Fluorescent probe capable of detecting sulfur dioxide derivative and viscosity and synthesis method

Abstract

The invention belongs to the technical field of fluorescent probes, and particularly discloses a fluorescent probe capable of detecting sulfur dioxide derivatives and viscosity. The invention also discloses a synthesis method of the fluorescent probe. The fluorescent probe provided by the invention has the advantages of simple synthesis route, mild reaction conditions, simple and convenient post-treatment, good selectivity, high sensitivity and the like, and can be used for dual detection of in-vivo and in-vitro SO2 derivatives and viscosity.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a fluorescent probe capable of detecting sulfur dioxide derivatives and viscosity and a synthesis method thereof. Background Art

[0002] Sulfur dioxide (SO2), as an important environmental and industrial pollutant, is widely present in nature and various industrial processes, such as volcanic eruptions, forest fires, and the burning of fossil fuels. SO2 not only pollutes the environment, but also poses a threat to human health, can cause respiratory diseases, and is even associated with the occurrence of various cancers. In addition, SO2 is also used in food processing and agriculture, such as as a preservative and antioxidant. Therefore, accurate detection and testing of the levels of SO2 and its derivatives is crucial for environmental protection and public health.

[0003] In recent years, fluorescent probe technology has shown great potential in the field of SO2 detection due to its high sensitivity, high selectivity and visual detection. Fluorescent probes can change their fluorescence properties when reacting with target molecules, thereby realizing the detection of SO2. However, most existing fluorescent probes can only detect a single target and cannot simultaneously detect SO2 and its derivatives and other important parameters in biological systems, such as viscosity. Summary of the invention

[0004] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a fluorescent probe and a synthesis method for detecting sulfur dioxide derivatives and viscosity. The fluorescent probe provided by the present invention can not only detect SO2 derivatives with high sensitivity and selectivity, but also respond to viscosity changes, and has the advantages of good selectivity, low toxicity, and good biocompatibility, and provides a useful mitochondrial-targeted fluorescent probe for studying intracellular SO2 derivatives and viscosity at the subcellular level. The fluorescent probe provided by the present invention has mitochondrial targeting ability, which can make its application in biological systems more accurate, and provides a new perspective and tool for the study of cell microenvironment, and has important environmental detection and biomedical research value.

[0005] One aspect of the present invention relates to a fluorescent probe capable of detecting sulfur dioxide derivatives and viscosity, wherein the chemical structural formula of the fluorescent probe is:

[0006] The fluorescent probe is named "HVTI", and its Chinese name is: ((E)-2-(2-(4-hydroxynaphthalen-1-yl)vinyl)-1,1,3-trimethyl-1H-benzo[E]indol-3-ium).

[0007] Preferably, the raw material of the fluorescent probe includes a first raw material I, and the structural formula of the first raw material I is;

[0008] Another aspect of the present invention relates to a method for synthesizing the fluorescent probe capable of detecting sulfur dioxide derivatives and viscosity, comprising the following steps:

[0009] S1. Synthetic intermediate compound I: 1,2,3,3-tetramethyl-3H-indol-1-ium:

[0010] 5-9 mMol of 2,3,3-trimethyl-3H-indole, 1-9 mL of iodomethane, and 30-180 mL of anhydrous ethanol are stirred at 25-38°C for 12-18 hours. After the reaction is completed, the mixture is filtered and washed with cold saturated brine to obtain a yellow solid, namely 1,2,3,3-tetramethyl-3H-indol-1-ium (I);

[0011] S2. Synthesis of ((E)-2-(2-(4-hydroxynaphthalen-1-yl)vinyl)-1,1,3-trimethyl-1H-benzo[E]indol-3-ium) (HVTI) (II):

[0012] 2-5 mMol of intermediate compound I, 2-5 mMol of 4-hydroxy-1-naphthaldehyde, 100-180 mL of anhydrous ethanol and 0.05-0.15 mMol of piperidine are heated to reflux at 76-89°C in a N2 atmosphere for 12-24 h. After the reaction is completed, the solvent is removed by vacuum distillation, and then the mixture is extracted with dichloromethane, washed with saturated brine and purified by column chromatography to obtain a black solid HVTI (II).

