A fluorescent molecular probe based on triphenylamine derivative and preparation method and application thereof

Abstract

The application discloses a fluorescent molecular probe based on triphenylamine derivative and a preparation method and application thereof, and belongs to the technical field of fluorescent molecular probes. The application solves the problems of low sensitivity, slow response speed and poor selectivity of the current fluorescent molecular probe for H2S level detection. The application uses 5-(4-(diphenylamine)phenyl) thiophene-2-carboxaldehyde and 4-amino-3-hydroxybenzoic acid to prepare a triphenylamine derivative, and the derivative can be used as a fluorescent molecular probe HBA for detecting H2S. In the detection process, H2S is coordinated with the carbon-nitrogen double bond in HBA and the oxygen in the hydroxyl group, a cyclization reaction occurs, a six-membered ring is formed, and fluorescence quenching is caused. The HBA has the advantages of low detection limit, high selectivity and anti-interference, fast response speed, low cytotoxicity, excellent membrane permeability, can realize real-time fluorescent imaging of cells, and can be used for detecting H2S level in non-alcoholic fatty liver disease.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent molecular probe technology, specifically relating to a fluorescent molecular probe based on a triphenylamine derivative, its preparation method, and its application. Background Technology

[0002] Nonalcoholic fatty liver disease (NAFLD), a chronic liver disease closely related to metabolic disorders, is becoming increasingly prevalent globally. Studies have shown that the development of NAFLD is closely related to abnormal H2S levels, and excessive H2S can affect liver function; mice with high-fat diet-induced hepatic steatosis exhibit increased intrahepatic H2S. Therefore, cellular H2S is considered a potential biomarker for NAFLD. Thus, establishing a safe, effective, and rapid method for detecting H2S levels in NAFLD is of great significance.

[0003] Fluorescence imaging technology is widely used for the dynamic monitoring of cellular bioactive molecules, especially fluorescent molecular probe detection technology. It offers advantages over traditional detection methods such as ion chromatography, atomic absorption spectroscopy, and electrochemical analysis, including high selectivity, low detection limits, rapid response, ease of operation, and low cost, making it widely applicable for in vivo H2S detection. Currently, many researchers have developed fluorescent molecular probes for detecting H2S levels. For example, Fang Bin et al. developed a mitochondrial-targeted H2S-activated fluorescent molecular probe and constructed a liver ischemia-reperfusion injury (HIRI) model, which can monitor in vivo and in vitro mitochondrial H2S levels. Yang Xiaopeng et al. synthesized a dual-site fluorescent probe, rDNA-1, which can track and detect H2S level changes during liver injury in real time. However, current fluorescent molecular probes still suffer from drawbacks such as low sensitivity, slow response speed, and poor selectivity in detecting H2S level changes induced by NAFLD. In recent years, triphenylamine-based fluorescent groups have attracted widespread attention due to their simple structure, excellent optical properties, and low synthesis cost. Furthermore, compared to traditional aggregation-induced fluorescence quenching (ACQ) fluorescent materials, aggregation-induced emission (AIE) fluorescent materials have significant advantages, overcoming the fluorescence quenching problem caused by aggregation in physiological environments. This provides new opportunities for AIE fluorescent materials in various sensing applications. Therefore, it is necessary to develop an AIE fluorescent molecular probe based on triphenylamine derivatives to achieve specific detection of H2S in NAFLD. Summary of the Invention

[0004] In order to overcome the problems of low sensitivity, slow response speed and poor selectivity of current fluorescent molecular probes used for H2S level detection, this invention provides a fluorescent molecular probe based on triphenylamine derivatives, its preparation method and application.

[0005] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution: One objective of this invention is to provide a triphenylamine derivative, the structural formula of which is as follows: .

[0006] The second objective of this invention is to provide a method for preparing the above-mentioned triphenylamine derivative. The method involves dissolving 5-(4-(diphenylamine)phenyl)thiophene-2-carboxaldehyde and 4-amino-3-hydroxybenzoic acid in a solvent, heating and refluxing the mixture, and after the reaction is complete, filtering and recrystallizing the product to obtain the triphenylamine derivative.

[0007] Further specifying, the preparation method of 5-(4-(diphenylamine)phenyl)thiophene-2-carboxaldehyde is as follows: triphenylamine boric acid, 5-bromothiophene-2-carboxaldehyde, potassium carbonate and tetra(triphenylphosphine)palladium are added to a solvent and heated under nitrogen protection and refluxed.

