CN116640838A - Method for breaking poisson distribution to form reaction compartment group - Google Patents

Abstract

The present application relates to a method for breaking the Poisson distribution rule to form a reaction compartment group, the method comprising: preparing a group of substances to be detected, which is composed of more than two substances to be detected; preparing a group of targeted reaction complexes, the target The target reaction complex group is composed of two or more targeting reaction complexes, which include a carrier, a targeting ligand corresponding to the substance to be detected connected to the carrier, and a target ligand corresponding to the substance to be detected connected to the carrier. The reaction reagents, optionally including label molecules corresponding to the substances to be detected, are connected to the carrier.

Description

A method for breaking Poisson distribution to form reaction compartment groups

Technical Field

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

Background Art

Cell heterogeneity is a universal life phenomenon. As an independent living individual, the properties and differences exhibited by single cells play a vital role in the development of the entire life system. Every tissue and organ in the human body contains multiple cell types, and each type of cell changes with the different states of life activities of the organism. If thousands of single cells are studied, the heterogeneity information between cells will be blurred. Therefore, understanding the working principles of complex organisms and understanding the life functions and immune responses of each cell type are extremely important for revealing the working mechanisms of human tissues and organs and the laws of gene regulation. For example, malignant tumors that cause cancer in humans are highly heterogeneous tissues composed of tumor cells of multiple phenotypes, while real malignant cells are mixed with normal cells and often only account for a small part of the entire tissue. Therefore, single cell analysis can determine which cells are resistant to drugs and which cells are easy to metastasize, which plays an important role in guiding precision medication, predicting the course of disease and clinical guidance.

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

Summary of the invention

At present, there are many technical difficulties 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 cytometry. Fluorescence-activated flow cytometry can scan thousands of cells through multi-spectral channels at the same time. It has 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 required cell subpopulations, and can also realize multi-parameter analysis. It has great advantages in the analysis of actual samples. Therefore, using fluorescence-activated flow cytometry to separate single cells and separate single cells in 96 or 384 microplates for subsequent analysis is currently one of the most widely used technologies in the field of single-cell analysis (Jaitin, et al., Science, 343.776-779; Bagnoli, et al., Nature communications, 9.2937.)

The second technical challenge is the amplification of trace contents in single cells, and how to label each cell during sample preparation, i.e., introduce cell coding, in order to achieve high-throughput single-cell sequencing. And due to the deviation in the amplification process, how to label each transcript information in a single cell, i.e., introduce molecular coding, and how to integrate cell coding with molecular coding to achieve accurate quantification of cell contents has been the focus of methodological innovation for researchers in recent years. In recent years, researchers have developed a technology of coded microspheres that can be used to label high-throughput single-cell inclusion information.

The third technical difficulty lies in achieving high-throughput one-to-one pairing of single cells and single microspheres in microplates. The fluorescence-activated flow sorting method currently widely used for particle sorting is not suitable for the sorting of encoded microspheres. First, the encoded microspheres are expensive. Since fluorescence-activated flow sorting requires a large amount of background microspheres, this method will cause a large amount of reagent waste. Second, the size of the microspheres is often not matched with the consumables of flow sorting. Third, the sorting efficiency is low, and the number of cells and microspheres sorted per unit time is limited, making it impossible to achieve efficient, high-throughput, one-to-one rapid pairing of single cells and single microspheres, which is far from the clinical requirement for the total number of cells. Fourth, the sorted single microspheres are easily broken.

At present, there is no public technology that can integrate high-throughput capture of single cells, unbiased amplification of trace single-cell contents, and comprehensive analysis of single-cell contents. However, there have been public reports on the use of microfluidics technology to analyze single-cell transcriptomes at high throughput. For example, the method of combining droplet microfluidics and encoded microspheres reported in the Cell article (Macosko et al., 2015, Cell: 161, 1202-1214; Klein et al., 2015, Cell 161, 1187-1201) uses a droplet microfluidics method based on the Poisson distribution principle to pair and capture single cells and single microspheres. The mRNA released by single cell lysis is captured by the paired encoded microspheres, and then the single cell mRNA information is encoded and amplified through reverse transcription and amplification. The expression of a large number of large cell mRNAs is analyzed through high-throughput sequencing and bioinformatics methods. The capture of cells and microspheres in this method is based on the Poisson distribution principle. Most of the droplets do not have cells, and only ~1% of the droplets contain single cells. Combined with the Poisson distribution of microspheres, the effective analysis targets are further reduced, and 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), due to the small number of cells in the sample (10-100/mL blood), this method cannot be used to achieve single cell analysis. These technologies are limited to the analysis of single cell mRNA, and other single cell contents cannot be analyzed, such as genomes, miRNAs, proteomes, methylated DNA, metabolites, liposomes, phospholipids, etc. None of the currently disclosed technologies involve high-throughput analysis.

Microfluidic chips are a new and rapidly developing field in recent years. They use microchannels with different structures and external force fields in various forms to manipulate, process and control microfluids or samples on a microscopic scale, thereby realizing the integration of some or even all functions of traditional laboratories on a chip. However, the limitations of conventional microfluidic chips are also very obvious. They need to design pumps and valves inside the chip to cooperate with external fluid control equipment with complex operations. The technical threshold is high, and a chip is difficult to reuse. When different reaction reagents are needed to perform multi-level and multi-scale analysis on the same sample, a large amount of chip production costs are consumed (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 previous sequencing methods analyze actual samples, the currently reported sequencing methods based on coded microspheres need to first use fluorescence-activated flow cytometry to sort out the target cells and then transfer them to various analysis platforms. The information of cell contents will change with the changes in the cell's environment, resulting in the sequencing information reflected in the final sequencing results being different from the information in the actual environment of the cell at that time.

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

This application mainly focuses on the third type of technical difficulties mentioned above, namely, solving the one-to-one pairing problem of single cells and reaction reagents when realizing high-throughput analysis of multiple cells, while increasing the co-capture rate of droplets for cells and microspheres, solving the inefficiency caused by the "double Poisson distribution".

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

1. A method for forming a reaction compartment group, the method comprising:

preparing a group of objects to be detected, which consists of two or more objects to be detected;

Preparing a group of targeted reaction complexes, wherein the group of targeted reaction complexes is composed of two or more targeted reaction complexes, wherein the targeted reaction complexes include a carrier, a targeting ligand corresponding to the object to be detected connected to the carrier, a reaction reagent corresponding to the object to be detected connected to the carrier, and optionally a label molecule corresponding to the object to be detected connected to the carrier;

Contacting the group of objects to be detected with the group of targeted reaction complexes to form a group of conjugates;

The reaction compartment groups are formed by the conjugates themselves or by the vehicles encapsulating the conjugates.

