Detection of molecular-cellular interactions based on proximity labelling techniques using fluorescein conjugates

Abstract

The present application relates to the technical field of proximity labeling, and particularly provides a method for detecting the interaction between molecules and cells based on a fluorescein conjugate, wherein the fluorescein conjugate is used as a catalyst to co-culture with cells, the excess fluorescein conjugate is cleaned, a substrate is added to the cells after co-culture to perform a reaction, the fluorescein is activated by light, and the fluorescein catalyzes the labeling of other molecules around the fluorescein. The labeling technology is widely applicable and based on commercially available fluorescein conjugate as a photosensitive catalyst for labeling, and can be applied in the field of detecting the interaction between molecules and cells and the detection kit using the method. The present application solves the limitations of the catalyst in the existing proximity labeling technology field, greatly reduces the cost, is simple to use and suitable for a platform for a variety of detection objects, and can be applied to different molecular and cellular research.

Description

Technical Field

[0001] This invention relates to the field of proximity labeling technology, and more particularly to a proximity labeling method based on fluorescein conjugates, the application of this method in the detection of molecular-cell interactions, and a kit for detecting molecular-cell interactions using this method. Background Technology

[0002] The basic principle of proximity labeling technology is to fuse enzymes with proximity labeling functions (such as APEX2, TurboID, etc.) with the target protein, and then use enzyme-catalyzed covalent modification to label neighboring proteins with tags such as biotin. Finally, biotin-labeled proteins are enriched using avidin magnetic beads for mass spectrometry identification, analyzing the interactions between the target protein and neighboring protein information. Proximity labeling technology is crucial for elucidating molecular and cellular interactions.

[0003] Among existing proximity labeling methods, enzymes such as APEX and BioID require gene editing, making them difficult to apply to non-protein targets and clinical samples. Photocatalysts based on metal complexes or photosensitizers require specialized chemical synthesis and coupling, such as relying on organic photocatalysts like Eosin Y to activate photoprobes or using spatially targeted iridium complexes to absorb visible light photon energy and transfer it to the probe. (See Microenvironment mapping via Dexter energy transfer on immune cells, JACOB B. GERI et al., Science, Vol. 367, No. 6482, pp. 1091-10976, March 6, 2020; and Multiscale photocatalytic proximity labeling reveals cell surface neighbors on and between cells, ZHI LIN et al., Science, Vol. 385, No. 6706, July 19, 2024). Current labeling catalysts suffer from limitations such as the need for customized genetic editing or chemical synthesis, lack simple and easy-to-use platforms applicable to multiple detection targets, are inaccessible to most laboratories, and are costly. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a proximity labeling method based on widely available and readily available commercial fluorescein conjugates as catalysts, which solves the limitations of existing labeled catalysts and makes them easier to apply to different molecular and cellular studies; at the same time, it provides the application of this method in the field of detecting molecular and cellular interactions and the detection kit using this method.

[0005] In this invention, "fluorescein conjugate" refers to a complex formed by chemically coupling a fluorescent dye with a specific molecule (such as an antibody or protein). "FITC conjugate" refers to a compound formed by chemically combining fluorescein isothiocyanate (FITC) with other biomolecules. "FAM conjugate" refers to a compound formed by combining a carboxyfluorescein label (FAM) with a specific molecule (such as a polypeptide, protein, or polysaccharide). "Fluorescein conjugate" refers to a complex formed by chemically linking fluorescein with other molecules.

[0006] In this invention, "biotinylation" refers to the process of covalently linking biotin to proteins, nucleic acids, or other molecules.

[0007] Other undefined terms in this invention should be understood in accordance with their common meaning in this technical field.

[0008] This invention provides a method for proximity labeling based on fluorescein as a catalyst: First, purchase or synthesize FITC conjugates or other photosensitive catalyst conjugates such as FAM conjugates and Fluorescein conjugates according to experimental needs.

[0009] The fluorescein conjugate was co-incubated with target cells, including tumor cells, immune cells, epithelial cells, mesenchymal cells, nerve cells, or similar cells. The incubation time and the concentration of the fluorescein conjugate were adjusted according to different experiments. For cholesterol-FITC, the incubation time was 15 min and the concentration was 0.05 mg / mL; for WGA-FITC, the incubation time was 15 min and the concentration was 0.1 mg / mL; for antibody-FITC, the incubation time was 30 min and the concentration was 1-10 µg / mL; for aptamer-FAM, the incubation time was 1 h and the concentration was 1.16 μM; for fluorescein-chloroalkane, the incubation time was 30 min and the concentration was 100 nM; and for siRNA-FAM, the incubation time was 6 h and the concentration was 0.1 μM.

