A responsive fluorescent probe targeting cell membrane and preparation method and application thereof

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a cell membrane-targeting responsive fluorescent probe, its preparation method, and its application. The structure of the fluorescent probe is shown below. When this responsive fluorescent probe is used to detect T cell activation, after 20 minutes of incubation, the fluorescence intensity of activated Psi+ cells is 1.6 times that of CD69+ cells and 2.9 times that of CD25+ cells. The response sensitivity far exceeds that of traditional biomarkers, and the fluorescence signal intensity is positively correlated with the degree of activation, resulting in high quantitative accuracy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a cell membrane-targeting responsive fluorescent probe, its preparation method, and its application. Background Technology

[0002] The immune system, as the body's core defense system, is closely related to the development and progression of exogenous infectious diseases, tumors, aging, and other chronic diseases due to changes in its function. The weakening of immune surveillance directly participates in the progression of chronic diseases, and the clinical benefits of immunotherapy (such as complete remission in some patients with advanced cancer) further highlight the necessity of comprehensive immune function evaluation—an evaluation closely related to disease course, treatment efficacy, and prognosis. Current mainstream immune cell detection relies on fluorescently labeled antibodies combined with flow cytometry, but existing methods monitor only one or a few biological markers, failing to comprehensively reflect the activation state of cells. This results in insufficient sensitivity and limited quantitative accuracy, lacking a simple, efficient, and accurate evaluation method that comprehensively reflects the activation state of immune cells, making it difficult to meet the urgent clinical needs for early disease diagnosis and real-time monitoring of treatment efficacy.

[0003] Existing immune function evaluation technologies have multiple limitations: cutting-edge technologies such as next-generation sequencing (nucleic acid level methylation mapping, TCR / BCR diversity detection) and flow cytometry (multi-marker identification) can provide comprehensive analysis, but they are complex to operate, costly to detect, and rely on special equipment or reagents, and are still in the research stage and cannot be widely used; traditional peripheral blood biochemical tests can only reflect changes in the number / proportion of lymphocytes, and have low sensitivity for early disease diagnosis; imaging diagnosis has a lag when used to evaluate the efficacy of immunotherapy, and even false negatives occur in the early stages, which cannot guide clinical medication in a timely manner; in addition, the activation markers commonly used in existing flow cytometry detection (such as CD69 and CD25) have insufficient response sensitivity, making it difficult to accurately quantify the degree of immune cell activation, which limits the reliability of evaluation results.

[0004] The cell membrane, as a vital organelle, maintains the stability of the intracellular environment. Composed of different types of phospholipids, cholesterol, and proteins, the cell membrane possesses complex biophysical properties (polarity, viscosity, and electrostatic properties, etc.), among which viscosity plays a crucial role in biological processes such as membrane fluidity, transmembrane transport, and biological signal transduction. While changes in cell membrane viscosity have been associated with various diseases, their relationship with immune cell activation remains completely unknown. Biological experiments have confirmed that activated immune cells form immune synapses, thereby triggering protein signal transduction. Metabolomics evidence indicates that the composition and metabolism of phospholipid structures in the cell membranes of activated immune cells undergo significant changes, suggesting a close correlation between changes in cell membrane viscosity and immune cell activation. Therefore, developing a functional fluorescent probe that reflects and identifies activated immune cells would provide a more sensitive, convenient, and rapid detection method for assessing the functional state of immune cells. Summary of the Invention

[0005] Based on this, the present invention provides a fluorescent probe targeting the cell membrane, its preparation method and application, which can realize the detection of the activation state of T cells.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: This invention provides a cell membrane-responsive fluorescent probe, the structure of which is shown below: , where n is any integer from 0 to 7.

[0007] Preferably, in the above fluorescent probe, n is 0, 1, 4 or 7.

[0008] Another aspect of the present invention provides an intermediate, the structural formula of which is shown below: , where the value of n is the same as that of the above-mentioned responsive fluorescent probes.

[0009] In another aspect, the present invention provides a method for preparing the above-mentioned responsive fluorescent probe, the method comprising: (1) Mix the compound shown in Formula I and the compound shown in Formula II, and heat the mixture to obtain the compound shown in Formula III; (2) Mix the compound shown in Formula III and the compound shown in Formula IV, and heat the mixture to obtain a responsive fluorescent probe; The structural formulas of compounds shown in Formula I, Formula II, Formula III, and Formula IV are shown below: The compound shown in Formula I: ; The compound shown in Formula II: ; Compounds shown in Formula III: ; Compounds shown in Formula IV: ; The value of n is the same as that of the responsive fluorescent probes described above.

