Water-soluble near-infrared fluorescent probe for detecting viscosity of biological system as well as preparation method and application of water-soluble near-infrared fluorescent probe

Abstract

The invention discloses a water-soluble near-infrared fluorescent probe for detecting viscosity of a biological system as well as a preparation method and application of the water-soluble near-infrared fluorescent probe. The fluorescent probe is prepared by condensing 8-hydroxyjulolidine and 4-methyl-1-(triethylene glycol monomethyl ether) quinolinium salt, the structural formula is shown in the specification, and the probe has a remarkable Stokes shift characteristic and also has the characteristics of high response speed, high selectivity, excellent biocompatibility and the like. The probe can be used for effectively monitoring viscosity changes of stress-induced onion endoepidermis cells and mung bean slice cells and dynamic changes of nystatin-induced viscosity in juvenile zebrafish bodies, and can be applied to imaging analysis of whole mung bean plants and peanut plants. Besides, the obtained fluorescent probe is simple to prepare, high in yield, mature in synthetic route, low in cost and suitable for amplified synthesis and actual production application, and a reliable tool is provided for actual application in the fields of biomedicine, agricultural monitoring and the like.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis technology, specifically relating to a water-soluble near-infrared fluorescent probe for detecting the viscosity of biological systems, its preparation method, and its application. Background Technology

[0002] Viscosity, as an important physical property of intracellular biological tissues, plays a crucial role in maintaining normal physiological functions. In animal systems, abnormal cell viscosity is significantly associated with various pathological processes, including inflammatory responses, diabetes, malignant tumors, mitochondrial dysfunction, atherosclerosis, and neurodegenerative diseases (such as Alzheimer's disease). In plant systems, environmental stresses (such as pathogen infection and drought conditions) directly lead to changes in cell viscosity. For example, pathogen infection can cause the accumulation of viscous substances in the plant's vascular system, hindering the transport of water and nutrients; drought stress can increase cytoplasmic viscosity, thereby interfering with normal metabolic activities.

[0003] Traditional viscosity measurement techniques (including capillary, rotation, ultrasonic, falling ball, and vibration methods) are widely used in industry, but their limitations in detection principles make them unsuitable for viscosity monitoring in living biological systems. In recent years, with the development of near-infrared fluorescence imaging technology, fluorescent probes based on molecular rotor mechanisms have provided a new solution for viscosity detection in biological systems. These probes have large tissue penetration depths and low autofluorescence interference, enabling high-resolution, real-time dynamic detection of living tissues. However, most viscosity monitoring probes suffer from poor water solubility, short emission wavelengths, and aggregation quenching, which limit their application in complex biological systems. Water-soluble near-infrared probes, due to their ability to effectively reduce non-specific adsorption of biomolecules and ensure uniform distribution in aqueous media, thus improving detection accuracy, show broad application prospects. Therefore, designing and developing viscosity-sensitive probes that combine sensitivity, selectivity, and water solubility is particularly important.

[0004] The target product of this application has been characterized. Studies have shown that in buffer solutions, the compound can selectively recognize viscosity, while other ions have almost no interference with the recognition process. Summary of the Invention

[0005] To overcome the problems in the prior art, this invention provides a water-soluble near-infrared fluorescent probe for detecting the viscosity of biological systems, its preparation method, and its applications. This fluorescent probe exhibits a relatively stable viscosity response, good linearity, and selectivity, and has been successfully used for fluorescence imaging of plants (sections and whole plants) and zebrafish.

[0006] The present invention also provides a method for preparing the above-mentioned fluorescent probe and its application.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A water-soluble near-infrared fluorescent probe for detecting the viscosity of biological systems, wherein the molecular formula of the fluorescent probe is C0. 30 H 37 N2O4 + The structural formula is as follows:

[0009]

[0010] The synthetic route for preparing the water-soluble near-infrared fluorescent probe for viscosity detection described above is as follows:

[0011]

[0012] Specifically, it includes the following steps:

[0013] (1) m-aminophenol, 1-bromo-3-chloropropane and sodium carbonate were reacted in DMF to give compound 2;

[0014] (2) Compound 2 and phosphorus oxychloride react in DMF to give compound 3;

[0015] (3) 4-methylquinoline and 1-bromo-2-(2-2-(2-methoxyethoxy)ethoxy)ethane were reacted in acetonitrile to give compound 5;

[0016] (4) Compound 3 and compound 5 were reacted in anhydrous ethanol to obtain the fluorescent probe HJA-MQ-D.

[0017] Preferably, the specific preparation process of compound 2 in step (1) is as follows: under nitrogen protection, m-aminophenol, 1-bromo-3-chloropropane and sodium carbonate are mixed in DMF, stirred at 80-100°C until the reaction is complete, cooled to room temperature, extracted with ethyl acetate, rotary evaporated, dried and column chromatography are used to obtain the solid, which is compound 2.