[0010] After washing away excess luciferin conjugate, the substrate is added to the co-incubated cells for reaction. More preferably, excess free conjugate can be washed away with PBS, and the cells can be resuspended in PBS containing phenolic substrate, aryl azide substrate, or aniline substrate.

[0011] Illumination labeling time varies from 5 min to 1 h, adjusted according to experimental needs. LED lamps or lasers can be used as the light source. For cholesterol-FITC, LED labeling for 10-60 min produces a noticeable signal; for WGA-FITC, laser labeling for 5 min produces a noticeable signal; for antibody-FITC, LED labeling for 5-30 min; for aptamer-FAM, LED labeling for 60 min; for fluorescein-chloroalkane, LED labeling for 30 min; and for siRNA-FAM, LED labeling for 30 min.

[0012] Through the above steps, protein biotinylation can be achieved based on luciferin conjugate catalysis, and labeling can be performed in a radius range of 50nm-300nm.

[0013] Furthermore, the remaining substrate can be washed away with PBS, and subsequent operations can be performed as needed, such as immunofluorescence staining, flow cytometry, mass spectrometry, or single-cell sequencing. In some cases, substrate washing is unnecessary and does not affect the labeling radius and analytical applications of this invention.

[0014] Furthermore, this invention verifies that fluorescein conjugates can be used for labeling, identifies labeling sites, and uses various fluorescein conjugates for labeling in various scenarios. Examples of fluorescein conjugates used in embodiments of this invention include WGA-FITC conjugates, antibody-FITC, cholesterol-FITC, aptamer-FAM, siRNA-FAM, and fluorescein-chloroalkane.

[0015] Furthermore, this invention has been validated using proteomics or single-cell sequencing. Specific proteomics procedures can be found in Examples 8, 9, and 14, while specific single-cell sequencing procedures can be found in Example 10.

[0016] In the embodiments provided by this invention, the application of proximity labeling technology based on fluorescein conjugates in molecular detection is further provided. The molecules include small molecules, proteins, DNA, RNA, sugars, etc.

[0017] Furthermore, this invention provides the application of proximity labeling technology based on fluorescein conjugates in the detection of cell interactions.

[0018] The method of this invention is simple, easy to use, readily implementable, and highly cost-effective. It can also be used as a detection kit. Depending on experimental needs, we can develop different types of detection kits, such as siRNA-interacting protein detection kits, clinical sample cell interaction detection kits, membrane proteomics detection kits, etc.

[0019] All reagent kits include the following components: ① LED light or laser, preferably with a power greater than or equal to 19mW / cm². 2 ② Fluorescein conjugates, such as siRNA-FAM for siRNA interaction protein detection kits; specific antibody-FITC for clinical sample cell interaction detection kits; cholesterol-FITC for membrane proteomics detection kits, etc.; ③ Labeled substrates: biotin-phenol, aryl-azido-biotin, and biotin-aniline are three types of substrates; 4 Washing solution, such as PBS solution, is used to wash away excess fluorescein conjugates.

[0020] All of the above components can be purchased as commercially available products. The usage method involves incubating the conjugate, adding the labeled substrate, and then applying light for labeling. Specific steps for different kits are detailed in the respective examples. After labeling using various kits, customers can combine them with mass spectrometry and single-cell sequencing technologies for subsequent detection and analysis.

[0021] The beneficial effects of this invention are: (1) FITC-ID is readily available because it uses widely available fluorescein conjugates, making it easier to obtain compared to existing methods that require custom synthesis and conjugation.

[0022] (2) It has dual functions. The fluorescent catalyst can perform both fluorescence imaging and proximity biotinylation, which can verify the correct targeting and positioning of the catalyst.

[0023] (3) Low cytotoxicity. Fluorescein is a commonly used fluorescent dye in laboratories and by those skilled in the art. It has shown low cytotoxicity and its safety has been recognized.

[0024] (4) High versatility, compatible with different configurations such as small molecules, proteins / antibodies, nucleic acids, and chemifluorescein conjugates, and the selection of multiple substrates (biotin-phenol, aryl-azido-biotin, and biotin-aniline, etc.) enables multi-scale analysis. In the embodiments of this invention, biotin-phenol (Example 4), aryl-azido-biotin (Example 3), and biotin-aniline (Example 11) all achieved effective labeling signals. In comparison, biotin-phenol has lower reactivity and a weaker signal, but a larger labeling radius; aryl-azido-biotin has higher reactivity and a stronger labeling signal under the same conditions, but a relatively smaller labeling radius, and its labeled amino acid residues are mainly lysine. Biotin-aniline has the highest labeling efficiency and the strongest labeling signal under the same conditions, and its labeled amino acid residues are mainly histidine. The appropriate substrate can be selected according to requirements during use.

