WO2023093887A1

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

Info

Publication number: WO2023093887A1
Application number: PCT/CN2022/134738
Authority: WO
Inventors: 张经纬; 景祥益; 梁雪
Original assignee: 复旦大学
Priority date: 2021-11-26
Filing date: 2022-11-28
Publication date: 2023-06-01
Also published as: CN116640838A

Abstract

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

Description

A Method of Breaking Poisson Distribution to Form Reacting Compartment Clusters

technical field

The application relates to the field of high-throughput chemical analysis, in particular to a targeted reaction complex and their use in breaking Poisson distribution to form reaction compartment groups.

Background technique

The heterogeneity of cells is a ubiquitous life phenomenon. As an independent living entity, the properties and differences displayed by a single cell play a vital role in the development of the entire living system. Every tissue and organ in the human body contains a variety of cell types, and each type of cell will change with the life activity of the organism. If thousands of single cells are studied, it will be blurred Therefore, it is extremely important to understand the working principles of complex organisms, the life functions and immune responses of each cell type to reveal the working mechanism of human tissues and organs and the laws of gene regulation. For example, malignant tumors that cause human cancer are highly heterogeneous tissues, composed of tumor cells of various phenotypes, and the real malignant cells are mixed with normal cells, often accounting for only a small part of the entire tissue, so single-cell The analysis can determine which cells are drug-resistant and which cells are easy to transfer, which plays an important role in guiding precise drug use, predicting the course of disease development, and clinical guidance.

In seemingly homogeneous cell subpopulations, expression may also vary between single cells. Although the genome fundamentally determines cell behavior such as transcription or translation, gene expression is a random molecular process that is closely related to cellular Growth time and space are related, so that the analysis of the genome cannot accurately reflect the differences in the actual behavior of cells; as the main bearer of life activities, proteins have a direct impact on cell differences, dynamics and functions, but proteins in single cells Horizontal quantitative analysis, protein amplification, and efficient reading of protein sequences are always huge technical barriers; while RNA acts downstream of DNA and upstream of proteins, it has gradually become a powerful tool for indirectly judging gene expression and protein abundance. Analysis of the transcriptome can reveal the heterogeneity and randomness of genetic material and its expression at the single-cell level.

Contents of the invention

At present, there are many technical problems in high-throughput single-cell sequencing technology: one is the separation of high-throughput single cells. The most widely used high-throughput single-cell separation method is mainly completed by fluorescence-activated cell flow sorting. Flow cytometry can scan thousands of cells through multispectral channels at the same time, with high throughput and fast speed, and can accurately locate the position of single cell sorting; it can sort specific and non-specific cells to obtain the desired cells Subgroups can also achieve multi-parameter analysis, which has great advantages in the analysis of actual samples. Therefore, fluorescence-activated flow sorting is used to separate single cells, so that single cells can be separated in 96 or 384 microwell plates for subsequent analysis. Analysis is currently one of the most widely used techniques in the field of single-cell analysis (Jaitin, et al., Science, 343.776-779; Bagnoli, et al., Nature communications, 9.2937.)

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. First, the cost of encoding microspheres is expensive. Since fluorescence-activated flow cytometry sorting needs to consume a large amount of background microspheres, this method will cause a lot of waste of reagents. Second, the size of the microspheres often does not match the flow sorting consumables. Third, The sorting efficiency is low, and the number of cells and microspheres sorted per unit time is limited. It is impossible to achieve efficient high-throughput single cell and single microsphere one-to-one rapid pairing, which is far from the clinical requirements for the total number of cells. The selected single microspheres are easily broken.

Currently, there is no published technology that can integrate high-throughput capture of single cells, unbiased amplification of tiny single-cell contents, and comprehensive analysis of single-cell contents. However, there have been published reports of high-throughput analysis of single-cell transcriptomes using microfluidic technology. For example, the Cell article (Macosko et al., 2015, Cell: 161, 1202-1214; Klein et al., 2015, Cell 161, 1187-1201) reported the method of combining droplet microfluidics and encoding microspheres, based on Poor The principle of loose distribution uses the droplet microfluidic method to pair capture single cells and single microspheres. The mRNA released by single cell cleavage is captured by the paired coding microspheres, and then reverse transcription and amplification are performed to encode the single cell mRNA information. With amplification, high-throughput sequencing and bioinformatics methods were used to analyze the expression of large cell mRNAs. The capture of cells and microspheres in this method is based on the principle of Poisson distribution. Most of the droplets have no cells, and only ~1% of the droplets contain single cells. Combined with the Poisson distribution of microspheres, the effective analysis target is further reduced. Only a small number of cells in a large number of actual samples can be analyzed, which may ignore some important individual cells in the sample. In addition, this strategy is only suitable for samples with a large number of analysis objects. For some rare cells (such as circulating tumor cells), the number of cells in the sample is too small (10-100/mL blood), and this method cannot be used to achieve single-cell analysis. . These techniques are limited to the analysis of single-cell mRNA, and other single-cell contents cannot be analyzed, such as genome, miRNA, proteome, methylated DNA, metabolites, liposomes, phospholipids, etc. None of the currently disclosed technologies involves high-throughput analysis.

Microfluidic chip is an emerging field that has developed rapidly and matured in recent years. It uses microchannels of various structures and various forms of external force fields to manipulate, process and control microfluids or samples on a microscopic scale, thereby It realizes the integration of some or even all functions of traditional laboratories on one chip. However, the limitations of conventional microfluidic chips are also very obvious. It needs to design pumps and valves inside the chip to cooperate with external fluid control equipment with complex operations. The technical threshold is high, and it is difficult to reuse a chip. The reaction reagents need to consume a lot of chip production costs when performing multi-level and multi-scale analysis of the same sample (Macosko et al., 2015, Cell, 161, 1202-1214; Klein et al., 2015, Cell, 161, 1187- 1201; Han et al., 2018, Cell, 172, 1091–1107).

The fourth potential technical problem is that when the previous sequencing methods analyze actual samples, the currently reported sequencing methods based on encoded microspheres need to use fluorescence-activated flow cytometry to sort the target cells first, and then transfer them to each cell. On the analysis platform, the information of the cell contents will change as the environment of the cells changes, which may lead to deviations between the sequencing information reflected in the final sequencing results and the information in the real environment of the cells at that time.

The fifth technical difficulty lies in the separation of rare cells. When the number of cells to be analyzed is very rare and the independent transcriptome information of each single cell needs to be analyzed, traditional techniques based on capillary picking, gradient dilution, or laser cutting require labor costs. High, time-consuming, labor-intensive, and low throughput limit the high-throughput rapid separation and sequencing analysis of rare cells.

This application mainly focuses on the third type of technical problem above, that is, to solve the problem of one-to-one pairing of single cells and reaction reagents when realizing high-throughput analysis of multiple cells, and at the same time increase the number of droplet pairs between cells and microspheres. The high co-capture rate solves the inefficiency caused by the "double Poisson distribution".

In order to solve the above technical problems, the application provides:

1. A method of forming a population of reaction compartments, the method comprising:

Prepare a group of substances to be detected, which is composed of more than two substances to be detected;

Prepare a targeting reaction complex group, the targeting reaction complex group is composed of more than two targeting reaction complexes, the targeting reaction complex includes a carrier, a targeting ligand corresponding to the substance to be detected connected to the carrier base, a reaction reagent corresponding to the substance to be detected connected to the carrier, optionally including a label molecule connected to the carrier corresponding to the substance to be detected;

using the population of substances to be detected to be contacted with the population of targeting reaction complexes to form a population of conjugates;

A population of reaction compartments is formed by the conjugate itself or by surrounding the conjugate with a vehicle.

2. The method according to item 1, wherein the analyte is selected from one or more of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles, and cells, and by The complexes they form, most preferably the analytes are cells.

3. The method as described in item 1, wherein the shape of the carrier is selected from one or more of cube shape, tetrahedron shape, spherical shape, ellipsoid shape, octopus shape, bowl shape, red blood cell shape; most preferably bowl shape and/or red blood cells.

4. The method according to item 3, wherein the quantity of the carrier is 4 times or more than the quantity of the substance to be detected, preferably 10 times or more than the quantity of the substance to be detected, most preferably the quantity of the substance to be detected 10 to 30 times the number of objects.

5. The method as described in item 3 or 4, wherein the maximum diameter of the carrier is more than 1 time that of the substance to be detected, preferably 3 times or more than the maximum diameter of the substance to be detected, most preferably its maximum diameter is 4 times to 10 times that of the detected object.

6. The method according to any one of items 3 to 5, wherein the carrier is made of a polymer or a small molecule, preferably a polymer carrier, more preferably a polystyrene carrier made of polystyrene or hydrogel , further the polymer carrier is made of polystyrene, most preferably the polymer is ferromagnetic or paramagnetic.

