CN116179668A

CN116179668A - Targeting reaction complexes and their use in targeted multiplex assays

Info

Publication number: CN116179668A
Application number: CN202211501282.4A
Authority: CN
Inventors: 张经纬; 景祥益; 梁雪
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2021-11-26
Filing date: 2022-11-28
Publication date: 2023-05-30
Also published as: WO2023093886A1

Abstract

The present application relates to a targeting reaction complex for targeting and analyzing an analyte, comprising: a carrier; a targeting ligand for the analyte attached to the carrier; a reagent for detecting an analyte attached to a carrier; and a tag molecule attached to the carrier corresponding to the analyte. Furthermore, the present application relates to a population of targeted reaction complexes formed therefrom, a population of reaction compartments and methods of using them for high throughput analysis.

Description

Targeting reaction complexes and their use in targeted multiplex assays

Technical Field

The present application relates to the field of high throughput chemical analysis, in particular to targeting reaction complexes and their use in targeted multiplex detection.

Background

Cell heterogeneity is a ubiquitous life phenomenon, single cells act as independently active living individuals, and the nature and variability exhibited plays a vital role in the development of the overall life system. Each tissue and organ in a human body covers various cell types, and each type of cell changes according to different life activity states of the organism, if thousands of single cells are studied, heterogeneity information among the cells is obscured, so that understanding the working principle of a complex organism, understanding the life function and immune response of each cell type are extremely important for revealing the mechanism of the work of the tissue and organ of the human body and the regulation of genes. For example, malignant tumors causing cancer of human bodies are highly heterogeneous tissues and consist of tumor cells with various phenotypes, and real malignant cells and normal cells are mixed and often occupy only a small part of the whole tissues, so that single-cell analysis can be performed to judge which cells have drug resistance and which cells are easy to transfer, and the method has important roles in the fields of guiding accurate medication, predicting disease course development, clinical guidance and the like.

In a cell subset which looks homogeneous, the expression between single cells may be different, and the genome fundamentally determines the behavior of the cells such as transcription or translation, but the gene expression is a random molecular process, and is related to the growth time and space of the cells, so that the analysis of the genome cannot accurately reflect the difference of the actual behaviors between the cells; proteins are taken as main contributors to vital activities and directly affect cell variability, dynamics and functions, but quantitative analysis of proteins at single cell level, protein amplification and efficient reading of protein sequences are always huge technical barriers; RNA acts on the downstream of DNA and upstream of protein, and has become a powerful tool for indirectly judging the gene expression condition and the protein abundance, so that analysis of transcriptomes can reveal genetic material and the heterogeneity and randomness of expression of genetic material at the single cell level.

Disclosure of Invention

The existing high-throughput single-cell sequencing technology has a plurality of technical problems: the method has the advantages that firstly, the high-flux single cell separation is realized by the fluorescence activated cell flow separation method which is most widely applied, thousands of cells can be scanned through a multispectral channel by the fluorescence activated flow separation method, the flux is high, the speed is high, and the single cell separation position can be accurately positioned; the separation of specific and non-specific cells to obtain the desired cell subpopulations can be performed, and multiparameter analysis can be realized, which has great advantages in the analysis of actual samples, so that the separation of single cells by fluorescence-activated flow separation for subsequent analysis in 96 or 384 microwell plates is one of the most widely used techniques in the field of single cell analysis at present (jaisin, et al Science,343.776-779;Ba gnoli,et al, nature communications, 9.2937.)

The second technical challenge is the amplification of the trace content of single-cell, and how to label each cell during sample preparation, i.e., introduce cell coding, in order to achieve high throughput single-cell sequencing; and because of the deviation in the amplification process, how to mark each transcript information in a single cell, namely, introduce a molecular code, and how to integrate the cell code and the molecular code, so as to realize the accurate quantification of the cell content is the key point of methodology innovation in recent years of scientific researchers. In recent years, researchers have developed a technique for encoding microspheres that can be used for the labeling of high-throughput single-cell content information.

The third technical problem is that the high-flux one-to-one pairing of single cells and single microspheres in a micro-pore plate is realized, the fluorescent activated flow type sorting widely used for sorting microparticles at present is not suitable for sorting of coded microspheres, firstly, the cost of the coded microspheres is high, a large amount of background microspheres are required to be consumed for fluorescent activated flow type sorting, a large amount of reagents are wasted in the mode, the size of the microspheres is often not matched with the consumable of flow type sorting, thirdly, the sorting efficiency is low, the sorting quantity of cells and microspheres in unit time is limited, the high-efficiency one-to-one quick pairing of single cells and single microspheres cannot be realized, the requirement on the total number of cells is far from clinic, and fourthly, the sorted single microspheres are easy to break.

There is no technology disclosed that can integrate high throughput single cell capture, unbiased amplification of minute amounts of single cell content, and comprehensive analysis of single cell content. However, high throughput analysis of single cell transcriptomes using microfluidic techniques has been reported. Such as those reported in Cell paper (Macosko et al 2015, cell:161, 1202-1214; klein et al 2015,Cell 161,1187-1201) for combining droplet microfluidics and encoded microspheres, the method of droplet microfluidics is used for capturing single cells and single microspheres in pairs based on poisson distribution principle, mRNA released by single Cell cleavage is captured by the encoded microspheres paired with the single cells, single Cell mRNA information is encoded and amplified through reverse transcription and amplification, and the expression of a large number of large Cell mRNA is analyzed by high throughput sequencing and bioinformatics methods. The method is based on the poisson distribution principle, the majority of liquid drops have no cells, only 1% of liquid drops contain single cells, and then the poisson distribution of the microspheres is combined, so that the effective analysis target is further reduced, and only a small part of cells in a large number of actual samples can be analyzed, so that some important cell individuals in the samples can be ignored. In addition, the strategy is only suitable for analyzing samples with a large number of objects, and for some rare cells (such as circulating tumor cells), single-cell analysis cannot be realized by the method due to the fact that the number of cells in the samples is too small (10-100/mL blood). These techniques are limited to 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 presently disclosed techniques involve high throughput analysis.

The microfluidic chip is an emerging field in recent years and rapidly develops to a mature field, and utilizes micro-channels with different structures and external force fields with various forms to manipulate, process and control micro-fluid or sample on a micro scale, so that the integration of partial or even complete functions of a traditional laboratory on one chip is realized. However, the limitations of conventional microfluidic chips are also evident in that pumps, valves, and external fluid control devices with complex operations need to be designed inside the chip, the technical threshold is high, and one chip is difficult to reuse, and a large amount of chip manufacturing costs are required to perform multi-level, multi-scale analysis on the same sample with different reagents (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 in the previous sequencing method, when analyzing the actual sample, the currently reported sequencing method based on the encoded microsphere needs to firstly select the target cells by fluorescence activation flow, and then transfer the target cells to each analysis platform, and the cell content information changes along with the change of the environment where the cells are located, so that the sequencing information reflected by the final sequencing result may deviate from the information in the real environment where the cells are located at the moment.

The fifth technical problem is that the separation of rare cells, when the number of cells to be analyzed is very rare and independent transcriptome information of each single cell needs to be analyzed, the traditional technology based on capillary tube picking, gradient dilution or laser cutting is high in labor cost, time-consuming, labor-consuming and low in flux, and high-flux rapid separation and sequencing analysis of the rare cells are limited.

The application focuses mainly on the third technical problem, namely, the problem of one-to-one pairing of single cells and reaction reagents is solved when high-throughput analysis of a plurality of cell types is realized; meanwhile, the reaction reagent has the function of cell identification, and compared with the traditional flow separation and single cell analysis method, the method simplifies the flow of high-throughput analysis of a plurality of types of cells.

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

1. a targeting reaction complex for targeting and analyzing an analyte, comprising:

a carrier;

a targeting ligand for the analyte attached to the carrier;

a reagent for detecting an analyte attached to a carrier; and

a tag molecule linked to a carrier and corresponding to the analyte.

2. The targeting reaction complex according to item 1, wherein the analyte is selected from one or more of proteins, nucleic acids, saccharides, lipids, metabolites, polypeptides, bacteria, viruses, organelles and cells, and complexes formed by them, most preferably the analyte is a cell.

3. The targeted reaction complex of item 1, wherein the analysis is selected from one or more of spectroscopic detection, sequencing, mass spectrometry detection, picture capture, electrical signal detection.

4. The targeting reaction complex according to item 1, wherein the carrier is composed of a polymer or a small molecule, preferably a polymer carrier, further preferably a polystyrene carrier, further preferably the carrier has a diameter of 1 μm to 100 μm, further preferably the carrier shape is one or two or more selected from the group consisting of a square, a tetrahedron, a sphere, an ellipsoid, a bowl, a red blood sphere; most preferably bowl-shaped and/or red blood bulb-shaped; it is further preferred that the carrier has a diameter particle size distribution coefficient CV of less than 20%, and it is further preferred that the surface of the carrier is coated with a mechanical buffer coating; most preferably the surface of the support is coated with a hydrogel coating.

