the analyte linked to the carrier.
  • the targeted reaction complex according to item 1 wherein the analyte is selected from one or both of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles and cells In the above, and complexes formed therefrom, it is most preferred that the analyte is a cell.
  • the carrier is composed of a polymer or a small molecule, preferably a polymer carrier, more preferably a polystyrene carrier, and more preferably the carrier has a diameter of 1 ⁇ m -100 ⁇ m
  • the shape of the carrier is selected from one or more of cube shape, tetrahedron shape, sphere, ellipsoid, bowl shape, red blood cell shape; most preferably bowl shape and/or red blood cell shape; further preferably the carrier
  • the diameter particle size distribution coefficient CV is less than 20%.
  • the surface of the carrier is coated with a mechanical buffer coating; most preferably, the surface of the carrier is coated with a hydrogel coating.
  • 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, glycosylated peptides, polysaccharides, soluble receptors, steroids, hormones, mitogens, antigens, superantigens, growth factors, cytokines, leptin, viral proteins, cell adhesion molecules, chemokines, streptavidin Biotin and its analogs, biotin and its analogs, antibodies, antibody fragments, single chain variable fragments (scFv), nanobodies, T cell receptors, major histocompatibility complex (MHC) molecules, antigenic peptides- One or two or more of MHC molecular complex (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
  • the label molecule is selected from natural or artificial information molecules, including: oligonucleotide barcodes, oligopeptide or polypeptide barcodes, composed of natural bases and LNA, Nucleotides composed of unnatural bases such as PNA and XNA, oligosaccharide or polysaccharide barcodes, chromophoric groups and auxochrome groups, metal atoms or ions, small molecules with distinguishable molecular weights, blocks One or more of polymers, covalent linkages between polymers and backbone molecules, and complexes formed between them.
  • natural or artificial information molecules including: oligonucleotide barcodes, oligopeptide or polypeptide barcodes, composed of natural bases and LNA, Nucleotides composed of unnatural bases such as PNA and XNA, oligosaccharide or polysaccharide barcodes, chromophoric groups and auxochrome groups, metal atoms or ions, small molecules with distinguishable molecular weights,
  • reaction reagent is an oligonucleotide primer, an enzyme or a small molecule.
  • connection is selected from covalent bonds, metallic bonds, ionic bonds, van der Waals forces, including hydrogen bonds, mechanical bonds, halogen bonds, chalcogen bonds, Interaction, intercalation, overlapping, cation– ⁇ bond, anion– ⁇ bond, salt bridge, secondary bond between nonmetal atoms, secondary bond between metal atom and nonmetal atom, aurophilic interaction, argentophilic interaction, double hydrogen bond and The secondary key of the golden key.
  • the targeting reaction complex 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 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 connection of the targeting ligand
  • the targeting reaction complex according to item 4 wherein the small molecule constituting the carrier is one or more of the targeting ligand, the reaction reagent, and the tag molecule.
  • a group of targeting reaction complexes for targeting and analyzing more than two analytes comprising two or more targeting reaction complexes according to any one of items 1-10, wherein,
  • the targeting ligands included in each reaction complex are different from each other;
  • each reaction complex comprises different reagents from each other;
  • each reaction complex comprises different tag molecules from each other.
  • the conjugate forms a reaction compartment by itself or forms a reaction compartment around the conjugate through a medium; the analyte is labeled according to the reaction between the label molecule and the reaction reagent; optionally based on the The label is used to analyze the analyte.
  • the medium is an oily medium, preferably a fluorinated oily medium, or a solid medium, preferably a microwell plate.
  • a reaction compartment comprising:
  • a reaction complex according to any one of items 1 to 10 targeting said analyte is provided.
  • reaction compartment according to item 17 further comprising a medium surrounding a conjugate formed by binding of at least one of said reaction complexes to said analyte.
  • each reaction compartment comprises an analyte and a reaction complex, the analyte and reaction complex being bound within the reaction compartment or separate state.
  • a group of reaction compartments comprising two or more reaction compartments according to item 17 or 18.
  • the targeting ligands included in the reaction complexes in each reaction compartment are different from each other;
  • reaction complexes in each reaction compartment comprise different reaction reagents from each other;
  • reaction complexes in each reaction compartment comprise different tag molecules from each other.
  • Adopting the technical scheme of the present application while realizing high-throughput analysis of multiple types of analytes (such as cells), the one-to-one pairing problem of analytes (single cells) and carriers (single microspheres) is solved , while increasing the co-capture rate of the medium (droplets) on cells and microspheres, which solves the low efficiency problem caused by the "double Poisson distribution" phenomenon.
  • erythrocyte-shaped and bowl-shaped carriers such as microspheres
  • their steric hindrance effect is used to further ensure the realization of "one-to-one pairing of single cell and single microsphere".
  • the targeting complex group used in this application has at least two reaction reagents, realizing multiplex "high-throughput analysis".
  • Figure 1A shows a schematic diagram of the composition of the targeting reaction complex according to the present application, wherein the label can be composed of two parts: a "second label” containing the information of the "targeting ligand", and a label for different analytes (such as cells) "tag molecule";
  • Figure 1B shows the hydrogel-encapsulated microspheres: DNA-encoded microspheres with targeted analysis can be targeted to different subpopulations in mixed cells for single-cell analysis after encapsulation in hydrogel;
  • Figure 1C shows that the transparent microspheres can carry PCR primers targeting human or mouse cells on the hydrogel layer, combined with ligands targeting human or mouse, to realize the analysis of cells of different species.
  • Fig. 2A shows a schematic diagram of the formation of target reaction complex groups of more than two analytes
  • Fig. 2B shows a schematic diagram of the preformed combination of multiple analytes and target reaction complex groups.
  • Figure 3 shows a schematic flow diagram of forming a reaction compartment surrounded by a high-throughput medium after the analyte and the targeted reaction complex form a conjugate.
  • Figure 4A shows a schematic flow diagram of the application of multiple targeting reaction complexes to analyze complex systems
  • Figure 4B shows a schematic diagram of the formation of target reaction complexes and immune cell subsets respectively
  • Figure 4C shows DNA-encoded microspheres encapsulated from hydrogel Prep-to-immune system multiplex workflow.
  • Figure 5A shows how B cell and T cell specific primers are loaded onto B cell assay beads and T cell assay beads, respectively;
  • Figure 5B shows the results of staining polyT sequences with probes with FAM
  • Figure 5C shows the results of staining the BCR sequence with a probe with Cy3
  • Figure 5D shows the resulting graph of the staining of water
  • Figure 5E shows the results of staining microspheres with probes containing non-specific sequences.
  • Figure 6A shows the results of staining polyT sequences with probes with FAM
  • Figure 6B shows the results of staining the TCR sequence with a probe with Cy3
  • Figure 6C shows the resulting graph of the staining of water
  • Figure 6D shows the results of staining microspheres with probes containing non-specific sequences.
  • Figure 7 shows the operation diagram of hydrogel encapsulation of microspheres by microfluidics.
  • Fig. 8A shows the operation diagram of the microfluidic device system to generate droplets encapsulating the target capture carrier and cell conjugate
  • Figure 8B shows that the first fluid (phase I solution, the combination of targeted capture carrier and cell), the second fluid (phase II solution, reverse transcription solution), Continuous phase (the carrier oil is a fluorinated oil and contains a surfactant, eg PFPE-PEG-PFPE (perfluoropolyether-polyethylene glycol-perfluoropolyether) triblock copolymer).
  • the carrier oil is a fluorinated oil and contains a surfactant, eg PFPE-PEG-PFPE (perfluoropolyether-polyethylene glycol-perfluoropolyether) triblock copolymer).
  • Figure 9 shows the library after fragmentation of cDNA amplification products.
  • FIG. 10A shows immune system single-cell transcriptome data
  • Figure 10B shows the dendrogram and TCR two-dimensional map data of single-cell TCR
  • FIG. 10C shows the D50 data of single cell TCR
  • Figure 10D shows dendrogram data of single-cell BCRs
  • Figure 10E shows the unique CDR3 distribution of single-cell BCRs.
  • Figure 11 shows the results of incubation binding of hydrogel-coated microspheres containing targeting ligands to target cells.
  • Figure 12 shows the results of oscillating emulsification, high throughput to form independent compartments; after separation, the droplets are tiled for PCR reaction.
  • Figure 13A shows the results of qPCR analysis in independent droplets of human and mouse after PCR thermal cycle
  • Figure 13B shows the real-time detection of droplets, and the expression of cellular mRNA in each droplet presents different Ct values
  • Fig. 13C shows the statistical results of Ct values of qPCR of different GAPDH genes of human and mouse in the droplet.
  • Figure 14A shows that the analyte and the target reaction complex remain bound to each other in the reaction compartment (white arrow);
  • Figure 14B shows the state of the analyte and the targeted reaction complex separated from each other (white arrow) or bound to each other (gray arrow) in the reaction compartment;
  • Figure 14C shows that analytes are populations of cells interacting with each other, binding to targeted reaction complexes to form reaction compartments.
  • 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 a targeted reaction complex in the first aspect.
  • a targeting reaction complex for targeting and analyzing an analyte comprising:
  • a targeting ligand for the analyte linked to the carrier
  • reaction reagent linked to the carrier for detecting the analyte
  • a tag molecule corresponding to the analyte linked to the carrier is A tag molecule corresponding to the analyte linked to the carrier.
  • 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 1A 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 commonly, the carrier is made of a polymer, preferably polystyrene; in Figure 1B, the DNA-encoded microspheres carry both A) and B) as follows, where A) is used to label single cells with oligonucleotides Tag molecules; B) Reverse transcript sequences are part of the reagents for single cell analysis.
  • the hydrogel coating coated on the outside of DNA-encoded microspheres is not only a mechanical buffer layer, but also carries targeting ligands represented by antibodies.
  • the analyte is selected from one or more of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles and cells, and complexes formed by them , most preferably the analyte is a cell.
  • 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).
  • the analysis is selected from one or more of spectroscopic detection, sequencing, mass spectrometry detection, picture capture, and electrical signal detection.
  • spectroscopic detection/picture capture sometimes specifically refers to fluorescence analysis.
  • Fluorescence analysis refers to the use of certain substances in an excited state after being irradiated by electromagnetic waves, and the molecules in the excited state undergo a de-excitation process of collision and emission.
  • the fluorescence that occurs can reflect the characteristics of the substance, and can be used for qualitative or quantitative analysis. Since some substances do not emit fluorescence (or the fluorescence is very weak), it is necessary to convert the non-fluorescence-emitting substances into those that can emit fluorescence.
  • reagents such as fluorescent dyes
  • reactive reagents can sometimes be Specifically refers to a fluorescent reagent that can cause the analyte to fluoresce.
  • sequencing sometimes specifically refers to nucleic acid sequencing, which includes at least DNA sequencing and RNA sequencing.
  • massively parallel sequencing technology Massively Parallel Signature Sequencing, MPSS
  • polymerase cloning Polymerase cloning
  • 454pyro-sequencing 454pyro-sequencing
  • Illumina (Solexa) sequencing ABI SOLiD sequencing
  • ion semiconductor Sequencing Ion semiconductor sequencing
  • DNA nanoball sequencing DNA nanoball sequencing
  • nanopore sequencing Oxford Nanopore sequencer
  • the transcriptome sequencing technology used in the embodiments of the present application belongs to Illumina (Solexa) sequencing or DNA nanoball sequencing based on reversibly terminated fluorescent bases.
