Preparation method and application of fluorescent probe for quantitative detection of pH value in solution and cell

Abstract

The application discloses a fluorescent probe for quantitatively detecting solution and intracellular pH value and a preparation method and application thereof, and belongs to the technical field of fluorescent probe preparation.The fluorescent probe is a benzindole fluorescent compound, and the specific chemical structure is shown in the following formula.The fluorescent compound can be used as the fluorescent probe, and is used for solution pH value detection and reflecting intracellular pH value change.Compared with a probe for qualitatively researching the relative strength of pH value, the probe can realize quantitative detection of the solution and intracellular environment pH value.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe preparation technology, specifically relating to a method for preparing and applying a fluorescent probe for quantitatively detecting the pH value of solutions and cells. Background Technology

[0002] pH, also known as the hydrogen ion activity index, is a scale for the activity of hydrogen ions in a solution, and is generally considered a measure of the acidity or alkalinity of a solution. The closer the pH value is to 0, the more acidic the solution; conversely, the closer it is to 14, the more alkaline the solution. At room temperature, a solution with pH=7 is considered neutral. Measuring the pH value of a solution can determine its chemical properties, allowing for better control and regulation of chemical reactions. pH also reflects changes in the intracellular microenvironment and is closely related to biomolecular interactions, metabolite diffusion, and signal transduction. Therefore, pH is widely used in environmental protection, the food industry, and biomedicine.

[0003] Currently, the main methods for detecting pH values ​​include: 1. Acid-base indicators, pH meters, pH test strips, glass electrode method, ion-selective electrode method, and conductivity method. The glass electrode method offers advantages such as high accuracy, fast response, and a wide measurement range. However, because the electrode is easily contaminated and damaged, this method requires periodic calibration. 2. The ion-selective electrode method offers accurate and reliable measurement results and a wide range of applications. However, the preparation of ion-selective electrodes is relatively complex and costly. 3. The conductivity method is simple and easy to implement, requiring only a general conductivity instrument and not a specialized pH measuring instrument. However, this method is not accurate enough for certain solutions, such as weak acid-weak base solutions.

[0004] Organic fluorescent probes based on fluorescence imaging technology have become a powerful technique for in-situ, non-destructive, and independent visualization of pH changes. Currently, pH-responsive fluorescent compounds primarily qualitatively study the relative strength of pH values ​​through changes in fluorescence intensity, with a detection range generally between 2.0 and 7.0. Probes capable of detecting and quantifying pH in alkaline environments are relatively few. Therefore, developing a fluorescent probe that can quantify solution pH, achieve pH detection in aqueous solutions and cells, and possesses good stability, along with its application forms, is of great significance for the quantitative detection of pH in aqueous solutions and cells. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a method for preparing and applying a fluorescent probe for quantitatively detecting pH in solutions and cells. This invention addresses the technical bottlenecks of existing fluorescent probes, such as their difficulty in quantitatively detecting pH in aqueous solutions and insufficient stability during the detection process, by developing a highly efficient and stable fluorescent probe. By constructing the ratiometric fluorescent probe NYY-07, this invention successfully achieved the quantitative detection of pH in aqueous solutions and successfully applied it to localize mitochondria in cells. This fluorescent probe provides a new strategy and technical means for biomedical diagnostics.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention discloses a method for preparing a fluorescent probe for quantitative detection of pH values ​​in solutions and cells. The fluorescent probe is a benzoindole fluorescent compound, and its specific chemical structure is shown below: .

