Fluorescent probe as well as preparation method and application thereof

Abstract

The invention relates to the field of fluorescent probes, in particular to a fluorescent probe as well as a preparation method and application thereof. The fluorescent probe is based on a fluorescence resonance energy transfer (FRET) mechanism, has the excellent characteristics of quick response, sensitivity, high selectivity, no fluorescence crosstalk, near-infrared emission and the like, and can be used for quickly quantifying the content of SO2 in food and environmental water as well as sensitive and high-selectivity SO2 imaging of animal and plant cells and zebra fish living bodies; and SO2 and viscosity change in a living body can be monitored at the same time in a zero-crosstalk manner.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probes, specifically a fluorescent probe, its preparation method, and its application. Background Technology

[0002] Sulfur dioxide (SO2), an important environmentally and biologically active molecule, has a wide range of sources, including natural and anthropogenic processes such as the combustion of sulfur-containing fuels, emissions from chemical synthesis, volcanic activity, and the use of food additives (preservatives and bleaching agents). In biological systems, SO2 is often expressed as sulfite (SO32-). 2⁻ ) and bisulfite (HSO) 3⁻ It exists in the form of soluble derivatives such as , and enters the body circulation through dietary intake or air inhalation.

[0003] Studies have shown that exposure to excessive exogenous SO2 can cause damage to the respiratory mucosa and oxidative stress imbalance, leading to chronic inflammation, neurodegenerative diseases, and vascular endothelial dysfunction. It is noteworthy that SO2 in the body not only originates from external intake but can also be generated through endogenous pathways. For example, specific sulfur-containing metabolites (such as hydrogen sulfide and cysteine) are converted into SO2 through oxidation reactions under the action of enzymes such as thioredoxin reductase, and participate in cell signal transduction and redox balance regulation. However, abnormal accumulation of endogenous SO2 may lead to pathological effects such as DNA damage and protein function inhibition, thereby causing cardiovascular and cerebrovascular diseases such as hypertension, atherosclerosis, and myocardial damage; respiratory diseases such as asthma and chronic obstructive pulmonary disease; metabolic diseases such as diabetes and fatty liver; and neurological diseases such as Alzheimer's disease and Parkinson's disease. On the other hand, intracellular viscosity is a core parameter reflecting the physical state of the cytoplasm, profoundly affecting the metabolism, immunity, and disease progression of organisms. Abnormal intracellular viscosity is closely related to various diseases, including neurodegenerative diseases, cancer metastasis and differentiation, cardiovascular diseases, and metabolic diseases. Therefore, real-time monitoring of changes in cell viscosity is crucial for assessing disease progression and understanding disease mechanisms. Given the significant harm that abnormal concentrations of sulfur dioxide and cell viscosity pose to the human body, developing rapid, sensitive, and highly selective detection methods for the quantitative detection of SO2 residues in food and for monitoring abnormal changes in intracellular SO2 and viscosity has significant practical and social implications. Summary of the Invention

[0004] To achieve the above objectives, the present invention aims to provide a fluorescent probe, its preparation method, and its applications. This fluorescent probe is a fluorescent probe for targeted detection of SO2 and simultaneous zero-crosstalk monitoring of SO2 and viscosity changes in organisms. Utilizing the excellent characteristics of the fluorescent probe of the present invention, such as rapid molecular response, high sensitivity, high selectivity, zero crosstalk fluorescence, and near-infrared emission (around 760 nm), it can be applied to rapidly quantify SO2 content in food and environmental water bodies, and to provide sensitive and highly selective SO2 imaging in organisms such as animal and plant cells and live zebrafish, as well as simultaneous zero-crosstalk monitoring of SO2 and viscosity changes in organisms. This provides a theoretical basis and practical guidance for ensuring food safety and assisting in the diagnosis of human diseases, and can solve the problems in the background art. The present invention provides the following technical solution: A fluorescent probe has the following structure: , The abbreviation for this fluorescent probe is PPTC.

