Labeling method for characterizing cell-cell contact and use thereof

Abstract

A chemical labeling system HIM-IP for detecting cell-cell interaction. With regard to the system, a catcher cell and a prey cell are constructed by means of modifying tyrosinase and phenol on the surfaces of two cells to be detected, respectively, and labeling is then performed by means of using biotin or single-stranded DNA carrying a nucleophilic group as a molecular tag. The system can not only perform detection by means of traditional flow cytometry, but can also be compatible with commercialized single-cell transcriptome sequencing platforms, thereby enabling a correlation between cell-cell interaction information and transcriptome information at the single-cell level. The system has the characteristics that complex genetic engineering modifications to cells is not required, the whole modification and labeling process is simple and efficient, and the sensitivity of detecting cell-cell interaction is high. By means of integrating single-cell sequencing technology, in-depth exploration of the biological mechanisms underlying the regulations of cell-cell interaction is facilitated.

Description

A labeling method for characterizing intercellular contacts and its application

[0001] This application requires the following:

[0002] Priority to the prior application, patent application number 202311145586.6, filed with the State Intellectual Property Office of China on September 6, 2023, entitled “A labeling method for characterizing intercellular contacts and its application”;

[0003] The entire content of this prior application is incorporated into this application by reference. Technical Field

[0004] The present invention relates to the field of biological detection, and in particular to a labeling method for characterizing intercellular contact and an application thereof. Background Art

[0005] In recent years, more and more studies have found that intercellular contact-type interactions play a vital biological role in the regulation of many life activities. For example, physical contact between neurons is the basis for electrochemical signal transduction in the central nervous system; the entire process of T cells crossing the endothelial cell barrier from the blood vessel wall, migrating to the lymphoid tissue, and finally being sensitized by antigen-presenting cells is inseparable from the signal regulation mediated by intercellular contact; the formation of tissue or physiological barrier structures is also inseparable from the physical contact between adjacent cells and the signal transduction triggered by such interactions. However, the regulatory mechanism of intercellular interactions is extremely complex, and the biological significance behind many cell contact patterns remains unknown.

[0006] Against this backdrop, various biotechnologies for tracking or detecting intercellular interactions have emerged. Due to the complexity and dynamic nature of intercellular interactions, developing methods to characterize these biological events is extremely challenging. Currently developed detection technologies primarily include chemical labeling and single-cell sequencing. Chemical labeling methods typically utilize enzymatic or photocatalytic reactions to label intercellular interactions, offering the advantages of high sensitivity and specificity. For example, Peng Wu et al. reported a fucosyltransferase-based chemical labeling strategy FucoID to detect contacts 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 photocatalytic labeling technology μMap based on Dexter energy transfer and achieved high-resolution labeling of cell-to-cell interacting 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-to-cell interactions at the flow cytometry level and cannot characterize cell-to-cell interactions at the single-cell scale or genetic level.

[0007] In contrast, single-cell sequencing can perform high-throughput, multi-parameter analysis of interacting cells of specific subpopulations based on the current single-cell transcriptome platform, and can deeply reveal the regulatory mechanisms behind cell-to-cell interactions at the genetic level. However, the current single-cell sequencing method cannot distinguish the level of cell-to-cell interactions, and can usually only analyze cell samples that have already adhered to each other, ignoring the biological information of weak cell-to-cell interactions, and is not as sensitive as the chemical labeling method. Therefore, how to retain the high specificity and sensitivity of chemical labeling while incorporating the advantages of single-cell sequencing technology has important scientific and application value for further understanding of cell-to-cell interactions. However, related characterization technologies have not yet been reported.

[0008] Summary of the Invention

[0009] The present invention aims to develop a labeling method for detecting intercellular contacts (hereinafter referred to as HIM-IP). This labeling technology is compatible with traditional flow cytometry detection methods and integrates single-cell transcriptome sequencing modules. Compared with existing chemical labeling strategies, HIM-IP can achieve high-throughput, parameterized characterization of intercellular interactions at the single-cell and gene levels. Compared with previously reported single-cell sequencing technologies, HIM-IP can distinguish between intercellular interactions of varying strengths, with higher detection sensitivity and specificity.

[0010] First aspect

[0011] The present invention provides a system for detecting cell-cell contact or interaction, the system comprising:

[0012] 1) a catcher cell, wherein the surface of the catcher cell is modified with tyrosinase;

[0013] 2) prey cells, the surfaces of the prey cells being modified with phenol groups;

[0014] 3) Nucleophilic molecular tags.

[0015] According to an embodiment of the present invention, 1) in the catcher cell, tyrosinase is connected to the surface of the catcher cell by coupling;

[0016] Preferably, the tyrosinase is linked to the surface of the catcher cell via a click chemistry reaction (e.g., a copper-free click chemistry reaction);

[0017] Further preferably, tyrosinase is linked to the catcher cell surface via a click chemistry reaction between an azide group and an alkyne ring.

[0018] According to an embodiment of the present invention, the alkyne ring is a 3-9 membered alkyne ring, preferably a 7-9 membered alkyne ring, such as cyclooctyne.

[0019] According to an embodiment of the present invention, 1) the preparation method of the catcher cells is as follows:

[0020] i) modifying the surface of the catcher cell with reactive groups (e.g., azide groups);

[0021] ii) preparing a tyrosinase modified with a reactive group (e.g., an alkyne ring);

[0022] iii) co-incubating 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 their surfaces.

