Fluorescent molecular probe based on triphenylamine derivative as well as preparation method and application of fluorescent molecular probe

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent molecular probes, and in particular relates to a fluorescent molecular probe based on triphenylamine derivatives, and a preparation method and application thereof. Background Art

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

[0003] Fluorescence imaging technology is widely used to dynamically monitor bioactive molecules in cells, particularly fluorescent molecular probes. These techniques offer 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. Consequently, they are widely used 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 mitochondria-targeted H2S-activated fluorescent molecular probe and constructed a liver ischemia-reperfusion injury (HIRI) model capable of monitoring mitochondrial H2S levels in vitro and in vivo. Yang Xiaopeng et al. synthesized a dual-site fluorescent probe, rDNA-1, that can track and detect changes in H2S levels in real time during liver injury. However, current fluorescent molecular probes still suffer from low sensitivity, slow response, and poor selectivity for detecting changes in H2S levels caused 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. In addition, compared with traditional aggregation-induced fluorescence quenching (ACQ) fluorescent materials, aggregation-induced emission (AIE) fluorescent materials have significant advantages. They can overcome the problem of fluorescence quenching caused by aggregation of ACQ materials in physiological environments, which 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 for H2S level detection, the present invention provides a fluorescent molecular probe based on triphenylamine derivatives, a preparation method and application thereof.

[0005] In order to solve the above technical problems, the present invention is implemented through the following technical solutions:

[0006] One of the objects of the present invention is to provide a triphenylamine derivative having the following structural formula:

[0007]

[0008] A second object of the present invention is to provide a method for preparing the above-mentioned triphenylamine derivative, which comprises dissolving 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde and 3-amino-4-hydroxybenzoic acid in a solvent, heating under reflux for reaction, and filtering and recrystallizing the product after the reaction to obtain the triphenylamine derivative.

[0009] It is further defined that the preparation method of 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde is: adding triphenylamineboric acid, 5-bromothiophene-2-carboxaldehyde, potassium carbonate and tetrakis(triphenylphosphine)palladium to a solvent, and heating under reflux for reaction under nitrogen protection.

[0010] It is further specified that the molar ratio of triphenylamine boric acid, 5-bromothiophene-2-carboxaldehyde, potassium carbonate and tetrakis(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°C, and the reaction time is 6-8h.

[0011] It is further defined that the molar ratio of 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde to 3-amino-4-hydroxybenzoic acid is 1:(1.2-1.5).

[0012] It is further defined that the solvent is ultra-dry ethanol.

[0013] It is further defined that the heating temperature is 65-75° C. and the reaction time is 4-6 h.

[0014] A third object of the present invention is to provide a use of the above triphenylamine derivative as a fluorescent molecular probe.

[0015] It is further defined that the fluorescent molecular probe is used to detect hydrogen sulfide.

[0016] The beneficial effects of the present invention are:

[0017] The triphenylamine derivatives provided by the present invention can be used as fluorescent molecular probes (HBAs). These probes exhibit high selectivity, high sensitivity, rapid response, low cytotoxicity, and strong fluorescence emission. Furthermore, the color of the HBA fluorescent molecular probe changes significantly before and after reaction with H2S, enabling visual detection of H2S levels in NAFLD. Compared to existing technologies, the present invention also offers the following advantages:

[0018] (1) The fluorescent molecular probe HBA of the present invention contains a triphenylamine group, a lipophilic, photostable, and strong electron donor that emits intense fluorescence. During H2S detection, H2S coordinates with the C=N double bond and the oxygen in the hydroxyl group in the fluorescent molecular probe HBA, undergoing a cyclization reaction to form a six-membered ring containing a -SO- functional group. This causes 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 fluorescent molecular probe HBA to detect H2S levels in NAFLD.

[0019] (2) The fluorescent molecular probe HBA of the present invention has AIE characteristics, with a detection limit as low as 0.45nM and a fluorescence quantum yield of 18.51%, which can achieve rapid response; when other competing analytes are present in the system, the specific response signal (fluorescence quenching effect) of the probe HBA to H2S does not significantly attenuate, indicating that the probe can still maintain a highly selective recognition ability for the target H2S in a complex coexistence system. In addition, when H2S is present, the HBA fluorescence signal changes, while the introduction of other analytes does not trigger a change in the HBA fluorescence intensity, indicating that the specific interaction between the fluorescent molecular probe HBA and H2S has anti-interference characteristics. This further illustrates that the fluorescent molecular probe HBA maintains high selectivity and anti-interference characteristics in systems where other analytes are present, providing a reliable technical means for H2S analysis in complex biological environments.

