A water-soluble naphthaleneimide-based fluorescent dye and its preparation method and application

Abstract

The present invention discloses a water-soluble naphthalimide-based fluorescent dye, its preparation method, and application. The molecular structure of the naphthalimide-based fluorescent dye is as follows: The present invention also specifically discloses a preparation method for the water-soluble naphthalimide-based fluorescent dye and its application in detecting solution and intracellular viscosity. The naphthalimide-based fluorescent dye prepared by the present invention is soluble in aqueous solution, exhibits strong stability in viscosity response, and is not interfered with by other biomolecules.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescence analysis and detection, and particularly relates to a water-soluble naphthaleneimide-based fluorescent dye and a preparation method and application thereof. Background Art

[0002] Viscosity, a key variable in the biological microenvironment, has a significant impact on the metabolic activity of organisms. At the cellular level, changes in viscosity are a barometer of physiological and pathological states. It not only affects the efficiency of cellular metabolism but is also directly related to the normal functioning of cells. Abnormal viscosity changes can trigger microenvironmental imbalances, which in turn can act as a catalyst for a range of complex diseases such as cystic kidney disease, malignant tumors, Alzheimer's disease, and Parkinson's disease. In view of this, technologies that can monitor viscosity fluctuations in living cells in real time are particularly important in biological and medical research. They are of irreplaceable value for early diagnosis of diseases, formulation of treatment strategies, and implementation of preventive measures. Although scientists have made significant progress in viscosity measurement technology in recent years, with diverse and innovative methods, technical means that can achieve both high precision and comprehensiveness remain scarce, providing researchers with broad research space and challenges.

[0003] Currently, traditional methods for measuring viscosity primarily include vibrating disk viscometers, capillary viscometers, and vibrating string viscometers. However, these methods have significant limitations. They require tedious sample pretreatment and can irreversibly damage the sample's structure and composition. Furthermore, they are primarily suitable for macroscopic measurements and are inadequate for precise measurements at the cellular level. Their single-mode nature limits their application in complex biological systems. In contrast, fluorescent probe technology offers significant advantages, including cost-effectiveness, ease of use, high sensitivity, strong selectivity, and excellent stability. Crucially, fluorescent probes enable direct, non-destructive monitoring of target substances within living cells. However, most existing fluorescent viscosity probes lack complete water solubility, a significant drawback for monitoring biological microenvironments. Given that the intracellular environment is primarily aqueous, developing water-soluble fluorescent dyes to precisely track dynamic changes in cellular viscosity would undoubtedly greatly expand their potential for biomedical applications, providing powerful technical support for real-time monitoring of cellular states and in-depth investigations of disease mechanisms. Therefore, the development of new water-soluble fluorescent probes can not only overcome the shortcomings of traditional methods, but also accurately adapt to the microenvironment detection needs in the aqueous environment of the organism. It has immeasurable application value in revealing the biological significance of changes in cell viscosity and potential disease associations. Summary of the Invention

[0004] In response to the above technical problems, the present invention provides a water-soluble naphthaleneimide-based fluorescent dye and a preparation method thereof. The naphthaleneimide-based fluorescent dye prepared by this method can be dissolved in an aqueous solution, has strong stability in response to viscosity, and is not interfered with by other biological molecules.

[0005] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0006] A water-soluble naphthalene imide-based fluorescent dye (NPP-2N+) is a water-soluble fluorescent probe with the following molecular structure:

[0007]

[0008] The preparation method of the water-soluble naphthalene imide-based fluorescent dye of the present invention comprises the following specific steps:

[0009] Step S1, dissolving 4-bromo-1,8-naphthalene anhydride and 3-amino-1-propanol in ethanol, reacting at 80-100° C. under inert gas protection, and standing at room temperature after the reaction is complete to precipitate a solid, which is filtered, washed with icy ethanol, and then collected and dried to obtain a yellow-brown solid, namely, intermediate compound 1;

[0010] Step S2, dissolving the intermediate compound 1 obtained in step S1, 4-formylphenylboronic acid, potassium carbonate and tetrakistriphenylphosphine palladium in a mixed solution of tetrahydrofuran and water (Wahaha drinking purified water, all aqueous solutions referred to below are Wahaha drinking purified water unless otherwise specified), reacting at 70-80° C. under inert gas protection, standing at room temperature after the reaction is complete, adding ethyl acetate, washing with saturated sodium chloride aqueous solution, removing the organic phase and purifying by column chromatography to obtain intermediate compound 2;