7. The method according to item 1, wherein the targeting ligand can be natural or artificial, selected from nucleic acids comprising locked nucleic acids and XNA and their analogs, aptamers, small peptides, polypeptides, glycosyl peptides, polysaccharides, soluble receptors, steroids, hormones, mitogens, antigens, superantigens, growth factors, cytokines, leptin, viral proteins, cell adhesion molecules, chemokines, streptavidin, and the like Biotin and its analogues, antibodies, antibody fragments, single chain variable fragments (scFv), nanobodies, T cell receptors, major histocompatibility complex (MHC) molecules, antigenic peptide-MHC molecule complexes One or more of (pMHC), DNA binding protein, RNA binding protein, intracellular or cell surface receptor ligands, and multiple ligands, composite ligands, and coupling ligands formed by them.

8. The method as described in item 1, wherein the label molecule is selected from natural or artificial information molecules, including: oligonucleotide barcodes, oligopeptide or polypeptide barcodes, natural bases and LNA, PNA, XNA, etc. Nucleotides composed of unnatural bases, oligosaccharide or polysaccharide barcodes, chromophoric and auxochrome groups, metal atoms or ions, small molecules with distinguishable molecular weights, block polymers, polymeric One or more than two kinds of covalent linkages between substances and backbone molecules and the complex formed between them.

9. The method according to item 1, wherein the reaction reagent is an oligonucleotide primer, an enzyme or a small molecule.

10. The method according to item 1, wherein the carrier diameter particle size distribution coefficient CV is less than 20%.

11. The method as described in item 10, wherein, the surface of the carrier is coated with a mechanical buffer coating; further preferably coated with a hydrogel coating; most preferably the hydrogel coating has a ferromagnetic or paramagnetic particles.

12. The method according to item 1, wherein the connection is selected from covalent bonds, metallic bonds, ionic bonds, van der Waals forces, including hydrogen bonds, mechanical bonds, halogen bonds, chalcogen bonds, amophilic interactions, intercalation, Overlapping, cation–π bonds, anion–π bonds, salt bridges, secondary bonds between nonmetal atoms, secondary bonds between metal atoms and nonmetal atoms, aurophilic interactions, argentophilic interactions, double hydrogen bonds, and secondary bonds of gold bonds level key.

13. The method according to item 1, wherein the targeting ligand is connected to the carrier through the connection of the reaction reagent, the connection of the label molecule or the connection of the linker; the reaction reagent and The connection of the carrier is through the connection of the targeting ligand, the connection of the label molecule or the connection of the linker; the connection of the label molecule and the carrier is through the connection of the targeting ligand, Ligation via the reagent or via a linker.

14. The method according to item 6, wherein the small molecule is one or more of the targeting ligand, the reaction reagent, and the label molecule.

15. The method according to item 1, wherein the medium is an oily medium, preferably a fluorinated oily medium, or a solid medium, preferably a microwell plate.

16. The method according to item 1, wherein the two or more substances to be detected can be the same or not, and the two or more targeting reaction complexes can be the same or not, when the When the two or more substances to be detected are different and the two or more targeting reaction complexes are different, the targeting reaction complex includes label molecules corresponding to the substances to be detected connected to the carrier.

Beneficial technical effects realized by the technical solution of the present application

By adopting the technical scheme of the present application, the one-to-one pairing problem of the analyte (single cell) and the carrier (single microsphere) is solved while realizing high-throughput analysis of multiple analytes (such as cells), At the same time, the co-capture rate of the medium (droplet) on the cells and microspheres is increased, which solves the problem of inefficiency caused by the "double Poisson distribution" phenomenon that occurs when the analyte is paired with the carrier. By using erythrocyte-shaped and bowl-shaped carriers (such as polystyrene microspheres) and carriers with a large size and a large number relative to the object to be detected (such as cells), the steric hindrance effect is used to further ensure that the "single cell and single cell The realization of the purpose of "one-to-one pairing of microspheres". By coating a layer of buffer layer (hydrogel layer) on the surface of the carrier (such as polystyrene microspheres), the danger of cells being crushed during the process of combining cells with microspheres is avoided, and the loss of the substance to be detected is reduced. The targeting complex group used in the present application has at least one reaction reagent, which realizes high-throughput analysis of the substance to be detected.

Description of drawings

Figure 1 shows a schematic diagram of the composition of the targeting reaction complex according to the present application; the targeting reaction complex includes a carrier, a targeting ligand corresponding to the substance to be detected connected to the carrier, and a ligand corresponding to the substance to be detected connected to the carrier. Reaction reagents optionally include label molecules corresponding to the substances to be detected connected to the carrier.

Figure 2A shows that the hard microspheres may crush the cells when they are combined with the cells; Figure 2B shows that the hydrogel coating on the surface of the hard microspheres has a protective effect on the cells; Hydrogel-coated targeting-response complexes (DNA-encoded microspheres coated with hydrogel coatings).

Figure 3A shows the hydrogel encapsulation by pipetting the microspheres; Figure 3B shows the image of the hydrogel after gel formation and demulsification.

Figure 4A shows that the diameter of the microsphere is larger than that of the cell, and the steric hindrance effect helps the combination of the microsphere and the cell at 1:1; it is difficult for a cell to combine with two or more microspheres; Figure 4B shows that the diameter of the microsphere is smaller than that of the cell, and the formation of Multiple microspheres bound to one cell, or aggregates of cells and microspheres.

Figure 5A shows the preparation of hydrogel-coated magnetic microspheres; Figure 5B shows the magnetic purification after demulsification; Figure 5C shows the hydrogel-coated magnetic microspheres, and magnetic purification after demulsification; Under the microscope, the antibody modified on the surface of the hydrogel layer after activation can be observed (the outer layer is bright white); in Figure 5E, the white arrow refers to the formation of the antibody-modified hydrogel-coated microspheres through incubation with cells1: 1 combined.

Figure 6A-C shows the bowl-shaped carrier, and the steric hindrance effect helps the microspheres to combine with the cells at 1:1; it is difficult for a cell to combine with two or more bowl-shaped or red blood cell-shaped microspheres; The generation of bowl-shaped aqueous two-phase and ultraviolet crosslinking are carried out here; Figure 6B shows the bowl-shaped particles; Figure 6C shows the 1:1 combination of bowl-shaped particles and cells.

Figure 7A shows the finished hydrogel microspheres prepared by microfluidic equipment; Figure 7B shows that in the hydrogel microspheres, the label molecule and polyT are on the same reverse transcription primer; Figure 7C shows the hydrogel microspheres aggregate in droplets.

Figure 8A shows modified microspheres with anti-human-CD298, human β2 microglobulin, mouse CD45, and mouse MHC class I antibodies, which can be used as universal binding microspheres to bind cells; Figure 8B shows the water The gel microspheres bind to the cells to form a 1:1 complex.

Figure 9A is a flow chart of the entire high-throughput analysis process (targeted reaction complexes are pre-combined with analytes and then packaged into high-throughput compartments); Figure 9B, when the number of microspheres is 10 times or more than that of cells , can form a 1:1 combination, and there is a probability of 1/100 to observe that one cell binds to two microspheres; Poisson distribution improves the capture rate of cells; Figure 9D shows that in some cases, the target reaction complex and the bound cells will be separated in the compartment; Figure 9E shows that the analyte is a pair of cells interacting with each other.

Figure 10A-C shows the process of controlling the water/oil liquid in the microfluidics to form a water-in-oil compartment through a syringe pump; where Figure 10B shows the high-throughput formation of a microfluidic device; Figure 10C shows a microsphere The complex with cells enters the microfluidic intersection, and high-throughput droplets are generated; Figure 10D shows the results of single-cell analysis; shows the results of single-cell analysis, and human and mouse cell subpopulations can be separated.

Figure 11A shows the following steps: the first step of hydrogel encapsulation of microspheres, and cross-linked streptavidin; the second step of cell biotinylation; the third step, the biotinylated cells with streptavidin Incubation and binding of avidin; the fourth step, high-throughput packaging of the complexes of microspheres and cells to form compartments, release of reaction reagents in the compartments, and labeling of cells in the compartments; Figure 11B shows the Gel-encapsulated target-tagged beads modified with fluorescent streptavidin; Figure 11C shows biotinylated cells; Figure 11D shows hydrogel beads bound to cells; Figure 11E shows manual pipette tips The compartments are formed outside the hydrogel-wrapped microspheres by pipetting, and the cells in the compartments are lysed to release the mRNA therein.

Detailed ways

Specific embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although specific embodiments of the present application are shown in the drawings, it should be understood that the present application may be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that the present application can be more thoroughly understood, and the scope of the present application can be fully conveyed to those skilled in the art.

It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art should understand that they may use different terms to refer to the same component. The specification and claims do not use differences in nouns as a way of distinguishing components, but use differences in functions of components as a criterion for distinguishing. "Includes" or "comprises" mentioned throughout the specification and claims is an open term, so it should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred implementation mode for implementing the application, but the description is for the purpose of the general principles of the specification, and is not intended to limit the scope of the application. The scope of protection of the present application should be defined by the appended claims.