[0023] According to an embodiment of the present invention, the reactive groups on the surface of the catcher cells are capable of undergoing a coupling reaction with the reactive groups modified on the tyrosinase.

[0024] According to an embodiment of the present invention, ii) the alkyne ring-modified tyrosinase is a cyclooctyne-modified tyrosinase, preferably a dibenzocyclooctyne-modified tyrosinase, more preferably a dibenzocyclooctyne-polyethylene glycol (DBCO-PEG)-modified tyrosinase.

[0025] According to an embodiment of the present invention, iii) the concentration of the alkyne ring-modified tyrosinase is 50 μg / mL-1000 μg / mL, for example, 200 μg / mL.

[0026] According to an embodiment of the present invention, i) modifying the surface of the catcher cell with an azide group adopts any one of the following methods:

[0027] a) culturing the catcher cells in a culture medium to obtain surface-azidated catcher cells; the culture medium comprises at least one component selected from tetraacylated N-azidoacetylgalactosamine (Ac4GalNAz), tetraacylated N-azidoacetylmannosamine (Ac4ManNAz), and azidated sialic acid; preferably, the culture time is 24-72 hours;

[0028] b) reacting azide-PEG-NHS ester with the catcher cells to obtain surface-azidated catcher cells; the reaction time is, for example, 30 minutes;

[0029] Preferably, the reaction is carried out in a buffer, such as PBS buffer;

[0030] c) reacting p-azidoethylphenol and tyrosinase with the catcher cells to obtain catcher cells with surface azide modification; the reaction time is, for example, 10 minutes;

[0031] Preferably, the reaction is carried out in a buffer, such as PBS buffer.

[0032] According to an embodiment of the present invention, in method c), the concentration of p-azidoethylphenol is 1 μM-20 μM, for example, 10 μM.

[0033] According to an embodiment of the present invention, in method c), the concentration of tyrosinase is 5 μg / mL-80 μg / mL, for example, 20 μg / mL.

[0034] Because both cells and tyrosine kinases have commonly used reaction sites on their surfaces, such as amino groups and sulfhydryl groups, direct bifunctional reagents used to connect cells and tyrosine kinases through these commonly used reaction sites can result in cross-linking reactions between cells and between tyrosine kinases, significantly reducing the controllability of the reaction. The method for preparing catcher cells employed in the present invention utilizes coupling reactions between reactive groups on the catcher cell surface and modified reactive groups on tyrosinase (e.g., coupling reactions between alkyne rings and azides) to modify the tyrosine kinase onto the catcher cell surface.

[0035] According to an embodiment of the present invention, 2) the method for constructing prey cells is as follows:

[0036] reacting a reagent containing a phenol group with prey cells to obtain prey cells with surfaces modified with phenol groups;

[0037] Preferably, the reagent containing a phenol group is selected from the group consisting of sodium 1-((3-(4-hydroxyphenyl)propionyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate (SHPP), 3,4-dihydroxyphenylpropyl acid-N-succinimide ester, hydroxyphenylethyl acid-N-succinimide ester and 3,4-dihydroxyphenylethyl acid-N-succinimide ester;

[0038] Preferably, the reaction time is, for example, 30 minutes; the reaction temperature is, for example, room temperature;

[0039] Preferably, the reaction is carried out in a buffer, such as PBS buffer.

[0040] According to an embodiment of the present invention, the nucleophilic molecular tag comprises a nucleophilic group and a tag.

[0041] According to an embodiment of the present invention, 3) the nucleophilic group in the nucleophilic molecular tag is selected from amino, thiol and aniline.

[0042] According to an embodiment of the present invention, the tag in 3) the nucleophilic molecular tag is selected from fluorescent molecules, biotin or its derivatives, oligonucleotides (such as single-stranded DNA), etc.

[0043] According to an embodiment of the present invention, 3) the nucleophilic molecular tag is selected from any one of the following:

[0044] According to an embodiment of the present invention, 3) the nucleophilic molecular tag is selected from a single-stranded DNA molecule whose 5' end is modified with an amino group, a thiol group or an aniline group.

[0045] According to an embodiment of the present invention, the base sequence of the single-stranded DNA has the following general structure:

[0046] 5'-(N)x+capture sequence-3';

[0047] Among them, N represents any of the four bases A, T, C, and G;

[0048] The value of x is any natural number between 10 and 100.

[0049] According to an embodiment of the present invention, the capture sequence is selected from any one of the following:

[0050] Sequence 1: 5'-AAAAAAAAAAAAAAA(A)y-3'; where y is any natural number between 0 and 15.

[0051] Sequence 2: 5′-GCTCACCTATTAGCGGCTAAGG-3′;

[0052] Sequence 3: 5′-GCTTTAAGGCCGGTCCTAGCAA-3′.

[0053] The base sequence of single-stranded DNA has the above-mentioned general structure and can meet the compatibility requirements of current commercial single-cell transcriptome sequencing platforms.

[0054] According to different application scenarios or detection targets or detection purposes, the catcher cells and prey cells are selected from any pair of cells that need to interact with each other to be detected, such as animal cells.

[0055] According to an embodiment of the present invention, both the trapper cells and the prey cells are Jurkat cells.

[0056] According to an embodiment of the present invention, the catcher cells are CAR-T cells and the prey cells are tumor cells.