[0011] Step S3, dissolving 3-bromopropyltrimethylammonium bromide and 4-methylpyridine in N,N-dimethylformamide, stirring and reacting at 100° C. under inert gas protection, during the reaction, a white solid is generated. After cooling to room temperature, the solid is filtered, washed with N,N-dimethylformamide and dichloromethane, respectively, and the solid is collected and dried to obtain intermediate compound 3;

[0012] Step S4, dissolving the intermediate compound 2 obtained in step S2 and the intermediate compound 3 obtained in step S3 in an ethanol solution, then adding piperidine, and reacting at 80-90° C. under the protection of an inert gas. After the reaction is complete, the mixture is allowed to stand at room temperature to precipitate a solid. The solid is filtered, washed with ice ethanol, and then collected and dried to obtain a brown solid, namely, a water-soluble naphthalene imide fluorescent dye NPP-2N+.

[0013] It is further defined that the molar ratio of 4-bromo-1,8-naphthalene anhydride to 3-amino-1-propanol in step S1 is 1:1 to 1.4.

[0014] It is further defined that the molar ratio of the intermediate compound 1, 4-formylphenylboronic acid, potassium carbonate and tetrakistriphenylphosphine palladium in step S2 is 1:1-1.2:2.5-3.5:0.03-0.05, and the volume ratio of tetrahydrofuran and water in the mixed solution of tetrahydrofuran and water is 2-4:1.

[0015] It is further defined that the molar ratio of the intermediate compound 2 to the intermediate compound 3 in step S4 is 1:1 to 1.2.

[0016] The specific synthesis route of the water-soluble naphthalene imide-based fluorescent dye of the present invention is as follows:

[0017]

[0018] The water-soluble naphthaleneimide-based fluorescent dye of the present invention is used for fluorescence imaging detection of cell viscosity under different incubation conditions.

[0019] The water-soluble naphthaleneimide-based fluorescent dye of the present invention is used for detecting the viscosity of a solution or a cell.

[0020] The fluorescence detection mechanism of this invention is as follows: Due to the presence of a highly rotatable group within the molecular structure of the naphthalimide-based fluorescent dye, a twisted intramolecular charge transfer (TICT) occurs in low-viscosity solvents, forming a twisted intramolecular charge transfer state (TICT state), which suppresses fluorescence emission. However, as the viscosity of the system increases, the inhibition of the molecular rotation ability gradually increases, hindering the internal transformation of the molecule, resulting in a change in fluorescence intensity and the observation of a strong fluorescence signal.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0022] 1. The water-soluble naphthalene imide-based fluorescent dye prepared by the present invention has a novel structure, a simple synthesis method, and the target compound NPP-2N+ can be obtained in the post-treatment process without the need for column chromatography separation and purification.

[0023] 2. The water-soluble naphthaleneimide-based fluorescent dye prepared by the present invention has excellent water solubility. In the process of preparing the analysis and detection mother solution, no organic solvent is required for solubilization, and NPP-2N+ can be directly prepared into a 5mM mother solution.

[0024] 3. The water-soluble naphthalene imide-based fluorescent dye prepared by the present invention has excellent stability. Common metal ions, anions and different solution pH values ​​will not affect the fluorescence performance of NPP-2N+ for detecting solution viscosity.

[0025] 4. The water-soluble naphthaleneimide-based fluorescent dye prepared by the present invention can exhibit different spectral properties in response to changes in solvent viscosity with different gradients, and can be used to detect qualitative and quantitative changes in solution viscosity with good sensitivity.

[0026] 5. The viscosity detection performance of the water-soluble naphthaleneimide-based fluorescent dye prepared by the present invention is not affected by organic small molecules, DNA, and phospholipid bilayer compounds such as DOPC ((R)-2,3-bis(oleoyloxy)propyl(2-(trimethylammonium)ethyl)phosphate), and can be used to detect changes in intracellular viscosity under different incubation conditions. It has broad application prospects in the field of biological viscosity microenvironment detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the N+ nuclear magnetic resonance hydrogen spectrum of the fluorescent dye NPP-2 prepared in Example (the solvent is DMSO-d6).