[0008] Furthermore, the molar ratio of triphenylamine boric acid, 5-bromothiophene-2-carboxaldehyde, potassium carbonate, and tetra(triphenylphosphine)palladium is (1~15):12:20:1; the solvent is a mixed solution of tetrahydrofuran and water in a volume ratio of 1:1; the heating temperature is 65~75℃, and the reaction time is 6~8h.

[0009] Further specified, the molar ratio of 5-(4-(diphenylamine)phenyl)thiophene-2-carboxaldehyde and 4-amino-3-hydroxybenzoic acid is 1:(1.2~1.5).

[0010] Further specifying, the solvent is ultra-dry ethanol.

[0011] Further specified, the heating temperature is 75~85℃, and the reaction time is 4~6h.

[0012] A third objective of this invention is to provide an application of the above-mentioned triphenylamine derivative as a fluorescent molecular probe.

[0013] Further specifying, the fluorescent molecular probe is used to detect hydrogen sulfide.

[0014] The beneficial effects of this invention are as follows: The triphenylamine derivative provided by this invention can be used as a fluorescent molecular probe HBA. This HBA probe exhibits high selectivity, high sensitivity, rapid response, low cytotoxicity, and strong fluorescence emission. Furthermore, the color of the fluorescent molecular probe HBA changes significantly before and after reacting with H2S, enabling visual detection of H2S levels in NAFLD. Compared with existing technologies, this invention also has the following advantages: (1) The fluorescent molecular probe HBA of the present invention contains a triphenylamine group, which is a strong electron-donating group with lipophilicity and photostability, and can emit strong fluorescence. During the H2S detection process, H2S coordinates with the C=N double bond and oxygen in the hydroxyl group of the fluorescent molecular probe HBA, and undergoes a cyclization reaction to form a six-membered ring containing a -SO- functional group, causing the fluorescence color to change from bright yellow to colorless, i.e., fluorescence quenching. This molecular recognition process provides a stable signal output basis for the detection of H2S level in NAFLD by the fluorescent molecular probe HBA.

[0015] (2) The fluorescent molecular probe HBA of the present invention has AIE characteristics, with a detection limit as low as 0.45 nM and a fluorescence quantum yield of 18.51%, enabling rapid response. When other competing analytes are present in the system, the specific response signal of probe HBA to H2S (fluorescence quenching effect) does not decrease significantly, indicating that the probe can maintain high selectivity for the target analyte H2S in complex coexisting systems. In addition, the HBA fluorescence signal changes when H2S is present, but the introduction of other analytes does not cause a change in HBA fluorescence intensity, indicating that the specific interaction between the fluorescent molecular probe HBA and H2S has anti-interference characteristics. This further demonstrates that the fluorescent molecular probe HBA maintains high selectivity and anti-interference characteristics in systems with other analytes, providing a reliable technical means for H2S analysis in complex biological environments.

[0016] (3) The fluorescent molecular probe HBA of the present invention has low cytotoxicity and excellent membrane permeability, enabling real-time fluorescence imaging of cells and demonstrating good biocompatibility and imaging resolution at the in vivo level. In addition, the fluorescence intensity of HBA has a good linear relationship with H2S concentration, and can be used to detect H2S levels in NAFLD. Attached Figure Description

