A lysosome-targeting fluorescent probe for recognizing viscosity, polarity and onoo - and a preparation method and application thereof

Abstract

Lysosome-targeting fluorescent probe for recognizing viscosity, polarity and onoo ‑ and its preparation method and application, relate to a fluorescent probe and its preparation method and application.The present application is to solve the problem that the existing fluorescent probe cannot simultaneously respond to polarity, viscosity and ONOO ‑ , and the synthesis steps of the multi-modal probe are complicated, the preparation method of the fluorescent probe: 3, 3, 5-trimethoxy-3-cyclohexenone is reacted with 1, 3-propylenedinitrile to obtain compound TMEM; compound TMEM is reacted with 4-morpholinyl benzaldehyde to obtain the target compound PVN-Lyso.The viscosity, polarity and ONOO ‑ multifunctional fluorescent probe is applied to the sensing detection of viscosity, polarity and ONOO ‑ in a cell microenvironment system, and the present application is applied to the technical field of analytical chemistry.

Description

Technical Field

[0001] This invention relates to a lysosome-targeted recognition of viscosity, polarity, and ONOO. - Fluorescent probes, their preparation methods, and applications. Background Technology

[0002] Lysosomes, as important organelles in eukaryotic cells, play a crucial role not only in the processing of cellular metabolites but also in signal transduction, metabolic regulation, cell repair, autophagy, and apoptosis. The stability of lysosomes, including viscosity and polarity, is indispensable for the normal function of lysosomal membrane proteins, a prerequisite for efficient autophagy, and a fundamental condition for degradation and effective signal transduction. Changes in lysosomal polarity can disrupt key physiological processes such as cell proliferation and differentiation, potentially leading to cell death. Alterations in lysosomal viscosity have become a key driver of cardiovascular disease, neurodegenerative diseases, and even cancer. Simultaneously, reactive oxygen species (ROS) produced in the body are essential for maintaining cellular function and redox balance; particularly reactive oxygen species (ROS) such as peroxynitrite (ONO2). - As a redox messenger, it coordinates the lysosomal autophagy pathway.

[0003] Although used to detect polarity, viscosity, and ONOO - Numerous methods have emerged, but most still rely on invasive procedures. The ability to achieve real-time, in-situ, and non-invasive detection remains a critical unmet need. Fluorescent probes have become indispensable tools in pathophysiology and environmental monitoring, favored for their ease of operation, low cost, lack of sample pretreatment requirements, and ability to perform real-time monitoring and in-situ tracking. Therefore, there is an urgent need to develop a simple synthetic probe capable of simultaneously responding to polarity, viscosity, and ONOO. - Multimodal probes will greatly facilitate the early detection and prevention of related diseases.

[0004] Dicyanoisophorones, as electron-withdrawing groups commonly used to construct fluorescent probes, have become highly promising candidate systems for fluorescence imaging applications due to their excellent photophysical properties, large Stokes shift, good biocompatibility, and low toxicity. To date, numerous probes have been developed for detecting polarity, viscosity, and ONOO. - Fluorescent probes exist, but most have complex synthetic routes and limited imaging depth. Furthermore, fluorescent probes capable of simultaneously and in real-time quantification of these three parameters and possessing lysosome-specific targeting capabilities are extremely rare. Therefore, this paper aims to design a fluorescent probe that is simple to synthesize and can achieve targeting of polarity, viscosity, and ONOO. - Fluorescent probe tools for detection are of great significance for environmental protection and disease prevention. Summary of the Invention

[0005] This invention aims to solve the problem that existing fluorescent probes cannot simultaneously respond to polarity, viscosity, and ONOO. - Furthermore, addressing the cumbersome synthesis steps of multimodal probes, this paper provides a lysosomal targeted recognition method for viscosity, polarity, and ONOO. - Fluorescent probes, their preparation methods, and applications.

[0006] This invention provides a lysosomal targeted recognition method for viscosity, polarity, and ONOO. - The structural formula of the fluorescent probe is as follows:

[0007] .