[0057] 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 + T cells.

[0058] Second aspect

[0059] The present invention also provides a method for detecting cell-cell contact or interaction, comprising:

[0060] 1) Construction of catcher cells: tyrosinase is modified on the surface of the catcher cells;

[0061] 2) Constructing prey cells: modifying the surface of prey cells with phenol groups;

[0062] 3) The trapper cells, prey cells, and nucleophilic molecular tags are mixed and reacted, and then detected. According to an embodiment of the present invention, 1) in the trapper cells, tyrosinase is connected to the surface of the trapper cells by coupling;

[0063] Preferably, the tyrosinase is linked to the surface of the catcher cell via a click chemistry reaction (e.g., a copper-free click chemistry reaction);

[0064] Further preferably, tyrosinase is linked to the catcher cell surface via a click chemistry reaction between an azide group and an alkyne ring.

[0065] According to an embodiment of the present invention, the alkyne ring is a 3-9 membered alkyne ring, preferably a 7-9 membered alkyne ring, such as cyclooctyne.

[0066] According to an embodiment of the present invention, 1) the method for constructing catcher cells is as follows:

[0067] i) modifying the surface of the catcher cell with reactive groups (e.g., azide groups);

[0068] ii) preparing a tyrosinase modified with a reactive group (e.g., an alkyne ring);

[0069] 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 their surfaces.

[0070] According to an embodiment of the present invention, the reactive groups on the surface of the catcher cells are capable of undergoing a coupling reaction with the reactive groups modified on the tyrosinase.

[0071] According to an embodiment of the present invention, ii) the alkyne ring-modified tyrosinase is a cyclooctyne-modified tyrosinase, preferably a dibenzocyclooctyne-modified tyrosinase, more preferably a dibenzocyclooctyne-polyethylene glycol (DBCO-PEG)-modified tyrosinase.

[0072] According to an embodiment of the present invention, iii) the concentration of the alkyne ring-modified tyrosinase is 50 μg / mL-1000 μg / mL, for example, 200 μg / mL.

[0073] According to an embodiment of the present invention, i) modifying the surface of the catcher cell with an azide group adopts any one of the following methods:

[0074] a) culturing the catcher cells in a culture medium to obtain surface-azidated catcher cells; the culture medium comprises at least one component selected from tetraacylated N-azidoacetylgalactosamine (Ac4GalNAz), tetraacylated N-azidoacetylmannosamine (Ac4ManNAz), and azidated sialic acid; preferably, the culture time is 24-72 hours;

[0075] b) reacting azide-PEG-NHS ester with the catcher cells to obtain surface-azidated catcher cells; the reaction time is, for example, 30 minutes;

[0076] Preferably, the reaction is carried out in a buffer, such as PBS buffer;

[0077] c) reacting p-azidoethylphenol and tyrosinase with the catcher cells to obtain catcher cells with surface azide modification; the reaction time is, for example, 10 minutes;

[0078] Preferably, the reaction is carried out in a buffer, such as PBS buffer.

[0079] According to an embodiment of the present invention, in method c), the concentration of p-azidoethylphenol is 1 μM-20 μM, for example, 10 μM.

[0080] According to an embodiment of the present invention, in method c), the concentration of tyrosinase is 5 μg / mL-80 μg / mL, for example, 20 μg / mL.

[0081] According to an embodiment of the present invention, 2) the method for constructing prey cells is as follows:

[0082] reacting a reagent containing a phenol group with prey cells to obtain prey cells with surfaces modified with phenol groups;

[0083] Preferably, the reagent containing a phenol group is selected from the group consisting of sodium 1-((3-(4-hydroxyphenyl)propionyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate (SHPP), 3,4-dihydroxyphenylpropyl acid-N-succinimide ester, hydroxyphenylethyl acid-N-succinimide ester and 3,4-dihydroxyphenylethyl acid-N-succinimide ester;

[0084] Preferably, the reaction time is, for example, 30 minutes; the reaction temperature is, for example, room temperature;

[0085] Preferably, the reaction is carried out in a buffer, such as PBS buffer. According to an embodiment of the present invention, the nucleophilic molecular tag comprises a nucleophilic group and a tag.

[0086] According to an embodiment of the present invention, the nucleophilic group in the nucleophilic molecular tag is selected from amino, sulfhydryl and aniline.

[0087] According to an embodiment of the present invention, the tag in the nucleophilic molecular tag is selected from fluorescent molecules, biotin or its derivatives, oligonucleotides (such as single-stranded DNA), etc.

[0088] According to an embodiment of the present invention, the nucleophilic molecular tag is selected from any one of the following:

[0089] According to an embodiment of the present invention, the nucleophilic molecular tag is selected from single-stranded DNA molecules whose 5' end is modified with an amino group, a thiol group or an aniline group.

[0090] According to an embodiment of the present invention, the base sequence of the single-stranded DNA has the following general structure:

[0091] 5'-(N)x+capture sequence-3';

[0092] Among them, N represents any of the four bases A, T, C, and G;

[0093] The value of x is any natural number between 10 and 100.

[0094] According to an embodiment of the present invention, the capture sequence is selected from any one of the following:

[0095] Sequence 1: 5'-AAAAAAAAAAAAAAA(A)y-3'; where y is any natural number between 0 and 15.