[0007] Includes the following steps, S1. 4-Bromo-1,8-dinaphthoic anhydride and 2-aminoethylmorpholine were added to anhydrous ethanol solution and heated to reflux. After the reaction was completed, the mixture was filtered to obtain compound N-1, wherein N-1 is 4-bromo-N-(2-morpholinylethyl)naphthalimide. S2. 4-Bromo-N-(2-morpholinylethyl)naphthalimide and 4-piperazin-1-benzaldehyde were added to ethylene glycol monomethyl ether and refluxed. The solvent was then evaporated to dryness and separated by column chromatography to obtain compound N-2, wherein N-2 is 4-piperazinyl-(4-benzaldehyde)-N-(2-morpholinylethyl)naphthalimide; S3. 2-Iodoethane and 1,2,2-trimethyl-1H-benzo[e]indole were added to ultra-dry acetonitrile and refluxed. The solvent was then evaporated to dryness and the compound N-3 was obtained by column chromatography. The N-3 is 3-(2-hydroxyethyl)-1,1,2-trimethyl-1H-benzo[e]indole-3-onium. S4. Compounds N-2 and N-3 and 100 μL of piperidine were added to anhydrous ethanol solution and refluxed. The solvent was then evaporated by rotary evaporation, and the fluorescent probe NYY-07 was obtained by column chromatography. NYY-07 is (E)-3-(2-hydroxyethyl)-1,1-dimethyl-2-[4-(4-{2-(2-morpholinylethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-6-yl}piperazin-1-yl)styryl]-1H-benzo[e]indole-3-onium.

[0008] Further, in step S1, the molar ratio of 4-bromo-1,8-dinaphthoic anhydride to 2-aminoethylmorpholine is 0.8:1.

[0009] Further, in step S2, the eluent phase purified by column chromatography consists of dichloromethane and methanol in a volume ratio of 25:1, and the molar ratio of 4-bromo-N-(2-morpholinylethyl)naphthalimide to 4-piperazine benzaldehyde is 1:1.

[0010] Further, in step S4, the eluent phase for column chromatography purification consists of dichloromethane and methanol in a volume ratio of 20:1, and the molar ratio of 4-piperazinyl-(4-benzaldehyde)-N-(2-morpholinylethyl)naphthalimide to compound N-3 is 1:1.

[0011] Furthermore, the fluorescent probe according to claim 1 is used in neurodegenerative diseases, ischemia-reperfusion injury, and tumor chemotherapy monitoring.

[0012] Furthermore, a method for quantitative detection of pH value based on dual excitation wavelength ratio calibration of a fluorescent probe includes the following steps: P1. Dissolve the fluorescent probe in chromatographic grade dimethyl sulfoxide to prepare a solution with a concentration of 1×10⁻ ... 5 M's probe stock solution; P2. Prepare standard buffer solutions with different pH values, covering a pH range of 4.0-7.0. The buffer solutions are PBS buffer or HEPES buffer, with an ionic strength of 0.01-0.1 M. P3. Add an equal volume of probe stock solution to each standard pH buffer solution to bring the final probe concentration to 1×10⁻⁻⁻⁻⁶. 5 M, mix well and let stand for 5-10 minutes; P4. Measure the fluorescence emission spectra at excitation wavelengths λ1 = 450 nm and λ2 = 525 nm, and record the fluorescence intensities I1 and I2 at emission wavelengths λ_em1 = 556 nm and λ_em2 = 625 nm, respectively. P5. Calculate the ratio value R = I2 / I1, and establish a calibration curve between R and pH value. The calibration curve satisfies a linear relationship in the pH range of 4.1-5.5: R = a×pH + b, where a is the slope, b is the intercept, and the coefficient of determination R²≥0.99. P6. Substitute the R value obtained from the test solution or cell sample under the same conditions into the calibration curve to calculate the accurate pH value.

[0013] Furthermore, a method for simultaneously and quantitatively detecting pH value and microenvironment polarity / viscosity using the fluorescent probe of claim 6 includes the following steps: P1. Dissolve the fluorescent probe in the test solution or cell culture medium to achieve a final probe concentration of 1×10⁻ 6 M to 1×10⁻ 5 M; P2. Under the first excitation mode A: excitation wavelength λ_exA = 450 nm, and fluorescence intensity I_A at emission wavelength λ_emA = 556 nm is detected; P3. Under the second excitation mode B: excitation wavelength λ_exB = 525 nm, and fluorescence intensity I_B at emission wavelength λ_emB = 625 nm is detected; P4. Calculate the ratio value R = I_B / I_A, and obtain the pH value based on the calibration curve; P5. Simultaneously, the fluorescence lifetime τ or fluorescence intensity I_B at the emission wavelength of 625 nm under excitation mode B is used as an independent response parameter of polarity / viscosity, wherein: - When the microenvironment viscosity increases, I_B is enhanced and τ is prolonged; - When the microenvironment polarity decreases, I_B is enhanced and τ is prolonged. P6. The pH response and the polarity / viscosity response are orthogonal to each other. That is, pH changes only affect the ratio value R and not the absolute value of τ, and viscosity / polarity changes only affect I_B and τ and not the relative proportion of I_A.