[0028] Figure 2 This is the N+ NMR carbon spectrum of the fluorescent dye NPP-2 prepared in Example (the solvent is DMSO-d6).

[0029] Figure 3 This is the high-resolution mass spectrum of the fluorescent dye NPP-2N+ prepared in Example (the solvent is CH3OH).

[0030] Figure 4 The absorption spectra of aqueous solutions of the fluorescent dye NPP-2N+ with different concentrations prepared in the examples are shown.

[0031] Figure 5 The emission spectra of aqueous solutions of the fluorescent dye NPP-2N+ with different concentrations prepared in the examples are shown.

[0032] Figure 6 The fluorescence response of the aqueous solution of the fluorescent dye NPP-2N+ prepared in the example to various metal ions.

[0033] Figure 7 The fluorescence response of the aqueous solution of the fluorescent dye NPP-2N+ prepared in the example to various anions.

[0034] Figure 8 The fluorescence responsiveness of the aqueous solution of the fluorescent dye NPP-2N+ prepared in the example to various active small molecules.

[0035] Figure 9 The fluorescence response mechanism of the fluorescent dye NPP-2N+ prepared in the example to the solution viscosity.

[0036] Figure 10 The fluorescence stability of the fluorescent dye NPP-2N+ prepared in this example.

[0037] Figure 11 This figure shows the fluorescence stability of the fluorescent dye NPP-2N+ prepared in the example in solutions with different pH values.

[0038] Figure 12 This is the fluorescence spectrum of the fluorescent dye NPP-2N+ prepared in the example in aqueous solutions with different viscosities.

[0039] Figure 13 The relationship between the fluorescence intensity of the fluorescent dye NPP-2N+ prepared in this example at 480 nm and the viscosity of the aqueous solution.

[0040] Figure 14 These are live cell fluorescence imaging images of the fluorescent dye NPP-2N+ prepared in the example under different incubation conditions.

[0041] Figure 15 The relative fluorescence intensity of the fluorescent dye NPP-2N+ prepared in the example was measured in living cells under different incubation conditions. DETAILED DESCRIPTION

[0042] The above contents of the present invention are further described in detail below through examples, but this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above contents of the present invention fall within the scope of the present invention.

[0043] Example

[0044] The chemical reagents, solvents, and active small molecules used in the preparation of the fluorescent dye NPP-2N+ in the present invention were all of analytical grade. During the structural confirmation and analytical performance testing of the fluorescent dye NPP-2N+, a DTX-600 nuclear magnetic resonance spectrometer from Bruke was used, with deuterated dimethyl sulfoxide as the solvent and TMS as the internal standard to record the hydrogen and carbon nuclear magnetic resonance spectra. High-resolution mass spectrometric data were recorded using a Q-Exactive HR-MS mass spectrometer from Bruke, USA. Fluorescence spectra were recorded using an F-7000 fluorescence spectrometer from Hitachi, Japan, and UV spectra were recorded using a T9CS dual-beam UV-visible spectrophotometer from Beijing Puxi General.

[0045] A method for preparing a water-soluble naphthaleneimide-based fluorescent dye NPP-2N+, comprising the following steps:

[0046] Step S1, preparation of intermediate compound 1, ie, 6-bromo-2-(3-hydroxypropyl)-1H-benzo[d,e]isoquinoline-1,3(2H)-dione:

[0047] 4-Bromo-1,8-naphthalene anhydride (1.97 g, 7.00 mmol) and 3-amino-1-propanol (0.66 mL, 8.40 mmol) were added to a 100 mL two-necked round-bottom flask, and then anhydrous ethanol (30 mL) was added. The mixture was stirred at 90°C under nitrogen for 2 hours. After the reaction was completed, the reaction system was cooled to room temperature. A large amount of solid precipitated. The solid was filtered through a Buchner funnel, washed with icy ethanol, and then collected and dried to obtain a yellow-brown solid, intermediate compound 1 (2.00 g), with a yield of 85.48%.

