Fluorescent probe based on phenothiazine structure and preparation method thereof

Abstract

The invention belongs to the field of fluorescent probe detection, and particularly relates to a phenothiazine structure-based fluorescent probe and a preparation method thereof, the fluorescent probe takes 2, 4-dinitrophenyl as an H2S recognition unit and a fluorescence quenching agent, shows excellent H2S detection capability including large Stokes shift, remarkable sensitivity and good selectivity, and can be used for detecting H2S. The fluorescent probe can be effectively used for fluorescence imaging of endogenous H2S in HeLa cells and detection of H2S released by food spoilage. According to the preparation method, the novel fluorescent probe for detecting hydrogen sulfide is constructed through simple and efficient synthesis steps, H2S can be sensitively detected in actual food samples and living cells, and the novel fluorescent probe has great market application value.

Description

Technical Field

[0001] The invention belongs to the field of fluorescent probe detection, and particularly relates to a fluorescent probe based on a phenothiazine structure and a preparation method thereof. Background Art

[0002] Hydrogen sulfide is a toxic gas with a rotten egg smell and is the third gas signal after carbon monoxide (CO) and nitric oxide (NO). H2S can affect the signal transduction process in cells, thereby controlling the cell function, so it has important research significance in the fields of biology and medicine. In mammals, endogenous H2S is mainly produced by some enzymes catalyzing sulfur-containing substrates, such as cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), 3-mercaptopyruvate sulfotransferase (3-MST) and cysteine ​​aminotransferase (CAT). Many studies have shown that H2S may be involved in various physiological processes in the body, especially vascular regulation, anti-oxidation, anti-apoptosis and anti-inflammation. In other words, abnormal H2S levels can induce a variety of diseases, including diabetes, cirrhosis, Alzheimer's disease and Down syndrome. Because the concentration of H2S in organisms is closely related to our health, a reliable method for detecting H2S concentration is urgently needed to be studied and developed by biological and chemical technologies.

[0003] To date, researchers have tried traditional methods for detecting hydrogen sulfide, including colorimetry, potentiometrics, polarography, and gas chromatography. However, these methods are complex, low in sensitivity, and low in accuracy, and are not suitable for detecting hydrogen sulfide in biological systems. Fluorescent probes have the advantages of being non-invasive, simple to operate, high in precision, and strong in real-time imaging capabilities. They are favored by researchers and are recommended for monitoring H2S in complex dynamic physiological environments. To date, the design strategies of fluorescent probes are mainly based on specific reaction mechanisms, such as nucleophilic reactions, reduction reactions of nitro and azide groups, Michael addition reactions, and thiolysis reactions of dinitrophenyl ethers. Despite the excellent results achieved for the design of fluorescent probes, there are still some limitations that need to be addressed. Many current probes have long response times, and H2S produced by tissues is diffusive and variable, making it difficult to detect. In addition, some probes have high detection limits, which limits their applicability in tissues that produce lower concentrations of H2S. Summary of the invention

[0004] The present invention provides a fluorescent probe based on a phenothiazine structure and a preparation method thereof, so as to solve the problems of long response time, high detection difficulty and poor practicality of the probe in the prior art.

[0005] To achieve the above object, the present invention proposes the following technical solutions:

[0006] A fluorescent probe based on a phenothiazine structure, wherein the molecular structure of the fluorescent probe is:

[0007]

[0008] A method for preparing the fluorescent probe based on the phenothiazine structure according to claim 1, comprising the following steps:

[0009] Step 1, obtaining a first compound based on raw materials;

[0010] Step 2, obtaining a second compound based on the first compound;

[0011] Step 3, obtaining a third compound based on the second compound;

[0012] Step 4, dissolving compound 3, 2-aminothiophenol and sodium metabisulfite in an organic solvent to obtain a mixture, heating the mixture and cooling it to room temperature, adding distilled water, extracting and combining the organic layers, washing, drying and filtering, removing the solvent under reduced pressure, and purifying the residue by silica gel column chromatography to obtain a fourth compound as a yellow solid;

[0013] Step 5, add the fourth compound, 2,4-dinitrofluorobenzene, an organic base or an inorganic base and a dry organic solvent into a round-bottom flask, stir at room temperature overnight, after the reaction is completed, pour the solution into ice water, extract and combine the organic layers, wash, dry and filter, remove the solvent under reduced pressure, and purify the residue by silica gel column chromatography to obtain a fluorescent probe.