[0014] Furthermore, a method for distinguishing between apoptosis and autophagy based on the method described in claim 7 is characterized by comprising the following steps: P1. Co-incubate the fluorescent probe with the cells to be tested at a concentration of 1×10⁻⁻ 6 M, incubation time is 15-30 minutes, so that the probe is targeted and enriched in the cell mitochondria; P2. Using a confocal microscope or flow cytometer, red channel fluorescence signals were acquired at an excitation wavelength of 488 nm, with an emission band of 590-650 nm. P3. Perform time-series imaging on the cells and record the following parameters at different time points t: - Mean fluorescence intensity I_mito(t) in the mitochondrial region - Average fluorescence intensity I_cyto(t) in the cytoplasm - Calculate the mitochondrial / cytoplasmic fluorescence intensity ratio R_sub(t) = I_mito(t) / I_cyto(t); P4. Establish a two-parameter dynamic variation curve: - pH parameter: Mitochondrial pH value pH_mito(t) is calculated from R_sub(t) based on the calibration curve; - Viscosity parameter: The change in mitochondrial matrix viscosity η(t) is reflected by the absolute fluorescence intensity change ΔI / Δt of I_mito(t); P5. Differentiate between apoptosis and autophagy based on the following criteria: - if dpH_mito / dt < -0.05 pH unit / min and dη / dt > threshold V_apo, representing a sharp increase in viscosity, it is determined as early apoptosis; - if dpH_mito / dt < -0.02 pH unit / min and 0 < dη / dt < threshold V_auto, representing a slight increase in viscosity, it is determined as an autophagy process; - wherein V_apo = 3×V_auto, and V_auto is pre-calibrated by a positive control experiment of starvation-induced autophagy.

[0015] Further, a method for dual-parameter imaging of pH and viscosity in deep in vivo tissues using the fluorescent probe of claim 1, comprising the following steps: P1, dissolving the fluorescent probe in normal saline or PBS buffer to prepare a stock injection solution with a concentration of 1×10⁻ 4 M; P2, introducing the probe into a living animal model by means of tail vein injection or local injection, with an injection dose of 5-10 mg / kg body weight; P3, after waiting for 30-60 minutes for the probe to enrich in the target tissue, imaging is performed using a two-photon excitation fluorescence microscope or a photoacoustic imaging system: - Two-photon excitation wavelength: 800-900 nm; - Emission detection bands: 600-700 nm, 700-800 nm; said 600-700 nm corresponds to a pH response channel; said 700-800 nm corresponds to a viscosity response channel; P4, converting the fluorescence ratio R = I_700-800 / I_600-700 in the imaging region into a pH distribution map using a calibration curve; P5, obtaining a spatial distribution map of tissue viscosity / polarity using fluorescence lifetime imaging technology; P6, spatially superimposing the pH distribution map and the viscosity distribution map to construct a dual-parameter functional imaging map of the tissue microenvironment.

[0016] The beneficial effects of the present invention are: 1. The fluorescent compound described in the present invention can be used as a fluorescent probe for detecting pH value in solution and reflecting changes in intracellular pH value. Compared with probes that qualitatively study the relative strength of pH, this probe can achieve quantitative detection of pH value in solutions and intracellular environments. In addition, the fluorescent compound described in the present invention can accurately target intracellular mitochondria, further realize dynamic detection of pH changes in organelles, and provide potential technical means for clinical research and disease diagnosis and treatment.