5. The targeting response complex according to item 1, wherein the targeting ligand may be natural or artificial, 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, leptin, 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 and multiplex ligands, complexing ligands, coupling ligands formed jointly thereof.

6. The targeting reaction complex according to item 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.

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

8. The targeting reaction complex according to item 1, wherein the linkage is selected from the group consisting of covalent bonds, metallic bonds, ionic bonds _、 Van der Waals forces, including hydrogen bonding, mechanical bonding, halogen bonding, chalcogenide bonding, gold philic bonding, intercalation, overlap, cation-pi bonding, anion-pi bonding, salt bridging, secondary bonds between nonmetallic atoms, secondary bonds between metallic atoms and nonmetallic atoms, gold philic bonding, silver philic bonding, double hydrogen bonding, and secondary bonds of gold bonds.

9. The targeting reaction complex according to item 1, wherein the targeting ligand is linked to the carrier by a linkage of the reactive agent, by a linkage of the tag molecule or by a linkage of a linker; the attachment of the reactive agent to the carrier is through the attachment of the targeting ligand, through the attachment of the tag molecule or through a linker; the attachment of the tag molecule to the carrier is via attachment of the targeting ligand, via attachment of the reactive agent or via attachment of a linker.

10. The targeting reaction complex according to item 4, wherein the small molecule constituting the carrier is one or more of the targeting ligand, the reaction reagent, and the tag molecule.

11. A population of targeting reaction complexes for targeting and analyzing two or more analytes, comprising two or more targeting reaction complexes according to any of items 1-10, wherein,

the targeting ligands included in each reaction complex are different from each other;

preferably, each of the reaction complexes comprises a different reactant from each other;

it is further preferred that the tag molecules comprised by each reaction complex are different from each other.

12. The population of targeting reaction complexes of item 11, wherein the targeting reaction complexes further comprise a second tag molecule corresponding to the targeting ligand.

13. A method for analyzing an analyte using the reaction complex according to any one of items 1 to 10 or the reaction complex group according to item 11 or 12,

which comprises the following steps:

allowing an analyte to interact with the targeted reaction complex of any one of items 1-10 or the population of reaction complexes of items 11 or 12 to form a conjugate;

the conjugate itself forming a reaction compartment or the conjugate being surrounded by a vehicle forming a reaction compartment; labeling the analyte according to the reaction of the tag molecule and the reactant; optionally analyzing the analyte based on the label.

14. The method of item 13, wherein the amount of analyte interacting with the targeted reaction complex of any one of items 1-10 or the population of reaction complexes of items 11 or 12 and the assay process are controlled such that only one analyte-containing conjugate per conjugate is assayed.

15. The method of claim 13, wherein the vehicle is an oily medium, preferably a fluorooily medium, or a solid medium, preferably a microplate.

16. The method of item 13, wherein, when analyzing using the population of reaction complexes according to item 11 or 12, information of the targeting ligand is provided based on the second tag molecule to confirm the type of analyte.

17. A reaction compartment, comprising:

an analyte;

a reaction complex according to any one of items 1 to 10 targeted to the analyte.

18. The reaction compartment of claim 17, further comprising a vehicle surrounding at least one of the reaction complexes with a conjugate generated by binding of the analyte.

19. The reaction compartment of claim 17 or 18, wherein each reaction compartment comprises one analyte and one reaction complex, the analyte and reaction complex being in a bound state or a separated state within the reaction compartment.

20. A reaction compartment population comprising two or more reaction compartments according to item 17 or 18.

21. The reaction compartment group according to item 20, wherein, in two or more reaction compartments according to item 17 or 18,

the targeting ligands comprised by the reaction complexes in each reaction compartment are different from each other;

preferably, the reactants included in the reactant composition in each reaction compartment are different from each other;

it is further preferred that the tag molecules comprised by the reaction complexes in each reaction compartment are different from each other.

22. The reaction compartment population of claim 17, wherein the targeted reaction complex further comprises a second tag molecule corresponding to the targeting ligand.

23. Use of a reaction compartment population according to any one of claims 17 to 22 for the analysis of a population of analytes.

24. The use of item 23, wherein the population of analytes is a population of cells.

Beneficial technical effects achieved by the application

By adopting the technical scheme, the problem of one-to-one pairing of the analytes (single cells) and the carriers (single microspheres) is solved while high-throughput analysis of the analytes (such as the cells) of a plurality of types is realized, the co-capture rate of the carriers (liquid drops) to the cells and the microspheres is increased, and the problem of inefficiency caused by the phenomenon of double poisson distribution is solved. By using erythrocyte-shaped and bowl-shaped carriers (e.g. microspheres), the steric hindrance effect of them is exploited to further ensure the realization of a "one-to-one pairing of single cells with single microspheres". By coating the surface of the carrier (e.g., microsphere) with a buffer layer (hydrogel layer), the risk of crushing the cells during their binding to the microsphere is avoided and the loss of the analyte to be detected is reduced. The targeted complex population employed in the present application has at least two reagents that enable multiplexed "high throughput analysis".

Drawings

FIG. 1A shows a schematic of the composition of a targeting reaction complex according to the present application, wherein the tag may consist of two parts: a "second tag" comprising "targeting ligand" information, and a "tag molecule" that labels a different analyte (e.g., a cell);

fig. 1B shows hydrogel-coated microspheres: after the DNA coding microsphere with targeting analysis is wrapped by hydrogel, different subgroups in the mixed cells can be targeted for single cell analysis;

FIG. 1C shows that transparent microspheres can carry PCR primers targeting human or murine cells on a hydrogel layer and bind ligands targeting human or murine to effect analysis of cells of different species.

FIG. 2A shows a schematic representation of the composition of a population of targeted reaction complexes for more than two analytes; FIG. 2B shows a schematic of preformed conjugates of multiple analytes and targeted reaction complex populations.

FIG. 3 shows a schematic flow chart of the formation of a reaction compartment by high throughput vector surrounding after formation of a conjugate of an analyte with a targeted reaction complex.

FIG. 4A shows a schematic flow diagram of a complex system for analysis using multiple targeted reaction complexes; FIG. 4B shows a schematic representation of the formation of conjugates of the targeting response complex and the immune cell subpopulation, respectively; FIG. 4C shows a flow from hydrogel-coated DNA encoding microsphere preparation to multiplex analysis of the immune system.

FIG. 5A shows how B-cell and T-cell specific primers are loaded onto B-cell and T-cell assay microspheres, respectively;

FIG. 5B shows a graph of the results of staining a polyT sequence with a probe with FAM;

FIG. 5C shows a graph of the result of staining BCR sequences with a probe with Cy 3;

FIG. 5D shows a graph of the result of dyeing with water;

FIG. 5E shows a graph of the result of staining a microsphere with a probe containing a non-specific sequence.

FIG. 6A shows a graph of the result of staining a polyT sequence with a probe with FAM;

FIG. 6B shows a graph of the results of staining a TCR sequence with a Cy 3-bearing probe;

FIG. 6C shows a graph of the results of dyeing with water;

FIG. 6D shows a graph of the result of staining microspheres with probes containing non-specific sequences.

Figure 7 shows an operational diagram of hydrogel encapsulation of microspheres by microfluidic control.

FIG. 8A shows an operational diagram of a microfluidic device system for generating droplets of a coating of a targeted capture carrier with a cell conjugate;

fig. 8B shows injection of a first fluid (phase I solution, targeted capture carrier to cell conjugate), a second fluid (phase II solution, reverse transcription solution), a continuous phase (carrier oil is fluorinated oil and comprises a surfactant, such as PFPE-PEG-PFPE (perfluoropolyether-polyethylene glycol-perfluoropolyether) triblock copolymer) into a microfluidic chip by a microfluidic device system.

FIG. 9 shows the library after cleavage of cDNA amplification products.

FIG. 10A shows immune system single cell transcriptome data;

FIG. 10B shows a tree view of single cell TCR and TCR two-dimensional map data;

FIG. 10C shows D50 data for single cell TCR;

FIG. 10D shows the dendrogram data for single cell BCR;

fig. 10E shows unique CDR3 distribution of single cell BCR.

FIG. 11 shows a graph of the results of incubation binding of targeting ligand-containing hydrogel-coated microspheres to target cells.

FIG. 12 shows oscillating emulsification with high throughput forming compartments independent of each other; and (3) a graph of the result of the PCR reaction by tiling the liquid drops after separation.

