Novel fluorescent probe based on triphenylamine and its preparation method and application

Abstract

The present invention discloses a novel fluorescent probe based on triphenylamine, a preparation method and application thereof, and the preparation method comprises the following steps: S1. dissolving 4-methoxyaniline and 1-bromo-4-iodobenzene in solvent A, then adding copper iodide, 1,10-phenanthroline and potassium tert-butoxide to react to obtain compound 1; S2. dissolving compound 1 obtained by S1, (5-formylthiophene-2-yl)boric acid and anhydrous potassium carbonate in solvent B, then adding PdCl2 (dppf) to react to obtain compound 2; S3. dissolving compound 2 obtained by S2 and malononitrile in solvent C, then adding triethylamine to react to obtain a TPA-CN probe. The TPA-CN probe is used to monitor exogenous and endogenous SO2 and viscosity changes in living cells. The present invention can perform dual imaging of SO2 and viscosity in biological systems and food samples.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical detection, and in particular to a novel fluorescent probe based on triphenylamine, a preparation method thereof and an application thereof. Background Art

[0002] Sulfur dioxide (SO2) is a toxic environmental pollutant notorious for its unpleasant odor, mainly derived from the burning of fossil fuels and industrial development. SO2 is easily converted into sulfite and bisulfite (SO3 2- / HSO3 - ) derivatives. Since SO2 derivatives have excellent antiseptic, antioxidant and antibacterial abilities, they are often used as additives in food, beverage and pharmaceutical fields to protect products during production and storage. Therefore, SO2 can be absorbed by the human body through various pathways, and excessive intake of SO2 derivatives also seriously affects human health. In addition, as an important gaseous signaling molecule, SO2 can not only be produced in living systems through enzymatic reactions of sulfur-containing amino acids and hydrogen sulfide, but also plays a vital role in regulating the physiological functions of organisms. Normal levels of endogenous SO2 derivatives can regulate redox balance, inflammatory response, vascular function, etc. However, abnormal levels of SO2 derivatives may lead to the occurrence of certain diseases, such as cardiovascular disease, respiratory disease, nervous system disorders, cancer, etc. Therefore, it is of great significance to develop a method that can detect SO2 derivatives in real time with high selectivity and high sensitivity in living systems and real foods.

[0003] As a parameter of the intracellular microenvironment, viscosity is associated with numerous physiological activities, such as membrane fluidity, interactions between biomolecules, signal regulation, and diffusion-mediated cellular processes. Changes in the viscosity of biological systems can serve as an indicator of changes in the relevant physiological environment and are important for identifying related diseases. Abnormal viscosity levels can disrupt cellular function and cause diseases such as diabetes, cardiovascular disease, and cancer. Furthermore, studies suggest that viscosity and SO₂ levels may be linked to organelle dysfunction and apoptosis. Therefore, dual SO₂ and viscosity measurements are crucial for biological applications.

[0004] Compared to traditional methods for detecting SO₂ levels and viscosity, fluorescence imaging has attracted widespread attention due to its noninvasive, highly sensitive, easy-to-use, and real-time imaging capabilities. Currently, a variety of fluorescent probes have been developed for SO₂ or viscosity detection. However, most of these probes recognize individual analytes by turning fluorescence patterns on or off, making them susceptible to interference from test conditions and environmental factors. Ratiometric fluorescent probes can mitigate these adverse effects. Furthermore, some bifunctional fluorescent probes capable of simultaneously detecting SO₂ and viscosity have been reported. Unfortunately, most probes are primarily limited by aggregation quenching and relatively small Stokes shifts, restricting their further application in in vivo SO₂ and viscosity detection. In contrast, aggregation-induced emission (AIE) materials offer significant advantages in addressing these issues. Their considerable stability, high quantum yield, normal aggregate-state fluorescence, and strong photostability make them highly promising for imaging applications. Triphenylamine, a typical AIE molecule, is commonly used in the design of AIE probes due to its strong intramolecular rotation ability and twist intramolecular charge transfer (TICT) mechanism. AIE probes that can simultaneously detect SO2 and viscosity are relatively rare. Therefore, ratiometric fluorescent probes with AIE properties have great potential for monitoring SO2 and viscosity in complex environments. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a novel fluorescent probe based on triphenylamine and a preparation method and application thereof in view of the deficiencies in the above-mentioned prior art.

