A bisulfite and polar fluorescent probe and its preparation method and application

Abstract

The present invention discloses a fluorescent probe capable of simultaneously detecting bisulfite and polarity, a preparation method thereof, and its application in biological imaging, and belongs to the field of chemical analysis and detection technology. The probe BDMOB can simultaneously detect changes in polarity and bisulfite content. As the polarity of the solution increases, the green channel of the probe will be enhanced and the red channel will be weakened, and the fluctuation of the polarity of the solution can be detected in a dual-channel manner with high sensitivity. When the bisulfite content increases, the fluorescence intensity of the probe in the orange channel will gradually increase from nothing to something, and it is a good "off-on" type fluorescent probe. The probe has good anti-interference and biocompatibility, and can be used to distinguish normal cells from cancer cells, monitor changes in endogenous and exogenous bisulfite content, and accurately track changes in the microenvironment before and after inflammation and ferroptosis cells, and has been successfully applied in vivo, opening up a new path for the study of abnormal conditions such as inflammation and ferroptosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry and relates to a preparation method of a fluorescent probe capable of simultaneously detecting bisulfite and polarity and its application in in vivo imaging of inflammation and ferroptosis. Background Art

[0002] Sulfur dioxide and its derivatives serve as food antioxidants and preservatives, and are widely used in vegetables, fruits, foods, winemaking, pharmaceuticals, and other fields. However, abnormal levels of sulfur dioxide and its derivatives have been linked to cancer, respiratory diseases, neurological disorders, cardiovascular disease, and inflammation. Commonly used SO2 detection methods are costly and time-consuming. Fluorescent probes, due to their high spatiotemporal resolution and noninvasive monitoring capabilities, are powerful tools for real-time monitoring of biological systems.

[0003] Furthermore, in biological systems, polarity not only develops and maintains cellular homeostasis but also serves as an integral component of the cellular microenvironment. Cells undergo spatial organization and protein composition activities, including differentiation, migration, and proliferation, and their polarity changes. Abnormal changes in intracellular polarity can lead to numerous diseases, such as diabetes, Alzheimer's disease, diabetes, cirrhosis, inflammation, and cancer. Therefore, monitoring changes in polarity within organisms is crucial.

[0004] Currently, most probes can only detect changes in a single microenvironment within a cell, which is not conducive to multi-factor analysis of disease models. Conventional fluorescent probes for detecting bisulfite in existing technologies are generally highly cytotoxic and cannot be used in actual medical treatment. In summary, we hope to design a dual-function fluorescent probe that can accurately capture bisulfite and polarity changes in cells and organisms. This probe can generate signals when abnormal conditions occur in the body, allowing for more sensitive and accurate perception of disease occurrence, and providing assistance in the prevention, diagnosis, and treatment of diseases. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method for preparing a fluorescent probe capable of simultaneously detecting bisulfite and polarity and its application in bioimaging, which has the characteristics of a simple synthetic route, good selectivity, and high sensitivity, and can effectively detect bisulfite and polarity at physiological levels.

[0006] The present invention provides a fluorescent probe having the following structure:

[0007]

[0008] The fluorescent probe synthesis in the present invention is as follows:

[0009]

[0010] A1: 2-(Benzo[d]thiazol-2-yl)-4-methylphenol

[0011] A2: 3-(Benzo[d]thiazol-2-yl)-2-hydroxy-5-methylbenzaldehyde

[0012] A3: 3-Acetyl-8-(benzo[d]thiazol-2-yl)-6-methyl-2H-benzopyran-2-one

[0013] BDMOB: 8′-(Benzo[d]thiazol-2-yl)-7-(diethylamino)-6′-methyl-2′-oxo-2′H-[2,3′-benzopyrano]-1-oxonium perchlorate

[0014] The preparation steps of the fluorescent probe BDMOB are as follows:

[0015] Step 1: Preparation of intermediate A1

[0016] Accurately weigh 5-methylsalicylaldehyde using an electronic balance, and transfer 2-aminobenzenethiol to a 50 mL round-bottom flask. Add N,N-dimethylformamide and sonicate to dissolve the solid. Under argon, gradually raise the temperature to 180°C and react for 2 hours. After the reaction, cool to room temperature and pour into 30 mL of ice water. A large amount of precipitate will immediately form. Filter and wash with distilled water. Collect the precipitate and dry it in a vacuum oven for 12 hours to obtain an off-white solid A1 (93.2%).