[0014] The molecular structure of the raw material is:

[0015]

[0016] The molecular structural formula of the first compound is:

[0017]

[0018] The molecular structural formula of the second compound is:

[0019]

[0020] The molecular structural formula of the third compound is:

[0021]

[0022] The molecular structural formula of the fourth compound is:

[0023]

[0024] Preferably, in step 4, the mixture is heated at 80-160° C. for 2-24 hours. Preferably, in step 4, an organic solvent immiscible with water is used to extract and combine the organic layers. Preferably, in step 4, saturated brine is used for washing.

[0025] Preferably, in step 4, drying is performed using a desiccant.

[0026] Preferably, in step 5, an organic solvent that is immiscible with water is used to extract and combine the organic layers.

[0027] Preferably, in step 5, saturated brine is used for washing.

[0028] Preferably, in step 5, drying is performed using a desiccant.

[0029] The present invention is beneficial in that:

[0030] The present invention proposes a fluorescent probe based on a phenothiazine structure. The fluorescent probe uses 2,4-dinitrobenzene molecules as H2S recognition units and fluorescence quenchers, and exhibits excellent detection capabilities for H2S, including a large Stokes shift, significant sensitivity and good selectivity. The probe can be effectively used for fluorescence imaging of endogenous H2S in HeLa cells and detecting H2S released by food spoilage.

[0031] The present invention also proposes a method for preparing a fluorescent probe, and constructs a new fluorescent probe for detecting hydrogen sulfide with simple and efficient synthesis steps. The probe can sensitively detect H2S in actual food samples and living cells, and has great market application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0033] Figure 1 Schematic diagram of the detection of H2S by a fluorescent probe X based on phenothiazine structure;

[0034] Figure 2 The figure is a flow chart of a method for preparing a fluorescent probe X based on a phenothiazine structure;

[0035] Figure 3 Schematic diagram of the H2S recognition mechanism of a fluorescent probe X based on a phenothiazine structure;

[0036] Figure 4 The UV absorption spectra and fluorescence spectra of compound 4, probe X, probe X (10 μM) and Na2S (100 μM);

[0037] Figure 5 is the relationship diagram of the response time of X to Na2S and the pH-dependent fluorescence response of Na2S;

[0038] Figure 6a The selectivity test result of fluorescent probe X when Na2S is introduced into the probe X solution;

[0039] Figure 6b Na2S will cause an obvious "on" fluorescence reaction, and the results of the selectivity test of fluorescent probe X are shown in the figure;

[0040] Figure 6c This is a graph showing the selectivity test results of fluorescent probe X in the presence of a competing analyte;

[0041] Figure 7a This is the result of the sensitivity test of the fluorescent probe X when the concentration of Na2S increases;

[0042] Figure 7b The sensitivity test result of fluorescent probe X with the increase of Na2S concentration is shown in the figure;

[0043] Figure 7c This is a schematic diagram of the regression equation of the sensitivity test results of the fluorescent probe X;

[0044] Figure 8 This is a schematic diagram of the results of detecting food spoilage using fluorescent probe X;

[0045] Fig. 9 Schematic diagram of fluorescence imaging results of living cells using fluorescent probe X. DETAILED DESCRIPTION

[0046] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

[0047] The following detailed description is an exemplary description, which is intended to provide further detailed description of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. The terms used in the present invention are only for describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present invention.

[0048] See also Figure 2 As shown, the present invention provides a fluorescent probe based on a phenothiazine structure, the principle is as follows Figure 1 As shown, the molecular structure formula of the fluorescent probe is:

[0049]

[0050] The present invention also provides a method for preparing a fluorescent probe based on a phenothiazine structure, which specifically comprises the following steps:

[0051] Compound 1 was obtained based on the raw materials:

[0052] 2-Methoxyphenothiazine (2.29 g, 10 mmol), NaOH (0.4 g, 10 mmol) and iodomethane (2.13 mg, 15 mmol) were dissolved in 20.0 mL of dimethyl sulfoxide, and the mixture was heated at 65°C for 12 hours. After cooling to room temperature, the mixture was extracted with CH2Cl2, and the organic phase was dried with Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, eluent: petroleum ether / DCM = 5 / 1) to obtain white solid compound 1 (2.21 g, 91%);

