Cell surface cytokine enrichment system and method based on biological coupling strategy

Abstract

The invention belongs to the technical field of biology, and provides a system and a method for enriching to-be-detected cytokines on the surface of a cell based on a biological coupling strategy, so as to enrich the to-be-detected cytokines on the surface of the cell and ensure that a capture and detection probe is stably constructed on a cell membrane and is not internalized. Depending on a cell membrane surface biological coupling strategy of efficient binding force of biotin and streptavidin, Sulfo-NHS-LC-Biotin with negative sulfo groups is adopted to weaken the permeability of a cell membrane, effective biotinylation of cell membrane protein is realized, a capture probe and a detection probe with streptavidin are combined with the biotinylated cell membrane protein, and the biotinylated cell membrane protein is detected. According to the present invention, the probe is ensured to be stably constructed on the cell membrane, the internalization is avoided, the capture probe is specifically combined with the cell surface to-be-detected cell factor to achieve enrichment, the accuracy and the reliability of the cell surface to-be-detected cell factor enrichment are improved, the operation is simple, and the powerful tool is provided for the cell biology research and the clinical diagnosis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method and system for enriching cytokines to be tested on the surface of cells. Background Art

[0002] In cell biology research and clinical diagnosis, accurate detection of cytokines on the cell surface is of great significance. However, current methods face many challenges in achieving the enrichment of cytokines on the cell surface, such as difficulty in ensuring that the capture and detection probes are stably constructed on the cell membrane and are not internalized. Summary of the invention

[0003] The present invention relies on the high efficiency of biotin and streptavidin to propose a bioconjugation strategy on the cell membrane surface. Sulfo-NHS-LC-Biotin is a sulfonated N-hydroxysulfosuccinimide ester (NHS-B) of biotin. The presence of its negatively charged sulfonyl group weakens the permeability of the cell membrane and can effectively biotinylate cell membrane proteins. This bioconjugation strategy can effectively prevent nanoparticles from being internalized into cells.

[0004] The present invention provides a method for enriching cytokines to be detected on the cell surface, comprising the following steps: Step S1, biotinylating cell membrane proteins using Sulfo-NHS-LC-Biotin, wherein Sulfo-NHS-LC-Biotin is a sulfonated N-hydroxysulfosuccinimide ester of biotin, and its negatively charged sulfonyl group reduces the permeability of the cell membrane, thereby effectively biotinylating the cell membrane proteins; Step S2, combining the capture probe and the detection probe with streptavidin with the biotinylated cell membrane protein, and utilizing the efficient binding force of biotin and streptavidin to ensure that the probe is stably constructed on the cell membrane and is not internalized; Step S3, the capture probe specifically binds to the cytokine to be tested on the cell surface, thereby achieving enrichment of the cytokine to be tested on the cell surface.

[0005] In certain embodiments, the probe is a gold nanoparticle probe or a silver nanoparticle probe.

[0006] The present invention has the following effects compared with the prior art: 1) It effectively ensures that the capture and detection probes can be stably constructed on the cell membrane, avoiding the problem of internalization and improving the accuracy and reliability of enrichment of the cytokines to be tested; 2) The operation steps are clear and easy to implement, providing an efficient method for the detection and analysis of cytokines to be tested on the cell surface; 3) The high binding capacity of biotin and streptavidin is utilized to ensure the stability and repeatability of the experiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] In order to more clearly illustrate the specific implementation manner of the present invention or the technical solution in the prior art, the drawings required for describing the specific implementation manner are briefly introduced below.

[0008] Figure 1 This is a schematic diagram of the sandwich structure in Example 3.

[0009] Figure 2 This is the confocal fluorescence imaging image of the cells in Example 6.

[0010] Figure 3 The activity of VEGF secreted by MCF-7 cells in Example 6 after being gradually functionalized by NHS-B / streptavidin and CPs.

[0011] Figure 4 This is the characterization of the silica nanoparticles and nanoprobes prepared in Example 5.

[0012] Figure 5 This is the fluorescence confocal detection of VEGF concentration in Example 6.

[0013] Figure 6 The VEGF secretion levels of the three cell lines in Example 6 are shown.