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

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

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

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

The acquisition methods of various biological materials described in the examples are only to provide an experimental acquisition method to achieve the purpose of specific disclosure, and should not be a limitation on the source of biological materials in this application. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and ethics can be replaced according to the tips in the examples.

The present application provides methods of forming a population of reaction compartments.

In a specific embodiment, a method of forming a population of reaction compartments is provided, the method comprising:

Here, "compartment" means providing a specific reaction space for a specific chemical reaction in which reactants can react with each other without reacting with reactants in adjacent compartments, however, Depending on the type, the product of the compartment may exchange substances with the product of the adjacent compartment (for example: dyes, surfactants, etc.).

In the context of this specification, "targeting" complies with the general definition in the field of biotechnology, typically antibody-antigen affinity reaction, affinity reaction of complementary nucleic acid sequences.

In the context of this specification, "connection" should be understood in a broad sense, at least including various connections through molecular bonds and without molecular bonds.

Figure 1 shows the components of the targeting reaction complex, where the structural relationship of targeting ligands, label molecules and reaction reagents respectively connected to the "carrier" is shown. Among them, the "carrier" can be composed of the same molecules as the targeting ligand, label and reaction reagent, that is, it can be composed of small molecules. More usually, the carrier consists of a polymer, preferably polystyrene.

In yet another specific embodiment, a method for forming a reaction compartment population is provided, wherein the analyte is selected from the group consisting of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles, and cells One or more than two, and complexes formed by them, most preferably the analytes are cells.

In the context of this specification, "cell" conforms to the general definition in the field of biology, which includes at least prokaryotic cells and eukaryotic cells. For the application of the technology of this application, it can sometimes specifically refer to differentiated cell populations of multicellular organisms, For example, immune cell populations include at least lymphoid B cells, lymphoid T cells, and NK cells.

In the context of this specification, "organelle" conforms to the general definition in the field of biology, and organelle is generally considered to be a microstructure or micro-organ scattered in the cytoplasm with a certain shape and function. They make up the basic structure of cells and enable cells to work and run normally. The organelles in the cell mainly include: mitochondria, endoplasmic reticulum, centrosome, chloroplast, Golgi apparatus, ribosome, etc.

In the context of this specification, "complex" refers to two or more substances that interact with each other and have a certain binding strength. These substances can be composed of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria , viruses, organelles, and cells. For example: after incubation of HepG2 with T cells that recognize the cell line (genetically modified to carry the T cell receptor gene targeting the "major histocompatibility complex-AFP158 peptide"), cell-to-cell interactions will form; Specific examples are: complexes of bacteria and bacteriophages (viruses), complexes of cells and membrane proteins (polypeptides), and complexes of ribosomes and RNA (nucleic acids).

In a specific embodiment, a method for forming a group of reaction compartments is provided, wherein the shape of the carrier is selected from one of cube shape, tetrahedron shape, sphere shape, ellipsoid shape, octopus shape, bowl shape, and red blood spheroid shape Or two or more; most preferably bowl-shaped and/or red blood cell-shaped. "Bowl shape" specifically refers to a concave spherical shape, which is provided with a concave surface on the plane side of the "hemisphere". When the substance to be detected is bound to the concave surface of the carrier, due to the steric hindrance effect, the substance to be detected Will not bind to another carrier. The concave spherical shape specifically refers to a spherical circular plane with a concave surface. When the substance to be detected is bound to the concave surface of the carrier, the substance to be detected will not be bound to another carrier due to steric hindrance. The "red blood cell" carrier has two concave surfaces. When the substance to be detected is bound to the upper concave surface or the lower concave surface of the carrier, the substance to be detected will not bind to another carrier due to steric hindrance.

In yet another specific embodiment, a method for forming a group of reaction compartments is provided, wherein the amount of the carrier is more than 10 times the amount of the substance to be detected, most preferably 10 times the amount of the substance to be detected times to 50 times. Specifically, the quantity of the carrier can be 10 times, 12 times, 14 times, 16 times, 18 times, 20 times, 22 times, 24 times, 26 times, 28 times, 30 times, 32x, 34x, 36x, 38x, 40x, 42x, 44x, 46x, 48x or 50x. It is desirable to control the amount of analyte interacting with the aforementioned targeted reaction complex or population of aforementioned reaction complexes and the assay process so that only one analyte per conjugate is analyzed. In the embodiment of the present application, specifically, when the number of microspheres carrying the reaction reagent is 10-20 times the number of analyzed cells, each microsphere has a high probability of binding to only one cell according to the Poisson distribution.

In the context of this specification, the meaning of "Poisson distribution" is to describe the number of microspheres or cells randomly appearing in a unit space; the meaning of "double Poisson distribution" is to describe the number of microspheres and cells randomly appearing in a unit space quantity. In an embodiment, if there is a phenomenon that the number of microspheres and cells in a unit space is greater than the double Poisson distribution, it can be considered as "breaking the Poisson distribution".

In a specific embodiment, a method for forming a group of reaction compartments is provided, wherein the maximum diameter of the carrier is more than 2 times that of the substance to be detected, and most preferably its maximum diameter is 3 times to 10 times that of the substance to be detected. times. Specifically, the maximum diameter of the carrier may be 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times that of the substance to be detected. In the context of this specification, "diameter" specifically refers to the distance between the center of a plane figure or a solid (such as a circle, a conical section, a sphere, a cube) and two points on an edge). In the case of using erythrocyte-shaped and bowl-shaped carriers, due to the steric hindrance effect, the 1:1 combination of the carrier and the analyte is realized to the greatest extent. Especially when erythrocyte-shaped microspheres and immune cells are used, and the diameter of erythrocyte-shaped microspheres is 3 times to 30 times that of immune cells, the 1:1 combination of the carrier and the substance to be detected is realized to the greatest extent.

Specifically, for example, 10 times, 12 times, 14 times, 16 times, 18 times, 20 times, 22 times, 24 times, 26 times more than the substance to be detected can be used while using the "bowl-shaped" or "red blood cell"-shaped carrier. times, 28 times, 30 times, 32 times, 34 times, 36 times, 38 times, 40 times, 42 times, 44 times, 46 times, 48 times or 50 times the number of carriers; Simultaneous use of 3 to 10 times the carrier size of the substance to be detected, for example, 10 times, 12 times, 14 times, 16 times, 18 times, 20 times, 22 times, 24 times, 26 times, 28 times more than the substance to be detected , 30 times, 32 times, 34 times, 36 times, 38 times, 40 times, 42 times, 44 times, 46 times, 48 times or 50 times the number of carriers; Times, 12 times, 14 times, 16 times, 18 times, 20 times, 22 times, 24 times, 26 times, 28 times, 30 times, 32 times, 34 times, 36 times, 38 times, 40 times, 42 times, 44 times, 46 times, 48 times or 50 times the number of carriers while using "bowl-shaped" or "red blood cell"-shaped carriers; times, 18 times, 20 times, 22 times, 24 times, 26 times, 28 times, 30 times, 32 times, 34 times, 36 times, 38 times, 40 times, 42 times, 44 times, 46 times, 48 times or 50 times the number of carriers while using "bowl-shaped" or "red blood cell"-shaped carriers, and using a carrier size with a maximum diameter of 3 to 10 times the size of the object to be detected; Probability of combining only one cell" technical effect.

In yet another embodiment, there is provided a method of forming a population of reaction compartments, wherein said carrier is made of polystyrene or small molecules, most preferably made of polystyrene.

In a specific embodiment, a method for forming a population of reaction compartments is provided, wherein the targeting ligand can be natural or artificial, selected from nucleic acids including locked nucleic acids and XNA and their analogs, adapters small peptides, polypeptides, glycosylated peptides, polysaccharides, soluble receptors, steroids, hormones, mitogens, antigens, superantigens, growth factors, cytokines, leptin, viral proteins, cell adhesion molecules, chemokines , streptavidin and its analogs, biotin and its analogs, antibodies, antibody fragments, single-chain variable fragments (scFv), nanobodies, T cell receptors, major histocompatibility complex (MHC) Molecule, antigenic peptide-MHC molecular complex (pMHC), DNA-binding protein, RNA-binding protein, one or more than two of intracellular or cell surface receptor ligands, and multiple ligands and complex ligands formed by them , Coupling base. In the context of this specification, "antibody" conforms to the general definition in the field of biology. Specifically, an antibody refers to a protective protein produced by the body due to stimulation by an antigen. It is a large Y-shaped protein secreted by plasma cells (effector B cells) and used by the immune system to identify and neutralize foreign substances such as bacteria and viruses. It is only found in body fluids such as blood in vertebrates, and The cell membrane surface of its B cells. Antibodies recognize a unique feature of a particular foreign object, called an antigen. In the technical solution of the present application, for example, surface receptors of lymphocytes can be used as antigens to prepare corresponding antibodies, which are chemically modified on the surface of microspheres to separate lymphocyte populations. In the context of this specification, "aptamer" conforms to the general definition in the field of biology, and in the technical solution of this application, it may specifically refer to nucleic acid aptamers. Aptamer is a DNA (deoxyribonucleic acid), RNA (ribonucleic acid) sequence, XNA (nucleic acid analog) or peptide. Usually, it is an oligonucleotide fragment obtained from a nucleic acid molecule library by using an in vitro screening technique - Systematic evolution of ligands by exponential enrichment (SELEX). Nucleic acid aptamers can bind to a variety of target substances with high specificity and high selectivity, so they are widely used in the field of biosensors. When the nucleic acid aptamer specifically binds to the target substance, the configuration of the nucleic acid aptamer itself will change accordingly. In the technical solution of the present application, when the analyte is a nucleic acid fragment, its nucleic acid aptamer can be selected and chemically modified and fixed on the surface of the microsphere to separate cells.