FIG. 13A shows a graph of the results of the formation of an in-independent droplet qPCR analysis of human and murine after PCR thermal cycling;

FIG. 13B shows real-time detection of droplets, where the amount of cellular mRNA expression in each droplet exhibits a different Ct value;

FIG. 13C shows a graph of the results of statistics of Ct values for human and murine different GAPDH genes qPCR in droplets.

FIG. 14A shows the state where the analyte and the targeting reaction complex remain bound to each other in the reaction compartment (white arrow);

FIG. 14B shows the state where the analyte and the targeting reaction complex are separated from each other (white arrow) or bound to each other (gray arrow) in the reaction compartment;

FIG. 14C shows analytes as populations of cells interacting with each other, forming a reaction compartment upon binding to a targeting reaction complex.

Detailed Description

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

It should be noted that certain terms are used throughout the description and claims to refer to particular components. Those of skill in the art will understand that a person may refer to the same component by different names. The description and claims do not identify differences in terms of components, but rather differences in terms of the functionality of the components. As used throughout the specification and claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description hereinafter sets forth a preferred embodiment for practicing the invention, but is not intended to limit the scope of the invention, as the description proceeds with reference to the general principles of the description. The scope of the invention is 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 is not purposefully formulated into the composition and/or is present as a contaminant or in trace amounts only. Thus, the total amount of the specific components 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 a particular component is undetectable using 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 explicitly indicated to refer to only alternatives or alternatives are mutually exclusive, although the disclosure supports definitions of only alternatives and "and/or". As used herein, "another" may mean at least a second or more.

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

The various biomaterials described in the examples were obtained by merely providing an experimental route for achieving the objectives of the specific disclosure and should not be construed as limiting the source of biomaterials of the present invention. In fact, the source of the biological material used is broad, and any biological material that is available without violating law and ethics may be used instead as suggested in the examples.

The present application provides in a first aspect a targeted reaction complex.

In one embodiment, a targeting reaction complex for targeting and analyzing an analyte is provided, comprising:

a carrier;

a targeting ligand for the analyte attached to the carrier;

a reagent for detecting an analyte attached to a carrier; and

a tag molecule linked to a carrier and corresponding to the analyte.

In the context of the present specification, "targeting" is in accordance with the general definition of biotechnology, typically an antibody-antigen affinity reaction, an affinity reaction of complementary nucleic acid sequences.

In the context of the present specification, "connected" is to be understood broadly, and includes at least various connections through molecular bonds and connections not through molecular bonds.

FIG. 1A shows the components of a targeting reaction complex, where the structural relationship of the targeting ligand, tag molecule and reactant, respectively, attached to a "carrier" is shown. Wherein, the "carrier" may be composed of the same molecules as the targeting ligand, the tag and the reactive agent, i.e. may be composed of small molecules. More commonly, the support is composed of a polymer, preferably polystyrene; in FIG. 1B, the DNA encoding microsphere carries both A) and B), wherein A) is an oligonucleotide tag molecule for labeling a single cell; b) The reverse transcribed sequence is part of the single cell assay reagents. The hydrogel coating coated outside the DNA coding microsphere is not only a mechanical buffer layer, but also carries targeting ligands represented by antibodies.

In a specific embodiment, 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, and most preferably the analyte is a cell.

In the context of the present specification, "cells" are intended to be within the general definition of the field of biology, including at least prokaryotic and eukaryotic cells, and for the purposes of the technology of the present application may sometimes be referred to in particular as differentiated cell populations of multicellular organisms, such as immune cell populations, including at least lymphocytes B cells, lymphocytes T cells, NK cells.

In the context of this specification, an "organelle" is a general definition in the biological field, and an organelle (organelle) is generally considered to be a microstructure or micro-organ with a morphology and function interspersed within the cytoplasm. They constitute the basic structure of the cell, enabling the cell to function properly. The organelles in cells are mainly: mitochondria, endoplasmic reticulum, centrosome, chloroplast, golgi apparatus, ribosome, etc.

In the context of the present specification, a "complex" refers to two or more substances that interact with each other with a certain binding strength, which substances may consist of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles and cells. For example: hepG2, after incubation with T cells (genetically engineered, carrying T cell receptor genes targeting the "major histocompatibility Complex-AFP 158 peptide") recognizing the cell line, will form cell-to-cell interactions; specific examples are: complexes of bacteria and phages (viruses), complexes of cells and membrane proteins (polypeptides), and complexes of ribosomes and RNAs (nucleic acids).

In a specific embodiment, wherein the analysis is selected from one or more of spectroscopy detection, sequencing, mass spectrometry detection, picture capture, electrical signal detection.

In the context of this specification, spectroscopic detection/image capture is sometimes referred to as a fluorescence analysis method, which refers to a method that uses fluorescence, which reflects the characteristics of a substance, generated by a deexcitation process in which some substance is in an excited state after being irradiated with electromagnetic waves, and the excited state molecules undergo a collision and emission, to perform qualitative or quantitative analysis. Since some substances do not emit fluorescence (or fluorescence is weak) by themselves, it is necessary to convert the substances that do not emit fluorescence into substances that emit fluorescence. For example, by using certain reagents (such as fluorescent dyes) to form complexes with non-fluorescent materials, each complex being capable of emitting fluorescence, and then performing the assay; in the context of the present specification, a "reagent" may sometimes refer to a fluorescent reagent capable of causing fluorescence of an analyte.

In the context of the present specification, "sequencing" sometimes refers to nucleic acid sequencing, which includes at least DNA sequencing and RNA sequencing, and mainly includes the following according to development history, influence, sequencing principles and techniques, etc.: large-scale parallel sequencing techniques (Massively Parallel Signature Sequencing, MPSS), polymerase cloning (Polony Sequencing), 454pyro-sequencing (454 pyro-sequencing), sequencing-by-synthesis based on reversible terminating fluorescent bases Illumina (Solexa) sequencing, ABI SOLiD sequencing, ion semiconductor sequencing (Ion semiconductor sequencing), DNA nanosphere sequencing (DNA nanoball sequencing), nanopore sequencing (Oxford Nanopore sequencer), and the like. For example, transcriptome sequencing techniques used in the examples of the present application pertain to sequencing-by-synthesis Illumina (Solexa) sequencing or DNA nanosphere sequencing (DNA nanoball sequencing) based on reversibly terminating fluorescent bases.

In the context of the present specification, "mass spectrometry" sometimes refers to mass spectrometry or general mass spectrometry, which differs from traditional flow cytometry in two major points: the first and the second label systems are different, the former mainly uses various fluorophores as the label of the antibody, and the latter uses polymer (such as polypeptide, nucleic acid, etc.) or various metal elements as the label; and second, the difference of the detection system, the former uses a laser and a photomultiplier as detection means, and the latter uses a charge-mass ratio detection technology as detection means. For example, MALDI-TOF is used in embodiments of the present application.

The detection of the electric signal requires that ions flow from one side to the other side according to the charge difference between the ions, and the detection is realized by changing the current signals received by the electrodes at the two sides. There is no limitation in this application to the detection of electrical signals and any means and apparatus for detecting charge flow or differences that may be employed by those skilled in the art may be employed. For example, nanopore sequencing primarily uses electrophoresis to transport a sample of unknown sequence through a nanopore of about 1 diameter. The nanopore system generates a detectable current signal on the electrolyte by applying a fixed applied electric field, the magnitude of which is related to the pore size of the nanopore and the composition of the nucleic acid passing through the nanopore. When the nanopore aperture is small enough, the sample can cause unique current signal changes as it passes through the nanopore, and such a mechanism also makes sequencing using the nanopore an event possible. The magnitude of its current through a single nanopore can be defined as the amount of charge that passes through the nanopore per unit time.

In a specific embodiment, the carrier is composed of a polymer or a small molecule, preferably a polymer carrier, further preferably a polystyrene carrier, further preferably the diameter of the microsphere is 1mm to 100mm, further preferably the shape of the carrier is one or more selected from the group consisting of square, tetrahedron, sphere, ellipsoid, bowl, concave sphere, and red blood sphere; most preferably bowl-shaped, concave spherical and/or red blood spherical; it is further preferred that the microspheres have a diameter particle size distribution coefficient CV of less than 20%, and it is further preferred that the microspheres have a coating applied to their surface; most preferably coated with a hydrogel coating. In the context of the present specification, a "bowl-shape" refers in particular to a concave sphere, which is provided with a concave surface on one side of the plane of the "hemisphere", and when the object to be detected is bound to this concave surface of the carrier, the object to be detected is no longer bound to another carrier due to the steric hindrance effect. Concave spherical refers to a spherical round plane with a concave surface, when the object to be detected is combined with the concave surface of the carrier, the object to be detected is not combined with another carrier any more due to the steric hindrance effect. While the carrier of the "red blood sphere" 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 is not bound to the other carrier any more due to the steric hindrance effect.