[0014] Figure 7 This is Example 7, where fluorescence activated imaging-flow cytometry was used to detect VEGF secretion by population cells. DETAILED DESCRIPTION

[0016] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the implementation cases are only used to illustrate and explain the present invention, and are not used to limit the present invention described in detail in the claims. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional methods unless otherwise specified, and the test materials used can be obtained from commercial companies unless otherwise specified.

[0017] Example 1 Functionalization of cell surface with NHS-B and streptavidin When the cells were at 80% to 90% confluency, they were treated with ice-cold PBS (pH = 7.4) three times to remove ammonia-containing medium and proteins from the cells, and then incubated in Sulfo-NHS-LC-biotin PBS solution (2.0 mM, pH = 7.4) for 30 min at 4°C. After biotinylation, the cells were washed three times with PBS solution (pH = 7.4). Then, streptavidin was attached to the biotinylated cells. The cells were treated with 2.0 mM streptavidin in PBS (pH = 7.4) for 30 min at 4°C. Subsequently, the streptavidin-functionalized cells were washed three times with PBS to remove excess unbound molecules. Finally, streptavidin functionalization was completed on the cell surface for subsequent experiments.

[0018] Example 2 Cell viability assay The cells were cultured in a 20 mm glass dish for 24 h and then washed three times with cold PBS. Next, the cells were incubated with PBS solution (pH = 7.4) containing Sulfo-NHS-LC-Biotin and PBS solution containing SA at 4 °C for 30 min. Subsequently, the cells were rinsed three times with cold PBS solution and then stained with calcein-AM (2.0 μM) and PI (4.0 μM) for 15 min in the dark. Finally, the cells were washed three times with PBS solution and imaged with a fluorescence microscope (FV1000, Olympus). The excitation wavelengths of AM and PI were 488 nm and 543 nm, respectively, and the emission ranges of AM and PI were 510 nm to 530 nm and 630 nm to 700 nm, respectively.

[0019] Example 3 Validation of the immune sandwich structure When the confluence of cells cultured in 20 mm glass bottom dishes reached 80-90%, we treated the cells with Sulfo-NHS-LC-biotin and streptavidin according to the above steps and washed them three times with PBS (pH=7.4). Then, 1.0 mL DMEM medium, 110 μL FBS, 50 μL CPs labeled with human cytokeratin 20 Alexa Fluor 488 conjugated antibody (2.0 mg / mL), 50 μL RPs (2.0 mg / mL) and 0.8 μL PMA (5.0 mg / mL) were added to the cells successively and incubated for 2 h. Then, the cells were washed three times with PBS (pH=7.4) to remove excess probe molecules. Then 1.0 mL fresh PBS solution (pH=7.4) was added to the culture dish and prepared for fluorescence confocal microscopy imaging (FV1000, Olympus). Fluorescence confocal imaging of the three types of cells in the culture dish was taken (1024×1024 pixels, 20.0 μs / pixel, excitation wavelength of 488 nm, emission wavelength of 510-530 nm for CPs, and 630-660 nm for RPs).

[0020] Example 4 Silica and Ru(bpy)3 2+ Synthesis of doped silica SiO2 nanoparticles (NPs) with carboxyl surfaces were prepared by the reverse microemulsion method. Briefly, 1.77 mL of TX-100, 7.5 mL of cyclohexane, and 1.8 mL of n-hexanol were added to a flat-bottomed glass reaction bottle and stirred for 20 min. Then, 180 μL of H2O solution was dropped into the mixture, stirred for 20 min, 50 μL of TEOS was added, stirred for another 20 min, and then 60 μL of ammonia aqueous solution (29%) was added. The mixture was stirred for another 24 h at room temperature. Next, 50 μL of TOETAT solution (40% aqueous solution) was dropped into the mixture, stirred for another 24 h at room temperature, and acetone was added for demulsification. The mixture was centrifuged (11400 rpm, 10 min) and washed three times with anhydrous ethanol. Finally, it was redispersed in 10 mL of anhydrous ethanol for subsequent experiments. [Ru(bpy)3] doped 2+ The preparation method of the dye silica (F-SiO2) was the same as above, except that the above 180 μL of H2O solution contained 80 μL of 10 mM [Ru(bpy)3] 2+ Dye solution.