[0053] Compound 2 was obtained based on compound 1:

[0054] Under Ar atmosphere, dry 2.0 mL DMF was added dropwise to 1.0 mL POCl3 and cooled to 0°C. After stirring for 15 minutes, a DMF solution (10.0 mL) of compound 1 (2.43 g, 10 mmol) was added to the above mixture. The mixture was stirred at room temperature overnight. Then the mixture was poured into ice water. Stirring for about 2 hours, an orange precipitate was obtained. The crude product was filtered and purified by silica gel column chromatography with PE / EtOAc=5 / 1 as eluent to obtain a yellow solid 2 (2.48 g, 87%);

[0055] Compound 3 was obtained based on compound 2:

[0056] Compound 2 (1.36 g, 5 mmol) was dissolved in 10.0 mL of anhydrous DCM in an ice-water bath, and boron tribromide (1.6 mL, 16.5 mmol) was slowly added to the mixture. Then, the mixture was slowly warmed to room temperature and stirred overnight. The reaction mixture was quenched with ice water. The aqueous solution was extracted with dichloromethane (50×3), and the combined organic phases were washed with brine, dried over Na2SO4, and filtered. The crude product was a yellow solid 3 (771.9 mg, 60%);

[0057] The raw material is 2-methoxyphenothiazine, and its molecular structure is:

[0058]

[0059] The compound 1 is 2-methoxy-10-methyl-10H-phenothiazine, and its molecular structure is:

[0060]

[0061] The compound 2 is 2-methoxy-10-methyl-10H-phenothiazine-3-carbaldehyde, and its molecular structure is:

[0062]

[0063] The compound 3 is 2-hydroxy-10-methyl-10H-phenothiazine-3-carbaldehyde, and its molecular structure is:

[0064]

[0065] Compound 3, 2-aminobenzenethiol and sodium metabisulfite were dissolved in DMF, mixed, heated and cooled to room temperature, distilled water was added, organic layers were combined by CH2Cl2 extraction, washed with saturated brine, dried over anhydrous NaSO4 and filtered. After the solvent was removed under reduced pressure, the residue was purified by silica gel column chromatography (PE / CH2Cl2=2 / 1) to obtain compound 4 as a yellow solid.

[0066] The molecular structure of compound 4 is:

[0067]

[0068] Compound 4, 2,4-dinitrofluorobenzene, K2CO3 and dry DMF were added to a round-bottom flask and stirred overnight at room temperature. After the reaction was completed, the reaction solution was poured into ice water, extracted with CH2Cl2, the organic layers were combined, washed with saturated brine, dried with anhydrous Na2SO4 and filtered. After the solvent was removed under reduced pressure, the residue was purified by silica gel column chromatography (PE / EA=5 / 1) to obtain a yellow solid compound X, which was a fluorescent probe.

[0069] The 2,4-dinitrophenyl (DNP) ether bond of the probe X is rapidly broken in the presence of H2S, releasing a phenothiazine fluorophore that can emit strong fluorescence at a wavelength of 520 nm, such as Figure 3 shown.

[0070] Embodiment 1:

[0071] Based on the raw materials, compound 1 was obtained: 2-methoxyphenothiazine (2.29 g, 10 mmol), NaOH (0.4 g, 10 mmol) and iodomethane (2.13 mg, 15 mmol) were dissolved in 20.0 mL of dimethyl sulfoxide, and the mixture was heated at 65 ° C for 12 hours. Cooled to room temperature, extracted with CH2Cl2, the organic phase was dried with Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, eluent: petroleum ether / DCM = 5 / 1) to obtain white solid compound 1 (2.21 g, 91%);

[0072] Compound 2 was obtained based on compound 1: Under Ar atmosphere, dry 2.0 mL DMF was added dropwise to 1.0 mL POCl3 and cooled to below 0°C. After stirring for 15 minutes, a DMF solution (10.0 mL) of compound 1 (2.43 g, 10 mmol) was added to the above mixture. The mixture was stirred at room temperature overnight. Then the mixture was poured into ice water. Stirring for about 2 hours, an orange precipitate was obtained. The crude product was filtered and purified by silica gel column chromatography with PE / EtOAc=5 / 1 as the eluent to obtain a yellow solid 2 (2.48 g, 87%);