[0005] A method for preparing a fluorescent probe includes the following steps: Synthetic Intermediate 1: 5-Bromothiophene-2-carboxaldehyde (401.33 mg, 2.11 mmol) and 4-[4-(N-Boc)piperazin-1-yl]phenylboronic acid pinacol ester (778.32 mg, 2.01 mmol) were dissolved in anhydrous ethanol, followed by the addition of toluene (10 mL) and 2 M K₂CO₃ aqueous solution (5 mL) to obtain a mixture. Tetra(triphenylphosphine)palladium (120.85 mg, 0.11 mmol) was added to the mixture under a nitrogen atmosphere, and the mixture was stirred at 110 °C. After the reaction was completed by TLC monitoring, the reaction mixture was washed with saturated NaCl solution, extracted with ethyl acetate, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The resulting crude solid product was dissolved in 30 mL of dichloromethane and 15 mL of ethanol. The mixture was added to a solution of 1 mL trifluoroacetic acid, stirred overnight at room temperature, the solvent was removed under reduced pressure, the residue was poured into water and extracted with ethyl acetate, the organic phase was dried over anhydrous Na2SO4 and concentrated, and the residue was purified by silica gel column chromatography to give yellow intermediate 1. Synthetic intermediate 2: Under a nitrogen atmosphere, yellow intermediate 1 (274.8 mg, 1.01 mmol) and 1,2,3,3-tetramethyl-3H-indolium salt (208.9 mg, 1.20 mmol) were dissolved in 5 mL of anhydrous ethanol. Then, 2 drops of piperidine (86.2 mg, 1.02 mmol) were added to the solution. The reaction mixture was stirred at 65 °C for 6 hours to produce a precipitate. The precipitate was filtered under reduced pressure to separate the crude product. The crude product was then washed with cold ethanol and dried under vacuum to obtain dark red intermediate 2. Preparation of the finished product: 4-(1H-phenanthro[9,10-d]imidazol-2-yl)benzoic acid was dissolved in 5 mL of anhydrous dichloromethane. EDCI (201.2 mg, 1.04 mmol) and DMAP (199.8 mg, 1.64 mmol) were then added to the solution. The reaction mixture was stirred at room temperature for 30 minutes. Intermediate 2 (342.6 mg, 0.80 mmol) was then added dropwise. After the reaction was completed as monitored by TLC, the solvent was removed under normal pressure. The residue was washed three times with saturated brine, dried over anhydrous Na2SO4, and extracted with dichloromethane. The organic phase was then desolventized to remove the solvent, yielding the crude product. The crude product was purified by column chromatography to obtain a black solid probe PPTC. The fluorescent probe PPTC of this invention is obtained through a multi-step reaction involving Suzuki coupling, Knoevenagel condensation, and amidation. The synthetic route is shown below: 4-(1H-phenanthro[9,10-d]imidazol-2-yl)benzoic acid is derived from X. Zhang, W. Wu, Y. Wei, Y. Zhang, X. Nie, X. Sun, L. Lin, D. Yang, Y. Yan, A FRET-based multifunctional fluorescence probe for the simultaneous detection of sulfite and viscosity inliving cells, Bioorg Chem, 2024, 148, 107423.

[0006] As a further aspect of the present invention: the eluent used for silica gel column chromatography purification and crude product is a mixed solution of CH2Cl2 and MeOH with a volume ratio of 100:1.

[0007] The above-mentioned fluorescent probes are used in the monitoring of SO2 in food, environmental water, animal and plant cells, and live zebrafish, as well as in the simultaneous monitoring of SO2 and viscosity changes in animal cells and live zebrafish.

[0008] Compared with the prior art, the beneficial effects of the present invention are: The fluorescent probe of this invention is based on the fluorescence resonance energy transfer (FRET) mechanism and has excellent characteristics such as rapid response, high sensitivity, high selectivity, fluorescence crosstalk-free, and near-infrared emission. It can not only rapidly quantify SO2 content in food and environmental water, as well as perform sensitive and highly selective SO2 imaging in animal and plant cells and zebrafish, but also simultaneously monitor SO2 and viscosity changes in organisms with zero crosstalk. Attached Figure Description

[0009] Figure 1The probes PPTC and SO3 in Embodiment 1 of this invention 2- The spectrum of the reaction. From left to right: Figure 1 a, Figure 1 b and Figure 1 c.

[0010] Figure 2 The probe PPTC of Example 1 of this invention responds to SO3 under different pH conditions. 2- The results of the ratio fluorescence change.

[0011] Figure 3 The results of the selectivity and competitiveness test of the probe PPTC in Example 1 of this invention are shown. Figure 3 a is a ratio fluorescence bar graph of the probe PPTC after interaction with different analytes; Figure 3 b represents the color change of the probe PPTC under sunlight after it reacts with different analytes; Figure 3 c represents the interaction between the probe PPTC and SO3 in the presence of interfering substances. 2- Ratio fluorescence histogram after treatment; Figure 3 d represents the interaction between the probe PPTC and SO3 in the presence of interfering substances. 2- Color changes under sunlight after application.

[0012] Figure 4 The probes PPTC and SO3 in Embodiment 1 of this invention 2- The result of the ratio response. Figure 4 a represents the fluorescence titration spectrum; Figure 4 b is a linear relationship graph.

[0013] Figure 5 The viscosity response results of the probe PPTC in Example 1 of this invention are shown. Figure 5 a represents the fluorescence spectrum of the probe PPTC in response to viscosity; Figure 5 b represents the linear relationship between the fluorescence intensity of the probe PPTC and the response to glycerol concentration; Figure 5 c represents the visible light and fluorescence colors of the probe PPTC in glycerol at different concentrations.

[0014] Figure 6 This is a schematic diagram of the PPTC probe used in Example 1 of the present invention for detecting SO2 residues in food samples.