[0017] Step 2: Preparation of intermediate A2

[0018] Accurately weigh A1 and hexamethylenetetramine using an electronic balance and place them in a 50 mL round-bottom flask. Add trifluoroacetic acid and sonicate to dissolve the solid. Under argon, reflux the reaction mixture at 80°C overnight. After the reaction mixture cools, slowly add sodium hydroxide solution with stirring until no more precipitate forms. Collect the precipitate by filtration and purify it by silica gel column chromatography using dichloromethane as the developing solvent to obtain A2 (45.0%) as a yellow solid.

[0019] Step 3: Preparation of intermediate product A3

[0020] Weigh A2 and dissolve it in 20 mL of anhydrous ethanol. Add ethyl acetoacetate and piperidine to the reaction mixture. Heat the reaction mixture under reflux under argon and allow to react overnight. After the reaction is complete, cool to room temperature, filter the reaction mixture, and wash with a small amount of ethanol to obtain a yellow solid A3 (60.3%).

[0021] Step 4: Preparation of fluorescent probe BDMOB

[0022] Concentrated H₂SO₄ was added to a 50 mL round-bottom flask. Compound A3 was added dropwise to the concentrated H₂SO₄ in an ice-water bath. 4-Diethylaminosalicylaldehyde was added to the mixture in batches with vigorous stirring. The mixture was heated and refluxed at 90°C for 2 hours. The reaction mixture was cooled to room temperature and added dropwise to ice water with stirring. HClO₄ was added to produce a large precipitate. The precipitate was filtered, washed with cold water to remove the solvent, and vacuum dried for 24 hours to yield a purple-black solid fluorescent probe BDMOB (85.3%).

[0023] The response mechanism of the fluorescent probe of the present invention to bisulfite is as follows:

[0024]

[0025] The fluorescent probe BDMOB responds to bisulfite via a Michael addition reaction, as shown in the equation above. Utilizing a photoinduced electron transfer mechanism, the unbound bisulfite fluorescent probe exhibits virtually no fluorescence emission under specific excitation wavelengths. When bisulfite is added to the solution, it adds to the carbon-carbon double bond of the probe's pyranose structure, disrupting the probe's conjugation. This inhibits electron transfer and produces the corresponding bisulfite adduct, thereby achieving bisulfite response.

[0026] The detection mechanism of the fluorescent probe of the present invention for polarity is as follows:

[0027]

[0028] BDMOB uses benzopyran and benzothiazole as fluorophores, introduces strong electron-donating diethylamino and electron-withdrawing benzothiazole to form a D-π-A structure, which makes the fluorescent probe BDMOB exhibit solvatochromism and is highly sensitive to changes in polarity.

[0029] Figure 5 This is the UV-visible absorption spectrum of the probe BDMOB in mixed solutions of dimethylformamide and chloroform at different ratios. The absorption spectrum shows regular changes as the ratio of the mixed solution changes.

[0030] Figure 6 The ratio of the absorbance of the probe BDMOB at 450 nm and 550 nm in mixed solutions of dimethylformamide and chloroform at different ratios increases with the proportion of dimethylformamide. The inset shows the color of the solution under 365 nm handheld UV light, showing a clear soluchromic effect.

[0031] Figure 7 is the fluorescence emission spectrum of the probe BDMOB in the mixed solution of dimethylformamide and chloroform at different ratios (λ ex=450 nm), the fluorescence intensity of the green channel gradually increased with the increase of solution polarity.

[0032] Figure 8 is the fluorescence emission spectrum of the probe BDMOB in the mixed solution of dimethylformamide and chloroform at different ratios (λ ex =550 nm), and the fluorescence intensity of the red channel gradually weakened with the increase of solution polarity.

[0033] Figure 9 is the ratio of the fluorescence intensity of the probe BDMOB at 530 nm and 660 nm in a mixed solution of dimethylformamide and chloroform at different ratios (λ ex =450nm, 550nm), and its value increases with the increase of the proportion of dimethylformamide.

[0034] Figure 10 The color coordinates of the probe BDMOB at different polarities intuitively show the changes in the luminescent color in the solution.

[0035] Figure 11 is the fluorescence emission spectrum of the probe BDMOB after adding five equivalents of bisulfite (λ ex =480nm).