[0018] Preferably, the specific preparation process of compound 3 in step (2) is as follows: under nitrogen protection, compound 2 is added to phosphorus oxychloride and DMF, and stirred at 70-90°C until the reaction is complete. Then, distilled water is added to the mixture, stirred overnight, filtered, and dried to obtain the solid compound 3.

[0019] Preferably, the specific preparation process of compound 5 in step (3) is as follows: under nitrogen protection, 4-methylquinoline and 1-bromo-2-(2-2-(2-methoxyethoxy)ethoxy)ethane are mixed in acetonitrile and stirred and refluxed at 100-120°C until the reaction is complete. After the reaction is completed, a solid is precipitated upon cooling. The solid is washed with petroleum ether and dried to obtain compound 5.

[0020] Preferably, the preparation process of the fluorescent probe in step (4) is as follows: under nitrogen protection, compound 3 and compound 5 are dissolved in anhydrous ethanol, stirred and refluxed at 85-100°C until the reaction is complete, column chromatography is performed after the reaction is completed, and the obtained solid is dried under vacuum to obtain the fluorescent probe.

[0021] Preferably, in step (1), the molar ratio of m-aminophenol, 1-bromo-3-chloropropane and sodium carbonate is (1.5-2.5):1:(2-4).

[0022] Preferably, in step (2), the molar ratio of compound 2 to phosphorus oxychloride is 1:(1 to 1.5).

[0023] Preferably, in step (3), the molar ratio of 4-methylquinoline and 1-bromo-2-(2-2-(2-methoxyethoxy)ethoxy)ethane is 1:(3-4).

[0024] Preferably, in step (4), the molar ratio of compound 3 to compound 5 is 1:(1 to 1.5).

[0025] The above-mentioned water-soluble near-infrared fluorescent probe is used for the fluorescence detection of viscosity in biological systems.

[0026] Furthermore, for imaging plant cells, the cells need to be treated with a salt solution, heavy metal solution, oxidant solution, or sucrose solution before imaging processing. The concentrations of the salt solution and heavy metal solution are 30–100 μM, the concentration of the oxidant solution is 5–50 mM, and the concentration of the sucrose solution is 0.1–0.5 g / mL. Preferably, the salt is sodium chloride, and the heavy metal is Cd. 2+ The oxidant is H2O2. The plant cells are onion epidermal cells or mung bean sprout slice cells.

[0027] Specifically, onion epidermal cells are first stained in 1-10 μM probe solution for 3-10 minutes, then treated in salt solution, heavy metal solution or oxidant solution for 3-10 minutes, rinsed with water and then imaged.

[0028] Specifically, mung bean sprouts and cell slices were first stained in 1-10 μM probe solution for 3-10 minutes, then treated in sucrose solution for 3-10 minutes, rinsed with water, and then imaged.

[0029] Furthermore, for imaging the whole plant, treatment with a salt solution, heavy metal solution, or oxidant solution is required before imaging processing. The concentration of the salt solution is 30–100 μM, the concentration of the heavy metal solution is 100–250 μM, and the concentration of the oxidant solution is 5–100 mM. Preferably, the salt is sodium chloride, and the heavy metal is Cd. 2+ The oxidant is H2O2.

[0030] Specifically, germinated plant seeds are treated with a salt solution or heavy metal solution for 10 hours to 9 days, then soaked in a 1-10 μM fluorescent probe HJA-MQ-D solution for 0.5 to 2 hours, rinsed with water, and then imaged. The plant seeds refer to mung bean or peanut seeds.

[0031] Furthermore, for imaging zebrafish, the image needs to be treated with an antibiotic solution at a concentration of 5–20 μM before imaging processing. Preferably, the antibiotic is nystatin.

[0032] Specifically, zebrafish were pretreated in a culture medium containing nystatin solution for 20–40 minutes, then incubated in a culture medium containing 5–15 μM fluorescent probe solution for 20–40 minutes before imaging.

[0033] Compared with the prior art, the beneficial effects of the present invention are:

[0034] 1. The fluorescent probe described in this invention exhibits a significant fluorescence enhancement effect in high viscosity environments and has excellent anti-interference performance against common interfering ions, thus achieving specific response detection to viscosity changes.

[0035] 2. This probe is characterized by its simple synthesis, excellent water solubility, and good biocompatibility. It can be widely used for viscosity dynamic monitoring in animal and plant systems, and is particularly suitable for detecting viscosity changes in whole plant systems. It has important application value in the fields of biomedicine and agricultural science. Attached Figure Description

[0036] Figure 1 The hydrogen NMR spectrum of the water-soluble near-infrared fluorescent probe prepared in Example 1;

[0037] Figure 2 The carbon NMR spectrum of the water-soluble near-infrared fluorescent probe prepared in Example 1;

[0038] Figure 3 A comparison of the UV absorption spectra of the water-soluble near-infrared fluorescent probe prepared in Example 1 in low-viscosity and high-viscosity systems;

[0039] Figure 4 The fluorescence spectrum of the water-soluble near-infrared fluorescent probe prepared in Example 1 varies with viscosity.