[0020] (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 in vivo. Furthermore, the fluorescence intensity of HBA exhibits a good linear relationship with H2S concentration, making it suitable for detecting H2S levels in NAFLD. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The nuclear magnetic resonance of the intermediate synthesized in Example 1 of the present invention is 1 HNMR spectrum;

[0022] Figure 2 This is a high-resolution mass spectrum of the intermediate synthesized in Example 1 of the present invention;

[0023] Figure 3 The nuclear magnetic resonance of the fluorescent molecular probe HBA synthesized in Example 1 of the present invention is 1 HNMR spectrum;

[0024] Figure 4 The nuclear magnetic resonance of the fluorescent molecular probe HBA synthesized in Example 1 of the present invention is 13 CNMR images;

[0025] Figure 5This is a high-resolution mass spectrum of the fluorescent molecular probe HBA synthesized in Example 1 of the present invention;

[0026] Figure 6 This is a high-resolution mass spectrum of the fluorescent molecular probe HBA synthesized in Example 1 of the present invention after reacting with H2S;

[0027] Figure 7 The infrared spectrum and nuclear magnetic resonance of the fluorescent molecular probe HBA synthesized in Example 1 of the present invention after reacting with H2S 1 HNMR diagram, including (A) infrared spectrum, (B) nuclear magnetic resonance 1 HNMR spectrum;

[0028] Figure 8 This is a schematic diagram of the reaction principle between the fluorescent molecular probe HBA synthesized in Example 1 of the present invention and H2S;

[0029] Figure 9 The fluorescence spectra of the fluorescent molecular probe HBA synthesized in Example 1 of the present invention in DMSO / H2O mixtures with different water component contents are 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% respectively;

[0030] Figure 10 : These are fluorescence images of the fluorescent molecular probe HBA synthesized in Example 1 of the present invention in DMSO / H2O mixtures with different water component contents, wherein the water component contents are 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% (under 365nm ultraviolet light excitation);

[0031] Figure 11 This is the ultraviolet absorption spectrum of the fluorescent molecular probe HBA synthesized in Example 1 of the present invention before and after the reaction with H2S;

[0032] Figure 12 This is a graph showing the selectivity of the fluorescent molecular probe HBA synthesized in Example 1 of the present invention for H2S. The numbers 1 to 30 in (B) 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;

[0033] Figure 13 is the fluorescence spectrum of the fluorescent molecular probe HBA synthesized in Example 1 of the present invention as the fluorescence intensity changes with the H2S concentration;

[0034] Figure 14 is the linear relationship between the fluorescent molecular probe HBA synthesized in Example 1 of the present invention and the H2S concentration;

[0035] 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 the present invention with H2S;

[0036] Figure 16 Schematic diagram of the change in fluorescence intensity of the fluorescent molecular probe HBA synthesized in Example 1 of the present invention with pH before and after the reaction with H2S;

[0037] Figure 17 is the absolute fluorescence quantum yield of the fluorescent molecular probe HBA synthesized in Example 1 of the present invention before and after the reaction with H2S;

[0038] Figure 18 is the relative cell viability of HeLa cells in different concentrations of the fluorescent molecular probe HBA synthesized in Example 1;

[0039] Figure 19 Schematic diagram of the fluorescent molecular probe HBA synthesized in Example 1 of the present invention imaging H2S cells;

[0040] Figure 20 Oil red O staining and hematoxylin-eosin (H&E) staining of liver tissues of mice in the control group and NAFLD model group over time in Example 7;

[0041] Figure 21 The results of ALT and AST enzyme level measurements in the control group and NAFLD model group mice in Example 7;

[0042] Figure 22 These are the fluorescence imaging images and fluorescence intensities of 14-day-old mice and their organ tissues in the control group, probe group, exogenous hydrogen sulfide group, and NAFLD model group in Example 7, including (A) mouse fluorescence imaging, (B) mouse fluorescence intensity, (C) organ fluorescence imaging, and (D) liver tissue fluorescence intensity. DETAILED DESCRIPTION

[0043] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0044] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0045] Secondly, the term "one embodiment" or "embodiment" 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 various places throughout this specification does not necessarily refer to the same embodiment, nor does it constitute a separate or selective embodiment that is mutually exclusive of other embodiments.