[0096] Sequence 2: 5′-GCTCACCTATTAGCGGCTAAGG-3′;

[0097] Sequence 3: 5′-GCTTTAAGGCCGGTCCTAGCAA-3′.

[0098] The base sequence of single-stranded DNA has the above-mentioned general structure and can meet the compatibility requirements of current commercial single-cell transcriptome sequencing platforms.

[0099] According to an embodiment of the present invention, in 3), the reaction condition is incubation at 37° C. for 30 minutes.

[0100] 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.

[0101] According to an embodiment of the present invention, in 3), after the reaction, flow cytometry is used for detection or single-cell transcriptome sequencing is used.

[0102] Depending on different application scenarios or detection targets or detection purposes, the catcher cells and prey cells can be derived from any pair of cells that need to interact with each other to be detected, such as animal cells.

[0103] According to an embodiment of the present invention, both the trapper cells and the prey cells are Jurkat cells.

[0104] According to an embodiment of the present invention, the catcher cells are CAR-T cells and the prey cells are tumor cells.

[0105] 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 + T cells.

[0106] The third aspect

[0107] The present invention also provides a kit for detecting intercellular contact or interaction, the kit comprising:

[0108] 1) Reagents for constructing catcher cells;

[0109] 2) Reagents for constructing prey cells;

[0110] 3) Labeling reagents;

[0111] Wherein, the reagent for constructing the catcher cell is a reagent for introducing tyrosinase to the surface of the catcher cell;

[0112] The reagent for constructing the prey cell is a reagent for introducing a phenol group onto the surface of the prey cell;

[0113] The labeling reagent is a reagent containing a nucleophilic group and a tag.

[0114] According to an embodiment of the present invention, the nucleophilic group captures the o-quinone produced by the phenol group catalyzed by tyrosinase on the surface of the catcher cell, thereby completing the labeling of the prey cell.

[0115] According to an embodiment of the present invention, 1) the reagents for constructing catcher cells include a reagent for introducing reactive groups onto the surface of the catcher cells and tyrosinase modified with the reactive groups.

[0116] According to an embodiment of the present invention, the reactive groups on the surface of the catcher cells are capable of undergoing a coupling reaction with the reactive groups modified on the tyrosinase.

[0117] According to an embodiment of the present invention, the reactive groups on the surface of the catcher cells are azide groups.

[0118] According to an embodiment of the present invention, the modified reactive group on tyrosinase is an alkynyl group, such as an alkyne ring group.

[0119] According to an embodiment of the present invention, the reagent for introducing reactive groups into the surface of the catcher cell is selected from a reagent containing an azide group, for example, any one or more of tetraacylated N-azidoacetylgalactosamine (Ac4GalNAz), tetraacylated N-azidoacetylmannosamine (Ac4ManNAz), azidated sialic acid, azide-PEG-NHS ester, and p-azidoethylphenol.

[0120] According to an embodiment of the present invention, the reactive group-modified tyrosinase is an alkyne ring-modified tyrosinase, preferably a dibenzocyclooctyne-modified tyrosinase, such as a dibenzocyclooctyne-polyethylene glycol (DBCO-PEG)-modified tyrosinase.

[0121] According to an embodiment of the present invention, the cycloalkyne is a 3-9 membered alkyne ring, preferably a 7-9 membered alkyne ring, such as cyclooctyne.

[0122] 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-dihydroxyphenylpropyl acid-N-succinimide ester, hydroxyphenylethyl acid-N-succinimide ester and 3,4-dihydroxyphenylethyl acid-N-succinimide ester.

[0123] According to an embodiment of the present invention, 3) the nucleophilic group in the labeling reagent is selected from amino, thiol and aniline.

[0124] 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 (such as single-stranded DNA), etc.

[0125] According to an embodiment of the present invention, 3) the labeling agent is selected from any one of the following:

[0126] According to an embodiment of the present invention, 3) the labeling agent is selected from single-stranded DNA molecules whose 5' end is modified with an amino group, a thiol group or an aniline group.

[0127] According to an embodiment of the present invention, the base sequence of the single-stranded DNA has the following general structure:

[0128] 5'-(N)x+capture sequence-3';

[0129] Among them, N represents any of the four bases A, T, C, and G;

[0130] The value of x is any natural number between 10 and 100.

[0131] According to an embodiment of the present invention, the capture sequence is selected from any one of the following:

[0132] Sequence 1: 5'-AAAAAAAAAAAAAAA(A)y-3'; where y is any natural number between 0 and 15.

[0133] Sequence 2: 5′-GCTCACCTATTAGCGGCTAAGG-3′;

[0134] Sequence 3: 5′-GCTTTAAGGCCGGTCCTAGCAA-3′.

[0135] The kit optionally further comprises:

[0136] 5) Buffer, such as PBS buffer;

[0137] 6) Tyrosine kinase.

[0138] The present invention also provides use of the above kit in detecting intercellular contact or interaction. Beneficial effects

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

[0140] The HIM-IP designed by the present invention has the following advantages:

[0141] 1. The cell engineering methods involved in this invention all utilize chemical modification, eliminating the need for genetic modification. The entire modification and labeling process takes only about two hours, offering rapid and convenient technical advantages. This method is highly applicable to primary cells or difficult-to-engineer cell lines, demonstrating its universal applicability.