[0025] (5) Characterization of cell surface proteome based on FITC-ID (Examples 8, 9 and 14) and analysis of scRNA-seq of clinical samples (Example 10) demonstrate its powerful ability to reveal molecular and cellular organization of biological systems, providing key insights for biological research. Attached Figure Description

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0027] Figure 1 This is an in vitro labeling of BSA mediated by FITC, analyzed by immunoblotting. Samples containing BSA, FITC, and aryl-azide-biotin were irradiated with a blue LED at 30 mW·cm⁻² for 10 minutes. The reaction products were analyzed by SDS-PAGE electrophoresis and by streptavidin-HRP (top) or Ponceau staining (bottom).

[0028] Figure 2 This is the identification of the labeled sites. Left image: Schematic diagram showing biotinylated residues on BSA. Right image: Mass spectrum of a peptide containing labeled lysine (K*).

[0029] Figure 3 Pulse-tracking labeling with cholesterol-FITC was performed in live cells. Following the initial pulse of cholesterol-FITC, cells were optically labeled with aryl azide-biotin at specific time points (0, 1, 6, or 12 hours). HeLa cells were then stained with streptavidin-Cy3 and DAPI for fluorescence imaging.

[0030] Figure 4 This is fluorescence imaging with WGA-FITC labeling. MC38 cells were stained with WGA-FITC and then optically labeled with biotin-phenol. Fluorescence imaging was performed using cells stained with Nav-650 and DAPI.

[0031] Figure 5 Cell-cell interactions were detected using antibody-FITC assays. HEK293T cells were transfected with V5-CTLA4 or HA-CD86, stained with anti-V5 primary antibody and FITC secondary antibody, and co-incubated before labeling. Cells were then stained with streptavidin-Cy3 for fluorescence imaging. Cis and trans labeling (white arrows) were observed in FITC+ and FITC- cells, respectively.

[0032] Figure 6 Labeled cells were analyzed by flow cytometry. No trans-labeling signal was observed when V5-CTLA4 cells were co-incubated with untransfected CD86 HEK293T (-CD86), while cis-labeling on FITC+ cells remained unaffected. Samples without FITC antibody or light were used as controls.

[0033] Figure 7 This is CD3 aptamer-FAM labeling. Top image: Jurkat T or HEK293 cells were incubated with CD3 aptamer-FAM, then labeled with aryl-azido-biotin under blue LED illumination, and analyzed by flow cytometry. Biotinylation (streptavidin-Cy3) was observed on Jurkat T cells only in the presence of FAM and light, while no signal was observed on HEK293 cells. Bottom image: Fluorescence imaging of CD3 aptamer-FAM labeling. Left: Jurkat T cells biotinylated and stained with streptavidin-Cy3. Right: Dashed fluorescence intensity map showing co-localization between aptamer-FAM and streptavidin-Cy3.

[0034] Figure 8 The cells were labeled using fluorescein-chloroalkane ligands. Fluorescence imaging showed photo- and rapamycin-dependent FITC-ID in HEK293T cells co-transfected with two split HaloTag fragments. The full-length HaloTag was used as a positive control. Biotinylated proteins and nuclei were stained with streptavidin-Cy3 and DAPI, respectively.

[0035] Figure 9 HEK293T cells were labeled with siRNA-FAM for proteomics analysis. HEK293T cells were transfected with GAPDH siRNA–FAM, and labeled for 30 min after 6 hours. Samples without blue light, untransfected, and transfected with non-targeting siRNA-FAM were used as controls. Proteomics analysis was performed. Immunofluorescence showed biotinylation (streptavidin-Cy3) under both light and siRNA-FAM conditions. Mass spectrometry results showed that the GAPDH siRNA-FAM and Scramble siRNA-FAM groups were enriched in many RNA-binding proteins compared to the untransfected group.

[0036] Figure 10 PC9 cells were labeled with cholesterol-FITC and subjected to proteomics analysis. Gefitinib-treated and untreated PC9 cells were labeled with cholesterol-FITC after incubation for 1 hour. A sample without cholesterol-FITC was used as a negative control. Western blotting results showed that a clear labeling signal was generated under both light and in the presence of cholesterol-FITC. Mass spectrometry results showed that the label was enriched in many membrane proteins, and there were differences in the cell surface proteome between gefitinib-treated and untreated cells.