In yet another specific embodiment, a method for forming a group of reaction compartments is provided, wherein the tag molecules are selected from natural or artificial information molecules, including: oligonucleotide barcodes, oligopeptide or polypeptide barcodes, by Nucleotides, oligosaccharide or polysaccharide barcodes, chromophoric groups and auxochrome groups, metal atoms or ions, and molecular weights composed of natural bases and LNA, PNA, XNA and other unnatural bases can be distinguished One or more of the small molecules, block polymers, covalent links between polymers and backbone molecules, and complexes formed between them. In the context of this specification, a DNA barcode (DNA barcode) refers to a standard, sufficiently variable, easily amplified and relatively short DNA fragment that can represent the species in an organism. DNA barcoding has become an important tool in ecological research, not only for species identification, but also to help biologists further understand the interactions that occur within ecosystems. When an unknown species or part of a species is discovered, the researchers profile the DNA barcode of its tissue and compare it to other barcodes in an international database. A match with one of these would allow the researchers to confirm the species' identity. DNA barcoding technology is an emerging technology for rapid and accurate identification of species by using a conserved fragment in the DNA of organisms. Specific to the embodiment of this application, when the "analysis" is the transcriptome sequencing of immune cell populations, the use of DNA barcodes can deconvolute the analysis results after obtaining high-throughput analysis results of immune cell populations change.

In a specific embodiment, a method of forming a population of reaction compartments is provided, wherein the reaction reagent is an oligonucleotide primer, an enzyme or a small molecule. In this context, a reagent is a substance that reacts with an analyte and produces a signal that can be used for detection. In the context of this specification, "reagent" may sometimes specifically refer to a fluorescent reagent capable of causing the analyte to emit fluorescence. In the context of this specification, "primer" conforms to the general definition in the field of biotechnology, specifically, a primer refers to a macromolecule with a specific nucleotide sequence that stimulates synthesis at the initiation of nucleotide polymerization, A molecule that is hydrogen bonded to a reactant is called a primer. Primers are usually two artificially synthesized oligonucleotide sequences, one primer is complementary to a DNA template strand at one end of the target region, and the other primer is complementary to the other DNA template strand at the other end of the target region, and its function is to act as a nucleotide At the starting point of polymerization, nucleic acid polymerase can synthesize a new nucleic acid chain from its 3-end. In vitro artificially designed primers are widely used in polymerase chain reaction, sequencing and probe synthesis, etc. Specific to the embodiment of the present application, when the "analysis" is transcriptome sequencing, oligonucleotide primers can be used as "reaction reagents". In the context of this specification, "small molecules constituting reaction reagents" can be various small molecules in the field of chemical technology. When the "analysis" is fluorescence detection, in particular, the "small molecules" can emit fluorescence Small molecules such as FAM, VIC, etc.

In a specific embodiment, there is provided a method of forming a population of reaction compartments, wherein said carrier diameter has a particle size distribution coefficient CV of less than 20%. The English abbreviation here is defined as follows: CV=SD/average particle diameter, which can represent the width of the particle size distribution. Among them, SD: is the standard deviation (Standard Deviation), which is recorded as σ in statistics; CV is also called relative standard deviation (Coefficient of variation), and is recorded as α in statistics. Herein, the diameter of the microspheres used can be calculated by means and devices known to those skilled in the art, for example, it can be measured under a microscope and analyzed with image processing software, and can be measured by an instrument for measuring particle size (Bio-Rad T20 Cytometer) to determine, can also use the data provided by the microsphere supplier.

In a specific embodiment, there is provided a method of forming a population of reaction compartments, wherein the surface of the support is coated with a mechanical buffer coating; most preferably with a hydrogel coating. Here, the "mechanical buffer coating" should be regarded as a part of the "carrier", that is, the "targeting ligand", "reactive reagent" and "label molecule" described in this specification are all on the carrier The "mechanical buffer coating" is coated on the (non-coated mother core) before being connected to the carrier by, for example, chemical modification.

Alternatively, the tag molecule is first linked to the carrier, then coated with a mechanical buffer coating, and finally the reaction reagent and the targeting ligand are linked outside the mechanical buffer coating by, for example, chemical modification.

As an alternative, the reaction reagent is first connected to the carrier, then coated with a mechanical buffer coating, and finally the label molecule and the targeting ligand are connected outside the mechanical buffer coating by, for example, chemical modification.

As an alternative, the targeting ligand is first linked to the carrier, then a mechanical buffer coating is applied, and finally the reaction reagent and the label molecule are linked outside the mechanical buffer coating by, for example, chemical modification.

Alternatively, the label molecule and the reaction reagent are first linked to the carrier, then coated with a mechanical buffer coating, and finally the targeting ligand is linked outside the mechanical buffer coating by, for example, chemical modification.

Alternatively, the label molecule and the targeting ligand are first linked to the carrier, then coated with a mechanical buffer coating, and finally the reaction reagent is linked outside the mechanical buffer coating through, for example, chemical modification.

As an alternative, the reaction reagent and the targeting ligand are linked to the carrier first, then a mechanical buffer coating is applied, and finally the tag molecule is linked outside the mechanical buffer coating by, for example, chemical modification.

Alternatively, the label molecule, targeting ligand and label molecule are first linked to a carrier, and then a mechanical buffer coating is applied.

In a specific embodiment, there is provided a method of forming a population of reaction compartments, wherein the linkages are selected from covalent bonds, metallic bonds, ionic bonds, van der Waals forces, including hydrogen bonds, mechanical bonds, halogen bonds, sulfur Family bonds, aurophilic interactions, intercalation, overlapping, cation-π bonds, anion-π bonds, salt bridges, secondary bonds between non-metal atoms, secondary bonds between metal atoms and non-metal atoms, aurophilic interactions, argentophilic interactions , double hydrogen bonds and secondary bonds of gold bonds.

In a specific embodiment, a method for forming a group of reaction compartments is provided, wherein the linking of the targeting ligand to the carrier is through the linking of the reaction reagent, the linking of the tag molecule or the linking of the labeling molecule. The connection of the adapter; the connection of the reaction reagent and the carrier is through the connection of the targeting ligand, the connection of the label molecule or the connection of the linker; the connection of the label molecule and the carrier is through The linking of the targeting ligand, the linking through the reagent or the linking through the linker.

In a specific embodiment, a method for forming a group of reaction compartments is provided, wherein the small molecule is one or more of the targeting ligand, the reaction reagent, and the label molecule. That is, the targeting ligand, the reaction reagent, and the tag molecule themselves can form a part of the carrier.

In a specific embodiment, a method for forming a group of reaction compartments is provided, wherein the medium is an oily medium, preferably a fluorinated oily medium, or a solid medium, preferably a microwell plate. The reason for using an oily medium as the "vehicle" of the "compartment" is that the oily medium can effectively block charged nucleic acid molecules from crossing different "compartments". After marking, there will be no cross-contamination of products passing through the "compartment".

In a specific embodiment, a method of forming a population of reaction compartments is provided, wherein,

The two or more substances to be detected can be the same or different,

And the two or more targeting reaction complexes can be the same or not,

When the two or more substances to be detected are different and the two or more targeting reaction complexes are different, the targeting reaction complex includes label molecules corresponding to the substances to be detected connected to the carrier.

In a specific embodiment, in the above-mentioned method for forming a group of reaction compartments, the two or more substances to be detected are the same, and the two or more targeted reaction complexes are the same.

In a specific embodiment, in the above method for forming a group of reaction compartments, the two or more substances to be detected are the same, and the two or more target reaction complexes are different.

In a specific embodiment, in the above method for forming a group of reaction compartments, the two or more substances to be detected are different, and the two or more targeted reaction complexes are the same.

In a specific embodiment, in the above-mentioned method for forming a group of reaction compartments, the two or more substances to be detected are different, and the two or more targeting reaction complexes are different; and the targeting The reaction complex includes a label molecule corresponding to the detection substance connected to the carrier.

Example part

Example 1 Preparation of a carrier (microsphere wrapped with a hydrogel layer) with a mechanical buffer coating

Polystyrene microspheres (Nanomicro Technology Uni-PS-30) can be customized by Chemgenes Corp to form microspheres with DNA coded (label) reverse transcription polyT primers (reaction reagents).