[0048] Step S2, preparation of intermediate compound 2, namely 4-(2-(3-hydroxypropyl)-1,3-dioxo-2,3-dihydro-1H-benzo[d,e]isoquinolin-6-yl)benzaldehyde:

[0049] In a 100 mL two-necked round-bottom flask, intermediate compound 1 (1 g, 2.99 mmol), 4-formylphenylboronic acid (0.50 g, 2.99 mmol), tetrakistriphenylphosphine palladium (0.14 g, 0.12 mmol), and anhydrous potassium carbonate (1.24 g, 8.98 mmol) were added, respectively. The atmosphere was purged with nitrogen three times under vacuum, and then 30 mL of tetrahydrofuran and 10 mL of water were added. After another three vacuum changes with nitrogen, the reaction mixture was stirred at 77°C under nitrogen protection for 16 hours. After completion of the reaction, the reaction system was cooled to room temperature, and the reaction solution was poured into 50 mL of water and extracted with ethyl acetate (15 mL × 3). The organic phase was collected and washed with saturated brine (30 mL × 3), and the organic phase was dried over anhydrous sodium sulfate for 10 minutes. After drying, the solid was removed by filtration, and the liquid was rotary evaporated under vacuum to remove ethyl acetate. After drying, the crude product of intermediate compound 2 was obtained, which was purified by column chromatography (petroleum ether:ethyl acetate) to obtain intermediate compound 2 with a yield of 85.45%.

[0050] Step S3, intermediate compound 3, i.e., intermediate compound 3, is prepared by the synthesis method disclosed in the literature (J.Org.Chem.2008,73,6587-6594):

[0051] 3-Bromopropyltrimethylammonium bromide and 4-methylpyridine were dissolved in N,N-dimethylformamide and stirred at 100°C under nitrogen for 2 hours. A large amount of white solid was produced during the reaction. After cooling to room temperature, the solid was filtered and washed with N,N-dimethylformamide and dichloromethane. The solid was collected and dried to obtain intermediate compound 3.

[0052] (4) Preparation of fluorescent dye NPP-2N+:

[0053] In a 100 mL two-necked round-bottom flask, intermediate compound 2 (0.20 g, 0.56 mmol) and intermediate compound 3 (197.00 mg, 0.56 mmol) were added, followed by 20 mL of anhydrous ethanol and 3 drops of piperidine. The atmosphere was then purged with nitrogen three times under vacuum. The reaction mixture was incubated at 85°C under nitrogen for 12 hours. After completion, a precipitate formed. The hot reaction mixture was filtered through a Buchner funnel. The solid was washed with a small amount of ethanol and then dried to obtain a dark yellow solid, the fluorescent dye NPP-2N, with a yield of 56.84%.

[0054] Fluorescent dye NPP-2N+ nuclear magnetic resonance determination: 1 H NMR (600MHz, DMSO) δ9.07(d,J=6.5Hz,2H),8.58(t,J=6.5Hz,2H),8.40(d,J=6.6Hz,2H),8.3 0(d,J=8.5Hz,1H),8.24(d,J=16.3Hz,1H),8.01(d,J=8.2Hz,2H),7.89(dd,J=16.6,7.6Hz,2H ),7.77–7.71(m,3H),4.64(t,J=7.3Hz,2H),4.53(t,J=5.1Hz,1H),4.17–4.14(m,2H),3.53(d d,J=11.6,6.1Hz,2H),3.46–3.42(m,2H),3.12(s,9H),2.50–2.45(m,2H),1.85–1.81(m,2H); 13 CNMR(151MHz,DMSO)δ163.92(s),163.70(s),153.58(s),145.56(s),145.10(s),140. 85(s),140.62(s),135.83(s),132.44(s),131.41(s),131.16(s),130.85(s),129.61 (s), 129.02 (s), 128.52 (d, J = 17.2 Hz), 128.18 (s), 124.65 (d, J = 9.1 Hz), 123.07 (s), 122.16 (s), 62.26 (s), 59.48 (s), 57.20 (s), 52.98 (s), 38.16 (s), 31.45 (s), 24.60 (s). Figure 1 As shown, the carbon NMR spectrum is as follows Figure 2 shown.