[0013] The third aspect of the present invention relates to the use of the above fluorescent probe in imaging exogenous SO2 in living cells, wherein the cells are Raw 264.7 cells.

[0014] A fourth aspect of the present invention relates to the use of the above fluorescent probe in detecting SO2 derivatives and viscosity in zebrafish.

[0015] The fifth aspect of the present invention relates to the use of the above fluorescent probe in locating mitochondria.

[0016] The testing principle of the fluorescent probe HVTI provided by the present invention for SO2 derivatives is as follows: after HVTI reacts with SO2 derivatives, the fluorescence emission wavelength blue-shifts, and the solution color changes from purple to colorless, thereby realizing visible detection of SO2 derivatives; and quantitative analysis of SO2 derivatives is realized by measuring the change in the fluorescence intensity of the probe.

[0017] The viscosity test principle of the fluorescent probe HVTI provided by the present invention is: the response of HVTI to viscosity is based on the twisted intramolecular charge transfer (TICT) mechanism. The naphthalene ring and indole in the HVTI structure can rotate freely in a low viscosity environment, resulting in non-radiative consumption of excited state energy and showing very weak fluorescence. In a high viscosity solution, the free rotation of the probe is inhibited, thereby inhibiting the TICT process, resulting in the recovery and enhancement of the probe fluorescence, showing very strong fluorescence.

[0018] When the fluorescent probe HVTI responds to viscosity and SO2 derivatives, its fluorescence emission peaks are located at 614nm and 456nm, respectively. This result shows that there is a significant emission difference of 158nm between the maximum emission peaks of viscosity and SO2 derivatives, which is very beneficial for fluorescent probes to image viscosity and SO2 derivatives in biological systems in different emission channels.

[0019] The detection limit of HVTI for the SO2 derivative NaHSO3 is 0.51 μM.

[0020] The logarithm of HVTI fluorescence intensity (log I 614 ) is linearly related to the logarithm of viscosity (logη) in the range of 1.53-312.84cp (R 2 =0.9905).

[0021] The present invention also provides the above-mentioned fluorescent probe HVTI for detecting SO2 derivatives and viscosity. Experiments have shown that the fluorescent probe provided by the present invention can be applied to the detection of SO2 derivatives and viscosity in living cells and zebrafish. The fluorescent probe HVTI has the characteristics of good selectivity, low toxicity and good biocompatibility, and provides a useful mitochondrial targeted fluorescent probe for studying intracellular SO2 derivatives and viscosity at the subcellular level.

[0022] As an important parameter in the cell microenvironment, viscosity is closely related to a variety of biological processes and disease states. Changes in intracellular viscosity can affect the diffusion of substances, the movement of organelles, and the transmission of cell signals. The fluorescent probe HVTI provided by the present invention can simultaneously detect SO2 derivatives and viscosity, which will provide a powerful tool for studying cell physiology and pathology, and has important environmental detection and biomedical research value.

[0023] The fluorescent probe HVTI provided by the present invention has a simple synthesis route, mild reaction conditions, simple and convenient post-processing, can successfully label and detect target molecules, has obvious changes in fluorescence intensity, and is easy to quantitatively analyze; it has good biocompatibility and is suitable for imaging research of living cells and organisms.

[0024] The detection limit of the fluorescent probe HVTI provided by the present invention for NaHSO3 is 0.51 μM. The logarithm of the fluorescence intensity of HVTI (log I 614 ) is linearly related to the logarithm of viscosity (logη) in the range of 1.53-312.84cp (R 2 =0.9905).

[0025] The fluorescent probe HVTI fluorescent probe provided by the present invention has excellent mitochondrial targeting ability and can effectively locate mitochondria.

[0026] The fluorescent probe HVTI provided by the present invention can quickly enter the body of zebrafish and effectively detect SO2 derivatives and viscosity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the synthesis method of the fluorescent probe HVTI.

[0028] Figure 2 The fluorescent probe HVTI 1 H NMR and 13 C NMR spectrum.

[0029] Figure 3 The fluorescence emission spectra of the fluorescent probe HVTI after reacting with different concentrations of NaHSO3 and the linear relationship between NaHSO3 and fluorescence intensity between 30-150μM; (a) Fluorescence emission spectra of the fluorescent probe HVTI (10μM) after reacting with different concentrations of NaHSO3 (0-180μM); (b) The linear relationship between the fluorescence intensity of the fluorescent probe HVTI and NaHSO3 (30-150μM).