[0036] Figure 12 is the fluorescence intensity of the probe BDMOB at 560 nm after adding bisulfite (λ ex =480nm), and there is a good linear relationship.

[0037] Figure 13 is the photostability of the probe BDMOB itself and its response time after adding different equivalents of bisulfite (λ ex =480 nm), after the addition of bisulfite, the fluorescence intensity gradually increased and stabilized after 30 minutes.

[0038] Figure 14 is the fluorescence intensity of the probe BDMOB itself at 560 nm at different pH values ​​and its fluorescence intensity after adding ten equivalents of bisulfite (λ ex =480 nm), the probe BDMOB can effectively monitor the presence of bisulfite in the pH range of 5-11.

[0039] Figure 15 The cytotoxicity of the probe BDMOB is low and has good biocompatibility.

[0040] Figure 16 This is confocal imaging of HeLa cells after the probe BDMOB was co-incubated with different concentrations of bisulfite. As the bisulfite concentration increased, the fluorescence intensity of the orange channel gradually increased.

[0041] Figure 17 The bar graph shows the relative fluorescence intensity of cells after the probe BDMOB was co-incubated with different concentrations of bisulfite, which can intuitively show the relationship between the fluorescence intensity and the concentration of added bisulfite.

[0042] Figure 18 This is the fluorescence intensity analysis of a single cell section after the probe BDMOB was co-incubated with different concentrations of bisulfite, which intuitively shows the fluorescence distribution within the cell.

[0043] Figure 19 This confocal image shows endogenous bisulfite in cells incubated with the probe BDMOB. Group A serves as a blank control. Groups B and C lack endogenous bisulfite production and exhibit no fluorescence. Group D, which does produce bisulfite endogenously, shows a significant increase in fluorescence intensity in the orange channel. However, after the addition of an inhibitor, which inhibits endogenous bisulfite production, fluorescence disappears. This demonstrates that the probe BDMOB can sensitively and rapidly detect endogenous bisulfite production.

[0044] Figure 20 Confocal imaging of ferroptosis cells co-incubated with the probe BDMOB. Glutathione peroxidase inhibitors can cause cell ferroptosis. Glutathione peroxidase inhibitors increase the fluorescence intensity of the green and orange channels, and the fluorescence intensity of the green and orange channels decreases in the presence of ferroptosis inhibitors.

[0045] Figure 21 The fluorescence intensity of each channel when the probe BDMOB was co-incubated with ferroptotic cells is shown, which intuitively shows the changes in the fluorescence intensity of the green channel, orange channel, and red channel.

[0046] Figure 22 Confocal imaging of the probe BDMOB co-incubated with inflammatory cells. Lipopolysaccharide causes cell inflammation, its content increases, the fluorescence intensity of the green channel weakens, and the fluorescence intensity of the orange and red channels gradually increases. The addition of apocynin restores the fluorescence intensity of the green channel and weakens the fluorescence intensity of the orange and red channels.

[0047] Figure 23 The fluorescence intensity of each channel when the probe BDMOB was co-incubated with inflammatory cells is shown, which intuitively shows the changes in the fluorescence intensity of the green channel, orange channel, and red channel.

[0048] Figure 24This is confocal imaging of the probe BDMOB in normal human cervical epithelial cells and cervical cancer cells. In normal human cervical epithelial cells, the fluorescence intensity of the green channel is strong and the fluorescence intensity of the red channel is weak; in cervical cancer cells, the fluorescence intensity of the green channel is weak and the fluorescence intensity of the red channel is strong.

[0049] Figure 25 The fluorescence intensity of each channel after the probe BDMOB was co-incubated with human normal cervical epithelial cells and cervical cancer cells, which intuitively shows the changes in the fluorescence intensity of the green channel and the red channel.

[0050] Figure 26 Confocal imaging of the probe BDMOB in nude mice induced with ferroptosis showed that the fluorescence of the green and orange light channels was enhanced in the ferroptotic nude mice. After drug intervention to alleviate ferroptosis, the fluorescence of the green and orange light channels was weakened to varying degrees, and the red channel was enhanced.

[0051] Figure 27 The fluorescence intensity of each channel of the probe BDMOB in nude mice induced with ferroptosis is shown, which intuitively shows the changes in the fluorescence intensity of the green channel, orange channel, and red channel.

