Water-soluble naphthalimide fluorescent dye as well as preparation method and application thereof

Abstract

The invention discloses a water-soluble naphthalimide-based fluorescent dye as well as a preparation method and application thereof. The molecular structural formula of the naphthalimide-based fluorescent dye is as follows: # imgabs0 #. The invention also specifically discloses a preparation method of the water-soluble naphthalimide-based fluorescent dye and application of the water-soluble naphthalimide-based fluorescent dye in detection of solution and intracellular viscosity. The naphthalimide-based fluorescent dye prepared by the invention can be dissolved in an aqueous solution, and has the characteristics of strong stability and no interference by other biomolecules on the response to viscosity.

Description

Technical Field

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

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

[0003] At present, the traditional means of detecting viscosity mainly include vibrating disk viscometers, capillary viscometers and vibrating string viscometers, etc. However, these methods have significant limitations. They not only require cumbersome sample pretreatment processes, but also cause irreversible damage to the structure and composition of the sample itself. In addition, they are mainly suitable for the detection of macroscopic samples, and are difficult to be competent for precise measurement tasks at the cellular level. Moreover, the single detection mode limits its application in complex biological systems. In contrast, fluorescent probe technology has shown significant advantages, such as high cost-effectiveness, simple operation, high sensitivity, strong selectivity and good stability. The most important thing is that fluorescent probes can achieve direct and non-destructive monitoring of target substances in living cells. However, most of the existing viscosity fluorescent probes are not completely water-soluble, which has become a major shortcoming in monitoring biological microenvironments. Given that the intracellular environment is mainly aqueous medium, the development of water-soluble fluorescent dyes to accurately track the dynamic changes of cell viscosity will undoubtedly greatly expand its application potential in the biomedical field and provide strong technical support for real-time monitoring of cell status and in-depth research on 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 view of the above technical problems, the present invention provides a water-soluble naphthylimide-based fluorescent dye and a preparation method thereof. The naphthylimide-based fluorescent dye prepared by the method can be dissolved in an aqueous solution, has a strong stability in response to viscosity and is not interfered by other biological molecules.

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

[0006] A water-soluble naphthalene imide-based fluorescent dye (NPP-2N+), which is a water-soluble fluorescent probe, has 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 the protection of an inert gas, and standing at room temperature after the reaction is complete to precipitate a solid, which is filtered, washed with ice 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 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 organic phase and purifying by column chromatography to obtain an intermediate compound 2;

[0011] Step S3, dissolving 3-bromopropyltrimethylammonium bromide and 4-methylpyridine in N,N-dimethylformamide, stirring and reacting at 100° C. under the protection of an inert gas, a white solid is generated during the reaction, and after cooling to room temperature, the solid is filtered, and the solid is washed with N,N-dimethylformamide and dichloromethane respectively, and the solid is collected and dried to obtain an 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, adding piperidine, reacting at 80-90° C. under the protection of an inert gas, and standing at room temperature after the reaction is complete to precipitate a solid, and 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 naphthylimide-based fluorescent dye of the present invention is used for fluorescence imaging detection of cell viscosity under different incubation conditions.

[0019] The water-soluble naphthylimide-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 the present invention is as follows: Due to the presence of a group with a high degree of spatial rotation ability in the molecular structure of the naphthylimide-based fluorescent dye, there is an intramolecular twisted charge transfer (Twisted Intramolecular Charge Transfer, TICT) in a low-viscosity solvent, forming a twisted intramolecular charge transfer state (TICT state), thereby inhibiting fluorescence emission. However, as the viscosity of the system increases, the inhibition of the molecular spatial rotation ability gradually increases, hindering the internal transformation of the molecule, resulting in a change in fluorescence intensity, thereby observing 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 naphthalene imide-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 dissolution, and NPP-2N+ can be directly prepared into a 5mM mother solution.

[0024] 3. The water-soluble naphthylimide-based fluorescent dye prepared by the present invention has excellent stability, and common metal ions, anions and different solution pH values ​​will not affect the fluorescent properties of the NPP-2N+ detection solution viscosity.

[0025] 4. The water-soluble naphthylimide-based fluorescent dye prepared by the present invention can show different spectral properties for changes in solvents with different gradient viscosities, and can be used for qualitative and quantitative change detection of solution viscosity with good sensitivity.