[0010] In another aspect, the present invention provides a reagent for detecting T cell activation status, which includes the above-mentioned responsive fluorescent probe.

[0011] Preferably, the T cell activation status detection reagent further includes one or more of phosphate buffer solution, ACK lysis buffer, or fixative.

[0012] In another aspect, the present invention provides the application of the above-mentioned responsive fluorescent probe in detecting the cell activation state of T cells.

[0013] Preferably, in the above applications, the responsive fluorescent probe detects the cell activation state of T cells by targeting the cell membrane of T cells.

[0014] More preferably, in the above applications, the responsive fluorescent probe detects the cell activation state of T cells by detecting the viscosity of the T cell membrane.

[0015] Preferably, in the above applications, the T cells are CD3+ T cells.

[0016] The beneficial effects of this invention include: When detecting T cell activation, the responsive fluorescent probe (Psi) provided by this invention shows that the fluorescence intensity of Psi+ cells in the activated group after 4 hours of incubation is 1.6 times that of CD69+ cells and 2.9 times that of CD25+ cells, demonstrating a response sensitivity far exceeding that of traditional markers. Furthermore, the fluorescence signal intensity is positively correlated with the degree of activation, resulting in high quantitative accuracy. The probe labeling requires no washing, and detection can be performed with a short incubation time of 20 minutes, making the method simple. Attached Figure Description

[0017] Figure 1 Structural characteristics and synthetic routes of reactive fluorescent probes (Psi); Figure 2 The fluorescence response spectra of a reactive fluorescent probe (Psi) under different viscosity conditions are shown. Figure 3 Flow cytometry results of immune cells using responsive fluorescent probes (Psi) with different structures; Figure 4 The results of flow cytometry detection of different immune cells in blood by a responsive fluorescent probe (Psi); Figure 5 Flow cytometry detection of a reactive fluorescent probe (Psi) method in an immune cell activation model; Figure 6This study investigates the relationship between the fluorescence intensity of the cell membrane and phospholipid metabolism after activation of immune cells by a responsive fluorescent probe (Psi). Detailed Implementation

[0018] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0019] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0020] In a first aspect, embodiments of the present invention provide a cell membrane-targeting responsive fluorescent probe (Psi), the structure of which is shown below: , where n is any integer from 0 to 7.

[0021] It should be noted that, in addition to its lipophilic structure, the fluorescent probe of this invention features a negatively charged hydrophilic sulfonic acid group (in contrast to the positively charged group), which significantly enhances its binding force to immune cell membranes, enabling rapid, wash-free, one-step fluorescent recognition and detection. Before and after immune cell activation, cell membrane viscosity increases; after 20 minutes of labeling with this probe, it can be directly quantified and detected using flow cytometry. Specifically, the fluorescent probe of this invention is an amphiphilic molecule with a rotatable double bond structure. Free probe molecules experience fluorescence quenching due to double bond rotation. When the lipophilic end (dimethylamine portion) is embedded in the cell membrane, double bond rotation is restricted, resulting in weak fluorescence emission. As cell membrane viscosity increases, the fluorescence intensity further enhances, producing AIE (Aggregation-induced effects) efficacy. Furthermore, the introduction of the sulfonic acid group increases the water solubility of the fluorescent probe, preventing self-aggregation in a biological aqueous environment and thus avoiding non-specific fluorescence. Compared to probes modified with other positively charged groups at the same position, at the same staining concentration and staining time, the sulfonic acid-modified probe Psi shows a higher staining positivity rate for immune cells.

[0022] It should also be noted that T cell activation is closely related to cell membrane phospholipid metabolism. Changes in probe fluorescence intensity directly depend on phospholipase-mediated phospholipid metabolism and are specifically located on the cell membrane. This allows for precise reflection of the metabolic state during T cell activation and can be used for real-time monitoring of phospholipid metabolism during T cell activation.

[0023] In some specific examples, n in the fluorescent probes described above is 0, 1, 4, or 7.

