Dual-function fluorescent probe for simultaneously detecting viscosity and peroxynitrite as well as preparation method and application of dual-function fluorescent probe

Abstract

The invention discloses a dual-function fluorescent probe for simultaneously detecting viscosity and peroxynitrite as well as a preparation method and application of the dual-function fluorescent probe, and belongs to the technical field of fluorescent probes. According to the bifunctional fluorescent probe, naphthalimide and hemicyanine are connected through a carbon-carbon double bond, and viscosity and peroxynitrite are detected at the same time through double recognition sites. Due to free intramolecular rotation between naphthalimide and hemicyanine, an intramolecular charge transfer effect is twisted, and the probe is non-fluorescent. The fluorescence can be enhanced by two different ways: (i) rotation is limited by increasing viscosity, thereby generating strong near-infrared fluorescence; and (ii) carbon-carbon double bonds are destroyed after peroxynitrite is added, so that an intramolecular charge transfer mechanism is caused, and the fluorescence ratio is obviously enhanced. The probe has the advantages of good selectivity, rapid response and the like when being used for detecting viscosity and peroxynitrite, and has a huge application prospect in the technical fields of analytical chemistry, life science, biomedical treatment and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a dual-function fluorescent probe for simultaneously detecting viscosity and peroxynitrite, and a preparation method and application thereof. Background Art

[0002] Viscosity is an important parameter that determines the cell microenvironment. Its abnormal changes often affect the molecular interactions, material transport, chemical signal transduction and metabolic processes within the cell. Studies have shown that fluctuations in the level of reactive nitrogen species (RNS) in cells are closely related to changes in viscosity. Among them, peroxynitrite (ONOO - ) is one of the indispensable RNS, which is composed of nitric oxide (·NO) and superoxide radical (O2 ·- ) is endogenously produced through a rapid diffusion-controlled reaction. It can change viscosity by oxidizing cellular biomolecules such as proteins, thiols, lipids, and nucleic acids, ultimately affecting the physiological functions of normal cells and often leading to cell apoptosis or necrosis. Therefore, the development of an efficient method that can simultaneously detect peroxynitrite and viscosity changes is of great significance for revealing key processes in biological systems.

[0003] In recent years, fluorescent probes have become a widely used analytical detection tool due to their simple operation, low cost, high sensitivity, non-invasiveness and high spatiotemporal resolution. However, most of the existing fluorescent probes can only detect viscosity or peroxynitrite independently, and work in the non-near-infrared band (emission wavelength less than 650nm), which is easily interfered by biological background fluorescence, limiting their application in complex biological systems. In view of the important role of peroxynitrite and viscosity in various physiological processes and diseases, it is urgent to develop a new type of near-infrared fluorescent probe that can simultaneously realize real-time dynamic monitoring of viscosity and peroxynitrite in complex biological environments. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide a dual-function fluorescent probe for simultaneously detecting viscosity and peroxynitrite and a preparation method and application thereof.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A dual-function fluorescent probe for simultaneously detecting viscosity and peroxynitrite, the structural formula of which is shown below:

[0007]

[0008] This bifunctional fluorescent probe connects naphthalimide and hemicyanine via a carbon-carbon double bond (-C=C-) and simultaneously detects viscosity and peroxynitrite via dual recognition sites. The probe is non-fluorescent due to the twisted intramolecular charge transfer (TICT) effect caused by the free intramolecular rotation between naphthalimide and hemicyanine. Its fluorescence can be enhanced in two different ways: (i) by increasing viscosity to restrict rotation, thereby generating strong near-infrared fluorescence (λ em =675nm); (ii) by adding peroxynitrite, the carbon-carbon double bond is destroyed, resulting in an intramolecular charge transfer (ICT) mechanism, and the fluorescence (λ em =550 / 675nm) ratio is significantly enhanced.