[0006] In order to solve the above technical problems, the technical solution of the present invention is:

[0007] A novel triphenylamine-based fluorescent probe having the following structural formula:

[0008]

[0009] See also Figure 1 A method for preparing the novel fluorescent probe based on triphenylamine as described above comprises the following steps:

[0010] S1. 4-methoxyaniline and 1-bromo-4-iodobenzene are dissolved in solvent A, and then copper iodide, 1,10-phenanthroline, and potassium tert-butoxide are added to react to obtain compound 1;

[0011] S2. Compound 1 obtained in S1, (5-formylthiophen-2-yl)boronic acid and anhydrous potassium carbonate are dissolved in solvent B, and then PdCl2(dppf) is added to react to obtain compound 2;

[0012] S3. Compound 2 obtained in S2 and malononitrile are dissolved in solvent C, and then triethylamine is added to react to obtain a TPA-CN probe.

[0013] As a preferred embodiment, the molar ratio of the 4-methoxyaniline, the 1-bromo-4-iodobenzene, the copper iodide, the 1,10-phenanthroline and the potassium tert-butoxide in S1 is 20:50:3:3:60.

[0014] As a preferred embodiment, the solvent A in S1 is toluene.

[0015] As a preferred embodiment, the molar ratio of the compound 1, the (5-formylthiophene-2-yl)boric acid, the anhydrous potassium carbonate and the PdCl2(dppf) in S2 is 1:3:10:0.03.

[0016] As a preferred embodiment, the solvent B in S2 is a mixture of toluene and methanol in a volume ratio of 1:1.

[0017] As a preferred embodiment, the molar ratio of the compound 2, the malononitrile and the triethylamine in S3 is 1:3:1.

[0018] As a preferred embodiment, the solvent C is dichloromethane.

[0019] A novel triphenylamine-based fluorescent probe as described above is used to monitor exogenous and endogenous SO2 and viscosity changes in living cells.

[0020] A novel triphenylamine-based fluorescent probe as described above was used to detect SO2 in water and food.

[0021] See also Figure 2 The new triphenylamine-based fluorescent probe has an ADA structure, with triphenylamine as the electron donor, thiophene as a π spacer to extend the conjugated structure, and malononitrile as the electron acceptor. As the viscosity increases, the intramolecular rotation of the new triphenylamine-based fluorescent probe is restricted, resulting in a significant increase in the red fluorescence intensity of the new triphenylamine-based fluorescent probe.

[0022] See also Figure 2 , a novel triphenylamine-based fluorescent probe specifically responds to SO2 derivatives via an addition reaction on the C=C double bond between thiophene and malononitrile, resulting in hindered intramolecular charge transfer (ICT) process and emitting blue fluorescence of the donor.

[0023] The beneficial effects of the present invention are as follows: the novel triphenylamine-based fluorescent probe (TPA-CN probe) described in the present invention can perform dual imaging of SO2 and viscosity in biological systems and food samples. The TPA-CN probe achieves viscosity response through the TICT mechanism and SO2 response through the ICT mechanism. The TPA-CN probe has a fast response rate to SO2 (90s), good sensitivity (0.12μM), wide pH adaptability, good selectivity, and good anti-interference ability. The TPA-CN probe has low cytotoxicity and good biocompatibility in biological environments, and can detect viscosity changes of tumor cells as well as endogenous and exogenous SO2. In addition, the TPA-CN probe has been successfully used to detect SO2 in actual samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The synthetic route of the novel triphenylamine-based fluorescent probe of the present invention is as follows;

[0025] Figure 2 This is a diagram showing the detection principle of the novel triphenylamine-based fluorescent probe of the present invention;

[0026] Figure 3 is the H NMR spectrum of compound 1 of the present invention;

[0027] Figure 4 is the H NMR spectrum of compound 2 of the present invention;