[0024] It should be noted that the value of n in the fluorescent probe of this invention can be 0, 1, 4, or 7, i.e., C n H 2n+1 The group can preferably be H, methyl, butyl, or heptyl. More preferably, it is 1, i.e., C. n H 2n+1 The group is methyl.

[0025] Secondly, embodiments of the present invention provide an intermediate, the structural formula of which is shown below: , where the value of n is the same as that of the above-mentioned responsive fluorescent probes.

[0026] Thirdly, embodiments of the present invention provide a method for preparing the above-mentioned fluorescent probe, the method comprising: (1) Mix the compound shown in Formula I and the compound shown in Formula II, and heat the mixture to obtain the compound shown in Formula III; (2) Mix the compound shown in Formula III and the compound shown in Formula IV, and heat the mixture to obtain a responsive fluorescent probe; The structural formulas of compounds shown in Formula I, Formula II, Formula III, and Formula IV are shown below: The compound shown in Formula I: ; The compound shown in Formula II: ; Compounds shown in Formula III: ; Compounds shown in Formula IV: ; The value of n is the same as that of the responsive fluorescent probes described above.

[0027] It should be noted that the synthesis steps of the responsive fluorescent probe in this invention are simple, and the probe synthesis can be completed in just two steps.

[0028] Fourthly, embodiments of the present invention provide a reagent for detecting T cell activation status, which includes the above-mentioned responsive fluorescent probe.

[0029] It should be noted that cells with different characteristics have different cell membrane compositions. After immune cell activation, the phospholipid structure of the cell membrane changes the local cell membrane viscosity, leading to signal transduction. The fluorescent probe further enhances the fluorescence emission signal due to its AIE efficacy. Therefore, this probe staining speed is fast, and the free probe in the liquid does not produce signal background interference, achieving wash-free imaging and flow cytometry quantitative detection, realizing a one-step detection of immune cell activation.

[0030] In some specific examples, the T cell activation status detection reagent mentioned above also includes one or more of phosphate buffer solution, ACK lysis buffer, or fixative.

[0031] Fifthly, embodiments of the present invention provide an application of the above-mentioned fluorescent probe in detecting the cell activation state of T cells.

[0032] In some specific examples, in the above applications, responsive fluorescent probes detect the cell activation status of T cells by targeting the cell membrane of T cells.

[0033] It should be noted that the responsive fluorescent probe in this invention is related to the phospholipid metabolism of the cell membrane in detecting the activation state of T cells.

[0034] In some specific examples, in the above applications, responsive fluorescent probes detect the cell activation status of T cells by detecting the viscosity of the T cell membrane.

[0035] It should be noted that, in environmental viscosity detection, the fluorescent probe provided by this invention exhibits a 7081-fold increase in fluorescence signal at 582 nm when the environmental viscosity increases from 1 cp to 1499 cp, demonstrating high sensitivity and specificity, and accurately covering the detection requirements across a wide range of viscosity levels. In tumor peripheral blood, the changes in immune cells are complex. As tumor progression occurs, the proportion of lymphocytes decreases, with a decrease in the number of CD4+ T cells and a slight increase in CD8+ T cells. The fluorescent probe of this invention, combined with cell population markers of different subgroups within CD8+ cells, shows different positive proportions in the T naive, T central memory, and T effector subgroups. These proportions are correlated with activation and proliferation signals, and the probe exhibits higher sensitivity.

[0036] In some specific examples, the T cells in the above applications are CD3+ T cells.

[0037] It should be noted that the fluorescent probe provided by this invention has a positive rate of ≥60% for CD3+ T cells and for CD11b cells in immune cell recognition. +With a cell positivity rate ≤10%, clear targeting, good cell compatibility, and no obvious toxicity, this probe can accurately distinguish CD3+ T cells. Furthermore, in cell membrane phospholipid metabolism studies, this probe specifically locates on the cell membrane, and changes in fluorescence intensity directly depend on phospholipase-mediated phospholipid metabolism. It can accurately reflect the metabolic state related to changes in cell membrane viscosity, serving as a "visual sensor" for phospholipid metabolism reflecting the increased local cell membrane viscosity caused by immune cell activation. Simultaneously, this probe is applicable to various T cell types, including mouse immune cells and human peripheral blood lymphocytes, is compatible with flow cytometry and fluorescence microscopy, can be stored at 0–4℃, is easy to operate, and has a wide range of applications and is easy to promote.