[0142] 2. All chemical reagents involved in the HIM-IP technical solution (including tyrosinase, SHPP, biotin tags, and DNA tags, etc.) can be obtained through commercial channels, and basically no self-synthesis is required, 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 marker for intercellular interactions, enabling the correlation of intercellular interaction information with transcriptomic information at the single-cell level. This technical feature overcomes the limitation of current chemical-labeled intercellular interaction detection, which can only analyze at the cell population level, and effectively integrates the powerful advantages of single-cell sequencing technology in revealing the mechanisms of intercellular interactions. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0145] FIG2 is an imaging result of the azidated Jurkat cells in Example 1 after being stained with DBCO-Cy5 and Hoechst33342.

[0146] FIG3 is a confocal imaging result of Jurkat cells with tyrosinase modified on their surfaces in Example 1.

[0147] FIG4 shows the cell surface azidation modification level in dependence on azidoethylphenol concentration in Example 2.

[0148] FIG5 shows the tyrosinase concentration-dependent cell surface azide modification levels in Example 3.

[0149] FIG6 shows the modification level of tyrosinase in Jurkat cells after treatment with different concentrations of Ac4GaINAz in Example 4.

[0150] FIG. 7 shows the effect of tyrosinase and SHPP modification on biotin labeling efficiency in the HIM-IP system of Example 5.

[0151] FIG8 is a graph showing the correlation 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 is a graph showing the correlation between the proportion of catcher cells and the proportion of biotin-labeled prey cells in the interaction system between CAR-T and Ramos or K562 cells in Example 6.

[0153] FIG10 shows the flow cytometry results of T cell labeling by HIM-IP after co-incubation of BMDCs presented with different polypeptides with T cells in Example 7.

[0154] Figure 11 shows the single-cell sequencing results of CAR-T and Ramos after single-stranded DNA interaction labeling in Example 8. DETAILED DESCRIPTION

[0155] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations 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 encompassed within the scope of protection that the present invention is intended to protect.

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

[0157] HIM-IP: a labeling method for detecting cell-cell contacts.

[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: bioconjugation reagent, PEG stands for polyethylene glycol, and NHS stands for N-hydroxysuccinimide.

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

[0163] Example 1

[0164] Construction of catcher cells based on Jurkat cells

[0165] (1) Collect 2 million Jurkat cells by centrifugation, wash twice with PBS, and resuspend in 490 μL PBS. Add 5 μL of p-azidoethylphenol solution (1 mM) and 5 μL of tyrosinase solution (2 mg / mL) to the cell suspension to a final concentration of 10 μM p-azidoethylphenol and 20 μg / mL tyrosinase, respectively. After gentle vortexing, shake the mixture 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. In this step, a small amount of cells can be removed for Hoechst 33342 and dibenzocyclooctyne-Cy5 (DBCO-Cy5) staining, and fluorescence confocal imaging can be used to detect the azide modification of the cell surface.

[0167] (3) Mix the washed cell suspension from the previous step with a solution of DBCO-PEG-modified tyrosinase (also labeled with FITC) to a final concentration of 200 μg / mL. Gently mix the cells and incubate at room temperature for 30 minutes.

[0168] (4) After the reaction is completed, the cells are collected by centrifugation and washed 2-3 times. After staining with Hoechst 33342, the modification of tyrosinase on the cell surface is observed under a fluorescence confocal microscope.

[0169] Conclusion: As shown in Figure 2, the surface of the azidated Jurkat cells was significantly labeled with red fluorescence by DBCO-Cy5, confirming the successful azidation. As shown in Figure 3, FITC-labeled tyrosinase was distributed on the surface of the Jurkat cells, confirming the successful modification of tyrosinase on the Jurkat cell membrane.

[0170] Example 2

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

[0172] The operation steps were the same as steps (1) and (2) in Example 1, except that the final concentrations of p-azidoethylphenol in step (1) were 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 measured by flow cytometry.

[0173] Conclusion: As shown in FIG4 , increasing the modification concentration of p-azidoethylphenol can increase 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 operation 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 measured by flow cytometry.

[0177] Conclusion: As shown in FIG5 , increasing the modification concentration of tyrosinase can increase the degree of azidation modification on the surface of Jurkat cells.

[0178] Example 4

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

[0180] (1) Jurkat cells were collected by centrifugation, and the cell culture medium was replaced with complete culture medium containing 0, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM Ac4GalNAz, and the culture was continued for 72 hours.

[0181] (2) After the culture was completed, Jurkat cells were collected and reacted with DBCO-PEG-modified tyrosinase (FITC-labeled), and then analyzed by flow cytometry.

[0182] Conclusion: As shown in Figure 6, the density of azidation modification on the Jurkat cell surface increases with increasing Ac4GalNAz concentration. These data demonstrate that Ac4GalNAz can be used to azidate the Jurkat cell surface and subsequently label tyrosinase.

[0183] Example 5

[0184] HIM-IP was used to detect the interaction between Jurkat cells at the flow cytometry level:

[0185] Jurkat cells easily aggregate during culture, indicating strong contact-type interactions between these cells. The following are the experimental steps for HIM-IP to detect interactions between Jurkat cells:

[0186] (1) 10 million Jurkat cells were collected by centrifugation, washed twice with PBS, resuspended, and divided into two equal parts, one for constructing catcher cells and the other for constructing prey cells.