[0040] Figure 5 The linear relationship between the fluorescence emission wavelength and intensity at 698 nm of the water-soluble near-infrared fluorescent probe prepared in Example 1;

[0041] Figure 6 The fluorescence spectra of the water-soluble near-infrared fluorescent probe prepared in Example 1 as a function of time in low-viscosity and high-viscosity systems;

[0042] Figure 7 The fluorescence spectrum relationship of the water-soluble near-infrared fluorescent probe prepared in Example 1 with pH changes in different viscosity systems is shown in the figure.

[0043] Figure 8 The fluorescence spectra of the water-soluble near-infrared fluorescent probe prepared in Example 1 in different polar solvents;

[0044] Figure 9 Different ions (in the figure: from left to right, 1, Zn) 2+ ,2.Ni 2+ 3.K + ,4.Co 2+ 5.Ba 2+ 6.Mn 2+ 7.Fe 3+ 8.Cu 2+ 9.Cd 2+ ,10.Na + ,11.Ca 2+ ,12.N2H4,13.S 2- ,14.F - ,15.Cl - ,16.Br - ,17.ClO - ,18.HSO3 - A bar chart comparing the fluorescence intensity of the fluorescent probe prepared in Example 1 with that of Proline, Glutamic acid, Lysine, Arginine, Tryptophan, Phenylalanine, Cysteine, Glutathione, Homocysteine, and Blank at 698.0 nm.

[0045] Figure 10 The water-soluble near-infrared fluorescent probe prepared in Example 1 was used to test its ability to identify viscosity interference. (In the figure: from left to right, 1. Zn) 2+ ,2.Ni 2+ 3.K + ,4.Co 2+ 5.Ba 2+ 6.Mn 2+ 7.Fe 3+ 8.Cu 2+ 9.Cd 2+ ,10.Na + ,11.Ca 2+ ,12.N2H4,13.S 2- ,14.F- ,15.Cl - ,16.Br - ,17.ClO - ,18.HSO3 - ,19.Proline,20.Glutamic acid,21.Lysine,22.Arginine,23.Tryptophan,24.Phenylalanine,25.Cysteine,26.Glutathione,27.Homocysteine,28.Blank);

[0046] Figure 11 The image shows a comparison of the water solubility of the water-soluble near-infrared fluorescent probe with two other probes. (A) shows the chemical structures of HJA-MQ-M, HJA-MQ-E, and HJA-MQ-D. (B) shows the fluorescence intensity versus concentration curves of the three probes (with water as the solvent). (C) shows the linear fitting results of the fluorescence intensity versus concentration of the three probes in the low concentration region (0-400 μM).

[0047] Figure 12 These are bioimaging images of water-soluble near-infrared fluorescent probes, where (A) is a bioimaging image of onion inner epidermis after pretreatment with different salt concentrations, and (a) is a fluorescence quantification image of onion inner epidermis in A.

[0048] Figure 13 This is a bioimaging image of a water-soluble near-infrared fluorescent probe, where (B) shows the results after passing through different Cd... 2+ Bioimaging images of onion inner epidermis after concentration pretreatment, (b) is the fluorescence quantification image of onion inner epidermis in B;

[0049] Figure 14 These are bioimaging images of water-soluble near-infrared fluorescent probes, where (C) is a bioimaging image of the inner epidermis of onions after pretreatment with different H2O2 concentrations, and (c) is a fluorescence quantification image of the inner epidermis of onions in C.

[0050] Figure 15 These are bioimaging images of water-soluble near-infrared fluorescent probes, where (A) is a bioimaging image of mung bean slice cells after sucrose pretreatment, and (a) is a fluorescence quantification image of mung bean slice cells in A.

[0051] Figure 16 This is a whole-plant imaging image of a water-soluble near-infrared fluorescent probe, where (A) shows mung bean seeds exposed to heavy metal cadmium ions (Cd). 2+ (A) Experimental treatment procedure for stress, (B) and (C) are whole-plant fluorescence imaging results after 10 hours and 5 days of treatment with different concentrations of cadmium ions (0, 30, 50, 70, 100 μM), respectively, and (D) and (E) whole-plant imaging fluorescence quantification diagram.

[0052] Figure 17 This is a whole-plant imaging image of a water-soluble near-infrared fluorescent probe, where (A) shows peanut seeds exposed to heavy metal cadmium ions (Cd). 2+ (A) Experimental treatment procedure for stress, (B) and (C) are whole-plant fluorescence imaging results after 10 hours and 5 days of treatment with different concentrations of cadmium ions (0, 30, 50, 70, 100 μM), respectively, and (D) and (E) whole-plant imaging fluorescence quantification diagram.

[0053] Figure 18 The images show whole-plant imaging of water-soluble near-infrared fluorescent probes. (A) shows the experimental treatment process of mung bean seeds under salt stress. (B) and (C) are whole-plant fluorescence imaging results after 10 hours and 7 days of treatment with different concentrations of NaCl solution (0, 100, 150, 200, 250 mM), respectively. (D) and (E) are whole-plant fluorescence quantification images.

