A labeling method for characterizing cell-cell contacts and uses thereof

Abstract

The application discloses a chemical labeling system HIM-IP for detecting cell-cell interaction. The system is constructed by modifying tyrosinase and phenol on the surfaces of two cells to be detected respectively, constructing a trapper cell and a prey cell, and then using biotin or single-stranded DNA with a nucleophilic group as a molecular tag for labeling. The system can not only be detected by traditional flow cytometry, but also be compatible with commercial single-cell transcriptome sequencing platforms, realizing the correlation of cell-cell interaction information and transcriptome information at the single-cell level. The system does not need complex genetic engineering modification of cells, the whole modification and labeling process is simple and efficient, and the detection sensitivity of cell-cell interaction is high. By integrating single-cell sequencing technology, the biological mechanism of cell-cell interaction regulation can be further excavated.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and more specifically to a labeling method for characterizing intercellular contact and its application. Background Technology

[0002] In recent years, an increasing number of studies have discovered that cell-cell contact interactions play a crucial biological role in the regulation of many life activities. For example, physical contact between neurons is the basis of electrochemical signal transduction in the central nervous system; the entire process of T cells crossing the endothelial barrier from the blood vessel wall, migrating to lymphoid tissues, and ultimately being sensitized by antigen-presenting cells is inseparable from cell-cell contact-mediated signal regulation; the formation of tissue or physiological barrier structures also depends on physical contact between neighboring cells and the signal transduction triggered by this interaction. However, the regulatory mechanisms of cell-cell interactions are extremely complex, and the biological significance behind many cell contact patterns remains unknown.

[0003] Against this research backdrop, various biotechnologies for tracing or detecting cell-cell interactions have emerged. Due to the complexity and dynamic nature of cell-cell interactions, developing characterization methods for these biological events is extremely challenging. Currently developed detection technologies mainly include chemical labeling and single-cell sequencing. Chemical labeling typically utilizes enzymatic or photocatalytic reactions to label cell-cell interactions, offering advantages such as high sensitivity and specificity. For example, Peng Wu et al. reported a fucosyltransferase-based chemical labeling strategy, FucoID, for detecting contact between T cells and DC cells (Liu, Z.; Li, JP; Chen, M.; Wu, M.; Shi, Y.; Li, W.; Teijaro, JR; Wu, P. Detecting tumor antigen-specific T cells via interaction-dependent fucosyl-biotinylation. Cell, 2020, 183, 1117.); MacMillan et al. developed a dexter energy transfer-based photocatalytic labeling technology, μMap, and achieved high-resolution labeling of intercellular interaction proteins (Geri, JB; Oakley, JV; Reyes-Robles, T.; Wang, T.; McCarver, SJ; White, CH; Rodriguez-Rivera, FP; Parker Jr., DL; Hett, EC; Fadeyi, OO; Oslund, RC; MacMillan, DWC Microenvironment mapping via Dexter energy transfer on immune cells. Science 2020, 367, 1091. However, chemical labeling methods can usually only analyze cell-cell interactions at the flow cytometry level, and cannot characterize cell-cell interactions at the single-cell scale and gene level.

[0004] In contrast, single-cell sequencing, based on current single-cell transcriptomics platforms, allows for high-throughput, multi-parameter analysis of specific subpopulations of interacting cells, revealing the regulatory mechanisms behind cell-cell interactions at the gene level. However, current single-cell sequencing methods cannot distinguish the level of cell-cell interactions, typically only analyzing samples of cells that have already adhered to each other, ignoring the biological information of weak cell-cell interactions, and are less sensitive than chemical labeling methods. Therefore, how to retain the high specificity and sensitivity of chemical labeling methods while incorporating the advantages of single-cell sequencing technology has significant scientific and applied value for further understanding of cell-cell interactions. However, relevant characterization techniques have not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to develop a labeling method (hereinafter referred to as HIM-IP) for detecting intercellular interactions. This labeling technology can integrate a single-cell transcriptome sequencing module while being compatible with traditional flow cytometry detection methods. Compared with existing chemical labeling strategies, HIM-IP can achieve high-throughput, parameterized characterization of intercellular interactions at the single-cell and gene levels; and compared with reported single-cell sequencing technologies, HIM-IP can distinguish between intercellular interactions of different intensities, exhibiting higher detection sensitivity and specificity.

[0006] First aspect

[0007] This invention provides a system for detecting intercellular contact or interaction, the system comprising:

[0008] 1) A catcher cell, wherein the surface of the catcher cell is modified with tyrosinase;

[0009] 2) Prey cells, the surface of which is modified with phenolic groups;

[0010] 3) Nucleophilic molecular tags.

[0011] According to an embodiment of the present invention, 1) in the catcher cell, tyrosinase is coupled to the surface of the catcher cell;

[0012] Preferably, tyrosinase is attached to the surface of catcher cells via a click chemistry reaction (e.g., a copper-free click chemistry reaction);

[0013] More preferably, the tyrosinase is attached to the surface of the catcher cell via a click chemical reaction between the azide group and the alkyne ring.

[0014] According to embodiments of the present invention, the alkyne ring is a 3-9 membered alkyne ring, preferably a 7-9 membered alkyne ring, such as cyclooctyne.

[0015] According to an embodiment of the present invention, 1) the method for preparing catcher cells is as follows:

[0016] i) Modify the surface of catcher cells with reactive groups (e.g., azide groups);

[0017] ii) Prepare tyrosinases modified with reactive groups (e.g., alkyne rings);

[0018] iii) Co-incubate the tyrosinase modified with the reactive group in step i) with the catcher cells modified with the reactive group in step ii) to obtain catcher cells with tyrosinase modified on the surface.

[0019] According to an embodiment of the present invention, the reactive groups on the surface of the catcher cell can undergo a coupling reaction with the reactive groups modified on the tyrosinase.

[0020] According to an embodiment of the present invention, ii) the alkyne-modified tyrosinase is a cyclooctyne-modified tyrosinase, preferably a dibenzocyclooctyne-modified tyrosinase, more preferably a dibenzocyclooctyne-polyethylene glycol (DBCO-PEG)-modified tyrosinase.

[0021] According to an embodiment of the present invention, iii) the concentration of the alkyne-modified tyrosinase is 50 μg / mL to 1000 μg / mL, for example 200 μg / mL.