  • mass spectrometry sometimes specifically refers to mass spectrometry or ordinary mass spectrometry.
  • mass spectrometry has two main differences: first, the labeling system Different, the former mainly uses various fluorescent groups as antibody labels, while the latter uses polymers (such as polypeptides, nucleic acids, etc.) or various metal elements as labels; second, the detection system is different, the former uses lasers and photoelectric The multiplier tube is used as the detection method, while the latter uses the charge-to-mass ratio detection technology as the detection method. For example, the examples of this application used MALDI-TOF.
  • nanopore sequencing mainly uses electrophoresis to transport a sample of unknown sequence through a pore with a diameter of about 1 nanometer. This nanopore system will generate a detectable current signal on the electrolyte by applying a fixed external electric field, and the strength of the current signal passing through the nanopore will be related to the size of the nanopore and the composition of the nucleic acid passing through the nanopore .
  • the magnitude of the current passing through a single nanopore can be defined as the amount of charge passing through this nanopore per unit time.
  • the carrier is composed of a polymer or a small molecule, preferably a polymer carrier, more preferably a polystyrene carrier, more preferably the diameter of the microsphere is 1mm-100mm, and more preferably the carrier
  • the shape is selected from one or more of cube, tetrahedron, sphere, ellipsoid, bowl, concave sphere, red blood spheroid; most preferably bowl, concave sphere and/or red blood spheroid; further preferably the shape of the microsphere
  • the diameter particle size distribution coefficient CV is less than 20%, and it is further preferred that the surface of the microsphere is coated with a coating; most preferably, it is coated with a hydrogel coating.
  • bowl shape specifically refers to a concave spherical shape, which is provided with a concave surface on one side of the plane 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. 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.
  • the diameter of the microsphere can be 5mm-95mm, preferably 10mm-90mm; specifically, it can be 10mm, 20mm, 30mm, 40mm, 50mm, 60mm, 70mm, 80mm, 90mm; preferably its diameter is greater than or equal to the analyte
  • the diameter is most preferably 1 to 100 times the diameter of the analyte.
  • "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.
  • 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.
  • the diameter particle size distribution coefficient CV is 20%.
  • 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 hydrogel is preferably made of polyacrylamide.
  • the hydrogel coating acts as a mechanical buffer layer.
  • the thickness of the hydrogel coating is ideally 100nm-5 ⁇ m, specifically 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 1 ⁇ m, 2 ⁇ m, 3 ⁇ m, 4 ⁇ m, 5 ⁇ m; especially in
  • the carrier uses red blood spheroid microspheres with a diameter of 25-35 microns, and the test object is immune cells.
  • a particularly good technology has been obtained to prevent immune cells from being crushed by the microspheres when combined. Effect.
  • 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 contained in the carrier ( After the “mechanical buffer coating” is coated on the uncoated core), it is 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.
  • the core of the carrier may be a 10mm, 20mm, 30mm, 40mm, 50mm, 60mm, 70mm, 80mm or 90mm polystyrene carrier.
  • the core of the support is a 30 micron diameter polystyrene support.
  • a polystyrene support with a core diameter of 30 microns is used, and 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 1 ⁇ m, 2 ⁇ m, 3 ⁇ m, 4 ⁇ m, 5 ⁇ m can be used thick hydrogel coating.
  • the 30-micron-diameter mother core is coated with a 5-micron hydrogel layer by droplet microfluidics, finally forming a polystyrene carrier with a diameter of 40 microns.
  • a 40 micron polystyrene carrier carrying a 5 micron polyacrylamide layer of a DNA-encoded reverse transcription primer is coated by droplet microfluidics.
  • Diameter refers to the distance from the center of a plane figure or solid (such as circle, conical section, sphere, cube) to two points on the side.
  • Particle size distribution refers to the proportion of particles with different particle sizes in a group of particles, also known as particle dispersion. When expressed as a proportion of the number of particles, it is called the number distribution. After photographing by microscopic imaging, the particle size distribution can be obtained by performing image processing (for example: using ImageJ software) on the particles in the image.
  • a targeting ligand is a molecule used to target and bind to an analyte.
  • the targeting ligand can be natural or artificial, selected from nucleic acids and their analogs (locked nucleic acid, XNA, etc.), aptamers, small peptides, polypeptides, glycosylated peptides, Polysaccharides, soluble receptors, steroids, hormones, mitogens, antigens, superantigens, growth factors, cytokines, leptin, viral proteins, cell adhesion molecules, chemokines, streptavidin and its analogs, biological Antibodies and their analogs, antibodies, antibody fragments, single chain variable fragments (scFv), nanobodies, T cell receptors, major histocompatibility complex (MHC) molecules, antigenic peptide-MHC molecule complexes (pMHC) One or two or more of DNA-binding proteins, RNA-binding proteins, intracellular or cell surface receptor ligands, and multiple ligands, composite ligands, and coupling ligands formed by them.
  • the targeting ligand is an antibody that specifically recognizes such a cell.
  • the targeting ligand is an antibody that specifically recognizes a protein on the surface of the organelle.
  • 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.
  • label molecules are used to characterize different analytes, and different analytes in this paper can refer to different types of analytes, or can refer to specific different single analytes in the same type .
  • the label molecule is selected from natural or artificial information molecules, including: oligonucleotide barcodes, oligopeptide or polypeptide barcodes, natural bases and non-natural bases such as LNA, PNA, XNA One or two or more of the constituent nucleotides, oligosaccharide or polysaccharide barcodes, block polymers, covalent links between polymers and backbone molecules, and complexes formed between them.
  • 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.
  • the label molecule when the analyte is a cell, can be a sequence different from each other on the DNA-encoded microsphere; in a specific embodiment, when the analyte is a cell organelle, Tag molecules may be sequences that differ from each other on the DNA-encoded microspheres.
  • a reagent is a substance that reacts with an analyte and produces a signal that can be used for detection.
  • the reaction reagent is an oligonucleotide primer, an enzyme or a small molecule.
  • 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".
  • the reaction reagent can be a primer for reverse transcription to analyze mRNA in the cell.
  • the reaction reagent when the analyte is a cell organelle (such as mitochondria), can be a PCR primer to analyze a specific region in mitochondrial DNA. Reagents can be immobilized on the carrier, and part of the reaction components can also be added subsequently through solution.
  • 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, HEX, etc.
  • Linking refers to the interaction of targeting ligands, reagents, carriers and tags to form a whole.
  • 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, auphilic interactions, intercalation, overlapping, cationic- ⁇ 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.
  • 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 carrier
  • the connection is through the connection of the targeting ligand, the connection through the label molecule or the connection through the linker; the connection between the label molecule and the carrier is through the connection of the targeting ligand, through the reaction Connection of reagents or connection via adapters.
  • the small molecule constituting the carrier is one or more of the targeting ligand, the reaction reagent, and the tag molecule. That is, the targeting ligand, the reaction reagent, and the tag molecule themselves can form a part of the carrier.
  • DNA-encoded oligonucleotides can be connected to the polystyrene carrier through a linker; in a specific embodiment, polystyrene coated with acrylic hydrogel layer Vinyl carriers enable attachment of targeting ligands via EDC coupling chemistry.
  • the present application provides a group of targeted reaction complexes.
  • a targeting reaction complex group for targeting and analyzing more than two analytes which comprises more than two aforementioned targeting reaction complexes, wherein,
  • the targeting ligands included in each reaction complex are different from each other;
  • each reaction complex comprises different reagents from each other;
  • each reaction complex comprises different tag molecules from each other.
  • the targeting reaction complex further includes a second tag molecule corresponding to the targeting ligand.
  • the function of the second label molecule is to further divide the targeting ligands into different subgroups.
  • the reaction complex group has two different targeting ligands (such as antibodies); and has the same reaction reagent (such as poly T); and has the same label molecule (such as DNA encoding) .
  • the reaction complex group has two different targeting ligands (such as antibodies); and has two different reaction reagents (such as poly-T); and has the same label molecule (such as DNA coding).
  • the population of reactive complexes has two different targeting ligands (such as antibodies); and has the same reactive reagent (such as poly-T); and has two different labeling molecules (such as DNA coding).
  • the population of reactive complexes has two different targeting ligands (eg, antibodies); and has two different reactive reagents (eg, poly-T); and has two different labeling molecules (such as DNA codes).
  • targeting ligands eg, antibodies
  • reactive reagents eg, poly-T
  • labeling molecules such as DNA codes
  • the group of reactive complexes has more than three different targeting ligands (such as antibodies); and has one or two reactive reagents (such as poly-T); and has one or two The above tag molecule (eg DNA code).
  • the group of reactive complexes has more than three different targeting ligands (such as antibodies); and has one or two reactive reagents (such as poly-T); and has more than three different Tag molecules (eg DNA codes).
  • the group of reactive complexes has more than three different targeting ligands (such as antibodies); and has more than three different reactive reagents (such as poly T); and has one or two The above tag molecule (eg DNA code).
  • the group of reactive complexes has more than three different targeting ligands (such as antibodies); and has more than three different reactive reagents (such as poly T); and has more than three different Tag molecules (eg DNA codes).
  • targeting ligands such as antibodies
  • reactive reagents such as poly T
  • Tag molecules eg DNA codes
  • the present application provides methods for high-throughput analysis using the complexes described above.
  • the conjugate forms a reaction compartment by itself or forms a reaction compartment around the conjugate through a medium; according to the label
  • the analyte is labeled by reacting the molecule with the reagent; optionally the analyte is analyzed based on the label.
  • 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.).
  • the amount of the analyte interacting with the aforementioned targeting reaction complex or the population of the aforementioned reaction complexes and the analysis process are controlled such that only one analyte is present in each conjugate. for analysis.
  • 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.
  • the medium is an oily medium, preferably a fluorinated oily medium, or a solid medium, preferably a microwell plate.
  • oily medium preferably a fluorinated oily medium, or a solid medium, preferably a microwell plate.
  • the information of the targeting ligand is provided based on the second label molecule to confirm the type of the analyte.
  • the present application provides reaction compartments and populations thereof.
  • reaction compartment comprising:
  • reaction compartment further comprises a medium surrounding a conjugate formed by at least one of said reaction complexes bound to said analyte.
  • each reaction compartment comprises an analyte and a reaction complex, said analyte and reaction complex being either bound or separated within said reaction compartment.
  • binding state specifically refers to the bioaffinity state, including at least the antibody-antigen binding state or the binding state between the target nucleic acid and the aptamer.
  • a group of reaction compartments which comprises two or more of the aforementioned reaction compartments.
  • a group of reaction compartments is provided, wherein, in two or more aforementioned reaction compartments,
  • the targeting ligands included in the reaction complexes in each reaction compartment are different from each other;