2. The method of claim 1, wherein the analyte is selected from one or more of proteins, nucleic acids, sugars, lipids, metabolites, peptides, bacteria, viruses, organelles and cells, and complexes formed therefrom, and most preferably the analyte is a cell.

3. A method as described in item 1, wherein the shape of the carrier is selected from one or more of a cube, a tetrahedron, a sphere, an ellipsoid, an octopus, a bowl, and an erythrocyte; the most preferred shape is a bowl and/or an erythrocyte.

4. The method of claim 3, wherein the number of the carrier is 4 times or more of the number of the object to be detected, preferably 10 times or more of the number of the object to be detected, and most preferably 10 to 30 times the number of the object to be detected.

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

6. A method as described in any one of items 3 to 5, wherein the carrier is composed 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 has ferromagnetism or paramagnetism.

7. The method of claim 1, wherein the targeting ligand may be natural or artificial, and is 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 its analogs, biotin and its analogs, antibodies, antibody fragments, single-chain variable fragments (scFv), nanobodies, T cell receptors, major histocompatibility complex (MHC) molecules, antigen peptide-MHC molecule complexes (pMHC), DNA binding proteins, RNA binding proteins, one or more of intracellular or cell surface receptor ligands, and multiple ligands, composite ligands, and coupled ligands formed together 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, nucleotides composed of natural bases and non-natural bases such as LNA, PNA, XNA, oligosaccharide or polysaccharide barcodes, chromophores (chromophoric group) and auxochrome groups (auxochrome group), metal atoms or ions, small molecules with distinguishable molecular weight, block polymers, covalent linkages of polymers and backbone molecules, one or more of them and complexes formed therebetween.

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

10. The method according to claim 1, wherein the particle size distribution coefficient CV of the carrier diameter 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, it is coated with a hydrogel coating; most preferably, the hydrogel coating has ferromagnetic or paramagnetic particles.

12. A method as described in item 1, wherein the connection is selected from covalent bonds, metallic bonds, ionic bonds, van der Waals forces, secondary bonds including hydrogen bonds, mechanical bonds, halogen bonds, sulfide bonds, aurophilic effects, embedding, overlap, cation-π bonds, anion-π bonds, salt bridges, secondary bonds between non-metal atoms, secondary bonds between metal atoms and non-metal atoms, aurophilic effects, silver-philic effects, double hydrogen bonds and gold bonds.

13. A method as described in item 1, wherein the connection between the targeting ligand and the carrier is through the connection of the reaction reagent, through the connection of the label molecule or through the connection of a connector; the connection between the reaction reagent and the carrier is through the connection of the targeting ligand, through the connection of the label molecule or through the connection of a connector; the connection between the label molecule and the carrier is through the connection of the targeting ligand, through the connection of the reaction reagent or through the connection of a connector.

14. The method of 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 microplate.

16. A method as described in item 1, wherein the two or more objects to be detected may be the same or different, and the two or more targeted reaction complexes may be the same or different. When the two or more objects to be detected are different and the two or more targeted reaction complexes are different, the targeted reaction complex includes a label molecule corresponding to the object to be detected connected to a carrier.

Beneficial technical effects achieved by the technical solution of this application

By adopting the technical solution 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), and at the same time, the co-capture rate of the medium (droplet) for cells and microspheres is increased, and the inefficiency problem caused by the "double Poisson distribution" phenomenon that occurs when the analyte is paired with the carrier is solved. By using red blood cell-shaped and bowl-shaped carriers (such as polystyrene microspheres) and carriers that are larger in size and larger in number relative to the object to be detected (such as cells), the steric hindrance effect is used to further ensure the realization of the purpose of "one-to-one pairing of single cells and single microspheres". By coating a buffer layer (hydrogel layer) on the surface of the carrier (such as polystyrene microspheres), the risk of cells being crushed during the binding process between cells and microspheres is avoided, and the loss of the object to be detected is reduced. The targeted complex group used in the present application has at least one reaction reagent, which realizes high-throughput analysis of the object to be detected.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a schematic diagram of the composition of the targeted reaction complex according to the present application; the targeted reaction complex includes a carrier, a targeting ligand connected to the carrier corresponding to the object to be detected, a reaction reagent connected to the carrier corresponding to the object to be detected, and optionally includes a label molecule connected to the carrier corresponding to the object to be detected.

Figure 2A shows that the hard microspheres may crush the cells when combined with the cells; Figure 2B shows that the hydrogel coating wrapped on the surface of the hard microspheres has a protective effect on the cells; Figure 2C shows a targeted reaction complex coated with a hydrogel coating according to the present application (DNA-encoded microspheres coated with a hydrogel coating).

FIG3A shows the hydrogel encapsulation achieved by blowing microspheres with a pipette; FIG3B shows the image of the hydrogel after gelation and demulsification.

Figure 4A shows that the diameter of the microspheres is larger than that of the cells, and the steric effect helps the microspheres and cells to bind in a 1:1 ratio; it is difficult for a cell to bind with two or more microspheres; Figure 4B shows that when the microspheres are smaller than the cell diameter, multiple microspheres bind to one cell, or cells and microspheres aggregate.

Figure 5A shows the preparation of hydrogel-coated magnetic microspheres; Figure 5B shows magnetic purification after demulsification; Figure 5C shows hydrogel-coated magnetic microspheres, which are purified by magnetic means after demulsification; Figure 5D shows that under a fluorescence microscope, antibodies modified on the surface of the hydrogel layer after activation (bright white on the outer layer) can be observed; in Figure 5E, the white arrows indicate that the antibody-modified hydrogel-coated microspheres form a 1:1 binding with cells through incubation.

Figures 6A-C show bowl-shaped carriers. The steric effect helps the microspheres to bind to cells in a 1:1 ratio. It is difficult for a cell to bind to two or more bowl-shaped or red blood cell-shaped microspheres. Figure 6A shows the formation and UV cross-linking of a bowl-shaped aqueous double phase in a droplet. Figure 6B shows bowl-shaped particles. Figure 6C shows a 1:1 binding between bowl-shaped particles and cells.

FIG. 7A shows the finished hydrogel microspheres prepared using a microfluidic device; FIG. 7B shows the hydrogel microspheres in which the label molecule and polyT are on the same reverse transcription primer; and FIG. 7C shows the hydrogel microspheres polymerized in droplets.