[0038] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0039] Preparation Examples In the following embodiments, the synthesis route of the fluorescent probe is as follows: Figure 1 As shown.

[0040] Example 1: Synthesis and Characterization of Responsive Fluorescent Probes (Psi) (1) Synthesis of intermediate: 1 mmol of 2-(4-pyridyl)acetonitrile (the compound shown in Formula II) and 2 mmol of 4-dimethylaminobenzaldehyde (the compound shown in Formula I (n=1)) were dissolved in acetonitrile and reacted under nitrogen protection at a temperature of 90 °C for 6 h. After the reaction was completed, the reaction system was cooled and the yellow solid was collected by filtration, which is the intermediate (as shown in Formula III (n=1)). (2) Synthesis of fluorescent probe: 1 mmol of the intermediate synthesized in step (1) above and 1 mmol of 1,3-propanesulfonyl lactone (the compound shown in formula IV) were dissolved in anhydrous ethanol and heated under nitrogen protection at a temperature of 90°C for 4 h. After the reaction was completed, the solvent was removed by rotary evaporator. The crude product was subjected to silica gel column chromatography to obtain a dark red solid, which is the responsive fluorescent probe (as shown in formula V (n=1), also known as Psi).

[0041] (3) Characterization of the responsive fluorescent probe Psi The proton NMR spectrum, carbon NMR spectrum, and high-resolution mass spectrometry data of the fluorescent probe Psi prepared in Example 1 (n=1) are as follows: 1H NMR spectrum: 1H NMR (400 MHz, DMSO) δ 8.81 (d, J = 6.3 Hz, 2H), 8.41(s, 1H), 8.03 (d, J = 6.3 Hz, 2H), 7.78 (d, J = 8.2 Hz, 2H), 6.71 (d, J = 8.2Hz, 2H), 4.47 (t, J = 7.3 Hz, 2H), 3.01 (s, 6H), 2.38 (t, J = 7.5 Hz, 2H), 2.36 – 2.25 (m, 2H); Carbon NMR spectrum: 13 C NMR (100 MHz, DMSO) δ 157.05, 152.56, 150.37, 145.03,146.08, 128.71, 128.60, 124.62, 124.53, 124.06, 118.82, 116.93, 116,84,99.31, 56.33, 44.64, 41.33, 25.84; High-resolution mass spectrometry (HR-ESI-MS): m / z calcd for [M+H] + ([C) 19 H 22 N3O3S] + ):372.1376, found:372.1298.

[0042] Example 2: Spectral performance testing of a responsive fluorescent probe (Psi) (1) Preparation of stock solution for responsive fluorescent probe (Psi) The responsive fluorescent probe (Psi) prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 20 mM standard solution, namely the fluorescent probe stock solution. After being dispensed, it was stored at 0-4℃ for use. The fluorescence spectrum was tested in a 1 cm × 1 cm quartz cuvette. The fluorescent probe stock solution was diluted to 10 μM-20 μM in a mixed solvent of glycerol and methanol before fluorescence spectrum testing.

[0043] (2) Fluorescence response test of fluorescent probe Psi to changes in environmental viscosity: The probe stock solution was uniformly dispersed in glycerol-methanol (Gly-MeOH) mixed solutions with different volume ratios (glycerol to methanol volume ratios of 0:10, 2:8, 4:6, 6:4, 8:2, and 10:0, respectively). The fluorescence signal of the probe in solutions of different viscosities was measured using a fluorescence spectrophotometer (Shanghai Tianmei FL970). The excitation wavelength was 470 nm. The test results are as follows: Figure 2As shown, the results indicate that the fluorescence signal intensity of Psi near 582 nm increases with increasing ambient viscosity; when the ambient viscosity increases from 1 cp to 1499 cp, the fluorescence signal of Psi at 582 nm increases by 7081 times, indicating that the fluorescent probe can specifically detect viscosity changes.