[0022] According to an embodiment of the present invention, i) the modification of the catcher cell surface with azide groups is performed using any of the following methods:

[0023] a) Catcher cells are cultured in a culture medium to obtain azidated surface-modified catcher cells; the culture medium contains at least one component selected from tetraacylated N-azidoacetylgalactosamine (Ac4GalNAz), tetraacylated N-azidoacetylmannosamine (Ac4ManNAz), and azidosialic acid; preferably, the culture time is 24-72 hours.

[0024] b) Reacting azido-PEG-NHS ester with catcher cells to obtain catcher cells with surface azido-modified structure; reaction time, for example, 30 minutes;

[0025] Preferably, the reaction is carried out in a buffer solution, such as PBS buffer;

[0026] c) Reaction of p-azidoethylphenol and tyrosinase with catcher cells to obtain catcher cells with surface azidation modification; reaction time, for example, 10 minutes;

[0027] Preferably, the reaction is carried out in a buffer solution, such as PBS buffer.

[0028] According to an embodiment of the present invention, in method c), the concentration of azidoethylphenol is 1 μM-20 μM, for example 10 μM.

[0029] According to an embodiment of the present invention, in method c), the concentration of tyrosinase is 5 μg / mL to 80 μg / mL, for example 20 μg / mL.

[0030] Since both cells and tyrosine kinases have commonly used reaction sites, such as amino and thiol groups, directly linking cells and tyrosine kinases through these common reaction sites using bifunctional reagents would lead to cross-linking reactions between cells and between tyrosine kinases, significantly reducing the controllability of the reaction. The catcher cell preparation method used in this invention utilizes the coupling reaction between the reactive groups on the catcher cell surface and the reactive groups modified on the tyrosinase (e.g., the coupling reaction between the alkyne ring and azide), thereby modifying the catcher cell surface with tyrosine kinase.

[0031] According to an embodiment of the present invention, 2) the method for constructing prey cells is as follows:

[0032] Reaction of prey cells with reagents containing phenol groups yields prey cells with phenol group-modified surfaces.

[0033] Preferably, the reagent containing a phenolic group is selected from: sodium 1-((3-(4-hydroxyphenyl)propionyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate (SHPP), 3,4-dihydroxyphenylpropionic acid-N-succinimide ester, hydroxyphenylethyl acid-N-succinimide ester and 3,4-dihydroxyphenylethyl acid-N-succinimide ester;

[0034] Preferably, the reaction time is, for example, 30 minutes; the reaction temperature is, for example, room temperature;

[0035] Preferably, the reaction is carried out in a buffer solution, such as PBS buffer.

[0036] According to an embodiment of the present invention, the nucleophilic molecular tag comprises a nucleophilic group and a tag.

[0037] According to an embodiment of the present invention, 3) the nucleophilic group in the nucleophilic molecular tag is selected from amino, thiol and aniline.

[0038] According to an embodiment of the present invention, 3) the tag in the nucleophilic molecular tag is selected from fluorescent molecules, biotin or its derivatives, oligonucleotides (e.g., single-stranded DNA), etc.

[0039] According to an embodiment of the present invention, 3) the nucleophilic molecular tag is selected from any of the following:

[0040]

[0041] According to an embodiment of the present invention, 3) the nucleophilic molecular tag is selected from single-stranded DNA molecules with an amino, thiol or aniline group modified at the 5' end.

[0042] According to embodiments of the present invention, the base sequence of single-stranded DNA has the following general structure:

[0043] 5'-(N)x+capture sequence-3';

[0044] Where N represents any one of the four bases: A, T, C, and G;

[0045] The value of x is any natural number between 10 and 100.

[0046] According to an embodiment of the present invention, the capture sequence is selected from any of the following:

[0047] Sequence 1: 5'-AAAAAAAAAAAAAAA(A)y-3'; where y is any natural number between 0 and 15.

[0048] Sequence 2: 5'-GCTCACCTATTAGCGGCTAAGG-3';

[0049] Sequence 3: 5'-GCTTTAAGGCCGGTCCTAGCAA-3'.

[0050] The base sequence of single-stranded DNA has the above-mentioned universal structure, which can meet the compatibility requirements of current commercial single-cell transcriptome sequencing platforms.

[0051] Depending on the application scenario, detection target, or detection purpose, the catcher cell and prey cell are selected from any pair of cells that need to be detected to interact, such as animal cells.

[0052] According to an embodiment of the present invention, both the catcher cells and the prey cells are Jurkat cells.

[0053] According to an embodiment of the present invention, the catcher cell is a CAR-T cell, and the prey cell is a tumor cell.

[0054] According to an embodiment of the present invention, the catcher cells are mouse bone marrow-derived dendritic cells (BMDCs), and the prey cells are CD8 cells. + T cells.

[0055] Second aspect

[0056] The present invention also provides a method for detecting intercellular contact or interaction, the method comprising:

[0057] 1) Constructing catcher cells: Modifying the surface of catcher cells with tyrosinase;

[0058] 2) Constructing prey cells: Modifying the surface of prey cells with phenolic groups;

[0059] 3) The catcher cells, prey cells, and nucleophilic molecular tags are mixed and reacted, and the reaction is then detected. According to an embodiment of the present invention, 1) in the catcher cells, tyrosinase is coupled to the surface of the catcher cells;

[0060] Preferably, tyrosinase is attached to the surface of catcher cells via a click chemistry reaction (e.g., a copper-free click chemistry reaction);

[0061] More preferably, the tyrosinase is attached to the surface of the catcher cell via a click chemical reaction between the azide group and the alkyne ring.

[0062] According to embodiments of the present invention, the alkyne ring is a 3-9 membered alkyne ring, preferably a 7-9 membered alkyne ring, such as cyclooctyne.

[0063] According to an embodiment of the present invention, 1) the method for constructing catcher cells is as follows:

[0064] i) Modify the surface of catcher cells with reactive groups (e.g., azide groups);

[0065] ii) Prepare tyrosinases modified with reactive groups (e.g., alkyne rings);

[0066] iii) The tyrosinase modified with the reactive group in step i) is co-incubated with the catcher cells modified with the reactive group in step ii) to obtain catcher cells with tyrosinase modified on the surface.

[0067] According to an embodiment of the present invention, the reactive groups on the surface of the catcher cell can undergo a coupling reaction with the reactive groups modified on the tyrosinase.