  • reaction complexes in each reaction compartment comprise different reaction reagents from each other;
  • reaction complexes in each reaction compartment comprise different tag molecules from each other.
  • the targeting reaction complex further includes a second tag molecule corresponding to the targeting ligand.
  • reaction compartment consists of: an analyte; the aforementioned reaction complex targeted to said analyte.
  • a “reaction compartment” may serve as the smallest independent unit of a chemical reaction for analysis; that is, in different "reaction compartments” different chemical reactions may occur for analytical purposes.
  • the present application provides the use of the above reaction compartment.
  • the use of the aforementioned set of reaction compartments for analyzing a set of analytes is provided.
  • said population of analytes is a population of cells.
  • differentiated immune cell populations obtained from vertebrate blood or lymph fluid.
  • Example 1-7 An overview of the experimental procedure for the multiplexed single-cell analysis of the immune system in the peripheral blood of tumor-treated patients (see also Figure 4C):
  • Step 1 Prepare a reaction complex targeting specific subsets of cells in the immune system, which consists of DNA-encoded microspheres that can capture B-cell receptor (BCR) and T-cell receptor (TCR) genes.
  • BCR B-cell receptor
  • TCR T-cell receptor
  • the microsphere core is TOYOPEARL HW-65 (TOSOH Bioscience)
  • the DNA code synthesized on it is the label molecule
  • poly-T is the reaction reagent.
  • the second step the two kinds of microspheres are respectively encapsulated with hydrogel, and the antibody (targeting ligand) is modified to assist the combination of cell subpopulations.
  • the antibody targeting ligand
  • Step 3 Peripheral blood mononuclear cells are extracted from patient samples, incubated with a variety of microspheres with targeting antibodies and single cell reaction reagents, and then encapsulated by droplet microfluidics for high-throughput.
  • Step 4 Each microsphere in the droplet specifically enriches the specific RNA molecule in the target cell.
  • Step 5 After the sample library is built, high-throughput sequencing is performed. After the data is obtained, the bioinformatics data is clustered by targeting ligand tags and single-cell tags to obtain the overall information of the immune system.
  • Embodiment 1 has the preparation of multiple reaction reagent microspheres
  • Example 1.1 Preparation of Microspheres capable of Simultaneous Analysis of B Cell Receptor (BCR) and Transcriptome
  • microspheres (Chemgenes Corp.Cat# Macosko-2011-10) have a core of methyl methacrylate polymer (PMA) (carrier), and the surface is modified with polyT capture oligonucleotides encoded by DNA (label molecules). By performing chain extension on it, polyT (reaction reagent) and BCR sequence ( FIG. 5A ) (reaction reagent, which also has the function of the second label molecule) can be carried at the same time.
  • the microspheres can be used to analyze the transcriptome of single cells, and their BCR-specific capture oligonucleotides can be used to analyze B cell antibodies to the coding region.
  • ssDNA Design complementary single-stranded DNA
  • Splint A complementary splint sequence (Splint) was also designed with an 8-12 bp A repeat overhang (Shenggong).
  • Table 1 prepares a list of microsphere primers targeting B cell receptor (BCR) mRNA; DNA-encoded microspheres originally carrying poly-T sequences can be further added with BCR detection sequences by T4 ligase reaction.
  • BCR B cell receptor
  • Toehold primers were mixed in the desired ratio and the mixture was diluted to obtain the desired final probe concentration (for BCR sequence experiments, all toehold primers were mixed in equal proportions).
  • Table 4 Mixed toehold probe master mix configuration targeting BCR; multiple toehold probes configured as a mix.
  • Hybrid Toehold Probe (9.375 ⁇ M) 30ul 8.29 ⁇ M water 3.9ul the overall response 33.9ul the
  • microsphere ligation reactions can be performed in parallel. Wash the microspheres with 250 ⁇ L of Tris-EDTA, sodium dodecyl sulfate (TE-SDS) buffer; twice with Tris-EDTA-Tween 20 (TE-TW) buffer.
  • Tris-EDTA, sodium dodecyl sulfate (TE-SDS) buffer twice with Tris-EDTA-Tween 20 (TE-TW) buffer.
  • microspheres can be stored in TE-TW at 4°C, and the BCR sequences on the microspheres can be hybridized and verified by fluorescently labeled probes (Fig. 5B-5E).
  • Hybridization conditions 25uL lysis buffer + 25uL probe mixture + 20uL microspheres; set in Eppendorf ThermoMixer at 65°C for 5 minutes, 48°C for 8 minutes, 40°C for 8 minutes, 30°C for 8 minutes (1200 rpm). Wash 3 times with 200uL of cold 6x SSC buffer.
  • Example 1.2 Preparation of microspheres capable of simultaneous analysis of T cell receptor (TCR) and transcriptome
  • microspheres (Chemgenes Corp.Cat# Macosko-2011-10) have a core of methyl methacrylate polymer (PMA) (carrier), and the surface is modified with polyT capture oligonucleotides encoded by DNA (label molecules). By performing chain extension on it, polyT (reaction reagent) and TCR sequence ( FIG. 6A ) (reaction reagent, which also has the function of the second label molecule) can be carried at the same time.
  • the microspheres can be used to analyze the transcriptome of single cells, and their TCR-specific capture oligonucleotides can be used to analyze the T cell receptor sequence of T cells in the coding region.
  • ssDNA Design complementary single-stranded DNA
  • Splint was also designed with an 8-12 bp A repeat overhang (Shenggong).
  • Table 8 is a list of primers for preparing microspheres targeting TCR mRNA; TCR detection sequences can be further added to DNA-encoded microspheres originally carrying poly-T sequences by T4 ligase reaction.
  • Table 10 Incubation of TCR toehold probes, splint oligonucleotides, and microspheres; multiple toehold probes configured as a mixture.
  • Table 11 Mixed toehold probe master mix configuration targeting TCR; multiple toehold probes configured as a mixture.
  • Table 13 TCR sequence ligation reaction; preparation of DNA-encoded microspheres targeting the whole transcriptome and TCR-specific sequences.
  • microsphere ligation reactions can be performed in parallel. Wash the microspheres with 250 ⁇ L of Tris-EDTA, sodium dodecyl sulfate (TE-SDS) buffer; twice with Tris-EDTA-Tween 20 (TE-TW) buffer.
  • Tris-EDTA, sodium dodecyl sulfate (TE-SDS) buffer twice with Tris-EDTA-Tween 20 (TE-TW) buffer.
  • microspheres can be stored in TE-TW at 4°C, and the TCR sequences on the microspheres can be hybridized and verified by fluorescently labeled probes (Fig. 6A-6D).
  • Hybridization conditions 25uL lysis buffer + 25uL probe mixture + 20uL microspheres; set in Eppendorf ThermoMixer at 65°C for 5 minutes, 48°C for 8 minutes, 40°C for 8 minutes, 30°C for 8 minutes (1200 rpm). Wash 3 times with 200uL of cold 6x SSC buffer.
  • Embodiment 2.1 Encapsulation of hydrogel 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 2.2 Activation of hydrogel-coated microspheres and cross-linking targeting antibodies
  • Count cells 8.0*10 ⁇ 5 cells and incubate the microspheres in 200 ⁇ L PBS buffer containing 2mM EDTA, swirl the solution for 30 minutes
  • B cells are combined with hydrogel-coated microspheres targeting B cells, and PolyT and BCR-specific primers on the microspheres can conduct comprehensive and in-depth analysis of the mRNA of B cells; hydrocoagulation of T cells and targeting T cells Glue-coated microspheres combined with PolyT and TCR-specific primers on the microspheres can conduct comprehensive and in-depth analysis of T cell mRNA.
  • microspheres are coated with a vehicle (fluorine-containing oily medium) through a droplet microfluidic device.
  • Droplet generation (Fig. 8A, Fig. 8B): Droplets were produced using a microfluidic chip with a height of 100 ⁇ m and a nozzle width of 100 ⁇ m (source; through general photolithography and PDMS nanoimprinting processes, the microfluidic chip was independently prepared by the laboratory. chip) generated. Typical flow rates used were: cells (phase I) - 12 ⁇ L/h, transcriptome amplification mix (phase II) - 288 ⁇ L/h, and droplet formation oil (5% PEG-PFPE 2 HFE7500) - 600 ⁇ L/h.
  • the amplified product was interrupted using a DNA fragmentation kit (One-step DNA Lib Prep Kit for Illumina V2 (50ng Input DNA), RK20239). Use the Tn5 transposase method to fragment the DNA and then perform library amplification.
  • the PCR amplification procedure of the library is shown in the table below. After the library amplification is completed, use 1% agarose gel electrophoresis to verify the amplified library fragments. A typical library amplification The augmented product is shown in the figure ( Figure 9).
  • Example 7 Analysis of experimental results (from the hydrogel-coated microspheres of Example 2.2.)
  • T cells mainly analyze the TCR sequence, and other immune cells analyze the whole transcriptome; the sequencing results can not only obtain the target ligand information, but also obtain the detailed information of BCR, TCR, and other cells single-cell transcriptome-independent information. After clustering processing by targeting ligands and single-cell DNA codes, high-resolution information of immune cells is obtained (T cell analysis results include TCR information).
  • the sequencing data can be clustered and the clonality (Clonality) [ Figure 10B], D50 data [ Figure 10C]; through the second label of the targeting ligand, the Clustering of B cells on the sequencing data and performing BCR clonality ( Figure 10D) and unique CDR3 distribution data of BCR ( Figure 10E)
  • the first step prepare hydrogel-coated microspheres targeting specific human or mouse cells, and introduce droplet digital PCR (ddPCR) primers (reaction reagents) and sequence-specific probes for analysis of humans and mice into the hydrogel monomer.
  • ddPCR droplet digital PCR
  • needles labeling molecules
  • antibodies targeting ligands
  • Step 2 Mix human and mouse cells, incubate and combine with various microspheres with targeting antibodies and single-cell droplet ddPCR reagents, and perform high-throughput packaging through droplet microfluidics.
  • Step 4 Each microsphere within the droplet specifically amplifies a specific DNA region in the targeted cell.
  • Step 5 Obtain the overall information of the cell mixture through intra-droplet amplification fluorescence imaging.
  • TEBST buffer (20mM Tris-HCl pH 8.0, 274mM NaCl, 5.4mM KCl, 20mM EDTA, 0.2% Triton X-100), mix well.
  • Example 9 Mix the two hydrogel-coated microspheres generated in Example 9 carrying primers targeting 293T cells and 3T3 cell ligand primers 1:1, and rotate at room temperature for 15 minutes at 5 rpm
  • Count cells 8.0*10 ⁇ 5 cells and incubate the microspheres in 200 ⁇ L PBS buffer containing 2mM EDTA, swirl the solution for 30 minutes
  • Human 293T cells are combined with hydrogel-coated microspheres targeting 293T cells, and GAPDH_Human-specific primers on the microspheres can amplify and analyze human GAPDH genes; mouse 3T3 cells and hydrogel targeting 3T3 cells Glue-wrapped microspheres combined with GAPDH_Mouse-specific primers on the microspheres can amplify and analyze the mRNA of 3T3 cells ( Figure 11).
  • microspheres were encapsulated by shaking the vehicle (fluorinated oily medium). All chemicals were ordered from Sigma-Aldrich, Fisher Scientific and Roche.
  • Droplets were generated using a microfluidic chip using a 100 ⁇ m height and 100 ⁇ m wide nozzle. Typical flow rates used were: cells (phase I) - 12 ⁇ L/h, transcriptome amplification mix (phase II) - 288 ⁇ L/h, and droplet formation oil (5% PEG-PFPE 2 HFE7500) - 600 ⁇ L/h.
  • the technical solution of the present application meets the technical requirements of data splitting for single-cell analysis (targeted multiple detection).
  • the microsphere mixture can realize single-cell multiple sequencing analysis of immune cells.
  • the method does not need to purify each subpopulation of cells to be analyzed by cell enrichment or flow cytometry, which simplifies the operation steps.
  • the target reaction complex microspheres can not only recognize the cell object during design, deliver the special reaction reagent of the cell to the micro-droplet where the cell is located, but also perform subsequent accurate data disassembly through the label on the complex. point.
  • FIGS. 10A-10E The specific results are shown in FIGS. 10A-10E , wherein FIG. 10A shows the results of single-cell sequencing grouping of peripheral blood mononuclear cells.
  • the frequency of TCR sequences was additionally analyzed (10C), and the TCR clone distribution (10B) was visualized through the TCR dendrogram, and the VJ sequence distribution (10B) was visualized through the TCR two-dimensional map.
  • the frequency of BCR sequences was additionally analyzed for B cells in immune cell subsets (10D, 10E) (FIG. 10E), and the BCR dendrogram was visualized to visualize the distribution of BCR clones (10D).
  • the microsphere mixture can realize single-cell multiple fluorescence detection of human-mouse mixed cells.
  • single-cell qPCR in droplets can target different targets on different cancer cells, as well as different targets on human and mouse cells, achieving high-throughput detection of target GAPDH mRNA in each cell. Abundance characterization to obtain population heterogeneity information.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