[0052] Figure 28 Confocal imaging of the probe BDMOB in nude mice induced with inflammation. After lipopolysaccharide-induced inflammation in nude mice, the fluorescence intensity of the green channel weakened, while the fluorescence intensity of the orange and red channels increased. After the addition of apocynin (an anti-inflammatory drug), the fluorescence intensity of the green channel recovered, while the fluorescence intensity of the orange and red channels weakened.

[0053] Figure 29 The fluorescence intensity of each channel of the probe BDMOB in nude mice with induced inflammation is shown, which intuitively shows the changes in the fluorescence intensity of the green channel, orange channel, and red channel. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Preparation and design route of probe BDMOB.

[0055] Figure 2 Mechanism of bisulfite detection by probe BDMOB.

[0056] Figure 3 Mechanism of polarity detection by probe BDMOB.

[0057] Figure 4 Photophysical properties of probe BDMOB in different solvents (including maximum absorbance λ abs,max , maximum emission λ em,max , Stokes shift, molar absorptivity and fluorescence quantum yield (QY).

[0058] Figure 5UV-visible absorption spectra of probe BDMOB in mixed solutions of dimethylformamide and chloroform at different ratios.

[0059] Figure 6 The absorbance ratio of the probe BDMOB at 450 nm and 550 nm in mixed solutions of dimethylformamide and chloroform at different ratios. The inset shows the color of the solution under 365 nm handheld UV lamp illumination.

[0060] Figure 7 Fluorescence emission spectra of probe BDMOB in mixed solutions of dimethylformamide and chloroform at different ratios (λ ex =450nm).

[0061] Figure 8 Fluorescence emission spectra of probe BDMOB in mixed solutions of dimethylformamide and chloroform at different ratios (λ ex =550nm).

[0062] Figure 9 The ratio of the fluorescence intensity of the probe BDMOB at 530 nm and 660 nm in the mixed solution of dimethylformamide and chloroform at different ratios (λ ex =450nm, 550nm).

[0063] Figure 10 Color coordinates of probe BDMOB at different polarities.

[0064] Figure 11 The fluorescence emission spectrum of the probe BDMOB after adding five equivalents of bisulfite (λ ex =480nm).

[0065] Figure 12 The fluorescence intensity of the probe BDMOB at 560 nm after adding bisulfite (λ ex =480nm).

[0066] Figure 13 The photostability of the probe BDMOB itself and its response time after adding different equivalents of bisulfite (λ ex =480nm).

[0067] Figure 14 The fluorescence intensity of the probe BDMOB itself at 560 nm at different pH values ​​and the fluorescence intensity after adding ten equivalents of bisulfite (λ ex =480nm).

[0068] Figure 15 Cytotoxicity of the probe BDMOB.

[0069] Figure 16Confocal imaging of HeLa cells after incubation of the probe BDMOB with different concentrations of bisulfite.

[0070] Figure 17 Relative fluorescence intensity of cells after incubation with probe BDMOB and different concentrations of bisulfite.

[0071] Figure 18 Fluorescence intensity analysis of cell sections after incubation of the probe BDMOB with different concentrations of bisulfite.

[0072] Figure 19 Confocal imaging of endogenous bisulfite in cells after incubation with the probe BDMOB.

[0073] Figure 20 Confocal imaging of ferroptotic cells co-incubated with the probe BDMOB.

[0074] Figure 21 Fluorescence intensity of each channel when the probe BDMOB was co-incubated with ferroptotic cells.

[0075] Figure 22 Confocal imaging of inflammatory cells co-incubated with the probe BDMOB.

[0076] Figure 23 Fluorescence intensity of each channel when the probe BDMOB was co-incubated with inflammatory cells.

[0077] Figure 24 Confocal imaging of the probe BDMOB in human normal cervical epithelial cells and cervical cancer cells.

[0078] Figure 25 Fluorescence intensity of each channel of the probe BDMOB after co-incubation of normal human cervical epithelial cells and cervical cancer cells.

[0079] Figure 26 Confocal imaging of the probe BDMOB in nude mice induced with ferroptosis.

[0080] Figure 27 Fluorescence intensity of each channel of the probe BDMOB in nude mice induced with ferroptosis.

[0081] Figure 28 Confocal imaging of the probe BDMOB in nude mice with induced inflammation.

[0082] Figure 29 Fluorescence intensity of each channel of the probe BDMOB in nude mice with induced inflammation.