[0073] Compound 3 was obtained based on compound 2: Compound 2 (1.36 g, 5 mmol) was dissolved in 10.0 mL of anhydrous DCM in an ice-water bath, and boron tribromide (1.6 mL, 16.5 mmol) was slowly added to the mixture. Then, the mixture was slowly warmed to room temperature and stirred overnight. The reaction mixture was quenched with ice water. The aqueous solution was extracted with dichloromethane (50×3), and the combined organic phases were washed with brine, dried over Na2SO4, and filtered. The crude product was a yellow solid 3 (771.9 mg, 60%);

[0074] Compound 3 (257.3 mg, 1 mmol), 2-aminothiophenol (125.2 mg, 1 mmol) and sodium metabisulfite (190.1 mg, 1 mmol) were dissolved in 10 mL of DMF, and the mixture was heated at 80-160°C for 2-24 hours. After cooling to room temperature, 30 mL of distilled water was added, and the organic layers were combined by CH2Cl2 extraction, washed with saturated brine, dried over anhydrous NaSO4 and filtered. After removing the solvent under reduced pressure, the residue was purified by silica gel column chromatography (PE / CH2Cl2=2 / 1) to obtain compound 4 (286.3 mg, 79%) as a yellow solid.

[0075] NMR spectroscopy analysis:

[0076] 1 H NMR (400MHz, CDCl3) δ12.66(s,1H),7.92(d,J=8.0Hz,1H),7.87(d,J=8.0Hz,1H),7.51-7.45(m,1H),7.38(s,1H ),7.38-7.34(m,1H),7.23-7.14(m,2H),6.98(t,J=7.4Hz,1H),6.87(d,J=8.0Hz,1H),6.54(s,1H),3.43(s,3H). 13C NMR (101MHz, CDCl3) δ158.84,158.78,151.85,151.54,144.35,132.20,127.57,127.20,126. 64,125.88,125.10,123.10,123.09,121.67,121.49,114.68,111.56,106.19,103.25,35.75.

[0077] High resolution mass spectrometry:

[0078] HRMS(ESI,m / z):calced:[C 20 H 14 N2OS2+H] + =363.0626.Found:[C 20 H 14 N2OS2+H] + =363.0511.

[0080] Compound 4 (181.2 mg, 0.5 mmol), 2,4-dinitrofluorobenzene (186.1 mg, 1 mmol), K2CO3 (207.3 mg, 1.5 mmol) and 5 mL of dry DMF were added to a 50 mL round-bottom flask and stirred overnight at room temperature. After the reaction was completed, the reaction solution was poured into 50 mL of ice water and extracted with CH2Cl2. The organic layers were combined, washed with saturated brine, dried over anhydrous Na2SO4 and filtered. After the solvent was removed under reduced pressure, the residue was purified by silica gel column chromatography (PE / EA=5 / 1) to obtain a yellow solid compound X (237.8 mg, 90%), which was the fluorescent probe.

[0081] NMR spectroscopy analysis:

[0082] 1 H NMR (400MHz, CDCl3) δ8.92 (d, J=2.8Hz, 1H), 8.23 ​​(dd, J=9.2, 2.8Hz, 1H), 8.19 (s ,1H),7.96(d,J=8.0Hz,1H),7.80(dd,J=8.0,0.4Hz,1H),7.45(td,J=7.5,1.2Hz ,1H),7.34(td,J=7.2,1.2Hz,1H),7.25-7.16(m,2H),7.03(td,J=7.2,1.2Hz,1H ),6.97(d,J=9.2Hz,1H),6.86(dd,J=8.1,0.8Hz,1H),6.57(s,1H),3.37(s,3H). 13C NMR (101MHz, CDCl3) δ160.20,155.81,152.65,150.42,149.52,143.69,141.73,139.36,135.09,129.07,128.13,127. 98,127.50,126.51,125.38,123.84,123.76,123.08,123.05,122.26,121.42,120.23,117.40,114.77,107.36,35.79.