[0055] Fluorescent dye NPP-2N+ high-resolution mass spectrometry: HR-ESI-MS calculation for M-2Br - -(CH3)3NH+ :475.2022, found 475.2026. High-resolution mass spectrum Figure 3 shown.

[0056] NPP-2N+ concentration response UV absorption and fluorescence emission intensity measurement experiment:

[0057] Accurately weigh 0.0070 g of the fluorescent dye NPP-2N+ prepared above using an analytical balance and dissolve it in an aqueous solution to prepare a 5.00 mM stock solution for later use. Use a pipette to measure 0 μL, 8 μL, 16 μL, 24 μL, 32 μL, 40 μL, 48 μL, 56 μL, 64 μL, 72 μL, and 80 μL of the stock solution into different 5 mL centrifuge tubes. Then, add an appropriate amount of water to each centrifuge tube to make a final volume of 4 mL, thus preparing aqueous solutions with NPP-2N+ concentrations of 0 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM, respectively. The absorption spectra of NPP-2N+ aqueous solutions with different concentrations under the same test conditions were examined using a UV-visible spectrophotometer, and the emission spectra of NPP-2N+ aqueous solutions with different concentrations under the same test conditions were examined using a fluorescence spectrometer. Figure 4 It can be seen that the prepared NPP-2N+ aqueous solution has a maximum absorption wavelength absorption peak at 373nm, and this absorption peak increases with the increase of the concentration of NPP-2N+ in the aqueous solution, and the absorption intensity continues to increase when the concentration reaches 100μM. Figure 5 It can be seen that the prepared NPP-2N+ aqueous solution has a fluorescence emission peak at 488nm under 366nm excitation conditions, and the emission intensity is strongest when the concentration of the NPP-2N+ aqueous solution is 10μM. When the concentration is 20μM, the emission intensity does not decrease significantly. However, if the concentration of the NPP-2N+ aqueous solution continues to increase, the emission intensity will decrease sharply. Therefore, unless otherwise specified, subsequent experiments will use an NPP-2N+ aqueous solution with a concentration of 20μM for testing.

[0058] NPP-2N+ fluorescence selectivity experiment:

[0059] 0.0070 g of the fluorescent dye NPP-2N+ prepared above was accurately weighed with an analytical balance and dissolved in an aqueous solution to prepare a 5.00 mM stock solution for later use. 16 μL of the stock solution was measured with a pipette and placed in different 5 mL centrifuge tubes. Then, 10 equivalents of various common cations (Li + ,Mg 2+ ,Ca 2+ ,Cr 3+ ,Mn 2+ ,Fe 2+ ,Fe3+ ,Co 2+ ,Ni 2+ ,Cu 2+ ,Zn 2+ ,Ag + ,Cd 2+ ,Hg 2+ ,Al 3+ ,Pb 2+ ,Ba 2+ ,Sr 2+ ,Cs 2+ ), anions (Cl - ,Br - ,I - ,CO3 2- ,NO3 - ,NO2 - ,SO4 2- ,SO3 2- ,HS - ,ClO - ) and active small molecules (Arginine, Serine, Urea, Glycine, Threonine, Glucose, Glutathione, Homocysteine, Cysteine, Glutamate, 1-Octanol, DNA, DOPC) and an appropriate amount of water to make the final volume 4mL; use a pipette to measure 16μL of the above mother solution and place it in a 5mL centrifuge tube, add an appropriate amount of propylene glycol to the centrifuge tube to make the final volume 4mL (Gly group); at the same time, set up a blank control group of NPP-2N+ aqueous solution. Then use a fluorescence spectrometer to examine the emission spectrum of the above solution system under the same test conditions. Figure 6 、 Figure 7 and Figure 8 It can be seen that the fluorescence emission intensity of the mixed aqueous solution system at 488nm did not change significantly compared with the fluorescence intensity of the blank control group, while the fluorescence emission intensity of the Gly group at 488nm was significantly enhanced compared with the fluorescence intensity of the blank control group. The enhanced fluorescence of the Gly group is because the viscosity of glycerol is much greater than that of water, such as Figure 9 As shown in the figure, high viscosity limits the free rotation of the fluorescent dye NPP-2N+ molecule, causing the excited state energy to return to the ground state through radiative transition. Therefore, the fluorescence emission intensity of NPP-2N+ is enhanced in high viscosity solutions.