[0009] The preparation method of the above-mentioned dual-function fluorescent probe comprises the following steps:

[0010] (1) Preparation of intermediate 1

[0011] 4-Bromo-1,8-naphthalene dicarboxylic anhydride and 4-(2-aminoethyl)-morpholine were dissolved in an organic solvent, and heated under reflux and stirred at 100°C for reaction; after the reaction, the reaction solution was cooled, filtered under reduced pressure, and the obtained solid was washed and dried to obtain intermediate 1, whose structural formula is:

[0012]

[0013] (2) Preparation of Intermediate 2

[0014] The intermediate 1 (N-(2-morpholinoethyl)-4-bromo-1,8-naphthalimide and N-hydroxyphthalimide) was dissolved in an organic solvent, potassium carbonate was added, and the reaction was stirred at 80°C; after the reaction was completed, the mixture was cooled to room temperature, the pH was adjusted with dilute hydrochloric acid, the reaction solution was extracted, and the mixture was further dried and sanded, and purified to obtain the intermediate 2, whose structural formula is:

[0015]

[0016] (3) Preparation of Intermediate 3

[0017] The intermediate 2 (N-(2-morpholinoethyl)-4-hydroxy-1,8-naphthalimide and hexamethylenetetramine) was dissolved in an organic solvent, and heated under reflux and stirred at 120°C for reaction; after the reaction, the reaction liquid was cooled, and filtered under reduced pressure to obtain the intermediate 3, whose structural formula is:

[0018]

[0019] (4) Preparation of Viscosity and Peroxynitrite Dual-Function Fluorescent Probes

[0020] Under an inert gas atmosphere, the intermediate 3 (N-(2-morpholinoethyl)-4-hydroxy-3-formyl-1,8-naphthalimide) and 3-ethyl-1,1,2-trimethylbenzindolinium iodide are dissolved in an organic solvent and heated to 80° C. under the catalysis of piperidine; after the reaction is completed, the reaction solution is cooled to room temperature, and the reaction solution is subjected to vacuum rotary evaporation to obtain sand, and a red-purple solid is purified, namely the bifunctional fluorescent probe.

[0021] In step (1), the organic solvent is preferably anhydrous ethanol.

[0022] In step (1), the molar ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride to 4-(2-aminoethyl)-morpholine is 1:1.5.

[0023] In step (1), the heating and stirring reaction time is 6 hours.

[0024] In step (2), the organic solvent is preferably dimethyl sulfoxide.

[0025] In step (2), the molar ratio of the intermediate 1 to N-hydroxyphthalimide is 1:1.1.

[0026] In step (2), the molar ratio of the intermediate 1 to potassium carbonate is 1:3.

[0027] In step (2), the heating and stirring reaction time is 2 hours.

[0028] In step (2), the purification is performed by column chromatography, and the column chromatography conditions are as follows: dichloromethane and methanol are used as eluents in a volume ratio of 25:1; and the silica gel is 200-300 meshes.

[0029] In step (3), the organic solvent is preferably trifluoroacetic acid.

[0030] In step (3), the molar ratio of the intermediate 2 to hexamethylenetetramine is 1:2.

[0031] In step (3), the heating and stirring reaction time is 12 hours.

[0032] In step (4), the organic solvent is preferably anhydrous ethanol.

[0033] In step (4), the molar ratio of the intermediate 3 to 3-ethyl-1,1,2-trimethylbenzindolium iodide is 1:1.

[0034] In step (4), the heating and stirring reaction time is 12 hours.

[0035] In step (4), purification is performed by column chromatography, and the column chromatography conditions are as follows: dichloromethane and methanol are used as eluents, gradient elution is adopted, and the volume ratio is 40:1 to 10:1; and the silica gel is 200 to 300 meshes.

[0036] The present invention also provides the application of the dual-function fluorescent probe in the simultaneous detection of viscosity and peroxynitrite in cells and organisms.

[0037] Beneficial effects of the present invention:

[0038] 1. The present invention provides a new dual-function fluorescent probe that can realize dual recognition of viscosity and peroxynitrite.

[0039] 2. The fluorescent probe of the present invention has good selectivity and specifically recognizes viscosity and peroxynitrite, but has no response to other substances such as active oxygen, active nitrogen, and active sulfur.