[0083] High resolution mass spectrometry:

[0084] HRMS(ESI,m / z):calced:[C 26 H 16 N4O5S2+H] + =529.0640.Found:[C 26 H 16 N4O5S2+H] + =529.0626.

[0086] Example 2

[0087] The fluorescent probe is subjected to ultraviolet-visible spectrum and fluorescence spectrum testing, which specifically includes the following steps:

[0088] The UV-visible absorption spectrum of probe X was detected in PBS solution (PBS / DMSO=3:7, pH=7.4). The maximum absorption wavelength of the probe was 384nm. After adding 10.0 times the amount of Na2S, the maximum absorption peak red-shifted to 404nm, and the color of the solution also changed from light yellow to yellow ( Figure 4 ), at which point Na2S can be easily detected with the naked eye. It is noteworthy that the UV-visible absorption spectrum of the probe after reacting with 10.0 times the amount of Na2S is very similar to that of compound 4. Figure 4 As shown in Fig. 2b, the fluorescence emission of probe X in PBS solution (PBS / DMSO=3:7, pH=7.4) is very low. In contrast, after adding Na2S, the fluorescence intensity of probe X at a wavelength of 520 nm increased sharply, showing an obvious fluorescence "turn-on" effect, as shown in Fig. Figure 4 shown.

[0089] Example 3

[0090] The response time and the optimal pH range of the fluorescent probe are tested, which specifically includes the following steps:

[0091] Response time plays a vital role in fluorescent probes. In order to evaluate the response time of X to Na2S, the present invention detected the change of fluorescence intensity of X with Na2S. The change of fluorescence intensity at 520nm wavelength with time was observed, as shown in Fig. Figure 5 As shown in a. As the reaction time increases, the fluorescence intensity of the probe first increases rapidly, then slows down, and finally stabilizes after 30 minutes. At the same time, the present invention also explores the pH-dependent fluorescence reaction of the probe to Na2S. Figure 5 As shown in (b), in the absence of Na2S, the fluorescence intensity of the probe at a wavelength of 520 nm changes very little at different pH values ​​(from 4.0 to 11.0), indicating that the probe exhibits good stability over a wide pH range. After adding Na2S, the fluorescence response of the solution increases significantly, as shown in (c). Figure 5 shown.

[0092] Compared with weakly acidic environment, weakly alkaline environment is more conducive to the detection of hydrogen sulfide. The optimal detection range is between pH = 7 and 11. Considering the acidity and alkalinity of the biological environment, pH = 7.4 was selected in subsequent experiments.

[0093] Example 4

[0094] The specificity test of the fluorescent probe specifically comprises the following steps:

[0095] The present invention studies various biologically relevant species, including some representative ions and amino acids. When Na2S is introduced into the probe X solution, the absorption peak of the probe will be slightly red-shifted, while the presence of other ions and amino acids will not have a significant effect on the absorption ( Figure 6a ). In addition, only Na2S caused a clear "on" fluorescence response ( Figure 6b ), indicating that the probe is more selective for Na2S than other anions and biological species. Even in the presence of competing analytes, Na2S continued to produce similar fluorescence responses, demonstrating the specificity of the probe ( Figure 6c ). Therefore, probe X has good selectivity for Na2S and has the potential to detect Na2S in complex biological environments.

[0096] Example 5

[0097] The sensitivity test of the fluorescent probe specifically comprises the following steps:

[0098] The UV absorption spectrum of test X (10 μM) in PBS solution (PBS / DMSO=3:7, pH=7.4) is as follows: Figure 7a As shown in Figure 2, it was found that when the Na2S concentration increased from 0 μM to 500 μM, the absorption peak slightly red-shifted and the absorption at 404 nm gradually increased ( Figure 7a). In addition, the present invention conducted a fluorescence titration experiment to obtain the emission spectrum of X to Na2S. As the concentration of Na2S increased (5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 μM), as shown in FIG. Figure 7b After adding Na2S, the emission peak at 520nm also gradually increased. By plotting the linear relationship curve between the fluorescence intensity at 520nm and the Na2S concentration, a reliable calibration curve was obtained, and the regression equation was: 520 =4.6983[Na2S]+1.6588,R 2 =0.99( Figure 7c ). In the range of 0-100 μM, the fluorescence intensity is directly related to the Na2S concentration, and the detection limit is as low as 58 nM.