[0054] Figure 19 The images show whole-plant imaging of water-soluble near-infrared fluorescent probes. (A) shows the experimental treatment process of peanut seeds under salt stress. (B) and (C) are whole-plant fluorescence imaging results after 10 hours and 9 days of treatment with different concentrations of NaCl solution (0, 100, 150, 200, 250 mM), respectively. (D) and (E) are whole-plant fluorescence quantification images.

[0055] Figure 20 The images are bioimaging images of a water-soluble near-infrared fluorescent probe, where (A) is a bioimaging image of zebrafish treated with nystatin, and (a) is a fluorescence quantification image of the zebrafish in A. Detailed Implementation

[0056] The present invention will be further described below with reference to embodiments and accompanying drawings, but this is not intended to limit the invention.

[0057] Example 1

[0058] The preparation process of the fluorescent probe in this embodiment specifically includes the following steps:

[0059] (1) Preparation of compound 2

[0060] The synthesis route is as follows:

[0061]

[0062] Weigh out m-aminophenol (5.46 g, 50 mmol) and sodium carbonate (7.95 g, 75 mmol) and add them to a 100 mL round-bottom flask. Then, measure out 1-bromo-3-chloropropane (3 mL, 25 mmol) and N,N-dimethylformamide (20 mL) and add them sequentially to the flask. Under nitrogen protection, reflux at 90 °C. Monitor the reaction by TLC. The reaction is complete in about 12 h. After the reaction is complete, rotary evaporation and column chromatography (eluent: petroleum ether:ethyl acetate, volume ratio = 15:1) are performed to obtain a white powder compound 1 with a mass of 2.63 g and a yield of approximately 49%. The NMR information of compound 2 is as follows:

[0063] 1 H NMR(400MHz,Chloroform-d)δ6.66(d,J=8.0Hz,1H),6.06(d,J=8.0Hz,1H),4.61(d,J =5.5Hz,1H),3.09(q,J=5.1Hz,4H),2.68(dt,J=15.1,6.6Hz,4H),2.09–1.90(m,4H).

[0064] 13 C NMR (100MHz, Chloroform-d) δ151.74,143.94,126.73,114.31,107.81,102.91,50.21,49.54,27.21,22.38,21.62,20.96.

[0065] (2) Preparation of compound 3

[0066] The synthesis route is as follows:

[0067]

[0068] Under ice-water bath conditions, phosphorus oxychloride (0.6 mL, 6.4 mmol) was slowly added to 2 mL of DMF. The mixture was stirred for 15 minutes under nitrogen protection. Then, compound 2 (1.0 g, 5.3 mmol) was dissolved in 1 mL of DMF and slowly added to the cooled solution using a syringe. The mixture was reacted at room temperature for 30 minutes, then rapidly heated to 80 °C and reacted for another 30 minutes. After cooling to room temperature, the reaction solution was poured into 150 mL of water and stirred for 8 hours. After the reaction was complete, the mixture was filtered to obtain a light green powder compound 3 with a mass of 0.449 g and a yield of approximately 40%.

[0069] The NMR information for compound 3 is as follows:

[0070] 1H NMR (400MHz, DMSO-d6) δ11.80(s,1H),9.29(s,1H),6.90(s,1H),3.20(q,J=5.7H z, 4H), 2.54 (t, J = 6.3Hz, 2H), 2.47 (q, J = 5.1, 3.8Hz, 2H), 1.76 (q, J = 5.9Hz, 4H).

[0071] 13 C NMR (100MHz, DMSO-d6) δ192.08,158.87,149.68,131.39,113.84,110.47,104.64,49.99,49.54,26.99,21.50,20.41,19.73.

[0072] (3) Preparation of compound 5

[0073] The synthesis route is as follows:

[0074]

[0075] 0.68 mL (5 mmol) of 4-methylquinoline, 3.01 mL (15 mmol) of 1-bromo-2-(2-2-(2-methoxyethoxy)ethoxy)ethane, and 10 mL of acetonitrile were added sequentially to a flask, and the mixture was refluxed at 110 °C under nitrogen protection. The reaction was monitored by TLC and was completed in approximately 8 hours. After completion, the mixture was placed in a freeze-thawed container for recrystallization. The solution was filtered under vacuum after washing with petroleum ether to obtain a purple solid with a mass of 0.48 g, yielding approximately 38%.

[0076] The NMR information for compound 5 is as follows:

[0077] 1 H NMR (400MHz, DMSO-d6) δ9.48(d,J=6.1Hz,1H),8.81(d,J=9.0Hz,1H),8.68(dd,J=8.5,1.4Hz,1H),8.38(ddd,J=8.7,6.9,1.4Hz,1H),8.21(dd,J=11.1, 6.9Hz,2H),5.40(t,J=4.9Hz,2H),4.09(t,J=4.9Hz,2H),3.64(dd,J=3.9,2 .0Hz,2H),3.50(s,2H),3.44–3.42(m,2H),3.42–3.40(m,2H),3.15(s,3H).