一种靶向并分析待分析物的靶向反应复合物，其包括：载体；与载体连接的、待分析物的靶向配基；与载体连接的、用于检测待分析物的反应试剂；以及与载体连接的、与所述待分析物相对应的标签分子。还涉及由它形成的靶向反应复合物群、反应隔室群和用它们进行高通量分析的方法。

Description

靶向反应复合物及其在靶向多重检测中的用途 技术领域
本申请涉及高通量化学分析领域，具体涉及靶向反应复合物及其在靶向多重检测中的用途。
背景技术
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系统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。
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。
发明内容
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；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。
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。
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。
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)。
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。
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。
本申请主要聚焦于上面的第三类技术难题，即，在实现对多个种类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；同时由于反应试剂兼具细胞识别的功能，该方法相较传统的流式分选配合单细胞 分析方法，简化了多个种类细胞高通量分析的流程。
为了解决上述技术问题，本申请提供了：
1.一种靶向并分析待分析物的靶向反应复合物，其包括：
载体；
与载体连接的、待分析物的靶向配基；
与载体连接的、用于检测待分析物的反应试剂；以及
与载体连接的、与所述待分析物相对应的标签分子。
2.根据项1所述的靶向反应复合物，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。
3.根据项1所述的靶向反应复合物，其中，所述分析选自光谱学检测、测序、质谱检测、图片捕获、电信号检测中的一种或两种以上。
4.根据项1所述的靶向反应复合物，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，进一步优选所述载体的直径为1μm-100μm，进一步优选所述载体形状选自正方体形、四面体形，球形、椭球形、碗形、红血球形的一种或两种以上；最优选碗形和/或红血球形；进一步优选所述载体的直径粒径分布系数CV小于20％，进一步优选所述载体的表面涂覆有力学缓冲涂层；最优选所述载体的表面涂覆有水凝胶涂层。
5.根据项1所述的靶向反应复合物，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、促分裂原、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。
6.根据项1所述的靶向反应复合物，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。
7.根据项1所述的靶向反应复合物，其中，所述反应试剂是寡聚核苷酸引物、酶或小分子。
8.根据项1所述的靶向反应复合物，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。
9.根据项1所述的靶向反应复合物，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。
10.根据项4所述的靶向反应复合物，其中，其中所述构成载体的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。
11.一种用于靶向并分析两种以上待分析物的靶向反应复合物群，其包含两种以上根据项1-10的任一项所述的靶向反应复合物，其中，
每种反应复合物所包括的靶向配基彼此不同；
优选每种反应复合物所包括的反应试剂彼此不同；
进一步优选每种反应复合物所包括的标签分子彼此不同。
12.根据项11所述的靶向反应复合物群，其中，所述靶向反应复合物还包括与所述靶向配基相对应的第二标签分子。
13.使用根据项1-10中任一项所述的反应复合物或根据项11或12所述的反应复合物群分析待分析物的方法，
其包括以下步骤：
使待分析物与根据项1-10的任一项所述的靶向反应复合物或项11或12所述的反应复合物群相互作用形成结合物；
所述结合物自身形成反应隔室或通过媒介物围绕所述结合物形成反应隔室；根据所述标签分子和所述反应试剂进行的反应来对所述待分析物进 行标记；任选基于所述标记对所述待分析物进行分析。
14.根据项13所述的方法，其中，控制进行相互作用的待分析物与根据项1-10的任一项所述的靶向反应复合物或项11或12所述的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。
15.根据项13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。
16.根据项13所述的方法，其中，在使用根据项11或12所述的反应复合物群进行分析时，基于第二标签分子提供靶向配基的信息以确认待分析物的类型。
17.一种反应隔室，其包括：
待分析物；
靶向所述待分析物的根据项1至10的任一项所述的反应复合物。
18.根据项17所述的反应隔室，其还包括围绕至少一个所述反应复合物与所述待分析物结合所生成的结合物的媒介物。
19.根据项17或18所述的反应隔室，其中，每个反应隔室包括一个待分析物和一个反应复合物，所述待分析物和反应复合物在所述反应隔室内处于结合状态或者分开状态。
20.一种反应隔室群，其包含两种以上根据项17或18所述的反应隔室。
21.根据项20所述的反应隔室群，其中，在两种以上根据项17或18所述的反应隔室中，
每种反应隔室中的反应复合物所包括的靶向配基彼此不同；
优选每种反应隔室中的反应复合物所包括的反应试剂彼此不同；
进一步优选每种反应隔室中的反应复合物所包括的标签分子彼此不同。
22.根据项17所述的反应隔室群，其中，所述靶向反应复合物还包括与所述靶向配基相对应的第二标签分子。
23.根据项17～22中任一项所述的反应隔室群用于对待分析物群进行分析的用途。
24.根据项23所述的用途，其中，所述待分析物群是细胞群。
本申请实现的有益技术效果
采用本申请的技术方案，在实现对多个种类的待分析物(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“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(例如微球)，利用它们的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”的实现。通过在载体(例如微球)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两种反应试剂，实现了多路的“高通量分析”。
附图说明
图1A显示了根据本申请的靶向反应复合物的构成示意图，其中标签可由两部分组成：包含“靶向配基”信息的“第二标签”，以及标记不同待分析物(例如细胞)的“标签分子”；
图1B显示了水凝胶包裹的微球：带靶向分析的DNA编码微球经过水凝胶包裹后，可靶向混合细胞中的不同亚群，进行单细胞分析；
图1C显示了透明微球可在水凝胶层携带靶向人或鼠细胞的PCR引物，并结合靶向人或鼠的配基，实现对不同物种细胞的分析。
图2A显示了两种以上待分析物的靶向反应复合物群的构成示意图；图2B显示了多种待分析物与靶向反应复合物群预先形成结合物的示意图。
图3展示了待分析物与靶向反应复合物形成结合物后，通过高通量媒介物围绕形成反应隔室的流程示意图。
图4A显示应用多重靶向反应复合物分析复杂系统的流程示意图；图4B显示靶向反应复合物与免疫细胞亚群分别形成结合物的示意图；图4C显示从水凝胶包裹的DNA编码微球制备到免疫系统多重分析的流程。
图5A显示了如何将B细胞和T细胞特异性引物分别加载到B细胞分析微球和T细胞分析微球上；
图5B显示了对polyT序列用带FAM的探针进行染色的结果图；
图5C显示了对BCR序列用带Cy3的探针进行染色的结果图；
图5D显示了水的染色的结果图；
图5E显示了含非特异性序列的探针对微球进行染色的结果图。
图6A显示了对polyT序列用带FAM的探针进行染色的结果图；
图6B显示了对TCR序列用带Cy3的探针进行染色的结果图；
图6C显示了水的染色的结果图；
图6D显示了含非特异性序列的探针对微球进行染色的结果图。
图7显示了通过微流控进行微球的水凝胶包裹的操作图。
图8A显示了用微流体装置系统产生包裹靶向捕获载体与细胞结合物的液滴的操作图；
图8B显示了通过微流体装置系统向微流控芯片中注入第一种流体(I相溶液，靶向捕获载体与细胞的结合物)、第二种流体(II相溶液，逆转录溶液)、连续相(载体油为氟化油并且包含表面活性剂，例如PFPE-PEG-PFPE(全氟聚醚-聚乙二醇-全氟聚醚)三嵌段共聚物)。
图9显示了对cDNA扩增产物打断后的文库。
图10A显示了免疫系统单细胞转录组数据；
图10B显示了单细胞TCR的树图和TCR二维图数据；
图10C显示了单细胞TCR的D50数据；
图10D显示了单细胞BCR的树图数据；
图10E显示了单细胞BCR的独特CDR3分布。
图11显示了含靶向配基的水凝胶包裹微球与目标细胞的孵育结合的结果图。
图12显示了振荡乳化，高通量形成彼此独立隔室；分离后将液滴平铺进行PCR反应的结果图。
图13A显示了经过PCR热循环后，形成对人和鼠的独立液滴内qPCR分析的结果图；
图13B显示了对液滴的实时检测，各液滴内细胞mRNA表达量呈现出不同的Ct值；
图13C显示了对液滴中人与鼠不同GAPDH基因qPCR的Ct值统计的结果图。
图14A显示了待分析物与靶向反应复合物在反应隔室中保持相互结合的状态(白色箭头)；
图14B显示了待分析物与靶向反应复合物在反应隔室中相互分开(白色箭头)或彼此结合的状态(灰色箭头)；
图14C显示了待分析物为彼此相互作用的细胞群，与靶向反应复合物结合后形成反应隔室。
具体实施方式
下面将参照附图更详细地描述本发明的具体实施例。虽然附图中显示了本发明的具体实施例，然而应当理解，可以以各种形式实现本发明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发明，并且能够将本发明的范围完整的传达给本领域的技术人员。
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发明的较佳实施方式，然所述描述乃以说明书的一般原则为目的，并非用以限定本发明的范围。本发明的保护范围当视所附权利要求所界定者为准。
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。
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。
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。
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。
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，不应成为对本发明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。
本申请在第一方面提供了一种靶向反应复合物。
在一个具体实施方式中，提供了一种靶向并分析待分析物的靶向反应复合物，其包括：
载体；
与载体连接的、待分析物的靶向配基；
与载体连接的、用于检测待分析物的反应试剂；以及
与载体连接的、与所述待分析物相对应的标签分子。
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。
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。
图1A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；在图1B中，DNA编码微球同时携带了如下A)和B)，其中A)用以标记单细胞的寡核苷酸标签分子；B)逆转录序列是单细胞分析反应试剂的一部分。包覆在DNA编码微球外部的水凝胶涂层既是力学缓冲层，又携带以抗体为代表的靶向配基。