In particular, the diameter of the microspheres may be in the range 5mm to 95mm, preferably 10mm to 90mm; in particular 10mm, 20mm, 30mm, 40mm, 50mm, 60mm, 70mm, 80mm, 90mm; preferably having a diameter of greater than or equal to the diameter of the analyte, most preferably having a diameter of from 1 to 100 times the diameter of the analyte. In the context of this specification, "diameter" refers to the distance between two points on the edge from the center of a flat pattern or solid (e.g., circle, cone, sphere, cube). In the case of using erythrocyte-shaped and bowl-shaped carriers, a 1:1 combination of carrier and analyte is achieved to the greatest extent due to steric hindrance effects. Especially in the case of using erythrocyte-shaped microspheres and immune cells, and the diameters of the erythrocyte-shaped microspheres are 3 times to 30 times that of the immune cells, the 1:1 combination of the carrier and the object to be detected is realized to the greatest extent.

In this context, the diameter of the microspheres employed can be calculated in a manner and by means known to the person skilled in the art, for example by measuring under a microscope and analyzing with image processing software, by means of an instrument for determining the particle size (Bio-Rad T20 cell technology instrument), or by using data provided by the supplier of the microspheres.

In one embodiment, the diameter particle size distribution coefficient CV is 20%. The english abbreviations herein are defined as follows: cv=sd/average particle size, which may represent the width of the particle size distribution. Wherein SD: is the standard deviation (Standard Deviation) statistically and mathematically noted as σ; CV is also known as the relative standard deviation (Coefficient ofvariation) and is statistically and mathematically noted α.

The hydrogel is preferably made of polyacrylamide. The hydrogel coating functions as a mechanical buffer layer. The thickness of the hydrogel coating is desirably from 100nm to 5 μm, and may be specifically 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm; particularly, under the conditions that the carrier adopts red blood spherical microspheres with the diameter of 25-35 microns, the object to be detected is immune cells, and hydrogel with the thickness of 250nm-5 microns is used, the technical effect of preventing the immune cells from being broken by the microspheres during combination is achieved. The term "mechanical buffer coating" should be regarded as part of the term "support", i.e. the term "targeting ligand", "reactive agent" or "tag molecule" as described in the present specification is attached to the support (uncoated core) by, for example, chemical modification after the support has been coated with the "mechanical buffer coating".

Alternatively, the tag molecule is first attached to the support, followed by the application of a mechanical buffer coating, and finally the reaction agent and the targeting ligand are attached outside the mechanical buffer coating, e.g. by chemical modification.

Alternatively, the reactive agent is first attached to the support, followed by the application of a mechanical buffer coating, and finally the label molecule and the targeting ligand are attached outside the mechanical buffer coating, e.g. by chemical modification.

Alternatively, the targeting ligand is first attached to the support, followed by the application of a mechanical buffer coating, and finally the reaction agent and the tag molecule are attached outside the mechanical buffer coating, e.g. by chemical modification.

Alternatively, the tag molecule and the reactive agent are first attached to the support, followed by application of a mechanical buffer coating, and finally the targeting ligand is attached outside the mechanical buffer coating, e.g. by chemical modification.

Alternatively, the tag molecule and targeting ligand are first attached to the support, followed by application of a mechanical buffer coating, and finally the reaction agent is attached outside the mechanical buffer coating by, for example, chemical modification.

Alternatively, the reaction reagent and targeting ligand are first attached to the support, followed by application of a mechanical buffer coating, and finally the tag molecule is attached outside the mechanical buffer coating by, for example, chemical modification.

Alternatively, the tag molecule, targeting ligand and tag molecule are attached to a carrier first, followed by application of a mechanical buffer coating.

In a specific embodiment, the parent core of the support may be a 10mm, 20mm, 30mm, 40mm, 50mm, 60mm, 70mm, 80mm or 90mm polystyrene support. In a specific embodiment, the parent core of the support is a 30 micron diameter polystyrene support.

In one embodiment, a polystyrene support with a 30 micron diameter parent core is used, and hydrogel coatings of 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm thickness may be used.

In a specific embodiment, the 30 micron diameter master is coated with a 5 micron hydrogel layer by droplet microfluidic to ultimately form a 40 micron diameter polystyrene support.

In a specific embodiment, a 40 micron polystyrene carrier carrying a 5 micron polyacrylamide layer of DNA-encoding reverse transcription primers is microfluidic coated by droplets.

Diameter (diameter) refers to the distance from the center to two points on the sides through a flat pattern or solid (e.g., circle, cone, sphere, cube). The particle size distribution refers to the proportion of particles of different particle sizes in a population of particles, also referred to as the dispersion degree of the particles. When expressed as a proportion of the number of particles, it is referred to as a number distribution. After photographing by microscopic imaging, the particles in the image are subjected to image processing (e.g., using ImageJ software) to obtain a diameter particle size distribution.

In this context, a targeting ligand is a molecule that is used to target and bind to an analyte.

In yet another embodiment, the targeting ligand may be natural or artificial, selected from one or more of nucleic acids and analogues thereof (locked nucleic acids, XNA, etc.), 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, antigen peptide-MHC molecule complexes (pMHC), DNA binding proteins, RNA binding proteins, intracellular or cell surface receptor ligands, multiplex ligands, complex ligands, coupling ligands formed in common.

In yet another embodiment, where the analyte is a cell, the targeting ligand is an antibody that specifically recognizes such a cell.

In yet another embodiment, when the analyte is an organelle, the targeting ligand is an antibody that specifically recognizes a organelle surface protein.

In the context of the present specification, an "antibody" is in accordance with the general definition in the field of biology, in particular an antibody (anti) refers to a protein of the body that has a protective effect 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, viruses, etc., found only in body fluids such as vertebrate blood, and the cell membrane surface of B cells thereof. Antibodies recognize a unique feature of a particular foreign object, which is called an antigen. In the technical scheme of the application, the surface receptor of the lymphocyte can be used as an antigen to prepare corresponding antibodies, and the corresponding antibodies are chemically modified on the surface of the microsphere to play a role in separating lymphocyte groups.

In the context of the present specification, an "aptamer" corresponds to a general definition in the field of biology, and in the technical scheme of the present application, may particularly refer to a nucleic acid aptamer. The Aptamer (Aptamer) is a DNA (deoxyribonucleic acid), RNA (ribonucleic acid) sequence, XNA (nucleic acid analogue) or peptide. Oligonucleotide fragments obtained from a library of nucleic acid molecules are typically obtained using in vitro screening techniques, an exponential enrichment ligand system evolution technique (Systematic evolution ofligands by exponential enrichment, SELEX). The aptamer can be combined with various target substances with high specificity and high selectivity, so that the aptamer is widely applied to the field of biosensors. When the aptamer specifically binds to the target substance, the conformation of the aptamer itself changes. In the technical scheme of the application, when the analyte is a nucleic acid fragment, a nucleic acid aptamer of the analyte can be selected and fixed on the surface of the microsphere through chemical modification so as to play a role in separating cells.

In this context, a tag molecule is used to characterize different analytes, which in this context may refer to different classes of analytes, as well as to specific, non-universal, single analytes in the same class.

In yet another embodiment, the tag molecule is selected from natural or artificial information molecules comprising: one or more than two of oligonucleotide bar codes, oligopeptide or polypeptide bar codes, nucleotides composed of natural bases, LNA, PNA, XNA and other non-natural bases, oligosaccharide or polysaccharide bar codes, block polymers, covalent links of polymers and skeleton molecules and complexes formed between the two.

In the context of the present specification, a DNA barcode (DNA barcode) refers to a standard, sufficiently mutated, easily amplified and relatively short DNA fragment capable of representing the species in an organism. DNA barcodes have become an important tool for ecological research, not only for species identification, but also to help biologists further understand the interactions that occur within the ecosystem. Upon finding an unknown species or portion of a species, researchers delineate the DNA barcodes of their tissues and then compare them to other barcodes in the international database. If matched with one of them, the researcher can confirm the identity of this species. DNA barcode technology is an emerging technology that utilizes a segment of conserved fragments in the DNA of an organism to rapidly and accurately identify species. In particular to the examples herein, when the "analysis" is transcriptome sequencing for an immune cell population, the use of DNA barcodes allows deconvolution of the analysis results after high throughput analysis results for the immune cell population are obtained.

In a specific embodiment, when the analyte is a cell, the tag molecules may be sequences that differ from each other in sequence on the DNA encoding microspheres; in a specific embodiment, when the analyte is an organelle, the tag molecules may be sequences that differ from each other in sequence on the DNA-encoding microsphere.