[0040] 3. The fluorescent probe of the present invention has a significant near-infrared fluorescence "on" response to viscosity. Within the viscosity range of 1.0 to 1410cp, the fluorescence intensity is enhanced by about 400 times, showing a high response to viscosity changes.

[0041] 4. The fluorescent probe of the present invention has high sensitivity in identifying peroxynitrite, with a response time of about 10 minutes and a detection limit as low as 42 nM.

[0042] 5. The fluorescent probe of the present invention can be used to observe the changes of peroxynitrite and viscosity in SH-SY5Y cells and zebrafish in real time.

[0043] 6. The synthesis steps are simple, the raw materials are easily available, the reaction conditions are mild, and the yield is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The present invention will be further described below in conjunction with the accompanying drawings.

[0045] Figure 1 The invention discloses a synthetic route for preparing the bifunctional fluorescent probe Nap-Cy.

[0046] Figure 2 This is a test of the detection performance of the bifunctional fluorescent probe Nap-Cy of the present invention on peroxynitrite.

[0047] Among them, (A) and (B) are the absorption spectrum and fluorescence spectrum of the probe Nap-Cy before and after responding to peroxynitrite, respectively; (C) and (D) are the fluorescence spectrum and time response curve of the probe Nap-Cy in response to different concentrations of peroxynitrite, respectively; (E) and (F) are the selectivity and pH stability tests of the probe Nap-Cy, respectively.

[0048] Figure 3This is a test of the viscosity detection performance of the bifunctional fluorescent probe Nap-Cy of the present invention.

[0049] Figure 4 This is the fluorescence imaging of endogenous peroxynitrite in SH-SY5Y cells by the bifunctional fluorescent probe Nap-Cy of the present invention.

[0050] Figure 5 This is the fluorescence imaging of exogenous peroxynitrite in zebrafish by the dual-functional fluorescent probe Nap-Cy of the present invention.

[0051] Figure 6 This is the fluorescence imaging of SH-SY5Y cell viscosity by the bifunctional fluorescent probe Nap-Cy of the present invention.

[0052] Figure 7 This is the fluorescence imaging of zebrafish viscosity using the dual-function fluorescent probe Nap-Cy of the present invention. DETAILED DESCRIPTION

[0053] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] Example 1 Synthesis of bifunctional fluorescent probe Nap-Cy

[0055] The synthesis route of the bifunctional fluorescent probe Nap-Cy in this example is as follows Figure 1 As shown, the specific synthesis steps are as follows:

[0056] (1) Synthesis of intermediate 1

[0057] Accurately weigh 4-bromo-1,8-naphthalene dicarboxylic anhydride (2.77 g, 10 mmol) into a 200 mL eggplant-shaped bottle, then add 100 mL of anhydrous ethanol, ultrasonicate for 10 min to make it evenly dispersed, then add 4-(2-aminoethyl)-morpholine (1.97 mL, 15 mmol) into the eggplant-shaped bottle, and reflux the above reaction solution at 100 ° C for 6 h. TLC spot plate monitors the progress of the reaction, and after the reaction is completed, the precipitate is collected by filtration and washed three times to obtain N-(2-morpholinoethyl)-4-bromo-1,8-naphthalene dicarboximide with a yield of 89%.

[0058] (2) Synthesis of Intermediate 2

[0059] Weigh N-(2-morpholinoethyl)4-bromo-1,8-naphthalimide (1.05 g, 2.7 mmol) and N-hydroxyphthalimide (489 mg, 3 mmol) accurately into a 50 mL round-bottom flask, then add 13 mL of ultra-dry dimethyl sulfoxide, ultrasonicate for 10 min to make it dispersed evenly, then add potassium carbonate (1.12 g, 8.1 mmol) as a catalyst and base, and reflux the above reaction solution at 80 ° C for 2 h. The reaction solution changes from yellow to dark red, and the reaction is continued for 1 h. After the reaction is completed, cool to room temperature, transfer the reaction solution to a beaker, add 100 mL of deionized water, and adjust the pH to 6 with 1 M dilute hydrochloric acid. Subsequently, the product was extracted three times with 50 mL of dichloromethane, and the organic phase was collected and rotary evaporated at low temperature and reduced pressure to obtain a bright yellow solid, which was separated and purified by silica gel column chromatography (200-300 mesh) using dichloromethane and methanol (v / v, 25:1) as eluents to obtain N-(2-morpholinoethyl)-4-hydroxy-1,8-naphthalene dicarboximide with a yield of 84%.