FIG8A shows that the modified microspheres with anti-human-CD298, human β2 microglobulin, mouse CD45 and mouse MHC class 1 antibodies can be used as universal binding microspheres to bind to cells; FIG8B shows that the hydrogel microspheres bind to cells to form a 1:1 complex.

Figure 9A is a flow chart of the entire high-throughput analysis process (the targeted reaction complex is pre-bound to the analyte and then encapsulated in the high-throughput compartment); Figure 9B, when the number of microspheres is 10 times or more than that of cells, a 1:1 binding can be formed, and there is approximately a 1/100 probability of observing one cell bound to two microspheres; Figure 9C shows that after the compartment is formed, each cell has a targeted reaction complex microsphere bound to it, thereby breaking the Poisson distribution and increasing the cell capture rate; Figure 9D shows that in some cases, the targeted reaction complex and the bound cell will be separated in the compartment; Figure 9E shows that the analyte is a pair of cells that interact with each other.

Figures 10A-D show the process of forming oil-in-water compartments by controlling the water/oil liquid in the microfluidic through an injection pump; Figure 10B shows the high-throughput compartment formation of the microfluidic device; Figure 10C shows the microsphere and cell complex entering the microfluidic cross-intersection and high-throughput droplet generation; Figure 10D shows the results of single-cell analysis; showing the results of single-cell analysis, human and mouse cell subpopulations can be separated.

Figure 11A shows the following steps: the first step is to encapsulate the microspheres with hydrogel and cross-link streptavidin; the second step is to biotinylate the cells; the third step is to combine the biotinylated cells with incubation with streptavidin; the fourth step is to encapsulate the complex of microspheres and cells in a high-throughput manner to form compartments, release the reaction reagents in the compartments, and label the cells in the compartments; Figure 11B shows hydrogel-encapsulated targeted label microspheres modified with fluorescent streptavidin; Figure 11C shows biotinylated cells; Figure 11D shows hydrogel microspheres bound to cells; Figure 11E shows a manual gun tip blowing on the outside of the hydrogel-encapsulated microspheres to form compartments, and lyse the cells in the compartments to release the mRNA therein.

DETAILED DESCRIPTION

The specific embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although the specific embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of the components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" is an open term, so it should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of implementing the present application, but the description is based on the general principles of the specification and is not used to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims.

As used herein, "substantially free" with respect to a particular component is used herein to mean that the particular component is not purposefully formulated into the composition and/or is present only as a contaminant or in trace amounts. Thus, the total amount of the particular component resulting from any accidental contamination of the composition is less than 0.05%, preferably less than 0.01%. Most preferred are compositions wherein the amount of the 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, when used with the word "comprising", the word "a" or "an" may mean one or more than one.

The term "or" is used in the claims to mean "and/or" unless explicitly stated to refer only to alternatives or the alternatives are mutually exclusive, although the present disclosure supports a definition referring only to alternatives and "and/or." As used herein, "another" can mean at least a second or more.

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

The methods for obtaining various biological materials described in the examples are only provided as an experimental method to achieve the specific disclosed purpose, and should not be construed as limiting the sources of biological materials in this application. In fact, the sources of biological materials used are extensive, and any biological material that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions in the examples.

The present application provides a method for forming a cluster of reaction compartments.

In one embodiment, a method for forming a reaction compartment group is provided, the method comprising:

preparing a group of objects to be detected, which consists of two or more objects to be detected;

Preparing a group of targeted reaction complexes, wherein the group of targeted reaction complexes is composed of two or more targeted reaction complexes, wherein the targeted reaction complexes include a carrier, a targeting ligand corresponding to the object to be detected connected to the carrier, a reaction reagent corresponding to the object to be detected connected to the carrier, and optionally a label molecule corresponding to the object to be detected connected to the carrier;

Contacting the group of objects to be detected with the group of targeted reaction complexes to form a group of conjugates;

The reaction compartment groups are formed by the conjugates themselves or by the vehicles encapsulating the conjugates.

Here, "compartment" means providing a specific reaction space for a specific chemical reaction, within which reactants can react with each other but not with reactants in adjacent compartments. However, the products of the compartment may exchange substances with the products of adjacent compartments depending on their type (for example, dyes, surfactants, etc.).

In the context of this specification, "targeting" conforms to 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, including at least various connections via molecular bonds and connections not via molecular bonds.

FIG1 shows the components of the targeted reaction complex, which shows the structural relationship between the targeting ligand, the label molecule and the reaction reagent, respectively connected to the "carrier". The "carrier" can be composed of the same molecules as the targeting ligand, the label and the reaction reagent, that is, it can be composed of small molecules. More commonly, the carrier is composed of a polymer, preferably polystyrene.

In another specific embodiment, a method for forming a reaction compartment group is provided, wherein the analyte is selected from one or more of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles and cells, and complexes formed therefrom, and most preferably the analyte is a cell.

In the context of this specification, "cell" conforms to the general definition in the field of biology, which at least includes prokaryotic cells and eukaryotic cells. For the purpose of the technology of this application, it can sometimes specifically refer to a differentiated cell population of a multicellular organism, such as an immune cell population, which at least includes lymphocyte B cells, lymphocyte T cells, and NK cells.

In the context of this specification, "organelle" conforms to the general definition in the field of biology. Organelles are generally considered to be microstructures or microorgans with certain shapes and functions scattered in the cytoplasm. They constitute the basic structure of cells, allowing cells to work and operate normally. The main organelles in cells are: mitochondria, endoplasmic reticulum, centrosomes, chloroplasts, Golgi bodies, ribosomes, 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, HepG2 will form cell-to-cell interactions after incubation with T cells that recognize this cell line (after genetic modification, carrying T cell receptor genes targeting "major histocompatibility complex-AFP158 peptide segment"); specific examples include: 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 reaction compartment group is provided, wherein the shape of the carrier is selected from one or more of a cube, a tetrahedron, a sphere, an ellipsoid, an octopus, a bowl, and an erythrocyte; bowl-shaped and/or erythrocyte-shaped are most preferred. "Bowl-shaped" refers specifically to a concave sphere, which has a concave surface on one side of the plane of the "hemisphere". When the object to be detected is bound to the concave surface of the carrier, the object to be detected will not be bound to another carrier due to the steric hindrance effect. A concave sphere refers specifically to a spherical circular plane with a concave surface. When the object to be detected is bound to the concave surface of the carrier, the object to be detected will not be bound to another carrier due to the steric hindrance effect. The "erythrocyte-shaped" carrier has two concave surfaces. When the object to be detected is bound to the upper concave surface or the lower concave surface of the carrier, the object to be detected will not be bound to another carrier due to the steric hindrance effect.