在一个具体实施方式中，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。
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。
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。
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。
在一个具体实施方式中，其中，所述分析选自光谱学检测、测序、质谱检测、图片捕获、电信号检测中的一种或两种以上。
在本说明书的上下文中，光谱学检测/图片捕获有时特指荧光分析法，荧光分析法是指利用某些物质被电磁波照射后处于激发态，激发态分子经历一个碰撞及发射的去激发过程所发生的能反映出该物质特性的荧光，可以进行定性或定量分析的方法。由于有些物质本身不发射荧光(或荧光很弱)，这就需要把不发射荧光的物质转化成能发射荧光的物质。例如用某些试剂(如荧光染料)，使其与不发射荧光的物质生成络合物，各种络合物能发射荧光，再进行测定；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，“测序”有时特指核酸测序，其至少包括DNA测序和RNA测序，根据发展历史、影响力、测序原理和技术不同等，主要有以下几种：大规模平行测序技术(Massively Parallel Signature Sequencing,MPSS)、聚合酶克隆(Polony Sequencing)、454焦磷酸测序(454pyro-sequencing)、基于可逆终止荧光碱基的边合成边测序Illumina(Solexa)sequencing、ABI SOLiD sequencing、离子半导体测序(Ion semiconductor sequencing)、DNA纳米球测序(DNA nanoball sequencing)、纳米孔测序(Oxford Nanopore测序仪)等。例如，本申请的实施例所使用的转录组测序技术就属于基于可逆终止荧光碱基的边合成边测序Illumina(Solexa)sequencing或DNA纳米球测序(DNA nanoball sequencing)。
在本说明书的上下文中，“质谱检测”有时特指质谱流式法或普通质谱分析，所述质谱流式法和传统流式细胞术相比，主要有两点不同：第一、标签系统的不同，前者主要使用各种荧光基团作为抗体的标签，后者则使用高分子(例如：多肽、核酸等)或各种金属元素作为标签；第二、检测系统的不同，前者使用激光器和光电倍增管作为检测手段，而后者使用荷质比 检测技术作为检测手段。例如，本申请的实施例所使用的是MALDI-TOF。
电信号检测需要离子根据它们之间的电荷差异从一边流向另一边，通过两侧电极接收到的电流信号发生变化实现检测。在本申请中对于电信号检测没有任何限定可以采用任何本领域技术人员可以采用的检测电荷流动或差异的方式和装置。例如，纳米孔测序主要是利用电泳的方式将一个未知序列的样品输送穿过一个直径约1纳米孔。这个纳米孔系统会借由施以固定的外加电场在电解液上产生的可检测电流信号，其穿过纳米孔的电流信号强弱则会与纳米孔孔径大小及通过此纳米孔核酸的组成有关。当此纳米孔孔径够狭小时，样品穿过此纳米孔道时便能造成独特的电流信号改变，这样的机制也让使用纳米孔测序一事变得可能。其穿过单个纳米孔的电流大小可定义为单位时间内通过这个纳米孔的电荷量。
在一个具体实施方式中，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，进一步优选所述微球的直径为1mm-100mm，进一步优选所述载体形状选自正方体形、四面体形，球形、椭球形、碗形、凹球形、红血球形的一种或两种以上；最优选碗形、凹球形和/或红血球形；进一步优选所述微球的直径粒径分布系数CV小于20％，进一步优选微球表面涂覆有涂层；最优选涂覆有水凝胶涂层。在本说明书的上下文中，“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。
具体地，所述微球的直径可以在5mm-95mm、优选10mm-90mm；具体地可以为10mm、20mm、30mm、40mm、50mm、60mm、70mm、80mm、90mm；优选其直径大于等于待分析物的直径，最优选其直径为待分析物直径的1倍至100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。
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。
在一个具体实施方式中，直径粒径分布系数CV为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。
所述水凝胶优选由聚丙烯酰胺制成。所述水凝胶涂层的作用是力学缓冲层。所述水凝胶涂层的厚度理想地为100nm-5μm，具体地可以为100nm、150nm、200nm、250nm、300nm、350nm、400nm、450nm、500nm、1μm、2μm、3μm、4μm、5μm；尤其在载体选用25-35微米直径的红血球形微球、待检测物为免疫细胞、使用250nm-5μm厚度水凝胶的情况下，取得了特别良好的防止免疫细胞在结合时被微球击碎的技术效果。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”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。
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。
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。
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。
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。
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。
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。
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。
在一个具体实施方式中，载体的母核可以为10mm、20mm、30mm、40mm、50mm、60mm、70mm、80mm或90mm的聚苯乙烯载体。在一个具体的实施方式中，载体的母核为30微米直径的聚苯乙烯载体。
在一个具体实施方式中，使用母核为30微米直径的聚苯乙烯载体，并可以使用100nm、150nm、200nm、250nm、300nm、350nm、400nm、450nm、500nm、1μm、2μm、3μm、4μm、5μm厚度的水凝胶涂层。
在一个具体的实施方式中，通过液滴微流控对该30微米直径的母核包覆5微米水凝胶层，最终形成直径40微米的聚苯乙烯载体。
在一个具体的实施方式中，通过液滴微流控包覆携带了DNA编码逆转录引物的5微米聚丙烯酰胺层的40微米的聚苯乙烯载体。
直径(diameter)，是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。粒径分布是指某一粒子的群体中，不同粒径的粒子所占比例，亦称为粒子的分散度。以粒子的个数所占的比例表示时，称为个数分布。通过显微成像拍照后，对图像中的颗粒进行图像处理(如：使用ImageJ软件)可得到直径粒径分布。
在本文中，靶向配基是用于靶向待分析物并与之结合的分子。
在又一具体实施方式中，所述靶向配基可以是天然或人造的，选自核酸及其类似物(锁核酸、XNA等)、适配体、小肽、多肽、糖基化肽、多糖、可溶性受体、类固醇、荷尔蒙、促分裂原、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。
在又一具体实施方式中，当待分析物是细胞的时候，靶向配基为特异性识别这种细胞的抗体。
在又一具体实施方式中，当待分析物是细胞器的时候，靶向配基为特异性识别细胞器表面蛋白的抗体。
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，被免疫系统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——指数富集的配体系统进化技术(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。
在本文中，标签分子是用于表征不同的待分析物，在本文中不同的待分析物可以是指不同种类的待分析物，也可以是指同一种类中的具体的不通的单个待分析物。
在再一具体实施方案中，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、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系统内发生的相互作用。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。
在一个具体的实施方式中，当待分析物是细胞的时候，标签分子可以是在DNA编码微球上彼此序列不同的序列；在一个具体的实施方式中，当待分析物是细胞器的时候，标签分子可以是在DNA编码微球上彼此序列不同的序列。
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。在另一具体实施方式中，所述反应试剂是寡聚核苷酸引物、酶或小分子。
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”。
在一个具体的实施方式中，当待分析物是细胞的时候，反应试剂可以是逆转录的引物用以分析细胞中的mRNA。
在一个具体的实施方式中，当待分析物是细胞器(例如线粒体)的时候，反应试剂可以是PCR的引物用以分析线粒体DNA中的特定区域。反应试剂可以固定在载体上，部分反应组分也可以后续通过溶液添加。
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、HEX等。
“连接”指的是将靶向配基、反应试剂、载体和标签通过相互作用形成一个整体。在一个具体实施方式中，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。
在又一具体实施方式中，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。
在一个具体实施方式中，其中所述构成载体的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。
在一个具体的实施方式中，在聚苯乙烯载体上能通过连接基团(linker)连接DNA编码寡核苷酸；在一个具体的实施方式中，包裹了带丙烯酸的水凝胶层的聚苯乙烯载体能通过EDC偶联化学，实现与靶向配基的连接。
本申请在第二方面提供了一种靶向反应复合物群。
在一个具体实施方式中，提供了一种用于靶向并分析两种以上待分析物的靶向反应复合物群，其包含两种以上前述靶向反应复合物，其中，
每种反应复合物所包括的靶向配基彼此不同；
优选每种反应复合物所包括的反应试剂彼此不同；
进一步优选每种反应复合物所包括的标签分子彼此不同。
在又一具体实施方式中，所述靶向反应复合物还包括与所述靶向配基相对应的第二标签分子。在此，所述第二标签分子的作用是将靶向配基进一步划分为不同的亚群。
在一个具体实施方式中，所述反应复合物群具有两种不同靶向配基(例如抗体)；且具有同一种反应试剂(例如多聚T)；且具有同一种标 签分子(例如DNA编码)。
在一个具体实施方式中，所述反应复合物群具有两种不同靶向配基(例如抗体)；且具有两种不同的反应试剂(例如多聚T)；且具有同一种标签分子(例如DNA编码)。
在一个具体实施方式中，所述反应复合物群具有两种不同靶向配基(例如抗体)；且具有同一种反应试剂(例如多聚T)；且具有两种不同的标签分子(例如DNA编码)。
在一个具体实施方式中，所述反应复合物群具有两种不同靶向配基(例如抗体)；且具有两种不同的反应试剂(例如多聚T)；且具有两种不同的标签分子(例如DNA编码)。
在一个具体实施方式中，所述反应复合物群具有三种以上不同靶向配基(例如抗体)；且具有一种或两种反应试剂(例如多聚T)；且具有一种或两种以上标签分子(例如DNA编码)。
在一个具体实施方式中，所述反应复合物群具有三种以上不同靶向配基(例如抗体)；且具有一种或两种反应试剂(例如多聚T)；且具有三种以上不同的标签分子(例如DNA编码)。