1. Pipette 1mL of DNA-encoded polyT microspheres stored in ethanol into a 2ml medium centrifuge tube;

2. Remove the supernatant, add 0.2ml PBS buffer (Teknova) to wash the microspheres, repeat 2 times;

3. Take 10 μl of microspheres and count in the cell counting plate;

4. Add all the remaining microspheres into the polyacrylamide (PAA) solution described in Example 1, and mix well at the same time;

5. Pipette the entire solution in step 4 and add it to the fluorine-containing oil phase, and pipette with a 200 μl pipette for 3-4 minutes (Figure 3A).

6. Stand at room temperature for 4 hours.

7. Remove the lower layer of oil with a pipette.

8. Add 20% (v/v) perfluorooctyl alcohol (PFO) in 100uL HFE oil to a 2ml collection tube as a chemical demulsifier;

9. After mixing, spin the 2ml collection tube at 2000g for 2 minutes. Remove the PFO/HFE supernatant by pipetting. Repeat 1x;

10. Remove the supernatant by pipetting, add 1ml of PBS containing 0.2% Tween20 to remove the surfactant/solution. Repeat 2x;

11. Add 1mL TEBST buffer (20mM Tris-HCl pH 8.0, 274mM NaCl, 5.4mM KCl, 20mM EDTA, 0.2% Triton X-100), mix well.

12. Spin at 3000x g for 3 minutes. Remove the supernatant by pipetting. Repeat 3x.

13. Resuspend in 1ml TEBST (Figure 3B). The microspheres can be stored indefinitely at 4°C in this solution.

On the microsphere (carrier) coated with the hydrogel layer, the targeting ligand, label or reaction reagent can be loaded in advance or afterwards.

Example 2 (1) Formation of conjugates

Weigh 10 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (abbreviation: EDC) and 5 mg of N-hydroxysuccinimide (NHS), respectively with 200 μL of MES buffer (pH 6.5) dissolve.

Mix the microspheres with carboxyl hydrogel coating and EDC/NHS solution produced in Example 1, and rotate at room temperature for 15 minutes

Incubate the microspheres with rotation at 5 rpm for 5 minutes

· Remove the supernatant

Resuspend microspheres in 500 μL PBS buffer

Add 200 μL of 500mg/ml Anti human CD20 (Biolegend) to the bead solution

· Spin the microspheres for 2 hours at RT

Spin the microspheres down at 5 rpm for 5 minutes

Count cells 8.0*10^5 cells and incubate with antibody-modified (targeting ligand) hydrogel-coated microspheres in 200 μL PBS buffer and 2 mM EDTA, rotate the solution for 30 minutes

Collect the antibody-modified microspheres, remove all the supernatant and count the cells in the supernatant, and observe the formation of a 1:1 conjugate between the targeting ligand-modified microspheres and the cells (Figure 4A)

Since cells bind microspheres, the probability of having both microspheres and cells per unit volume breaks the Poisson distribution.

Embodiment 2 (two) The impact of carrier size on the combination of carrier and cell (as a comparative example)

There are many reports of affinity magnetic bead sorting cells in previous literature and commercial product descriptions (for example: Miltenyi's

MicroBead Technology, Thermo Fisher’s affinity Dyna Bead products, etc.: https://www.miltenyibiotec.com/US-en/products/macs-cell-separation/cell-sep aration-reagents/realease-microbeads.html), These magnetic beads are smaller in diameter than cells (ie, in the range of 100 nm to 2.7 microns).

This example is a comparative example, and the purpose of the experiment is to verify that if the size of the microsphere is too small, it will affect the combination with the cells. When the number is greater than the number of cells, a phenomenon in which multiple microspheres combine with one cell will be formed.

Steps to activate microspheres:

1. Count the carboxyl-containing microspheres (Polysciences Inc. cat#18133) in 500ul MES buffer. The carboxyl groups on the microspheres can be combined with cells after activation (targeting ligand): 5.0*10^6 microspheres (10uL microspheres in 90uL PBS buffer (detection microsphere concentration: 6.72x10^5/mL, Transfer ~744uL microspheres→5.0x10^6)

2. Add 7.7mg (standard protocol 0.5mg/100uL) EDC and 8.0mg NHS to 744uL microsphere resuspension MES buffer;

3. Mix the microspheres and EDC/NHS solution, swirling the solution for 20 minutes at room temperature, mixing gently during 10-minute intervals;

4. Spin the microspheres at a speed of 500rpm for 5 minutes (or use a magnetic separator);

5. Remove the supernatant (lose some microspheres in the process);

6. Wash twice with 750uL DPBS buffer, pH=7.0

7. Resuspend in 300uL DPBS buffer; measure the microsphere concentration (1.35x10^6/mL), add an additional 99uL to reach 1x10^6 microspheres/mL in DPBS

8. Centrifuge and divide 100uL into 4 test tubes (test for various microsphere: cell ratios, respectively 1:1, 1:5)

Steps for cell binding:

1. Replace the solution into DPBS+1mM EDTA buffer for cell microsphere binding reaction

2. Rotate the microspheres at room temperature for 2 hours; during the process t=0min, 30min, 1h sampling inspection

3. Observation under a microscope

This experiment is a comparative example, and the conclusion drawn is that it is very important that the size of the microsphere is larger than the cell.

Because as shown in this comparative example: if the diameter of the microsphere is smaller than the cell, multiple microspheres will combine with one cell, or even multiple microspheres and multiple microspheres will form aggregates (Figure 4B), which cannot be broken. Poisson distribution at the cell end during cell analysis; and when the microsphere diameter is greater than or equal to the cell diameter (in this example, it is greater than twice the cell diameter), the steric hindrance effect makes it difficult for the microsphere to combine with the second cell, thus breaking Poisson distribution.

Example 3 (1) Preparation of carrier (microsphere) with separation function (magnetic nanoparticle layer) and binding experiment with cells

1. Prepare hydrogel-wrapped microspheres with a magnetic inner core by the method of Example 2 (Fig. 5A-C): the microsphere structure is a polystyrene inner core, coated with magnetic nanoparticles, and the outer layer is polypropylene with carboxyl groups Amide hydrogel layer (see Example 2 for PAA solution formulation and polymerization method)

2. Add 5mg EDC and 1.2mg NHS to 100μL MES buffer;

3. Rotate for 15 minutes, spin down the microsphere at RT for 5 minutes and spin for 5 minutes to remove the supernatant;

4. Resuspend the microspheres in 500mg/μL PBS buffer;

5. Add 200 microspheres of anti-human CD2 (Biolegend, targeting ligand) to rotate;

6. Incubate the microspheres at room temperature for 2 hours;

7. Spin and centrifuge the microspheres for 5 minutes (Figure 5D);

8. Count 8.0*10^5 microspheres and different numbers of cells in 200 μL PBS buffer and rotate for 30 minutes; the steps for measuring the number of cells are:

a. Measured Jurkat cell concentration (7.62x10^5mL)*3.5mL

b. Resuspend cells in 100uL PBS+2mM EDTA to reach 1x10^6/100uL～1x10^7 cells/mL

c. For experiments with various microsphere:cell ratios, aliquot cells into each rxn tube (100uL Jurkat cells, 40uL Jurkat cells+160uL DPBS, 20uL Jurkat cells+180uL DPBS).

9. Use a magnet to collect the microspheres, and it can be observed that the cells and the microspheres are combined at a ratio of 1:1 (Figure 5E), which indicates that the Poisson distribution law is broken at the cell end.

Experimental conclusion: When the microspheres are pre-combined with cells, the cell capture efficiency can be improved and the Poisson distribution can be broken.

Example 3 (2) The influence of the shape of the carrier on the combination of the carrier and the cells

The purpose of this experiment is to prove that the bowl-shaped microspheres can bind to cells and break the Poisson distribution law by using the steric hindrance effect.

Step 1: Fabrication of bowl-shaped microspheres (carrier)

For the dispersed phase, will contain 6.3% w/v biotinylated polyethylene glycol, 4.5% w/v gelatin and 1.5% w/v lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) A homogeneous precursor solution dissolved in DPBS was injected parallelized and emulsified step by step at a rate of 8 μL/min. Novec 7500 containing 2% Pico-Surf was injected into the continuous phase at 16 μL/min. Single-phase generated PEG/gelatin droplets flowed through Tygon tubes (0.03" ID, 0.0625" OD, Murdock) immersed in a 4°C water bath for temperature-induced phase separation of PEG and gelatin. Adjust the length of the tubing to ensure complete phase separation (~60 cm, 10 min incubation). The phase-separated droplet stream was introduced into a PDMS reservoir submerged in a water bath at 4 °C and exposed to UV light (200 mW/cm ² ) for 1–3 seconds near the outlet region of the reservoir for polymerization. Upon UV irradiation, the photocrosslinkable PEG component formed a polymer network, while the gelatin component remained unpolymerized (see Figure 6A for details). The cross-linked particles are collected and oil and gelatin-rich droplets are removed in a series of washing steps. First remove the surfactant oil by pipetting. A solution of 20% v/v perfluorooctyl alcohol (PFO, Sigma) in Novec 7500 and milli-q water was added sequentially to break the emulsion and transfer the particles to the aqueous phase. The samples were centrifuged at 2500 x g for 1 min to remove the oily phase. The microspheres were washed twice with Novec 7500 to remove remaining PFO and surfactant. After removing the oil layer with a pipette, the residual oil was washed three times with hexane (Sigma). The samples were then washed three times with 70% ethanol, followed by two milliliters of water to remove gelatin. Sterilize the pellets by incubating overnight in 70% ethanol before use. Bowl-shaped particles under the microscope (as shown in Figure 6B).