[0017] Other advantages, objectives, and features of the invention will be set forth in the following description and will be apparent to those skilled in the art in some respects, or may be learned by practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0018] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 The synthetic route of the fluorescent probe of this invention is shown below; Figure 2 The above is the proton NMR spectrum of the fluorescent probe of this invention. Figure 3 This is the carbon NMR spectrum of the fluorescent probe of the present invention; Figure 4 This is a high-resolution mass spectrum of the fluorescent probe of the present invention; Figure 5 (a) is the fluorescence emission spectrum of the fluorescent probe of the present invention in PBS buffer solutions with different pH values ​​at an excitation wavelength of 450 nm; (b) is the fluorescence emission spectrum of the fluorescent probe of the present invention in PBS buffer solutions with different pH values ​​at an excitation wavelength of 525 nm. Figure 6 (a) is a graph showing the change in the ratio of fluorescence intensity emitted by the fluorescent probe of the present invention at an excitation wavelength of 450 nm; (b) is a graph showing the linear relationship of the ratio of fluorescence intensity emitted by the fluorescent probe NYY-07 of the present invention at an excitation wavelength of 450 nm. Figure 7 The effect of cations and anions in the solution on the pH detection performance of the fluorescent probe; Figure 8 The graph shows the reversibility test results of the fluorescent probe of the present invention in aqueous solutions at different pH values. Figure 9 (a) Red channel of the fluorescent probe of the present invention in the mitochondria of HeLa cells; (b) Red channel of the red mitochondrial dye Mito-Tracker Deep Red FM in HeLa cells; (c) Bright field imaging of mitochondria in HeLa cells; (d) Overlay of the probe NYY-07 and dye Mito-Tracker Deep Red FM of the present invention in the mitochondria of HeLa cells; (e) Fluorescence intensity distribution of the two probes in the linear region; (f) Intensity correlation diagram of the two probes of the present invention.

[0019] Figure 10(a) is a washing image of HeLa cells after incubation with the fluorescent probe of the present invention; (b) is a washing image of HeLa cells after incubation with the fluorescent probe of the present invention DMEM+PS for 2 h; (c) is a fluorescence bar graph after cell imaging. Detailed Implementation

[0020] like Figure 1-10 As shown, this invention discloses a method for preparing and applying a fluorescent probe for quantitatively detecting pH values ​​in solutions and cells.

[0021] Example 1 S1. Take a 50 mL round-bottom flask and add 4-bromo-1,8-dinaphthoic anhydride (275 mg, 1 mmol) and 2-aminoethylmorpholine (130 mg, 1 mmol) to 5 mL of ethanol solution. Heat to 80 °C and reflux for 8 h. After the reaction is completed, filter to obtain compound N-1 (330 mg, yield 86%). The N-1 is 4-bromo-N-(2-morpholinylethyl)naphthalimide. S2. Compound N-1 (388 mg, 1 mmol) and 4-piperazin-1-benzaldehyde (190 mg, 1 mmol) were added to 3 mL of ethylene glycol monomethyl ether and heated to 80 °C under reflux for 8 h. The solvent was then evaporated to dryness, and compound N-2 (568 mg, 72% yield) was obtained by column chromatography. The N-2 was 4-piperazinyl-(4-benzaldehyde)-N-(2-morpholinylethyl)naphthalimide. S3. Under a nitrogen atmosphere, 2-iodoethanol (432 mg, 1 mmol) and 1,2,2-trimethyl-1H-benzo[e]indole (525 mg, 1 mmol) were added to ultra-dry acetonitrile and heated to 80 °C under reflux for 48 h. The mixture was then washed with ice-cold ethanol and filtered to obtain gray-purple crystalline compound N-3 (491 mg, 77% yield). N-3 is 3-(2-hydroxyethyl)-1,1,2-trimethyl-1H-benzo[e]indole-3-onium. S3. Compound N-2 (498 mg, 1 mmol), piperidine (100 μL) and compound N-3 (301 mg, 1 mmol) were added to anhydrous ethanol solution and heated to 80 °C and refluxed for 8 h. The solvent was then evaporated to dryness, and column chromatography was used to obtain a reddish-brown compound, which is the fluorescent probe NYY-07 (331 mg, yield 51%) for detecting pH changes.