[0028] Figure 5 is the H NMR spectrum of the TPA-CN probe of the present invention;

[0029] Figure 6 This is a high-resolution mass spectrum of compound 1 of the present invention;

[0030] Figure 7 This is a high-resolution mass spectrum of compound 2 of the present invention;

[0031] Figure 8 This is a high-resolution mass spectrum of the TPA-CN probe of the present invention;

[0032] Figure 9 is the AIE characteristic diagram of the TPA-CN probe of the present invention;

[0033] Figure 10 This is the absorption spectrum of the TPA-CN probe of the present invention in response to SO2;

[0034] Figure 11 The fluorescence spectrum of the TPA-CN probe of the present invention in response to SO2;

[0035] Figure 12 This is a high-resolution mass spectrum of the TPA-CN probe of the present invention after responding to SO2;

[0036] Figure 13 This is a graph showing the dependence of the TPA-CN probe of the present invention on SO2 concentration;

[0037] Figure 14 is the fluorescence intensity ratio of the probe TPA-CN of the present invention (I 458 / I 704 ) and SO2 concentration;

[0038] Figure 15 This is a time-dependent spectrum of the TPA-CN probe of the present invention and SO2;

[0039] Figure 16 This is a relationship diagram of the effect of pH on the TPA-CN probe of the present invention;

[0040] Figure 17 This is a selectivity experiment diagram of the TPA-CN probe of the present invention;

[0041] Figure 18 This is an anti-interference experiment diagram of the TPA-CN probe in the present invention;

[0042] Figure 19 The fluorescence spectrum of the TPA-CN probe of the present invention in a mixture of methanol and glycerol at different ratios is shown;

[0043] Figure 20 The fluorescence spectra of the TPA-CN probe of the present invention in different solvents;

[0044] Figure 21 This is a graph showing the cytotoxicity of the TPA-CN probe of the present invention in HeLa cells;

[0045] Figure 22 The uptake of the TPA-CN probe of the present invention in HeLa cells;

[0046] Figure 23 The present invention is a graph showing the response of the TPA-CN probe to exogenous SO2 in HeLa cells;

[0047] Figure 24 This is a graph showing the response of the TPA-CN probe to endogenous SO2 in HeLa cells;

[0048] Figure 25 This is a graph showing the response changes of the TPA-CN probe to viscosity in HeLa cells according to the present invention. DETAILED DESCRIPTION

[0049] The structural principle and working principle of the present invention are further described in detail below with reference to the accompanying drawings.

[0050] Example

[0051] Synthesis of a new fluorescent probe based on triphenylamine

[0052] S1. Under stirring conditions, 1.23g of 4-methoxyaniline and 7.07g of 1-bromo-4-iodobenzene were dissolved in 20mL of toluene. Then 285mg of copper iodide, 270mg of 1,10-phenanthroline and 3.36g of potassium tert-butoxide were added. Under a nitrogen atmosphere, refluxed for 12 hours, then cooled to room temperature, concentrated by vacuum distillation to obtain a crude product, which was extracted with dichloromethane, dried over anhydrous sodium sulfate, and concentrated under reduced pressure on a rotary evaporator. The product was purified by column chromatography using petroleum ether as an eluent to obtain 2.1g of compound 1 as a white solid with a yield of 48.5%;

[0053] S2. 866 mg of compound 1, 936 mg of (5-formylthiophen-2-yl)boronic acid, and 2.76 g of anhydrous potassium carbonate were dissolved in a mixture of 10 mL of toluene and 10 mL of methanol and stirred under a nitrogen atmosphere for several minutes. 42 mg of PdCl2(dppf) was added, and the mixture was heated to 75°C under a nitrogen atmosphere for 12 hours. After the reaction was complete, the mixture was cooled to room temperature and concentrated under reduced pressure to obtain a crude product, which was then extracted with dichloromethane, dried over anhydrous sodium sulfate, and concentrated under reduced pressure on a rotary evaporator. The product was purified by column chromatography using a mixture of petroleum ether and ethyl acetate in a volume ratio of 10:1 as eluent to obtain 630 mg of compound 2 as a yellow solid in a 63.6% yield.