[0060] NPP-2N+ fluorescence stability experiment:

[0061] Use an analytical balance to accurately weigh 0.0070g of the fluorescent dye NPP-2N+ prepared above and dissolve it in an aqueous solution to prepare a 5.00mM stock solution for use. Use a pipette to measure 16μL of the above stock solution and place it in different 5mL centrifuge tubes. Then add appropriate amounts of water or glycerol to the centrifuge tubes to make the final volume 4mL. Use a fluorescence spectrometer to examine the stability of the emission spectrum of NPP-2N+ in different solutions under the same test conditions. Figure 10 It can be seen that the emission intensity at 488 nm of the prepared NPP-2N+ aqueous solution or propylene glycol solution does not decrease significantly after 20 cycles of excitation at 366 nm, indicating that NPP-2N+ has high stability.

[0062] Use a pipette to measure 16 μL of the above mother solution and place it in different 5 mL centrifuge tubes. Then add appropriate amounts of PBS buffer solutions with different pH values ​​or mixed solutions of PBS buffer solutions with different pH values ​​(5% by volume) and glycerol (95% by volume) to the centrifuge tubes to make the final volume 4 mL. Use a fluorescence spectrometer to examine the emission intensity of NPP-2N+ at 488 nm in different solutions under the same test conditions. Figure 11 It can be seen that the fluorescence emission spectrum of NPP-2N+ is not significantly affected in high-viscosity and low-viscosity solutions with different pH values, and the fluorescence intensity of the high-viscosity solution at the same pH value is much higher than that of the low-viscosity solution at 488 nm.

[0063] Quantitative fluorescence detection experiment of NPP-2N+ on solution viscosity:

[0064] Use an analytical balance to accurately weigh 0.0070g of the fluorescent dye NPP-2N+ prepared above and dissolve it in an aqueous solution to prepare a 5.00mM stock solution for use. Use a pipette to measure 16μL of the above stock solution and place it in different 5mL centrifuge tubes. Then add appropriate amounts of water (volume fraction of 100% to 0%) and glycerol (volume fraction of 0% to 100%) to the centrifuge tubes to make a final volume of 4mL. Use a fluorescence spectrometer to examine the emission spectra of NPP-2N+ in solutions of different viscosities under the same test conditions. Figure 12 It can be seen that the emission intensity of the prepared fluorescent dye NPP-2N+ solutions with different viscosities at 488nm increases with the increase of solution viscosity, and there is a certain relationship between the emission intensity of the fluorescent dye NPP-2N+ at 480nm and the solution viscosity ( Figure 13 ), indicating that NPP-2N+ can be used to quantitatively determine the viscosity of the solution.

[0065] Fluorescence imaging applications of NPP-2N+ for detecting viscosity of living cells:

[0066] Accurately weigh 0.0070 g of the fluorescent dye NPP-2N+ prepared above using an analytical balance and dissolve it in aqueous solution to prepare a 5.00 mM stock solution. Using a pipette, transfer 16 μL of this stock solution to a 10 mL centrifuge tube. Then, add an appropriate amount of cell culture medium to the tube to a final volume of 8 mL, thus preparing a cell culture solution containing 10 μM fluorescent dye NPP-2N+. HeLa cells were seeded into confocal imaging culture dishes, and 1 mL of the appropriate culture medium (containing 10 wt% fetal bovine serum) was added. The cells were incubated in a constant temperature incubator (5% CO2, 37°C) for 24 hours. The cells were then washed three times with 1 mL of PBS and incubated with culture medium containing 10 μM NPP-2N+. After 30 minutes, the cells were washed three times with 1 mL of PBS. In the 4°C experimental group, cells were first incubated at 4°C for 30 minutes, then washed three times with PBS (1 mL), and then incubated with the culture medium containing NPP-2N+ (10 μM). After 30 minutes, the cells were washed three times with PBS (1 mL). 2+ In the experimental group, cells were first treated with Cu 2+ The cells were incubated with the culture medium containing NPP-2N+ (10 μM) inhibitor for 30 minutes, then washed with PBS (1 mL) for 3 times, and then incubated with the cells in the culture medium containing NPP-2N+ (10 μM). After 30 minutes, the cells were washed with PBS (1 mL) for 3 times. Finally, the fluorescence distribution in the cells was observed under a confocal microscope. Figure 14 and Figure 15 It can be seen that NPP-2N+ in the control group, 4℃ experimental group and Cu 2+ The cells in the experimental groups showed different fluorescence intensities, which were related to the intracellular viscosity. As the viscosity of the intracellular environment increased, the fluorescence intensity of the fluorescent dye NPP-2N+ in the cells also increased, indicating that the fluorescent dye NPP-2N+ can be used for qualitative detection of intracellular viscosity.