[0017] Figure 1 The nuclear magnetic resonance of the intermediate synthesized in Example 1 of this invention 1 HNMR spectrum; Figure 2 This is a high-resolution mass spectrum of the intermediate synthesized in Example 1 of this invention; Figure 3 The nuclear magnetic resonance (NMR) of the fluorescent molecular probe HBA synthesized in Example 1 of this invention. 1 HNMR spectrum; Figure 4 The nuclear magnetic resonance (NMR) of the fluorescent molecular probe HBA synthesized in Example 1 of this invention. 13 CNMR plot; Figure 5 This is a high-resolution mass spectrum of the fluorescent molecular probe HBA synthesized in Example 1 of this invention; Figure 6This is a high-resolution mass spectrum of the fluorescent molecular probe HBA synthesized in Example 1 of this invention after reacting with H2S; Figure 7 The infrared spectrum and nuclear magnetic resonance spectrum of the fluorescent molecular probe HBA synthesized in Example 1 of this invention after reacting with H2S are shown. 1 HNMR spectra, where (A) is the infrared spectrum and (B) is the nuclear magnetic resonance spectrum. 1 HNMR spectrum; Figure 8 This is a schematic diagram illustrating the reaction principle of the fluorescent molecular probe HBA synthesized in Example 1 of this invention with H2S; Figure 9 The fluorescence spectra of the fluorescent molecular probe HBA synthesized in Example 1 of this invention are shown in DMSO / H2O mixtures with different water content, wherein the water content is 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%. Figure 10 These are fluorescence images of the fluorescent molecular probe HBA synthesized in Example 1 of this invention in DMSO / H2O mixtures with different water content, wherein the water content is 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% (under 365nm UV excitation). Figure 11 This is the UV absorption spectrum of the fluorescent molecular probe HBA synthesized in Example 1 of this invention before and after the reaction with H2S; Figure 12 This is a selectivity diagram of the fluorescent molecular probe HBA synthesized in Example 1 of this invention for H2S. In (B), numbers 1 to 30 represent: HBA, S 2- F - Cl - ,Br - I - SCN - CH3COO - BF4 - CO3 2- HCO3 - NO3 - SO3 2- HSO3 - HPO4 2- CN - ,ClO - S2O3 2- CrO4 2- SO4 2- Gly, Arg, Lys, Ala, Asp, Phe, ERY, SMZ, SM, and CAP; Figure 13 This is the fluorescence spectrum of the fluorescent molecular probe HBA synthesized in Example 1 of the present invention, showing the change in fluorescence intensity with H2S concentration; Figure 14 This is the linear relationship between the fluorescent molecular probe HBA synthesized in Example 1 of this invention and the concentration of H2S; Figure 15 This is a schematic diagram showing the change in fluorescence intensity over time before and after the reaction of the fluorescent molecular probe HBA synthesized in Example 1 of this invention with H2S. Figure 16 This is a schematic diagram showing the change in fluorescence intensity of the fluorescent molecular probe HBA synthesized in Example 1 of the present invention before and after the reaction with H2S as a function of pH. Figure 17 This refers to the absolute fluorescence quantum yield of the fluorescent molecular probe HBA synthesized in Example 1 of this invention before and after the reaction with H2S; Figure 18 The relative cell viability of HeLa cells in different concentrations of the fluorescent molecular probe HBA synthesized in Example 1; Figure 19 This is a schematic diagram of H2S cell imaging using the fluorescent molecular probe HBA synthesized in Example 1 of the present invention; Figure 20 These are Oil Red O staining and hematoxylin-eosin (H&E) staining images of liver tissue from the control group and NAFLD model group mice over time in Example 7. Figure 21 These are the ALT and AST enzyme levels measured in the control group and NAFLD model group mice in Example 7; Figure 22 The images show fluorescence images and fluorescence intensities of mice and their organs and tissues in the control group, probe group, exogenous hydrogen sulfide group, and NAFLD model group at 14 days after the event in Example 7. Among them, (A) mouse fluorescence image, (B) mouse fluorescence intensity, (C) organ fluorescence image, and (D) liver tissue fluorescence intensity. Detailed Implementation

[0018] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0019] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0020] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0022] In the following examples, comparative examples, and detection experiments 1 H nuclear magnetic resonance spectrum and 13 C10 NMR spectra were acquired using an AVANCE 600MHz spectrometer; pH measurements were performed using a pH-FE20 acidity meter from Mettler Toledo Shanghai; high-resolution mass spectrometry (HRMS) was obtained using a Waters Xevo UPLC / G2-SQ Tof MS spectrometer; UV-Vis spectra were obtained using a Shimadzu UV-2550 spectrometer; fluorescence spectra were obtained using a Hitachi F-4700 fluorescence spectrometer; FT-IR spectra were measured using an Irtracer-100 (Shimadzu, Japan) by dispersing the samples in a potassium bromide dish; biological imaging was performed using a laser scanning confocal microscope (Leica TCS SP8); and live animal imaging was performed using a Shanghai Tianneng Tianlong ABL X5 PRO instrument.

[0023] In this invention, M represents mol / L, mM represents mmol / L, and μM represents μmol / L.

[0024] Example 1 A method for synthesizing HBA, a fluorescent molecular probe based on a triphenylamine derivative, includes the following steps: S1. Triphenylamine boric acid (3.47 g, 12 mmol), 5-bromothiophene-2-carboxaldehyde (1.91 g, 10 mmol), potassium carbonate (2.49 g, 18 mmol), and tetra(triphenylphosphine)palladium (0.139 g, 0.12 mmol) were added to 30 mL of a tetrahydrofuran / water (1 / 1, v / v) mixture. The mixture was refluxed at 70 °C under nitrogen protection until the starting material spot disappeared on TLC. The reaction was then stopped, and the reaction system was cooled to room temperature. The mixture was extracted three times with dichloromethane (30 mL × 3 times). The organic layer was then extracted three times with saturated sodium chloride solution (15 mL × 3 times). The organic layer was separated, dried with anhydrous sodium sulfate, and the solvent was evaporated to dryness to obtain a viscous black crude product. Finally, the product was purified by column chromatography to obtain a green solid, which was the intermediate 5-(4-(diphenylamine)phenyl)thiophene-2-carboxaldehyde. S2. Dissolve the intermediate (100 mg, 0.28 mmol) in 20 mL of ultra-dry ethanol, add 4-amino-3-hydroxybenzoic acid (45 mg, 0.30 mmol), heat to reflux at 75-85 °C for 4.5 h until the reaction is complete as monitored by TLC, stop the reaction, cool the reaction system to room temperature, a large amount of precipitate is produced, filter, and then wash three times with hot ethanol (20 mL) to obtain an orange-red solid powder, namely the fluorescent molecular probe (HBA) based on triphenylamine derivative, with a yield of 89.2% and a melting point of 268.5-269.7 °C.