[0008] This invention provides a lysosomal targeted recognition method for viscosity, polarity, and ONOO. - The preparation method of the fluorescent probe is as follows:

[0009] I. The reaction of 3,3,5-trimethoxy-3-cyclohexenone with 1,3-malononitrile yields compound TMEM:

[0010] 3,5,5-trimethoxy-3-cyclohexenone and 1,3-malononitrile were dissolved in ethanol, and piperidine was added. The mixture was heated to reflux and reacted for 10-12 h. The reaction was detected by a TCL plate. After the reaction was complete, the mixture was cooled to room temperature and deionized water was added to the solution. A precipitate was formed. The precipitate was filtered, washed, and dried to obtain compound TMEM.

[0011] The molar ratio of 3,5,5-trimethoxy-3-cyclohexenone, 1,3-malononitrile to piperidine is 1.0~1.05:1.0~1.5:0.1~0.15; the molar volume ratio of 3,5,5-trimethoxy-3-cyclohexenone to ethanol is 1.0~1.1 mmol:2.0~2.2 mL.

[0012] II. The reaction of compound TMEM with 4-morpholinobenzaldehyde yields the target compound PVN-Lyso:

[0013] Compound TMEM, 4-morpholinobenzaldehyde, and piperidine were added to a round-bottom flask containing acetonitrile. The mixture was heated for 2–4 hours, cooled to room temperature, and then cooled with deionized water. A precipitate was formed. The precipitate was filtered, washed, and dried to obtain the target compound, thus completing the preparation of the fluorescent probe. The molar ratio of compound TMEM, 4-morpholinobenzaldehyde, and piperidine was 1.0–1.05:1.0–1.05:0.1–0.15; the molar volume ratio of compound TMEM to acetonitrile was 1.0–1.1 mmol:15.0–16.5 mL.

[0014] This invention relates to viscosity, polarity, and ONOO. - Application of multifunctional fluorescent probes in cellular microenvironment systems for measuring viscosity, polarity, and ONOO- The sensing detection includes fluorescence detection and visual colorimetric detection.

[0015] This invention is fluorescent The reaction formula for the preparation of the optical probe is as follows:

[0016] Among them, PVN-Lyso is a lysoso-targeted recognition of viscosity, polarity, and ONOO. - Fluorescent probes.

[0017] The beneficial effects of this invention are:

[0018] (1) The synthesis of probes can be completed in just two steps, and the raw materials are economical and the post-processing is relatively simple;

[0019] (2) This invention enables probes to target lysosomes to recognize viscosity, polarity, and ONOO. - It has good sensing and detection capabilities, good selectivity, strong resistance to interference from other metal ions, anions, amino acids and reactive oxygen species, and low detection limit;

[0020] (3) Significant changes in fluorescence color can be observed under ultraviolet light, and viscosity and polarity can be identified through different fluorescence color regions. It is a fluorescent probe with chromogenic sensing function.

[0021] (4) This probe is effective against viscosity, polarity and ONOO. - It has a short response time and a strong fluorescence signal. It can achieve rapid, real-time detection of viscosity and polarity. Furthermore, this invention can be applied to the detection of viscosity, polarity, and ONOO within lysosomes in cells. - Fluorescence imaging is used for identification. Therefore, this invention provides a simple, rapid, and sensitive method for identifying viscosity, polarity, and ONOO. - Detection reagents and imaging tools for monitoring changes in indicators under pathophysiological conditions have broad application prospects. Attached Figure Description

[0022] Figure 1 Fluorescence spectra of probe PVN-Lyso in 1,4-dioxane / PBS solutions of different volume fractions;

[0023] Figure 2 Selectivity experiment of PVN-Lyso (10 μM) in PBS solution;

[0024] Figure 3 The effect of coexisting ions on the fluorescence intensity of PVN-Lyso (10 μM) under different polarity conditions;

[0025] Figure 4 To investigate the effect of coexisting substances on viscosity measurement under a fixed glycerol ratio;

[0026] Figure 5 The probe is PVN-Lyso (concentration of 1×10⁻⁶). -5 Fluorescence spectra of H₂O / Glycerol systems with different mol / L ratios;

[0027] Figure 6 To determine the concentrations of ONOO in a glycerol / PBS (6 / 4, v / v) system. - Effects on PVN-Lyso fluorescence spectra;

[0028] Figure 7 Confocal fluorescence imaging analysis of PVN-Lyso in four cell types: A549, BEAS-2B, HepG2, and H8.