[0015] Figure 7 This is a schematic diagram of the PPTC probe used to construct a mobile phone sensing platform according to Embodiment 1 of the present invention.

[0016] Figure 8 The results show the cytotoxicity of the probe PPTC in Example 1 of this invention.

[0017] Figure 9This is a diagram showing the application results of the probe PPTC from Example 1 of the present invention in onion epidermal cell imaging.

[0018] Figure 10 The image shows the results of the application of the probe PPTC in HeLa cells for imaging of exogenous and endogenous SO2, as described in Example 1 of this invention. Figure 10 Figure a shows the results of the exogenous SO2 imaging application; Figure 10 Figure b shows the results of endogenous SO2 imaging application.

[0019] Figure 11 This is an imaging image of the changes in endogenous SO2 and viscosity in HeLa cells and zebrafish using the probe PPTC from Example 1 of the present invention. Figure 11 a is an imaging diagram showing changes in endogenous SO2 and viscosity in HeLa cells; Figure 11 b is an image showing changes in endogenous SO2 and viscosity in zebrafish.

[0020] Figure 12 This is a diagram showing the effect of the probe PPTC in rice germ according to Example 1 of the present invention. Figure 12 a is an imaging diagram showing the changes in SO2 and viscosity under exogenous SO2; Figure 12 b is an image showing the changes in SO2 and viscosity under high salt stress. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] This invention provides a near-infrared zero-crosstalk dual-response ratio fluorescent probe based on the fluorescence resonance energy transfer (FRET) mechanism, and its applications in various scenarios such as SO2 content detection in food and environmental water, monitoring and imaging of SO2 concentration changes in animal and plant cells and zebrafish, and simultaneous monitoring and imaging of SO2 and viscosity changes in animal cells and zebrafish. The fluorescent probe is constructed using 2-phenyl-phenanthimidazole as the energy donor and fluorophore, and piperazine-phenyl-thiophene-indole salt as the energy acceptor, to achieve FRET fluorescence emission of the probe. It has the following characteristics: (1) Due to its own FRET mechanism, the fluorescent probe molecule produces fluorescence emission at close to 800 nm (lem=760 nm). Under the action of sulfite, SO3 2-The probe undergoes a nucleophilic addition reaction with the carbon-carbon double bond of the thiophene ethylene moiety. At the same time, the fluorescence at 760 nm is quenched and a strong fluorescence emission at 510 nm is generated. The ratio-type dual-wavelength fluorescence signal enables the fluorescent probe to have a self-calibration function, thereby eliminating the interference of background fluorescence in the sample and the test environment, and making the fluorescent probe have higher test sensitivity and anti-interference characteristics; (2) The two fluorescence emission channels of the fluorescent probe are completely separated, and the fluorescence emission wavelength difference Dl = 283 nm, which is significantly different from the phenomenon of cross-overlapping of the two emission peaks of the currently reported FRET fluorescent probe, realizing simultaneous zero crosstalk detection of SO2 and viscosity; (3) The probe has high specificity and anti-interference ability for SO2 detection, high sensitivity (detection limit 61.9 nM), fast response (4 min), low cytotoxicity and excellent luminescence characteristics and photostability in a physiological environment of pH 7.4; (4) In the viscosity response mode, as the viscosity of the system increases, the TICT effect in the fluorescent probe molecule is suppressed, and the fluorescence at 760 nm is reduced. Near-infrared emission fluorescence intensity shows a significant linear correlation with viscosity change; (5) This fluorescent probe can realize fluorescence detection applications in multiple scenarios: quantitative detection of SO2 content in food and environmental water, SO2 imaging in animal and plant cells and zebrafish, and simultaneous monitoring and imaging of SO2 and viscosity changes in animal and plant cells, zebrafish, and plant microenvironment.