[0022] The specific proton NMR spectrum of the fluorescent probe NYY-07 is as follows: Figure 2 As shown, the carbon NMR spectrum is as follows: Figure 3 As shown, high-resolution mass spectrometry is as follows Figure 4 As shown.

[0023] 1H NMR (400 MHz, CDCl3) δ 8.53 (d, J = 7.4 Hz, 1H), 8.48 (d, J = 8.0Hz, 1H), 8.41 (d, J = 8.5 Hz, 1H), 7.96 (d, J = 8.8 Hz, 1H), 7.75 (d, J = 8.1Hz, 1H), 7.68 (d, J = 8.3 Hz, 2H), 7.40 (d, J = 8.1 Hz, 3H), 7.23 (s, 2H), 7.08 (d, J = 8.6 Hz, 1H), 6.95 (d, J = 8.3 Hz, 2H), 6.82 (d, J = 15.8 Hz,1H), 6.20 (d, J = 16.0 Hz, 1H), 4.28 (t, J = 7.1 Hz, 2H), 3.78 (d, J = 6.8Hz, 2H), 3.64 (s, 4H), 3.42 (d, J = 38.1 Hz, 8H), 2.65 (t, J = 7.1 Hz, 2H), 2.56 (s, 4H), 2.16 (t, J = 7.7 Hz, 2H), 1.74 (s, 6H). 13 C NMR (151 MHz, CDCl3)δ 163.46, 162.98, 154.70, 149.68, 146.56, 131.55, 131.06, 130.22, 129.29,129.19, 128.90, 128.36, 128.03, 127.58, 126.82, 125.24, 124.89, 122.26,122.07, 121.72, 121.13, 116.05, 115.19, 114.10, 112.95, 109.44, 65.88, 62.34,59.39, 55.13, 52.69, 52.41, 51.96, 49.05, 48.27, 35.94, 30.91, 30.48, 29.11, 25.39, 21.67, 20.53, 20.03, 13.18, 13.11. HRMS theoretical values ​​[C] 46 H 48 N5O4] + =734.3690, Measured value =734.3707, Error =2.31 ppm verify: Quantitative detection of pH value using fluorescent probe NYY-07 in aqueous solution: The fluorescent probe NYY-07 was dissolved in 10 ml of chromatographic grade DMSO to prepare a solution with a concentration of 10. -5 A solution of M was prepared, and PBS buffer solutions with different pH values ​​were prepared using 1 M NaOH and 1 M HCl. Subsequently, 1.98 mL of PBS buffer solution with different pH values ​​and 20 μL of probe solution were added to the centrifuge tubes, mixed thoroughly, and then fluorescence was tested. The fluorescence test parameters were: excitation wavelengths of 450 nm and 525 nm, and excitation and emission slits were both set to 10 nm.

[0024] The obtained fluorescence spectrum is as follows Figure 5 As shown, the fluorescence intensity of the NYY-07 fluorescent probe gradually decreases with increasing pH at an emission wavelength of 625 nm, while gradually increasing with increasing pH at an emission wavelength of 556 nm, exhibiting a certain regular change. Similarly, at excitation wavelengths of 525 nm and emission wavelengths of 625 nm, the fluorescence intensity gradually decreases with increasing pH, also exhibiting a regular change. Therefore, the NYY-07 probe can achieve quantitative detection of pH in aqueous solutions.

[0025] Then calculate I 625 nm / I 556 The value of nm, such as Figure 6 As shown, within the pH range of 4.4-5.2, the fluorescence intensity ratio exhibits an excellent relationship with pH value, with a coefficient of determination as high as R² = 0.993 after fitting analysis. Based on this high-confidence fitting curve, the pKa value of the probe in this acidic region can be accurately calculated. The calculated pKa value is 4.818, as shown in Table 1. It was found that within the pH range of 4.1-5.5, this probe has extremely high resolution for minute fluctuations in H+, and can be used to accurately monitor changes in the acidic microenvironment in solution.

[0026] Table 1 Comparison table of pH range detection for probe NYY-07 with PM-Mor-OH and Rh-NorCy.