[0025] The synthetic route for the intermediate 5-(4-(diphenylamine)phenyl)thiophene-2-carboxaldehyde in this embodiment is as follows:

[0026] Nuclear magnetic resonance spectroscopy of the intermediate ( 1 H NMR) detection, results are as follows Figure 1 As shown, its structural characterization is as follows: 1 ¹H NMR (600 MHz, DMSO) δ 9.87 (s, 1H), 8.00 (d, J = 4.0 Hz, 1H), 7.71–7.66 (m, 2H), 7.60 (d, J = 4.0 Hz, 1H), 7.36 (t, J = 7.9 Hz, 4H), 7.13 (t, J = 7.4 Hz, 2H), 7.10 (d, J = 7.6 Hz, 4H), 6.96 (d, J = 8.7 Hz, 2H); High-resolution mass spectrometry (HRMS) was used to detect the intermediate, and the results are as follows: Figure 2 As shown.

[0027] The HBA molecular formula obtained in this embodiment is C. 30 H 22 N2O3S was used to perform nuclear magnetic resonance spectroscopy on the HBA obtained in this embodiment. 1 H NMR) detection, results are as follows Figure 3 As shown, its structural characterization is as follows: 1 ¹H NMR (600MHz, DMSO) δ 12.73–12.45 (m, 1H), 10.02 (s, 1H), 8.85 (s, 1H), 7.71–7.66 (m, 5H), 7.51 (d, J = 3.8Hz, 1H), 7.38–7.32 (m, 4H), 7.13–7.06 (m, 6H), 6.99 (dd, J = 8.4, 4.4Hz, 3H); NMR spectra of the HBA obtained in this embodiment were analyzed (…). 13 (C NMR) detection, results are as follows Figure 4 As shown, its structural characterization is as follows: 13C10 NMR (151 MHz, DMSO) δ 167.59, 155.44, 154.67, 148.51, 148.19, 147.10, 141.17, 138.40, 135.61, 130.19, 129.02, 127.37, 127.20, 125.14, 124.25, 123.89, 122.84, 122.46, 122.13; High-resolution mass spectrometry (HRMS) detection of HBA yielded the following results: Figure 5 As shown. From Figures 1-5 As can be seen, the intermediate and HBA were successfully prepared.

[0028] The HBA synthesized in this embodiment was mixed with a DMSO / H2O mixture (2 / 8, v / v, 0.01 M Hepes, pH=7.4) to obtain the test solution, in which the HBA concentration was 1×10⁻⁶. -5 mol / L, prepare multiple portions of the test solution for later use.

[0029] from Figure 5 The positive ion peak of HBA was found to be at m / z 491.1430 ([HBA+H)). + ). Figure 6 The HRMS spectral data after the interaction of HBA and H2S are shown. Figure 6 A new mass-to-charge ratio signal m / z was discovered and recorded as 521.0990 ([HBA+H2S+H)). + ).

[0030] The FT-IR test results before and after the interaction of HBA and H2S are as follows: Figure 7 As shown in (A), at 1661cm -1 The peak at 1123 cm⁻¹ is due to the functional groups of the C=N double bond in HBA. The absorption of the C=N double bond is weakened upon the introduction of H₂S. The probe HBA reacts with H₂S to form stretching vibrations of the -SO- and -CO- bonds, resulting in a peak at 1123 cm⁻¹. -1 and 966cm -1 Broad peaks were generated at each location. FT-IR results indicate that the probe HBA underwent a cyclization reaction with H2S, forming the HBA-H2S compound.