[0029] Figure 8 To evaluate the ability of PVN-Lyso to monitor LPS-induced changes in intracellular viscosity for cell imaging experiments;

[0030] Figure 9 To evaluate the ability of PVN-Lyso to monitor the ability of DMSO treatment of A549 to induce changes in cell polarity in cell imaging experiments;

[0031] Figure 10 To evaluate the ability of PVN-Lyso to distinguish cancer cells from normal cells in cell imaging experiments;

[0032] Figure 11 The probe PVN-Lyso was used to detect changes in the microenvironment in live zebrafish using fluorescence. Detailed Implementation

[0033] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0034] Specific Implementation Method 1: This implementation method involves lysosome-targeted recognition of viscosity, polarity, and ONOO. - The structural formula of the fluorescent probe is as follows:

[0035] .

[0036] The detection of viscosity, polarity, and ONOO described in this embodiment - The fluorescent probe is denoted as probe PVN-Lyso. The probe itself is non-fluorescent due to the TICT mechanism. When the viscosity of the fluorescent probe PVN-Lyso increases or its polarity decreases, the TICT mechanism is suppressed, thereby leading to the enhancement of fluorescence and the change of fluorescence color in the detection system.

[0037] Specific Implementation Method Two: This implementation method involves lysosome-targeted recognition of viscosity, polarity, and ONOO.- The preparation method of the fluorescent probe is as follows:

[0038] I. The reaction of 3,3,5-trimethoxy-3-cyclohexenone with 1,3-malononitrile yields compound TMEM:

[0039] 3,5,5-trimethoxy-3-cyclohexenone and 1,3-malononitrile were dissolved in ethanol, and piperidine was added. The mixture was heated to reflux and reacted for 10-12 h. The reaction was detected by a TCL plate. After the reaction was complete, the mixture was cooled to room temperature and deionized water was added to the solution. A precipitate was formed. The precipitate was filtered, washed, and dried to obtain compound TMEM.

[0040] The molar ratio of 3,5,5-trimethoxy-3-cyclohexenone, 1,3-malononitrile to piperidine is 1.0~1.05:1.0~1.5:0.1~0.15; the molar volume ratio of 3,5,5-trimethoxy-3-cyclohexenone to ethanol is 1.0~1.1 mmol:2.0~2.2 mL.

[0041] II. The reaction of compound TMEM with 4-morpholinobenzaldehyde yields the target compound PVN-Lyso:

[0042] Compound TMEM, 4-morpholinobenzaldehyde, and piperidine were added to a round-bottom flask containing acetonitrile. The mixture was heated for 2–4 hours, cooled to room temperature, and then cooled with deionized water. A precipitate was formed. The precipitate was filtered, washed, and dried to obtain the target compound, thus completing the preparation of the fluorescent probe. The molar ratio of compound TMEM, 4-morpholinobenzaldehyde, and piperidine was 1.0–1.05:1.0–1.05:0.1–0.15; the molar volume ratio of compound TMEM to acetonitrile was 1.0–1.1 mmol:15.0–16.5 mL.

[0043] Specific Implementation Method 3: This implementation method differs from Specific Implementation Method 2 in that the washing described in steps one and two involves washing with deionized water five times. Everything else is the same as in Specific Implementation Method 2.

[0044] Specific Implementation Method Four: This implementation method differs from Specific Implementation Method Two or Three in that the heating temperature described in steps one and two is 75~85℃. Everything else is the same as in Specific Implementation Method Two or Three.

[0045] Specific Embodiment Five: This embodiment differs from Specific Embodiments Two to Four in that the molar ratio of 3,5,5-trimethoxy-3-cyclohexenone, 1,3-malonadionitrile, and piperidine is 1:1.5:0.1. Everything else is the same as in Specific Embodiments Two to Four.

[0046] Specific Implementation Method Six: This implementation method differs from Specific Implementation Methods Two to Five in that the molar volume of 3,5,5-trimethoxy-3-cyclohexenone and ethanol is 1 mmol: 2 mL. Everything else is the same as in Specific Implementation Methods Two to Five.