Step 2: Binding of bowl-shaped microspheres to cells

Bowl-shaped microspheres were modified with AF350 streptavidin (Fisher Scientific) (targeting ligand) prior to use. The bowl is washed with cell culture medium and concentrated in a conical tube. The amount of concentrated bowl-shaped microspheres calculated to cover the entire pore area with a monolayer of bowl-shaped particles is 4.8 μL/cm ² for 55 μm bowl-shaped microspheres, respectively. Transfer and disperse the calculated amount of bowl-shaped particles in the orifice plate. The wells with a monolayer of bowl-shaped microspheres were seeded with Jurkat cells at a ratio of 1:1 between target cells and bowl-shaped microspheres. Pipette the solution in the well several times to evenly disperse the cells and microspheres throughout the well area and make the cells adhere to the bowl-shaped microspheres. To remove non-attached cells, samples were filtered through a 20 μm reversible cell strainer (CellTrics) and washed with a solution consisting of 0.5% bovine serum albumin (BSA), 1% P/S, and DPBS containing 0.05% Pluronic F-127. "Wash Buffer" washes. Next, invert the cell strainer and wash with wash buffer to recover the bowl-shaped particles in the conical tube. Centrifuge the bowl-shaped pellet to remove wash buffer. The association of bowl-shaped particles with cells was observed by microscopy (see Figure 6C for details).

Experimental conclusion: When the microspheres are bowl-shaped, the 1:1 combination of microspheres and cells can be better achieved (see Figure 6C for details). Compared with Example 2 (2), the steric hindrance effect makes it difficult for the microspheres to combine with the second cell, thereby breaking the Poisson distribution.

Example 4 Preparation of a universal carrier: the carrier (microsphere) carries a variety of targeting ligands: anti-human-CD298, human β2 microglobulin, mouse CD45 and mouse MHC class I antibody.

(These are antibodies, right? Would it be better to add the word "anti"? Yes)

1. Commercial microspheres (Chemgenes Corp. Cat#Macosko-2011-10) contain DNA-encoded polyT capture oligonucleotides (reaction reagents and labels). It is coated with a carboxyl-containing hydrogel coating by droplet microfluidics, and the microspheres are counted in 500 μL MES buffer 8.0*10^5 microspheres

2. Add 5mg EDC and 1.2mg NHS to 100μL MES buffer

3. Mix the microspheres and EDC/NHS solution, and rotate the solution for 15 minutes at room temperature

4. Centrifuge the microspheres at 5 rpm for 5 minutes

5. Remove the supernatant

6. Resuspend the microspheres in 500 μL PBS buffer

7. Add 200 μL of 500 mg/mL human-CD298, human β2 microglobulin, mouse CD45 and mouse MHC class I to the microsphere solution (targeting ligand)

8. Spin the microspheres at room temperature for 2 hours

9. Centrifuge the microspheres at 5 rpm for 5 minutes

10. Count cells 8.0*10^5 cells and incubate with beads in 200 μL PBS buffer containing 2mM EDTA, swirl the solution for 30 minutes

11. Collect the microspheres using a magnet and remove all supernatant (Figure 2C).

Experimental conclusion: By loading various antibodies (targeting ligands) on the hydrogel-coated microspheres (carriers) containing DNA-encoded (labeled) reverse transcription primers (reaction reagents), the range of cell binding of the microspheres can be further expanded .

Example 5 Preparation of hydrogel microsphere carrier (the carrier is a solid hydrogel microsphere without a coating layer)

Unless otherwise specified, the chemical reagents in the following examples were purchased from Sigma Aldrich.

1. Prepare a polyacrylamide (PAA) solution consisting of 6.2% acrylamide, 0.18% N,N'-methylenebis(acrylamide), 0.3% ammonium persulfate, and 0.6% sodium acrylate. This solution was filled into a 1 ml syringe (BD) with a 28G needle.

2. Prepare an insoluble continuous phase composed of 5% (w/w) fluorosurfactant and 1% N,N,nn-tetramethylethyldiamine (TEMED) in hydrofluoroether (HFE) oil, and use for droplet formation and stabilization. Fill the solution into a new 1ml syringe.

3. Collect 1ml droplets in a 15ml collection tube and incubate at room temperature for 3 hours for polymerization. After incubation, remove the underlying oil with a pipette.

4. Add 20% (v/v) perfluorooctyl alcohol (PFO) in 1ml HFE oil to a 15ml collection tube as a chemical demulsifier

5. After mixing, spin the 15ml collection tube at 2000x g for 2 minutes. Remove the PFO/HFE supernatant by pipetting. Repeat 1x.

6. Remove the supernatant by pipetting, add 1ml of PBS containing 0.2% Tween20 to remove the surfactant/solution. Repeat 2x.

7. Add 1mL TEBST buffer (20mM Tris-HCl pH 8.0, 274mM NaCl, 5.4mM KCl, 20mM EDTA, 0.2% Triton X-100), mix well.

8. Spin at 3000x g for 3 minutes. Remove the supernatant by pipetting. Repeat 3x.

9. Resuspend in 1ml TEBST (Figure 7A). The microspheres can be stored indefinitely at 4°C in this solution.

Example 6 The reaction reagents and label molecules carried by the prepared microspheres can be combined into one substance (that is, the 5'-Acrydite-CTA CAC GAC GCT CTT CCG ATC T NNNNT ₂₈ VN primer has dual functions of label molecules and reaction reagents) ; In the sequence, V represents a mixture of G, C, and A; N represents a mixture of A, G, C, and T.

Using the microfluidic system in Example 6 to prepare 40% acrylic acid-N,N'-methylenebis(acrylamide) (37.5:1), 10% ammonium persulfate, 10X Gel buffer (100uL 1M Tris HCl pH=7.5; 20uL pH=8 0.5M EDTA; 150uL 1M NaCl; 730uL ddH ₂ O), 2X Oligo solution (100uM), polyacrylamide (PAA) solution formed hydrogel microspheres (formula shown in Figure 7B ). Wherein the Oligo sequence is: 5'-Acrydite-CTA CAC GAC GCT CTT CCG ATC T NNNNT ₂₈ VN (SEQ ID NO: 1).

Load the oligomer-containing solution for droplet generation. When the droplet is unstable, take a picture of the droplet to determine the droplet size. After the run, the oil was removed and TEMED was added. The oil contained 0.4% TEMED. To pour the oil back into the bead emulsion, the microspheres were solidified at 37 degrees (FIG. 7C).

Here, one substance (primer) has two functions (label molecule and reaction reagent).

Example 7 Preparation of hydrogel microspheres targeting cells and combining with cells to form complexes

Get the hydrogel microspheres prepared in Example 7 (the carboxyl group comes from the acrylic acid monomer), and count 8.0*10^5 microspheres in 500 μL of MES buffer

Add 5 mg EDC and 1.2 mg NHS to 100 μL MES buffer

· Mix the microspheres and EDC/NHS solution, swirling the solution for 15 minutes at room temperature

Centrifuge the microspheres at 5 rpm for 5 minutes

· Remove the supernatant

Resuspend microspheres in 500 μL PBS buffer

Mix 200 μL microspheres containing various reaction reagents with 500 mg/mL anti-CD45 antibody (BioLegend, targeting ligand)

Spin the microspheres for 2 hours at room temperature (Figure 8A)

Centrifuge the microspheres at 5 rpm for 5 minutes

Count cells 8.0*10^5 PBMC cells Incubate with beads in 200 μL PBS buffer containing 2mM EDTA, swirl the solution for 30 minutes

Collect the microspheres by gravity centrifugation and remove all supernatant (Figure 8B)

This example proves that when the reaction reagent and the label molecule belong to the same substance, the microspheres carrying the targeting ligand can also bind to cells.

Example 8 High-throughput formation of compartments and single-cell RNA sequencing (using the microspheres combined with human and mouse universal antibodies in Example 4)

The microspheres (Fig. 2C) and cell complexes were encapsulated with a vehicle (fluorine-containing oily medium) through a droplet microfluidic device (Fig. 10A). As shown in FIG. 10A , the complex of single cells and microspheres (from Example 4) was encapsulated into droplets using a custom-designed microfluidic device. The device connects two aqueous phases before they separate into discrete droplets. Laminar flow prevents mixing of two aqueous input phases prior to droplet formation, one flow phase containing lysate and other buffers, and the other flow phase containing cells in complex with DNA-encoded microspheres suspended in buffer. Droplet reaction compartments were generated using a microfluidic chip using a 90 μm height and 80 μm wide nozzle. Typical flow rates used are: ~200 μL/h for lysate-rich phase, ~100 μL/h for cell-enriched phase complexed with DNA-encoded microspheres and ~600 μL/h for droplet stabilization oil (2% PEG-PFPE ₂ HFE7500). h (FIG. 10B).