[0031] To investigate the mechanism of interaction between HBA and H2S, nuclear magnetic resonance spectroscopy was performed on the system before and after the interaction between HBA and H2S. 1 H NMR detection (the reaction system of HBA and H2S was obtained by adding H2S to the test solution, with the amount of H2S added being 0.5 times and 2.0 times the molar amount of HBA, respectively), results are as follows: Figure 7As shown in (B), the proton signals at δ8.83 ppm (Ha) and δ7.67 ppm (Hb) are due to the hydrogen in the hydroxyl group and the hydrogen in the carbon-nitrogen double bond of the fluorescent molecular probe HBA, respectively. It was also observed that as the amount of H2S added increased, the proton signal of Ha significantly weakened and broadened, and shifted to a lower field. Furthermore, with increasing H2S addition, the proton signal (Hb) located at the C=N double bond split and weakened. These results indicate that H2S coordinates with the oxygen in the C=N double bond and hydroxyl group of the fluorescent molecular probe HBA, forming a cyclization reaction to generate a six-membered ring, leading to the fluorescence quenching of HBA.

[0032] The reaction process between probe HBA and H2S is as follows: Figure 8 As shown, the fluorescent molecular probe HBA contains a triphenylamine group, a strong electron-donating group with lipophilicity and photostability, thus exhibiting strong fluorescence. Upon the addition of H₂S, the probe HBA reacts with H₂S to form a six-membered ring containing a -SO₄⁻ functional group, leading to fluorescence quenching.

[0033] Example 2 AIE characteristics study of probe HBA: The HBA synthesized in Example 1 was added to a mixture of DMSO and H2O in different volume ratios to obtain solutions 1 to 11 (all with an HBA concentration of 1×10⁻⁶). --5 The water content (fw) in the DMSO / H2O mixtures were 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%, respectively.

[0034] Figure 9 The diagram illustrates the variation of HBA fluorescence intensity with the water content in the DMSO / H2O mixture. It shows that, regardless of whether the UV irradiation is at 365 nm or 420 nm, the fluorescence intensity of HBA in pure aqueous solution is stronger than that in pure DMSO solvent. When the water content (fw) in the DMSO / H2O mixture is below 60%, the fluorescence intensity of HBA is weak. When fw increases to 70%, the fluorescence intensity of HBA significantly increases, reaching its maximum at fw of 80%. The test results indicate that the HBA prepared in this example possesses AIE characteristics. The fluorescence images of HBA under 365 nm UV excitation in DMSO / H2O mixtures with different water contents are shown below. Figure 10 As shown.

[0035] Example 3 HBA's selective testing for H2S: H2S (10.0 times the equivalent of HBA) was added to the test solution in Example 1. The UV absorption spectra of the test solution and the test solution after adding H2S are as follows: Figure 11As shown, in the absence of H2S, the absorption peak of HBA appears at 420 nm. After adding H2S, the maximum absorption peak blue-shifts to 410 nm, and its absorbance increases slightly.

[0036] H2S and other analytes (anions, amino acids, and antibiotics) were added to the test solution in Example 1: F - Cl - ,Br - I - SCN - CH3COO - BF4 - CO3 2- HCO3 - NO3 - SO3 2- HSO3 - HPO4 2- CN - ,ClO - S2O3 2- CrO4 2- SO4 2- Glycine (Gly), Arginine (Arg), Lysine (Lys), Alanine (Ala), Asparagine (Asp), Phenylalanine (Phe), Erythromycin (ERY), Sulfamethoxazole (SMZ), Streptomycin (SM), Chloramphenicol (CAP), and S 2- The amount of H2S and other analytes added was 10.0 times the equivalent of HBA. The fluorescence spectra of the test solution and the solution after adding H2S and other analytes are as follows: Figure 12 As shown in (A), the probe HBA itself exhibits significant fluorescence emission at 528 nm, and the solution displays bright yellow fluorescence. Upon addition of H2S, the fluorescence of the probe HBA is quenched, the color darkens, and the fluorescence intensity decreases significantly. However, after the addition of other analytes, HBA still exhibits strong fluorescence intensity, indicating that other analytes have no effect on HBA.

[0037] Figure 12 (B) shows the fluorescence intensity at 420 nm of the test solution and after adding H2S and other analytes to the test solution. It can be seen that the introduction of other substances did not interfere with the recognition of H2S by the probe HBA. This indicates that the probe HBA has a good anti-interference ability for H2S recognition, and the presence of other analytes in the system will not affect its recognition effect.

[0038] The above test results show that H2S can quench the fluorescence of probe HBA, demonstrating its uniqueness compared to other analytes. Other analytes did not interfere with the recognition of H2S by probe HBA, indicating that probe HBA has excellent anti-interference ability for H2S recognition, and the presence of other analytes in the system does not affect its recognition performance. Therefore, probe HBA can be used as a specific fluorescent molecular probe for detecting H2S.