In another specific embodiment, a method for forming a reaction compartment group is provided, wherein the number of the carrier is more than 10 times the number of the object to be detected, and most preferably 10 to 50 times the number of the object to be detected. Specifically, the number 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, 32 times, 34 times, 36 times, 38 times, 40 times, 42 times, 44 times, 46 times, 48 times or 50 times the number of the object to be detected. It is desirable to control the amount of the analyte to be interacted with the aforementioned targeted reaction complex or the aforementioned reaction complex group and the analysis process so that the conjugate containing only one analyte in each conjugate is analyzed. In an embodiment of the present application, specifically, when the number of microspheres carrying the reaction reagent is 10 times-20 times the number of cells to be analyzed, each microsphere is only combined with one cell with a high probability according to the Poisson distribution.

In the context of this specification, "Poisson distribution" means describing the number of microspheres or cells that appear randomly in a unit space; "double Poisson distribution" means describing the number of microspheres and cells that appear randomly in a unit space. In an embodiment, if the number of microspheres and cells that appear simultaneously in a unit space is greater than the double Poisson distribution, it can be considered to "break the Poisson distribution".

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

Specifically, a "bowl-shaped" or "red blood cell" shaped carrier can be used, for example, with 10 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 than the object to be detected; specifically, a "bowl-shaped" or "red blood cell" shaped carrier can be used with a maximum diameter of 10 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, 4 The amount of carriers used may be, for example, 10 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 more than the amount of carriers used to detect the object; specifically, the amount of carriers used may be, for example, 10 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 more than the amount of carrier 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 can be used while using "bowl-shaped" or "red blood cell" shaped carriers; specifically, "bowl-shaped" or "red blood cell" shaped carriers can be used while using, for example, 10 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 to be detected, and a carrier size with a maximum diameter of 3 to 10 times that of the object to be detected can be used; so as to ultimately achieve the technical effect of "each microsphere has a high probability of binding to only one cell according to the Poisson distribution".

In yet another specific embodiment, a method for forming a cluster of reaction compartments is provided, wherein the carrier is made of polystyrene or small molecules, most preferably made of polystyrene.

In one embodiment, a method for forming a reaction compartment group is provided, 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 its analogs, biotin and its analogs, antibodies, antibody fragments, single chain variable fragments (scFv), nanobodies, T cell receptors, major histocompatibility complex (MHC) molecules, antigen peptide-MHC molecule complexes (pMHC), DNA binding proteins, RNA binding proteins, one or more of intracellular or cell surface receptor ligands, and multiple ligands, composite ligands, and coupled ligands formed by them. In the context of this specification, "antibody" conforms to the general definition in the field of biology. Specifically, antibody refers to a protein with a protective effect produced by the body due to the stimulation of 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 of vertebrates and on the cell membrane surface of their B cells. Antibodies can recognize a unique feature of a specific foreign object, and the foreign target is called an antigen. In the technical scheme of the present application, for example, the 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, "aptamers" conform to the general definition in the field of biology. In the technical scheme of the present application, they can specifically refer to nucleic acid aptamers. Nucleic acid aptamers (Aptamer) are a section of DNA (deoxyribonucleic acid), RNA (ribonucleic acid) sequence, XNA (nucleic acid analog) or peptide. It is usually an oligonucleotide fragment obtained from a nucleic acid molecule library using an in vitro screening technology, namely, systematic evolution of ligands by exponential enrichment (SELEX). Nucleic acid aptamers can bind to a variety of target substances with high specificity and selectivity, and are therefore widely used in the field of biosensors. When nucleic acid aptamers bind specifically to target substances, the configuration of the nucleic acid aptamers themselves 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 play the role of separating cells.

In another specific embodiment, a method for forming a reaction compartment group is provided, wherein the label molecule is selected from natural or artificial information molecules, including: oligonucleotide barcodes, oligopeptide or polypeptide barcodes, nucleotides composed of natural bases and non-natural bases such as LNA, PNA, XNA, oligosaccharide or polysaccharide barcodes, chromophores (chromophoric group) and auxochrome groups (auxochrome group), metal atoms or ions, small molecules with distinguishable molecular weights, block polymers, polymers and backbone molecules covalently linked, one or more of them, and complexes formed therebetween. In the context of this specification, a DNA barcode refers to a standard, sufficiently variable, easily amplified and relatively short DNA fragment in an organism that can represent the species. DNA barcodes have become an important tool for ecological research, not only for species identification, but also to help biologists further understand the interactions occurring within the ecosystem. When an unknown species or part of a species is discovered, researchers describe the DNA barcode of its tissue and then compare it with other barcodes in an international database. If it matches one of them, the researchers can confirm the identity of the species. DNA barcoding technology is an emerging technology that uses a conserved segment in the DNA of an organism to quickly and accurately identify species. Specifically, in the embodiments of the present application, when the "analysis" is transcriptome sequencing of an immune cell population, the use of DNA barcoding can deconvolute these analysis results after obtaining high-throughput analysis results of the immune cell population.

In a specific embodiment, a method for forming a reaction compartment group is provided, wherein the reaction reagent is an oligonucleotide primer, an enzyme or a small molecule. In this article, the reaction reagent is used to react with the analyte and produce a substance that can be used to detect a signal. In the context of this specification, "reaction reagent" can sometimes refer specifically to a fluorescent reagent that can cause the analyte to emit fluorescence. In the context of this specification, "primer" meets the general definition in the field of biotechnology. Specifically, primer refers to a macromolecule with a specific nucleotide sequence that is stimulated to synthesize at the beginning of nucleotide polymerization, and is connected to the reactant in the form of hydrogen bonds. Such a molecule is called a primer. Primers are usually two sections of 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 another DNA template strand at the other end of the target region. Its function is to serve as the starting point of nucleotide polymerization, and nucleic acid polymerase can start synthesizing new nucleic acid chains from its 3 ends. In vitro artificially designed primers are widely used in polymerase chain reaction, sequencing and probe synthesis, etc. Specifically to the embodiments 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" are small molecules that can emit fluorescence, such as FAM, VIC, etc.