Herein, a reagent is a substance that is used to react with an analyte and produce a signal that can be used to detect the signal. In another embodiment, the reactant is an oligonucleotide primer, an enzyme, or a small molecule.

In the context of the present specification, a "primer" is intended to be in accordance with the general definition of the field of biotechnology, in particular a primer which is a macromolecule which stimulates synthesis at the start of nucleotide polymerization, a macromolecule having a specific nucleotide sequence, which is linked to a reactant in the form of hydrogen bonds, such a molecule being referred to as a primer. Primers are typically two oligonucleotide sequences that are synthesized artificially, one primer being complementary to one DNA template strand at one end of the target region and the other primer being complementary to the other DNA template strand at the other end of the target region, and function as a starting point for nucleotide polymerization, from the 3-terminus of which a nucleic acid polymerase can begin to synthesize a new nucleic acid strand. Primers designed manually in vitro are widely used for polymerase chain reaction, sequencing, probe synthesis and the like. In particular to the examples herein, oligonucleotide primers may be employed as "reagents" when the "analysis" is transcriptome sequencing.

In a specific embodiment, where the analyte is a cell, the reagent may be a reverse transcribed primer for analysis of mRNA in the cell.

In a specific embodiment, where the analyte is an organelle (e.g., a mitochondria), the reagent may be a primer of a PCR to analyze a specific region in mitochondrial DNA. The reagents may be immobilized on a carrier and part of the reaction components may be added later by means of a solution.

In the context of the present specification, a "small molecule constituting a reactive agent" may be various small molecules in the field of chemical technology, and in particular, when the "analysis" is a fluorescence detection, the "small molecule" is a small molecule that can emit fluorescence, such as FAM, HEX, or the like.

"ligating" means that the targeting ligand, reactant, carrier and tag are formed into one entity by interaction. In a specific embodiment, the linkage is selected from the group consisting of covalent bonds, metallic bonds, ionic bonds, van der Waals forces, secondary bonds including hydrogen bonds, mechanical bonds, halogen bonds, chalcogenide bonds, enophilic bonds, intercalation, superposition, cation-pi bonds, anion-pi bonds, salt bridges, non-metallic interatomic secondary bonds, metallic and non-metallic interatomic secondary bonds, enophilic bonds, bishydrogen bonds, and gold bonds.

In yet another embodiment, the targeting ligand is linked to the carrier by a linkage of the reactive agent, by a linkage of the tag molecule or by a linker; the attachment of the reactive agent to the carrier is through the attachment of the targeting ligand, through the attachment of the tag molecule or through a linker; the attachment of the tag molecule to the carrier is via attachment of the targeting ligand, via attachment of the reactive agent or via attachment of a linker.

In a specific embodiment, the small molecules comprising the carrier are one or more of the targeting ligand, the reactive agent, and the tag molecule. That is, the targeting ligand, the reactive agent, the tag molecule itself may form part of a carrier.

In a specific embodiment, the DNA encoding oligonucleotide can be attached to a polystyrene support via a linker (linker); in a specific embodiment, the polystyrene carrier encapsulating the acrylic hydrogel layer can be linked to the targeting ligand by EDC coupling chemistry.

The present application provides in a second aspect a targeted reaction complex population.

In one embodiment, a population of targeting reaction complexes for targeting and analyzing two or more analytes is provided, comprising two or more of the foregoing targeting reaction complexes, wherein,

In yet another embodiment, the targeting reaction complex further comprises a second tag molecule corresponding to the targeting ligand. Here, the role of the second tag molecule is to further divide the targeting ligand into different subgroups.

In one embodiment, the population of reaction complexes has two different targeting ligands (e.g., antibodies); and having the same reactant (e.g., poly-T); and have the same tag molecule (e.g., DNA encoding).

In one embodiment, the population of reaction complexes has two different targeting ligands (e.g., antibodies); and having two different reactants (e.g., poly-T); and have the same tag molecule (e.g., DNA encoding).

In one embodiment, the population of reaction complexes has two different targeting ligands (e.g., antibodies); and having the same reactant (e.g., poly-T); and have two different tag molecules (e.g., DNA encoding).

In one embodiment, the population of reaction complexes has two different targeting ligands (e.g., antibodies); and having two different reactants (e.g., poly-T); and have two different tag molecules (e.g., DNA encoding).

In a specific embodiment, the population of reaction complexes has more than three different targeting ligands (e.g., antibodies); and having one or two reactive agents (e.g., poly-T); and have one or more tag molecules (e.g., DNA encoding).

In a specific embodiment, the population of reaction complexes has more than three different targeting ligands (e.g., antibodies); and having one or two reactive agents (e.g., poly-T); and have three or more different tag molecules (e.g., DNA encoding).

In a specific embodiment, the population of reaction complexes has more than three different targeting ligands (e.g., antibodies); and has more than three different reactants (e.g., poly-T); and have one or more tag molecules (e.g., DNA encoding).

In a specific embodiment, the population of reaction complexes has more than three different targeting ligands (e.g., antibodies); and has more than three different reactants (e.g., poly-T); and have three or more different tag molecules (e.g., DNA encoding).

The present application provides in a third aspect methods of performing high throughput assays using the complexes described above.

In one embodiment, a method of analyzing an analyte using the aforementioned reaction complexes or groups of reaction complexes is provided,

which comprises the following steps:

allowing the analyte to interact with the targeted reactant complex or the population of reactant complexes to form a conjugate; the conjugate itself forming a reaction compartment or the conjugate being surrounded by a vehicle forming a reaction compartment; labeling the analyte according to the reaction of the tag molecule and the reactant; optionally analyzing the analyte based on the label.

Herein, the term "compartment" means that a specific reaction space is provided for a specific chemical reaction, in which reactants can react with each other without reacting with reactants of an adjacent compartment, but the product of the compartment may be subject to mass exchange with the product of the adjacent compartment (e.g., dye, surfactant, etc.) depending on the type thereof.

In a specific embodiment, the amount of analyte interacting with the targeted reaction complex or groups of reaction complexes and the analysis process are controlled such that only one analyte-containing conjugate per conjugate is analyzed. In the examples of the present application, in particular, when the number of microspheres carrying the reaction reagent is 10 to 20 times the number of cells to be analyzed, each microsphere binds only one cell with a high probability according to the poisson distribution.

In a further embodiment, the vehicle is an oily medium, preferably a fluorooily medium, or a solid medium, preferably a microplate. The reason for using an oily medium as a "vehicle" for the "compartment" is that the oily medium effectively blocks charged nucleic acid molecules from crossing different "compartments", with the benefit that reagents carrying different analyte tags do not cross-contaminate the "compartment" products after labeling the cell contents.

In one embodiment, information is provided for targeting ligands based on the second tag molecule to confirm the type of analyte when the aforementioned reaction complex population is used for analysis.

The present application provides in a fourth aspect reaction compartments and clusters thereof.

In one embodiment, there is provided a reaction compartment comprising:

an analyte; the aforementioned reaction complexes targeted to the analyte.

In yet another embodiment, the reaction compartment further comprises a vehicle surrounding at least one of the reaction complexes with the binding agent formed by binding of the analyte.

In a specific embodiment, each reaction compartment comprises one analyte and one reaction complex, which are in a bound state or in a separated state within the reaction compartment.

In the context of the present specification, the "binding state" refers to a bioaffinity state, including at least an antibody-antigen binding state or a binding state of a target nucleic acid and an aptamer.

In yet another embodiment, a reaction compartment group is provided comprising two or more of the aforementioned reaction compartments.

In a specific embodiment, a reaction compartment group is provided, wherein, in two or more of the aforementioned reaction compartments,

In a specific embodiment, the targeting reaction complex further comprises a second tag molecule corresponding to the targeting ligand.

In the context of the present specification, the reaction compartment consists of: an analyte; the aforementioned reaction complexes targeted to the analyte. The "reaction compartment" may be the smallest individual unit of chemical reaction for analysis; that is, in different "reaction compartments" different chemical reactions may take place for analytical purposes.

The present application provides in a fifth aspect the use of a reaction compartment as described above.

In a specific embodiment, there is provided the use of the aforementioned reaction compartment population for the analysis of a population of analytes.

In yet another embodiment, the population of analytes is a population of cells. In particular a population of differentiated immune cells obtained from a vertebrate blood sample or lymph fluid.

In this application, the definitions described for the various aspects are generic.

Examples section

For the first part of examples (examples 1-7): summary of experimental procedures for multiplex single cell analysis of the immune system in peripheral blood of tumor-treated patients (see also fig. 4C):

the first step: a reaction complex is prepared that targets a specific subset of cells of the immune system, the complex consisting of DNA encoding microspheres that capture B Cell Receptor (BCR), T Cell Receptor (TCR) genes. Wherein the microsphere core is TOYOPEARL HW-65 (TOSOH Bioscience), the DNA synthesized above is encoded into a tag molecule, and the poly T is a reactant.