[0068] According to an embodiment of the present invention, ii) the alkyne-modified tyrosinase is a cyclooctyne-modified tyrosinase, preferably a dibenzocyclooctyne-modified tyrosinase, more preferably a dibenzocyclooctyne-polyethylene glycol (DBCO-PEG)-modified tyrosinase.

[0069] According to an embodiment of the present invention, iii) the concentration of the alkyne-modified tyrosinase is 50 μg / mL to 1000 μg / mL, for example 200 μg / mL.

[0070] According to an embodiment of the present invention, i) the modification of the catcher cell surface with azide groups is performed using any of the following methods:

[0071] a) Catcher cells are cultured in a culture medium to obtain azidated surface-modified catcher cells; the culture medium contains at least one component selected from tetraacylated N-azidoacetylgalactosamine (Ac4GalNAz), tetraacylated N-azidoacetylmannosamine (Ac4ManNAz), and azidosialic acid; preferably, the culture time is 24-72 hours.

[0072] b) Reacting azido-PEG-NHS ester with catcher cells to obtain catcher cells with surface azido-modified structure; reaction time, for example, 30 minutes;

[0073] Preferably, the reaction is carried out in a buffer solution, such as PBS buffer;

[0074] c) Reaction of p-azidoethylphenol and tyrosinase with catcher cells to obtain catcher cells with surface azidation modification; reaction time, for example, 10 minutes;

[0075] Preferably, the reaction is carried out in a buffer solution, such as PBS buffer.

[0076] According to an embodiment of the present invention, in method c), the concentration of azidoethylphenol is 1 μM-20 μM, for example 10 μM.

[0077] According to an embodiment of the present invention, in method c), the concentration of tyrosinase is 5 μg / mL to 80 μg / mL, for example 20 μg / mL.

[0078] According to an embodiment of the present invention, 2) the method for constructing prey cells is as follows:

[0079] Reaction of prey cells with reagents containing phenol groups yields prey cells with phenol group-modified surfaces.

[0080] Preferably, the reagent containing a phenolic group is selected from: sodium 1-((3-(4-hydroxyphenyl)propionyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate (SHPP), 3,4-dihydroxyphenylpropionic acid-N-succinimide ester, hydroxyphenylethyl acid-N-succinimide ester and 3,4-dihydroxyphenylethyl acid-N-succinimide ester;

[0081] Preferably, the reaction time is, for example, 30 minutes; the reaction temperature is, for example, room temperature;

[0082] Preferably, the reaction is carried out in a buffer solution, such as PBS buffer. According to an embodiment of the invention, the nucleophilic molecular tag comprises a nucleophilic group and a tag.

[0083] According to an embodiment of the present invention, the nucleophilic group in the nucleophilic molecular tag is selected from amino, thiol and aniline.

[0084] According to embodiments of the present invention, the tag in the nucleophilic molecular tag is selected from fluorescent molecules, biotin or its derivatives, oligonucleotides (e.g., single-stranded DNA), etc.

[0085] According to an embodiment of the present invention, the nucleophilic molecular tag is selected from any of the following:

[0086]

[0087] According to an embodiment of the present invention, the nucleophilic molecular tag is selected from single-stranded DNA molecules with an amino, thiol, or aniline group modified at the 5' end.

[0088] According to embodiments of the present invention, the base sequence of single-stranded DNA has the following general structure:

[0089] 5'-(N)x+capture sequence-3';

[0090] Where N represents any one of the four bases: A, T, C, and G;

[0091] The value of x is any natural number between 10 and 100.

[0092] According to an embodiment of the present invention, the capture sequence is selected from any of the following:

[0093] Sequence 1: 5'-AAAAAAAAAAAAAAA(A)y-3'; where y is any natural number between 0 and 15.

[0094] Sequence 2: 5'-GCTCACCTATTAGCGGCTAAGG-3';

[0095] Sequence 3: 5'-GCTTTAAGGCCGGTCCTAGCAA-3'.

[0096] The base sequence of single-stranded DNA has the above-mentioned universal structure, which can meet the compatibility requirements of current commercial single-cell transcriptome sequencing platforms.

[0097] According to an embodiment of the present invention, in step 3), the reaction condition is incubation at 37°C for 30 minutes.

[0098] According to an embodiment of the present invention, in 3), the concentration of the nucleophilic molecular tag is 10 μM-200 μM, for example 100 μM.

[0099] According to an embodiment of the present invention, in step 3), the reaction is detected by flow cytometry or by single-cell transcriptome sequencing.

[0100] Depending on the application scenario, detection target, or detection purpose, the catcher cell and prey cell can be derived from any pair of cells whose interaction needs to be detected, such as animal cells.

[0101] According to an embodiment of the present invention, both the catcher cells and the prey cells are Jurkat cells.

[0102] According to an embodiment of the present invention, the catcher cell is a CAR-T cell, and the prey cell is a tumor cell.

[0103] According to an embodiment of the present invention, the catcher cells are mouse bone marrow-derived dendritic cells (BMDCs), and the prey cells are CD8 cells. + T cells.

[0104] Third aspect

[0105] The present invention also provides a kit for detecting intercellular contact or interaction, the kit comprising:

[0106] 1) Reagents for constructing catcher cells;

[0107] 2) Reagents for constructing prey cells;

[0108] 3) Labeling reagents;

[0109] Among them, the reagent for constructing catcher cells is a reagent that introduces tyrosinase onto the surface of catcher cells;

[0110] The reagent used to construct prey cells is one that introduces phenolic groups onto the surface of the prey cells;

[0111] The labeling reagent is a reagent containing a nucleophilic group and a tag.

[0112] According to an embodiment of the present invention, a nucleophilic group captures an ortho-quinone produced on the surface of a predator cell by tyrosinase-catalyzed phenolic groups, thereby completing the marking of the prey cell.

[0113] According to an embodiment of the present invention, 1) the reagents for constructing catcher cells include reagents for introducing reactive groups onto the surface of catcher cells and reactive group-modified tyrosinases.

[0114] According to an embodiment of the present invention, the reactive groups on the surface of the catcher cell can undergo a coupling reaction with the reactive groups modified on the tyrosinase.

[0115] According to an embodiment of the present invention, the reactive group on the surface of the catcher cell is an azide group.

[0116] According to an embodiment of the present invention, the reactive group modified on the tyrosinase is an alkynyl group, such as an alkynyl ring group.