[0044] Example 3: Efficacy of responsive fluorescent probes (Psi) for immune cell recognition Three structurally similar fluorescent probes, under the same experimental conditions, showed that the Psi probe outperformed PsiA and PsiB (structures shown in [link to PsiA and PsiB]). Figure 3 The highest recognition efficacy was observed for immune cells. Specific testing was conducted as follows: Frozen E6-1 cells were removed from liquid nitrogen, rapidly thawed in a 37°C water bath, and then 5 mL of RPMI-1640 complete culture medium was added. The cells were centrifuged at 1000 rpm for 5 min (room temperature), and the supernatant was discarded. Cells were resuspended in complete culture medium, seeded into cell culture flasks, and cultured in a CO2 incubator until the logarithmic growth phase. Cells were collected, resuspended in PBS + 2% FBS, and adjusted to a final volume of 100 μL. Fluorescent probes Psi, PsiA, and PsiB (10 μM) were added, and the cells were incubated at 4°C for 20 min in the dark. After incubation, the cells were washed once with 1 mL of flow cytometry staining buffer (PBS + 2% FBS), and finally resuspended in 300 μL of flow cytometry staining buffer. The cells were then analyzed using a flow cytometer (CytoFLEX). Results are as follows: Figure 3 As shown, Psi has a significantly higher recognition efficiency for immune cells than the other two probes (PsiA and PsiB).

[0045] Example 4: Recognition of peripheral blood CD3+ T cells by responsive fluorescent probes (Psi) 200 μL of blood was collected from the mouse orbital cavity and added to a centrifuge tube containing EDTA. The mixture was stirred to prevent coagulation. Three volumes of ACK lysis buffer were added, and the mixture was incubated at room temperature for 15 min. Then, it was centrifuged at 300 × g for 5 min at 4 °C. The supernatant was discarded to obtain immune cells. The immune cells were washed twice with PBS + 2% FBS, and the concentration was adjusted to 1 × 10⁻⁶ cells / mL. 6 Cells were prepared at a concentration of 100 μL / mL, with a final volume of 100 μL. Following the antibody instructions, fluorescently labeled antibodies PE anti-mouse-CD3 Antibody (Biolegend, catalog number 100206) and APC anti-mouse-CD11b Antibody (Biolegend, catalog number 101206), and 1 μL of 1 mM fluorescent probe Psi (prepared in Example 1) were added. The mixture was incubated at 4°C for 20 min in the dark. After incubation, the cells were washed once with 1 mL of flow cytometry staining buffer (PBS + 2% FBS), and finally resuspended in 300 μL of flow cytometry staining buffer. The cells were then analyzed using a flow cytometer (CytoFLEX). Results showed that... Figure 4As shown, CD3 + T cell probe positivity rate ≥60%, CD11b + A probe positivity rate of ≤10% in cells indicates that the chemical probes primarily recognize CD3. + T cells.

[0046] Example 5: Detection of activated lymphocytes in human peripheral blood using responsive fluorescent probes (Psi). Take a 1.5 mL sterile centrifuge tube, add 200 μL of EDTA-anticoagulated peripheral blood, then add 800 μL of ACK erythrocyte lysis buffer (blood:lysis buffer (volume ratio) = 1:4, to ensure complete erythrocyte lysis), gently invert to mix, and incubate at room temperature in the dark for 5 minutes (observe the liquid change from red to clear, indicating complete erythrocyte lysis). Add 5 times the volume of PBS to stop lysis, centrifuge, wash, and resuspend in complete culture medium. Count the cells and adjust the cell concentration to 1 × 10⁻⁶. 6 Cells / mL; Add 1 mL of cell suspension to each well, add PMA (final concentration 20 ng / mL), and incubate at 37°C and 5% CO2 for 5 h; Coat the culture plate with anti-CD3 antibody beforehand (overnight at 4°C), discard the coating, add 1 mL of cell suspension and anti-CD28 antibody (final concentration 2 μg / mL), and incubate at 37°C and 5% CO2 for 5 h; After incubation, collect the cells, resuspend them in PBS + 2% FBS and adjust the final volume to 100 μL, and add fluorescently labeled antibody APC anti-human CD69 Antibody (Biolegend, catalog number 3) according to the antibody instructions. 10905), PE anti-human CD25 Antibody (Biolegend, catalog number 302605), and fluorescent probe Psi staining solution (take 1 μL of stock solution, dilute with PBS to a final concentration of 10 μM), mix gently; incubate at 4°C in the dark for 20 min; after incubation, add 1 mL of flow cytometry staining buffer (PBS + 2% FBS), centrifuge at 1000 rpm for 5 min, discard the supernatant; repeat washing twice, and finally resuspend the cells in 300 μL of flow cytometry staining buffer (PBS + 2% FBS); place the flow cytometry tube on the instrument sample loading stage, and collect 1×10⁹ cells per sample. 4 -1×10 5 Individual cells; lymphocyte populations were delineated using forward scattered light (FSC) and side scattered light (SSC) (excluding cell debris), and then CD69 cells were delineated within the lymphocyte population. + CD25 + Psi + Cell proportion and mean fluorescence intensity (MFI); each sample was tested three times, and the raw fluorescence signal data were recorded and saved in FCS format for subsequent analysis; CD25 in the control group and activation group were calculated using FlowJo software. + CD69 +Psi + Mean fluorescence intensity (MFI) of cells.