在一个具体实施方式中，所述反应复合物群具有三种以上不同靶向配基(例如抗体)；且具有三种以上不同的反应试剂(例如多聚T)；且具有一种或两种以上标签分子(例如DNA编码)。
在一个具体实施方式中，所述反应复合物群具有三种以上不同靶向配基(例如抗体)；且具有三种以上不同的反应试剂(例如多聚T)；且具有三种以上不同的标签分子(例如DNA编码)。
本申请在第三方面提供了使用上述复合物进行高通量分析的方法。
在一个具体实施方式中，提供了使用前述的反应复合物或前述反应复合物群分析待分析物的方法，
其包括以下步骤：
使待分析物与前述靶向反应复合物或前述反应复合物群相互作用形成结合物；所述结合物自身形成反应隔室或通过媒介物围绕所述结合物形成反应隔室；根据所述标签分子和所述反应试剂进行的反应来对所述待分析物进行标记；任选基于所述标记对所述待分析物进行分析。
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)。
在一个具体实施方式中，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。
在又一具体实施方式中，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。
在一个具体实施方式中，在使用前述反应复合物群进行分析时，基于第二标签分子提供靶向配基的信息以确认待分析物的类型。
本申请在第四方面提供了反应隔室及其群。
在一个具体实施方式中，提供了一种反应隔室，其包括：
待分析物；靶向所述待分析物的前述反应复合物。
在又一具体实施方式中，该反应隔室还包括围绕至少一个所述反应复合物与所述待分析物结合所生成的结合物的媒介物。
在一个具体实施方式中，每个反应隔室包括一个待分析物和一个反应复合物，所述待分析物和反应复合物在所述反应隔室内处于结合状态或者分开状态。
在本说明书的上下文中，“结合状态”特指生物亲和状态，至少包括抗体-抗原结合状态或者靶核酸与适配体的结合状态。
在又一具体实施方式中，提供了一种反应隔室群，其包含两种以上前述反应隔室。
在一个具体实施方式中，提供了一种反应隔室群，其中，在两种以上前述反应隔室中，
每种反应隔室中的反应复合物所包括的靶向配基彼此不同；
优选每种反应隔室中的反应复合物所包括的反应试剂彼此不同；
进一步优选每种反应隔室中的反应复合物所包括的标签分子彼此不同。
在一个具体实施方式中，所述靶向反应复合物还包括与所述靶向配基相对应的第二标签分子。
在本说明书的上下文中，所述反应隔室由下述组成：待分析物；靶向所述待分析物的前述反应复合物。“反应隔室”可以作为用于分析的化学反应的最小独立单位；即，在不同的“反应隔室”中，可以为了分析的目的发生不同的化学反应。
本申请在第五方面提供了上述反应隔室的用途。
在一个具体实施方式中，提供了前述反应隔室群用于对待分析物群进行分析的用途。
在又一具体实施方式中，所述待分析物群是细胞群。特别是取自脊椎动物血样或淋巴液的、经分化的免疫细胞群。
在本申请中，针对各个方面描述的定义是可以通用的。
实施例部分
针对第一部分实施例(实施例1-7)：对肿瘤治疗患者的外周血中的免疫系统进行多重单细胞分析的实验流程概述(也见于图4C)：
第一步：制备靶向免疫系统特定亚群细胞的反应复合物，该复合物由可捕获B细胞受体(BCR)、T细胞受体(TCR)基因的DNA编码微球构成。其中微球母核为TOYOPEARL HW-65(TOSOH Bioscience)，上面合成的DNA编码为标签分子，多聚T为反应试剂。
第二步：对这两种微球分别进行水凝胶包裹，并修饰抗体(靶向配基)以辅助细胞亚群的结合。由此组成载体、反应试剂、靶向配基、标签分子四合一的整体。
第三步：从患者样本中提取外周血单个核细胞，与带靶向抗体及单细胞反应试剂的多种微球进行孵育结合后通过液滴微流控进行高通量包裹。
第四步：液滴内各微球对靶向细胞中的特定RNA分子特异性富集。
第五步：该样本建库后进行高通量测序，获取数据后通过靶向配基标 签及单细胞标签将生信数据聚类，获取免疫系统整体信息。
实施例1带有多种反应试剂微球的制备
实施例1.1.能够同时分析B细胞受体(BCR)和转录组微球的制备
商业化微球(Chemgenes Corp.Cat# Macosko-2011-10)母核为甲基丙烯酸甲酯聚合物(PMA)(载体)，表面修饰有DNA编码(标签分子)的polyT捕获寡核苷酸，通过对其进行链延伸，可同时携带polyT(反应试剂)与BCR序列(图5A)(反应试剂、兼具第二标签分子功能)。该微球可用于分析单细胞的转录组，其BCR特异性捕获寡核苷酸，可用于分析编码区域的B细胞抗体。
设计互补的单链DNA(ssDNA)定制引物序列添加了5'磷酸修饰的感兴趣区域，称为立足点探针。还设计互补夹板序列(Splint)也带有8-12bp的A重复突出端(生工)。
Figure PCTCN2022134736-appb-000001
Figure PCTCN2022134736-appb-000002
表1制备靶向B细胞受体(BCR)mRNA的微球引物列表；通过T4连接酶反应可将原本携带多聚T序列的DNA编码微球进一步添加BCR检测序列。
在浓度为500μM的Tris-EDTA(TE)缓冲液中重新悬浮所有寡核苷酸
Figure PCTCN2022134736-appb-000003
表2 BCR引物母液的配置；用水或TE溶液将引物稀释到目标浓度。
混合等体积(20uL)的定制引物和夹板寡核苷酸50mM NaCl并转移至PCR条管。将反应混合物加热至95℃(3分钟)并冷却以慢速(-0.1℃/s)升温至14℃。这将创建双链立足点探针用于连接外部引物与微球上的序列(200uL管)
Figure PCTCN2022134736-appb-000004
Figure PCTCN2022134736-appb-000005
表3 BCR立足点探针、夹板寡核苷酸与微球的孵育；将多重立足点探针配置为混合物。
用TE缓冲液稀释每个立足点探针，以获得100μM的最终浓度。
以所需比例混合立足点探针(toeholdprimer)并稀释混合物以获得所需的最终探针浓度(对于BCR序列实验，所有的立足点探针都混合在等比例)。
Figure PCTCN2022134736-appb-000006
表4靶向BCR的混合立足点探针母液配置；将多重立足点探针配置为混合物。
混合立足点探针(3.125μM) 体积 终浓度
混合立足点探针(9.375μM) 30ul 8.29μM
水 3.9ul  
总反应 33.9ul  
表5靶向BCR的混合立足点探针母液的稀释；将多重立足点探针混合液调制到目标浓度。
将16μL的这种混合探针混合物与40μL的PEG-4000(50％w/v)混合，40μL T4 DNA连接酶缓冲液、72μL水和2μL T4 DNA连接酶(1.5毫升管)。
标记 反应管A
混合探针溶液 16*1.5＝24μL
PEG-4000(50％w/v) 40*1.5＝60μL
T4 DNA连接酶缓冲液(NEB) 40*1.5＝60μL
Water 72*1.5＝108μL
T4 DNA连接酶(NEB) 2*1.5＝3μL
总反应 170*1.5＝255μL
表6 BCR序列连接反应；制备靶向全转录组和BCR特异性序列的DNA编码微球。
将12000个微球与上述连接混合物混合，在Eppendorf ThermoMixer中设置37℃下孵育1小时(每15秒以1800rpm振荡混合)。
通过在65℃下加热反应混合物3分钟来灭活酶
通过置于冰水中至少1分钟来淬灭反应混合物。
要获得所需数量的polyT+BCR序列的微球，可平行进行6-10个微球连接反应。用250μL Tris-EDTA、十二烷基硫酸钠(TE-SDS)缓冲液清洗微球；两次使用Tris-EDTA-Tween 20(TE-TW)缓冲。
这些处理好的微球可在4℃下储存在TE-TW中，通过荧光标记的探针可杂交并验证微球上的BCR序列(图5B-5E)。
探针杂交步骤如下：
配置探针的母液：
Figure PCTCN2022134736-appb-000007
表7探针母液的配置；配置终浓度为2μM的探针溶液。
杂交条件：25uL裂解缓冲液+25uL探针混合物+20uL微球；在Eppendorf ThermoMixer中设置65℃5分钟，48℃8分钟，40℃8分钟，30℃8分钟(1200转)。用200uL冷6x SSC缓冲液洗涤3次。
实施例1.2.能够同时分析T细胞受体(TCR)和转录组微球的制备
商业化微球(Chemgenes Corp.Cat# Macosko-2011-10)母核为甲基丙烯酸甲酯聚合物(PMA)(载体)，表面修饰有DNA编码(标签分子)的polyT捕获寡核苷酸，通过对其进行链延伸，可同时携带polyT(反应试剂)与TCR序列(图6A)(反应试剂、兼具第二标签分子功能)。该微球可用于分析单细胞的转录组，其TCR特异性捕获寡核苷酸，可用于分析编码区域的T细胞的T细胞受体序列。
设计互补的单链DNA(ssDNA)定制引物序列添加了5'磷酸修饰的感兴趣区域。还设计互补夹板序列(Splint)也带有8-12bp的A重复突出端(生工)。
Figure PCTCN2022134736-appb-000008
表8为制备靶向T细胞受体(TCR)mRNA的微球引物列表；通过T4连接酶反应可将原本携带多聚T序列的DNA编码微球进一步添加TCR检测序列。
在浓度为500μM的Tris-EDTA(TE)缓冲液中重新悬浮所有寡核苷酸
Figure PCTCN2022134736-appb-000009
表9 TCR引物母液的配置；用水或TE溶液将引物稀释到目标浓度。
混合等体积(20uL)的定制引物和夹板寡核苷酸50mM NaCl并转移至PCR条管。将反应混合物加热至95℃(3分钟)并冷却以慢速(-0.1℃/s)升温至14℃。这将创建双链立足点探针用于连接外部引物与微球上的序列(200uL管)
Figure PCTCN2022134736-appb-000010
表10 TCR立足点探针、夹板寡核苷酸与微球的孵育；将多重立足点探针配置为混合物。
用TE缓冲液稀释每个立足点探针，以获得100μM的最终浓度。
以所需比例混合toehold探针并稀释混合物以获得所需的最终探针浓度(对于TCR序列实验，所有的立足点探针都混合在等比例)。
Figure PCTCN2022134736-appb-000011
表11靶向TCR的混合立足点探针母液配置；将多重立足点探针配置为混合物。
混合立足点探针(3.125μM) 体积 终浓度
混合立足点探针(9.375μM) 30ul 8.29μM
水 3.9ul  
总反应 33.9ul  
表12靶向TCR的混合立足点探针母液的稀释；将多重立足点探针混合液调制到目标浓度。
将16μL的这种混合探针混合物与40μL的PEG-4000(50％w/v)混合，40μL T4 DNA连接酶缓冲液、72μL水和2μL T4 DNA连接酶(1.5毫升管)。
标记 反应管A
混合探针溶液 16*1.5＝24μL
PEG-4000(50％w/v) 40*1.5＝60μL
T4 DNA连接酶缓冲液(NEB) 40*1.5＝60μL
Water 72*1.5＝108μL
T4 DNA连接酶(NEB) 2*1.5＝3μL
总反应 170*1.5＝255μL
表13 TCR序列连接反应；制备靶向全转录组和TCR特异性序列的DNA编码微球。
将12000个微球与上述连接混合物混合，在Eppendorf ThermoMixer中设置37℃下孵育1小时(每15秒以1800rpm振荡混合)。
通过在65℃下加热反应混合物3分钟来灭活酶
通过置于冰水中至少1分钟来淬灭反应混合物。
要获得所需数量的polyT+TCR序列的微球，可平行进行6-10个微球连接反应。用250μL Tris-EDTA、十二烷基硫酸钠(TE-SDS)缓冲液清洗微球；两次使用Tris-EDTA-Tween 20(TE-TW)缓冲。
这些处理好的微球可在4℃下储存在TE-TW中，通过荧光标记的探针可杂交并验证微球上的TCR序列(图6A-6D)。
探针杂交步骤如下：
配置探针的母液：
Figure PCTCN2022134736-appb-000012
表14探针母液的配置；配置终浓度为2μM的探针溶液。
杂交条件：25uL裂解缓冲液+25uL探针混合物+20uL微球；在Eppendorf ThermoMixer中设置65℃5分钟，48℃8分钟，40℃8分钟，30℃8分钟(1200转)。用200uL冷6x SSC缓冲液洗涤3次。
实施例2对微球的水凝胶包裹，并修饰靶向B细胞与T细胞的抗体
实施例2.1.水凝胶微球的包裹
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。将此溶液与实施例1.1.、实施例1.2.的微球分别单独混合，各装入带有28G针头的1ml注射器中。
2、在氢氟醚(HFE)油中制备5％(w/w)含氟表面活性剂和1％N,N,N,N-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(图7)。