[0117] According to an embodiment of the present invention, the reagent for introducing reactive groups onto the surface of catcher cells is selected from reagents containing azido groups, such as any one or more of tetraacylated N-azidoacetylgalactosamine (Ac4GalNAz), tetraacylated N-azidoacetylmannosamine (Ac4ManNAz), azidosialic acid, azido-PEG-NHS ester, and p-azidoethylphenol.

[0118] According to an embodiment of the present invention, the reactive group-modified tyrosinase is an alkyne-modified tyrosinase, preferably a dibenzocyclooctyne-modified tyrosinase, such as a dibenzocyclooctyne-polyethylene glycol (DBCO-PEG)-modified tyrosinase.

[0119] According to embodiments of the present invention, the cycloyne is a 3-9 membered yne ring, preferably a 7-9 membered yne ring, such as cyclooctyne.

[0120] According to an embodiment of the present invention, 2) the reagent for constructing prey cells is selected from sodium 1-((3-(4-hydroxyphenyl)propionyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate (SHPP), 3,4-dihydroxyphenylpropionic acid-N-succinimidyl ester, hydroxyphenylethyl acid-N-succinimidyl ester and 3,4-dihydroxyphenylethyl acid-N-succinimidyl ester.

[0121] According to an embodiment of the present invention, 3) the nucleophilic group in the labeling reagent is selected from amino, mercapto and aniline.

[0122] According to an embodiment of the present invention, 3) the label in the labeling reagent is selected from fluorescent molecules, biotin or its derivatives, oligonucleotides (e.g., single-stranded DNA), etc.

[0123] According to an embodiment of the present invention, 3) the labeling reagent is selected from any of the following:

[0124]

[0125] According to an embodiment of the present invention, 3) the labeling reagent is selected from single-stranded DNA molecules with an amino, thiol or aniline group modified at the 5' end.

[0126] According to embodiments of the present invention, the base sequence of single-stranded DNA has the following general structure:

[0127] 5'-(N)x+capture sequence-3';

[0128] Where N represents any one of the four bases: A, T, C, and G;

[0129] The value of x is any natural number between 10 and 100.

[0130] According to an embodiment of the present invention, the capture sequence is selected from any of the following:

[0131] Sequence 1: 5'-AAAAAAAAAAAAAAA(A)y-3'; where y is any natural number between 0 and 15.

[0132] Sequence 2: 5'-GCTCACCTATTAGCGGCTAAGG-3';

[0133] Sequence 3: 5'-GCTTTAAGGCCGGTCCTAGCAA-3'.

[0134] The kit may optionally further include:

[0135] 5) Buffer solutions, such as PBS buffer;

[0136] 6) Tyrosine kinase.

[0137] The present invention also provides the application of the above-described kit in detecting intercellular contact or interaction.

[0138] Beneficial effects

[0139] The basic principle of the intercellular interaction labeling involved in this invention is as follows: phenolic groups can be oxidized by dissolved oxygen molecules in the environment to highly reactive benzoquinone groups under the catalysis of tyrosinase. These benzoquinone groups can then be covalently coupled to nucleophilic groups (such as amino, thiol, or aniline). In the HIM-IP system designed in this invention, tyrosinase and phenolic groups are modified on the surfaces of the catcher cell and prey cell, respectively. Only when the two cells come into close contact can the tyrosinase recognize and catalyze the oxidation of the phenolic groups, thereby coupling the nucleophilic tag to the reaction site. Therefore, only prey cells that come into contact with the catcher cell will be chemically labeled.

[0140] The HIM-IP designed in this invention has the following advantages:

[0141] 1. The cell engineering modifications involved in this invention all employ chemical modification methods, eliminating the need for genetic modification of cells. The entire modification and labeling process takes only about 2 hours, offering rapid and convenient technical advantages. It is highly suitable for primary cells or cell lines that are difficult to genetically engineer, demonstrating strong versatility.

[0142] 2. All chemical reagents involved in the HIM-IP technology solution (including tyrosinase, SHPP, biotin tags, and DNA tags, etc.) can be obtained through commercial channels, and there is basically no need to synthesize them in-house, making it easy to promote and apply the technology.

[0143] 3. The greatest advantage of the technical solution involved in this invention lies in the introduction of single-stranded DNA as a molecular tag for intercellular interactions, enabling the correlation between information on intercellular interactions and transcriptomics information at the single-cell level. This technical feature overcomes the limitation of current chemically labeled intercellular interaction detection, which can only be analyzed at the cell population level, and effectively integrates the powerful advantages of single-cell sequencing technology in revealing the mechanisms of intercellular interactions. Attached Figure Description

[0144] Figure 1 This is a schematic diagram of the HIM-IP system.

[0145] Figure 2 This is an imaging result of the azide-treated Jurkat cells in Example 1 after staining with DBCO-Cy5 and Hoechst33342.

[0146] Figure 3 This is a confocal imaging result of Jurkat cells with tyrosinase modified on their surface, as shown in Example 1.

[0147] Figure 4 The cell surface azidation modification level in Example 2 is a concentration-dependent expression of azidoethylphenol.

[0148] Figure 5 The cell surface azide modification level is dependent on the tyrosinase concentration in Example 3.

[0149] Figure 6 The modification level of tyrosinase in Jurkat cells treated with different concentrations of Ac4GaINAz in Example 4 is shown.

[0150] Figure 7 To determine whether tyrosinase and SHPP modification in the HIM-IP system of Example 5 affect the biotin labeling efficiency.

[0151] Figure 8 This is a correlation diagram showing the relationship between the proportion of catcher cells and the proportion of biotin-labeled prey cells in the Jurkat cell interaction system in Example 5.

[0152] Figure 9 This is a correlation diagram showing the relationship between the proportion of catcher cells and the proportion of biotin-labeled prey cells in the CAR-T cell-Ramos or K562 cell interaction system in Example 6.

[0153] Figure 10 The flow cytometry results of HIM-IP labeling of T cells after co-incubation of BMDCs with different polypeptide presentations in Example 7 with T cells.

[0154] Figure 11 The results are from single-cell sequencing of CAR-T and Ramos cells after single-stranded DNA interaction labeling in Example 8. Detailed Implementation

[0155] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0156] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0157] HIM-IP:

[0158] Ac4GalNAz: Tetraacylated N-azidoacetylgalactosamine.

[0159] Ac4ManNAz: Tetraacylated N-azidoacetylmannosamine.