[0023] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0024] I. Example 1 Synthetic Intermediate 1: 5-Bromothiophene-2-carboxaldehyde (401.33 mg, 2.11 mmol) and 4-[4-(N-Boc)piperazin-1-yl]phenylboronic acid pinacol ester (778.32 mg, 2.01 mmol) were dissolved in anhydrous ethanol, followed by the addition of toluene (10 mL) and 2 M K₂CO₃ aqueous solution (5 mL) to obtain a mixture. Tetra(triphenylphosphine)palladium (120.85 mg, 0.11 mmol) was added to the mixture under a nitrogen atmosphere, and the mixture was stirred at 110 °C. After the reaction was completed by TLC monitoring, the reaction mixture was washed with saturated NaCl solution, extracted with ethyl acetate, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The resulting crude solid product was dissolved in 30 mL of dichloromethane and 15 mL of ethanol. The mixture was added to a solution of 1 mL trifluoroacetic acid and stirred overnight at room temperature. The solvent was removed under reduced pressure, and the residue was poured into water and extracted with ethyl acetate. The organic phase was dried over anhydrous Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (eluent was a mixed solution of CH2Cl2 and MeOH in a volume ratio of 100:1) to give yellow intermediate 1 (193.55 mg, 71.13%). 1H NMR (500 MHz, DMSO-d6) δ 9.60 (s, 1H), 7.74−7.73 (s, 1H), 7.68−7.65(m, 2H), 7.41−7.40 (m, 1H), 6.84−6.82 (m, 2H), 3.34−3.26 (m, 4H), 3.08−3.01(m, 4H), 1.97−1.94 (m, 1H). 13 C NMR (125 MHz, DMSO-d6) δ 182.41, 152.37, 150.31, 142.58, 137.31, 127.83, 127.61, 124.55, 113.33, 49.14, 45.78. HRMS(ESI): Theoretical C 15 H 16 N2OS [M] + , 272.0983, Test value: 272.0984; Synthetic intermediate 2: Under a nitrogen atmosphere, yellow intermediate 1 (274.8 mg, 1.01 mmol) and 1,2,3,3-tetramethyl-3H-indolium salt (208.9 mg, 1.20 mmol) were dissolved in 5 mL of anhydrous ethanol. Then, 2 drops of piperidine (86.2 mg, 1.02 mmol) were added. The reaction mixture was stirred at 65 °C for 6 hours, and a precipitate was formed. The precipitate was filtered under reduced pressure to separate the crude product. The crude product was then washed with cold ethanol and dried under vacuum to obtain dark red intermediate 2 (329.7 mg, 76.2%). 1 H NMR(500 MHz, DMSO-d6) δ 7.89 (m, 1H), 7.64−7.48 (m, 7H), 7.40−7.35 (m, 2H), 6.86−6.80 (m, 2H), 4.23 (s, 3H), 3.35−3.25 (m, 8H), 1.97−1.94 (m, 1H), 1.70 (s, 6H). 13 C NMR (125 MHz, CDCl3) δ 168.48, 150.03, 147.36, 145.23, 141.43, 141.23, 140.30, 131.12, 129.30, 127.95, 127.89, 125.70, 125.52, 125.07, 117.66, 113.66, 112.60, 48.67, 44.63, 42.49, 36.71, 27.55. HRMS(ESI): Theoretical C27 H30N3S[M] + Test value: 428.2155; Preparation of the finished product: 4-(1H-phenanthro[9,10-d]imidazol-2-yl)benzoic acid (from X. Zhang, W. Wu, Y. Wei, Y. Zhang, X. Nie, X. Sun, L. Lin, D. Yang, Y. Yan, A FRET-based multifunctional fluorescence probe for the simultaneous detection of sulfite and viscosity in living cells, Bioorg Chem, 2024, 148, 107423) was dissolved in 5 mL of anhydrous dichloromethane. Then, EDCI (201.2 mg, 1.04 mmol) and DMAP (199.8 mg, 1.64 mmol) were added to the solution. The reaction mixture was stirred at room temperature for 30 minutes. Then, intermediate 2 (342.6 mg, 0.80 mmol) was added dropwise. After the reaction was completed by TLC monitoring, the solvent was removed under normal pressure. The residue was washed three times with saturated brine, dried with anhydrous Na2SO4, and extracted with dichloromethane. The organic phase was then desolventized to remove the solvent, yielding the crude product. The crude product was purified by column chromatography (using a mixed solution of CH2Cl2 and MeOH at a volume ratio of 100:1) to obtain a black probe PPTC solid (131.3 mg, 33.7%). 1 H NMR (500MHz, DMSO-d6) δ13.58 (s, 1H), 8.91−8.89 (m, 1H), 8.87−8.85 (m, 1H), 8.64−8.56 (m, 4H), 8.42−8.40 (m, 1Hs), 8.21−8.20 (m, 1H), 8.13−8.12 (m, 1hs), 7.83−7.54 (m, 15H), 7.11−7.09 (m, 2H), 6.98−6.97 (m, 2H), 4.03 (s, 3H), 3.18 (m, 8H), 1.77 (s, 6H). 13C NMR (126 MHz, DMSO-d6) δ 180.60, 169.23, 155.72, 152.02, 148.78, 143.57, 142.40, 139.65, 136.53, 128.34, 127.95, 126.55, 123.23, 115.68, 114.91, 109.45, 107.43, 51.98, 34.19, 26.13. HRMS (ESI): Theoretical C 49 H 42 N5OS[M] + :748.3105, Test value: 748.3108.

[0025] II. Optical performance testing of the PPTC solid probe obtained in Example 1: (1) Preparation of various mother liquors: Accurately weigh a certain amount of probe PPTC and dissolve it in methanol to obtain a 1.0 mM probe mother liquor; accurately weigh Na2SO3 and other interfering substances (such as NaF, NaCl, NaBr, NaI, Na2SO4, Na2CO3, NaNO3, NaNO2, CH3COONa, NaSCN, MgCl2, FeCl2, FeCl3, CuCl2, CaCl2,ONOO - Dissolving substances such as Cys, Hcy, H2O2, Na2S, NaClO, Na2S2O3, Ala, Gly, Leu, Met, Thr, Pro, Tyr, Asp, Phe, and Trp in ultrapure water yields a 10 mM mother liquor (except for GSH, which is 100 mM).