[0078] 13C NMR(100MHz,DMSO-d6)δ159.34,149.65,137.39,135.44,130.03,129.30,127 .57,122.72,120.02,71.61,70.31,70.05,69.96,68.23,58.51,56.92,20.26.

[0079] (1) Preparation of probe HJA-MQ-D

[0080] The synthesis route is as follows:

[0081]

[0082] Compound 3 (123.13 mg, 0.567 mmol) and compound 5 (209.95 mg, 0.567 mmol) were weighed and added to a 100 mL round-bottom flask. 10 mL of ethanol was then added, and the mixture was refluxed at 90 °C under nitrogen protection. The reaction was monitored by TLC and completed in approximately 12 hours. After completion, the mixture was rotary evaporated and separated by column chromatography (dichloromethane:methanol volume ratio = 80:1) to obtain a blue solid. The solid was then dried under vacuum to obtain a probe with a mass of 102 mg, yielding approximately 32%. The 1H and 1C NMR spectra of the fluorescent probe prepared in this example are shown below. Figure 1 and Figure 2 The specific NMR data are as follows:

[0083] 1 H NMR (400MHz, Methanol-d4) δ8.45(d,J=8.6Hz,1H),8.40(d,J=6.9Hz,1H),8.08(d,J=15.2Hz,1H),8.00(d,J=8. 8Hz,1H),7.88–7.82(m,1H),7.68–7.61(m,2H),7.44(d,J=15.1Hz,1H),7.16(s,1H),4.70(t,J=4.8Hz,2H),3.85 (t,J=4.7Hz,2H),3.49(dd,J=5.6,2.9Hz,2H),3.43–3.40(m,2H),3.35(dd,J=6.0,3.3Hz,2H),3.31–3.28(m,2H ),3.21(dt,J=4.0,2.0Hz,4H),3.17(s,3H),2.61(t,J=6.2Hz,2H),2.51(t,J=6.4Hz,2H),1.84(t,J=6.1Hz,4H).

[0084] 13C NMR(100MHz,Methanol-d4)δ155.26,154.41,148.35,145.08,141.30,138.46,133.92,127.49,126.62,126.06,125.82,117.85,11 6.00,112.75,111.10,109.75,106.65,71.51,70.32,70.08,69.91,67.94,57.71,55.05,50.04,49.25,27.05,21.58,20.77,20.67.

[0085] The application tests of the fluorescent probe prepared in this embodiment are as follows:

[0086] 1) Preparation of stock solution for testing

[0087] a. Fluorescent probe sample solution for viscosity detection (1.00 × 10⁻⁶) -3 Preparation of (mol / L): Accurately weigh 0.0057 g (M = 569.5) of the fluorescent probe and dissolve it in 10 mL of dimethyl sulfoxide to prepare a concentration of 1.00 × 10⁻⁶ mol / L. -3 A solution of mol / L.

[0088] b. All amino acids and ions were prepared with deionized water to a concentration of 1.0 × 10⁻⁶. -2 A solution of mol / L.

[0089] c. Preparation of PBS buffer solution (10mM, pH=7.4):

[0090] Accurately weigh 8.0 g NaCl (M = 58), 0.2 g KCl (M = 74), 1.44 g Na₂HPO₄ (M = 144), and 0.24 g KH₂PO₄ (M = 136), dissolve them in 800 mL of distilled water, and use a solution with a concentration of 1.0 × 10⁻⁶. -2 Adjust the pH of the above solution to 7.4 with a mol / L hydrochloric acid aqueous solution, and then bring the volume to 1L with distilled water.

[0091] 2) Experiment on the spectral properties of the probe

[0092] Using a pipette, 3 mL of PBS buffer and 3 mL of glycerol were respectively transferred to simulate low-viscosity and high-viscosity environments. Then, 30 μL of probe stock solution (1.00 × 10⁻⁶) was added. -3 (mol / L). After shaking well to ensure no bubbles are present, its UV absorption spectrum was measured at room temperature (parameters: scan range 350-900 nm, fast scan speed, 1 nm interval). Results are as follows: Figure 3 ,Depend on Figure 3It can be seen that the probe exhibits a significant red shift in glycerol, with its maximum absorption peak shifting from 606 nm to 638 nm. Glycerol-PBS solutions of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% were prepared using PBS buffer and glycerol at different volume ratios (v / v). 30 μL of probe stock solution (1.00 × 10⁻⁶) was added to each solution. -3 (mol / L). After shaking until no bubbles are present, the fluorescence intensity change of the solution was measured at room temperature (parameters: excitation wavelength 610 nm, excitation slit width 10.0 nm, emission slit width 10.0 nm). Results are as follows: Figure 4 As shown, with the increase of system viscosity, the fluorescence intensity of the solution system at 697.6 nm gradually increases. According to... The Hoffmann equation log I = C + x logη gives the linear equation for HJA-MQ-D as: log I 697.6 = 1.51588 + 0.7668logη, where R 2 =0.99727, probe HJA-MQ-D in the viscosity range of 0-100% Gly, log I 697.6 There is a good linear relationship between it and logη, as shown in the experimental results. Figure 5 As shown.