[0037] Figure 11Human tumor samples were labeled with antibody-FITC and analyzed using single-cell sequencing. Flow cytometry analysis revealed a higher proportion of cis and trans labeling in the antibody-FITC-labeled and light-exposed group. Single-cell RNA sequencing data analysis revealed extensive interactions between T cells and various cell types, including tumor cells, macrophages, endothelial cells, B cells, and dendritic cells, within the tumor microenvironment.

[0038] Figure 12 This is an in vitro labeling of FITC-mediated RNase A using immunoblotting. Samples containing RNase A, FITC, and biotin-aniline were irradiated with a blue LED at 30 mW·cm⁻² for 5 minutes. The reaction products were analyzed by SDS-PAGE electrophoresis and by streptavidin-HRP (top) or Ponceau staining (bottom).

[0039] Figure 13 Cell-cell interactions were detected using an antibody-FITC assay. HEK293T cells were transfected with V5-PDL1-P2A-zsGreen or HA-PD1-P2A-mCherry, stained with anti-V5 primary antibody and FITC secondary antibody, and then labeled with biotin-aniline after co-incubation. Cells were stained with streptavidin-AF647 for fluorescence imaging. Cis and trans labeling were observed in green (V5-PDL1) and red (HA-PD1) cells, respectively. It was noted that trans labeling was significantly reduced in the absence of ligand PD1 (a). Furthermore, the higher the PD1 content, the more pronounced the trans labeling (b).

[0040] Figure 14 This is a HER2-FITC labeling assay. SKOV3 cells were first incubated with anti-HER2 primary antibody and FITC secondary antibody staining, and then labeled with biotin-aniline light. Cells were stained with streptavidin-cy3 and DAPI for fluorescence imaging or streptavidin-HRP for immunoblotting analysis.

[0041] Figure 15 The antibody HER2-FITC was used to label proteins surrounding HER2, followed by proteomics analysis. A sample without HER2-FITC was used as a negative control. Mass spectrometry results showed that the labeling enriched many membrane proteins, most of which are known proteins surrounding HER2.

[0042] Figure 16 This experiment identified the available wavelength range for FITC as a catalyst. A sample without HER2-FITC was used as a negative control. Quantified biotinylation labeling signals showed that 450-530 nm is the available labeling wavelength range for FITC as a catalyst. Detailed Implementation

[0043] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0044] Unless otherwise specified, the experimental methods used in the embodiments are conventional methods. Unless otherwise specified, the materials and reagents used are commercially available. Unless otherwise specified, the abbreviations or acronyms used are understood by those skilled in the art to have a specific meaning in this invention based on the specific circumstances.

[0045] Example 1: Fluorescein-mediated in vitro protein labeling Experimental Procedure: Mix 10 μM BSA, 20 μM FITC, and 100 μM aryl-azide-biotin in PBS to a reaction volume of 200 μL. Irradiate with a 30 mW·cm−2 blue LED at room temperature for 10 minutes. A group without blue light serves as a negative control. The reaction products are analyzed by 12% SDS-PAGE electrophoresis, followed by streptavidin-HRP blotting or Ponceau S staining (see attached diagram). Figure 1 .

[0046] Experimental results: FITC, as a catalyst, can effectively biotinylate BSA protein under blue LED irradiation. FITC can achieve photoactivated biotinylation in vitro.

[0047] Example 2: Mass spectrometry analysis of labeled sites Experimental procedure: Mix 2 mg BSA, 1.25 mM FITC, and 100 μM aryl-azide-biotin in PBS to a reaction volume of 200 μL. Irradiate the mixture with a 30 mW·cm−2 blue LED at room temperature for 10 minutes. A sample without blue light was used as a negative control. Identify the labeled sites using mass spectrometry.

[0048] Experimental results: Biotinylation of multiple solvent-exposed nucleophilic residues (such as Lys, His, and Cys) was identified in in vitro labeled BSA, further confirming the presence of reactive nitroene intermediates (see appendix). Figure 2 .

[0049] Example 3: Cholesterol-FITC labeling Experimental Procedure: HeLa cells were seeded in 24-well plates. After washing the cells once with PBS, they were incubated in DMEM at 37°C with 0.05 mg / mL cholesterol-FITC for 15 minutes. After incubation, the cells were washed three times with PBS and cultured in complete medium for 0, 1, 6, or 12 hours. They were then washed once more with PBS and incubated in PBS at room temperature with 200 μM aryl-azide-biotin for 5 minutes. The cells were then labeled with blue light for 60 minutes. Samples without blue light or FITC were used as negative controls.