[0039] Example 4 HBA detection limit determination: Different amounts of H2S were added to the test solution in Example 1 to make the H2S concentration range from 0 to 100 μM (i.e., the amount of H2S added was 0 to 10.0 times the equivalent of HBA). The changes in HBA fluorescence intensity under different H2S concentrations were detected. Figure 13 As shown, the addition of different concentrations of H2S significantly reduced the fluorescence intensity of HBA at 528 nm, and the fluorescence intensity gradually weakened with increasing H2S concentration. Furthermore, as... Figure 14 As shown, within the H2S concentration range of 10–60 μM, there is a strong linear relationship between the HBA fluorescence intensity and the H2S concentration, R 2 The value is 0.9913.

[0040] The detection limit is an important indicator of probe sensitivity and can be calculated using the following equation.

[0041]

[0042] Where SD refers to the standard deviation of the blank sample, K is the signal-to-noise ratio (usually 3), and S is the slope of the linear fitting curve between the analyte concentration and the probe fluorescence intensity obtained from the probe concentration titration experiment.

[0043] Calculations show that the detection limit for H2S using HBA in this invention is 0.45 nM.

[0044] Example 5 Time response study of probe HBA to H2S: 50 μL of 0.01 M H2S solution was added to the test solution in Example 1. The fluorescence intensity of the test solution and the solution after adding H2S were measured respectively. The results are as follows: Figure 15 As shown in the figure, without the addition of H2S, the fluorescent molecular probe HBA showed no significant fluctuation in fluorescence intensity within 10 minutes, indicating good stability of HBA. After the addition of H2S, the fluorescence intensity immediately decreased and remained stable over time within 10 minutes without fluctuation, indicating that the probe HBA exhibits a fast response time and relatively stable fluorescence signal when detecting H2S levels, which is beneficial to improving the accuracy of H2S test results.

[0045] This embodiment also included spectral response tests of the probe HBA at different pH values ​​(2.0~12.0), such as... Figure 16 As shown in the figure, the fluorescence signal of probe HBA is relatively stable within the pH range of 6.0 to 8.0. After the addition of H2S, the fluorescence intensity of probe HBA decreases, but remains relatively stable within the neutral and weakly alkaline range (pH 6.0~8.0), indicating that probe HBA can effectively recognize H2S in a suitable physiological environment (pH 6.0~8.0).

[0046] In this embodiment, to determine the optical properties of the probe HBA and investigate the reasons for its rapid response behavior to H2S, the absolute fluorescence quantum yield of HBA before and after the addition of H2S was measured in the spectral range of 500–750 nm. The results are as follows: Figure 17 As shown, the final results were calculated using Fluoracel software. The probe HBA exhibited a high quantum yield of 18.51%. Upon the addition of H2S, H2S coordinated with the C=N double bond and oxygen in the hydroxyl group of HBA in the probe, forming a cyclization reaction to generate a six-membered ring. The formation of the six-membered ring disrupted the rigid planar conformation of the probe HBA, leading to a decrease in quantum yield to 9.68%. These results demonstrate that HBA possesses excellent optical properties, including a high fluorescence quantum yield and stable fluorescence response, which explains its rapid response during H2S level detection.

[0047] Example 6 Cytotoxicity of probe HBA and its imaging effect on H2S cells: The effect of HBA on HeLa cell viability was determined using the CCK-8 assay, and the cytotoxicity of HBA was analyzed. The specific methods are as follows: Human cervical cancer cells (HeLa cells) in the logarithmic growth phase were collected, adjusted to an appropriate concentration, counted, and then distributed at a concentration of 1.5 × 10⁻⁶ cells per well. 4Cells were seeded at a density of 100 μL per well into 96-well plates. After seeding, the cells were pre-cultured in a humidified incubator at 37°C and 5% CO2 for 12 h to allow them to adhere and reach a stable state. The original culture medium was then removed, and fresh culture medium containing different concentrations of HBA (0, 5, 10, 15, 20, 25 μM) was added. The 0 μM group served as a negative control without HBA (containing only culture medium or solvent). The cells were incubated for another 12 h at 37°C and 5% CO2. The HBA-containing medium was then aspirated, and the cells were gently washed three times with sterile phosphate-buffered saline (PBS) to remove any residual probe. Fresh culture medium containing 10% CCK-8 reagent (100 μL / well) was added to each well, ensuring even coverage of the cell layer. The cells were incubated in the dark at 37°C and 5% CO2 for 2 h. The absorbance (OD value) of each well was measured using an enzyme-linked immunosorbent assay (ELISA) microplate reader at a wavelength of 450 nm.