[0047] Specific Embodiment Seven: This embodiment differs from Specific Embodiments Two to Six in that the molar ratio of compound TMEM, 4-morpholinobenzaldehyde, and piperidine is 1:1:0.1. Everything else is the same as in Specific Embodiments Two to Six.

[0048] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Methods Two to Seven in that the molar volume ratio of compound TMEM to acetonitrile is 1 mmol: 15 mL. Everything else is the same as in Specific Implementation Methods Two to Seven.

[0049] Specific Implementation Method Nine: This implementation method involves lysosome-targeted recognition of viscosity, polarity, and ONOO. - Fluorescent probes are used in cellular microenvironment systems to study viscosity, polarity, and ONOO. - Sensor detection.

[0050] This embodiment utilizes the different fluorescence intensities of probes under varying viscosity conditions as an important means of detecting microenvironment viscosity. Furthermore, cell imaging analysis enables the detection of viscosity changes within cells.

[0051] This embodiment utilizes the different fluorescence intensities of probes under varying polarity conditions as an important means of detecting microenvironment polarity. Furthermore, cell imaging analysis can be used to detect polarity changes within cells.

[0052] This embodiment can also use cell fluorescence imaging technology to visualize ONOO within cellular lysosomes. - Perform fluorescence diagnostic identification.

[0053] Specific Implementation Method Ten: This implementation method differs from Specific Implementation Method Nine in that the sensing detection is fluorescence detection or visual colorimetric detection. Everything else is the same as in Specific Implementation Method Nine.

[0054] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0055] Example 1: Preparation of the fluorescent probe PVN-Lyso of the target compound:

[0056] (1) Synthesis of compound TMEM:

[0057]

[0058] 3,5,5-Trimethyl-3-cyclohexenone (3.8790, 28 mmol) was dissolved in 60 mL of C2H5OH, followed by the addition of malononitrile (1.28 g, 35.4 mmol) and piperidine (0.353 g, 2.8 mmol). The mixture was heated to reflux at 80 °C for 12 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature, poured into cold water, and the precipitate was filtered. The resulting filter cake was washed several times with water and dried to give compound TMEM as a coffee-colored solid with a yield of 48.47%.

[0059] (2) Synthesis of the target compound:

[0060]

[0061] Compound TMEM (0.1863 g, 1 mmol) and 4-morpholinobenzaldehyde (0.1912 g, 1.0 mmol) were added to 15 mL of acetonitrile. Piperidine (0.013 g, 0.1 mmol) was added dropwise, and the mixture was heated to reflux at 80 °C for 2 h. The reaction was monitored using a TCL plate. After the reaction was complete, the mixture was cooled to room temperature, poured into 10 mL of ice water, filtered under reduced pressure, and dried to obtain the target compound PVN-Lyso in 29.4% yield.

[0062] Example 2: Application of the fluorescent probe PVN-Lyso

[0063] The fluorescent probe PVN-Lyso synthesized in Example 1 was added to a 1,4-dioxane / PBS solution, and the concentration of the fluorescent probe PVN-Lyso in the system was 1×10⁻⁶. -5 The fluorescence spectra of 1,4-dioxane / PBS at different volume fractions were detected using a spectrophotometer at mol / L. Figure 1 Figure 1(a) shows the fluorescence spectrum of PVN-Lyso with a 1,4-dioxane volume fraction ranging from 5% to 65%. As can be seen from Figure 1(a), when the 1,4-dioxane volume fraction is within the range of 5%–65%, the fluorescence intensity of the system gradually increases with decreasing polarity. Figure 1 (b) It can be seen that the fluorescence intensity of PVN-Lyso at 670 nm has a good linear relationship with the proportion of 1,4-dioxane in the 1,4-dioxane / PBS mixture (R2 = 0.9890), indicating that PVN-Lyso can achieve quantitative analysis of solvent polarity in the range of 5%–65%.