[0030] Figure 4 Effect of HVTI on viscosity and HSO3 - (a) Absorption spectrum of probe HVTI (10 μM) and its response to viscosity and HSO3- (180 μM); (b) Emission spectrum of probe HVTI before and after reaction with sulfite in PBS solution (10% DMSO, pH 7.4); (c) Emission spectrum of probe HVTI before and after adding 90% glycerol.

[0031] Figure 5 The fluorescent probe HVTI is used to bind to various common analytes (including SO3 2- , HSO3 - , K + , Na + , Ca 2+ ,Mg 2+ , Zn 2 + , Cu 2+ , Ni2+ , Fe 3+ ,Cr 3+ , Cl - , NO2 - , NO3 - , SCN - ,Br - , S2O3 2- , SO4 2- , S 2- , CH3COO - ,CO3 2- , ClO - , H2O2, Cys, Hcy, GSH, Lys, Glu, Arg) were identified by UV and fluorescence spectra. The horizontal axis is wavelength, the vertical axis is light intensity, the concentration of the compound is 10μM, various common analytes (including SO3 2- , HSO3 - , K + , Na + , Ca 2+ ,Mg 2+ , Zn 2+ , Cu 2+ , Ni 2+ , Fe 3+ ,Cr 3+ , Cl - , NO2 - , NO3 - , SCN - ,Br - , S2O3 2- , SO4 2- , S 2- , CH3COO - ,CO3 2- , ClO - , H2O2, Cys, Hcy, GSH, Lys, Glu, Arg) concentration is 10μM; (a) Fluorescent probe HVTI (10μM) for HSO3 - and other analytes (10 mM); (b) fluorescence intensity of the probe to different analytes.

[0032] Figure 6 The fluorescence spectrum of the fluorescent probe HVTI (10 μM) in different viscosity systems and the linear relationship between the fluorescence intensity log I614 and logη.

[0033] Figure 7 The viability of Raw 264.7 cells after incubation with different concentrations of probes.

[0034] Figure 8 For colocalization experiments.

[0035] Fig. 9 Cell fluorescence imaging of fluorescent probes detecting endogenous and exogenous SO2 derivatives.

[0036] Fig.10 Fluorescence imaging of Raw 264.7 cells after treatment with nystatin (10 μM).

[0037] Fig.11 Fluorescence imaging of sulfur dioxide and viscosity in zebrafish using fluorescent probes.

[0038] Fig.12 As a fluorescent probe with HSO3 - Mass spectrometry after the reaction.

[0039] Fig.13 The fluorescence probe HVTI for HSO3 - exist 1 Sensing mechanism in H NMR spectroscopy. DETAILED DESCRIPTION

[0040] The following is further described in detail through specific implementation methods:

[0041] Example: Synthesis of fluorescent probe HVTI

[0042] The synthesis steps are as follows Figure 1 As shown, the following steps are specifically involved:

[0043] S1. Synthesis of intermediate compound I: 1,2,3,3-tetramethyl-3H-indol-1-ium

[0044] In a round-bottom flask equipped with a condenser and a stirrer, add 5-9 mMol of 2,3,3-trimethyl-3H-indole (M=159.23, CAS:1640-39-7) and 1-9 mL of iodomethane (density=2.28 g / mL, M=141.94, CAS:74-88-4). Add 30-180 mL of anhydrous ethanol as a solvent and stir at 25-38°C for 12-18 hours. Detect the reaction progress by thin layer chromatography (TLC). After the reaction is completed, filter and wash with cold saturated brine to obtain a yellow solid, i.e. 1,2,3,3-tetramethyl-3H-indol-1-ium (I).

[0045] In a specific embodiment of the present invention, the amount of 2,3,3-trimethyl-3H-indole is 5 mMol, the amount of methyl iodide is 1 mL, the molar ratio of 2,3,3-trimethyl-3H-indole to methyl iodide is 1:1, the volume of anhydrous ethanol is 30 mL, the reaction temperature is 30° C., and the reaction time is 12 h.