[0099] Embodiment 6:

[0100] Using the fluorescent probe to judge spoiled food specifically includes the following steps:

[0101] Select several 20g portions of fresh pork and several eggs, seal the 20g pork and the probe solution in a 50mL conical flask, seal the eggs and the probe solution in a 50mL conical flask, and set up a 50mL conical flask with only the probe solution as a control group.

[0102] The above conical flask was kept at room temperature, and the phenomenon was observed and photographed at different times under natural light and handheld 365nm ultraviolet light. The results are as follows Figure 8 shown.

[0103] After 2 days of incubation at room temperature, the color of the probe X solution in the pork and egg samples increased slightly and gradually deepened over time. After 5 days, the deterioration of the two food samples became more obvious, and the yellow fluorescence of probe X increased. This observation indicates that H2S is released during the deterioration of food.

[0104] The probe solution in the control group did not change after 5 days, indicating that probe X has super stability in the buffer solution.

[0105] Embodiment 7:

[0106] Using the fluorescent probe to perform fluorescence imaging on living cells specifically includes the following steps:

[0107] MME medium was used to culture HeLa cells. Four groups of experiments were performed simultaneously.

[0108] The control group cells were not treated.

[0109] The second group of untreated cells were incubated with 10 μM Probe X for 1 hour, and then the cells were washed three times with PBS. Fluorescence imaging was then performed.

[0110] The third group of cells were pretreated with 200 μM N-ethylmaleimide (NEM) for 1 hour to inhibit endogenous H2S production. The cells were then incubated with 100 μM Na2S and 10 μM Probe X for 1 hour and imaged in the same way.

[0111] The fourth group of cells were pretreated with 200 μM N-ethylmaleimide (NEM) for 1 hour, then incubated with 10 μM probe X for 1 hour, washed three times with PBS, and finally subjected to fluorescence imaging.

[0112] The results are as follows Fig. 9 As shown, no fluorescence was observed in the green channel for control cells because they were not treated at all.

[0113] The first group of cells underwent fluorescence analysis of endogenous H2S, e.g. Fig. 9 As shown in A.1, obvious green fluorescence appeared in the corresponding channel.

[0114] The second group of cells was subjected to fluorescence detection of exogenous H2S. On the basis of inhibiting endogenous H2S production, after incubation with Na2S (100μM) and probe X (10μM) for 1 hour, strong fluorescence was observed in the green channel ( Fig. 9 B.1).

[0115] After continuous incubation with NEM (200 μM) and probe X (10 μM) for 1 hour, fluorescence imaging was performed on the third group of cells, and fluorescence was also observed. These research results show that the probe is not only suitable for observing H2S introduced into and outside the cell, but also can effectively identify naturally occurring H2S and has excellent cell membrane permeability.

[0116] Tests have shown that the probe has extremely high sensitivity and specificity and can be used for fluorescence imaging of living cells to identify externally introduced and naturally occurring H2S.

[0117] The present invention provides a fluorescent probe based on a phenothiazine structure, which exhibits excellent detection capability for H2S, including a large Stokes shift (116 nm), significant sensitivity (58 nM) and good selectivity.

[0118] The present invention also proposes a method for preparing a fluorescent probe, and constructs a new fluorescent probe for detecting hydrogen sulfide with simple and efficient synthesis steps. The probe can sensitively detect H2S in actual food samples and living cells, and has great market application value.

[0119] The present invention uses 2,4-dinitrobenzene (DNP) molecules as H2S recognition units and fluorescence quenchers to construct a phenothiazine-based H2S fluorescent probe X. The presence of DNP inhibits the fluorescence of the probe. On the contrary, after adding Na2S, the dinitroanisole of the probe undergoes a thiolation reaction, thereby emitting a strong yellow fluorescence at a wavelength of 520nm, accompanied by a large Stokes shift (116nm). In particular, the process has good selectivity for H2S with high nucleophilicity and is not affected by other analytes (such as anions and thiols). In addition, probe X also has high sensitivity and excellent selectivity, and can be effectively used for fluorescence imaging of endogenous H2S in HeLa cells, as well as detecting H2S released by food corruption, and has great application prospects in the fields of food and biomedicine.