[0083] Specific implementation examples

[0084] Example 1: Synthesis of Compound A1

[0085] 5-Methylsalicylaldehyde (585.0 mg, 4.3 mmol) was accurately weighed using an electronic balance. 2-Aminobenzenethiol (460.0 μL, 4.3 mmol) was transferred to a 50 mL round-bottom flask. 6 mL of DMF was added and the mixture was heated under argon. After the reaction, the mixture was poured into ice water to precipitate. The precipitate was filtered, washed, and dried to obtain an off-white solid, A1 (965.4 mg, 4.0 mmol), with a yield of 93.2%.

[0086] Example 2: Synthesis of Compound A2

[0087] A1 (965.0 mg, 4.0 mmol) and hexamethylenetetramine (560.8 mg, 4.0 mmol) were accurately weighed using an electronic balance and placed in a 50 mL round-bottom flask. 20 mL of trifluoroacetic acid was added and the mixture was heated under argon overnight. After the reaction, NaOH solution was added. The precipitate was collected by filtration and purified by silica gel column chromatography to obtain a yellow solid A2 (484.3 mg, 1.8 mmol) in a 45.0% yield.

[0088] Example 3: Synthesis of Compound A3

[0089] A2 (269.1 mg, 1.0 mmol) was weighed and dissolved in 20 mL of anhydrous ethanol. Ethyl acetoacetate (256.0 μL, 2.0 mmol) and piperidine (80.0 μL, 0.8 mmol) were added to the reaction mixture. The mixture was heated to reflux under argon and allowed to react overnight. A3 (202.3 mg, 0.6 mmol) was obtained by filtration and washing to obtain a yellow solid in a yield of 60.3%.

[0090] Example 4: Synthesis of probe BDMOB

[0091] Concentrated H2SO4 (4.4 mL, 80.0 mmol) was added to a 50 mL round-bottom flask. Compound A3 (1.0 mmol, 335.4 mg) was added dropwise to the concentrated H2SO4 in an ice-water bath. 4-Diethylaminosalicylaldehyde (1.2 mmol, 231.9 mg) was added to the mixed solution and heated to reflux. The mixture was cooled to room temperature, added to ice water (30 mL), and HClO4 (0.4 mL) was added to produce a large amount of precipitate. The precipitate was filtered, washed with cold water to remove the solvent, and vacuum dried for 24 hours to obtain the purple-black solid product BDMOB (420.7 mg, 85.3% yield). 1HNMR(600MHz,DMSO)δ9.07(s,1H),8.66(d,J=7.9Hz,1H),8.48(s,1H),8.18(dd,J=14.6,7.8Hz,2H),7.97(dd,J=17.0,8.7Hz,2H) ,7.78(s,1H),7.49(t,J=8.3Hz,2H),7.42(t,J=7.5Hz,1H),7.08(s,1H),3.71(d,J=49.9Hz,4H),2.48(s,3H),1.31–1.21(m,6H). 13 C NMR (151MHz, DMSO) δ159.7,159.6,158.6,157.5,156,3,151.9,149.6,148.5,146.5,136.1,135.5,135.1,133.4, 133.3,127.2,126.2,123.4,122.8,121.5,120.6,120.3,119.8,117.0,112.7,96.3,29.5,29.4,20.8,13.7,12.6.

[0092] Example 5: Probe BDMOB for monitoring bisulfite and polarity in solution and its application in bioimaging