[0026] (2) Selectively add 30 μL of each of the above-mentioned mother solutions to a cuvette, add methanol-phosphate buffer (methanol and phosphate in volume ratio of 1:9) to a final volume of 3.0 mL, and use it to test the optical properties of the probe PPTC.

[0027] ① UV-Vis spectroscopy and fluorescence response kinetics: PPTC probe added to SO3 2- Before and after (probe stock solution 30 μL, SO3) 2- The UV-Vis spectrum and fluorescence response kinetics spectrum of the mother liquor (0 or 30 μL, total solution volume 3.0 mL) are shown in the figure. Figure 1 ;from Figure 1 As can be seen from a, the probe itself exhibits two relatively strong absorption peaks in the 400-700 nm range, with SO3... 2- With the addition of [a substance], the absorption peak with the largest absorption wavelength decreased significantly, accompanied by a significant increase in the absorption peak in the 300-400 nm range, and the solution color faded from purple to colorless; on the other hand, from [a different perspective]... Figure 1 As can be seen from b, under 430 nm excitation, with SO3 2- With the addition of [the substance], the red fluorescence emission intensity of the probe PPTC at 760 nm rapidly decayed to a low constant value, from [the point where] Figure 1 As can be seen from c, the intensity of the green fluorescence emission peak at 510 nm rises rapidly and reaches a plateau value after 4 minutes when the interaction between the two reaches saturation. Moreover, the fluorescence intensity at 510 nm is 12 times that at 760 nm.

[0028] ② Probes PPTC and SO3 under different pH conditions 2- fluorescence response The probe PPTC on SO3 under different pH conditions 2- The fluorescence response is shown in Figure 2 ,from Figure 2 It can be seen that without the addition of SO3 2- Previously, the fluorescence ratio (I510 / I760) of the probe PPTC itself showed almost no fluctuation and remained at a low value within the pH range of 4.0-10.0; after adding SO3... 2- Subsequently, within the pH range of 6.0-8.0, SO3 2- After interaction with PPTC, the I510 / I760 ratio increased significantly and reached a plateau value in a physiological pH environment (pH=7.4). Figure 2 The results show that the PPTC probe can effectively identify SO3 under physiological environmental conditions. 2- It also has the potential to track changes in SO2 concentration in living cells.

[0029] ③ Ion selectivity and ion competition (interference resistance) Take probes PPTC and SO3 respectively 2- (or 30 μL of each of the 33 interfering substances) or take probes PPTC and SO3. 2- 30 μL each of the stock solutions of 33 interfering substances were added to a fluorescence cuvette, and methanol-phosphate buffer (volume ratio: 1:9) was added to bring the volume to 3.0 mL. Under excitation at 430 nm, the fluorescence ratios of the probe PPTC at 510 nm and 760 nm were obtained to test the ion selectivity and ion competition of the probe PPTC. The results are shown in [Figure number missing]. Figure 3 .from Figure 3 As shown in a, the probe PPTC only interacts with SO3. 2- It exhibits a specific response, but hardly responds to other ions; from Figure 3 As can be seen from c, only those containing SO3 2- The solution in the reagent bottle decolorized, which also verified the interaction between the probe PPTC and SO3. 2- High specificity of the response. From Figure 3 b shows that in SO32- When coexisting with 33 other interfering substances, the probe PPTC and SO3 2- The interaction between the probe PPTC and SO3 is completely unaffected by interfering substances, indicating that the interaction between the probe PPTC and SO3 is... 2- Its response exhibits high anti-interference capabilities, from Figure 3 As seen in d, the color change observed in the competitive experiment further confirms that the probe PPTC responds to SO3. 2- It has strong anti-interference capabilities.

[0030] ④ Fluorescent titration Take 30 μL of the probe PPTC stock solution and different volumes of SO3 respectively. 2- The stock solution was placed in a fluorescent cuvette, and methanol-phosphate buffer (volume ratio: 1:9) was added to bring the volume to 3.0 mL, so that the SO3 concentration in the test system was within acceptable limits. 2- The concentration was 0-10 eq of the probe PPTC, and the results are shown in [the table]. Figure 4 .from Figure 4 As shown in Figure a, under excitation at 430 nm, the fluorescence at 760 nm gradually weakens, while the fluorescence at 510 nm amplifies sharply, and the fluorescence color changes from red to blue-green. Simultaneously, after adding different volumes of SO32-, the fluorescence intensity of the probe PPTC at 510 nm and 760 nm was recorded, and the ratio between the two was obtained. From... Figure 4 As can be seen from b, SO3 2- Within the range of 0-70 μM, the fluorescence ratio of the probe PPTC showed a good linear relationship with the concentration of SO32-, with a linear regression equation of y = 0.207x - 0.1055. The probe PPTC exhibited a strong linear relationship with the concentration of SO32-. 2- The detection limit was 61.88 nM (LOD=3α / k), indicating that the probe PPTC is effective against SO3. 2- The detection has high sensitivity.