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。
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
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。
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。
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。
8、3000x g旋转3分钟。移液去除上清液。重复3x。
9、重悬于1ml TEBST中。此溶液可在4℃下无限期保存
实施例2.2.水凝胶包裹微球的活化并交联靶向抗体
1、取200μL水凝胶包裹的微球(1:2)，用1ml PBS(ph7.4)缓冲液洗涤水凝胶微球。
2.使用MES缓冲液(ph 6.5)重悬水凝胶包裹的微球，快速涡旋。
3.称重10mg EDC和5mg NHS，分别用200μL MES缓冲液(ph6.5)溶解，快速加入水凝胶微球。
4.室温孵育0.5h。活化羧基
5.用ph 7.4的PBS缓冲液清洗水凝胶包裹的微球，重复2X。
6.重悬1ml PBS缓冲液(ph 7.4)。
注:清洗水凝胶包裹的微球需要更快，避免失去功效。
7.对于分析B细胞的水凝胶包裹微球，在步骤6后加入50μL CD20抗体(Abcam)；对于分析T细胞的水凝胶包裹微球，在步骤6后加入50μL CD2抗体(Abcam)。在室温孵育4h。
8.用pH 7.4的PBS缓冲液洗涤水凝胶包裹的微球，重复5次。除去未反应的CD20或CD2抗体(Abcam)。该微球上偶联的抗体为靶向配基。
9.重悬200μL PBS缓冲液(pH 7.4)，保存4℃
实施例3结合物的形成
将实施例2.2中生成的携带靶向T细胞和B细胞配基的两种水凝胶包裹DNA编码微球1：1进行混合，在室温下以5rpm旋转15分钟
·去除上层清液
·在500μL PBS缓冲液中重悬微球
·计数细胞8.0*10^5细胞与微球孵育在200μL含2mM EDTA的PBS缓冲液，旋转溶液30分钟
·去除所有上清计数上清中细胞
·B细胞与靶向B细胞的水凝胶包裹微球结合，微球上PolyT与BCR特异性的引物，可对B细胞的mRNA进行全面深入的分析；T细胞与靶向T细胞的水凝胶包裹微球结合，微球上PolyT与TCR特异性的引物，可对T细胞的mRNA进行全面深入的分析。
实施例4隔室形成
通过液滴微流控设备用媒介物(含氟油性介质)来包裹微球。
所有化学品均从Sigma-Aldrich、Fisher Scientific和Roche订购。使用1.2mM dNTP、polyT引物、1U/μL RNaseOUT、5mM DDT、9mM MgCl 2、0.25U/ml逆转录酶、1X DNA聚合酶预混液、1％(v/v)吐温-20、1mg/ml牛血清蛋白、2％(v/v)PEG-6000。
液滴生成(图8A、图8B)：液滴是使用使用100μm高度和100μm宽喷嘴的微流控芯片(来源；通过一般光刻和PDMS纳米压印的流程，由实验室自主制备微流控芯片)产生的。使用的典型流速为：细胞(I相)-12μL/h，转录组扩增混合物(II相)-288μL/h，和液滴生成油(5％PEG-PFPE 2HFE7500)-600μL/h。
实施例5 mRNA逆转录及扩增
将乳化生成的液滴收集在200ul管中，进行PCR程序，具体PCR程序如下表所示：
步骤 温度 时间 循环次数
1 60℃ 3min 1X
2 50℃ 1h 1X
3 98℃ 3min 1X
4 98℃ 20s 19-20X
  67℃ 20s  
  72℃ 6min  
6 15℃ - 1X
表15单细胞cDNA扩增温控程序；对标记好的cDNA分子进行扩增
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
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。
实施例6扩增产物的打断及建库
使用DNA打断试剂盒(One-step DNA Lib Prep Kit for Illumina V2(50ng Input DNA),RK20239)对扩增产物进行打断。利用Tn5转座酶法进行DNA打断后进行文库扩增，文库PCR扩增程序如下表所示，文库扩增完成后使用1％琼脂糖凝胶电泳验证扩增文库片段，一个典型的文库扩增产物如图(图9)。
步骤 温度 时间 循环次数
1 72℃ 3min 1X
2 98℃ 30s 1X
3 98℃ 15s 7X
  60℃ 30s  
  72℃ 1min  
4 72℃ 5min 1X
5 15℃ - 1X
表16单细胞cDNA Illumina文库的构建；将打断后的cDNA文库进行扩增，添加测序接头。
实施例7分析实验结果(来自于实施例2.2.的水凝胶包裹微球)
在微球上进行靶向mRNA捕获：T细胞主要分析TCR序列、其他免疫细胞分析全转录组；测序结果中既能得到靶向配基信息，又能得到BCR、TCR的详细信息，以及其他细胞的单细胞转录组独立信息。通过靶向配基和单细胞DNA编码进行聚类处理后，得到免疫细胞的高分辨率信息(T细胞分析结果含TCR信息)。
免疫系统的单细胞转录组数据见图10A。
通过靶向配基的第二标签，可对测序数据进行T细胞的聚类并进行克隆性(Clonality)【图10B】、D50数据【图10C】；通过靶向配基的第二标签，可对测序数据进行B细胞的聚类并进行BCR克隆性(Clonality)【图10D】、BCR的独特CDR3分布数据【图10E】
针对第二部分实施例(实施例8-11)对人鼠混合细胞进行各自的数字PCR分析的实验流程概述：
第一步：制备靶向特定人或鼠细胞的水凝胶包裹微球，在水凝胶单体中引入分析人、鼠的液滴数字PCR(ddPCR)引物(反应试剂)和序列特异性探针(标签分子)，并修饰抗体(靶向配基)以辅助人或鼠细胞的特异性结合。
第二步：将人与鼠细胞混合，与带靶向抗体及单细胞液滴ddPCR试剂的多种微球进行孵育结合后，通过液滴微流控进行高通量包裹。
第四步：液滴内各微球对靶向细胞中的特定DNA区域进行特异性扩增。
第五步：通过液滴内扩增荧光成像，获取细胞混合物的整体信息。
实施例8.分别制备携带PCR扩增反应试剂的水凝胶包裹微球
1、制备由6.2％丙烯酰胺，0.18％N,N'-双(丙稀酰)胱胺，0.3％过硫酸铵，0.6％丙烯酸钠，带10μM双键的引物(携带5’-Acrydite)(反应试剂)组成的聚丙烯酰胺(PAA)溶液。
对于HEK293T的分析，分别采用以下引物序列。
Figure PCTCN2022134736-appb-000013
Figure PCTCN2022134736-appb-000014
表17靶向人源GAPDH序列的PCR引物
对于3T3细胞
Figure PCTCN2022134736-appb-000015
表18靶向鼠源GAPDH序列的PCR引物
2、将此溶液与微球(TOSOH HW65)分别单独混合，添加在氢氟醚(HFE)油中制备5％(w/w)含氟表面活性剂和1％N,N,N,N-四甲基乙基二胺(TEMED)组成的不溶连续相，用于吹打液滴的生成和稳定。
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。
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
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。
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。
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。
8、3000x g旋转3分钟。移液去除上清液。重复3x。
9、重悬于1ml TEBST中。此溶液可在4℃下无限期保存(图1C)。
实施例9水凝胶包裹微球的活化并交联靶向抗体
1、取200μL水凝胶包裹的微球(1:2)，用1ml PBS(ph7.4)缓冲液洗涤水凝胶微球。
2.使用MES缓冲液(ph 6.5)重悬水凝胶包裹的微球，快速涡旋。
3.称重10mg EDC和5mg NHS，分别用200μL MES缓冲液(ph6.5)溶解，快速加入水凝胶微球。
4.室温孵育0.5h。活化羧基
5.用ph 7.4的PBS缓冲液清洗水凝胶包裹的微球，重复2X。
6.重悬1ml PBS缓冲液(pH 7.4)。
注:清洗水凝胶包裹的微球需要更快，避免失去功效。
7.对于分析人细胞的水凝胶包裹微球，在步骤6后加入50μL人-CD298、人β2微球蛋白抗体(Abcam)(靶向配基)，并偶联靶向人GAPDH的探针(标签分子)；对于分析鼠细胞的水凝胶包裹微球，在步骤6后加入50μL鼠CD45以及鼠MHC class I抗体(Abcam)(靶向配基)，并偶联靶向鼠GAPDH的探针(标签分子)。在室温孵育4h。
Figure PCTCN2022134736-appb-000016
表19区分人与鼠细胞的探针(标签分子)
8.用pH 7.4的PBS缓冲液洗涤水凝胶包裹的微球，重复5次。除去未反应的抗体。
9.重悬200μL PBS缓冲液(pH 7.4)，保存4℃
实施例10结合物的形成
将实施例9中生成的携带靶向293T细胞引物和3T3细胞配基引物的两种水凝胶包裹微球1：1进行混合，在室温下以5rpm旋转15分钟
·去除上层清液
·在500μL PBS缓冲液中重悬微球
·计数细胞8.0*10^5细胞与微球孵育在200μL含2mM EDTA的PBS缓冲液，旋转溶液30分钟
·去除所有上清计数上清中细胞
·人的293T细胞与靶向293T细胞的水凝胶包裹微球结合，微球上GAPDH_Human特异性的引物，可对人的GAPDH基因进行扩增分 析；鼠3T3细胞与靶向3T3细胞的水凝胶包裹微球结合，微球上GAPDH_Mouse特异性的引物，可对3T3细胞的mRNA进行扩增分析(图11)。
实施例11隔室形成
通过振荡媒介物(含氟油性介质)来包裹微球。所有化学品均从Sigma-Aldrich、Fisher Scientific和Roche订购。
液滴生成：液滴是使用使用100μm高度和100μm宽喷嘴的微流控芯片产生的。使用的典型流速为：细胞(I相)-12μL/h，转录组扩增混合物(II相)-288μL/h，和液滴生成油(5％PEG-PFPE 2HFE7500)-600μL/h。
水相中加入2.4mM dNTP、2U/μL RNaseOUT、20mM MgCl 2、0.5U/ml逆转录酶、2X DNA聚合酶预混液、2％(v/v)吐温-20、2mg/ml牛血清蛋白、4％(v/v)PEG-6000，60mM DTT的水溶液、400nM人和鼠的探针。
在桌面振荡仪上进行乳化(图12)。
将生成的液滴用EP管收集后置于PCR仪上，选择下列温控程序：
步骤 温度 时间 循环次数
1 72℃ 3min 1X
2 98℃ 30s 1X
3 98℃ 15s 35X
  60℃ 30s  
  72℃ 1min  
4 72℃ 5min 1X
5 4℃ - 永久
表20数字PCR扩增程序；释放水凝胶微球上的引物和探针，并对人和鼠的细胞基因组进行靶向扩增和检测
PCR过程通过Sniper QX40进行成像，监测FAM和HEX通道(图13A)，考察不同液滴内各细胞的mRNA表达异质性(图13B、13C)。
实施例总结：
本申请的技术方案满足了进行单细胞分析(靶向多重检测)的数据拆分的技术要求。
在第一部分实施例中，通过一系列合成了多种靶向反应的复合物微球，该微球混合物可实现对免疫细胞的单细胞多重测序分析。该方法无需用通过细胞富集或流式细胞仪对待分析细胞的各亚群进行纯化，简化了操作步骤。同时，靶向反应复合物微球在设计时既能识别细胞对象，将该细胞的特种反应试剂递送至该细胞所在的微液滴中，又能通过复合物上的标签进行后续精确的数据拆分。具体的结果见于图10A-10E，其中图10A显示了外周血单核细胞的单细胞测序分群结果。对于免疫细胞亚群中的T细胞额外分析了其TCR序列的频率(10C)，通过TCR树图得到可视化的TCR克隆分布(10B)，通过TCR二维图得到可视化的VJ序列分布(10B)。对免疫细胞亚群中的B细胞(10D、10E)额外分析了BCR序列的频率(图10E)，BCR树图得到可视化的BCR克隆分布(10D)。
在第二部分实施例中，通过一系列合成了多种靶向反应的复合物微球，该微球混合物可实现对人鼠混合细胞的单细胞多重荧光检测。如图13A-13C所示，通过液滴内单细胞qPCR可针对不同癌细胞上的不同靶标，以及针对人和鼠细胞上的不同靶标，实现对各细胞内的目标GAPDH mRNA进行高通量的丰度表征，得到群体的异质性信息。
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。