List of Microfluidic Device Components

(1) Inverted Microscope

(2) Pressure controller + 3 flow sensors / three syringe pumps

(3) 3 falcon tubes/3mL syringes

(4) Magnetic stirring system

(5) Microfluidic conduits for connection of experimental device components

(6) Microfluidic accessories and connectors

(7) PDMS co-flow microfluidic droplet generation device

(8) 100 micron cell strainer for beads

(9) 40 micron cell strainer for cells

(10) counting room

Bill of Materials:

Table 1 List of reaction materials for single-cell transcriptome sequencing

1. Wash the microspheres with 30mL 100% EtOH X2 (from Example 8)

2. Wash the microspheres with 30mL TE/TW buffer X2

3. Resuspend the microspheres in 10ml TE/TW buffer

4. Pass through a 100um filter (@4°C for long-term storage)

5. For single-cell transcriptome analysis, wash the complexes formed by microspheres and cells with saline flush

6. Resuspend the complex formed by washing microspheres and cells in physiological saline, the concentration is about 120 complexes/μL

7. Use a 6.4mm magnetic stirrer to fill the suspension of the cell-microsphere complex into a 3ml plastic syringe; the lysate is loaded from another needle tube (10% Sarkosyl normal saline)

8. Fill the droplet generating oil into a 10ml plastic syringe

9. Connect the syringe to a 125um co-flow device with 0.38mm inner diameter tubing

10. Keeping Microsphere-Cell Complexes in Suspension by Continuous Magnetic Stirring

Place the collected droplets under a microscope for observation (Figure 10C)

11. Add 30ml 6X SSC @ room temperature

12. Remove oil from the bottom of each aliquot drop with a P1000 pipette

13. Add 600ul perfluoro-1-octanol

14. Shake the tube vigorously by hand for ~20s

15. Rotate 1,000g～1min

16. Keep samples on ice to reduce dissociation of annealed mRNA

17. Remove about 5ml of supernatant above the oil-water layer

18. Wash the microspheres with another 30ml of 6X SSC buffer at room temperature

19. Transfer the aqueous layer to a new tube

20. Rotate 1,000g～1min

21. Transfer the microspheres to a non-stick 1.5ml tube

22. Wash twice with 1ml 6X SSC buffer

23. Wash once with 300ul 5x Maxima H-RT buffer

24. For 90,000 microspheres, add 200ul RT mixture:

i.1X Maxima RT Buffer

ii. 4% Ficoll PM-400

iii. 1mM dNTP

iv.1U/ul ribonuclease inhibitor

v.2.5uM TSO

vi.10U/ul Maxima H-RT

25. Incubate at room temperature for ~30min

26. Incubate at 42°C for ~90min

27. Wash once with 1ml 1X TE: with 0.5% sodium lauryl sulfate

28. Wash the microspheres twice with 1ml TE/TW buffer

29. Wash the microsphere once with 10mM Tris pH=7.5

30. Resuspend the microspheres in 200ul exonuclease I mixture

1X Exonuclease I Buffer (NEB)

1U/ul Exonuclease I (NEB)

31. Incubate @37℃～45min

32. Wash once with 1mL TE/SDS

33. Wash twice with 1 mL TE/TW

34. Wash once with ddH ₂ O

35. Resuspend in ddH ₂ O

36. PCR amplified aliquots of 1000 microspheres with a volume of 50uL each

1x Hifi HotStart Readymix(Roche)

0.8uM TSO primer

37. The PCR program is as follows:

温度temperature	时间time	循环cycle	循环数number of cycles
95℃95°C	3min3min	the	the
the	the	the	the
98℃98°C		20s20s	11	44
65℃65°C		45s45s	11	the
72℃72°C		3min3min	11	the
the	the	the	the
98℃98°C	20s20s	22	Xx
67℃67°C	20s20s	22	the
72℃72°C	3min3min	22	the
the	the	the	the
72℃72°C	5min5min	the	the

Table 2 PCR reaction program for cDNA amplification

38. Preparation of 3'-end cDNA fragments for sequencing

a. Place quartered 600pg cDNA into four standard Nextera XT labeling reactions

b. Use P5-TSO-hybrid and Nextera_N701 instead of the oligonucleotides provided in the kit

Table 3 PCR reaction program for library construction

The amplified library was sent to a third-party sequencing service company for sequencing.

Embodiment nine Analyzing the experimental result of embodiment eight

Applicants use bioinformatics tools such as Seurat to process raw sequencing results, and cells with low overall expression or high mitochondrial ratio are removed. For clustering, principal component analysis was used, followed by k-means clustering to identify distinct cell states. t-Distributed Stochastic Neighborhood Embedding (t-SNE) was used to visualize cell clusters. For meta-clustering, expression matrices from all hosts were pooled from four experiments using Seurat. Cell transcripts with fewer than 2000 cells were excluded from the analysis, and cells with fewer than 100 unique genes detected were excluded from the analysis (Fig. 10D).

Example 10 Microspheres with another targeting ligand (streptavidin)

The purpose of this experiment: Streptavidin is attached to the hydrogel coating of the microspheres to bind to the cells with surface-modified biotin. The experimental process is shown in Figure 11A:

Step 1: Hydrogel Bead Functionalization of Microspheres

1. Microspheres in __1.12_μL 1×PBST (__8.90x10^3_microspheres/μL)～5000-10k microspheres (carrier)

Use 600 μl of MES buffer (pH=4.7, 0.1 M) to resuspend the hydrogel beads to remove the buffer.

2. EDC (10mg) and NHS (5mg) were dissolved in 200μl MES buffer respectively, and the stock solution was added to the washed microspheres to reach the final concentration

物质substance	重量(g)Weight (g)
EDCEDC	0.010.01
磺基-NHSSulfo-NHS	0.0050.005

Table 4 Microsphere Activation Reagents

sample

200uL MES containing 10mg/ml EDC

200uL MES containing 5mg/ml Sulfo-NHS

3. The solution was vortexed for 30s, placed on a horizontal shaker (650rpm) and shaken at room temperature for 30min.

4. Centrifuge the pellet in PBST, rinse back and forth four times

5. Add NeutrAvidin stock solution (5mg/ml) to the granules to make the final protein concentration 1mg/mL, and incubate on a horizontal shaker for 2.5h (650rpm, room temperature).

the	样本sample	对照组control group
NeutrAvidin_μlNeutrAvidin_μl	100100	//
荧光链霉亲和素(2μg/ml)_μLFluorescent streptavidin (2μg/ml)_μL	//	22

Modification conditions of table 5 streptavidin

6. The particles were washed 4 times and stored in PBST at 4°C for long-term storage (Figure 11B). The NeutrAvidin on the microspheres is a targeting ligand, which can be targetedly combined with downstream cells modified with biotin.

Step 2: Biotinylation of 293T cells (operate at 4°C throughout the process)

1. Mammalian cells were stained with Calcein AM (ThermoFisher #C3099) in 1 mL PBS containing 0.04% BSA.

2. Prepare a 6x6cm ² Petri dish with 90-95% confluent cells

3. Wash the cells 3 times with ice-cold PBS (pH 8.0) to remove the amine-containing medium and proteins in the cells. Quickly remove PBS (Note: If cells are growing in suspension, use 1 x 10 cells per mL of biotin solution prepared in step 3. Do not exceed 4 x 10 total cells per labeling reaction)

Note: Do not keep the PBS in contact with the cells for more than 5 s to prevent the cells from rounding and falling off.

4. Gently scrape the cells into the solution, suspend the cells in 1ml PBS (pH 8.0), the concentration is about 25×10^6 cells/mL.

5. 1000_μL 293T cell in 1×PBS(__1.90x106_cell per mL)～10^6cell

6. Add 1.0 mg Sulfo-NHS-SS-Biotin (no weighing) reagent per ml of cell suspension (yielding ~2mM biotin reagent).

Note: Prepare a 10 mM solution of Sulfo-NHS-SS-Biotin immediately before use as follows:

Sulfo-NHS-SS-Biotin: Put 1mg into a microcupule and add 164μL of ultrapure water.

The reaction mixture was incubated at 4°C for 30 minutes.

Note: Incubation at 4 °C may reduce the active internalization of the biotin reagent.

7. Add PBS to the cell pellet, and gently pipette the cells up and down 3 times with a serological pipette. Centrifuge at 500 x g for 1 min and discard the supernatant.

NOTE: Quench the reaction and remove excess biotin reagent and by-products (Figure 11C).