[0031] ⑤ Viscosity response 30 μL of the PPTC probe stock solution and glycerol solutions of different volume fractions were added to a fluorescent cuvette to make a total solution volume of 3.0 mL. The results are shown in the figure. Figure 5 .from Figure 5 As can be seen from a, under 430 nm excitation, the fluorescence at 760 nm gradually increases. Figure 5 As can be seen from b, the fluorescence intensity of the probe PPTC has a good linear relationship with the viscosity of glycerol; at the same time, from Figure 5 As shown in Figure c, the response of the probe PPTC to glycerol exhibits a clear visual change. With the increase of glycerol concentration in the system, the color of the probe PPTC under visible and ultraviolet light changes from purple to light purple to purplish-red and from blue to dark red to bright red. Figure 5The results show that the PPTC probe has a good response to viscosity and can monitor slight changes in the viscosity of the system.

[0032] III. Multi-scenario Applications of Probes (1) Quantitative detection of SO2 residues in food samples The actual samples to be tested, including dried plums, raisins, dried strawberries, dried white fungus, dried shiitake mushrooms, and dried black fungus, were pulverized using a tissue homogenizer. 5.0 g of the sample powder was weighed, added to 5 mL of ultrapure water, and sonicated for 60 min. The mixture was then filtered, and the resulting filtrate was used as the sample test solution. 30 μL each of the food sample solution and the probe PPTC were added to a cuvette, and methanol-phosphate buffer (methanol to phosphate, volume ratio 1:9) was added to a final volume of 3.0 mL. The ratio fluorescence value of the probe solution was measured under excitation at a wavelength of 430 nm. Figure 4 The linear regression equation for b yielded the residual SO2 value in the sample; different concentrations of SO3 were added to the sample test solution. 2- The solution was used to test the SO2 recovery rate in food samples according to the aforementioned procedure. The food samples and procedures are described in [link to relevant documentation]. Figure 6 The test results are shown in Table 1.

[0033] Table 1. Results of SO2 content testing using probes in actual food samples and spiked samples.

[0034] The results in Table 1 show that the PPTC probe can sensitively detect the residual SO2 content in actual food and pharmaceutical products, and has a high recovery rate (95.33-103.33%) for the detection of SO2 content in spiked food samples, indicating that the PPTC probe can provide a feasible and reliable method for the detection of SO2 in complex matrices.

[0035] (2) Construction of portable mobile phone sensing platform and detection of SO2 in environmental water bodies The probe PPTC solution (0.5 mL, 1 mM, dissolved in DMSO) and agarose (500 mg) were dispersed in ultrapure water (20 mL) and stirred at 85 °C for 30 min. The mixture was then poured into a mold and allowed to solidify at room temperature for 1 h to form a PPTC-agarose hydrogel. The hydrogel sensor was immersed in different concentrations of Na₂SO₃ solution (0–200 µM) for 30 min, rinsed with deionized water, and the sample images were captured under natural light. The RGB values ​​of the images were extracted using a smartphone platform, and the blue-green intensity ratio (B / G) was calculated as the detection signal. A standard working curve was plotted with the B / G ratio as the ordinate and the Na₂SO₃ concentration as the abscissa. The same procedure was used to detect environmental water samples, and the sulfite content was quantified using the standard curve. All experiments were performed in triplicate. The standard working curve and testing procedure are described in [link to standard curve description]. Figure 7 The measurement results are shown in Table 2.

[0036] Table 2. Detection results of SO2 content in environmental water bodies and spiked samples using a portable mobile phone sensing platform.

[0037] As shown in Figure 7 and Table 2, the composite PPTC hydrogel sensor changes from purple to colorless and transparent after exposure to SO2 solution, demonstrating excellent visual colorimetric detection capability. Furthermore, the B / G value shows a strong linear correlation with SO2 concentration (0-200 µM) (y = −0.0023x + 1.5418, R2 = 0.9944). Simultaneously, the constructed portable sensing platform was used to detect environmental water samples from different sources, including tap water, swimming pool water, and lake water. Figure 7 The results in Table 2 indicate that the composite PPTC hydrogel sensor can serve as a portable, highly sensitive device for on-site, real-time, and visualized quantitative detection of SO2 in environmental water samples.

[0038] (3) Cytotoxicity of probe PPTC The results were evaluated using the MTT assay. Figure 8 .from Figure 8 As can be seen, even after HeLa cells were incubated in PPTC at a maximum concentration of 50 µM for 24 hours, the cell survival rate was still over 80%. Figure 8 The results indicate that PPTC has good biocompatibility and low cytotoxicity, making it suitable and safe for use in imaging experiments of live cells and zebrafish.