[0046] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods, and instruments used are conventional in the art and can be obtained commercially by those skilled in the art unless otherwise specified.

[0047] In the following examples, comparative examples and test experiments 1 H NMR spectroscopy and 13 C nuclear magnetic resonance spectra were obtained using an AVANCE 600 MHz spectrometer; pH measurements were performed using a pH-FE20 acidity meter from Mettler-Toledo (Shanghai); high-resolution mass spectra (HRMS) were obtained using a Waters Xevo UPLC / G2-SQ Tof MS spectrometer; UV-visible 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 sample 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 X5PRO instrument.

[0048] In the present invention, M represents mol / L, mM represents mmol / L, and μM represents μmol / L.

[0049] Example 1

[0050] A method for synthesizing a fluorescent molecular probe HBA based on a triphenylamine derivative comprises the following steps:

[0051] S1, triphenylamine boric acid (3.47g, 12mmol), 5-bromothiophene-2-carboxaldehyde (1.91g, 10mmol), potassium carbonate (2.49g, 18mmol) and tetrakis (triphenylphosphine) palladium (0.139g, 0.12mmol) are added in 30mL tetrahydrofuran / water (1 / 1, v / v) mixed solution, under nitrogen protection, reflux reaction at 70 ℃, until TLC point plate monitors that raw material spot disappears, stop reaction, reaction system is cooled to room temperature, with dichloromethane (30mL×3 times) extraction three times, take out organic layer, organic layer is extracted three times with saturated sodium chloride solution (15mL×3 times), separate organic layer, add anhydrous sodium sulfate drying, solvent is spin-dried and obtains sticky black crude product.Finally, separation and purification is carried out by column chromatography, obtain green solid, be intermediate 5-(4-(diphenylamine) phenyl) thiophene-2-carboxaldehyde;

[0052] S2. The intermediate (100 mg, 0.28 mmol) was dissolved in 20 mL of ultra-dry ethanol, 3-amino-4-hydroxybenzoic acid (45 mg, 0.30 mmol) was added, and the reaction was heated under reflux at 75-85 ° C for 4.5 h until the reaction was complete as monitored by TLC plate. The reaction was stopped and the reaction system was cooled to room temperature. A large amount of precipitate was produced. It was filtered and then rinsed three times with hot ethanol (20 mL) to obtain an orange-red solid powder, i.e., a fluorescent molecular probe (HBA) based on a triphenylamine derivative, with a yield of 89.2% and a melting point of HBA of 268.5-269.7 ° C.

[0053] The synthetic route of the intermediate 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde in this embodiment is as follows:

[0054]

[0055] The intermediate was subjected to NMR spectroscopy ( 1 H NMR) detection, the results are as follows Figure 1 As shown, its structural characterization is as follows: 1 H 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 performed on the intermediate, and the results were as follows: Figure 2 shown.

[0056] The molecular formula of HBA obtained in this example is C 30 H 22N2O3S, the HBA obtained in this example was subjected to nuclear magnetic resonance spectroscopy ( 1 H NMR) detection, the results are as follows Figure 3 As shown, its structural characterization is as follows: 1 H NMR (600 MHz, 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 = 3.8 Hz, 1H), 7.38-7.32 (m, 4H), 7.13-7.06 (m, 6H), 6.99 (dd, J = 8.4, 4.4 Hz, 3H); The HBA obtained in this example was subjected to nuclear magnetic resonance spectroscopy ( 13 C NMR) detection, the results are as follows Figure 4 As shown, its structural characterization is as follows: 13 CNMR (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; HBA was detected by high-resolution mass spectrometry (HRMS), and the results were as follows: Figure 5 As shown. Figures 1 to 5 It can be seen that the intermediate and HBA were successfully prepared.