[0093] Using a pipette, transfer 3 mL of PBS buffer solution and 3 mL of glycerol to simulate low and high viscosity environments, respectively, and add 30 μL of probe stock solution (1.00 × 10⁻⁶). -3 (mol / L). Shake well until no bubbles are present, and test every three minutes at room temperature. The results are as follows. Figure 6 As shown, the probe is highly stable in both low-viscosity and high-viscosity systems. Then, using a pipette, 3 mL of glycerol-PBS systems at different pH values ​​(glycerol volume ratios of 30%, 50%, and 80%, respectively; the PBS buffer was first adjusted to the corresponding pH with 1 mol / L hydrochloric acid or 1 mol / L NaOH solution, and then mixed with glycerol at the required volume ratio) were added, along with 30 μL (1.00 × 10⁻⁶). -3 The probe stock solution was prepared by shaking until no bubbles were present. The fluorescence intensity (697.6 nm) was measured at room temperature, and the results are as follows: Figure 7 As shown. By Figure 7It can be seen that the fluorescence intensity of the probe system remains stable at pH < 7.4, and begins to decrease when pH > 7.4. Therefore, this probe has strong anti-interference ability in acidic and weakly alkaline (pH < 7.4) systems. Using a pipette, 3 mL of glycerol and other solvents of different polarities (including PBS, DMSO, DMF, dichloromethane, tetrahydrofuran, acetone, methanol, ethanol, acetonitrile, and ethyl acetate) were added to 30 μL of probe stock solution (1.00 × 10⁻⁶). -3 (mol / L). Shake well until no bubbles are present, and perform fluorescence testing at room temperature. The results are as follows. Figure 8 As shown, the probe is not affected by solvent polarity.

[0094] Use a pipette to transfer 3 mL of PBS buffer and add 30 μL (1.00 × 10⁻⁶) to the solution. -3 1 mol / L probe stock solution was added, followed by the addition of analyte (30 μL, 1.00 × 10⁻⁶ mol / L) in sequence. -2 mol / L)(1.Zn 2+ ,2.Ni 2+ 3.K + ,4.Co 2+ 5.Ba 2+ 6.Mn 2+ 7.Fe 3 + 8.Cu 2+ 9.Cd 2+ 10.Na + ,11.Ca 2+ , 12.N2H4, 13.S 2- 14.F - 15.Cl - ,16.Br - 17.ClO - 18.HSO3 - 19. Proline, 20. Glutamic acid, 21. Lysine, 22. Arginine, 23. Tryptophan, 24. Phenylalanine, 25. Cysteine, 26. Glutathione, 27. Homocysteine, 28. Glycerol), their fluorescence spectra were detected, and the results are as follows. Figure 9 The bar chart shows the fluorescence intensity of the probe at the maximum emission wavelength of 697.6 nm after being combined with other ions and amino acids. It can be seen that the fluorescence emission of the probe is significantly enhanced in the high viscosity system, while the fluorescence intensity does not change significantly after the addition of other ions.

[0095] Use a pipette to transfer 3 mL of glycerol-PBS system (glycerol volume ratio 50%), and add 30 μL (1.00 × 10⁻⁶) of PBS. - 31 mol / L) probe stock solution, then add 30 μL (1.00 × 10⁻⁶ mol / L) of the solution. -2 mol / L) interfering substance (1. Zn 2+ ,2.Ni 2+ 3.K + ,4.Co 2+ 5.Ba 2+ 6.Mn 2+ 7.Fe 3+ 8.Cu 2+ 9.Cd 2+ 10.Na + ,11.Ca 2+ , 12.N2H4, 13.S 2- 14.F - 15.Cl - ,16.Br - 17.ClO - 18.HSO3 - 19. Proline, 20. Glutamic acid, 21. Lysine, 22. Arginine, 23. Tryptophan, 24. Phenylalanine, 25. Cysteine, 26. Glutathione, 27. Homocysteine, 28. Glycerol), mixed thoroughly, and subjected to fluorescence spectroscopy scanning. The fluorescence intensity at 697.6 nm was plotted as a bar chart. Figure 10 It can be seen that the remaining ions and amino acids have very little interference with the probe, and basically do not react. Therefore, this indicates that the probe responds well to glycerol in the presence of common anions, cations, and amino acid interfering substances, and has strong anti-interference ability against a variety of common anions and cations, demonstrating the excellent performance of the fluorescent probe.