[0027] Resistance of fluorescent probe NYY-07 to pH interference in aqueous solution: Given the complex and diverse intracellular environment, various biomolecules and ions present in living cells may interfere with the accurate measurement of pH by probes. Further investigation revealed that some metal cations, heavy metal ions, transition metal ions, and some common anion probes, present under physiological conditions at pH=4.1 and pH=5.5, exhibit high selectivity for biomolecules and ions at physiological concentrations.

[0028] The fluorescent probe NYY-07 was dissolved in 10 ml of chromatographic grade DMSO to prepare a solution with a concentration of 10. -5 A solution of M was prepared, and PBS buffer solutions with different pH values ​​were prepared using 1 M NaOH and 1 M HCl. Next, add 1.96 mL of PBS buffer solution with different pH values ​​and 20 μL of probe solution to the centrifuge tube, followed by 20 μL of different ion solutions. After mixing well, perform fluorescence testing. The excitation and emission slits are both set to 10 nm.

[0029] like Figure 7 As shown, the fluorescent probe NYY-07 still exhibits good anti-interference performance in pH detection even in the presence of interfering ions, through ratiometric detection.

[0030] Reversibility of the fluorescent probe NYY-07 with respect to pH in aqueous solution: To achieve real-time monitoring of pH changes in living tissues and cells, the fluorescence intensity of the fluorescent probe must have excellent reversibility in response to pH.

[0031] The pH of the solution was adjusted between 4.1 and 5.5 using small volumes of HCl (1 M) and NaOH (1 M) to measure the reversibility of the probe. The excitation wavelength was 450 nm, and both the excitation and emission slits were set to 10 nm for fluorescence spectroscopy detection.

[0032] like Figure 8 As shown, the results clearly demonstrate that the pH measurement process of the solution by probe is reversible. Furthermore, the pH of the solution reaches stability within a very short time.

[0033] Imaging of the fluorescent probe NYY-07 in live cells: The fluorescent probe was co-stained with the commercially available red mitochondrial dye Mito Tracker Deep Red to evaluate the mitochondrial staining ability of the probe. The detection conditions were: excitation wavelength of 488 nm and emission wavelength between 590 and 650 nm.

[0034] The results are as follows Figure 9As shown, the probe image and the Mito Tracker Deep Red image overlapped well, with a Pearson colocalization coefficient of 0.94, indicating that the probe can specifically stain mitochondria in living cells. Furthermore, in the presence of the probe, mitochondria were observed to appear as tiny rods.

[0035] Starvation induction in cells by fluorescent probe NYY-07: HeLa cells were incubated with a 1 μM probe for 30 min, and then cultured in nutrient-rich medium and serum-free medium.

[0036] like Figure 10 As shown, autophagy occurs in HeLa cells under starvation conditions, leading to a decrease in mitochondrial pH. This can be clearly observed through microscopic fluorescence imaging of HeLa cell autophagy. Compared to well-nourished cells, HeLa cells starved for 2 hours exhibit stronger fluorescence intensity.

[0037] The fluorescent probe of this invention enables quantitative detection of pH in aqueous solutions and can effectively locate pH within mitochondria to achieve intracellular pH detection. The detection method provided by this invention is simple to operate, uses inexpensive solvents, and allows for convenient post-processing.