[0093] Detection of bisulfite and polarity in solution: Figure 4 The photophysical properties of the prepared probe BDMOB in different solvents are shown in turn, including the maximum absorbance λ abs,max , maximum emission λ em,max , Stokes shift, molar absorption coefficient and fluorescence quantum yield Φ. The above results show that the fluorescence quantum yield of BDMOB is greatly improved after the addition of bisulfite. Figure 5 This is the UV-visible absorption spectrum of the probe BDMOB in a mixed solution of dimethylformamide and chloroform at different ratios. As the ratio of the mixed solution changes, the absorption spectrum shows regular changes. Figure 6 The ratio of the absorbance of the probe BDMOB at 450 nm and 550 nm in mixed solutions of dimethylformamide and chloroform at different ratios increases with the proportion of dimethylformamide. The inset shows the color of the solution under 365 nm handheld UV light, showing a clear soluchromic effect. Figure 7 is the fluorescence emission spectrum of the probe BDMOB in the mixed solution of dimethylformamide and chloroform at different ratios (λ ex =450 nm), the fluorescence intensity of the green channel gradually increased with the increase of solution polarity. Figure 8 is the fluorescence emission spectrum of the probe BDMOB in the mixed solution of dimethylformamide and chloroform at different ratios (λex =550 nm), and the fluorescence intensity of the red channel gradually weakened with the increase of solution polarity. Figure 9 is the ratio of the fluorescence intensity of the probe BDMOB at 530 nm and 660 nm in a mixed solution of dimethylformamide and chloroform at different ratios (λ ex =450nm, 550nm), and its value increases with the increase of the proportion of dimethylformamide. Figure 10 The color coordinates of the probe BDMOB at different polarities intuitively show the changes in the luminescent color in the solution. Figure 11 is the fluorescence emission spectrum of the probe BDMOB after adding five equivalents of bisulfite (λ ex =480 nm), the addition of bisulfite will enhance the fluorescence intensity of the orange channel. Figure 12 is the fluorescence intensity of the probe BDMOB at 560 nm after adding bisulfite (λ ex =480 nm), and there was a good linear relationship between the amount of added bisulfite and the fluorescence intensity at 650 nm. Figure 13 is the photostability of the probe BDMOB itself and its response time after adding different equivalents of bisulfite (λ ex =480 nm), after the addition of bisulfite, the fluorescence intensity gradually increased and stabilized after 30 minutes. Figure 14 is the fluorescence intensity of the probe BDMOB itself at 560 nm at different pH values ​​and its fluorescence intensity after adding ten equivalents of bisulfite (λ ex =480 nm), the probe BDMOB can effectively monitor the presence of bisulfite in the pH range of 5-11.

[0094] Bisulfite and Polarity Detection Bioimaging Applications: Figure 15 The figure shows the cytotoxicity of the probe BDMOB. As can be seen from the figure, HeLa cells incubated with different concentrations of BDMOB (0μM, 2μM, 5μM, 10μM, 20μM, and 40μM) have high cell survival rates. This indicates that the probe BDMOB has low cytotoxicity and good biocompatibility. Figure 16 This is confocal imaging of HeLa cells after the probe BDMOB was co-incubated with different concentrations of bisulfite. As the concentration of exogenous bisulfite increased, the fluorescence intensity of the orange channel gradually increased, which was consistent with the spectral law. Figure 17 The bar graph shows the relative fluorescence intensity of cells after the probe BDMOB was co-incubated with different concentrations of bisulfite, which can intuitively show the relationship between the fluorescence intensity and the concentration of added bisulfite. Figure 18 This is the fluorescence intensity analysis of a single cell section after the probe BDMOB was co-incubated with different concentrations of bisulfite, which intuitively shows the fluorescence distribution within the cell.

[0095] Endogenous bisulfite cell imaging: Figure 19 This confocal image shows endogenous bisulfite in cells incubated with the probe BDMOB. Group A serves as a blank control. Groups B and C lack endogenous bisulfite production, resulting in no fluorescence in the orange channel. Group D, which does produce endogenous bisulfite, significantly increases the fluorescence intensity of the orange channel. However, after the addition of a thiosulfate transsulfurase inhibitor, which inhibits endogenous bisulfite production, the fluorescence disappears. This demonstrates that the probe BDMOB can sensitively and rapidly detect endogenous bisulfite production.

[0096] Imaging of ferroptosis cell models: Figure 20 Confocal imaging of ferroptosis cells co-incubated with the probe BDMOB. Glutathione peroxidase inhibitors can cause cell ferroptosis. Glutathione peroxidase inhibitors increase the fluorescence intensity of the green and orange channels, and the fluorescence intensity of the green and orange channels decreases in the presence of ferroptosis inhibitors. Figure 21 The fluorescence intensity of each channel of the probe BDMOB co-incubated with ferroptotic cells is shown, visually demonstrating the changes in fluorescence intensity in the green, orange, and red channels. This indicates that the probe BDMOB can be used to detect different degrees of iron removal reactions caused by glutathione peroxidase inhibitor stimulation of cells.