[0160] SHPP: Sodium 1-((3-(4-hydroxyphenyl)propionyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate.

[0161] Azide-PEG-NHS:

[0162] p-Azidoethylphenol: 4-(2-Azidoethyl)phenol, CAS: 74447-34-0.

[0163] Example 1

[0164] Constructing catcher cells using Jurkat cells as a model

[0165] (1) Collect 2 million Jurkat cells by centrifugation, wash twice with PBS, and resuspend in 490 μL PBS. Add 5 μL of 1 mM azidoethylphenol solution and 5 μL of 2 mg / mL tyrosinase solution to the cell suspension to make the final concentrations of 10 μM azidoethylphenol and 20 μg / mL tyrosinase, respectively. After gentle vortexing, incubate at room temperature for 10 minutes.

[0166] (2) After the reaction is complete, the cells are collected by centrifugation, washed three times with PBS and resuspended. This step allows a small number of cells to be taken for Hoechst 33342 and dibenzocyclooctylene-Cy5 (DBCO-Cy5) staining, and the azidation modification of the cell surface can be detected by fluorescence confocal imaging.

[0167] (3) Mix the cell suspension washed in the previous step with the DBCO-PEG modified tyrosinase (also labeled with FITC) solution to make the final concentration of tyrosinase in the system 200 μg / mL. After gently mixing the cells, incubate at room temperature with shaking for 30 minutes.

[0168] (4) After the reaction, the cells were collected by centrifugation and washed 2-3 times. After staining with Hoechst33342, the modification of tyrosinase on the cell surface was observed under a fluorescence confocal microscope.

[0169] Conclusion: From the appendix Figure 2 As can be seen, the surface of Jurkat cells modified with azide showed significant red fluorescence labeled with DBCO-Cy5, proving that the azide modification was successful. (From the attached...) Figure 3 It can be seen that FITC-labeled tyrosinase is distributed on the surface of Jurkat cells, proving that the tyrosinase modification on the Jurkat cell membrane was successful.

[0170] Example 2

[0171] Optimization of azide modification conditions in catcher cell construction:

[0172] The operating steps were the same as steps (1) and (2) in Example 1, except that the final concentration of p-azidoethylphenol in step (1) was set to 1 μM, 2 μM, 5 μM, 10 μM, and 20 μM. The fluorescence signal intensity of DBCO-Cy5 on the surface of Jurkat cells was counted by flow cytometry.

[0173] Conclusion: From the appendix Figure 4 It can be seen that increasing the concentration of azidoethylphenol modification can improve the degree of azidation modification on the surface of Jurkat cells.

[0174] Example 3

[0175] Optimization of azide modification conditions in catcher cell construction:

[0176] The operating steps were the same as steps (1) and (2) in Example 1, except that the final concentration of tyrosinase in step (1) was set to 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, and 80 μg / mL. The fluorescence signal intensity of DBCO-Cy5 on the surface of Jurkat cells was counted by flow cytometry.

[0177] Conclusion: From the appendix Figure 5 It can be seen that increasing the concentration of tyrosinase modification can enhance the degree of azide modification on the surface of Jurkat cells.

[0178] Example 4

[0179] Cell surface azide modification strategy based on Ac4GalNAz:

[0180] (1) Collect Jurkat cells by centrifugation, replace the cell culture medium with complete medium containing 0, 5 μM, 10 μM, 20 μM, 50 μM and 100 μM Ac4GalNAz, and continue culturing for 72 hours.

[0181] (2) After culture, Jurkat cells were collected and reacted with DBCO-PEG modified tyrosinase (FITC labeling), and then flow cytometry analysis was performed.

[0182] Conclusion: From the appendix Figure 6 It can be seen that the density of azide modification on the surface of Jurkat cells increases with increasing Ac4GalNAz concentration. These data demonstrate that Ac4GalNAz can be used for azide modification of the Jurkat cell surface and subsequent tyrosinase labeling.

[0183] Example 5

[0184] HIM-IP for detecting Jurkat cell interactions at the flow cytometry level:

[0185] Jurkat cells readily aggregate during culture, indicating strong contact-type interactions among these cells. The experimental procedure for HIM-IP to detect these interactions in Jurkat cells is as follows:

[0186] (1) Collect 10 million Jurkat cells by centrifugation, wash twice with PBS and resuspend, then divide into two equal parts, one part for constructing catcher cells and the other part for constructing prey cells.

[0187] (2) To construct catcher cells, 5 μL of 1 mM p-azidoethylphenol solution and 5 μL of tyrosinase solution (2 mg / mL) were added to the cell suspension, bringing the final concentrations of p-azidoethylphenol and tyrosinase to 10 μM and 20 μg / mL, respectively. After gentle vortexing, the reaction was carried out at room temperature for 10 minutes. After the reaction, the cells were washed three times by centrifugation, resuspended in 500 μL PBS, and then DBCO-PEG-modified tyrosinase was added to bring the final concentration of tyrosinase in the system to 200 μg / mL. After gentle mixing of the cells, the reaction was carried out at room temperature for 30 minutes. After the reaction, the cells were washed three times with PBS and set aside for use. The catcher cells in the control group were unmodified Jurkat cells.

[0188] (3) To construct prey cells, SHPP was added to the Jurkat cell suspension to a final concentration of 4 μM. After a brief vortex, the cell suspension was incubated at room temperature for 30 minutes with shaking. After the reaction, the cells were washed once by centrifugation, and then 500 μL of Hoechst 33342 staining solution (5 μg / mL) was added, and staining was performed at room temperature for 8 minutes. After staining, the cells were washed three times by centrifugation and set aside for use. The prey cells of the control group were Jurkat cells that were not modified with SHPP but stained with Hoechst 33342.

[0189] (4) Next, catcher cells and prey cells were mixed in different ratios, and aniline-biotin-labeled cells were added to a final concentration of 100 μM. After incubation at 37°C for 30 minutes, the cells were collected by centrifugation, washed three times with PBS, and then FITC-labeled streptavidin staining solution (final concentration 20 μg / mL) was added. After staining at room temperature for 10 minutes and washing three times with PBS, flow cytometry characterization was performed.

[0190] Conclusion: From the appendix Figure 7It can be seen that the lack of tyrosinase modification in catcher cells or the lack of SHPP modification in prey cells will prevent the HIM-IP system from properly detecting intercellular interactions. The efficiency of intercellular interaction labeling is highest only when both elements are present simultaneously. (From the appendix...) Figure 8 It can be seen that by changing the proportion of catcher cells in the system, the proportion of prey cells labeled with biotin also changes accordingly. This result proves that HIM-IP has sufficient sensitivity to detect cell subpopulations that are in intercellular contact in the system.