[0046] S2. Synthesis of ((E)-2-(2-(4-hydroxynaphthalen-1-yl)vinyl)-1,1,3-trimethyl-1H-benzo[E]indol-3-ium) (HVTI) (II)

[0047] 2-5mMol intermediate compound I and 2-5mMol 4-hydroxy-1-naphthaldehyde were added to a round-bottom flask, dissolved in 100-180mL anhydrous ethanol solvent, 0.05-0.15mMol piperidine was added as a catalyst, and heated to reflux at 76-89°C for 12-24h in a N2 atmosphere. The reaction was detected by TLC, and after the reaction was completed, the solvent was removed by vacuum distillation, and then extracted with dichloromethane (DCM), washed with saturated brine, and purified by column chromatography (methanol: DCM = 1: 10-30) to obtain black solid HVTI (II).

[0048] In a specific embodiment of the present invention, the amount of intermediate compound I is 2mMol and the amount of 4-hydroxy-1-naphthaldehyde is 2mMol (the molar ratio of intermediate compound I and 4-hydroxy-naphthaldehyde is 1:1), the volume of anhydrous ethanol is 100mL, the amount of piperidine is 0.05mMol, and the mixture is heated to reflux at 78°C for 12h in a N2 atmosphere. The reaction is detected by TLC, and after the reaction is completed, the solvent is removed by vacuum distillation, and then extracted with dichloromethane (DCM), washed with saturated brine, and purified by column chromatography (methanol: DCM = 1:20) to obtain 0.4733g of black solid HVTI (II), with a yield of 52%.

[0049] Figure 2 The fluorescent probe HVTI 1 H NMR and 13 C NMR spectrum. Figure 2 As shown:

[0050] HVTI 1 H NMR (400MHz, DMSO-d6) δ8.77(d,J=15.2hz,1H),8.56(d,J=8.8hz,1H),8.34

[0051] (d,J=8.5hz,1H),8.26(d,J=8.2hz,1H),7.76(t,J=8.0hz,2H),7.67(d,J=8.0hz,1H),7.56(dt,J=14.7,7 .7hz,2H),7.45(t,J=7.5hz,1H),7.34(d,J=15.2hz,1H),6.93(d,J=8.8hz,1H),3.97(s,3H),1.82(s,6H).

[0052] 13C NMR(101MHz,DMSO-d6)δ177.58,167.32,145.58,142.94,142.54,134.69,133.04,130.14,129.18,127.37,1 26.45,126.13,124.40,123.09,122.87,120.58,113.51,113.38,106.50,50.88,33.54,27.34.MS(ESI): m / z found 328.17,calculated for[C 23 H 22 NO] + :328.17.

[0053] Examples 1-5

[0054] The main difference between the synthetic methods provided in Examples 1-5 is:

[0055] Table 1: Main differences between the synthetic methods provided in Examples 1-5

[0056]

[0057] By comparing the above synthesis methods, it can be seen that the yield of the fluorescent probe HVTI synthesized by the method of Example 2 is the highest, which is 56.58% (82%×69%).

[0058] Experiment 1: Mechanism of fluorescent probe HVTI recognizing NaHSO3

[0059] Dissolve PBS solid powder in deionized water and adjust the volume to 2L in a brown volumetric flask to obtain a 10mM PBS buffer solution with a pH of 7.4. Accurately weigh the fluorescent probe HVTI, dissolve the fluorescent probe HVTI in DMSO to obtain a 1mM stock solution, and prepare the stock solution of the analyte NaHSO3 (100mM) in deionized water and store at 4°C for later use. The test concentrations of the fluorescent probes HVTI and are 10μM, and the test concentration of the analyte NaHSO3 is 10mM. In the PBS buffer solution (10mM, pH=7.4) system, the concentration of the fluorescent probe HVTI is 10μM, and then 10mM of the newly prepared NaHSO3 solution is added thereto, and the solution is allowed to stand at room temperature for 30min. After the reaction of NaHSO3 with the fluorescent probe HVTI, LC-MS confirms the formation of an HVTI-HSO3 adduct, in which the peak at m / z value 410.08 corresponds to [HVTI-HSO3+H] + .