[0048] Set up background control wells (containing only CCK-8 reagent and culture medium, without cells) to subtract background interference.

[0049] The formula for calculating relative cell viability is as follows:

[0050] Among them, background OD 450 This indicates the absorbance of the wells containing only CCK-8 reagent and no cells in the culture medium.

[0051] Relative cell viability results as follows Figure 18 As shown, when the HBA concentration is 0~25μM, the survival rate of HeLa cells remains above 95%, indicating that the probe HBA has extremely low biotoxicity.

[0052] HeLa cells were seeded in 24-well plates containing 10% fetal bovine serum, penicillin (100 μg / mL), and streptomycin (100 μg / mL) and allowed to adhere overnight in a 5% carbon dioxide environment. Subsequently, the cultured HeLa cells were divided into four groups. The first group served as a control and was cultured in HBA solution for 1 h. The second group was co-incubated with HBA medium containing 10 μM hydrogen sulfide for 1 h. The third and fourth groups were co-incubated with HBA medium containing 50 and 100 μM H2S, respectively, for 1 h. The HeLa cells were then washed twice with buffer, and coverslips containing the cells were placed on a slide for confocal microscopy observation. The results are as follows: Figure 19 As shown, HeLa cells co-incubated with HBA for 1 h exhibited significant green fluorescence. However, with increasing H2S concentration, the green fluorescence signal of HeLa cells co-incubated with HBA+H2S for 1 h gradually weakened. This is evident from… Figure 19(B) It can also be seen that this indicates that the HBA probe can quickly detect H2S in living cells and that the HBA probe does not affect cell imaging, and can be used in biological experiments.

[0053] Example 7 Application of HBA probe in detecting H2S in NAFLD mouse model: In this experiment, 7-8 week old female mice purchased from Liaoning Changsheng Biotechnology Co., Ltd. were used as experimental subjects. All experiments were approved by the Animal Welfare and Ethics Experiment Professional Committee of Northeast Agricultural University. During the experiment, all mice were properly cared for and euthanized. They were provided with unlimited food and water in a sterile environment, and the ambient temperature was controlled within a 12-hour light / dark cycle.

[0054] The mice in this embodiment were grouped as follows: Group 1 was the control group, fed for 0 days and intraperitoneally injected with PBS solution; Group 2 was the probe group, fed for 0 days and intraperitoneally injected with HBA (100 μL, 0.1 mM); Group 3 was the exogenous hydrogen sulfide group, fed for 0 days and injected with H2S (100 μL, 1 mM), followed by another injection of HBA (100 μL, 0.1 mM) 12 hours later; Group 4 was the NAFLD model group, i.e., the endogenous hydrogen sulfide group, which was fed for 7, 14, and 21 days respectively, and all were intraperitoneally injected with HBA (100 μL, 0.1 mM). The NAFLD model was established as follows: a methionine-choline-deficient (MCD) diet combined with dexamethasone injection was used to establish the NAFLD mouse model. Mice were continuously fed a diet containing 60% MCD and intraperitoneally injected with dexamethasone (10 mg / kg / day). The mice were equipped with λ... ex = 488nm and λ em The Tanon ABL X5 PRO real-time animal imaging system with a 535nm fluorescence filter captured fluorescence images of mice in the control group, probe group, exogenous hydrogen sulfide group, and NAFLD model group at day 14. Control group mice were anesthetized on day 0 and injected with PBS solution via the tail vein. Probe group mice were anesthetized on day 0 and injected with H2S (100μL, 1mM) intraperitoneally. Exogenous hydrogen sulfide group mice were anesthetized on day 0 and injected with H2S (100μL, 1mM) via the tail vein. 12 hours later, HBA (100μL, 0.1mM) was injected again. NAFLD model group mice on days 7, 14, and 21 were anesthetized and injected with HBA (100μL, 0.1mM) via the tail vein. 2 hours later, the mice were euthanized, and their major organs (heart, liver, kidney, spleen, and lung) were collected for fluorescence imaging. Liver tissue from control and NAFLD model group mice was collected for Oil Red O staining and hematoxylin-eosin (H&E) staining.

[0055] The liver tissue staining images of the control group and NAFLD model group mice in this embodiment are as follows: Figure 20 As shown, liver pathological changes were assessed by Oil Red O staining and hematoxylin-eosin (H&E) staining. It can be seen that, compared with normally fed mice, the accumulation of lipid droplets in the liver tissue of NAFLD model mice increased significantly over time (the positive area of ​​Oil Red O staining expanded, and round vacuoles of varying sizes were observed in hepatocytes by H&E staining, which is due to the accumulation of lipid droplets), indicating that the NAFLD symptoms of mice gradually worsened.