[0120] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

[0121] Those skilled in the art will appreciate that embodiments of the present invention may be provided as methods, systems, or computer program products. Therefore, the present invention may take the form of a complete hardware embodiment, a complete software embodiment, or an embodiment combining software and hardware. Moreover, the present invention may take the form of a computer program product implemented on one or more computer-usable storage media (including but not limited to disk storage, CD-ROM, optical storage, etc.) containing computer-usable program code.

[0122] The present invention is described with reference to flowcharts and / or block diagrams of methods, devices (systems), and computer program products according to embodiments of the present invention. It should be understood that each process and / or block in the flowchart and / or block diagram, as well as the combination of processes and / or blocks in the flowchart and / or block diagram, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, a special-purpose computer, an embedded processor, or other programmable data processing device to produce a machine, so that the instructions executed by the processor of the computer or other programmable data processing device generate instructions for implementing the processes in the flowchart and / or block diagram. Figure 1 A process or multiple processes and / or boxes Figure 1 A device that provides the functions specified in a block or multiple blocks.

[0123] These computer program instructions may also be stored in a computer-readable memory capable of directing a computer or other programmable data processing device to operate in a specific manner, so that the instructions stored in the computer-readable memory produce an article of manufacture comprising an instruction device, which implements the process Figure 1 A process or multiple processes and / or boxes Figure 1 A function specified in one or more boxes.

[0124] These computer program instructions can also be loaded onto a computer or other programmable data processing device so that a series of operating steps are executed on the computer or other programmable device to produce a computer-implemented process, thereby providing instructions for implementing the process. Figure 1 A process or multiple processes and / or boxes Figure 1 A step that specifies a function in one or more boxes.

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A fluorescent probe based on a phenothiazine structure, characterized in that: The molecular structure of the fluorescent probe is:

2. A method for preparing the fluorescent probe based on the phenothiazine structure according to claim 1, characterized in that: The steps include: Step 1, obtaining a first compound based on raw materials; Step 2, obtaining a second compound based on the first compound; Step 3, obtaining a third compound based on the second compound; Step 4, dissolving compound 3, 2-aminothiophenol and sodium metabisulfite in an organic solvent to obtain a mixture, heating the mixture and cooling it to room temperature, adding distilled water, extracting and combining the organic layers, washing, drying and filtering, removing the solvent under reduced pressure, and purifying the residue by silica gel column chromatography to obtain a fourth compound as a yellow solid; Step 5, adding the fourth compound, 2,4-dinitrofluorobenzene, an organic base or an inorganic base and a dry organic solvent into a round-bottom flask, stirring at room temperature overnight, after the reaction is completed, pouring the solution into ice water, extracting and combining the organic layers, washing, drying and filtering, removing the solvent under reduced pressure, and purifying the residue by silica gel column chromatography to obtain a fluorescent probe; The molecular structure of the raw material is: The molecular structural formula of the first compound is: The molecular structural formula of the second compound is: The molecular structural formula of the third compound is: The molecular structural formula of the fourth compound is:

3. The method for preparing a fluorescent probe based on a phenothiazine structure according to claim 2, characterized in that: In the step 4, the mixture is heated at 80-160° C. for 2-24 hours.

4. The method for preparing a fluorescent probe based on a phenothiazine structure according to claim 2, characterized in that: In the step 4, an organic solvent immiscible with water is used to extract and combine the organic layers.

5. The method for preparing a fluorescent probe based on a phenothiazine structure according to claim 2, characterized in that: In the step 4, saturated brine is used for washing.

6. The method for preparing a fluorescent probe based on a phenothiazine structure according to claim 2, characterized in that: In the step 4, drying is performed using a desiccant.

7. The method for preparing a fluorescent probe based on a phenothiazine structure according to claim 2, characterized in that: In the step 5, an organic solvent immiscible with water is used to extract and combine the organic layers.

8. The method for preparing a fluorescent probe based on a phenothiazine structure according to claim 2, characterized in that: In the step 5, saturated brine is used for washing.

9. The method for preparing a fluorescent probe based on a phenothiazine structure according to claim 2, characterized in that: In the step 5, drying is performed using a desiccant.

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