[0064] The fluorescent probe PVN-Lyso synthesized in Example 1 was prepared at a concentration of 1×10⁻⁶. -5The fluorescence selectivity of 1 mol / L PBS solution for different ions (anions, cations, and reactive oxygen species) was detected by spectrophotometer. Figure 2 This study investigated the selectivity of PVN-Lyso (10 μM) in PBS solution. No significant fluorescence emission was observed with PVN-Lyso alone or with the addition of most of the analytes mentioned above; however, a significant enhancement in fluorescence signal was only observed with the addition of 1,4-dioxane or glycerol. These results indicate that PVN-Lyso exhibits high selectivity to changes in polarity / viscosity, and its fluorescence response is not easily interfered with by other biologically related substances. Figure 2 The coexisting ions detected are labeled as follows: 1, Pb 2+ ; 2, Co 2+ 3, Cu 2+ ; 4,Fe 2+ ; 5, Fe 3+ 6, Zn 2+ 7, Ba 2+ ; 8, Mg 2+ ; 9, Ni 2+ ; 10, Mn 2+ ; 11, Ca 2+ ; 12, Ga 3+ ;13, In 3+ ; 14, Ag + 15, Hg + 16, Cd 2+ ; 17, Al 3+ ; 18, Cys; 19, Ser; 20, Lys; 21, Arg; 22, His; 23, Asp; 24, Gln; 25, Ala; 26, Glu; 27, GSH; 28, HS - ; 29,CH3COO - ; 30, Br - 31, I - 32, BF4 - 33, HPO4 2- 34, NO3 - 35, HCO3 - 36, HSO4 - 37, NO2 - ; 38, ClO - 39, S2O5 2- ; 40, S2O3 2- 41, HSO3 - 42, SCN- 43, CO3 2- ; 44,H2PO4 - 45, H2O2; 46, 1 O2; 47, • OH; 48, ONOO - ; 49, 1,4-dioxane (40%); 50,glycerol (40%); 51, PVN-Lyso;

[0065] Figure 3 The effect of coexisting ions on the fluorescence intensity of PVN-Lyso (10 μM) under different polarity conditions. Figure 3 (a) shows the effect of co-existing oxygen and amino acids on the fluorescence intensity of PVN-Lyso; Figure 3 (b) shows the effect of coexisting cations on the fluorescence intensity of PVN-Lyso; Figure 3 (c) shows the effect of coexisting anionic acids on the fluorescence intensity of PVN-Lyso. As can be seen from the figure, the presence of the above-mentioned interfering substances did not cause significant changes in the fluorescence signal in media of different polarities, indicating that PVN-Lyso has a high specificity in response to polarity changes and is not easily affected by other components in complex matrices.

[0066] Figure 4 This describes the effect of coexisting substances on viscosity measurement under a fixed glycerol ratio. Figure 4 (a) shows the effect of co-existing oxygen and amino acids on the fluorescence intensity of PVN-Lyso; Figure 4 (b) shows the effect of coexisting cations on the fluorescence intensity of PVN-Lyso; Figure 4 (c) shows the effect of coexisting anionic acids on the fluorescence intensity of PVN-Lyso. Figure 4 The winning bid for "PVN-Lyso" is 1.0×10 -5 The fluorescence intensity of the probe at mol / L concentration in systems with different viscosities is shown. Under the same interference conditions, increasing the glycerol ratio (i.e., increasing viscosity) significantly enhances the fluorescence signal contrast. The fluorescence intensity of the system is compared when the probe PVN-Lyso and the detection ion coexist at the same concentration. As shown in the figure, PVN-Lyso exhibits high selectivity for viscosity, and its fluorescence response is largely unaffected by other common biological substances, making it suitable for viscosity detection in complex environments.

[0067] Figure 5 The probe is PVN-Lyso (the concentration of the probe in the system is 1×10⁻⁶). -5 Fluorescence spectra of H₂O / Glycerol in different proportions (mol / L) Figure 5As shown, the PVN-Lyso probe exhibits a weak fluorescence emission signal in a low-viscosity medium. With the gradual increase of the glycerol proportion, the fluorescence intensity shows a significant increasing trend. When the glycerol volume fraction reaches 90%, its fluorescence intensity is approximately 20,000 times stronger than that of the pure PBS system. Simultaneously, under sunlight, the solution color gradually changes from pale yellow to orange-yellow, and under a 365nm ultraviolet lamp, the solution color changes from dark red fluorescence to bright red fluorescence, enabling visual qualitative detection of viscosity.