[0057] The HBA synthesized in this example was mixed with a DMSO / H2O mixture (2 / 8, v / v, 0.01 M Hepes, pH = 7.4) to obtain a test solution. The HBA concentration in the test solution was 1×10 -5 mol / L, prepare multiple test solutions for later use.

[0058] from Figure 5 The positive ion peak of HBA was found to be m / z 491.1430 ([HBA+H] + ). Figure 6 This is the HRMS spectrum detection spectrum after the reaction of HBA and H2S. Figure 6 A new mass-to-charge ratio signal m / z 521.0990 ([HBA+H2S+H] + ).

[0059] The FT-IR test results before and after the action of HBA and H2S are as follows: Figure 7 As shown in (A), at 1661cm -1The peak at 1123 cm is due to the C=N double bond functional group of HBA. When H2S is introduced, the absorption of the C=N double bond is weakened. The probe HBA reacts with H2S to form the stretching vibration of -SO- and -CO- bonds. -1 and 966cm -1 The results of FT-IR showed that the probe HBA and H2S underwent a cyclization reaction to form HBA-H2S compound.

[0060] In order to explore the mechanism of interaction between HBA and H2S, the system before and after the action of HBA and H2S was subjected to nuclear magnetic resonance spectroscopy ( 1 H NMR) detection (the reaction system of HBA and H2S is obtained by adding H2S to the test solution, and the amount of H2S added is 0.5 times and 2.0 times the molar number of HBA, respectively). The results are as follows Figure 7 As shown in (B), the proton signals at δ8.83 ppm (Ha) and δ7.67 ppm (Hb) are due to the hydrogen of the hydroxyl group and the hydrogen of the carbon-nitrogen double bond in the fluorescent molecular probe HBA, respectively. It can also be seen that when the amount of HS added increases, the proton signal of Ha significantly weakens and becomes broader, shifting downfield. Furthermore, with increasing HS addition, the proton signal at the C=N double bond (Hb) splits and weakens. These results indicate that HS coordinates with the oxygen of the C=N double bond and the hydroxyl group in the fluorescent molecular probe HBA, forming a cyclization reaction to form a six-membered ring, which leads to HBA fluorescence quenching.

[0061] The reaction process between the probe HBA and H2S is as follows Figure 8 The fluorescent molecular probe HBA contains a triphenylamine group, a lipophilic, photostable, and strong electron donor, resulting in strong fluorescence. When H2S is added, the probe HBA reacts with the H2S to form a six-membered ring containing a -SO- functional group, leading to fluorescence quenching.

[0062] Example 2

[0063] Study on AIE characteristics of probe HBA:

[0064] 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 HBA concentrations of 1×10 -5 M), the water fraction (fw) in the DMSO / H2O mixture was 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%, respectively.

[0065] Figure 9The figure is a schematic diagram showing the change of HBA fluorescence intensity with the proportion of water components in the DMSO / H2O mixture. It can be seen that, whether under 365nm or 420nm ultraviolet irradiation, the fluorescence intensity of HBA in pure aqueous solution is stronger than that in pure DMSO solvent. When the water component (fw) in the DMSO / H2O mixture is less than 60%, the fluorescence intensity of HBA is weak. When fw increases to 70%, the fluorescence intensity of HBA is significantly enhanced, reaching a maximum value when fw is 80%. The test results show that the HBA prepared in this embodiment has AIE characteristics. The fluorescence images of HBA in DMSO / H2O mixtures with different water components under 365nm ultraviolet light excitation are shown in Figure 2. Figure 10 shown.

[0066] Example 3

[0067] HBA selectivity test for H2S:

[0068] H2S was added to the test solution of Example 1 (the addition amount was 10.0 times the equivalent of HBA). The UV absorption spectra of the test solution and the solution after H2S was added were as follows: Figure 11 As shown in the figure, it can be seen that in the absence of H2S, the absorption peak of HBA appears at 420nm. After adding H2S, the maximum absorption peak blue-shifts to 410nm, and its absorption value increases slightly.