[0060] In order to further explore the detection mechanism, this experiment carefully studied the effect of NaHSO3 on the fluorescent probe HVTI. 1H NMR titration. 1 H NMR was measured at 20°C on a Bruker DPXⅢ400 NMR instrument, with DMSO-d6 as solvent and TMS as internal standard. A small amount of probe HVTI, about 30 mg, was dissolved in 0.6 mL DMSO-d6, and 20 μL was drawn with a 50 μL pipette, with a concentration of 1.00x10 -3 oeLh . L -1 The NaHSO3 DMSO-d6 solution was added to the existing probe HVTI DMSO-d6 solution and its 1 H NMR spectrum. Repeat the above process until NaHSO3 is in excess. Collect and analyze the NMR spectra of different host-guest ratios. It can be seen that with HSO3 - After the reaction, the proton signals of 6.93ppm (Ha) and 7.65ppm (Hb) in HVTI gradually disappeared and rose to 5.08ppm (Ha') and 6.50ppm (Hb') in HVTI-HSO3, respectively. This is due to the destruction of the conjugated structure between indole and the strong receptor. Therefore, the above results support the interaction between the probe and HSO3 - The reaction mechanism is based on a 1,4 addition reaction, which reacts with HSO3 via the C=C double bond. - The addition reaction of HSO3 - Quantitative detection of Fig.12 and Fig.13 ).

[0061] Experiment 2: Fluorescence emission spectrum experiment of fluorescent probe HVTI

[0062] The fluorescence spectra of the probes were measured using a Cary Eclipse fluorometer.

[0063] In PBS buffer (pH 7.4), different concentrations of NaHSO3 were added to the probe solution, and the changes in fluorescence intensity and maximum emission wavelength were observed. The emission wavelength of HVTI is 614nm. After the addition of NaHSO3, the conjugated system is destroyed, the fluorescence spectrum is blue-shifted, and the emission wavelength of HVTI is 456nm. When the probe reacts with different concentrations of NaHSO3, the fluorescence intensity also increases with the increase of NaHSO3 concentration. The fluorescence signal of the probe has a good linear relationship with the concentration of NaHSO3. When NaHSO3 is in the range of 40-160μM, the fluorescence response values ​​of HVTI and NaHSO3 show a good linear relationship, and the linear relationship is y=3.58402x-18.53962. According to the detection limit formula LOD=3σ / k, the detection limit of HVTI is 0.51μM ( Figure 3 , Figure 4 ).

[0064] Experiment 3: Determination of UV and fluorescence spectra

[0065] The concentration of the fluorescent probe was diluted to 2 × 10 -5 mol / L, 29 common analytes (including K + , Na + , Ca 2 + ,Mg 2+ , Zn 2+ , Cu 2+ , Ni 2+ , Fe 3+ ,Cr 3+ , Cl - , NO2 - , NO3 - , SCN - ,Br - , S2O3 2- , SO4 2- , S 2- , CH3COO - ,CO3 2- , ClO - ,H2O2,Cys,Hcy,GSH,Lys,Glu,Arg,SO3 2- , HSO3 - ) was prepared into 1×10 -5 mol / L solution. They were added to 1mL cuvettes in turn, and their UV spectra under the action of different analytes were measured to screen out probes that specifically recognize SO2 derivatives. The addition of other analytes did not cause obvious changes in the probe fluorescence spectrum. After adding NaHSO3, the solution color changed from purple to colorless, the fluorescence spectrum shifted to the blue, and the fluorescence intensity increased sharply, indicating that the fluorescent probe HVTI can specifically recognize SO2 and has a high selectivity ( Figure 5 ).

[0066] Experiment 4: Viscosity Test

[0067] HVTI's optical research on viscosity detection:

[0068] The test system is phosphate buffer solution (PBS) and glycerol. Under the condition of fixed 25°C, different ratios of glycerol and PBS are adjusted to prepare test systems with different viscosity values. The concentration of the fluorescent probe HVTI is 10μM. After the probe is placed in the test system and allowed to stand for 1h at 25°C, the fluorescence of the prepared solution is detected by a fluorescence spectrophotometer, with λ = 500nm and slit width: (5, 5). Absorption spectrum test of fluorescent probe HVTI: The synthesized fluorescent probe is dissolved in dimethyl sulfoxide (DMSO) to prepare a 1mM stock solution. An appropriate amount of NaHSO3 is added to the stock solution, and the changes in the absorption spectrum are recorded using a UV spectrophotometer.