Step 3: Streptavidinized microspheres capture 293T cells

1. Microspheres and cells in all experiments were resuspended in 100% PBS.

水凝胶包裹微球的浓度(/mL)Concentration of hydrogel-coated microspheres (/mL)	细胞浓度(/mL)Cell concentration (/mL)
1000010000	10001000

Concentration when table 6 cells were incubated with microspheres

2. Incubate at 4°C for 30 minutes with gentle agitation.

3. The total solution volume is ≈1mL. Prior to imaging, the solution was gently vortexed to remove non-adherent cells from the pellet.

Cells combine to form microspheres and capture cells 1:1 (Figure 11D)

Step 4: Encapsulate the hydrogel-encapsulated microspheres that have captured the cells.

Pipetting hydrogel-encapsulated microspheres to form droplet compartments

the	油相oil phase	水相water box	总体积total capacity	吹打时间blow time
样本sample	60ul60ul	30ul30ul	90ul90ul	3min3min

Table 7 Conditions for droplet generation

The aqueous phase contained 29U/mL proteinase K (NEB #P8107S) and 70mM DTT (Sigma #D9779) and mixed 10 pipette strokes. Take care to avoid creating air bubbles when mixing the cell-bound microspheres with the solution. Add 280 µL of 0.5% ionic Krytox's HFE 7500 oil 65 to the cell-bead complex and vortex horizontally at 3000RPM for 15 s, then vertically for 2 min using a custom vortexer. Remove oil from beneath the emulsion such that less than 100 µL of oil remains. Prior to lysis, the microsphere-cell emulsion was subsampled on a C-Chip disposable hemocytometer (Fisher Scientific #DHCN015), with each subsample containing 3.5 μL of emulsion per field. Chips were imaged in brightfield at 2X magnification. The remaining emulsion was enzymatically cleaved on a PCR thermocycler (Eppendorf Mastercycler Pro) at 65°C for 35 minutes with the lid temperature set at 105°C. After lysis was complete, fluorescence images were captured using a Nikon 2000 microscope (Thorlab M470L5) with 470 nm excitation light (FIG. 11E).

Example result summary:

The application lists three production techniques of targeting reaction complexes. These targeted reaction complexes can be combined with the analytes (cells): A) polymer microspheres coated with hydrogel; B) generally spherical hydrogel microspheres; C) bowl-shaped hydrogel microspheres .

Applying the targeted reaction complex of the present application can realize single-cell analysis, and improve the efficiency of cell analysis while breaking the Poisson distribution. The process involves two key steps: 1. The analyte represented by cells is incubated with the target reaction complex, and is effectively combined with the target ligand; 2. Through droplet microfluidics or tip pipetting, High-throughput reaction compartments can be formed to break the double Poisson distribution of cell analysis;

Tag molecules on the targeting reaction complex can be used to label the analytes during high-throughput reactions within the compartment. Other common reactants can enter the reaction compartment through the mixed solution.

The embodiment demonstrates that the size of spherical microspheres must be larger than that of cells to break the probability of cell binding more efficiently (double Poisson distribution). When the number of microspheres is 10-20 times the number of cells, the 1 :1 combination (see Figure 8A and Figure 8B for details); and when bowl-shaped hydrogel microspheres are used, the steric hindrance effect is more significant, and the number of microspheres does not need to be too much in excess of the number of cells.

Once the target reaction complex is combined with the cells, the high-throughput generation of the reaction compartment (the picture after generating the compartment should be the most important: 9A is the most important concept map; 5E, 8B, 6C, and 9B are the reaction compartments Before the formation; 9C is after the formation of the reaction compartment, as shown in 9C: no reaction compartment was found in the droplet, there were only cells, but no microspheres; it meant that all the cells were analyzed, which meant that the moorings were broken Loose distribution) of cells for various depth analysis including cell lysis, mRNA capture or single cell sequencing.

Although the embodiments of the present application have been described above in conjunction with the accompanying drawings, the present application is not limited to the above-mentioned specific embodiments and application fields, and the above-mentioned specific embodiments are only illustrative, instructive, and not restrictive . Those skilled in the art can also make many forms under the enlightenment of this description and without departing from the protection scope of the claims of the application, and these all belong to the protection list of the application.

Claims

A method of forming a population of reaction compartments, the method comprising:

Prepare a group of substances to be detected, which is composed of more than two substances to be detected;

Prepare a targeting reaction complex group, the targeting reaction complex group is composed of more than two targeting reaction complexes, the targeting reaction complex includes a carrier, a targeting ligand corresponding to the substance to be detected connected to the carrier base, a reaction reagent corresponding to the substance to be detected connected to the carrier, optionally including a label molecule connected to the carrier corresponding to the substance to be detected;

using the population of substances to be detected to be contacted with the population of targeting reaction complexes to form a population of conjugates;

A population of reaction compartments is formed by the conjugate itself or by surrounding the conjugate with a vehicle.
The method according to claim 1, wherein the analyte is selected from one or more of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles and cells, and by them The complex formed, most preferably the analyte is a cell.
The method according to claim 1, wherein the shape of the carrier is selected from one or more of cube shape, tetrahedron shape, sphere, ellipsoid, octopus shape, bowl shape, red blood cell shape; most preferably bowl shape and / or red blood cells.
The method according to claim 3, wherein the quantity of the carrier is 4 times or more than the quantity of the substance to be detected, preferably 10 times or more than the quantity of the substance to be detected, most preferably the quantity of the substance to be detected 10 to 30 times the quantity.
The method according to claim 3 or 4, wherein the maximum diameter of the carrier is more than 1 time of the substance to be detected, preferably 3 times and above the maximum diameter of the substance to be detected, and most preferably its maximum diameter is 3 times that of the substance to be detected. 4 to 10 times that of objects.
The method according to any one of claims 3 to 5, wherein the carrier is made of a polymer or a small molecule, preferably a polymer carrier, more preferably a polystyrene carrier made of polystyrene or hydrogel, Further the polymer carrier is made of polystyrene, most preferably the polymer is ferromagnetic or paramagnetic.
The method according to claim 1, wherein the targeting ligand can be natural or artificial, selected from nucleic acids including locked nucleic acids and XNA and their analogs, aptamers, small peptides, polypeptides, glycosylated Peptides, polysaccharides, soluble receptors, steroids, hormones, mitogens, antigens, superantigens, growth factors, cytokines, leptin, viral proteins, cell adhesion molecules, chemokines, streptavidin and their analogs , biotin and its analogs, antibodies, antibody fragments, single-chain variable fragments (scFv), nanobodies, T cell receptors, major histocompatibility complex (MHC) molecules, antigenic peptide-MHC molecule complexes ( One or more of pMHC), DNA binding protein, RNA binding protein, intracellular or cell surface receptor ligands, and multiple ligands, composite ligands, and coupling ligands formed by them.
The method according to claim 1, wherein the label molecule is selected from natural or artificial information molecules, including: oligonucleotide barcodes, oligopeptide or polypeptide barcodes, natural bases and LNA, PNA, XNA, etc. Nucleotides composed of natural bases, oligosaccharide or polysaccharide barcodes, chromophoric groups and auxochrome groups, metal atoms or ions, small molecules with distinguishable molecular weights, block polymers, polymers One or more than two covalently linked substances with the backbone molecules and the complex formed between them.
The method of claim 1, wherein the reaction reagent is an oligonucleotide primer, an enzyme or a small molecule.
The method according to claim 1, wherein the carrier diameter particle size distribution coefficient CV is less than 20%.
The method according to claim 10, wherein the surface of the carrier is coated with a mechanical buffer coating; further preferably coated with a hydrogel coating; most preferably the hydrogel coating has ferromagnetic or paramagnetic particles .
The method of claim 1, wherein the linkage is selected from the group consisting of covalent bonds, metallic bonds, ionic bonds, van der Waals forces, including hydrogen bonds, mechanical bonds, halogen bonds, chalcogen bonds, alkophilic interactions, intercalation, overlapping , cation-π bond, anion-π bond, salt bridge, secondary bond between non-metal atoms, secondary bond between metal atom and non-metal atom, auphilic interaction, argentophilic interaction, secondary hydrogen bond and gold bond key.
The method according to claim 1, wherein the targeting ligand is connected to the carrier through the connection of the reaction reagent, the connection of the label molecule or the connection of the linker; the connection of the reaction reagent and the The connection of the carrier is through the connection of the targeting ligand, the connection of the label molecule or the connection of the linker; the connection of the label molecule and the carrier is through the connection of the targeting ligand, through The coupling of the reagents or via a linker.
The method according to claim 6, wherein the small molecule is one or more of the targeting ligand, the reaction reagent, and the label molecule.
The method according to claim 1, wherein the medium is an oily medium, preferably a fluorinated oily medium, or a solid medium, preferably a microwell plate.
The method according to claim 1, wherein the two or more substances to be detected can be the same or not, and the two or more targeting reaction complexes can be the same or not, when the two When the more than one detection substance is different and the two or more targeting reaction complexes are different, the targeting reaction complex includes a label molecule corresponding to the detection substance connected to the carrier.