[0069] H2S and other analytes (anions, amino acids and antibiotics) were added to the test solution of Example 1: - 、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 is 10.0 times the equivalent of HBA. The fluorescence spectra of the test solution and the test solution after adding H2S and other analytes are as follows: Figure 12 As shown in (A), the HBA probe exhibits significant fluorescence emission at 528 nm, with the solution exhibiting bright yellow fluorescence. However, upon addition of HS, the HBA probe's fluorescence is quenched, darkening in color and significantly decreasing in intensity. However, upon addition of other analytes, HBA still exhibits strong fluorescence intensity, indicating that other analytes have no effect on HBA.

[0070] 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 does not interfere with the recognition of H2S by the probe HBA, which indicates that the probe HBA has a good anti-interference ability for the recognition of H2S, and the presence of other analytes in the system will not affect its recognition effect.

[0071] These test results demonstrate that H2S quenches the fluorescence of the probe HBA, a unique property compared to other analytes. Other analytes do not interfere with H2S recognition by the probe HBA, demonstrating its robust anti-interference capability. The presence of other analytes in the system does not affect its recognition. Therefore, the probe HBA can be used as a specific fluorescent molecular probe for H2S detection.

[0072] Example 4

[0073] HBA detection limit determination:

[0074] Different amounts of H2S were added to the test solution of 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), and the changes in HBA fluorescence intensity under different H2S concentration conditions were detected. Figure 13 As shown in Figure 2, it can be seen that the addition of different concentrations of H2S leads to a significant decrease in the fluorescence intensity of HBA at 528 nm, and as the H2S concentration increases, the fluorescence intensity gradually weakens. Figure 14 As shown in Figure 2, when the H2S concentration range is 10-60 μM, there is a strong linear relationship between the HBA fluorescence intensity and the H2S concentration. 2 The value is 0.9913.

[0075] The detection limit is an important indicator of probe sensitivity and can be calculated according to the following equation:

[0076]

[0077] Where SD refers to the standard deviation of the blank sample, K is the signal-to-noise ratio, which is generally 3, and S is the slope of the linear fitting curve of the analyte concentration and probe fluorescence intensity obtained from the probe concentration titration test.

[0078] According to calculation, the detection limit of H2S detected by HBA in the present invention is 0.45 nM.

[0079] Example 5

[0080] Study on the time response of probe HBA to H2S:

[0081] 50 μL of 0.01M H2S solution was added to the test solution of Example 1, and the fluorescence intensity of the test solution and the test solution after H2S was added were detected. The results are as follows: Figure 15 As shown in the figure, when H2S was not added, the fluorescent molecular probe HBA showed no obvious fluctuation in fluorescence intensity within 10 minutes, indicating that HBA has good stability. After H2S was added, the fluorescence intensity dropped immediately and remained stable over time within 10 minutes without fluctuation. This shows that the probe HBA exhibits a fast response time when detecting H2S levels and the fluorescence signal is relatively stable, which is conducive to improving the accuracy of H2S test results.

[0082] This embodiment also conducted a spectrum response test of the probe HBA at different pH values ​​(2.0-12.0). 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 and 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).

[0083] In order to determine the optical properties of the probe HBA and explore the reasons for the rapid response behavior of H2S, the absolute fluorescence quantum yield of HBA before and after the addition of H2S was measured in the 500-750nm spectral range. Figure 17 The final results were calculated using Fluoracel software. The probe HBA exhibited a quantum yield as high as 18.51%. Upon addition of H2S, H2S coordinated with the C=N double bond and the oxygen in the hydroxyl group of the HBA probe, forming a cyclization reaction to form a six-membered ring. This six-membered ring formation disrupted the rigid planar conformation of the probe HBA, causing the quantum yield to drop to 9.68%. These results demonstrate that HBA possesses excellent optical properties, including a high fluorescence quantum yield and a stable fluorescence response, which contributes to its rapid response during H2S level detection.

[0084] Example 6

[0085] Study on the cytotoxicity of the probe HBA and its H2S cell imaging:

[0086] The CCK-8 assay was used to determine the effect of HBA on HeLa cell viability and analyze the cytotoxicity of HBA. The specific methods are as follows:

[0087] Human cervical cancer cells (HeLa cells) in logarithmic growth phase were adjusted to an appropriate concentration and counted before seeding into 96-well plates at a density of 1.5 × 10⁴ cells per well (final volume per well, typically 100 μL). The cells were pre-incubated in a humidified incubator at 37°C and 5% CO₂ for 12 h to allow attachment and stable growth. The culture medium was then removed and fresh culture medium containing various concentrations of HBA (0, 5, 10, 15, 20, and 25 μM) was added. The 0 μM group served as a negative control (containing culture medium or solvent alone) without HBA. The cells were incubated for another 12 h at 37°C and 5% CO₂. The HBA-containing culture medium was aspirated, and the cells were gently washed three times with sterile phosphate-buffered saline (PBS) to remove 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 a 37° C., 5% CO 2 incubator for 2 h in the dark, and the absorbance (OD value) of each well was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) microplate reader.