[0054] S3. Dissolve 495 mg of compound 2 and 198 mg of malononitrile in 10 mL of dichloromethane and stir. Add 150 μL of triethylamine and stir the reaction solution at room temperature for 6 hours. Then, dilute with dichloromethane, wash with water, dry over anhydrous sodium sulfate, and concentrate on a rotary evaporator. The product is purified by column chromatography using dichloromethane as the eluent to obtain 416 mg of the TPA-CN probe as a black solid, with a yield of 70.4%.

[0055] Test Example 1

[0056] Synthesis and characterization of TPA-CN probe

[0057] 1) Using a nuclear magnetic resonance instrument, the compound 1 prepared by Example S1, the compound 2 prepared by Example S2, and the TPA-CN probe prepared by Example S3 were subjected to nuclear magnetic resonance spectroscopy experiments. The results are as follows: Figure 3 、 Figure 4 and Figure 5 As shown;

[0058] 2) High-resolution mass spectrometry experiments were performed on compound 1 prepared by Example S1, compound 2 prepared by Example S2, and TPA-CN probe prepared by Example S3 using a dual-thermal mass spectrometer. The results are as follows: Figure 6 、 Figure 7 and Figure 8 As shown;

[0059] Depend on Figure 3-Figure 8 As can be seen, triphenylamine compounds have been used as fluorophores in various AIE probes due to their AIE properties and ease of synthesis and modification. Therefore, a 4-methoxytriphenylamine skeleton was selected as the electron donor, two thiophene groups were introduced to extend conjugation and electron donation, and a malononitrile unit was used as the electron acceptor. Malononitrile has strong electron-withdrawing ability, which favors Michael addition reactions at the cyano-vinyl double bond. Therefore, a near-infrared ratiometric SO2 fluorescent probe TPA-CN with an ADA structure was successfully designed and constructed based on the ICT mechanism.

[0060] Test Example 2

[0061] AIE properties of the TPA-CN probe

[0062] Experimental steps: 1.2 mg of the TPA-CN probe prepared in the example was dissolved in dimethyl sulfoxide (DMSO) to prepare test solutions with dimethyl sulfoxide contents of 0%, 10%, 20%, 30%, 40%, 45%, 50%, 60%, 70%, 80%, 90% and 95%, respectively. The TPA-CN concentration was 10 μM. The test solutions were tested using an emission spectrometer. The results are as follows: Figure 9 shown.

[0063] Depend on Figure 9 It can be seen that in the mixed solution with high DMSO content (more than 70%), the TPA-CN probe exhibits weak fluorescence. w ) increased from 40% to 95%, the fluorescence intensity of the TPA-CN probe at 704nm increased significantly, reaching a maximum value at 80% water content, 21.8 times that of pure DMSO. At high water fractions (90% and 95%), the fluorescence intensity decreased slightly due to the aggregation and dispersion of the TPA-CN probe. This change in fluorescence intensity is consistent with the AIE mechanism. The results show that the TPA-CN probe has obvious AIE properties in the DMSO / water system.

[0064] Test Example 3

[0065] Spectroscopic experiments of TPA-CN probe

[0066] Experimental steps: 1.2 mg of the TPA-CN probe prepared in the example was dissolved in DMSO to obtain a 1 mM probe stock solution, which was then diluted with PBS buffer (pH = 7.4) to obtain a 10 μM TPA-CN working solution (containing 50% DMSO). The TPA-CN working solution was tested using an absorption spectrometer. The results are as follows: Figure 10 As shown; then add HSO3 with a concentration of 200μM to the TPA-CN working solution - After the solution reacted for 3 minutes, a test solution (containing 50% DMSO) was obtained. The fluorescence spectrum of the test solution was measured using a fluorescence spectrometer at two excitation wavelengths: a 5×5 nm slit width (380 nm) and a 10×10 nm slit width (500 nm), recording emission wavelengths of 400-750 nm and 520-900 nm, respectively. The results are shown in Figure 2. Figure 11 As shown; then the high-resolution mass spectrometry experiment of the test solution was performed using a double-thermal mass spectrometer, and the results were as shown Figure 12 shown.