[0096] By introducing methyl and ethyl groups onto quinoline, two other probes with different structures, namely HJA-MQ-M and HJA-MQ-E, were synthesized. See details... Figure 11 In step A, accurately weigh two fluorescent probes and dissolve them in dimethyl sulfoxide to prepare a solution with a concentration of 1.00 × 10⁻⁶. -3 For a solution of mol / L, pipette 3 mL of purified water and add 30 μL of each of the three solutions (1.00 × 10⁻⁶ mol / L). -3 After rehydration with the probe stock solution (mol / L), a fluorescence assay was performed. See details below. Figure 11 The results showed that HJA-MQ-M, HJA-MQ-E, and HJA-MQ-D reached their maximum fluorescence intensity at concentrations of 250 μM, 200 μM, and 400 μM, respectively, indicating that HJA-MQ-D exhibited the best water solubility. Furthermore, this application also analyzed the linear relationship between fluorescence intensity and concentration for each probe within its peak range; see [link to relevant documentation] for details. Figure 11 In C, all three probes exhibited good linear response (R0). 2>0.99), but the probe HJA-MQ-D has the widest linear range, further confirming its excellent water solubility.

[0097] 3) Imaging of the probe

[0098] Onion inner epidermal cells were stained with the 5 μM HJA-MQ-D probe (stock solution diluted to 5 μM with DMSO) for 5 minutes. A control group and an experimental group were set up. The control group consisted of stained onion inner epidermal tissue; the experimental group consisted of stained onion inner epidermal tissue placed in different concentrations of NaCl aqueous solution (0, 30, 50, 100 mM), Cd, etc. 2+ Imaging was performed after rinsing with water in aqueous solutions (0, 30, 50, 70, 100 μM) and H₂O₂ aqueous solutions (0, 5, 10, 20, 50 mM) for 5 minutes in both groups. The results showed that the fluorescence intensity of onion inner epidermal cells in all three groups increased with increasing stress concentrations. Figure 12 , 13 14). In addition, mung bean sprout slices were used for testing. The tissues were stained with the 5 μM probe HJA-MQ-D for 5 minutes. A blank group and an experimental group were set up. The blank group consisted of stained mung bean sprout slices; the experimental group consisted of stained mung bean sprout slices placed in a 0.3 g / mL sucrose solution. Both groups were rinsed with water before imaging. The results showed that the fluorescence intensity of the mung bean sprout slices increased with increasing sucrose concentration. Figure 15 These results indicate that the HJA-MQ-D probe can effectively monitor changes in the cellular microenvironment.

[0099] To evaluate the application of the probe HJA-MQ-D in whole plants, this invention used mung beans and peanuts as plant models for testing. Dynamic viscosity changes in plant tissues were simulated by applying exogenous heavy metal and salt stress. The experiment was divided into a control group and an experimental group. First, mung bean and peanut seeds were disinfected with 75% alcohol for 30 seconds, rinsed three times with sterile water, and then cultured in the dark at room temperature. The seeds were then germinated in distilled water. The control group consisted of untreated plant seeds, while the experimental group had germinated seeds directly placed in solutions of different concentrations of Cd. 2+ Mung bean sprouts / seedlings and peanut sprouts / seedlings were obtained by culturing in aqueous solutions of 0, 30, 50, 70, and 100 μM and NaCl (0, 100, 150, 200, and 250 mM) for different durations. After culturing, all plant materials were immersed in a 5 μM (stock solution diluted to 5 μM with DMSO) fluorescent probe HJA-MQ-D solution for 1 hour, followed by rinsing off any residual probe solution with water, and finally imaging of the entire plant. Figure 16 Cd at different concentrations (0, 30, 50, 70, 100 μM) is shown. 2+ Imaging of the entire mung bean plant in solution 10 hours and 5 days after germination, and... Figure 17 This shows that in the same Cd 2+ Whole-plant imaging of peanuts 10 hours and 7 days after germination under different concentration gradients. The whole-plant imaging results show that both plants exhibit significant Cd concentrations. 2+ Concentration and time dependence: with Cd 2+ With increasing concentration (30→100μM) and prolonged exposure time (10 hours→5 / 7 days), the fluorescence intensity in plant tissues increased. This result confirms that the probe HJA-MQ-D can detect Cd. 2+ Stress-induced increase in viscosity within plant tissues, and the increase in fluorescence intensity were positively correlated with the intensity and duration of heavy metal stress. Figure 18 The images show the whole plant images of mung beans after germination for 10 hours and 7 days in NaCl solutions of different concentrations (0, 100, 150, 200, 250 mM). Figure 19 The images show whole-plant imaging of peanuts cultured under the same salt concentration gradient for 10 hours and 9 days. The whole-plant imaging results show that both plants exhibit significant salt concentration and time-dependent responses: with increasing salt concentration (100→250 mM), the fluorescence intensity in plant tissues significantly increased, and with prolonged treatment time, the fluorescence signal intensity also showed a clear increasing trend. This test result confirms that the probe HJA-MQ-D can monitor the increase in viscosity within plant tissues caused by salt stress, and that the increase in fluorescence intensity is positively correlated with the intensity and duration of salt stress.