And a second step of: the two microspheres were individually hydrogel coated and antibodies (targeting ligands) were modified to aid in binding of the cell subsets. Thus, the carrier, the reaction reagent, the targeting ligand and the tag molecule are integrated.

And a third step of: peripheral blood mononuclear cells are extracted from a patient sample, incubated and combined with a plurality of microspheres with targeting antibodies and single cell reaction reagents, and then subjected to high-flux encapsulation through droplet microfluidic.

Fourth step: each microsphere within the droplet is specifically enriched for a particular RNA molecule in the targeted cell.

Fifth step: after the sample is subjected to library establishment, high-throughput sequencing is carried out, and after data are obtained, raw information data are clustered through a targeting ligand tag and a single cell tag, so that the whole immune system information is obtained.

EXAMPLE 1 preparation of microspheres with various reactive Agents

Example 1.1 preparation of microspheres capable of simultaneously analyzing B Cell Receptor (BCR) and transcriptome

The commercial microsphere (Chemgenes corp. Cat# Macosko-2011-10) master is methyl methacrylate Polymer (PMA) (carrier), the surface of which is modified with a poly-t capture oligonucleotide of DNA code (tag molecule), and by chain extension, it can carry both poly-t (reactant) and BCR sequence (fig. 5A) (reactant, having the function of a second tag molecule). The microsphere can be used for analyzing transcriptome of single cells, and BCR specific capture oligonucleotides thereof can be used for analyzing B cell antibodies of coding regions.

Designing complementary single stranded DNA (ssDNA) custom primer sequences adds a 5' phosphate modified region of interest, known as a foothold probe. The complementary Splint sequence (Splint) was also designed with an A repeat overhang (Protect) of 8-12 bp.

Table 1 a list of microsphere primers targeting B Cell Receptor (BCR) mRNA was prepared; the DNA encoding microsphere originally carrying the multimeric T sequence can be further added with a BCR detection sequence through a T4 ligase reaction.

All oligonucleotides were resuspended in Tris-EDTA (TE) buffer at a concentration of 500. Mu.M

Table 2 configuration of BCR primer mother liquor; the primers were diluted to the target concentration with water or TE solution.

Equal volumes (20 uL) of custom primers and splint oligonucleotide 50mM NaCl were mixed and transferred to PCR tubes. The reaction mixture was heated to 95 ℃ (3 min) and cooled to raise the temperature to 14 ℃ at a slow rate (-0.1 ℃/s). This will create a double stranded foothold probe for ligating the external primer to the sequence on the microsphere (200 uL tube)

Table 3 incubation of BCR foothold probes, splint oligonucleotides with microspheres; multiple footing probes were configured as a mixture.

Each foothold probe was diluted with TE buffer to obtain a final concentration of 100. Mu.M.

The foothold probes (toehold primer) were mixed in the desired ratio and the mixture diluted to obtain the desired final probe concentration (all of the foothold probes were mixed in equal proportions for the BCR sequence experiment).

Table 4 mixed foothold probe stock configurations targeting BCR; multiple footing probes were configured as a mixture.

Table 5 dilution of mixed foothold probe stock targeted to BCR; the multiple foothold probe mixture is adjusted to a target concentration.

mu.L of this mixed probe mixture was mixed with 40. Mu.L of PEG-4000 (50% w/v), 40. Mu.LT 4DNA ligase buffer, 72. Mu.L of water and 2. Mu. L T4DNA ligase (1.5 ml tube).

Marking	Reaction tube A
		MixingProbe solution	16*1.5＝24μL
PEG-4000(50％w/v)	40*1.5＝60μL
		T4DNA ligase buffer (NEB)	40*1.5＝60μL
Water	72*1.5＝108μL
		T4DNA ligase (NEB)	2*1.5＝3μL
Total reaction	170*1.5＝255μL

Table 6BCR sequence ligation reactions; DNA encoding microspheres targeting whole transcriptome and BCR specific sequences were prepared.

12000 microspheres were mixed with the ligation mixture described above and incubated in an Eppendorf thermo Mixer set at 37℃for 1 hour (mixing shaking at 1800rpm every 15 seconds).

Enzyme inactivation by heating the reaction mixture at 65℃for 3 min

The reaction mixture was quenched by placing in ice water for at least 1 minute.

To obtain the desired number of microspheres of the polyT+BCR sequence, 6-10 microsphere ligation reactions can be performed in parallel. The microspheres were washed with 250. Mu.L Tris-EDTA, sodium dodecyl sulfate (TE-SDS) buffer; tris-EDTA-Tween 20 (TE-TW) buffer was used twice.

These treated microspheres can be stored in TE-TW at 4℃and BCR sequences on the microspheres can be hybridized and verified by fluorescent-labeled probes (FIGS. 5B-5E).

The probe hybridization steps are as follows:

mother liquor for preparing the probe:

solution	Concentration of mother liquor	Final concentration	Total volume (uL)
				PBS(pH7.5)	-	-	23.5
BSA	5mg/mL	0.1mg/ml	0.05uL
				ddH2O			49.5-23.5-0.05＝25.95
Complementary probe/negative control probe/water	100μM	2μM	1uL

Table 7 configuration of probe mother liquor; a probe solution was prepared at a final concentration of 2. Mu.M.

Hybridization conditions: 25uL lysis buffer+25 uL probe mix+20 uL microspheres; in Eppendorf ThermoMixer, 65℃for 5 minutes, 48℃for 8 minutes, 40℃for 8 minutes, 30℃for 8 minutes (1200 revolutions) were set. Wash 3 times with 200uL cold 6x SSC buffer.

Example 1.2 preparation of microspheres capable of simultaneously analyzing T Cell Receptor (TCR) and transcriptome

Commercial microsphere (Chemgenes corp. Cat# Macosko-2011-10) mother nucleus is methyl methacrylate Polymer (PMA) (carrier), surface is modified with DNA code (tag molecule) polyT capture oligonucleotide, and by chain extension, polyT (reactant) and TCR sequence (figure 6A) can be carried simultaneously (reactant, having the function of second tag molecule). The microsphere can be used for analyzing transcriptome of single cells, and TCR specific capture oligonucleotide of the microsphere can be used for analyzing T cell receptor sequences of T cells of a coding region.

Designing complementary single stranded DNA (ssDNA) custom primer sequences adds 5' phosphate modified regions of interest. The complementary Splint sequence (Splint) was also designed with an A repeat overhang (Protect) of 8-12 bp.

Table 8 is a list of microsphere primers prepared to target T Cell Receptor (TCR) mRNA; the TCR detection sequence can be further added to DNA encoding microspheres that otherwise carry multimeric T sequences by T4 ligase reaction.

Table 9 configuration of TCR primer mother liquor; the primers were diluted to the target concentration with water or TE solution.

Table 10 incubation of TCR foothold probes, splint oligonucleotides with microspheres; multiple footing probes were configured as a mixture.

The toehold probes were mixed in the desired ratio and the mixture diluted to achieve the desired final probe concentration (all foothold probes were mixed in equal proportions for TCR sequence experiments).

Table 11 mixed foothold probe stock configurations for targeting TCRs; multiple footing probes were configured as a mixture.

Mixed foothold probe (3.125 mu M)	Volume of	Final concentration
			Mixed foothold probe (9.375 mu M)	30ul	8.29μM
Water and its preparation method	3.9ul
			Total reaction	33.9ul

Table 12 dilution of mixed foothold probe stock of targeted TCRs; the multiple foothold probe mixture is adjusted to a target concentration.

Marking	Reaction tube A
		Mixed probe solution	16*1.5＝24μL
PEG-4000(50％w/v)	40*1.5＝60μL
		T4DNA ligase buffer (NEB)	40*1.5＝60μL
Water	72*1.5＝108μL
		T4DNA ligase (NEB)	2*1.5＝3μL
Total reaction	170*1.5＝255μL

Table 13TCR sequence ligation reactions; DNA encoding microspheres targeting whole transcriptome and TCR-specific sequences were prepared.

Enzyme inactivation by heating the reaction mixture at 65℃for 3 min

To obtain microspheres of the desired number of polyT+TCR sequences, 6-10 microsphere ligation reactions can be performed in parallel. The microspheres were washed with 250. Mu.L Tris-EDTA, sodium dodecyl sulfate (TE-SDS) buffer; tris-EDTA-Tween 20 (TE-TW) buffer was used twice.

These treated microspheres can be stored in TE-TW at 4℃and TCR sequences on the microspheres can be hybridized and verified by fluorescent labeled probes (FIGS. 6A-6D).