Claims (24)

  1. 一种靶向并分析待分析物的靶向反应复合物，其包括：
    载体；
    与载体连接的、待分析物的靶向配基；
    与载体连接的、用于检测待分析物的反应试剂；以及
    与载体连接的、与所述待分析物相对应的标签分子。
  2. 根据权利要求1所述的靶向反应复合物，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。
  3. 根据权利要求1所述的靶向反应复合物，其中，所述分析选自光谱学检测、测序、质谱检测、图片捕获、电信号检测中的一种或两种以上。
  4. 根据权利要求1所述的靶向反应复合物，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，进一步优选所述载体的直径为1μm-100μm，进一步优选所述载体形状选自正方体形、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；最优选碗形和/或红血球形；进一步优选所述载体的直径粒径分布系数CV小于20％，进一步优选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。
  5. 根据权利要求1所述的靶向反应复合物，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、促分裂原、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。
  6. 根据权利要求1所述的靶向反应复合物，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。
  7. 根据权利要求1所述的靶向反应复合物，其中，所述反应试剂是寡聚核苷酸引物、酶或小分子。
  8. 根据权利要求1所述的靶向反应复合物，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。
  9. 根据权利要求1所述的靶向反应复合物，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。
  10. 根据权利要求4所述的靶向反应复合物，其中，其中所述构成载体的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。
  11. 一种用于靶向并分析两种以上待分析物的靶向反应复合物群，其包 含两种以上根据权利要求1-10的任一项所述的靶向反应复合物，其中，
    每种反应复合物所包括的靶向配基彼此不同；
    优选每种反应复合物所包括的反应试剂彼此不同；
    进一步优选每种反应复合物所包括的标签分子彼此不同。
  12. 根据权利要求11所述的靶向反应复合物群，其中，所述靶向反应复合物还包括与所述靶向配基相对应的第二标签分子。
  13. 使用根据权利要求1-10中任一项所述的反应复合物或根据权利要求11或12所述的反应复合物群分析待分析物的方法，
    其包括以下步骤：
    使待分析物与根据权利要求1-10的任一项所述的靶向反应复合物或权利要求11或12所述的反应复合物群相互作用形成结合物；
    所述结合物自身形成反应隔室或通过媒介物围绕所述结合物形成反应隔室；
    根据所述标签分子和所述反应试剂进行的反应来对所述待分析物进行标记；
    任选基于所述标记对所述待分析物进行分析。
  14. 根据权利要求13所述的方法，其中，控制进行相互作用的待分析物与根据权利要求1-10的任一项所述的靶向反应复合物或权利要求11或12所述的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。
  15. 根据权利要求13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。
  16. 根据权利要求13所述的方法，其中，在使用根据权利要求11或12所述的反应复合物群进行分析时，基于第二标签分子提供靶向配基的信息以确认待分析物的类型。
  17. 一种反应隔室，其包括：
    待分析物；
    靶向所述待分析物的根据权利要求1至10的任一项所述的反应复合物。
  18. 根据权利要求17所述的反应隔室，其还包括围绕至少一个所述反应复合物与所述待分析物结合所生成的结合物的媒介物。
  19. 根据权利要求17或18所述的反应隔室，其中，每个反应隔室包括一个待分析物和一个反应复合物，所述待分析物和反应复合物在所述反应隔室内处于结合状态或者分开状态。
  20. 一种反应隔室群，其包含两种以上根据权利要求17或18所述的反应隔室。
  21. 根据权利要求20所述的反应隔室群，其中，在两种以上根据权利要求17或18所述的反应隔室中，
    每种反应隔室中的反应复合物所包括的靶向配基彼此不同；
    优选每种反应隔室中的反应复合物所包括的反应试剂彼此不同；
    进一步优选每种反应隔室中的反应复合物所包括的标签分子彼此不同。
  22. 根据权利要求17所述的反应隔室群，其中，所述靶向反应复合物还包括与所述靶向配基相对应的第二标签分子。
  23. 根据权利要求17～22中任一项所述的反应隔室群用于对待分析物群进行分析的用途。
  24. 根据权利要求23所述的用途，其中，所述待分析物群是细胞群。
PCT/CN2022/134736 2021-11-26 2022-11-28 靶向反应复合物及其在靶向多重检测中的用途 WO2023093886A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111418931.X 2021-11-26
CN202111418931 2021-11-26

Publications (1)

Publication Number Publication Date
WO2023093886A1 true WO2023093886A1 (zh) 2023-06-01

Family

ID=86437175

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/134736 WO2023093886A1 (zh) 2021-11-26 2022-11-28 靶向反应复合物及其在靶向多重检测中的用途

Country Status (2)

Country Link
CN (1) CN116179668A (zh)
WO (1) WO2023093886A1 (zh)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005062982A2 (en) * 2003-12-23 2005-07-14 Xing-Xiang Li Signal amplification methods for analyte detection
US20050191687A1 (en) * 2004-02-27 2005-09-01 Tianxin Wang Method for multiplexed analyte detection
CN104364652A (zh) * 2012-04-20 2015-02-18 Zbx公司 用于检测样品中的分析物的固相载体和方法
CN105137067A (zh) * 2009-11-09 2015-12-09 艾博生物医药(杭州)有限公司 一种检测装置
CN107223161A (zh) * 2014-12-15 2017-09-29 亿明达股份有限公司 用于基底上的单分子放置的组合物和方法
CN108474023A (zh) * 2016-01-22 2018-08-31 日本电气方案创新株式会社 靶标分析方法和用于该方法的靶标分析试剂盒
CN113624724A (zh) * 2020-05-07 2021-11-09 廖世奇 一种适配体分子信标对靶分子的多元检测分析方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005062982A2 (en) * 2003-12-23 2005-07-14 Xing-Xiang Li Signal amplification methods for analyte detection
US20050191687A1 (en) * 2004-02-27 2005-09-01 Tianxin Wang Method for multiplexed analyte detection
CN105137067A (zh) * 2009-11-09 2015-12-09 艾博生物医药(杭州)有限公司 一种检测装置
CN104364652A (zh) * 2012-04-20 2015-02-18 Zbx公司 用于检测样品中的分析物的固相载体和方法
CN107223161A (zh) * 2014-12-15 2017-09-29 亿明达股份有限公司 用于基底上的单分子放置的组合物和方法
CN108474023A (zh) * 2016-01-22 2018-08-31 日本电气方案创新株式会社 靶标分析方法和用于该方法的靶标分析试剂盒
CN113624724A (zh) * 2020-05-07 2021-11-09 廖世奇 一种适配体分子信标对靶分子的多元检测分析方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WEN, LAIXIN: "Application of Luminex Liquichip in Microbial Multiple Detection", OCCUPATION AND HEALTH, vol. 30, no. 16, 31 August 2014 (2014-08-31), XP009546685 *

Also Published As

Publication number Publication date
CN116179668A (zh) 2023-05-30

Similar Documents

Publication Publication Date Title
US11161087B2 (en) Methods and compositions for tagging and analyzing samples
US20230083422A1 (en) Methods and compositions for combinatorial barcoding
US11841371B2 (en) Proteomics and spatial patterning using antenna networks
CN107075543B (zh) 用于条形码化核酸的系统和方法
US20210163926A1 (en) Versatile amplicon single-cell droplet sequencing-based shotgun screening platform to accelerate functional genomics
US11866782B2 (en) Multi-omic analysis in monodisperse droplets
Xu et al. Microfluidic single‐cell multiomics analysis
WO2023093886A1 (zh) 靶向反应复合物及其在靶向多重检测中的用途
WO2023093887A1 (zh) 一种打破泊松分布形成反应隔室群的方法
US20240132958A1 (en) Multi-omic analysis in monodisperse droplets
US20220025430A1 (en) Sequence based imaging
US20220145285A1 (en) Compartment-Free Single Cell Genetic Analysis
WO2023225259A1 (en) Compositions and methods for characterizing antigen binding molecules from single cells
Azimzadeh et al. CRISPR-Powered Microfluidics in Diagnostics: A Review of Main Applications. Chemosensors 2022, 10, 3
WO2024015378A1 (en) Methods and systems for characterizing antigen-binding molecules expressed by immune cells
WO2024015856A1 (en) Compositions and methods for characterizing binding characteristics of antigen binding molecules from single cells
WO2023225201A1 (en) Compositions and methods for characterizing t cell, or t cell-like, receptors from single cells
WO2022245825A1 (en) Multi-omic analysis of extracellular vesicles in monodisperse droplets
WO2023114190A1 (en) Single-cell epigenomic profiling using fluidics and hydrogels

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22897967

Country of ref document: EP

Kind code of ref document: A1