[0050] Experimental Results: Co-localization of biotinylation and FITC fluorescence was observed at different time points, indicating that labeling is effective both inside and outside living cells. Robust labeling can be achieved with substrate concentrations of 100-200 μM and light exposure as short as 10 minutes. No biotin signal was detected in the absence of FITC, demonstrating that FITC acts as a catalyst in the labeling reaction (see Appendix). Figure 3 .

[0051] Example 4: WGA-FITC Marking Experiment Experimental Procedure: MC38 cells were seeded in 24-well plates and tested one day later. Cells were washed twice with PBS and then incubated in DMEM at 37°C with 0.1 mg / mL WGA-FITC for 15 minutes. After washing three times, they were incubated in PBS with 200 μM biotin-phenol for 5 minutes at room temperature. Irradiation was performed for 5 minutes using a Leica LED3 with a FITC filter at 100% output. Samples without blue light or FITC were set up as negative controls.

[0052] Experimental Results: The WGA-FITC group achieved photoactivated biotinylation, indicating that FITC can act as an effective photocatalyst for proximity labeling when coupled with proteins. No biotin signal was detected in the absence of FITC, demonstrating that FITC acts as a catalyst in the labeling reaction (see Appendix). Figure 4 .

[0053] Example 5: Antibody-FITC labeled cell interaction Experimental Procedure: HEK293 cells were seeded in plates and allowed to reach 60% confluence. V5-CTLA4 or HA-CD86 was transfected into the cells, respectively. After 24 hours, the cells were digested in PBS with 2 mM EDTA, washed twice, and stained with V5 Tag mouse antibody or HA-Tag rabbit monoclonal antibody for 1 hour at room temperature. After staining, the cells were washed twice and incubated with goat anti-mouse secondary antibody FITC and goat anti-rabbit secondary antibody AlexaFluor 647 for 30 minutes at room temperature. The cells were washed twice with PBS and then mixed with 200 μM aryl-azide-biotin at a density of 1 million cells / 200 μL in PBS. The cells were co-incubated for 1 hour and then irradiated with a 30 mW·cm−2 blue LED for 30 minutes at room temperature. Samples without light, FITC, or ligands were used as negative controls. After labeling, the cells were collected for flow cytometry analysis.

[0054] Experimental Results: >89% cis-labeling and >84% trans-labeling were observed in CTLA4+ and CD86+ cells, respectively. In the absence of ligand-receptor interaction, trans-labeling decreased by nearly 8-fold, while cis-labeling was almost unaffected, indicating that FITC can reliably recognize interacting cells. Biotin signaling was undetectable in the absence of FITC, demonstrating that FITC acts as a catalyst in the labeling reaction (see Appendix). Figure 5 and attached Figure 6 .

[0055] Example 6: CD3 aptamer - FAM tag Experimental Procedure: Use 1 million Jurkat T cells or HEK293 cells, 200 μL in volume. Block the sample with 10% goat serum at 4°C for 1 hour, then wash three times. Add CD3 aptamer-FAM (1.16 μM) and incubate on a rotor at 4°C for 1 hour. After washing twice, add 200 μM aryl-azide-biotin. Incubate the sample for 5 minutes, then irradiate with blue light for 60 minutes. Set up samples without blue light or FITC as negative controls.

[0056] Experimental Results: In Jurkat T cells, >90% of cells underwent biotinylation, and the degree of biotinylation was closely related to the intensity of luciferin. However, in HEK293 cells lacking CD3 expression, aptamer labeling was minimal (<3%), validating the specificity of this method. Confocal imaging showed precise overlap between biotin and luciferin signals, indicating limited diffusion of the nitrocellulose intermediate and a restricted labeling radius. No biotin signal was detected in the absence of FITC, demonstrating that FITC acts as a catalyst in the labeling reaction (see Appendix). Figure 7 .