[0060] (3) Synthesis of Intermediate 3

[0061] Accurately weigh N-(2-morpholinoethyl)-4-hydroxy-1,8-naphthalimide (489.45 mg, 1.5 mmol) and hexamethylenetetramine (420.57 mg, 3 mmol) and dissolve in 10 mL of trifluoroacetic acid, and reflux at 120 ° C for 12 h. After the reaction is completed, the solution is transferred to 30 mL of deionized water, stirred and placed in a 4 ° C refrigerator for 2 h, filtered and collected the precipitate, washed with distilled water several times and then vacuum dried. Finally, it was separated and purified by silica gel column chromatography (200-300 mesh) to obtain N-(2-morpholinoethyl)-4-hydroxy-3-formyl-1,8-naphthalimide with a yield of 68%.

[0062] (4) Synthesis of the bifunctional fluorescent probe Nap-Cy

[0063] Under an inert gas atmosphere, N-(2-morpholinoethyl)-4-hydroxy-3-formyl-1,8-naphthalimide (177 mg, 0.5 mmol) and 3-ethyl-1,1,2-trimethylbenzindolinium iodide (182.5 mg, 0.5 mmol) were accurately weighed and dissolved in 5 mL of anhydrous ethanol, followed by the addition of 100 μL of piperidine as a catalyst, and the reaction solution was reacted at 80°C for 12 h. After the reaction, the ethanol solvent was removed by vacuum distillation, and the product was separated and purified by 200-300 mesh silica gel column chromatography, using gradient elution, dichloromethane and methanol (v / v, 40:1-10:1) as eluents, to obtain the bifunctional fluorescent probe Nap-Cy with a yield of 48%.

[0064] 1H NMR(400MHz,DMSO-d6)δ8.62(s,1H),8.45(d,J=7.8Hz,1H),8.30(s,1H),8.28(d,J=3.5Hz,1H), 8.27(d,J=1.5Hz,1H),8.08(s,2H),7.83(d,J=8.9Hz,1H),7.67(s,1H),7.56(d,J=7.6Hz,1H),7. 52(d,J=6.1Hz,1H),7.48(d,J=7.7Hz,1H),5.73(s,1H),4.44(m,2H),4.13(d,J=6.9Hz,2H),3.5 3(t,J=4.6Hz,4H),2.54–2.51(m,2H),2.44(d,J=4.7Hz,4H),1.98(s,6H),1.46(d,J=7.1Hz,3H). 13 C NMR (101MHz, DMSO-d6) δ163.8,162.7,138.6,135.3,131.9,131.7,131.4,130.5,129.9,129.7,129.6,127.9,127. 2,125.6,124.5,122.4,121.5,119.1,117.0,111.9,66.2,55.9,54.9,53.4,51.8,36.3,26.9,12.6.HRMS(ESI):m / z calculated for Nap-Cy(C 36 H 36 N3O4 + ,[M–I - ] + ):574.2700,found:574.2695.

[0065] Example 2 Ultraviolet absorption and fluorescence spectrum test of dual-function fluorescent probe Nap-Cy

[0066] The fluorescent probe Nap-Cy prepared in Example 1 was dissolved in an ethanol solution to prepare a fluorescent probe stock solution with a concentration of 1.0 mM, and stored in a -80°C refrigerator. Then, a 10 μM test solution was prepared using ethanol and PBS solution at a volume ratio of 3:7. A stock solution of peroxynitrite was prepared using distilled water. In order to determine the maximum excitation and emission wavelengths of peroxynitrite detected by the probe Nap-Cy, we studied the changes in the UV-visible absorption and emission spectra before and after the reaction of peroxynitrite with the probe Nap-Cy. Figure 2As shown in A, in the absence of peroxynitrite, the probe Nap-Cy showed a maximum absorption peak at 590 nm, but after adding an appropriate amount of peroxynitrite (30 μM), the absorption peak at 590 nm gradually decreased. In addition, the probe Nap-Cy showed weak fluorescence emission at 675 nm, and the fluorescence quantum yield (Φ Nap-Cy ) was 0.0159. After adding peroxynitrite, the fluorescence intensity at 550nm was significantly enhanced (about 25 times), and the fluorescence quantum yield increased to 0.2148 ( Figure 2 B), the solution color changes from blue to yellow ( Figure 2 B, inset).