In a specific embodiment, a method for forming a reaction compartment group is provided, wherein the carrier diameter particle size distribution coefficient CV is less than 20%. The English abbreviations herein are defined as follows: CV = SD/average particle size, which can indicate the width of the particle size distribution. Wherein SD: is the standard deviation (Standard Deviation), which is statistically recorded as σ; CV is also called the relative standard deviation (Coefficient of variation), which is statistically recorded as α. In this article, the diameter of the microspheres used can be calculated using methods and devices known to those skilled in the art, such as being measured under a microscope and analyzed using image processing software, being measured using an instrument for measuring particle size (Bio-Rad T20 cell technology instrument), or using data provided by the microsphere supplier.

In one embodiment, a method for forming a reaction compartment group is provided, wherein the surface of the carrier is coated with a mechanical buffer coating; most preferably, it is coated with a hydrogel coating. Here, the "mechanical buffer coating" should be regarded as a part of the "carrier", that is, the "targeting ligand", "reaction reagent", and "label molecule" described in this specification are all connected to the carrier (uncoated mother core) after the "mechanical buffer coating" is coated on the carrier (uncoated mother core) through, for example, chemical modification.

As an alternative, 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 linked to the carrier, then coated with a mechanical buffer coating, and finally the tag molecule and the targeting ligand are linked outside the mechanical buffer coating by, for example, chemical modification.

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

As an alternative, the tag 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.

As an alternative, the tag 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 by, for example, chemical modification.

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

As an alternative, the tag molecule, targeting ligand and tag molecule are first linked to the carrier and then coated with a mechanical buffer coating.

In a specific embodiment, a method for forming a reaction compartment group is provided, wherein the connection is selected from covalent bonds, metal bonds, ionic bonds, van der Waals forces, secondary bonds including hydrogen bonds, mechanical bonds, halogen bonds, sulfide bonds, aurophilic effects, intercalation, overlap, cation-π bonds, anion-π bonds, salt bridges, secondary bonds between non-metal atoms, secondary bonds between metal atoms and non-metal atoms, aurophilic effects, silver-philic effects, double hydrogen bonds and gold bonds.

In a specific embodiment, a method for forming a reaction compartment group is provided, wherein the connection between the targeting ligand and the carrier is through the connection of the reaction reagent, through the connection of the label molecule or through the connection of a connector; the connection between the reaction reagent and the carrier is through the connection of the targeting ligand, through the connection of the label molecule or through the connection of a connector; the connection between the label molecule and the carrier is through the connection of the targeting ligand, through the connection of the reaction reagent or through the connection of a connector.

In a specific embodiment, a method for forming a reaction compartment group 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 label molecule themselves can form part of the carrier.

In one embodiment, a method for forming a reaction compartment group 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 "medium" of a "compartment" is that the oily medium can effectively block charged nucleic acid molecules from crossing different "compartments", and the benefit is that the reaction reagents carrying different analyte labels will not cross the "compartment" product to form cross contamination after labeling the cell content.

In one embodiment, a method for forming a reaction compartment group is provided, wherein:

The two or more objects to be detected may be the same or different.

The two or more targeted reaction complexes may be the same or different.

When the two or more objects to be detected are different and the two or more targeted reaction complexes are different, the targeted reaction complexes include a label molecule corresponding to the object to be detected and connected to a carrier.

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 targeted reaction complexes are the same.

In one 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 targeted reaction complexes are different.

In one 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 one embodiment, in the above method for forming a reaction compartment group, the two or more detectable substances are different, and the two or more targeted reaction complexes are different; and the targeted reaction complexes include a label molecule corresponding to the detectable substance connected to the carrier.

Examples

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

Polystyrene microspheres (Nanomicron Uni-PS-30) were customized by Chemgenes Corp to form microspheres with reverse transcription polyT primers (reaction reagents) with DNA coding (tags).

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

2. Remove the supernatant and add 0.2 ml PBS buffer (Teknova) to rinse the microspheres. Repeat twice.

3. Pipette 10 μl of microspheres and count them in a cell counting plate;

4. Add all the remaining microspheres to the polyacrylamide (PAA) solution described in Example 1 and mix thoroughly;

5. Pipette all the solution in step 4 and add it to the fluorinated oil phase. Use a 200 μl pipette to pipette for 3 to 4 minutes (Figure 3A).

6. Let stand at room temperature for 4 hours.

7. Use a pipette to remove the oil at the bottom.

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

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

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

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

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

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

The microspheres (carriers) coated with the hydrogel layer can be loaded with targeting ligands, labels or reaction reagents in advance or later.

Example 2 (I) Formation of the conjugate

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

The microspheres with carboxyl hydrogel coating produced in Example 1 were mixed with the EDC/NHS solution and rotated at room temperature for 15 minutes.

Incubate the microspheres at 5 rpm for 5 minutes

Remove the supernatant

Resuspend the microspheres in 500 μL PBS buffer

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

Rotate the microspheres at RT for 2 hours

Spin down the microspheres at 5 rpm for 5 minutes

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

Collect the antibody-modified microspheres, remove all supernatants, 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 to microspheres, the probability of having both microspheres and cells in a unit volume breaks the Poisson distribution.

Example 2 (II) Effect of carrier size on carrier-cell binding (as a comparative example)

There are many reports of affinity magnetic bead cell sorting in previous literature and commercial product descriptions (for example: Miltenyi Biotec MicroBead Technology, Thermo Fisher's affinity Dyna Bead products, etc.: https://www.miltenyibiotec.com/US-en/products/macs-cell-separation/cell-separation-reagents/realease-microbeads.html), the diameter of these magnetic beads is smaller than that of cells (i.e., between 100nm and 2.7 microns).

This example is a comparative example, and the purpose of the experiment is to verify the effect of too small microsphere size on its binding to cells. When the number of microspheres is greater than the number of cells, a phenomenon of multiple microspheres binding to one cell will occur.

Steps for activating microspheres:

1. Count the microspheres with carboxyl groups (Polysciences lnc.cat#18133) in 500ul MES buffer. The carboxyl groups on the microspheres can bind to cells after activation (targeting ligand): 5.0*10^6 microspheres (10uL microspheres in 90uL PBS buffer (detection microsphere concentration: 6.72×10^5/mL, transfer ~744uL microspheres → 5.0×10^6)

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

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

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

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

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

7. Resuspend in 300uL DPBS buffer; measure microsphere concentration (1.35×10^6/mL), add an additional 99uL to reach 1×10^6 microspheres/mL in DPBS

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

Steps for cell binding:

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

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

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 microspheres is larger than that of cells.