[0057] Example 7: fluorescein-chloroalkane ligand labeling Experimental Procedure: HEK293 cells were seeded in plates and allowed to reach 60% confluence. FRB-HaloTag156N-V5-CD4TM and FKBP-HaloTag156C-HA-CD4TM plasmids were co-transfected into the cells. Twenty-four hours after transfection, 400 nM rapamycin was added to the culture medium, and the cells were cultured for another 12 hours. The rapamycin was maintained in the culture medium throughout the subsequent experiments. Cells were stained with 100 nM fluorescein-chloroalkane HaloTagligand and washed twice with PBS. Cells were placed in PBS containing 200 μM aryl-azide-biotin or 500 μM biotin phenol and labeled by irradiation with a 30 mW·cm−2 blue LED for 30 minutes at room temperature. Samples without blue light, FITC, or rapamycin were used as negative controls.

[0058] Experimental Results: The chemogenetic version of FITC-ID based on split-HaloTag was successfully validated. Effective biotinylation labeling was only achieved in the presence of rapamycin, i.e., when the interaction between FKBP and FRB binds the two fragments of the split-HaloTag to form a complete functional HaloTag. In the negative control conditions lacking rapamycin (i.e., lack of interaction induction) or other key components (such as blue light, the catalyst FITC, and ligand), biotinylation labeling was significantly weakened or undetectable. This result demonstrates that this method can achieve precise labeling dependent on specific interactions, providing a powerful tool and methodological support for studying intermolecular interactions. It also further proves the flexibility and scalability of FITC-ID technology; by combining it with different technical strategies (such as split-HaloTag), it can meet more complex research needs, providing new pathways and possibilities for elucidating molecular interaction networks in biological systems. (See appendix) Figure 8 .

[0059] Example 8: siRNA-FAM tagging Experimental Procedure: HEK293 cells were cultured in 48-well plates and transfected with GAPDH siRNA-FAM at 60% confluence. Six hours after transfection, cells were washed once with PBS, incubated with 200 μM aryl-azide-biotin in PBS for 30 min, and then irradiated with a 30 mW·cm−2 blue LED at room temperature for 30 min. Samples without blue light, without the FITC catalyst, and without targeted siRNA-FAM were used as controls. Proteomics analysis was also performed.

[0060] Experimental results: FITC-ID was validated as a marker for proteins surrounding siRNA (see appendix). Figure 9 .

[0061] Example 9: Cholesterol-FITC labeled PC9 cells: Experimental Procedure: PC9 cells were pretreated with 600 nM gefitinib for 48 hours, then washed once with PBS, and incubated with 0.05 mg / mL cholesterol-FITC in DMEM at 37°C for 15 minutes. After incubation, the cells were washed three times, incubated with 200 μM aryl-azide-biotin in PBS at room temperature for 5 minutes, and then irradiated with blue light for 1 hour. A sample without cholesterol-FITC as a catalyst was used as a negative control. After cell lysis, biotinylated proteins were enriched from PC9 cell lysates using streptavidin magnetic beads, followed by trypsin digestion and mass spectrometry analysis.

[0062] Experimental Results: Mass spectrometry analysis identified >3200 proteins. Comparison of the cell surface proteomes of untreated and gefitinib-treated NSCLC cells revealed that gefitinib treatment resulted in an overall downregulation of cell adhesion molecules (especially integrins and cadherins), while proteins such as ERBB3, EFNA1, MUC1, and ALPP were upregulated. This demonstrates the specificity and sensitivity of FITC-ID in proteomics analysis, providing crucial biological insights. (See appendix) Figure 10 .

[0063] Example 10: Antibody-FITC labeling of human tumor samples: Sample Collection: Fresh tissue samples were obtained from hepatocellular carcinoma patients treated at Zhongshan Hospital of Fudan University. The study followed the protocol approved by the institutional review board, and written informed consent was obtained from all participants. Two untreated primary tumor samples were included in the analysis. Patient selection criteria included untreated primary tumors without significant underlying diseases (such as autoimmune diseases or acute infections).

[0064] Sample dissociation: Fresh HCC patient tumors were collected and cut into small pieces less than 1 mm in diameter. Cells were then agitated at 37°C for 20 minutes using a mild dissociation buffer containing 100 μg / mL liberase TL and 100 μg / mL DNase I in an RPMI 1640. Cells were then filtered through a 70 μm filter, washed with PBS, and centrifuged using a Ficoll density gradient for 20 minutes.

[0065] Sample labeling: Cell suspensions were isolated from HCC patient tumors and stained with mouse-derived antihCD3-APC in staining buffer for 30 minutes. After washing, the cells were stained with goat anti-mouse secondary antibody FITC for 30 minutes, washed again, and resuspended in PBS containing 200 μM aryl-azide-biotin. The cells were then plated in 24-well plates, incubated for 5 minutes, and irradiated with a 30 mW·cm−2 blue LED for 30 minutes. Samples without blue light or without the FITC catalyst were used as negative controls.