[0187] (2) To construct the catcher cells, 5 μL of p-azidoethylphenol solution (1 mM) and 5 μL of tyrosinase solution (2 mg / mL) were added to the cell suspension to make the final concentrations of p-azidoethylphenol and tyrosinase 10 μM and 20 μg / mL, respectively. After vortexing gently, the reaction was shaken at room temperature for 10 minutes. After the reaction was completed, the cells were washed three times by centrifugation and resuspended in 500 μL PBS, and then DBCO-PEG-modified tyrosinase was added to make the final concentration of tyrosinase in the system 200 μg / mL. After gently mixing the cells, the reaction was shaken at room temperature for 30 minutes. After the reaction was completed, the cells were washed three times with PBS and set aside. The catcher cells in the control group were Jurkat cells without any modification.

[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 placed at room temperature for a 30-minute shaking reaction. After the reaction, the cells were washed once by centrifugation, and then 500 μL of Hoechst 33342 dye solution (5 μg / mL) was added and stained at room temperature for 8 minutes. After the staining was completed, the cells were washed three times by centrifugation and set aside. The prey cells in the control group were Jurkat cells that were not modified with SHPP but were stained with Hoechst 33342.

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

[0190] Conclusion: As shown in Figure 7, the lack of tyrosinase modification in either the catcher cells or the prey cells results in the HIM-IP system being unable to properly detect cell-cell interactions. The efficiency of cell-cell interaction labeling is highest only when both components are present. Figure 8 shows that varying the ratio of catcher cells in the system also alters the proportion of prey cells labeled with biotin. This result demonstrates that the HIM-IP system is sensitive enough to detect cell subpopulations in contact within the system.

[0191] Example 6

[0192] HIM-IP is used to detect the interaction between CAR-T and tumor cells at the flow cytometry level. The specific operation 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, and Ramos and K562 cells are used to construct prey cells.

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

[0195] (3) To construct prey cells, SHPP was added to a Ramos cell or K562 cell suspension to a final concentration of 4 μM. After a brief vortex, the cell suspension was placed at room temperature for a 30-minute shake reaction. 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 stained at room temperature for 8 minutes. After the staining was completed, the cells were washed three times by centrifugation and set aside.

[0196] (4) Next, the modified CAR-T cells were mixed with Ramos cells or K562 cells at different quantitative ratios, and the aniline-biotin label 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. The cells were stained at room temperature for 10 minutes, washed three times with PBS, and then characterized by flow cytometry.

[0197] Conclusion: As shown in Figure 9, for the interaction system between CAR-T and Ramos cells, as the proportion of CAR-T cells continues to increase, the proportion of Ramos cells labeled with biotin also increases accordingly, proving that the cell-cell interaction is effectively labeled; in contrast, since K562 cells do not significantly interact with CAR-T cells themselves, the proportion of biotin-labeled cells is very low and does not change significantly with changes in the proportion of CAR-T cells in the system.

[0198] Example 7

[0199] HIM-IP detection of mouse BMDCs (mouse myeloid dendritic cells) and CD8 + T cell-cell contact and differentiation of cell-cell interactions of different intensities. The specific experimental procedures are as follows:

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

[0201] (2) To construct the catcher cells, 5 μL of p-azidoethylphenol solution (1 mM) and 5 μL of tyrosinase solution (2 mg / mL) were added to the cell suspension to make the final concentrations of p-azidoethylphenol and tyrosinase 10 μM and 20 μg / mL, respectively. After vortexing gently, the reaction was shaken 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 make the final concentration of tyrosinase in the system 200 μg / mL. After gently mixing the cells, the reaction was shaken at room temperature for 30 minutes. After the reaction was completed, the cells were washed three times with PBS, divided into four parts, 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, cells were washed three times with PBS and set aside.

[0202] (3) To construct prey cells, CD8 + Add SHPP to the T cell suspension to a final concentration of 4 μM. After brief vortexing, incubate the cell suspension at room temperature for 30 minutes. After the reaction, wash the cells three times by centrifugation and set aside.

[0203] (4) The treated BMDCs were mixed with CD8 + T cells were incubated at 37°C for 1.5 hours, followed by the addition of an aniline-biotin tag to a final concentration of 100 μM. After an additional 30-minute incubation, cells were washed three times with PBS and then stained with FITC-conjugated streptavidin (final concentration 20 μg / mL), Cy5-conjugated CD8 antibody, and Brilliant Violet 785-conjugated CD45.1 antibody. The cells were stained for 10 minutes, washed three times with staining buffer, and analyzed by flow cytometry.

[0204] Conclusion: As shown in Figure 10, due to the varying strengths of interactions between BMDCs and T cells presented by different peptides, the ratio of biotin labeling on T cells mediated by HIM-IP also changes accordingly: N4 > A2 > T4 > GP33. Therefore, HIM-IP can distinguish between cell-cell interactions of varying strengths.

[0205] Example 8

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

[0207] The specific experimental operations are 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 the catcher cells, 5 μL of p-azidoethylphenol solution (1 mM) and 5 μL of tyrosinase solution (2 mg / mL) were added to the cell suspension to make the final concentrations of p-azidoethylphenol and tyrosinase 10 μM and 20 μg / mL, respectively. After vortexing gently, the reaction was shaken at room temperature for 10 minutes. After the reaction was completed, the cells were washed three times by centrifugation, resuspended in 500 μL PBS, and then DBCO-PEG-modified tyrosinase was added to make the final concentration of tyrosinase in the system 200 μg / mL. After gently mixing the cells, the reaction was shaken at room temperature for 30 minutes. After the reaction was completed, the cells were washed three times with PBS and set aside.