[0038] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing a fluorescent probe for quantitative detection of pH in solutions and cells, characterized in that: The fluorescent probe is a benzoindole fluorescent compound, and its specific chemical structure is shown below: ； The fluorescent probe has a general formula (I) structure, wherein R¹ is morpholinoethyl or triphenylphosphinoethyl, R² is piperazinylphenyl, R³ is 1,1,2-trimethyl-1H-benzo[e]indole-3-onium, and the probe exhibits a ratiometric response in the pH range of 4.1-5.5, with a pKa of 4.8±0.1; Includes the following steps, S1. 4-Bromo-1,8-dinaphthoic anhydride and 2-aminoethylmorpholine were added to anhydrous ethanol solution and heated to reflux. After the reaction was completed, the mixture was filtered to obtain compound N-1, wherein N-1 is 4-bromo-N-(2-morpholinylethyl)naphthalimide. S2. 4-Bromo-N-(2-morpholinylethyl)naphthalimide and 4-piperazin-1-benzaldehyde were added to ethylene glycol monomethyl ether and refluxed. The solvent was then evaporated to dryness and separated by column chromatography to obtain compound N-2, wherein N-2 is 4-piperazinyl-(4-benzaldehyde)-N-(2-morpholinylethyl)naphthalimide; S3. 2-Iodoethane and 1,2,2-trimethyl-1H-benzo[e]indole were added to ultra-dry acetonitrile and refluxed. The solvent was then evaporated to dryness and the compound N-3 was obtained by column chromatography. The N-3 is 3-(2-hydroxyethyl)-1,1,2-trimethyl-1H-benzo[e]indole-3-onium. S4. Compounds N-2 and N-3 and 100 μL of piperidine were added to anhydrous ethanol solution and refluxed. The solvent was then evaporated by rotary evaporation, and the fluorescent probe NYY-07 was obtained by column chromatography. NYY-07 is (E)-3-(2-hydroxyethyl)-1,1-dimethyl-2-[4-(4-{2-(2-morpholinylethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-6-yl}piperazin-1-yl)styryl]-1H-benzo[e]indole-3-onium.

2. The method for preparing a fluorescent probe for quantitative detection of solution and intracellular pH value according to claim 1, characterized in that: In step S1, the molar ratio of 4-bromo-1,8-dinaphthoic anhydride to 2-aminoethylmorpholine is 0.8:

1.

3. The method for preparing a fluorescent probe for quantitative detection of solution and intracellular pH value according to claim 2, characterized in that: In step S2, the eluent for column chromatography purification consists of dichloromethane and methanol in a volume ratio of 25:1, and the molar ratio of 4-bromo-N-(2-morpholinylethyl)naphthalimide to 4-piperazine benzaldehyde is 1:

1.

4. The method for preparing a fluorescent probe for quantitative detection of solution and intracellular pH value according to claim 3, characterized in that: In step S4, the eluent for column chromatography purification consists of dichloromethane and methanol in a volume ratio of 20:1, and the molar ratio of 4-piperazinyl-(4-benzaldehyde)-N-(2-morpholinylethyl)naphthalimide to compound N-3 is 1:

1.

5. The application of the fluorescent probe according to claim 1 in neurodegenerative diseases, ischemia-reperfusion injury, and tumor chemotherapy monitoring.

6. A method for quantitative detection of pH value based on dual excitation wavelength ratio calibration of a fluorescent probe, characterized in that, Includes the following steps: P1. Dissolve the fluorescent probe in chromatographic grade dimethyl sulfoxide to prepare a solution with a concentration of 1×10⁻ ... 5 M's probe stock solution; P2. Prepare standard buffer solutions with different pH values, covering a pH range of 4.0-7.

0. The buffer solutions are PBS buffer or HEPES buffer, with an ionic strength of 0.01-0.1 M. P3. Add an equal volume of probe stock solution to each standard pH buffer solution to bring the final probe concentration to 1×10⁻⁻⁻⁻⁶. 5 M, mix well and let stand for 5-10 minutes; P4. Measure the fluorescence emission spectra at excitation wavelengths λ1 = 450 nm and λ2 = 525 nm, and record the fluorescence intensities I1 and I2 at emission wavelengths λ_em1 = 556 nm and λ_em2 = 625 nm, respectively. P5. Calculate the ratio value R = I2 / I1, and establish a calibration curve between R and pH value. The calibration curve satisfies a linear relationship in the pH range of 4.1-5.5: R = a×pH + b, where a is the slope, b is the intercept, and the coefficient of determination R²≥0.

99. P6. Substitute the R value obtained from the test solution or cell sample under the same conditions into the calibration curve to calculate the accurate pH value.