[0097] Imaging of inflammatory cell models: Figure 22 Confocal imaging of the probe BDMOB co-incubated with inflammatory cells. Lipopolysaccharide causes cell inflammation, its content increases, the fluorescence intensity of the green channel weakens, and the fluorescence intensity of the orange and red channels gradually increases. The addition of apocynin restores the fluorescence intensity of the green channel and weakens the fluorescence intensity of the orange and red channels. Figure 23 The fluorescence intensity of each channel of the probe BDMOB co-incubated with inflammatory cells is shown, which intuitively shows the changes in fluorescence intensity of the green channel, orange channel, and red channel. This shows that the probe BDMOB can respond to cellular inflammation caused by LPS intervention.

[0098] Imaging of normal and cancer cells: Figure 24 、 25 The following figure shows confocal fluorescence imaging and relative fluorescence intensity histograms of the BDMOB probe in different cell lines (normal human cervical epithelial cells and cervical cancer cells). After the addition of BDMOB, the fluorescence intensity in the green channel is strong and the fluorescence intensity in the red channel is weak in normal human cervical epithelial cells; in contrast, the fluorescence intensity in the green channel is weak and the fluorescence intensity in the red channel is strong in cervical cancer cells. This demonstrates the great potential of the BDMOB probe in distinguishing normal cells from cancer cells.

[0099] Imaging of ferroptosis nude mouse model: Figure 26 Figure 2 shows confocal imaging of the probe BDMOB in nude mice induced with ferroptosis and a histogram of the relative fluorescence intensity of each channel. Fluorescence in the green and orange channels increased in ferroptotic nude mice. After drug intervention to alleviate ferroptosis, fluorescence in the green and orange channels decreased to varying degrees, but did not return to initial levels, while the red channel increased. This demonstrates that the probe BDMOB can image and monitor fluctuations in polarity and bisulfite levels in ferroptotic nude mice in real time.

[0100] Imaging of nude mouse models of inflammation: Figure 28 、 29 The figure shows confocal imaging of the BDMOB probe in nude mice induced with inflammation, along with histograms of the relative fluorescence intensities of each channel. After lipopolysaccharide-induced inflammation in nude mice, the fluorescence intensity of the green channel decreased, while that of the orange and red channels increased. After the addition of apocynin (an anti-inflammatory drug), the fluorescence intensity of the green channel recovered, while that of the orange and red channels decreased. This demonstrates that the BDMOB probe is capable of real-time imaging and monitoring fluctuations in polarity and bisulfite levels in inflamed nude mice.

[0101] In summary, using a simple organic synthesis method, we have developed a fluorescent probe, BDMOB, capable of simultaneously detecting bisulfite and polarity, and successfully applied it in vivo. The prepared probe, BDMOB, exhibits high sensitivity, good biocompatibility, and strong anti-interference capabilities. Cell experiments demonstrated that the prepared probe has low cytotoxicity, can distinguish cancer cells from normal cells, and monitor changes in endogenous bisulfite levels within cells. Furthermore, using three distinct channels, it is possible to monitor the extent of inflammation and ferroptosis in cells and nude mice in real time, which is of great significance in the diagnosis and pharmacological intervention of abnormal conditions such as inflammation and ferroptosis.

Claims

1. A fluorescent probe BDMOB capable of simultaneously detecting bisulfite and polarity, having the structural formula:

2. A method for preparing a fluorescent probe BDMOB capable of simultaneously detecting bisulfite and polarity according to claim 1, comprising the following steps: 5-Methylsalicylaldehyde and 2-aminobenzenethiol were dissolved in N,N-dimethylformamide, sonicated, and heated under argon protection to react. After completion, ice water was added to form a precipitate, which was filtered, washed, collected, and dried to obtain an off-white solid A1. A1 and hexamethylenetetramine were ultrasonically dissolved in trifluoroacetic acid, heated under reflux overnight under argon protection, and sodium hydroxide solution was added to precipitate. The precipitate was collected by filtration and purified by silica gel column chromatography to obtain a yellow solid A2. A2, ethyl acetoacetate and piperidine were dissolved in anhydrous ethanol, heated under reflux overnight under argon protection, and filtered and washed to obtain a yellow solid A3; A3 was added dropwise to concentrated H2SO4 in an ice-water bath, 4-diethylamino salicylaldehyde was added and heated to reflux, the mixed solution was added dropwise to ice water, HClO4 was added to produce a precipitate, filtered, washed, and vacuum-dried to obtain a purple-black solid product BDMOB. The synthesis path is as follows:

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