[0067] The above embodiments describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A water-soluble naphthaleneimide-based fluorescent dye, characterized in that: The naphthaleneimide-based fluorescent dye is a water-soluble fluorescent probe, and its molecular structure is as follows: 。 2. A method for preparing the water-soluble naphthaleneimide-based fluorescent dye according to claim 1, characterized in that The specific steps are as follows: Step S1, dissolving 4-bromo-1,8-naphthalene anhydride and 3-amino-1-propanol in ethanol, reacting at 80-100° C. under inert gas protection, and standing at room temperature after the reaction is complete to precipitate a solid. The solid is filtered, washed with icy ethanol, and then collected and dried to obtain a yellow-brown solid, namely, intermediate compound 1. The structural formula of intermediate compound 1 is: ； Step S2, dissolving the intermediate compound 1 obtained in step S1, 4-formylphenylboronic acid, potassium carbonate and tetrakistriphenylphosphine palladium in a mixed solution of tetrahydrofuran and water, reacting at 70-80° C. under the protection of an inert gas, standing at room temperature after the reaction is complete, adding ethyl acetate, washing with a saturated sodium chloride aqueous solution, removing the solvent from the organic phase, and purifying to obtain the intermediate compound 2. The structural formula of the intermediate compound 2 is: ； Step S3, 3-bromopropyltrimethylammonium bromide and 4-methylpyridine are dissolved in N,N-dimethylformamide, and the mixture is stirred and reacted at 100° C. under inert gas protection. During the reaction, a white solid is generated. After cooling to room temperature, the solid is filtered and washed with N,N-dimethylformamide and dichloromethane respectively. The solid is collected and dried to obtain intermediate compound 3. The structural formula of intermediate compound 3 is: ； Step S4, dissolving the intermediate compound 2 obtained in step S2 and the intermediate compound 3 obtained in step S3 in an ethanol solution, adding piperidine, and reacting at 80-90° C. under the protection of an inert gas. After the reaction is complete, the mixture is allowed to stand at room temperature to precipitate a solid. The solid is filtered, washed with ice ethanol, and then collected and dried to obtain a brown solid, namely, a water-soluble naphthalene imide fluorescent dye NPP-2N+.

3. The method for preparing a water-soluble naphthaleneimide-based fluorescent dye according to claim 2, wherein: The molar ratio of 4-bromo-1,8-naphthalene anhydride to 3-amino-1-propanol in step S1 is 1:1 to 1.

4.

4. The method for preparing a water-soluble naphthaleneimide-based fluorescent dye according to claim 2, wherein: The molar ratio of the intermediate compound 1, 4-formylphenylboronic acid, potassium carbonate and tetrakistriphenylphosphine palladium in step S2 is 1:1~1.2:2.5~3.5:0.03~0.05, and the volume ratio of tetrahydrofuran and water in the mixed solution of tetrahydrofuran and water is 2~4:

1.

5. The method for preparing a water-soluble naphthaleneimide-based fluorescent dye according to claim 2, wherein: The molar ratio of the intermediate compound 2 to the intermediate compound 3 in step S4 is 1:1 to 1.

2.

6. A method for preparing the water-soluble naphthaleneimide-based fluorescent dye according to claim 1, characterized in that The specific synthetic route is as follows: 。 7. Use of the water-soluble naphthaleneimide-based fluorescent dye according to claim 1 in non-disease diagnosis or treatment by fluorescence imaging detection of cell viscosity under different incubation conditions.

8. Use of the water-soluble naphthaleneimide-based fluorescent dye according to claim 1 for detecting viscosity in solutions or cells for non-disease diagnosis or treatment.

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