[0039] (4) Imaging of exogenous SO2 in onion plant cells Onion epidermal cells were divided into five groups. Group 1 served as a control group, and cells were directly subjected to fluorescence imaging after washing with ultrapure water. Group 2 was immersed in 1 mL of ultrapure water containing 10 μM PPTC for 60 minutes, then washed twice with ultrapure water to remove excess PPTC before imaging. For groups 3, 4, and 5, onion epidermal cells were incubated in 1 mL of ultrapure water containing 10 μM PPTC for 60 minutes, washed twice with ultrapure water, and then treated with SO32– containing 25 μM, 50 μM, and 100 μM, respectively. After an additional 30 minutes of incubation, the cells were again washed twice with ultrapure water to remove excess SO32–. 2- Fluorescence imaging was then performed, and the results are shown in [the table]. Figure 9 .from Figure 9 As can be seen, both the green and red channels in the blank group show fluorescence, while the second group shows the long-wavelength fluorescence of the probe PPTC itself at 760 nm; with the addition of exogenous SO3 2- With increasing concentration, the fluorescence signal in the red channel (760 nm) of the PPTC probe gradually weakens and is quenched, while the fluorescence signal in the green channel (510 nm) gradually increases. Figure 9 The results showed that the probe PPTC could successfully enter the plant cell and had the ability to recognize SO2 in the plant cell.

[0040] (5) Imaging of endogenous and exogenous SO2 in animal cells (HeLa) Exogenous SO2 Imaging Assay: Cells were divided into four groups and treated as follows: Blank Group: Cells were washed with PBS and then added to fresh culture medium before imaging. Groups 2, 3, and 4: Cells were washed with PBS and then added to fresh culture medium and 10 μM PPTC, and incubated at 37℃ and 5% CO2 for 30 minutes. After removing the culture medium, 50, 100, and 200 μM SO3 were added, respectively. 2- Continue incubation for 30 minutes, wash cells with PBS, and then image.

[0041] Endogenous SO2 imaging experiment: The blank group was treated the same as the exogenous imaging group. Groups 2, 3, and 4: After washing cells with PBS, fresh culture medium was added, followed by the addition of 50 μM Na2S2O3, 50 μM GSH, and 50 μM each of Na2S2O3 and GSH, respectively. After incubation at 37℃ and 5% CO2 for 6 hours, 10 μM probe PPTC was added, followed by an additional 30 minutes of incubation before imaging. The results of the exogenous and endogenous SO2 imaging experiments are shown below. Figure 10 .

[0042] from Figure 10 As shown in Figure a, the blank group cells showed no fluorescence signal, while the cells showed fluorescence with the addition of exogenous SO3. 2-As the solution concentration increases, the fluorescence signal in the red channel of the cells gradually weakens and is quenched, while the fluorescence signal in the green channel gradually strengthens and illuminates; from Figure 10 As shown in b, the blank group showed no fluorescence signal, and the second and third groups, with Na2S2O3 and GSH added alone, only showed the red channel fluorescence signal of the probe PPTC itself at 760 nm; when Na2S2O3 and GSH were added simultaneously, the red channel signal of the cells in the third group disappeared, and the green channel fluorescence signal (510 nm) was lit. Figure 10 The results showed that the probe could efficiently monitor endogenous SO2 produced in cells induced by Na2S2O3 and GSH.

[0043] (6) Imaging of endogenous SO2 and viscosity changes in HeLa cells and live zebrafish HeLa cells and zebrafish were divided into four groups for the experiment. The first group was a blank group treated with ultrapure water; the second group was a control group with the probe added alone; and the third and fourth groups were experimental groups with different concentrations of lipopolysaccharide (LPS). Results are shown below. Figure 11 .from Figure 11 It can be seen that neither HeLa cells nor zebrafish in the blank group showed any fluorescence signal, while only the long-wavelength red channel signal of the probe PPTC itself was observed in the control group. However, both HeLa cells and zebrafish developed acute inflammation under the action of LPS. On the one hand, the viscosity of the cells and zebrafish increased sharply, resulting in the observation of gradually increasing red channel fluorescence signals (760 nm) in both. On the other hand, SO2 concentration was overexpressed in the cells and zebrafish, resulting in the observation of gradually increasing fluorescence signals (510 nm) in the green channel of both. Figure 11 The results show that the PPTC probe can be used as an effective monitoring tool to monitor changes in endogenous SO2 concentration and viscosity in cells and zebrafish microenvironments under zero crosstalk conditions, and has potential application value in predicting related diseases.