The probe hybridization steps are as follows:

mother liquor for preparing the probe:

Table 14 configuration of probe mother liquor; a probe solution was prepared at a final concentration of 2. Mu.M.

Example 2 hydrogel encapsulation of microspheres and modification of antibodies targeting B cells and T cells

EXAMPLE 2.1 encapsulation of hydrogel microspheres

1. A Polyacrylamide (PAA) solution consisting of 6.2% acrylamide, 0.18% N, N' -methylenebis (acrylamide), 0.3% ammonium persulfate, and 0.6% sodium acrylate was prepared. This solution was mixed with microspheres of example 1.1 and example 1.2 separately, each filled into 1ml syringes with 28G needles.

2. An insoluble continuous phase of 5% (w/w) fluorosurfactant and 1% n, n-tetramethyl ethylenediamine (TEMED) was prepared in Hydrofluoroether (HFE) oil for droplet generation and stabilization. The solution was loaded into a new 1ml syringe (fig. 7).

3. 1ml of the droplets were collected in a 15ml collection tube and incubated at room temperature for 3 hours for polymerization. After incubation, the lower layer of oil was removed with a pipette.

4. 20% (v/v) Perfluorooctanol (PFO) in 1ml HFE oil was added to a 15ml collection tube as a chemical demulsifier

5. After mixing, 15ml collection tubes 2000x g were spun for 2 minutes. The PFO/HFE supernatant was removed by pipetting. Repeat 1x.

6. The supernatant was removed by pipetting and 1ml of PBS containing 0.2% Tween20 was added to remove the surfactant/solution. Repeat 2x.

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

8. 3000x g for 3 minutes. The supernatant was removed by pipetting. Repeat 3x.

9. Resuspended in 1ml TEBST. The solution can be stored at 4deg.C for an indefinite period

Example 2.2 activation and Cross-linking of hydrogel-coated microspheres targeting antibodies

1. 200. Mu.L of hydrogel-coated microspheres (1:2) were taken and the hydrogel microspheres were washed with 1ml PBS (ph 7.4) buffer.

2. The hydrogel-coated microspheres were resuspended using MES buffer (ph 6.5) and vortexed rapidly.

3. 10mg of EDC and 5mg of NHS were weighed, dissolved in 200. Mu.LMES buffer (ph 6.5), and rapidly added to the hydrogel microspheres.

4. Incubate at room temperature for 0.5h. Activating carboxyl groups

5. Hydrogel-coated microspheres were washed with ph 7.4 PBS buffer and repeated 2X.

6. 1ml PBS buffer (ph 7.4) was resuspended.

The cleaning of the hydrogel-coated microspheres needs to be faster and avoids losing efficacy.

7. For hydrogel-coated microspheres for analysis of B cells, 50 μl of CD20 antibody (Abcam) was added after step 6; for the analysis of the hydrogel-coated microspheres of T cells, 50 μl of CD2 antibody (Abcam) was added after step 6. Incubate for 4h at room temperature.

8. The hydrogel-coated microspheres were washed with PBS buffer at pH 7.4 and repeated 5 times. Unreacted CD20 or CD2 antibodies (Abcam) were removed. The antibody coupled to the microsphere is a targeting ligand.

9. Re-suspending 200. Mu.LPBS buffer (pH 7.4) and storing at 4 DEG C

Example 3 formation of conjugates

Two hydrogels carrying targeted T cell and B cell ligands generated in example 2.2 were coated with DNA encoding microspheres 1:1, mixing and spinning at 5rpm for 15 minutes at room temperature

Removing the supernatant

Resuspension of the microspheres in 500. Mu.L PBS buffer

Count cells 8.0 x 10≡5 cells incubated with microspheres in 200. Mu.L of PBS buffer containing 2mM EDTA, and the solution was spun for 30 min

Remove cells from all supernatant counts

B cells are combined with hydrogel coated microspheres targeting the B cells, and primers specific to PolyT and BCR on the microspheres can perform comprehensive and deep analysis on mRNA of the B cells; t cells are combined with hydrogel coated microspheres of targeted T cells, and PolyT and TCR specific primers on the microspheres can carry out comprehensive and deep analysis on mRNA of the T cells.

Example 4 Compartment formation

The microspheres were encapsulated with a vehicle (fluorooily medium) by a droplet microfluidic device.

All chemicals were ordered from Sigma-Aldrich, fisher Scientific and Roche. Using 1.2mM dNTP, polyT primer, 1U/. Mu.L RNaseOUT, 5mM DDT, 9mM MgCl ₂ 0.25U/ml reverse transcriptase, 1 XDNA polymerase premix, 1% (v/v) Tween-20, 1mg/ml bovine serum albumin, 2% (v/v) PEG-6000.

Drop generation (fig. 8A, 8B): the droplets were micro-droplets using a nozzle with a height of 100 μm and a width of 100 μmFluidic chips (sources; microfluidic chips were prepared autonomously by the laboratory by means of a general photolithography and PDMS nanoimprint procedure). Typical flow rates used are: cell (phase I) -12. Mu.L/h, transcriptome amplification mix (phase II) -288. Mu.L/h, and drop-generating oil (5% PEG-PFPE) ₂ HFE7500)-600μL/h。

EXAMPLE 5 reverse transcription and amplification of mRNA

The droplets resulting from the emulsification were collected in 200ul tubes and subjected to a PCR procedure, the specific PCR procedure being shown in the following table:

table 15 single cell cDNA amplification temperature control procedure; amplifying the labeled cDNA molecules

20% (v/v) Perfluorooctanol (PFO) in 1ml HFE oil was added to a 15ml collection tube as a chemical demulsifier

After mixing, 15ml collection tubes 2000x g were spun for 2 minutes. The PFO/HFE supernatant was removed by pipetting. Repeat 1x.

Example 6 disruption of amplified products and pooling

The amplified product was disrupted using a DNA disruption kit (One-step DNA Lib Prep Kit for IlluminaV2 (50 ng Input DNA), RK 20239). Library amplification was performed after DNA disruption using Tn5 transposase, the library PCR amplification procedure is shown in the following table, and the amplified library fragments were verified by 1% agarose gel electrophoresis after library amplification was completed, and a typical library amplification product is shown in the figure (fig. 9).

Step (a)	Temperature (temperature)	Time	Number of cycles
				1	72℃	3min	1X
2	98℃	30s	1X
				3	98℃	15s	7X
	60℃	30s
					72℃	1min
4	72℃	5min	1X
				5	15℃	-	1X

Constructing a single-cell cDNA Illumina library; the cDNA library after cleavage was amplified and a sequencing adapter was added.

EXAMPLE 7 analytical test results (hydrogel-coated microspheres from example 2.2)

Targeted mRNA capture on microspheres: t cells analyze mainly TCR sequences and other immune cells analyze whole transcriptomes; the sequencing result can obtain the information of the targeting ligand and the detailed information of BCR and TCR and the independent information of single cell transcriptome of other cells. After clustering treatment by targeting ligand and single cell DNA coding, high resolution information (T cell analysis result contains TCR information) of immune cells is obtained.

Single cell transcriptome data for the immune system is shown in figure 10A.

Clustering of T cells and Clonality (Clonality) of the sequencing data by targeting the second tag of the ligand [ fig. 10B ], D50 data [ fig. 10C ]; by targeting the second tag of the ligand, the sequencing data can be clustered for B cells and BCR Clonality (Clonality) [ fig. 10D ], unique CDR3 distribution data of BCR [ fig. 10E ]

Summary of experimental procedures for individual digital PCR analysis of human murine mixed cells for the second section of the examples (examples 8-11):

the first step: hydrogel-coated microspheres targeting specific human or murine cells are prepared, drop-digital PCR (ddPCR) primers (reagents) and sequence-specific probes (tag molecules) for analysis of human, murine cells are introduced into the hydrogel monomers, and antibodies (targeting ligands) are modified to aid in specific binding of human or murine cells.

And a second step of: human and mouse cells are mixed, incubated and combined with a plurality of microspheres with targeting antibodies and single-cell droplet ddPCR reagents, and then high-throughput encapsulation is performed through droplet microfluidic.

Fourth step: each microsphere within the droplet specifically amplifies a specific DNA region in the targeted cell.

Fifth step: the overall information of the cell mixture was obtained by in-drop amplified fluorescence imaging.

Example 8. Hydrogel coated microspheres 1 carrying PCR amplification reagents were prepared separately, and a Polyacrylamide (PAA) solution consisting of 6.2% acrylamide, 0.18% N, N '-bis (acrylamide) cystamine, 0.3% ammonium persulfate, 0.6% sodium acrylate, and a primer carrying a double bond of 10. Mu.M (carrying 5' -Acrydite) (reagent) was prepared.