[0047] The results are as follows Figure 5 As shown, the results indicate that the proportion of positive cells recognized by the chemical probe Psi is positively correlated with the expression levels of activation markers CD69 and CD25. Furthermore, the MFI (Mean Function Index) of cells posited by the fluorescent probe Psi+ was significantly higher than that of cells posited by CD69. + CD25 + Cell percentage, specifically, at 4 hours of incubation, the percentage of Psi in the activated group (anti-CD3 / CD28). + Cellular MFI is CD69 + The response intensity of the membrane fluorescent probe to activation signals was 1.6 times that of CD69 cells and 2.9 times that of CD25+ cells, indicating that the response intensity of the membrane fluorescent probe to activation signals was much higher than that of CD69 and CD25 antibodies.

[0048] Example 6: Tests on the correlation between fluorescent probe enhancement effect and cell membrane phospholipid metabolism after T cell activation (1) Immune cell activation model: Frozen E6-1 cells were removed from liquid nitrogen, rapidly thawed in a 37°C water bath, and then 5 mL of RPMI-1640 complete culture medium was added. The cells were centrifuged at 1000 rpm for 5 min (room temperature), and the supernatant was discarded. The cells were resuspended in complete culture medium, seeded into 6-well culture dishes, and cultured in a CO2 incubator until the logarithmic growth phase. The cell concentration was adjusted to 1×10⁻⁶ cells / well with complete culture medium. 6 Cells / mL were seeded into new 6-well culture dishes (2 mL per well) according to the following groups: Control group: only complete culture medium was added, without adding activating stimulants, and then incubated in a 5% CO2 incubator at 37°C for 4-6 h; Activation group: the culture plate was coated with anti-CD3 antibody in advance (4°C overnight), the medium was discarded, and 1 mL of cell suspension and anti-CD28 antibody (final concentration 2 μg / mL) were added, and incubated at 37°C and 5% CO2 for 5 h; Blocking group: 1 mL of cell suspension was first added with 10 μM of sphingomyelinase inhibitor Malabaricone C (MCE, catalog number HY-N8518), and incubated in a 37°C and 5% CO2 incubator for 1 h, and then transferred to a culture plate coated with anti-CD3 antibody in advance, and anti-CD28 antibody (final concentration 2 μg / mL) was added, and incubated at 37°C and 5% CO2 for 5 h.

[0049] (2) Fluorescence microscopy observation: After incubation, the cells were centrifuged and the culture medium was carefully aspirated. 1 mL of PBS was added and the cells were gently washed three times, 5 min each time. 1 mL of 4% paraformaldehyde fixative was added to each well, and the cells were incubated at room temperature in the dark for 15 min (to fix the cell membrane structure and prevent marker diffusion). The fixative was aspirated, and the cells were washed three times with PBS, 5 min each time. 1 mL of fluorescent probe dilution buffer (10 μM, used to cover the slide) was added to each well, and the cells were incubated in the dark for 20 min. 3 washes with PBS were performed three times, 5 min each time, to remove unbound fluorescent probes. 1 mL of DAPI staining solution (final concentration 1 μg / mL) was added to each well, and the cells were incubated at room temperature in the dark for 5 min. 5-10 fields of view were randomly selected from each group, and bright-field images (to observe cell morphology), DAPI channel images (to locate the cell nucleus and define the cell membrane boundary), and PE / cy5 channel images (to observe the probe's localization in the cell) were acquired. Results are as follows: Figure 6 As shown in Figure A, the results indicate that the fluorescent probes are specifically localized on the cell membranes of T cells. Compared with the control group, the fluorescence intensity of the cell membrane probes in the activation group (anti-CD3 / anti-CD28) was significantly enhanced. In contrast, the fluorescence intensity of the cell membrane probes in the blocking group pretreated with the sphingomyelinase inhibitor Malabaricone C was significantly lower than that in the activation group.