[0067] Example 3 Fluorescence intensity response and kinetic analysis of the bifunctional fluorescent probe Nap-Cy to peroxynitrite

[0068] Use distilled water to prepare test solutions of different concentrations of peroxynitrite. Figure 2 As shown in C, the dose-dependent titration experiment shows that as the concentration of peroxynitrite increases, the fluorescence intensity of the probe Nap-Cy (10 μM) at 550 nm and 675 nm gradually increases. In the range of 0 to 60 μM, the fluorescence intensity ratio (F 550 / F 675 ) showed a good linear correlation with the concentration of peroxynitrite, the regression equation was y = 0.439 [peroxynitrite] + 0.13438, and the linear fitting constant was R 2 =0.9940. According to the 3σ / k equation (n=3), the detection limit (LOD) of the probe Nap-Cy for peroxynitrite is about 42 nM ( Figure 2 C, inset). In addition, we also evaluated the recognition kinetics of peroxynitrite by the probe Nap-Cy. Figure 2 As shown in D, after the addition of peroxynitrite (30 μM), the fluorescence intensity ratio of the probe Nap-Cy (F 550 / F 675 ) increased rapidly and reached a relative stability within 10 min. These results indicate that the probe Nap-Cy has excellent real-time responsiveness and high sensitivity in detecting peroxynitrite in vitro.

[0069] Example 4 Evaluation of the selectivity and pH stability of the bifunctional fluorescent probe Nap-Cy

[0070] Use distilled water to prepare the interfering substances (Na + , K + , Ca 2+ Mg 2+ 、Zn 2+ , Fe 2+ , Fe 3+ 、SO42- , NO3, CO3 2- 、H2O2、ClO - 、·OH、 1 O2, NO, NO 2- , NO, GSH, Cys, Hcy, S 2- , HS - 、SO3 2- and HSO3 - ) stock solution. Figure 2 As shown in E, the fluorescence intensity of the probe Nap-Cy (10 μM) changed only slightly after adding 500 μM of the interferents. It is worth noting that even in the presence of these interferents, the probe's response to 30 μM peroxynitrite was barely affected. Figure 2 F shows that in the absence of peroxynitrite, the fluorescence intensity ratio of the probe Nap-Cy in the pH range of 3–11 is relatively weak (F 550 / F 675 ). However, after adding 30 μM peroxynitrite, the probe responded significantly to peroxynitrite in the pH range of 5–11. These results indicate that the probe Nap-Cy not only has excellent anti-interference ability, but also can efficiently detect changes in peroxynitrite in the physiological pH range.

[0071] Example 5 Detection performance of bifunctional fluorescent probe Nap-Cy on viscosity

[0072] PBS buffer and glycerol were mixed into solution systems of different concentrations according to different volume ratios. Figure 3 As shown in A, the fluorescence emission of the probe Nap-Cy (10 μM) in PBS buffer solution with a wavelength of 675 nm is weak. As the proportion of glycerol in the glycerol / PBS mixture increases from 0% to 99%, the fluorescence intensity at a wavelength of 675 nm gradually increases, and eventually increases by about 400 times. At the same time, under a 365 nm handheld UV lamp, the fluorescence color of the solution gradually turns red ( Figure 3 C). In addition, the fluorescence intensity [log(I 675 nm)] and viscosity [log(η)] show a good linear relationship in the range of 2.5cp to 1150cp, with a correlation coefficient of 0.9696 ( Figure 3 B). This linear result indicates that the probe Nap-Cy is extremely sensitive to viscosity changes, and its excellent viscosity detection performance provides great application potential for the detection and imaging of viscosity changes in biological systems.