For HEK293T analysis, the following primer sequences were used, respectively.

Table 17 PCR primers targeting human GAPDH sequences for 3T3 cells

Table 18 PCR primers targeting murine GAPDH sequences

2. This solution was separately mixed with microspheres (TOSOH HW 65) and added to Hydrofluoroether (HFE) oil to prepare an insoluble continuous phase of 5% (w/w) fluorosurfactant and 1% N, N-tetramethyl ethylenediamine (TEMED) for droplet generation and stabilization.

8. 3000x g for 3 minutes. The supernatant was removed by pipetting. Repeat 3x.

9. Resuspended in 1ml TEBST. This solution can be stored indefinitely at 4℃ (FIG. 1C).

Example 9 activation and crosslinking of hydrogel-coated microspheres targeting antibodies

4. Incubate at room temperature for 0.5h. Activating carboxyl groups

5. Hydrogel-coated microspheres were washed with ph7.4 PBS buffer and repeated 2X.

6. 1ml PBS buffer (pH 7.4) was resuspended.

7. For hydrogel-coated microspheres to analyze human cells, 50 μl of human-CD 298, human β2 microglobulin antibody (Abcam) (targeting ligand), and conjugated with a probe (tag molecule) targeting human GAPDH were added after step 6; for analysis of hydrogel-coated microspheres of murine cells, 50 μl of murine CD45 and murine MHC class I antibody (Abcam) (targeting ligand) were added after step 6 and a probe (tag molecule) targeting murine GAPDH was conjugated. Incubate for 4h at room temperature.

Table 19 Probe (tag molecule) for distinguishing human and mouse cells

8. The hydrogel-coated microspheres were washed with PBS buffer at pH 7.4 and repeated 5 times. Unreacted antibodies were removed.

9. Re-suspending 200. Mu.LPBS buffer (pH 7.4) and storing at 4 DEG C

Example 10 formation of conjugate

Two hydrogels carrying the targeted 293T cell primers and 3T3 cell ligand primers generated in example 9 were packaged microsphere 1:1, mixing and spinning at 5rpm for 15 minutes at room temperature

Removing the supernatant

Resuspension of the microspheres in 500. Mu.L PBS buffer

Remove cells from all supernatant counts

Human 293T cells are combined with hydrogel coated microspheres targeting the 293T cells, and GAPDH_human specific primers on the microspheres can be used for amplification analysis of Human GAPDH genes; murine 3T3 cells were bound to hydrogel-coated microspheres targeting 3T3 cells, on which gapdh_mouse specific primers were used to perform an amplification assay on mRNA of 3T3 cells (fig. 11).

EXAMPLE 11 Compartment formation

The microspheres were encapsulated by shaking vehicle (fluorooily medium). All chemicals were derived from Sigma-

Aldrich, fisher Scientific and Roche subscriptions.

Droplet generation: droplets were generated using a microfluidic chip using a 100 μm height and 100 μm wide nozzle. Typical flow rates used are: cell (phase I) -12. Mu.L/h, transcriptome amplification mix (phase II) -288. Mu.L/h, and drop-generating oil (5% PEG-PFPE) ₂ HFE7500)-600μL/h。

Adding 2.4mM dNTP, 2U/. Mu.L RNaseOUT, 20mM MgCl into the water phase ₂ 0.5U/ml reverse transcriptase, 2 XDNA polymerase premix, 2% (v/v) Tween-20, 2mg/ml bovine serum albumin, 4% (v/v) PEG-6000, 60mM DTT in water, 400nM human and murine probes.

Emulsification was performed on a tabletop shaker (fig. 12).

The resulting droplets were collected with an EP tube and placed on a PCR instrument, and the following temperature control procedure was selected:

step (a)	Temperature (temperature)	Time	Number of cycles
				1	72℃	3min	1X
2	98℃	30s	1X
				3	98℃	15s	35X
	60℃	30s
					72℃	1min
4	72℃	5min	1X
				5	4℃	-	Permanent set

Table 20 digital PCR amplification procedure; releasing the primer and probe on the hydrogel microsphere, and carrying out targeted amplification and detection on human and mouse cell genome

The PCR process was imaged through Sniper QX40, monitoring the FAM and HEX channels (fig. 13A), and examining mRNA expression heterogeneity of each cell in different droplets (fig. 13B, 13C).

Summary of the examples:

the technical scheme of the application meets the technical requirement of data resolution of single cell analysis (targeted multiplex detection).

In the first example, single cell multiplex sequencing analysis of immune cells can be achieved by a series of complex microspheres that synthesize a variety of targeting reactions. The method does not need to purify each subgroup of cells to be analyzed by cell enrichment or flow cytometry, and simplifies the operation steps. Meanwhile, the target reaction compound microsphere can identify a cell object during design, deliver a special reaction reagent of the cell into a micro-droplet where the cell is located, and carry out subsequent accurate data resolution through a label on the compound. Specific results are shown in FIGS. 10A-10E, where FIG. 10A shows the single cell sequencing and grouping results of peripheral blood mononuclear cells. The frequency of their TCR sequences was additionally analyzed for T cells in immune cell subpopulations (10C), visualized TCR clone distribution was obtained by TCR profiling (10B), visualized VJ sequence distribution was obtained by TCR two-dimensional profiling (10B). The frequency of BCR sequences was additionally analyzed for B cells (10D, 10E) in the immune cell subpopulation (fig. 10E), and BCR dendrograms resulted in a visualized BCR clone distribution (10D).

In a second embodiment, single cell multiplex fluorescence detection of mixed cells of human and mouse can be achieved by a series of composite microspheres that synthesize a variety of targeting reactions. As shown in fig. 13A-13C, high-throughput abundance characterization of target GAPDH mRNA in each cell can be achieved by single-cell qPCR in droplets for different targets on different cancer cells, and for different targets on human and murine cells, resulting in heterogeneous population information.

Although embodiments of the present application have been described above with reference to the accompanying drawings, the present application is not limited to the specific embodiments and fields of application described above, which are merely illustrative, instructive, and not restrictive. Those skilled in the art, having the benefit of this disclosure, may make numerous forms, and equivalents thereof, without departing from the scope of the invention as defined by the claims.

Claims

a carrier;

a targeting ligand for the analyte attached to the carrier;

a reagent for detecting an analyte attached to a carrier; and

A tag molecule linked to a carrier and corresponding to the analyte.

2. The targeted reaction complex of 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 complexes formed therefrom, most preferably the analyte is a cell.

3. The targeted reaction complex of claim 1, wherein the analysis is selected from one or more of spectroscopic detection, sequencing, mass spectrometry detection, picture capture, electrical signal detection.

4. The targeting reaction complex according to claim 1, wherein the carrier is composed of a polymer or a small molecule, preferably a polymer carrier, further preferably a polystyrene carrier, further preferably the carrier has a diameter of 1 μm-100 μm, further preferably the carrier shape is selected from one or more of a square, a tetrahedron, a sphere, an ellipsoid, an octopus, a bowl, a red blood sphere; most preferably bowl-shaped and/or red blood bulb-shaped; further preferably the carrier has a diameter particle size distribution coefficient CV of less than 20%, further preferably the carrier surface is coated with a mechanical buffer coating; most preferably coated with a hydrogel coating.

5. The targeting response complex according to claim 1, wherein the targeting ligand may be natural or artificial, 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, leptin, 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 and their co-formed multiple ligands, complex ligands, coupling ligands.

6. The targeting reaction complex according to claim 1, wherein said 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.

7. The targeting reaction complex according to claim 1, wherein the reaction reagent is an oligonucleotide primer, an enzyme or a small molecule.

8. The targeting reaction complex according to claim 1, wherein said linkage is selected from the group consisting of covalent, metallic, ionic _、 Van der Waals forces, including hydrogen bonding, mechanical bonding, halogen bonding, chalcogenide bonding, gold philic bonding, intercalation, overlap, cation-pi bonding, anion-pi bonding, salt bridging, secondary bonds between nonmetallic atoms, secondary bonds between metallic atoms and nonmetallic atoms, gold philic bonding, silver philic bonding, double hydrogen bonding, and secondary bonds of gold bonds.

9. The targeting reaction complex according to claim 1, wherein the targeting ligand is linked to the carrier by a linkage of the reactive agent, by a linkage of the tag molecule or by a linker; the attachment of the reactive agent to the carrier is through the attachment of the targeting ligand, through the attachment of the tag molecule or through a linker; the attachment of the tag molecule to the carrier is via attachment of the targeting ligand, via attachment of the reactive agent or via attachment of a linker.

10. The targeting reaction complex according to claim 4, wherein the small molecules constituting the carrier are one or more of the targeting ligand, the reaction reagent, and the tag molecule.