[0191] Example 6

[0192] HIM-IP was used to detect the interaction between CAR-T cells and tumor cells at the flow cytometry level. The specific procedure is as follows:

[0193] (1) Prepare CD19-targeting CAR-T cells, Ramos cells (CD19-positive), and K562 cells (CD19-negative). Collect the cells by centrifugation, wash twice with PBS, and resuspend. CAR-T cells are used to construct catcher cells, while Ramos and K562 cells are used to construct prey cells.

[0194] (2) To construct catcher cells, 5 μL of 1 mM p-azidoethylphenol solution and 5 μL of tyrosinase solution (2 mg / mL) were added to the CAR-T cell suspension, bringing the final concentrations of p-azidoethylphenol and tyrosinase to 10 μM and 20 μg / mL, respectively. After gentle vortexing, the reaction was carried out at room temperature for 10 minutes. After the reaction, the cells were washed three times by centrifugation, resuspended in 500 μL PBS, and then DBCO-PEG-modified tyrosinase was added to bring the final concentration of tyrosinase in the system to 200 μg / mL. After gentle mixing of the cells, the reaction was carried out at room temperature for 30 minutes. After the reaction, the cells were washed three times with PBS and set aside for use.

[0195] (3) To construct prey cells, SHPP was added to Ramos or K562 cell suspensions to a final concentration of 4 μM. After a brief vortex, the cell suspension was incubated at room temperature for 30 minutes with shaking. After the reaction, the cells were washed once by centrifugation, and then 500 μL of Hoechst 33342 staining solution (5 μg / mL) was added, and staining was performed at room temperature for 8 minutes. After staining, the cells were washed three times by centrifugation and set aside for use.

[0196] (4) Next, the modified CAR-T cells were mixed with Ramos cells or K562 cells at different ratios, and aniline-biotin was added to a final concentration of 100 μM. After incubation at 37°C for 30 minutes, the cells were collected by centrifugation, washed three times with PBS, and then FITC-labeled streptavidin staining solution (final concentration 20 μg / mL) was added. After staining at room temperature for 10 minutes and washing three times with PBS, flow cytometry characterization was performed.

[0197] Conclusion: From the appendix Figure 9 It can be seen that for the CAR-T and Ramos cell interaction system, as the proportion of CAR-T cells increases, the proportion of Ramos cells labeled with biotin also increases accordingly, proving that the cell-to-cell interaction is effectively labeled. In contrast, K562 cells do not interact significantly with CAR-T cells, so the proportion of biotin labeled is very low and does not change significantly with the proportion of CAR-T cells in the system.

[0198] Example 7

[0199] HIM-IP detection of mouse BMDCs (mice myeloid dendritic cells) and CD8 + Inter-T cell contact was used to distinguish between cell-cell interactions of varying intensities. The specific experimental procedure is as follows:

[0200] (1) BMDCs were derived from the bone marrow of C57BL / 6 mice with CD45.1 and CD8. + T cells were extracted from the spleen of CD45.2-positive OT-1 mice. BMDCs were used to construct catcher cells, CD8+. + T cells are used to build prey cells.

[0201] (2) To construct catcher cells, 5 μL of 1 mM p-azidoethylphenol solution and 5 μL of tyrosinase solution (2 mg / mL) were added to the cell suspension to bring the final concentrations of p-azidoethylphenol and tyrosinase to 10 μM and 20 μg / mL, respectively. After gentle vortexing, the reaction was carried out at room temperature for 10 minutes. After the reaction, the cells were washed three times by centrifugation, resuspended in 500 μL PBS, and then DBCO-PEG modified tyrosinase was added to bring the final concentration of tyrosinase in the system to 200 μg / mL. After gentle mixing of the cells, the reaction was carried out at room temperature for 30 minutes. After the reaction, the cells were washed three times with PBS, divided into four aliquots, and incubated with the following peptides (37°C, 30 minutes): N4 peptide (SIINFEKL), A2 peptide (SAINFEKL), T4 peptide (SIITFEKL), and LCMV GP33 peptide (KAVYNFATM). After incubation, wash the cells three times with PBS and set aside for use.

[0202] (3) To construct prey cells, CD8 + SHPP was added to the T cell suspension to a final concentration of 4 μM. After a brief vortexing, the cell suspension was incubated at room temperature with shaking for 30 minutes. After the reaction, the cells were washed three times by centrifugation and ready for use.

[0203] (4) Combine the above-processed BMDCs with CD8 + T cells were co-incubated at 37°C for 1.5 hours, then aniline-biotin-tagged cells were added to a final concentration of 100 μM. After incubation for another 30 minutes, the cells were washed three times with PBS, followed by the addition of FITC-labeled streptavidin stain (final concentration 20 μg / mL), Cy5-labeled CD8 antibody, and Brilliant Violet 785-labeled CD45.1 antibody. After staining for 10 minutes and washing three times with staining buffer, flow cytometry characterization was performed.

[0204] Conclusion: From the appendix Figure 10 It can be seen that, due to the different interaction strengths between BMDCs presented by different peptides and T cells, the proportion of biotinylate-labeled T cells mediated by HIM-IP also changes accordingly, namely N4>A2>T4>GP33. Therefore, HIM-IP can distinguish between cell-cell interactions of different strengths.

[0205] Example 8

[0206] Detection of the interaction between CAR-T cells and tumor cells using a single-cell transcriptome sequencing platform

[0207] The specific experimental procedure is as follows:

[0208] (1) Prepare CD19-targeting CAR-T cells and Ramos cells (CD19-positive), collect the cells by centrifugation, wash twice with PBS and resuspend. CAR-T cells are used to construct catcher cells, and Ramos cells are used to construct prey cells.

[0209] (2) To construct catcher cells, 5 μL of 1 mM p-azidoethylphenol solution and 5 μL of tyrosinase solution (2 mg / mL) were added to the cell suspension, bringing the final concentrations of p-azidoethylphenol and tyrosinase to 10 μM and 20 μg / mL, respectively. After gentle vortexing, the reaction was carried out at room temperature for 10 minutes. After the reaction, the cells were washed three times by centrifugation, resuspended in 500 μL PBS, and then DBCO-PEG-modified tyrosinase was added to bring the final concentration of tyrosinase in the system to 200 μg / mL. After gentle mixing of the cells, the reaction was carried out at room temperature for 30 minutes. After the reaction, the cells were washed three times with PBS and set aside for use.