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

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

1. Prepare hydrogel-coated microspheres with a magnetic core using the method of Example 2 (FIG. 5A-C): The microsphere structure is a polystyrene core coated with magnetic nanoparticles, and the outer layer is a polyacrylamide hydrogel layer with carboxyl groups (the PAA solution formula and polymerization method are shown in Example 2)

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

3. Spin for 15 minutes, then spin the microspheres down 5 points at RT for 5 minutes to remove the supernatant;

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

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

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

7. The microspheres were centrifuged for 5 minutes (Figure 5D);

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

The steps to measure cell number are:

a. Measured Jurkat cell concentration (7.62×10^5mL)*3.5mL

b. Resuspend the cells in 100uL PBS + 2mM EDTA to reach 1×10^6/100uL～1×10^7 cells/mL

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

9. When the microspheres were collected using a magnet, it was observed that the cells and microspheres were bound to each other in a 1:1 ratio (Figure 5E), indicating that the Poisson distribution law was broken at the cell end.

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

Example 3 (II) Effect of carrier shape on carrier-cell binding

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

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

For the dispersed phase, a homogenous precursor solution containing 6.3% w/v biotinylated polyethylene glycol, 4.5% w/v gelatin, and 1.5% w/v lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) dissolved in DPBS was injected into a parallel emulsifier at a rate of 8 μL/min. A Novec 7500 containing 2% Pico-Surf was injected into the continuous phase at 16 μL/min. The resulting single-phase PEG/gelatin droplets flowed through a Tygon tube (0.03″ l.D., 0.0625″ OD, Murdock) immersed in a 4°C water bath for temperature-induced phase separation of PEG and gelatin. The length of the tubing was adjusted to ensure complete phase separation (approximately 60 cm, incubation for 10 minutes). The phase-separated droplet stream was introduced into a PDMS reservoir immersed in a 4°C water bath and exposed to ultraviolet light (200 mW/cm ² ) for 1-3 seconds near the reservoir outlet area for polymerization. Under UV irradiation, the photocrosslinkable PEG component forms a polymer network, while the gelatin component remains unpolymerized (see Figure 6A for details). The crosslinked particles are collected and the oil and gelatin-rich droplets are removed in a series of washing steps. The surfactant oil is first removed by pipetting. A 20% v/v solution of perfluorooctanol (PFO, Sigma) in Novec7500 and milli-q water are added successively to break the emulsion and transfer the particles to the aqueous phase. The sample was centrifuged at 2500×g for 1 minute to remove the oil phase. The microspheres were washed twice with Novec 7500 to remove the remaining PFO and surfactant. After removing the oil layer with a pipette, the residual oil was washed three times with hexane (Sigma). The sample was then washed three times with 70% ethanol and then twice with milli-q water to remove gelatin. The particles were sterilized by incubation in 70% ethanol overnight before use. Bowl-shaped particles under a microscope (as shown in Figure 6B).

Step 2: Bowl-shaped microspheres combined with cells

The bowl-shaped microspheres were modified with AF350 streptavidin (Fisher Scientific) before use (targeting ligand). The bowl-shaped microspheres were washed with cell culture medium and concentrated in a conical tube. The amount of concentrated bowl-shaped microspheres was calculated to cover the entire well area with a bowl-shaped particle monolayer, which was 4.8 μL/cm ² for 55 μm bowl-shaped microspheres. The calculated amount of bowl-shaped particles was transferred and dispersed in the well plate. Jurkat cells were inoculated into the wells with a bowl-shaped microsphere monolayer at a ratio of 1:1 with target cells and bowl-shaped microspheres. The solution in the well was aspirated several times to evenly disperse the cells and microspheres in the entire well area and to allow the cells to adhere to the bowl-shaped microspheres. In order to remove unattached cells, the sample was filtered with a 20 μm reversible cell filter (CellTrics) and washed with a "wash buffer" consisting of 0.5% bovine serum albumin (BSA), 1% P/S and DPBS containing 0.05% Pluronic F-127. Next, the cell strainer was inverted and washed with a wash buffer to recover the bowl-shaped particles in a conical tube. The bowl-shaped particles were centrifuged to remove the wash buffer. The bowl-shaped particles were observed to bind to the cells under a microscope (see Figure 6C for details).

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

Example 4 Preparation of universal carrier: The carrier (microsphere) carries a variety of targeting ligands: anti-human-CD298, human β2 microglobulin, mouse CD45 and mouse MHC class 1 antibody.

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

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

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

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 l to the microsphere solution (targeting ligand)

8. Rotate 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 in 200μL PBS buffer containing 2mM EDTA and incubate with beads. Rotate the solution for 30 minutes.

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

Experimental conclusion: By loading a variety of antibodies (targeting ligands) onto hydrogel-coated microspheres (carriers) containing reverse transcription primers (reaction reagents) with DNA coding (tags), the cell binding range 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. Load the solution into a 1 ml syringe (BD Company) with a 28G needle.

2. Prepare an insoluble continuous phase consisting of 5% (w/w) fluorinated surfactant and 1% N,N,nn-tetramethylethyldiamine (TEM ED) in hydrofluoroether (HFE) oil for droplet generation and stabilization. Load the solution into a new 1 ml syringe.

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

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

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

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

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

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

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

The reaction reagent and label molecule carried by the microspheres prepared in Example 6 can be combined into the same substance (i.e., the 5′-Acrydite-CTA CAC GAC GCT CTT CCG ATC T NNNNT ₂₈ VN primer has the dual functions of label molecule and reaction reagent); in the sequence, V represents a mixture of G, C, and A; N represents a mixture of four bases, A, G, C, and T.

The microfluidic system in Example 6 was used to prepare hydrogel microspheres formed by polyacrylamide (PAA) solution of 40% acrylic acid-N,N′-methylenebis(acrylamide) (37.5:1), 10% ammonium persulfate, 10X Gel buffer (100uL 1M Tris HCl pH=7.5; 20uL 0.5M EDTA pH=8; 150uL 1M NaCl; 730uL ddH ₂ O), 2X Oligo solution (100uM) (formulation as shown in FIG7B ), wherein the oligo sequence is: 5′-Acrydite-CTA CAC GACGCT CTT CCG ATC T NNNNT ₂₈ VN (SEQ ID NO: 1).

Load the solution containing oligomers to generate droplets. When the droplets are unstable, take a picture of the droplets to determine the droplet size. After the run, remove the oil and add TEMED, which contains 0.4% TEMED in the oil. To pour the oil back into the bead emulsion, place it at 37 degrees to solidify the microspheres (Figure 7C).