[0068] Figure 6 (a) is the probe PVN-Lyso to ONOO - Identification. In a glycerol / PBS (6 / 4, v / v) system, ONOO - It exhibits a significant quenching effect on the fluorescence of PVN-Lyso. Figure 6 In (b), ONOO - Within the concentration range of 20–40 μM, the fluorescence intensity of PVN-Lyso at 680 nm showed a good linear relationship with its concentration. As shown in the figure, PVN-Lyso can quantitatively detect ONOO. - concentration.

[0069] Figure 7 The PVN-Lyso probe was used for fluorescent recognition targeting lysosomes within cells. Four cell types—A549, BEAS-2B, HepG2, and H8—were selected as the research subjects. Figure 7 In the diagram, a, b, c, and d correspond to A549, BEAS-2B, H8, and HepG2, respectively. PVN-Lyso (10 μM) was co-incubated with commercial organelle labeling dyes (including lysososome, mitochondrial, and lipid droplet probes), and confocal fluorescence imaging analysis was performed. The colocalization analysis results of the four cell lines consistently showed that the fluorescence signal of PVN-Lyso had the highest overlap with the lysososome probe (Lyso-Tracker), and its Pearson colocalization coefficient was significantly higher than that of other organelle labeling dyes. The results indicate that PVN-Lyso can specifically target lysosomes and is suitable for monitoring the dynamic changes in viscosity and polarity within this organelle.

[0070] Figure 8The probe PVN-Lyso was applied to the fluorescent recognition of viscosity changes in cells. A549 cells were selected as the research subject. A549 cells were pretreated with different concentrations of LPS (0, 1, 5, and 10 μg / mL), followed by co-incubation with PVN-Lyso (10 μM) for 30 min. After washing three times with PBS, the cells were finally imaged under laser confocal fluorescence microscopy, and intracellular fluorescence emission was observed. The fluorescence imaging results showed that the fluorescence intensity gradually increased with increasing LPS concentration, further confirming that PVN-Lyso can sensitively respond to LPS-triggered intracellular viscosity changes.

[0071] Figure 9 The probe PVN-Lyso was applied to the fluorescence recognition of polarity changes in cells. A549 cells were selected as the research subject. A549 cells were pretreated with different concentrations of DMSO (0, 1, 5, and 10 μg / mL), followed by co-incubation with PVN-Lyso (10 μM) for 30 min. After washing three times with PBS, the cells were finally imaged under laser confocal fluorescence microscopy, and intracellular fluorescence emission was observed. The fluorescence imaging results showed that the fluorescence signal in the DMSO-treated groups was significantly enhanced, and the fluorescence intensity increased with increasing DMSO concentration. This indicates that PVN-Lyso4 can sensitively respond to the decrease in intracellular polarity induced by DMSO, and its fluorescence signal is positively correlated with the degree of cell polarity change, thus demonstrating the potential of this probe to dynamically monitor polarity changes in living cells.

[0072] Figure 10 This study investigated the application of the PVN-Lyso probe in fluorescence recognition to distinguish between cancer cells and normal cells. Two cancer cell lines (A549 and HepG2) and three normal cell lines (BEAS-2B, LO2, and H8) were selected as the research subjects. PVN-Lyso (10 µM) was co-incubated with both cancer cells (A549 and HepG2) and normal cells (BEAS-2B, LO2, and H8) for 30 min, followed by three washes with PBS. Finally, imaging was performed under laser confocal fluorescence microscopy, and intracellular fluorescence emission was observed. The fluorescence imaging results showed that the fluorescence intensity of both cancer cell lines was significantly higher than that of normal cells, indicating that PVN-Lyso can effectively distinguish between cancer cells and normal cells based on differences in cell viscosity and polarity.

[0073] Figure 11This study aimed to apply the probe PVN-Lyso to the fluorescence recognition of microenvironmental changes in live zebrafish. Zebrafish were selected as the research subject and pretreated with different concentrations of LPS (0, 1, and 10 μg / mL) for 4 h, followed by incubation with PVN-Lyso (10 μM) for 30 min. After washing three times with PBS, the cells were finally imaged under laser confocal fluorescence microscopy, and intracellular fluorescence emission was observed. The fluorescence imaging results showed that zebrafish treated with only the probe exhibited weak fluorescence in both channels; while zebrafish treated with LPS showed significantly enhanced fluorescence intensity in both polar and viscosity channels, with signal intensity increasing with increasing LPS concentration. This indicates that PVN-Lyso has the ability to sensitively respond to inflammation-induced microenvironmental changes in vivo.