[0210] (3) To construct prey cells, SHPP was added to the Ramos cell suspension to a final concentration of 4 μM. After a brief vortexing, the cell suspension was placed at room temperature and shaken for 30 minutes. After the reaction, the cells were washed three times by centrifugation and set aside for use.

[0211] (4) Next, the modified CAR-T cells were mixed with Ramos cells, and an amino-single-stranded DNA tag (sequence: 5'-CTACGGTGCCTTGAAGTGACATACGCTAAAAAAAAAAAAAAAAAAAAA-3') was added to bring the final concentration to 400 nM. After incubation at 37°C for 30 minutes, the cells were collected by centrifugation and washed three times with PBS. Then, the cells were sequenced and analyzed using the standardized 10X Genomics single-cell transcriptome sequencing protocol.

[0212] Conclusion: From the appendix Figure 11 As can be seen, the labeled single-stranded DNA on Ramos cells can be detected by single-cell sequencing technology. Furthermore, the levels of single-stranded DNA labeling differ among different Ramos cells, indicating significant heterogeneity in the interaction levels between these cells and CAR-T cells. These results demonstrate that the HIM-IP technology designed in this invention can use single-stranded DNA molecular tags to label intercellular interactions and analyze these interactions through single-cell transcriptome sequencing.

[0213] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A system for detecting intercellular contact or interaction, the system comprising: 1) A catcher cell, the surface of which is modified with tyrosinase; 2) Prey cells, the surface of which is modified with phenolic groups; 3) Nucleophilic molecular tags; In catcher cells, tyrosinase is attached to the surface of the catcher cell via a click chemical reaction between an azide group and an alkyne group; Nucleophilic molecular tags consist of nucleophilic groups and tags.

2. The system according to claim 1, characterized in that, 1) The preparation method of catcher cells is as follows: i) Modify the surface of catcher cells with reactive groups, wherein the reactive groups are azide groups; ii) Prepare a tyrosinase modified with a reactive group, wherein the reactive group is an alkynyl group; iii) Co-incubate the tyrosinase modified with the reactive group in step ii) with the catcher cells modified with the reactive group in step i) to obtain catcher cells with tyrosinase modified on the surface.

3. The system according to claim 2, characterized in that, In ii), the reactive group-modified tyrosinase is an alkyne ring-modified tyrosinase; the concentration of the alkyne ring-modified tyrosinase is 50 μg / mL-1000 μg / mL.

4. The system according to claim 2 or 3, characterized in that, i) Modify the surface of catcher cells with azide groups using any of the following methods: a) Catcher cells are cultured in a culture medium to obtain azidated surface-modified catcher cells; the culture medium contains at least one component selected from tetraacylated N-azidoacetylgalactosamine, tetraacylated N-azidoacetylmannosamine, and azidolated sialic acid; b) Reaction of azido-PEG-NHS ester with catcher cells yields catcher cells with surface azido-modified surfaces; c) Reaction of p-azidoethylphenol and tyrosinase with catcher cells yields catcher cells with surface azidation modification.

5. The system according to claim 4, characterized in that, In step c), the concentration of azidoethylphenol is 1 μM-20 μM; And / or, the concentration of tyrosinase is 5 μg / mL to 80 μg / mL.

6. The system according to any one of claims 1-3, characterized in that, 2) The method for constructing prey cells is as follows: Reaction of prey cells with reagents containing phenol groups yields prey cells with phenol group-modified surfaces.

7. The system according to claim 6, characterized in that, The reagent containing a phenolic group is selected from one or more of the following: sodium 1-((3-(4-hydroxyphenyl)propionyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate, 3,4-dihydroxyphenylpropionic acid-N-succinimide ester, hydroxyphenylethyl acid-N-succinimide ester and 3,4-dihydroxyphenylethyl acid-N-succinimide ester.

8. The system according to any one of claims 1-3, characterized in that, The nucleophilic group is selected from amino, mercapto, and aniline; And / or, 3) The tags in the nucleophilic molecular tags are selected from fluorescent molecules, biotin, and oligonucleotides.

9. The system according to claim 8, characterized in that, 3) The nucleophilic molecular tag is selected from any of the following: ； Alternatively, 3) Nucleophilic molecular tags are selected from single-stranded DNA molecules with amino, thiol, or aniline groups modified at the 5' end.

10. The system according to claim 9, characterized in that, The base sequence of single-stranded DNA has the following general structure: 5'-(N) x + Capture sequence -3'; Where N represents any one of the four bases A, T, C, and G; and x is any natural number between 10 and 100.

11. The system according to claim 10, characterized in that, The capture sequence can be selected from any of the following: Sequence 1: 5'-AAAAAAAAAAAAAAA(A)y-3'; where y is any natural number between 0 and 15; Sequence 2: 5'-GCTCACCTATTAGCGGCTAAGG-3'; Sequence 3: 5'-GCTTTAAGGCCGGTCCTAGCAA-3'.

12. The system according to any one of claims 1-3, characterized in that, The catcher cells and prey cells are selected from cells whose intercellular interactions need to be detected.

13. The system according to claim 12, characterized in that... Both the catcher cells and the prey cells are Jurkat cells; Alternatively, the catcher cells are CAR-T cells, and the prey cells are tumor cells; Alternatively, the catcher cells are dendritic cells derived from mouse bone marrow, and the prey cells are CD8+ cells. + T cells.

14. A method for detecting intercellular contact or interaction, the method comprising: 1) Constructing catcher cells: Modifying the surface of catcher cells with tyrosinase; 2) Constructing prey cells: Modifying the surface of prey cells with phenolic groups; 3) The catcher cells, prey cells, and nucleophilic molecular tags are mixed and reacted, and then detected. In catcher cells, tyrosinase is attached to the surface of the catcher cell via a click chemical reaction between an azide group and an alkyne group; Nucleophilic molecular tags consist of nucleophilic groups and tags.