[0066] Data Collection: After labeling, cells were collected for flow cytometry analysis or for single-cell sequencing. For single-cell sequencing samples, we performed subsequent CITE-seq analysis using streptavidin-conjugated barcode staining.

[0067] Experimental Results: Flow cytometry analysis validated the effectiveness of the labeling. A higher proportion of cis and trans labeling was observed in the antibody-FITC group, indicating that FITC-ID can reliably recognize interacting cells. Single-cell RNA sequencing data analysis revealed extensive interactions between T cells and various cell types (including tumor cells, macrophages, endothelial cells, B cells, and dendritic cells) in the tumor microenvironment. These results collectively demonstrate the application value and technical effectiveness of FITC-ID in human tumor sample research (see appendix). Figure 11 .

[0068] Example 11: In vitro protein labeling using biotin-aniline mediated by fluorescein Experimental Procedure: 10 μM RNase A, 20 μM FITC, and 100 μM biotin-aniline were mixed in PBS, with a reaction volume of 200 μL. The mixture was irradiated with a 30 mW·cm−2 blue LED at room temperature for 5 minutes. Groups without blue light, FITC, probe, or RNase A served as negative controls. Groups with additional 5 mM Trolox, 10 mM NaVc, and 10 mM NaN3 served as mechanistic controls, respectively. The reaction products were analyzed by 15% SDS-PAGE electrophoresis, followed by streptavidin-HRP blotting or Ponceau S staining (see attached diagram). Figure 12 .

[0069] Experimental results: FITC can effectively utilize biotin-aniline to biotinylate RNase A protein under blue LED irradiation. No labeling signal (biotin signal) can be detected when FITC is not present, proving that FITC acts as a catalyst in the labeling reaction. FITC can achieve photoactivated biotinylation in vitro.

[0070] Example 12: Antibody-FITC cell interaction using biotin-aniline labeling Experimental Procedure: HEK293 cells were seeded in plates and allowed to reach 60% confluence. V5-PDL1-P2A-zsGreen or HA-PD1-P2A-mCherry were transfected into the cells. After 24 hours, the cells were digested in PBS with 2 mM EDTA, washed twice, and stained with V5 Tag mouse antibody for 1 hour at room temperature. After staining, the cells were washed twice and incubated with goat anti-mouse secondary antibody FITC for 30 minutes at room temperature. The cells were washed twice with PBS and then mixed with 500 μM biotin-aniline at a density of 1 million cells / 200 μL in PBS. The cells were co-incubated for 1 hour and then irradiated with a 30 mW·cm−2 blue LED for 10 minutes at room temperature. Samples without light, FITC, or ligands were used as negative controls. After labeling, the cells were collected for flow cytometry analysis.

[0071] Experimental Results: >88% cis-labeling and >52% trans-labeling were observed in PDL1+ and PD1+ cells, respectively. In the absence of ligand-receptor interaction, trans-labeling was almost undetectable, while cis-labeling showed only a slight decrease, indicating that FITC-ID can reliably recognize interacting cells. The absence of FITC also meant that biotin signaling was undetectable, demonstrating that FITC acts as a catalyst in the labeling reaction (see Appendix). Figure 13 .

[0072] Example 13: HER2-FITC cell labeling experiment using biotin-aniline. Experimental Procedure: SKOV3 cells were seeded in 24-well plates and tested one day later. Cells were washed twice with PBS and then stained with HER2 mouse anti-cell antibody for 30 minutes at room temperature. After staining, the cells were washed three times and incubated with goat anti-mouse secondary antibody FITC for 30 minutes at room temperature. After washing three times, the cells were incubated with 500 μM biotin-aniline in PBS at room temperature. Then, the cells were irradiated with a 30 mW·cm−2 blue LED for 7 minutes. Samples without blue light or FITC were set up as negative controls. A separate amount of labeled SKOV3 cells were lysed using ripa buffer. The lysate was subjected to 12% SDS-PAGE electrophoresis and then Western blotted with streptavidin-HRP (see attached image). Figure 14 .

[0073] Experimental results: HER2-FITC can utilize biotin-aniline to label proteins, indicating that FITC can utilize biotin-aniline for cellular-level neighbor-to-neighbor labeling when coupled with proteins. No biotin signal was detected in the absence of FITC, demonstrating that FITC acts as a catalyst in the labeling reaction (see appendix). Figure 14 .