Claims

1. A lysosomal targeted recognition method for viscosity, polarity, and ONOO - The fluorescent probe is characterized by, The structural formula of the fluorescent probe is as follows: 。 2. The lysosome-targeted recognition of viscosity, polarity, and ONOO as described in claim 1 - The method for preparing the fluorescent probe is characterized by, The preparation method is as follows: I. The reaction of 3,3,5-trimethoxy-3-cyclohexenone with 1,3-malononitrile yields compound TMEM: 3,5,5-trimethoxy-3-cyclohexenone and 1,3-malononitrile were dissolved in ethanol, and piperidine was added. The mixture was heated to reflux and reacted for 10-12 h. The reaction was detected by a TCL plate. After the reaction was complete, the mixture was cooled to room temperature and deionized water was added to the solution. A precipitate was formed. The precipitate was filtered, washed, and dried to obtain compound TMEM. The molar ratio of 3,5,5-trimethoxy-3-cyclohexenone, 1,3-malononitrile to piperidine is 1.0~1.05:1.0~1.5:0.1~0.15; the molar volume ratio of 3,5,5-trimethoxy-3-cyclohexenone to ethanol is 1.0~1.1 mmol:2.0~2.2 mL. II. The reaction of compound TMEM with 4-morpholinobenzaldehyde yields the target compound PVN-Lyso: Compound TMEM, 4-morpholinobenzaldehyde, and piperidine were added to a round-bottom flask containing acetonitrile. The mixture was heated for 2–4 hours, cooled to room temperature, and then cooled with deionized water. A precipitate was formed. The precipitate was filtered, washed, and dried to obtain the target compound, thus completing the preparation of the fluorescent probe. The molar ratio of compound TMEM, 4-morpholinobenzaldehyde, and piperidine was 1.0–1.05:1.0–1.05:0.1–0.15; the molar volume ratio of compound TMEM to acetonitrile was 1.0–1.1 mmol:15.0–16.5 mL.

3. The lysosome-targeted recognition of viscosity, polarity, and ONOO as described in claim 2 - The method for preparing the fluorescent probe is characterized by, The washing described in steps one and two involves washing with deionized water five times.

4. The lysosome-targeted recognition of viscosity, polarity, and ONOO as described in claim 2 - The method for preparing the fluorescent probe is characterized by, The heating temperature described in steps one and two is 75~85℃.

5. A lysosome-targeted recognition method for viscosity, polarity, and ONOO as described in claim 2. - The method for preparing the fluorescent probe is characterized by, The molar ratio of 3,5,5-trimethoxy-3-cyclohexenone, 1,3-malononitrile, and piperidine is 1:1.5:0.

1.

6. The lysosome-targeted recognition of viscosity, polarity, and ONOO as described in claim 2 - The method for preparing the fluorescent probe is characterized by, The molar volume of 3,5,5-trimethoxy-3-cyclohexenone to ethanol is 1 mmol: 2 mL.

7. A lysosome-targeted recognition method for viscosity, polarity, and ONOO as described in claim 2. - The method for preparing the fluorescent probe is characterized by, The molar ratio of compound TMEM, 4-morpholinobenzaldehyde, and piperidine is 1:1:0.

1.

8. The lysosome-targeted recognition of viscosity, polarity, and ONOO as described in claim 2 - The method for preparing the fluorescent probe is characterized by, The molar volume ratio of compound TMEM to acetonitrile is 1 mmol: 15 mL.

9. The lysosomal targeted recognition of viscosity, polarity, and ONOO as described in claim 1 - Fluorescent probes are used in cellular microenvironment systems to study viscosity, polarity, and ONOO. - Sensor detection.

10. The application according to claim 9, characterized in that, The aforementioned sensing detection is either fluorescence detection or visual colorimetric detection.

Images