15. The method according to claim 14, characterized in that, 1) The method for constructing catcher cells is as follows: i) Modify the surface of catcher cells with reactive groups, wherein the reactive groups are azide groups; ii) Prepare a tyrosinase modified with a reactive group, wherein the reactive group is an alkynyl group; iii) The tyrosinase modified with the reactive group in step ii) is co-incubated with the catcher cells modified with the reactive group in step i) to obtain catcher cells with tyrosinase modified on the surface.

16. The method according to claim 15, characterized in that, In ii), the reactive group-modified tyrosinase is an alkyne ring-modified tyrosinase; the concentration of the alkyne ring-modified tyrosinase is 50 μg / mL-1000 μg / mL.

17. The method according to claim 14 or 15, characterized in that, i) Modify the surface of catcher cells with azide groups using any of the following methods: a) Catcher cells are cultured in a culture medium to obtain azidated surface-modified catcher cells; the culture medium contains at least one component selected from tetraacylated N-azidoacetylgalactosamine, tetraacylated N-azidoacetylmannosamine, and azidolated sialic acid; b) Reaction of azido-PEG-NHS ester with catcher cells yields catcher cells with surface azido-modified surfaces; c) Reaction of p-azidoethylphenol and tyrosinase with catcher cells yields catcher cells with surface azidation modification.

18. The method according to claim 17, characterized in that, The concentration of p-azidoethylphenol is 1 μM-20 μM; And / or, the concentration of tyrosinase is 5 μg / mL to 80 μg / mL.

19. The method according to any one of claims 14-16, characterized in that, 2) The method for constructing prey cells is as follows: Reaction of prey cells with reagents containing phenol groups yields prey cells with phenol group-modified surfaces.

20. The method according to claim 19, characterized in that, The reagent containing a phenolic group is selected from one or more of the following: sodium 1-((3-(4-hydroxyphenyl)propionyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate, 3,4-dihydroxyphenylpropionic acid-N-succinimide ester, hydroxyphenylethyl acid-N-succinimide ester and 3,4-dihydroxyphenylethyl acid-N-succinimide ester.

21. The method according to any one of claims 14-16, characterized in that, The nucleophilic groups in nucleophilic molecular tags are selected from amino, thiol, and aniline; And / or, the tags in the nucleophilic molecular tags are selected from fluorescent molecules, biotin, and oligonucleotides.

22. The method according to claim 21, characterized in that, Nucleophilic molecular tags are selected from any of the following: ； Alternatively, the nucleophilic molecular tag is selected from single-stranded DNA molecules with an amino, thiol, or aniline group modified at the 5' end.

23. The method according to claim 22, characterized in that, The base sequence of single-stranded DNA has the following general structure: 5'-(N)x + capture sequence-3'; Where N represents any one of the four bases A, T, C, and G; and x is any natural number between 10 and 100.

24. The method according to claim 23, characterized in that, The capture sequence can be selected from any of the following: Sequence 1: 5'-AAAAAAAAAAAAAAA(A)y-3'; where y is any natural number between 0 and 15; Sequence 2: 5'-GCTCACCTATTAGCGGCTAAGG-3'; Sequence 3: 5'-GCTTTAAGGCCGGTCCTAGCAA-3'.

25. The method according to any one of claims 14-16, characterized in that, The catcher cells and prey cells mentioned are derived from cells whose intercellular interactions need to be detected.

26. The method according to claim 25, characterized in that, Both the catcher cells and the prey cells are Jurkat cells; Alternatively, the catcher cells are CAR-T cells, and the prey cells are tumor cells; Alternatively, the catcher cells are dendritic cells derived from mouse bone marrow, and the prey cells are CD8+ cells. + T cells.

27. A kit for detecting intercellular contacts or interactions, said kit comprising: 1) Reagents for constructing catcher cells; 2) Reagents for constructing prey cells; 3) Labeling reagents; The reagent for constructing catcher cells is a reagent for introducing tyrosinase onto the surface of catcher cells. The reagent includes a reagent for introducing reactive groups onto the surface of catcher cells and tyrosinase modified with reactive groups. The reagent used to construct prey cells is one that introduces phenolic groups onto the surface of the prey cells; The labeling reagent is a reagent containing a nucleophilic group and a tag; The reactive groups on the surface of catcher cells are azide groups; The reactive group modified on tyrosinase is an alkynyl group.

28. The reagent kit according to claim 27, characterized in that, The reagents used to introduce reactive groups onto the surface of catcher cells are selected from reagents containing azide groups.

29. The reagent kit according to claim 28, characterized in that, The reagent containing the azido group is selected from any one or more of the following: tetraacylated N-azidoacetylgalactosamine (Ac4GalNAz), tetraacylated N-azidoacetylmannosamine (Ac4ManNAz), azidolated sialic acid, azido-PEG-NHS ester, and p-azidoethylphenol.

30. The kit according to claim 27, characterized in that, The reagents for constructing prey cells are selected from one or more of the following: sodium 1-((3-(4-hydroxyphenyl)propionyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate, 3,4-dihydroxyphenylpropionic acid-N-succinimidyl ester, hydroxyphenylethyl acid-N-succinimidyl ester and 3,4-dihydroxyphenylethyl acid-N-succinimidyl ester.

31. The reagent kit according to claim 27, characterized in that, The nucleophilic groups in the labeling reagents are selected from amino, mercapto, and aniline; And / or, the labels in the labeling reagents are selected from fluorescent molecules, biotin, and oligonucleotides.

32. The reagent kit according to claim 31, characterized in that, 3) The labeling reagent is selected from any of the following: ； Alternatively, the labeling reagent may be selected from single-stranded DNA molecules with amino, thiol, or aniline groups modified at the 5' end.

33. The reagent kit according to claim 32, characterized in that, The base sequence of single-stranded DNA has the following general structure: 5'-(N)x + capture sequence-3'; Where N represents any one of the four bases A, T, C, and G; and x is any natural number between 10 and 100.

34. The reagent kit according to claim 33, characterized in that, The capture sequence can be selected from any of the following: Sequence 1: 5'-AAAAAAAAAAAAAAA(A)y-3'; where y is any natural number between 0 and 15; Sequence 2: 5'-GCTCACCTATTAGCGGCTAAGG-3'; Sequence 3: 5'-GCTTTAAGGCCGGTCCTAGCAA-3'.

35. The reagent kit according to any one of claims 27-34, characterized in that, The kit may optionally further include any one or more of the following: 5) Buffer solution; 6) Tyrosine kinase.

36. The use of the kit according to any one of claims 27-35 in detecting intercellular contact or interaction.

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