[0069] In this experiment, the viscosity of the detection system was changed by changing the ratio of PBS to glycerol ( Figure 6 As the proportion of glycerol in the PBS-glycerol mixture increased (increased by 10 μM), the fluorescence intensity of the solution at 614 nm increased. The logarithm of the fluorescence intensity of HVTI (log I 614 ) is linearly related to the logarithm of viscosity (logη) in the range of 1.53-312.84cp (R 2 =0.9905).

[0070] In addition, this experiment also found that with the increase of viscosity, the fluorescence spectrum slightly red-shifted. These results show that the HVTI probe HVTI has a relatively sensitive fluorescence response to viscosity changes.

[0071] Experiment 5: Cell culture and cytotoxicity assay

[0072] RAW 264.7 cells (Wuhan Shane Biotechnology Co., Ltd.) were cultured in a special culture medium for RAW264.7 cells. The culture conditions were 37°C, 5% CO2, and 95% humidity. RAW 264.7 cells were seeded in a 96-well plate, and the culture medium was replaced after 24 hours of culture. The cells were then incubated with different concentrations of the fluorescent probe HVTI at 37°C for 24 hours. Subsequently, 100 μL of fresh culture medium containing MTT (5 mg / mL) was added to each well and incubated for 4 hours. Finally, the supernatant of each well was removed, 100 μL of DMSO solution was added to each well, and after shaking for 5 minutes, the light absorption value (OD value) was measured at 490 nm. After incubation of probes of different concentrations for 24 hours, the survival rate of Raw 264.7 cells was above 80%, indicating that the probe has low toxicity and good biocompatibility, and is suitable for SO2 and viscosity imaging of living cells ( Figure 7 ).

[0073] Experiment 6: Cell imaging experiment

[0074] The imaging ability of the fluorescent probe HVTI for intracellular and extracellular SO2 sources was investigated.

[0075] The fluorescent probe HVTI itself does not show obvious fluorescence in the blue channel. After adding NaHSO3 (100μM, 200μM) and incubating with the probe for 30 minutes, the blue channel shows obvious fluorescence, which is consistent with the above spectral properties. The experimental results show that the fluorescent probe HVTI can be used to image exogenous SO2 in living cells.

[0076] It is well known that mammalian hepatocytes produce endogenous SO2 derivatives through the reaction of glutathione (GSH) and thiosulfate (Na2S2O3) catalyzed by thiosulfate sulfotransferase (TST). The control group Raw 264.7 cells were imaged after being cultured with the fluorescent probe HVTI for 30 minutes. The experimental group was cultured with Na2S2O3 (250μM) and GSH (500μM) for 30 minutes, and then treated with the fluorescent probe HVTI for 30 minutes for imaging. The results showed that the production of endogenous SO2 derivatives can enhance blue fluorescence. In contrast, imaging after treatment with 500μM GSH for 30 minutes and 10μM fluorescent probe HVTI for 30 minutes showed that there was no obvious change in the images of Raw 264.7 cells ( Figure 8 ) The fluorescent probe HVTI has the ability to effectively image the intracellular SO2 source.

[0077] Experiment 7: Imaging the intracellular viscosity of the probe

[0078] Nystatin is a membrane permeability enhancer that can induce mitochondrial dysfunction and increase the viscosity of the cell microenvironment. This experiment detected the fluorescence changes of viscosity under the action of nystatin (NYs, Shanghai Guchen Biotechnology Co., Ltd., catalog number: GC11592-100mg), and the fluorescence intensity of the red channel was significantly enhanced. The specific experiment is as follows: the mother solution of nystatin was added to 1mL of cell culture medium containing Raw264.7 cells, and the DMSO solution of the probe HVTI was added after culturing at 37°C for 30min in a carbon dioxide incubator, and the incubation continued for 30min. The final concentration of the mycostatin in the cell culture medium is 10μM; the final concentration of the probe DMSO in the cell culture medium is 5μM. Raw 264.7 cells were imaged by bright field and red channel fluorescence. It was found that the fluorescence signal of the red channel was significantly enhanced before and after nystatin stimulation, indicating that the probe can perform fluorescence imaging of viscosity changes in cell mitochondria. The experimental results show that the fluorescent probe HVTI has the potential to detect viscosity changes, which is of great significance for the prevention and early diagnosis of diseases ( Fig. 9 ).