[0044] (7) Imaging of endogenous SO2 in rice germ Rice seeds were disinfected with 75% alcohol, rinsed with ultrapure water, and then soaked for 4 hours. Rice germ samples selected for SO2 imaging were divided into four groups: blank group, probe culture group, Na2S2O3 culture group, and NaCl culture group. Rice germ samples were treated with different concentrations of Na2S2O3 solution (100, 200 μM) for 24 hours, incubated with 10 μM PPTC probe for 60 min, and then imaged under an inverted fluorescence microscope. Similarly, different concentrations of NaCl solution (100, 200 μM) were used to simulate high salt stress; the results are shown in [Figure number missing]. Figure 12 .from Figure 12 As can be seen from a, with SO3 2–With increasing solution concentration, the fluorescence intensity of the red channel (760 nm) of the PPTC near-infrared emission probe gradually decreased, while the fluorescence intensity of the green channel (510 nm) gradually increased, indicating that the probe can sensitively detect changes in exogenous SO2 content in rice germ. Due to high salt stress, excessive water loss in rice germ triggers a series of inflammations, leading to increased germ viscosity and excessive SO2 expression. Figure 12 As shown in Figure b, the fluorescence intensity of the red channel gradually increases with the increase of sodium chloride concentration, indicating that the probe PPTC can simultaneously and sensitively detect changes in SO2 overexpression and viscosity in rice germ under high salt stress. This probe provides a new research method for studying plant growth under stress, and has significant potential application value, especially in studying the synergistic mechanism of changes in plant cell microenvironment and the regulation of gaseous signaling molecules under salt stress.

[0045] It should be noted that, in this invention, unless otherwise explicitly specified and limited, the terms "fixed," "set," etc., should be interpreted broadly, unless otherwise explicitly limited. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0046] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A fluorescent probe, characterized in that, Its structure is as follows: 。 2. A method for preparing a fluorescent probe, characterized in that, Includes the following steps: Synthetic Intermediate 1: 5-Bromothiophene-2-carboxaldehyde (401.33 mg, 2.11 mmol) and 4-[4-(N-Boc)piperazin-1-yl]phenylboronic acid pinacol ester (778.32 mg, 2.01 mmol) were dissolved in anhydrous ethanol, followed by the addition of toluene (10 mL) and 2 M K₂CO₃ aqueous solution (5 mL) to obtain a mixture. Tetra(triphenylphosphine)palladium (120.85 mg, 0.11 mmol) was added to the mixture under a nitrogen atmosphere, and the mixture was stirred at 110 °C. After the reaction was completed by TLC monitoring, the reaction mixture was washed with saturated NaCl solution, extracted with ethyl acetate, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The resulting crude solid product was dissolved in 30 mL of dichloromethane and 15 mL of ethanol. The mixture was added to a solution of 1 mL trifluoroacetic acid, stirred overnight at room temperature, the solvent was removed under reduced pressure, the residue was poured into water and extracted with ethyl acetate, the organic phase was dried over anhydrous Na2SO4 and concentrated, and the residue was purified by silica gel column chromatography to give yellow intermediate 1. Synthetic intermediate 2: Under a nitrogen atmosphere, yellow intermediate 1 (274.8 mg, 1.01 mmol) and 1,2,3,3-tetramethyl-3H-indolium salt (208.9 mg, 1.20 mmol) were dissolved in 5 mL of anhydrous ethanol. Then, 2 drops of piperidine (86.2 mg, 1.02 mmol) were added to the solution. The reaction mixture was stirred at 65 °C for 6 hours to produce a precipitate. The precipitate was filtered under reduced pressure to separate the crude product. The crude product was then washed with cold ethanol and dried under vacuum to obtain dark red intermediate 2. Preparation of the finished product: 4-(1H-phenanthro[9,10-d]imidazol-2-yl)benzoic acid was dissolved in 5 mL of anhydrous dichloromethane, and then EDCI (201.2 mg, 1.04 mmol) and DMAP (199.8 mg, 1.64 mmol) were added to the solution. The reaction mixture was stirred at room temperature for 30 minutes, and then intermediate 2 (342.6 mg, 0.80 mmol) was added dropwise. After the reaction was completed by TLC monitoring, the solvent was removed under normal pressure. The residue was washed three times with saturated brine, dried over anhydrous Na2SO4, and extracted with dichloromethane. The organic phase was then de-solventized under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to obtain a black probe PPTC solid.

3. The method for preparing the fluorescent probe according to claim 2, characterized in that, The eluent used for both silica gel column chromatography purification and crude product was a mixed solution of CH2Cl2 and MeOH in a volume ratio of 100:

1.

4. The application of a fluorescent probe prepared by any one of claims 2-3 in detecting SO2 content in food.

5. The application of a fluorescent probe prepared by any one of claims 2-3 in detecting SO2 content in environmental water.

6. The application of a fluorescent probe prepared by any one of claims 2-3 in detecting SO2 content in plant cells.

7. The application of a fluorescent probe prepared by any one of the methods described in claims 2-3 in the simultaneous monitoring of SO2 and viscosity changes in animal cells.

8. The application of a fluorescent probe prepared by any one of claims 2-3 in the simultaneous monitoring of SO2 and viscosity changes in live zebrafish.

9. The application of a fluorescent probe prepared by any one of claims 2-3 in detecting SO2 content in animal cells.

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