[0026] 5. The performance of the water-soluble naphthylimide-based fluorescent dye prepared by the present invention in detecting viscosity is not affected by organic small molecules, DNA, and phospholipid bilayer compounds DOPC ((R)-2,3-bis(oleoyloxy)propyl(2-(trimethylammonium)ethyl)phosphate), etc., and can be used to detect changes in intracellular viscosity under different incubation conditions, and 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 carbon nuclear magnetic resonance spectrum of the fluorescent dye NPP-2N+ prepared in the 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 This is the absorption spectra of aqueous solutions of the fluorescent dye NPP-2N+ with different concentrations prepared in the example.

[0031] Figure 5 This is the emission spectra of aqueous solutions of different concentrations of the fluorescent dye NPP-2N+ prepared in the example.

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

[0033] Figure 7 The fluorescence responsiveness 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] Fig. 9 The fluorescence response mechanism of the fluorescent dye NPP-2N+ prepared in the example to the solution viscosity.

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

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

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

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

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

[0041] Fig.15 The relative fluorescence intensity of the fluorescent dye NPP-2N+ prepared in the example of 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 the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0043] Example

[0044] The chemical reagents, solvents, active small molecules, etc. used in the process of preparing the fluorescent dye NPP-2N+ in the present invention are all analytically pure. In the process of structural confirmation and analytical performance testing of the fluorescent dye NPP-2N+, a DTX-600 nuclear magnetic resonance spectrometer of the company Bruke was used, the solvent was deuterated dimethyl sulfoxide, and TMS was used as the internal standard to record the nuclear magnetic resonance hydrogen spectrum and carbon spectrum. The Q-Exactive HR-MS mass spectrometer of the company Bruke in the United States was used to record high-resolution mass spectrum data. The F-7000 fluorescence spectrometer of Hitachi, Japan was used to record the fluorescence spectrum, and the T9CS double-beam ultraviolet-visible spectrophotometer of Beijing Puxi General Company was used to record the ultraviolet spectrum.

[0045] A method for preparing a water-soluble naphthalene imide-based fluorescent dye NPP-2N+, the specific steps are as follows:

[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 for 2 hours under nitrogen protection. After the reaction was completed, the reaction system was cooled to room temperature, and a large amount of solid precipitated. The solid was filtered through a Buchner funnel, washed with ice ethanol, and then collected and dried to obtain a yellow-brown solid, namely intermediate compound 1 (2.00 g), with a yield of 85.48%;

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

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

[0050] Step S3, intermediate compound 3, that is, 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 for 2 hours under nitrogen protection. A large amount of white solid was generated during the reaction. After cooling to room temperature, the solid was filtered and washed with N,N-dimethylformamide and dichloromethane respectively. The solid was collected and dried to obtain intermediate compound 3.

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

[0053] In a 100mL two-necked round-bottom flask, add intermediate compound 2 (0.20g, 0.56mmol) and intermediate compound 3 (197.00mg, 0.56mmol), add 20mL of anhydrous ethanol, then add 3 drops of piperidine, and replace nitrogen three times under vacuum. The reaction solution was reacted at 85°C for 12 hours under nitrogen protection. After the reaction was completed, a precipitate was generated. The reaction solution was filtered through a Buchner funnel while hot, and the solid was washed with a small amount of ethanol and collected and dried to obtain a dark yellow solid, namely 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). The H NMR spectrum is shown in 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 spectra are shown in Figure 3 shown.

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

[0057] 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 use. 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 above stock solution were measured with a pipette and placed in different 5 mL centrifuge tubes, and then appropriate amounts of water were added to the centrifuge tubes to make the final volume 4 mL, that is, 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 were prepared. The absorption spectra of NPP-2N+ aqueous solutions with different concentrations under the same test conditions were examined by UV-visible spectrophotometer, and the emission spectra of NPP-2N+ aqueous solutions with different concentrations under the same test conditions were examined by 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, and the emission intensity will decrease sharply if the concentration of the NPP-2N+ aqueous solution continues to increase. Therefore, if there is no special explanation, subsequent experiments will use an aqueous solution with a NPP-2N+ 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 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 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 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 fluorescence enhancement of the Gly group is because the viscosity of glycerol is much greater than that of water. Fig. 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 radiation 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 mother solution for use. Use a pipette to measure 16μL of the above mother solution and place it in different 5mL centrifuge tubes, then add appropriate amounts of water or propylene glycol 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. Fig.10 It can be seen that the emission intensity at 488nm of the prepared NPP-2N+ aqueous solution or propylene glycol solution under 366nm excitation conditions did not decrease significantly after 20 cycles of excitation, 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. Fig.11 It can be seen that in high-viscosity and low-viscosity solutions with different pH values, the fluorescence emission spectrum of NPP-2N+ is not significantly affected, and the fluorescence intensity of the high-viscosity solution with the same pH value at 488nm is much higher than that of the low-viscosity solution.