[0079] Experiment 8: Colocalization experiment

[0080] This experiment further confirmed the excellent mitochondrial targeting ability of the fluorescent probe HVTI through co-localization experiments in RAW 264.7 (Wuhan Shangen Biotechnology Co., Ltd.) cells.

[0081] After pretreatment with Nys (Shanghai Guchen Biotechnology Co., Ltd., Catalog No.: GC11592-100mg) for 30 minutes, RAW264.7 cells were cultured with the probe and mitochondrial green fluorescent probe (Mito Tracker Green, Shanghai Yaji Biotechnology Co., Ltd., Catalog No.: C1048, molecular weight 671.8) for 30 minutes. The red channel fluorescence of the fluorescent probe HVTI overlaps well with the green channel fluorescence, and the Pearson correlation coefficient of the fluorescent probe HVTI is 0.8452, indicating that the fluorescent probe HVTI can effectively locate mitochondria ( Fig.10 ).

[0082] Experiment 9: Zebrafish imaging experiment

[0083] In this experiment, 3-day-old zebrafish (zebrafish strain: VEGFR2:GFP, Fuzhou Baiweisi Biotechnology Co., Ltd.) were incubated with the fluorescent probe HVTI (10 μM), washed three times with PBS after 30 minutes, and then incubated with NaHSO3 (100 μM) for 30 minutes, and then washed with PBS for fluorescence imaging. The experimental results showed that the blue channel had strong fluorescence, and the red channel had no fluorescence signal.

[0084] Three-day-old zebrafish were pre-incubated with Nys (Shanghai Guchen Biotechnology Co., Ltd., Catalog No.: GC11592-100mg) (10μM) for 30 minutes, and then incubated with the fluorescent probe HVTI (10μM) for 30 minutes, and then washed with PBS three times for fluorescence imaging. The experimental results showed that the fluorescence intensity of the red channel was significantly enhanced, while the fluorescence intensity of the blue channel was almost non-existent.

[0085] The above experiments show that the fluorescent probe HVTI can quickly enter the zebrafish body and can be used to detect SO2 derivatives and viscosity in living animals ( Fig.11 ).

[0086] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A fluorescent probe capable of detecting sulfur dioxide derivatives and viscosity, characterized in that: The structural formula of the fluorescent probe is:

2. The fluorescent probe capable of detecting sulfur dioxide derivatives and viscosity according to claim 1, characterized in that: The raw materials of the fluorescent probe include a first raw material I, and the structural formula of the first raw material I is:

3. The method for synthesizing a fluorescent probe capable of detecting sulfur dioxide derivatives and viscosity according to claim 1, characterized in that: The following steps are involved: S1. Synthetic intermediate compound I: 1,2,3,3-tetramethyl-3H-indol-1-ium: 5-9 mMol of 2,3,3-trimethyl-3H-indole, 1-9 mL of iodomethane, and 30-180 mL of anhydrous ethanol are stirred at 25-38°C for 12-18 hours. After the reaction is completed, the mixture is filtered and washed with cold saturated brine to obtain a yellow solid, namely 1,2,3,3-tetramethyl-3H-indol-1-ium (I); S2. Synthesis of ((E)-2-(2-(4-hydroxynaphthalen-1-yl)vinyl)-1,1,3-trimethyl-1H-benzo[E]indol-3-ium) (HVTI) (II): 2-5 mMol of intermediate compound I, 2-5 mMol of 4-hydroxy-1-naphthaldehyde, 100-180 mL of anhydrous ethanol and 0.05-0.15 mMol of piperidine are heated to reflux at 76-89°C in a N2 atmosphere for 12-24 h. After the reaction is completed, the solvent is removed by vacuum distillation, and then the mixture is extracted with dichloromethane, washed with saturated brine and purified by column chromatography to obtain a black solid HVTI (II).

4. Use of the fluorescent probe according to claim 1 in imaging exogenous SO2 in living cells.

5. Use of the fluorescent probe according to claim 1 in detecting SO2 derivatives and viscosity in zebrafish.

6. Use of the fluorescent probe according to claim 1 in locating mitochondria.

Images