[0073] Example 6 Fluorescence imaging of endogenous peroxynitrite in SH-SY5Y cells using the bifunctional fluorescent probe Nap-Cy

[0074] SH-SY5Y cells were placed in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, and 2 mM L-alanyl-L-glutamine, and then cultured at 37°C in a humidified atmosphere containing 5% CO2 for 24 h. Figure 4 As shown in A1-A4, B and C, SH-SY5Y cells incubated with the probe Nap-Cy (10 μM) alone did not show obvious fluorescence signals in both the green and red channels. However, after SH-SY5Y cells were pre-stimulated with LPS / IFN-γ for 6 h and then incubated with the probe Nap-Cy for 0.5 h, a significant increase in fluorescence signals was observed in both channels ( Figure 4 A5-A8, B and C). To further verify that these fluorescence changes were mainly caused by endogenous peroxynitrite, we used the nitric oxide synthase (NOS) inhibitor 2,2,6,6-tetramethylpiperidinium-N-oxide (TEMPO) to inhibit the production of intracellular peroxynitrite. Figure 4 A9-A 12 , B, and C, it can be seen that the presence of TEMPO leads to a decrease in fluorescence intensity in SH-SY5Y cells. These observations indicate that the changes in fluorescence intensity before and after treatment are mainly attributed to the interaction of the probe with stimulated peroxynitrite, demonstrating the reliability of Nap-Cy in monitoring peroxynitrite in biological systems.

[0075] Example 6 Fluorescence imaging of exogenous peroxynitrite in zebrafish using the bifunctional fluorescent probe Nap-Cy

[0076] Zebrafish embryos were cultured in E3 medium containing 15mM NaCl, 0.5mM KCl, 1mM MgSO4, 1mM CaCl2, 0.15mM KH2PO4, 0.05mM Na2HPO4, 0.7mM NaHCO3 and 10-5% methylene blue, and the pH was adjusted to 7.5. Six-day-old zebrafish were used in the experiment, and three groups of zebrafish samples with different concentrations of peroxynitrite (0, 30, and 60μM) were prepared. Figure 5 As shown in AC, after incubation with the probe Nap-Cy for 0.5 h, confocal microscopy imaging showed that zebrafish without peroxynitrite had no fluorescence signals in both the green and red channels. As the concentration of peroxynitrite increased, the fluorescence signal gradually increased. These results indicate that the probe Nap-Cy can effectively enter the body of zebrafish and can be used to intuitively quantify the level of peroxynitrite in the body.

[0077] Example 7 Fluorescence imaging of SH-SY5Y cell viscosity by the bifunctional fluorescent probe Nap-Cy To evaluate whether the probe Nap-Cy can detect changes in intracellular viscosity, we induced changes in SH-SY5Y cell viscosity by starvation (incubation in medium without fetal bovine serum, -FBS) and nystatin treatment. Figure 6 As shown, SH-SY5Y cells pretreated with starvation or nystatin showed stronger fluorescence intensity in the red channel compared with cells incubated with the probe alone. This result suggests that the probe Nap-Cy can be used as a viscosity "on" near-infrared fluorescent probe for imaging changes in cell viscosity.

[0078] Example 8 Fluorescence imaging of zebrafish viscosity using the dual-function fluorescent probe Nap-Cy

[0079] In addition, we tested the ability of the probe Nap-Cy to detect viscosity changes in zebrafish using nystatin or monensin as viscosity stimulants. Figure 7 As shown, after co-incubation of nystatin or monensin with the probe Nap-Cy, the fluorescence signal in the red channel of zebrafish increased significantly, while zebrafish incubated with the probe Nap-Cy alone had almost no red fluorescence signal. These findings are very consistent with the observations in cells, confirming that the probe Nap-Cy can also effectively visualize viscosity fluctuations in zebrafish.