[0021] Example 5 Synthesis of Silica Nanoprobes Capture probe (CP) is a dual capture immunoprobe composed of human IL-8 / CXCL8 biotinylated antibody (Biotin-Ab) and VEGF monoclonal antibody (mAb VEGF ) is decorated on the surface of silica NPs. CP can simultaneously target overexpressed membrane proteins on the cell surface and VEGF factors secreted by cells. The reporter probe (RP) is a [Ru(bpy)3] 2+ Doped silica NPs, whose surface was modified with VEGF polyclonal antibody (pAb VEGF ), which is used to output fluorescence signals. The two probes are connected by VEGF secreted by single cells to form a sandwich structure. VEGF secreted by a single cell connects the two probes to form a sandwich structure on the cell membrane. It is worth noting that RPs enriched on the surface of the cell membrane are easily identified under fluorescence imaging due to their highly aggregated state. Based on the fluorescence intensity signal of RPs, the content of extracellular VEGF will be determined, while intracellular VEGF will not contribute to the signal.

[0022] The preparation process of CP is as follows: 1.0 mL of the above SiO2 solution (containing 2.0 mg of silica NP powder) was centrifuged to remove excess ethanol and redispersed in 1.0 mL of deionized water. Then, 10 μL of EDC (0.1 M) and 10 μL of NHS (0.1 M) were added and stirred for 1 h to activate the carboxyl groups on the surface of SiO2 nanoparticles. Unreacted molecules were removed by centrifugation (11400 rpm, 10 min) and then redispersed in 1.0 mL of deionized water. Next, 2.0 μL of biotinylated cell membrane protein antibody (B-Ab, 0.05 mg / mL) and 2.0 μL of vascular endothelial growth factor monoclonal antibody (mAb VEGF , 0.05 mg / mL) was added to the mixture and stirred at room temperature for 2 h, then the excess antibody was removed by centrifugation (11400 rpm, 10 min) and redispersed in 1.0 mL of deionized water. Finally, 98% ethanolamine was added and stirred for 20 min to block the activated carboxyl groups on the silica NPs. After washing again by centrifugation (11400 rpm, 10 min), the prepared SiO2@B-Ab / mAb VEGF NPs (CP) were suspended in 1.0 mL of deionized water and stored at 4 °C.

[0023] F-SiO2@pAb VEGF The preparation method of (RP) is the same as above, except that the SiO2 in CP is replaced by [Ru(bpy)3] 2+ In addition, the antibody used for RP is VEGF polyclonal antibody (pAbVEGF , 4.0 μL, 0.05 mg / mL). Finally, the obtained RP nanoparticles were stored at 4°C.

[0024] To reveal the location of CPs, the mAb was replaced by an antibody conjugated to human vascular endothelial growth factor Alexa Fluor 488. VEGF Decorate CPs to obtain fluorescence confocal imaging of CPs.

[0025] Example 6 Fluorescence confocal imaging to evaluate VEGF secretion by different cell populations When the cell confluence in 20 mm glass bottom dishes reached 80-90%, the cells were treated with Sulfo-NHS-LC-biotin and streptavidin according to the above steps, and washed three times with PBS (pH=7.4). Then, a mixture of 1.0 mL DMEM medium, 110 μL FBS, 50 μL CPs (2.0 mg / mL), 50 μL RP (2.0 mg / mL) and 0.8 μL PMA (5.0 mg / mL) was added to the cell culture dish and incubated for 2 h. Here, PMA can stimulate the cells to secrete VEGF. After washing the cells three times with PBS (pH=7.4) to remove the excess nanoprobes, 1.0 mL fresh PBS solution (pH=7.4) was added to the culture dish, and fluorescence confocal microscopy imaging (FV1000, Olympus) was performed. Fluorescence confocal imaging of three types of cells in culture dishes was taken (1024×1024 pixels, 20.0 μs / pixel, excitation wavelength 488 nm, emission wavelength 554-654 nm).