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

Example 7 Preparation of cell-targeting hydrogel microspheres and combining with cells to form a complex

Take the hydrogel microspheres prepared in Example 7 (the carboxyl groups come from acrylic acid monomers) and count 8.0*10^5 microspheres in 500 μL MES buffer.

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

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

Centrifuge the microspheres at 5 rpm for 5 minutes

Remove the supernatant

Resuspend the microspheres in 500 μL PBS buffer

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

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

Centrifuge the microspheres at 5 rpm for 5 minutes

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

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

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

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

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

List of Microfluidic Device Components

(1) Inverted microscope

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

(3) 3 falcon tubes/3mL syringes

(4) Magnetic stirring system

(5) Microfluidic conduits for connecting components of the experimental device

(6) Microfluidic accessories and connectors

(7) PDMS co-flow microfluidic droplet generation device

(8) 100 μm cell strainer for beads

(9) 40 micron cell strainer for cells

(10) Counting chamber

BOM:

Table 1 List of materials for single-cell transcriptome sequencing

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

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

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

4. Pass through 100um filter (@4℃ long-term storage)

5. For single-cell transcriptome analysis, wash the microsphere-cell complex with saline solution.

6. Resuspend and wash the complex formed by microspheres and cells in physiological saline, with a concentration of about 120 complexes/μL

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

8. Load the droplet generation oil into a 10 ml plastic syringe

9. Connect the syringe to the 125um isoflow device with 0.38mm inner diameter tubing

10. Keep the microsphere-cell complex in suspension by continuous magnetic stirring

The collected droplets were placed under a microscope for observation (Figure 10C)

11. Add 30 ml 6X SSC @ room temperature

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

13. Add 600ul perfluoro-1-octanol

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

15. Rotate at 1,000g for 1 minute

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

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

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

19. Transfer the aqueous layer to a new tube

20. Rotate at 1,000g for 1 minute

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

22. Wash twice with 1 ml 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 ~30 minutes

Incubate at 26.42°C for 90 minutes

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

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

29. Wash the microspheres once with 10 mM Tris pH = 7.5 30. Resuspend the microspheres in 200 ul of exonuclease I mixture

1X Exonuclease I Buffer (NEB)

1U/ul Exonuclease l (NEB)

31. Incubate @37℃～45min

32. Wash once with 1 mL TE/SDS

33. Wash twice with 1 mL TE/TW

34. Wash once with ddH ₂ O

35. Resuspend in ddH ₂ O

36. Aliquots of 1000 microspheres were amplified by PCR, each with a volume of 50uL 1x Hifi HotStart Readymix (Roche)

0.8uM TSO primer

37.The PCR procedure is as follows:

temperature time cycle Number of cycles 95℃ 3min 98℃ 20s 1 4 65℃ 45s 1 72℃ 3min 1 98℃ 20s 2 X 67℃ 20s 2 72℃ 3min 2 72℃ 5min

Table 2 PCR reaction program for cDNA amplification

38. Preparation of 3′-terminal cDNA fragments for sequencing

a. Place 600 pg cDNA in four equal portions into four standard Nextera XT labeling reactions

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

Table 3 PCR reaction program for library construction

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

Example 9 Analysis of the experimental results of Example 8

The applicant used bioinformatics tools such as Seurat to process the raw sequencing results, and cells with low overall expression or high mitochondrial ratios were removed. For clustering, principal component analysis was used, followed by k-means clustering to identify different cell states. t-distributed random neighbor embedding (t-SNE) was used to visualize cell clustering. For meta-clustering, expression matrices from all hosts were merged from four experiments using Seurat. Cell transcripts with fewer than 2,000 were excluded from the analysis, and unique genes detected in fewer than 100 cells were excluded from the analysis (Figure 10D).

Example 10 Microspheres with another targeting ligand (streptavidin)

The purpose of this experiment is to connect streptavidin to the hydrogel coating of the microspheres and bind to cells with biotin modified on the surface. The experimental process is shown in Figure 11A:

Step 1: Functionalization of hydrogel beads

1. 1.12 μL 1× PBST in microspheres (8.90×10^3 microspheres/μL) ~ 5000-10k microspheres (carrier)

The buffer was removed by resuspending the hydrogel beads using 600 μl of MES buffer (pH=4.7, 0.1 M).

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

substance Weight(g) EDC 0.01 Sulfo-NHS 0.005

Table 4 Microsphere activation reagent

sample

200uL MES containing 10mg/ml EDC

200uL MES containing 5mg/ml Sulfo-NHS

3. Vortex the solution for 30 seconds and place it on a horizontal oscillator (650 rpm) at room temperature for 30 minutes.

4. Centrifuge the pellet in PBST and rinse four times

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

sample Control group NeutrAvidin_μl 100 / Fluorescent streptavidin (2 μg/ml)_μL / 2

Table 5 Modification conditions of streptavidin

6. After rinsing the particles 4 times, they can be stored in PBST at 4°C for a long time (Figure 11B). The NeutrAvidin on the microspheres is a targeting ligand that can target and bind to downstream cells modified with biotin.

Step 2: Biotinylation of 293T cells (operated 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 6× ^6cm2 culture dishes with 90-95% confluent cells

3. Wash the cells 3 times with ice-cold PBS (pH 8.0) to remove amine-containing culture medium and proteins in the cells. Quickly remove PBS (Note: If the cells are grown in suspension, use 1×10^6 cells per ml of biotin solution prepared in step 3. The total number of cells per labeling reaction should not exceed 4×10^7)

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

4. Gently scrape the cells into the solution and suspend the cells in 1 ml PBS (pH 8.0) at a concentration of approximately 25×10^6 cells/mL.

5. 1000μL 293T cell in 1×PBS (1.90×106cell per mL)～10^6cell

6. Add 1.0 mg Sulfo-NHS-SS-Biotin (do not weigh) reagent per ml of cell suspension (yielding ~2 mM biotin reagent).

NOTE: Prepare a 10 mM solution of Sulfo-NHS-SS-Biotin immediately before use as follows: Sulfo-NHS-SS-Biotin: place 1 mg into a microtube and add 164 µL of ultrapure water.

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

Note: Incubation at 4 °C may reduce 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 × g for 1 minute and discard the supernatant.

Note: Quench the reaction and remove excess biotin reagent and byproducts (Figure 11C).