[0056] Blood samples were collected from the orbital cavity of mice in the control and NAFLD model groups to detect two key enzymes related to liver function: alanine aminotransferase (ALT) and aspartate aminotransferase (AST). ALT and AST are key biomarkers for assessing liver function and liver damage. Elevated ALT and AST levels indicate that hepatocellular damage has entered the bloodstream, leading to liver injury. ALT and AST enzyme level measurements were performed. Figure 21 As shown, with increasing feeding time, the levels of ALT and AST in the liver tissue of NAFLD mice were higher than those in the control group, indicating that the liver damage in NAFLD mice was more severe.

[0057] Oil Red O staining and hematoxylin-eosin (H&E) staining results, as well as ALT and AST enzyme level measurements, indicate that this embodiment successfully established a NAFLD mouse model.

[0058] The fluorescence imaging images and fluorescence signal intensity test results of mice in the control group, probe group, exogenous hydrogen sulfide group, and NAFLD model group at 14 days of age in this embodiment are as follows: Figure 22 (A) and Figure 22 As shown in (B) (where Control represents the control group), the mice injected with the HBA probe group showed the strongest fluorescence signal, while the fluorescence signal of the 14-day-old mice in the NAFLD model group was relatively weak. Furthermore, it can be seen that the fluorescence signal in the abdomen of the mice in the exogenous hydrogen sulfide group was significantly weaker than that in the second group. This indicates that the fluorescent molecular probe HBA of this invention can detect both endogenous and exogenous H2S.

[0059] The fluorescence imaging images of major organs (heart, liver, kidney, spleen, and lung) and the results of liver tissue fluorescence signal intensity tests in mice at 14 days of age in the control group, probe group, exogenous hydrogen sulfide group, and NAFLD model group are shown below. Figure 22 (C) and Figure 22(D) shows that the biological probe HBA mainly accumulates in the liver and lungs of mice. The probe group of mice injected with HBA showed the strongest organ fluorescence signal. The fluorescence signal of mice in the exogenous hydrogen sulfide group and NAFLD model group was weakened compared with the probe group. This indicates that excessive H2S is produced in mice during NAFLD, and also shows that the probe HBA of the present invention can be used to identify and detect H2S level.

[0060] The above results indicate that the HBA probe shows good potential in in vivo imaging, and can detect the level of endogenous H2S in mice, which can be used to detect abnormal H2S levels caused by NAFLD.

[0061] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A triphenylamine derivative, characterized in that, The structural formula of the triphenylamine derivative is as follows: 。 2. A method for preparing the triphenylamine derivative according to claim 1, characterized in that, The method involves dissolving 5-(4-(diphenylamine)phenyl)thiophene-2-carboxaldehyde and 4-amino-3-hydroxybenzoic acid in a solvent, heating under reflux, and then filtering and recrystallizing the product after the reaction to obtain a triphenylamine derivative.

3. The preparation method according to claim 2, characterized in that, The molar ratio of 5-(4-(diphenylamine)phenyl)thiophene-2-carboxaldehyde to 4-amino-3-hydroxybenzoic acid is 1:(1.2~1.5).

4. The preparation method according to claim 2, characterized in that, The solvent is ultra-dry ethanol; the heating temperature is 75~85℃, and the reaction time is 4~6h.

5. The preparation method according to claim 2, characterized in that, The preparation method of 5-(4-(diphenylamine)phenyl)thiophen-2-carboxaldehyde is as follows: triphenylamine boric acid, 5-bromothiophen-2-carboxaldehyde, potassium carbonate and tetra(triphenylphosphine)palladium are added to a solvent and heated under nitrogen protection and refluxed.

6. The preparation method according to claim 5, characterized in that, The molar ratio of triphenylamine boric acid, 5-bromothiophene-2-carboxaldehyde, potassium carbonate, and tetra(triphenylphosphine)palladium is (1~15):12:20:

1.

7. The preparation method according to claim 5, characterized in that, The solvent is a mixed solution of tetrahydrofuran and water in a volume ratio of 1:

1.

8. The preparation method according to claim 5, characterized in that, The heating temperature is 65~75℃, and the reaction time is 6~8h.

9. The application of a non-disease diagnostic and treatment method using the triphenylamine derivative of claim 1, characterized in that, This triphenylamine derivative is used as a fluorescent molecular probe.

10. The application according to claim 9, characterized in that, Fluorescent molecular probes are used to detect hydrogen sulfide.

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