7. A method for simultaneously and quantitatively detecting pH value and microenvironment polarity / viscosity using the fluorescent probe of claim 6, characterized in that, Includes the following steps: P1. Dissolve the fluorescent probe in the test solution or cell culture medium to achieve a final probe concentration of 1×10⁻⁻⁻⁶. 6 M to 1×10⁻ 5 M; P2. Under the first excitation mode A: excitation wavelength λ_exA = 450 nm, and fluorescence intensity I_A at emission wavelength λ_emA = 556 nm is detected; P3. Under the second excitation mode B: excitation wavelength λ_exB = 525 nm, and fluorescence intensity I_B at emission wavelength λ_emB = 625 nm is detected; P4. Calculate the ratio value R = I_B / I_A, and obtain the pH value based on the calibration curve; P5. Simultaneously, the fluorescence lifetime τ or fluorescence intensity I_B at the emission wavelength of 625 nm under excitation mode B is used as an independent response parameter of polarity / viscosity, where: - When the viscosity of the microenvironment increases, I_B is enhanced and τ is prolonged; - When the polarity of the microenvironment decreases, I_B is enhanced and τ is prolonged; P6. The pH response and the polarity / viscosity response are orthogonal to each other. That is, pH changes only affect the ratio value R and not the absolute value of τ, and viscosity / polarity changes only affect I_B and τ and not the relative proportion of I_A.

8. A method for distinguishing between apoptosis and autophagy based on the method described in claim 7, characterized in that, Includes the following steps: P1. Co-incubate the fluorescent probe with the cells to be tested at a concentration of 1×10⁻⁻ 6 M, incubation time is 15-30 minutes, so that the probe is targeted and enriched in the cell mitochondria; P2. Use a confocal microscope or flow cytometer to collect red channel fluorescence signals at an excitation wavelength of 488 nm and an emission band of 590-650 nm. P3. Perform time-series imaging on the cells and record the following parameters at different time points t: - Mean fluorescence intensity I_mito(t) in the mitochondrial region - Average fluorescence intensity I_cyto(t) in the cytoplasm - Calculate the mitochondrial / cytoplasmic fluorescence intensity ratio R_sub(t) = I_mito(t) / I_cyto(t); P4. Establish a two-parameter dynamic variation curve: - pH parameter: Mitochondrial pH value pH_mito(t) is calculated from R_sub(t) based on the calibration curve; - Viscosity parameter: The change in mitochondrial matrix viscosity η(t) is reflected by the absolute fluorescence intensity change ΔI / Δt of I_mito(t); P5. Differentiate between apoptosis and autophagy based on the following criteria: - If dpH_mito / dt < -0.05 pH units / min and dη / dt > threshold V_apo, it indicates a sharp increase in viscosity, which is then determined to be the early stage of apoptosis; - If dpH_mito / dt < -0.02 pH units / minute and 0 < dη / dt < threshold V_auto, it indicates a slight increase in viscosity, which is then determined to be the process of autophagy. - Where V_apo = 3×V_auto, and V_auto is pre-calibrated by a positive control experiment of starvation-induced autophagy.

9. A method for dual-parameter imaging of pH and viscosity in in vivo deep tissues using the fluorescent probe of claim 1, characterized in that, Includes the following steps: P1. Dissolve the fluorescent probe in physiological saline or PBS buffer to prepare a solution with a concentration of 1×10⁻⁻ 4 M's injection stock solution; P2. The probe is introduced into the live animal model via tail vein injection or local injection, with an injection dose of 5-10 mg / kg body weight. P3. After waiting 30-60 minutes for the probe to accumulate in the target tissue, image it using a two-photon excitation fluorescence microscope or photoacoustic imaging system: - Two-photon excitation wavelength: 800-900 nm; - Emission detection bands: 600-700 nm, 700-800 nm; the 600-700 nm band corresponds to the pH response channel; the 700-800 nm band corresponds to the viscosity response channel; P4. Using the calibration curve, convert the fluorescence ratio R = I_700-800 / I_600-700 in the imaging area into a pH distribution map; P5. Using fluorescence lifetime imaging technology, obtain a spatial distribution map of tissue viscosity / polarity; P6. Spatially overlay the pH distribution map and the viscosity distribution map to construct a dual-parameter functional imaging map of the tissue microenvironment.

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