[0026] Example 7 Fluorescence imaging-flow cytometry high-throughput analysis of VEGF secreted by different cell populations Streptavidin-modified cells cultured in 100 mm glass-bottom dishes were incubated with 2.7 mL DMEM medium, 300 μL FBS, 150 μL CPs (2 mg / mL), 150 μL RPs (2.0 mg / mL), and 3.0 μL PMA (5 mg / mL) for 2 h. The cells were washed three times with PBS (pH 7.4). Then, they were digested with trypsin (2.0 mL) for 2 min. The cells were resuspended in 1.0 mL PBS (pH = 7.4) and used in FlowSight Amnis imaging-flow cytometer (EMD Millipore, Germany). 50,000 cell events were analyzed for each cell line (excitation wavelength 488 nm, emission wavelength 642-745 nm, laser power = 60 mW).

[0027] In order to achieve the enrichment of the cytokines to be tested on the cell surface and ensure that the capture and detection probes are stably constructed on the cell membrane without being internalized, the present invention proposes a cell membrane surface bioconjugation strategy that relies on the efficient binding force of biotin and streptavidin. Sulfo-NHS-LC-Biotin (sulfonated N-hydroxysulfosuccinimide ester of biotin) with negatively charged sulfonyl groups is used to weaken the permeability of the cell membrane and achieve effective biotinylation of cell membrane proteins. The capture probe and detection probe with streptavidin are combined with the biotinylated cell membrane protein to ensure that the probe is stably constructed on the cell membrane to avoid being internalized, and the enrichment is achieved by the specific binding of the capture probe to the cytokines to be tested on the cell surface. The invention improves the accuracy and reliability of the enrichment of the cytokines to be tested on the cell surface, is simple to operate, and provides a powerful tool for cell biology research and clinical diagnosis.

Claims

1. A method for enriching cytokines to be tested on the cell surface, characterized in that: The following steps are involved: Step S1, biotinylating cell membrane proteins using Sulfo-NHS-LC-Biotin, wherein Sulfo-NHS-LC-Biotin is a sulfonated N-hydroxysulfosuccinimide ester of biotin, and its negatively charged sulfonyl group reduces the permeability of the cell membrane, thereby effectively biotinylating the cell membrane proteins; Step S2, combining the capture probe and the detection probe with streptavidin with the biotinylated cell membrane protein, and utilizing the efficient binding force of biotin and streptavidin to ensure that the probe is stably constructed on the cell membrane and is not internalized; Step S3, the capture probe specifically binds to the cytokine to be tested on the cell surface, thereby achieving enrichment of the cytokine to be tested on the cell surface.

2. The method for enriching the cell surface cytokines to be detected according to claim 1, characterized in that: The step of biotinylating cell membrane proteins using Sulfo-NHS-LC-Biotin includes reacting cells with Sulfo-NHS-LC-Biotin under appropriate conditions.

3. The method for enriching cell surface cytokines to be detected according to claim 1, characterized in that: In the step of combining the capture probe and the detection probe with streptavidin with the biotinylated cell membrane protein, the binding force of biotin and streptavidin is utilized to firmly bind the probe to the cell membrane, thereby improving the sensitivity and specificity of the detection.

4. A cell surface cytokine enrichment system, characterized in that: include: (1) Biotinylation module, used to biotinylate cell membrane proteins using Sulfo-NHS-LC-Biotin; (2) a probe binding module for binding the capture probe and detection probe with streptavidin to the biotinylated cell membrane protein; (3) A module for enriching the cytokine to be tested, which is used to specifically bind the cytokine to be tested on the cell surface through a capture probe, thereby achieving enrichment of the cytokine to be tested on the cell surface.

5. The cell surface cytokine enrichment system according to claim 4, characterized in that: The biotinylation module includes a reaction condition control unit for controlling the reaction conditions between cells and Sulfo-NHS-LC-Biotin.

6. The cell surface cytokine enrichment system according to claim 4, characterized in that: The probe binding module utilizes the efficient binding force of biotin and streptavidin to ensure that the probe is stably constructed on the cell membrane, avoids internalization, and improves the sensitivity and specificity of detection.

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