[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 mother solution for use. Use a pipette to measure 16μL of the above mother solution and place it in different 5mL centrifuge tubes. Then add appropriate amounts of water (volume fraction of 100% to 0%) and propylene glycol (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. Fig.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 ( Fig.13 ), indicating that NPP-2N+ can be used to quantitatively determine the viscosity of the solution.

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

[0066] 0.0070g of the fluorescent dye NPP-2N+ prepared above was accurately weighed with an analytical balance and dissolved in an aqueous solution to prepare a 5.00mM mother solution for use. 16μL of the above mother solution was measured with a pipette and placed in a 10mL centrifuge tube, and then an appropriate amount of cell culture medium was added to the centrifuge tube to make the final volume 8mL, that is, a cell culture medium solution with a concentration of 10μM of the fluorescent dye NPP-2N+ was prepared. HeLa cells were inoculated into a cell culture dish for confocal imaging, 1mL of the corresponding culture medium (containing 10wt% fetal bovine serum) was added, and cultured in a constant temperature incubator (volume fraction 5% CO2, 37°C) for 24 hours. Then washed 3 times with PBS (1mL), and then the culture medium containing NPP-2N+ (10μM) was added and incubated with the cells. After 30 minutes, the cells were washed 3 times with PBS (1mL). In the 4℃ experimental group, cells were first cultured at 4℃ for 30 minutes, then washed three times with PBS (1 mL), and then incubated with the cells in the 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 inhibitor (10 μM) in the culture medium for 30 minutes, then washed three times with PBS (1 mL), and then incubated with the cells in the culture medium containing NPP-2N+ (10 μM). After 30 minutes, the cells were washed three times with PBS (1 mL). Finally, the fluorescence distribution in the cells was observed under a confocal microscope. Fig.14 and Fig.15 It can be seen that NPP-2N+ in the control group, 4℃ experimental group and Cu 2+ The cells in the experimental group 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 naphthalene imide-based fluorescent dye, characterized in that: The naphthylimide-based fluorescent dye is a water-soluble fluorescent probe, and its molecular structure is as follows:

2. A method for preparing the water-soluble naphthalene imide-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 the protection of an inert gas, and standing at room temperature after the reaction is complete to precipitate a solid, which is filtered, washed with ice ethanol, and then collected and dried to obtain a yellow-brown solid, namely, intermediate compound 1; 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, adding ethyl acetate after standing at room temperature after the reaction is complete, washing with a saturated sodium chloride aqueous solution, removing the solvent from the organic phase, and purifying to obtain the intermediate compound 2; Step S3, dissolving 3-bromopropyltrimethylammonium bromide and 4-methylpyridine in N,N-dimethylformamide, stirring and reacting at 100° C. under the protection of an inert gas, a white solid is generated during the reaction, and after cooling to room temperature, the solid is filtered, and the solid is washed with N,N-dimethylformamide and dichloromethane respectively, and the solid is collected and dried to obtain an intermediate compound 3; 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, reacting at 80-90° C. under the protection of an inert gas, and standing at room temperature after the reaction is complete to precipitate a solid, and 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 naphthalene imide-based fluorescent dye according to claim 2, characterized in 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.

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

1.

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

2.

6. The method for preparing a water-soluble naphthalene imide-based fluorescent dye according to claim 2, characterized in that The specific synthetic route is as follows:

7. The water-soluble naphthylimide-based fluorescent dye according to claim 1 is used for fluorescence imaging detection of cell viscosity under different incubation conditions.

8. The water-soluble naphthylimide-based fluorescent dye according to claim 1 is used for detecting the viscosity of a solution or a cell.

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