[0088] Background control wells (containing only CCK-8 reagent and culture medium, without cells) were set up to deduct background interference.

[0089] The formula for calculating relative cell viability is as follows:

[0090]

[0091] Among them, background OD 450 The absorbance of wells containing only CCK-8 reagent and culture medium without cells is shown.

[0092] The relative cell viability results are as follows Figure 18 As shown, when the HBA concentration was 0-25 μM, the viability of HeLa cells remained above 95%, indicating that the probe HBA had extremely low biological toxicity.

[0093] HeLa cells were seeded in 24-well plates containing 10% fetal bovine serum, penicillin (100 μg / mL) and streptomycin (100 μg / mL), and the HeLa cells were adhered overnight in a 5% carbon dioxide environment. Subsequently, the cultured HeLa cells were divided into four groups. The first group served as the control group and was cultured in the bioprobe HBA solution for 1 hour. The second group was incubated with the bioprobe HBA medium containing 10 μM hydrogen sulfide for 1 hour. The third and fourth groups of cells were incubated with HBA medium containing 50 and 100 μM H2S, respectively, for 1 hour. The HeLa cells were then washed twice with buffer, and the coverslip containing the cells was placed on a slide for confocal microscopy observation. The results are shown in Figure 2. Figure 19 As shown in Figure 2, HeLa cells co-incubated with HBA for 1 hour showed obvious green fluorescence. As the concentration of H2S increased, the green fluorescence signal of HeLa cells co-incubated with HBA+H2S for 1 hour gradually weakened. Figure 19 (B) It can also be seen that this indicates that the probe HBA can quickly detect H2S in living cells, and the probe HBA does not affect cell imaging and can be used in biological experiments.

[0094] Example 7

[0095] Application of probe HBA for detecting H2S in NAFLD mouse model:

[0096] In this example, 7- to 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 Ethical Experimentation Committee of Northeast Agricultural University. During the experiments, all mice were properly cared for and euthanized. They received unlimited food and water in a sterile environment and were maintained under a 12-hour light / dark cycle.

[0097] The mice in this embodiment were divided into the following groups: the first group was the control group, which was fed for 0 days and intraperitoneally injected with PBS solution; the second group was the probe group, which was fed for 0 days and intraperitoneally injected with HBA (100 μL, 0.1 mM); the third group was the exogenous hydrogen sulfide group, which was fed for 0 days and injected with H2S (100 μL, 1 mM) and injected with HBA (100 μL, 0.1 mM) again 12 hours later; the fourth group was the NAFLD model group, that is, the endogenous hydrogen sulfide group. The mice in this group were fed for 7, 14 and 21 days, respectively, and were intraperitoneally injected with HBA (100 μL, 0.1 mM). The NAFLD model was established as follows: a NAFLD mouse model was established by a methionine-choline-deficient (MCD) diet combined with dexamethasone injection. The mice were continuously fed with a feed containing 60% MCD and intraperitoneally injected with dexamethasone (10 mg / kg / day). ex =488nm and λem The fluorescent images of mice in the control group, probe group, exogenous hydrogen sulfide group and NAFLD model group for 14 days were captured by the Tanon ABL X5PRO animal real-time imaging system with a fluorescence filter set of 535 nm. The control group mice were fed for 0 days, anesthetized and injected with PBS solution through the tail vein. The probe group mice were fed for 0 days, anesthetized and injected with H2S (100 μL, 1 mM) intraperitoneally. The mice in the exogenous hydrogen sulfide group were fed for 0 days, anesthetized and injected with H2S (100 μL, 1 mM) through the tail vein. Twelve hours later, HBA (100 μL, 0.1 mM) was injected again. The mice in the NAFLD model group were anesthetized and injected with HBA (100 μL, 0.1 mM) through the tail vein on days 7, 14, and 21. Two hours later, the mice were euthanized, and their major organs (heart, liver, kidney, spleen, and lungs) were collected for fluorescence images of the major organs. The liver tissues of the control group and NAFLD model group mice were collected for Oil Red O staining and hematoxylin-eosin (H&E) staining.