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

[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 a final concentration of 400 nM. After incubation at 37°C for 30 minutes, the cells were collected by centrifugation and washed three times with PBS. Next, the sequencing and subsequent library construction and sequencing analysis were performed according to the standardized 10X Genomics single-cell transcriptome sequencing process.

[0212] Conclusion: As can be seen from Figure 11, the labeled single-stranded DNA on Ramos cells can be detected by single-cell sequencing technology. In addition, the single-stranded DNA labeling levels of different Ramos cells vary, indicating that there is great heterogeneity in the level of interaction between these cells and CAR-T cells. The above results show that the HIM-IP technology designed by the present invention can use single-stranded DNA molecular tags to mark cell-to-cell interactions and analyze cell-to-cell interactions through single-cell transcriptome sequencing.

[0213] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A system for detecting cell-cell contact or interaction, the system comprising: 1) a catcher cell, wherein the surface of the catcher cell is modified with tyrosinase; 2) a prey cell, the surface of which is modified with a phenol group; 3) Nucleophilic molecular tags.

2. The system according to claim 1, characterized in that 1) In the catcher cell, tyrosinase is connected to the surface of the catcher cell by coupling; preferably, tyrosinase is connected to the surface of the catcher cell by click chemistry reaction; Preferably, the alkyne ring is a 3-9 membered alkyne ring; Preferably, 1) the preparation method of the catcher cells is as follows: i) modifying the surface of the catcher cell with reactive groups; ii) preparing a tyrosinase modified with a reactive group; iii) co-incubating 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. Preferably, the reactive groups on the surface of the catcher cells are capable of undergoing a coupling reaction with the reactive groups modified on the tyrosinase; Preferably, in ii), the reactive group-modified tyrosinase is an alkynyl-modified tyrosinase, preferably an alkyne-ring-modified tyrosinase, and more preferably a cyclooctyne-modified tyrosinase; Preferably, during the reaction in iii), the concentration of the alkynyl-modified tyrosinase is 50 μg / mL-1000 μg / mL; Preferably, i) modifying the catcher cell surface with azide groups adopts any of the following methods: a) placing the catcher cells in a culture medium to obtain catcher cells with surface azidation modification; the culture medium comprises at least one component selected from tetraacylated N-azidoacetylgalactosamine, tetraacylated N-azidoacetylmannosamine, and azidated sialic acid; b) reacting azide-PEG-NHS ester with the catcher cells to obtain catcher cells with surface azide modification; c) reacting p-azidoethylphenol and tyrosinase with the catcher cells to obtain catcher cells with surface azidation modification.

3. The system according to claim 1 or 2, characterized in that: 2) The construction method of prey cells is as follows: reacting a reagent containing a phenol group with the prey cells to obtain prey cells with surfaces modified with phenol groups; Preferably, the reagent containing a phenol 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; Preferably, 3) the nucleophilic molecular tag comprises a nucleophilic group and a tag; Preferably, the nucleophilic group is selected from amino, thiol and aniline; Preferably, 3) the label in the nucleophilic molecular label is selected from fluorescent molecules, biotin or its derivatives, oligonucleotides, etc.; Preferably, 3) the nucleophilic molecular tag is selected from any one of the following: Preferably, 3) the nucleophilic molecular tag is selected from a single-stranded DNA molecule whose 5' end is modified with an amino group, a thiol group or an aniline group; Preferably, the base sequence of the single-stranded DNA has the following general structure: 5'-(N)x+capture sequence-3'; Wherein, N represents any one of the four bases A, T, C, and G; the value of x is any natural number between 10 and 100; Preferably, the capture sequence is selected from any one 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′. Preferably, according to different application scenarios or detection targets or detection purposes, the catcher cells and prey cells are selected from any pair of cells that need to interact with each other to be detected; Preferably, both the catcher cells and the prey cells are Jurkat cells; Preferably, the catcher cells are CAR-T cells and the prey cells are tumor cells; Preferably, the catcher cells are dendritic cells derived from mouse bone marrow and the prey cells are CD8 + T cells.

4. A method for detecting cell-cell contact or interaction, the method comprising: 1) Construction of catcher cells: Modifying the surface of catcher cells with tyrosinase; 2) Constructing prey cells: modifying the surface of prey cells with phenol groups; 3) The catcher cells, prey cells and nucleophilic molecule tags are mixed and reacted, and then detected after the reaction.