[0050] (3) Flow cytometry detection: After incubation, the cells were centrifuged and the culture medium was carefully discarded. The cells were washed once with PBS + 2% FBS, and the concentration of immune cells was adjusted to 5 × 10⁻⁶. 5 Cells were cultured at a concentration of 100 μL / mL, with a final volume of 100 μL. Following the antibody instructions, fluorescently labeled antibodies PE anti-human-CD25 Antibody (Biolegend, catalog number 302605) and APC anti-human-CD69 Antibody (Biolegend, catalog number 310909) and fluorescent probe Psi (1 μL of stock solution diluted to a final concentration of 10 μM) were added. The cells were incubated at 4°C for 20 min in the dark. After incubation, the cells were washed once with 1 mL of flow cytometry staining buffer (PBS + 2% FBS), and finally resuspended in 300 μL of flow cytometry staining buffer. The cells were then analyzed using a flow cytometer (CytoFLEX). The results are as follows: Figure 6 B and Figure 6As shown in Figure C, the results indicate that the Anti-CD3 / CD28 functional antibody can effectively activate E6-1 cells, significantly increasing the mean fluorescence intensity (MFI) of the traditional activation markers CD25 and CD69 on the cell surface. After activation, the fluorescence intensity of the fluorescent probe Psi is significantly higher than that of CD25 and CD69, showing a more prominent response to cell activation. Pretreatment with the sphingomyelinase inhibitor Malabaricone C can effectively inhibit the fluorescence enhancement of Psi and the high expression of CD25 and CD69 by blocking the phospholipid metabolism pathway, confirming that phospholipid metabolism is a key regulatory link in the activation of immune cells.

[0051] The above results show that in the human T cell activation model, fluorescence microscopy revealed that the probe's fluorescence was located on the T cell membrane, and the local fluorescence intensity significantly increased after CD3+CD28 stimulation. This chemiluminescence enhancement signal was significantly reduced by sphingomyelinase inhibitors. Flow cytometry results further confirmed that the fluorescence enhancement effect of the Psi probe is activation-dependent and phospholipid metabolism-specific, and its sensitivity to immune cell activation is significantly better than that of CD25 and CD69.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A cell membrane-responsive fluorescent probe, the structure of which is shown below: wherein, n is any integer from 0 to 7.

2. The responsive fluorescent probe according to claim 1, characterized in that, n can be 0, 1, 4, or 7.

3. The intermediate, whose structural formula is shown below: ,in, The value of n is the same as that of the fluorescent probe described in claim 1 or 2.

4. The method for preparing the responsive fluorescent probe according to claim 1 or 2, characterized in that, Preparation methods include: (1) Mix the compound shown in Formula I and the compound shown in Formula II, and heat the mixture to obtain the compound shown in Formula III; (2) Mix the compound shown in Formula III and the compound shown in Formula IV, and heat the mixture to obtain a responsive fluorescent probe; The structural formulas of compounds shown in Formula I, Formula II, Formula III, and Formula IV are shown below: The compound shown in Formula I: ; The compound shown in Formula II: ; Compounds shown in Formula III: ; Compounds shown in Formula IV: ; Wherein, the value of n is the same as that of the fluorescent probe described in claim 1 or 2.

5. A reagent for detecting T cell activation status, characterized in that, Includes the responsive fluorescent probe as described in claim 1 or 2.

6. The T cell activation status detection reagent according to claim 5, characterized in that, T-cell activation status detection reagents also include one or more of phosphate buffered saline solution, ACK lysis buffer, or fixative.

7. The application of the responsive fluorescent probe according to claim 1 or 2 in detecting the cell activation state of T cells.

8. The application according to claim 7, characterized in that, Responsive fluorescent probes detect the cell activation status of T cells by targeting the cell membrane of T cells.

9. The application according to claim 8, characterized in that, Responsive fluorescent probes detect the cell activation status of T cells by detecting the viscosity of the T cell membrane.

10. The application according to any one of claims 7 to 9, characterized in that, The T cells are CD3+ T cells.

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