[0098] The liver tissue staining images of mice in the control group and NAFLD model group are shown in the following figure: Figure 20 As shown in the figure, the liver pathological changes were evaluated by Oil Red O staining and Hematoxylin-Eosin (H&E) staining. It can be seen that compared with the mice fed a normal diet, the accumulation of fat droplets in the liver tissue of the NAFLD model group mice increased significantly with the passage of feeding time (the positive area of ​​Oil Red O staining expanded, and H&E staining showed that round vacuoles of varying sizes appeared in the hepatocytes, which was caused by the accumulation of fat droplets), indicating that the symptoms of NAFLD in mice gradually worsened.

[0099] Blood samples were collected from the eye sockets 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 evaluating liver function and liver damage. Elevated ALT and AST enzyme levels indicate that liver cell damage enters the blood, leading to liver damage. ALT and AST enzyme level measurement results Figure 21 As the feeding time increased, the levels of ALT and AST in the liver tissue of NAFLD mice were higher than those in the control group, indicating that the degree of liver damage in NAFLD mice was higher.

[0100] The results of Oil Red O staining and Hematoxylin-Eosin (H&E) staining, as well as the measurement results of ALT and AST enzyme levels, showed that the NAFLD mouse model was successfully established in this example.

[0101] The fluorescence imaging and fluorescence signal intensity test results of 14-day-old mice in the control group, probe group, exogenous hydrogen sulfide group, and NAFLD model group are shown in Figure 2. Figure 22 (A) and Figure 22As shown in (B) (where Control represents the control group). The fluorescence signal was strongest in the HBA-injected group, while the fluorescence signal in the 14-day NAFLD model mice 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 weakened compared to the second group. This demonstrates that the fluorescent molecular probe HBA of the present invention can detect both endogenous and exogenous H2S.

[0102] The fluorescence imaging images of the main organs (heart, liver, kidney, spleen and lung) of the mice in the control group, probe group, exogenous hydrogen sulfide group and NAFLD model group on day 14 and the test results of the fluorescence signal intensity of the liver tissue are shown in the figure below: Figure 22 (C) and Figure 22 (D) The results showed that the biological probe HBA mainly accumulated in the liver and lungs of mice. The fluorescence signal of the organs of mice injected with HBA was the strongest. The fluorescence signal of mice in the exogenous hydrogen sulfide group and the NAFLD model group was weakened compared with the probe group. This indicates that H2S is overproduced in mice during NAFLD and also shows that the probe HBA of the present invention can be used to identify and detect H2S levels.

[0103] The above results indicate that the probe HBA shows good potential in in vivo imaging and is able to detect endogenous H2S levels in mice, and can be used to detect abnormal H2S levels caused by NAFLD.

[0104] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A triphenylamine derivative, characterized in that The triphenylamine derivative structural formula is as follows:

2. A method for preparing the triphenylamine derivative according to claim 1, characterized in that: The method comprises the following steps: dissolving 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde and 3-amino-4-hydroxybenzoic acid in a solvent, heating and refluxing the mixture for reaction, filtering and recrystallizing the product after the reaction is completed, and obtaining a triphenylamine derivative.

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

4. The preparation method according to claim 2, characterized in that The solvent is super dry ethanol; the heating temperature is 65-75°C, and the reaction time is 4-6 hours.

5. The preparation method according to claim 2, characterized in that The preparation method of 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde is as follows: triphenylamine boric acid, 5-bromothiophene-2-carboxaldehyde, potassium carbonate and tetrakis(triphenylphosphine)palladium are added to a solvent, and the mixture is heated under reflux for reaction under nitrogen protection.

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 tetrakis(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°C and the reaction time is 6-8h.

9. Use of the triphenylamine derivative according to claim 1, characterized in that: The triphenylamine derivative is used as a fluorescent molecular probe.

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

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