[0074] Example 14: HER2-FITC labeling of HER2-peripheral proteins in SKOV3 cells: Experimental Procedure: SKOV3 cells were coated with FITC antibody for HER2 protein using the same strategy as in Example 13, washed three times, and then incubated with 500 μM biotin-aniline in PBS at room temperature, followed by blue light irradiation for 5 minutes. A sample without the HER2–FITC catalyst was used as a negative control. After cell lysis, biotinylated proteins were enriched from SKOV3 cell lysates using streptavidin magnetic beads, followed by trypsin digestion and mass spectrometry analysis.

[0075] Experimental Results: Mass spectrometry identified 146 proteins interacting with HER2, of which >78% were cell membrane proteins. Known HER2 strong interacting proteins F11R, MPZL1, BCAM, and PTPRK were also detected by our method. This demonstrates the specificity and sensitivity of FITC-ID using biotin-aniline in resolving known protein interactions, providing crucial biological insights. (See appendix) Figure 15 .

[0076] Example 15: Wavelength Range Identification Experiment of FITC Catalyst Experimental Procedure: SKOV3 cells were coated with FITC antibody on HER2 protein using the same strategy as in Example 13, washed three times, and then incubated with 200 μM aryl-azido-biotin in PBS at room temperature. The cells were then irradiated for 30 min with different wavelengths of light (UV: 340-390 nm; violet: 425-445 nm; blue: 460-495 nm; cyan: 490-500 nm; green: 530-550 nm). A sample without the FITC catalyst was used as a negative control. The optimal wavelength range for the catalyst was determined by measuring the ratio of the biotinylated protein signal intensity in the FITC-coated group to that in the non-FITC group within the same wavelength range. (See Appendix) Figure 16 .

[0077] Experimental results: When the illumination wavelength is <450nm, there is no significant difference in biotinylation signal intensity between the group with and without the catalyst, proving that this wavelength range is not within the catalytic range of FITC. When the wavelength is >530nm, there is also no significant difference in biotinylation signal intensity between the group with and without the catalyst, proving that this wavelength range is also not within the catalytic range of FITC. However, when the wavelength is between 450-530nm, the signal intensity of the group with the FITC catalyst is more than 6 times stronger than that of the group without the FITC catalyst; this range is the usable wavelength range of FITC.

[0078] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A proximity labeling method based on fluorescein conjugates, characterized in that, The luciferin conjugate was used as a catalyst to co-incubate with cells. Excess luciferin conjugate was washed off, and the substrate was added to the co-incubated cells to carry out the reaction. Light irradiation activated the luciferin, which then catalyzed the labeling of other molecules around it.

2. The proximity marking method according to claim 1, characterized in that, The fluorescein conjugate is either a FITC conjugate or a FAM conjugate.

3. The proximity marking method according to claim 1, characterized in that, The substrate contains phenolic compounds, aryl azide compounds, or aniline compounds.

4. The proximity marking method according to claim 3, characterized in that, The substrate is an aniline compound.

5. The proximity marking method according to claim 1, characterized in that, The illumination marking time is from 5 minutes to 60 minutes.

6. The proximity marking method according to claim 1, characterized in that, The light source is an LED lamp or a laser with a wavelength range of 450-530nm.

7. The proximity marking method according to claim 1, characterized in that, The luciferin conjugate is incubated with cells for 15 minutes to 6 hours, and the concentration of the luciferin conjugate is 0.1 ug / ml to 0.1 mg / ml.

8. The proximity marking method according to claim 1, characterized in that, The marking radius ranges from 50 nm to 300 nm.

9. The proximity marking method according to claim 1, characterized in that, The cells include tumor cells, immune cells, epithelial cells, mesenchymal cells, or nerve cells.

10. The application of the proximity labeling method according to any one of claims 1 to 9 in the detection of molecular and cellular interactions.

11. The application according to claim 10, characterized in that, The molecules include small molecules, proteins, DNA, RNA, or sugars.

12. A kit for detecting molecular and cellular interactions, comprising a photosensitive catalyst, a substrate, a washing solution, and a light source; said photosensitive catalyst is a fluorescein conjugate.

13. The reagent kit according to claim 12, characterized in that, The fluorescein conjugate is either a FITC conjugate or a FAM conjugate.

14. The kit according to claim 12, characterized in that, The substrate contains phenolic compounds, aryl azide compounds, or aniline compounds.

15. The kit according to any one of claims 12 to 14, characterized in that, The kits include siRNA interaction protein detection kits, clinical sample cell interaction detection kits, or membrane proteomics detection kits.

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