5. The method according to claim 4, characterized in that 1) In the catcher cell, tyrosinase is connected to the surface of the catcher cell by coupling; Preferably, tyrosinase is linked to the surface of the catcher cell via a click chemistry reaction; Further preferably, tyrosinase is linked to the surface of the catcher cell via a click chemistry reaction between an azide group and an alkyne group; Preferably, the alkyne ring is a 3-9 membered alkyne ring; Preferably, 1) the method for constructing catcher cells is as follows: i) modifying the surface of the catcher cell with reactive groups; ii) preparing a tyrosinase modified with a reactive group; iii) co-incubating the tyrosinase modified with the reactive group in step i) with the catcher cell modified with the reactive group in step ii) to obtain the catcher cell with tyrosinase modified on the surface; Preferably, the reactive groups on the surface of the catcher cells are capable of undergoing a coupling reaction with the reactive groups modified on the tyrosinase; Preferably, in ii), the reactive group-modified tyrosinase is an alkynyl-modified tyrosinase, preferably an alkyne-ring-modified tyrosinase, and more preferably a cyclooctyne-modified tyrosinase; Preferably, during the reaction in iii), the concentration of the alkyne ring-modified tyrosinase is 50 μg / mL-1000 μg / mL; Preferably, i) modifying the catcher cell surface with azide groups adopts any of the following methods: a) placing the catcher cells in a culture medium to obtain catcher cells with surface azidation modification; the culture medium comprises at least one component selected from tetraacylated N-azidoacetylgalactosamine, tetraacylated N-azidoacetylmannosamine, and azidated sialic acid; b) reacting azide-PEG-NHS ester with the catcher cells to obtain catcher cells with surface azide modification; c) reacting p-azidoethylphenol and tyrosinase with the catcher cells to obtain catcher cells with surface azidation modification.

6. The method according to claim 4 or 5, characterized in that: 2) The construction method of prey cells is as follows: reacting a reagent containing a phenol group with the prey cells to obtain prey cells with surfaces modified with phenol groups; Preferably, the reagent containing a phenol 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; Preferably, the nucleophilic molecular tag comprises a nucleophilic group and a tag; Preferably, the nucleophilic group in the nucleophilic molecular tag is selected from amino, thiol and aniline; Preferably, the tag in the nucleophilic molecular tag is selected from fluorescent molecules, biotin or its derivatives, oligonucleotides, etc.; Preferably, the nucleophilic molecular tag is selected from any one of the following: Preferably, the nucleophilic molecular tag is selected from a single-stranded DNA molecule whose 5' end is modified with an amino group, a thiol group or an aniline group; Preferably, the base sequence of the single-stranded DNA has the following general structure: 5'-(N)x+capture sequence-3'; Wherein, N represents any one of the four bases A, T, C, and G; the value of x is any natural number between 10 and 100; Preferably, the capture sequence is selected from any one 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′. Preferably, in 3), the reaction conditions are incubation at 37°C for 30 minutes; Preferably, in 3), the concentration of the nucleophilic molecular tag is 10 μM-200 μM; Preferably, in 3), after the reaction, flow cytometry is used for detection or single-cell transcriptome sequencing is used; Preferably, according to different application scenarios or detection targets or detection purposes, the catcher cells and prey cells can be derived from any pair of cells that need to interact with each other to be detected; Preferably, both the catcher cells and the prey cells are Jurkat cells; Preferably, the catcher cells are CAR-T cells and the prey cells are tumor cells; Preferably, the catcher cells are dendritic cells derived from mouse bone marrow and the prey cells are CD8 + T cells.

7. A kit for detecting cell-cell contact or interaction, the kit comprising: 1) Reagents for constructing catcher cells; 2) Reagents for constructing prey cells; 3) labeling reagent; Wherein, the reagent for constructing the catcher cell is a reagent for introducing tyrosinase to the surface of the catcher cell; The reagent for constructing the prey cell is a reagent for introducing a phenol group onto the surface of the prey cell; The labeling reagent is a reagent containing a nucleophilic group and a tag.

8. The kit according to claim 7, characterized in that 1) Reagents for constructing catcher cells include reagents for introducing reactive groups onto the surface of catcher cells and tyrosinase modified with reactive groups; Preferably, the reactive groups on the surface of the catcher cells are capable of undergoing a coupling reaction with the reactive groups modified on the tyrosinase; Preferably, the reactive groups on the surface of the catcher cells are azide groups; Preferably, the modified reactive group on tyrosinase is an alkynyl group; Preferably, the reagent for introducing reactive groups to the surface of the catcher cells is selected from reagents containing an azide group, for example, any one or more selected from tetraacylated N-azidoacetylgalactosamine, tetraacylated N-azidoacetylmannosamine, azidated sialic acid, azide-PEG-NHS ester, and p-azidoethylphenol; Preferably, the reactive group-modified tyrosinase is an alkynyl-modified tyrosinase, preferably an alkyne-ring-modified tyrosinase, and more preferably a cyclooctyne-modified tyrosinase; Preferably, the cycloalkyne is a 3-9 membered alkyne ring; Preferably, 2) the reagent for constructing prey cells 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. Preferably, 3) the nucleophilic group in the labeling agent is selected from amino, sulfhydryl and aniline; Preferably, 3) the label in the labeling agent is selected from fluorescent molecules, biotin or its derivatives, oligonucleotides, etc.; Preferably, 3) the labeling agent is selected from any one of the following: Preferably, 3) the labeling agent is selected from a single-stranded DNA molecule whose 5' end is modified with an amino group, a thiol group or an aniline group; Preferably, the base sequence of the single-stranded DNA has the following general structure: 5'-(N)x+capture sequence-3'; Wherein, N represents any one of the four bases A, T, C, and G; the value of x is any natural number between 10 and 100; Preferably, the capture sequence is selected from any one 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′.

9. The kit according to claim 7 or 8, characterized in that The kit optionally further comprises any one or more of the following: 5) buffer; 6) Tyrosine kinase.

10. Use of the kit according to any one of claims 7 to 9 in detecting cell-cell contact or interaction.

Images