[0080] The above specific implementation method part specifically introduces the analytical method involved in the present invention. It should be noted that the above introduction is only to help those skilled in the art better understand the method and ideas of the present invention, rather than limiting the relevant content. Without departing from the principle of the present invention, those skilled in the art may also make appropriate adjustments or modifications to the present invention, and the above adjustments and modifications shall also fall within the scope of protection of the present invention.

Claims

1. A dual-function fluorescent probe for simultaneous detection of viscosity and peroxynitrite, characterized in that: Its structural formula is as follows:

2. The method for preparing a dual-function fluorescent probe for simultaneous detection of viscosity and peroxynitrite according to claim 1, characterized in that: The following steps are involved: (1) dissolving 4-bromo-1,8-naphthalene dicarboxylic anhydride and 4-(2-aminoethyl)-morpholine in an organic solvent, heating under reflux and stirring at 100° C. to react; after the reaction is completed, cooling the reaction solution, filtering under reduced pressure, washing and drying the obtained solid to obtain an intermediate 1; (2) Dissolve the intermediate 1 in an organic solvent, add potassium carbonate, and stir to react at 80° C. After the reaction is completed, cool to room temperature, adjust the pH with dilute hydrochloric acid, extract the reaction solution, further dry it, and make sand to purify it to obtain the intermediate 2; (3) Dissolving the intermediate 2 in an organic solvent, heating under reflux and stirring at 120° C. to react; after the reaction, cooling the reaction solution, and filtering under reduced pressure to obtain the intermediate 3; (4) Under an inert gas atmosphere, the intermediate 3 and 3-ethyl-1,1,2-trimethylbenzindolinium iodide are dissolved in an organic solvent and heated to 80° C. under the catalysis of piperidine; after the reaction is completed, the reaction solution is cooled to room temperature, and the reaction solution is subjected to vacuum rotary evaporation to obtain sand, and a red-purple solid is purified, namely the bifunctional fluorescent probe.

3. The method for preparing a dual-function fluorescent probe for simultaneous detection of viscosity and peroxynitrite according to claim 2, characterized in that: In step (1), the organic solvent is anhydrous ethanol; the molar ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride to 4-(2-aminoethyl)-morpholine is 1:1.5; and the heating and stirring reaction time is 6 hours.

4. The method for preparing a dual-function fluorescent probe for simultaneous detection of viscosity and peroxynitrite according to claim 2, characterized in that: In step (2), the organic solvent is dimethyl sulfoxide; the molar ratio of the intermediate 1 to N-hydroxyphthalimide is 1:1.1; the molar ratio of the intermediate 1 to potassium carbonate is 1:3; the heating and stirring reaction time is 2 hours; and purification is performed by column chromatography.

5. The method for preparing a dual-function fluorescent probe for simultaneous detection of viscosity and peroxynitrite according to claim 4, characterized in that: The column chromatography conditions are as follows: dichloromethane and methanol as eluents, with a volume ratio of 25:1; and silica gel of 200-300 meshes.

6. The method for preparing a dual-function fluorescent probe for simultaneous detection of viscosity and peroxynitrite according to claim 2, characterized in that: In step (3), the organic solvent is trifluoroacetic acid; the molar ratio of the intermediate 2 to hexamethylenetetramine is 1:2; and the heating and stirring reaction time is 12 hours.

7. The method for preparing a dual-function fluorescent probe for simultaneous detection of viscosity and peroxynitrite according to claim 2, characterized in that: In step (4), the organic solvent is anhydrous ethanol; the molar ratio of the intermediate 3 to 3-ethyl-1,1,2-trimethylbenzindolium iodide is 1:1; the heating and stirring reaction time is 12 hours; and purification is performed by column chromatography.

8. The method for preparing a dual-function fluorescent probe for simultaneous detection of viscosity and peroxynitrite according to claim 7, characterized in that: The column chromatography conditions are as follows: dichloromethane and methanol are used as eluents, gradient elution is adopted, and the volume ratio is 40:1-10:1; the silica gel is 200-300 meshes.

9. Use of the dual-function fluorescent probe according to claim 1 for simultaneous detection of viscosity and peroxynitrite in cells and organisms.

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