Step 3: Capture of 293T cells with streptavidin-labeled microspheres

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

Concentration of hydrogel-encapsulated microspheres (/mL) Cell concentration (/mL) 10000 1000

Table 6 Concentration of cells and microspheres during incubation

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

3. Total solution volume ≈ 1 mL. Before imaging, the solution was gently vortexed to remove non-adherent cells from the pellet. After cell binding, a 1:1 capture ratio of microspheres to cells was formed (Figure 11D)

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

The hydrogel-encapsulated microspheres were pipetted to form droplet compartments

Oil phase Water Phase Total volume Blowing time sample 60ul 30ul 90ul 3min

Table 7 Conditions for droplet generation

The aqueous phase contained 29U/mL proteinase K (NEB#P8107S) and 70mM DTT (Sigma#D9779) and mixed with 10 pipette strokes. Be careful to avoid creating bubbles when mixing the cell-bound microspheres with the solution. 280μL of 0.5% ionic Krytox HFE 7500 oil 65 was added to the cell-bead complex and vortexed horizontally at 3000RPM for 15 seconds, then vertically for 2 minutes using a custom vortexer. The oil was removed from under the emulsion so that less than 100μL of oil remained. Prior to lysis, the microsphere-cell emulsion was subsampled on a C-Chip disposable blood cell counter (Fisher Scientific#DHCN015), with each subsample containing 3.5μL of emulsion per field of view. The chip was imaged in bright field at 2x magnification. The remaining emulsion was subjected to enzymatic cleavage on a PCR thermal cycler (Eppendorf Mastercycler Pro) at 65° C. for 35 min with the lid temperature set to 105° C. After cleavage was complete, fluorescent images were captured using a Nikon 2000 microscope (Thorlab M470L5) with 470 nm excitation light ( FIG. 11E ).

Summary of Example Results:

This application lists three production processes of targeted reaction complexes. These targeted reaction complexes can bind to the object to be detected (cells): A) polymer microspheres wrapped with hydrogel coatings; B) general spherical hydrogel microspheres; C) bowl-shaped hydrogel microspheres.

The application of the targeted reaction complex of the present application can realize single-cell analysis, breaking the Poisson distribution while improving the efficiency of cell analysis. The process involves two key steps: 1. The analyte represented by cells is incubated with the targeted reaction complex and effectively bound through the targeted ligand; 2. Through droplet microfluidics or pipette tip blowing, the reaction compartment can be formed with high throughput to break the double Poisson distribution of cell analysis;

When high-throughput reactions are performed in the compartment, the label molecule on the targeted reaction complex can be used to label the analyte. 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 in order to more efficiently break the probability of cell binding (double Poisson distribution). When the number of microspheres is 10-20 times the number of cells, a 1:1 binding of microspheres and cells can be further achieved (see Figures 8A and 8B for details). When bowl-shaped hydrogel microspheres are used, the steric effect is more significant, and the number of microspheres does not need to be much greater than the number of cells.

Once the targeted reaction complex is bound to the cell, high-throughput generation of reaction compartments (the picture after the compartment is generated should be the most important: 9A is the most important conceptual picture; 5E, 8B, 6C, and 9B are before the reaction compartment is formed; 9C is after the reaction compartment is formed, as shown in 9C: no reaction compartment is found in the droplet, only cells but no microspheres; this means that all cells are analyzed, which means breaking the Poisson distribution) can perform various in-depth analyses of the cells (including cell lysis, mRNA capture, or single-cell sequencing).

Although the embodiments of the present application are described above in conjunction with the accompanying drawings, the present application is not limited to the above specific embodiments and application fields, and the above specific embodiments are merely illustrative and instructive, rather than restrictive. A person of ordinary skill in the art can also make many forms under the guidance of this specification and without departing from the scope of protection of the claims of the present application, all of which belong to the protection of the present application.

Claims (10)

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

preparing a group of objects to be detected, which consists of more than two objects to be detected;

Preparing a target reaction complex group consisting of more than two target reaction complexes, wherein the target reaction complex comprises a carrier, a target ligand connected to the carrier and corresponding to an object to be detected, a reaction reagent connected to the carrier and corresponding to the object to be detected, and optionally a tag molecule connected to the carrier and corresponding to the object to be detected;

contacting the population of target reaction complexes with the population of analytes to form a population of conjugates;

the reaction compartment group is formed by the conjugate itself or by the vehicle enveloping the conjugate.

2. The method of claim 1, wherein the analyte is selected from one or more of a protein, a nucleic acid, a sugar, a lipid, a metabolite, a polypeptide, a bacterium, a virus, an organelle, and a cell, and a complex formed thereof, most preferably the analyte is a cell.

3. The method of claim 1, wherein the shape of the carrier is selected from one or more of a cube, tetrahedron, sphere, ellipsoid, octopus, bowl, red blood sphere; most preferably bowl-shaped and/or red blood bulb-shaped.

4. A method according to claim 3, wherein the number of carriers is 4 times or more the number of the objects to be detected, preferably 10 times or more the number of the objects to be detected, most preferably 10 times to 30 times the number of the objects to be detected.

5. A method according to claim 3 or 4, wherein the carrier has a maximum diameter of 1 times or more the object to be detected, preferably 3 times or more the object to be detected, most preferably 4 to 10 times the object to be detected.

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

7. The method of claim 1, wherein the targeting ligand may be natural or artificial and is selected from one or more of nucleic acids including locked nucleic acids and XNA and analogues thereof, aptamers, small peptides, polypeptides, glycosylated peptides, polysaccharides, soluble receptors, steroids, hormones, mitogens, antigens, superantigens, growth factors, cytokines, leptins, viral proteins, cell adhesion molecules, chemokines, streptavidin and analogues thereof, biotin and analogues thereof, antibodies, antibody fragments, single chain variable fragments (scFv), nanobodies, T cell receptors, major Histocompatibility Complex (MHC) molecules, antigenic peptide-MHC molecule complexes (pMHC), DNA binding proteins, RNA binding proteins, intracellular or cell surface receptor ligands, multiplex ligands, complex ligands formed jointly thereof.

8. The method of claim 1, wherein the tag molecule is selected from natural or artificial information molecules comprising: oligonucleotide barcodes, oligopeptides or polypeptide barcodes, nucleotides composed of natural bases and LNA, PNA, XNA and other non-natural bases, oligosaccharide or polysaccharide barcodes, chromophores (chromophoric group) and color promoting groups (auxochrome groups), metal atoms or ions, small molecules with distinguishable molecular weights, block polymers, covalent links of polymers and backbone molecules, and complexes formed between them.

9. The method of claim 1, wherein the reaction reagent is an oligonucleotide primer, an enzyme, or a small molecule.

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