[0100] In a zebrafish model, this invention established three experimental groups: a blank control group consisting of 5-day-old zebrafish juveniles cultured normally; experimental group 1 consisting of zebrafish incubated for 30 minutes in 10 μM (stock solution diluted to 10 μM with DMSO) HJA-MQ-D solution; and experimental group 2 consisting of zebrafish pretreated with 10 μM nystatin (Nys) aqueous solution for 30 minutes, followed by incubation in 10 μM (stock solution diluted to 10 μM with DMSO) fluorescent probe HJA-MQ-D solution for another 30 minutes. All treatments were performed in a culture medium at 28°C under dark conditions to ensure probe stability. Experimental results are as follows: Figure 20 As shown, zebrafish in the blank control group did not show significant fluorescence signals; zebrafish in experimental group 1 treated with the fluorescent probe HJA-MQ-D showed weak fluorescence signals; while zebrafish in experimental group 2, pretreated with 10 μM Nys before probe staining, showed significantly enhanced fluorescence signals. This response phenomenon (blank group)

Claims

1. A water-soluble near-infrared fluorescent probe for detecting the viscosity of biological systems, characterized in that, The molecular formula of the fluorescent probe is C 30 H 37 N2O4 + The structural formula is as follows: HJA-MQ-D.

2. The method for preparing the water-soluble near-infrared fluorescent probe for detecting the viscosity of biological systems according to claim 1, characterized in that, The synthesis route is as follows: Specifically, the following steps are included: m-Aminophenol, 1-bromo-3-chloropropane and sodium carbonate react in DMF to give compound 2; Compound 2 and phosphorus oxychloride react in DMF to give compound 3; 4-Methylquinoline and 1-bromo-2-(2-2-(2-methoxyethoxy)ethoxy)ethane react in acetonitrile to give compound 5; Compounds 3 and 5 were reacted in anhydrous ethanol to obtain the fluorescent probe HJA-MQ-D.

3. The method for preparing a water-soluble near-infrared fluorescent probe for detecting the viscosity of biological systems according to claim 2, characterized in that, Specifically, the following steps are included: (1) Specific preparation process of compound 2: Under nitrogen protection, m-aminophenol, 1-bromo-3-chloropropane and sodium carbonate were mixed in DMF and stirred at 80~100 °C until the reaction was complete. After cooling to room temperature, the mixture was extracted with ethyl acetate, rotary evaporated, dried and column chromatography was used to obtain the solid, which is compound 2. (2) Specific preparation process of compound 3: Under nitrogen protection, compound 2 was added to phosphorus oxychloride and DMF, and stirred at 70~90℃ until the reaction was complete. Then, distilled water was added to the mixture, stirred overnight, filtered, and dried to obtain the solid, which is compound 3. (3) Specific preparation process of compound 5: Under nitrogen protection, 4-methylquinoline and 1-bromo-2-(2-2-(2-methoxyethoxy)ethoxy)ethane were mixed in acetonitrile and stirred and refluxed at 100~120℃ until the reaction was complete. After the reaction was completed, a solid was precipitated by cooling. The solid was washed with petroleum ether and dried to obtain compound 5. (4) The preparation process of the fluorescent probe is as follows: under nitrogen protection, compound 3 and compound 5 are dissolved in anhydrous ethanol, stirred and refluxed at 85~100℃ until the reaction is complete, column chromatography is performed after the reaction is completed, and the solid obtained is dried under vacuum to obtain the fluorescent probe.

4. The method for preparing a water-soluble near-infrared fluorescent probe for detecting the viscosity of a biological system according to claim 2 or 3, characterized in that, In step (1), the molar ratio of m-aminophenol, 1-bromo-3-chloropropane and sodium carbonate is (1.5~2.5):1:(2~4); in step (2), the molar ratio of compound 2 and phosphorus oxychloride is 1:(1~1.5); in step (3), the molar ratio of 4-methylquinoline and 1-bromo-2-(2-2-(2-methoxyethoxy)ethoxy)ethane is 1:(3~4); in step (4), the molar ratio of compound 3 and compound 5 is 1:(1~1.5).

5. An application of the water-soluble near-infrared fluorescent probe according to claim 1, characterized in that, Fluorescent detection of viscosity in biological systems.

6. The application according to claim 5, characterized in that, For imaging plant cells, the cells need to be treated with salt solution, heavy metal solution, oxidant solution or sucrose solution before imaging processing. The concentration of the salt solution or heavy metal solution is 30~100 μM, the concentration of the oxidant solution is 5~50 mM, and the concentration of the sucrose solution is 0.1~0.5 g / mL.

7. The application according to claim 5, characterized in that, For imaging of whole plants, the images need to be treated with a salt solution, a heavy metal solution, or an oxidant solution before imaging. The concentration of the salt solution is 30-100 μM, the concentration of the heavy metal solution is 100-250 μM, and the concentration of the oxidant solution is 5-100 mM.

8. The application according to claim 5, characterized in that, For imaging zebrafish, the images need to be treated with an antibiotic solution with a concentration of 5-20 μM before imaging processing.

Images