[0067] Depend on Figure 10 It can be seen that the TPA-CN probe has two different absorption peaks at 378nm and 550nm. It is worth noting that, compared with HSO3 - After the reaction, the absorption peak at 550 nm decreased significantly, while the absorption peak at 378 nm increased significantly. In addition, a visible color change from pink to colorless was observed, indicating that the TPA-CN probe can be used to identify SO2 in a colorimetric manner. Figure 11 It can be seen that adding HSO3 - After that, the fluorescence peak of TPA-CN probe under 500nm excitation is 704nm, and a new fluorescence signal of 458nm appears under 380nm excitation. Figure 12 It can be seen that at 794.4217[M+Na+H2O] - A peak at 37° is consistent with the target product, further verifying the nucleophilic addition mechanism of the probe. In conclusion, the TPA-CN probe can be applied to the dual emission ratiometric detection of SO2 derivatives.

[0068] Test Example 4

[0069] TPA-CN probe and HSO3 - Concentration change relationship experiment

[0070] Experimental steps: 1.2 mg of the TPA-CN probe prepared in Example 1 was dissolved in DMSO to obtain a 1 mM probe stock solution, which was then diluted with PBS buffer (pH = 7.4) to obtain a TPA-CN working solution. -The solution was added to the TPA-CN working solution to make the final concentration 10 μM. After reacting for 3 minutes, the detection solution (containing 50% DMSO) was obtained. The TPA-CN working solution was tested using an absorption spectrometer. The results were as follows: Figure 13 (a) As shown; the fluorescence spectrum of the test solution was measured using a fluorescence spectrometer at two excitation wavelengths: 5×5nm slit width (380nm) and 10×10nm slit width (500nm), recording emission wavelengths of 400-750nm and 520-900nm, respectively. The results are shown in Figure 13 (b) and Figure 13 (c) shows; recording the fluorescence intensity ratio (I 458 / I 704 ), the result is as follows Figure 13 (d) and Figure 14 shown.

[0071] Depend on Figure 13 It can be seen that with the increase of HSO3 - As the concentration increased from 0 μM to 200 μM, the absorption at 550 nm gradually decreased and blue-shifted to 504 nm, while the absorption peak at 378 nm slowly increased. Similarly, the fluorescence intensity of TPA-CN at 704 nm gradually weakened, while the fluorescence intensity at 458 nm was significantly enhanced. In addition, the ratio of fluorescence intensity before and after the reaction (I 458 / I 704 ) changes from 0.02 to 76.97, showing an increase of about 3848 times. Figure 14 It can be seen that I 458 / I 704 The logarithm of HSO3 - The concentration (0~50μM) showed a certain linear relationship (Y=0.03856X-1.368, R 2 =0.9917), and the lower limit of detection (LOD=3σ / k) was calculated to be 0.12 μM. These results indicate the sensitivity of TPA-CN probe for SO2 detection.

[0072] Test Example 5

[0073] TPA-CN probe and a certain concentration of HSO3 - and time-varying relationship experiments

[0074] Experimental steps: 1.2 mg of the TPA-CN probe prepared in Example 1 was dissolved in DMSO to obtain a 1 mM probe stock solution, which was then diluted with PBS buffer (pH = 7.4) to obtain a TPA-CN working solution; then HSO3 at a concentration of 200 μM was added to the TPA-CN working solution. -The solution was prepared to a final concentration of 10 μM, and the reaction was continued for 30 s, 60 s, 90 s, 120 s, 150 s, and 180 s to obtain a detection solution (containing 50% DMSO). The TPA-CN working solution was tested using an absorption spectrometer. The results were as follows: Figure 15 (a) As shown; the fluorescence spectrum of the test solution was measured using a fluorescence spectrometer at two excitation wavelengths: 5×5nm slit width (380nm) and 10×10nm slit width (500nm), recording emission wavelengths of 400-750nm and 520-900nm, respectively. The results are shown in Figure 15 (b) and Figure 15 (c) shows; recording the fluorescence intensity ratio (I 458 / I 704 ), the result is as follows Figure 15 (d) shown.

[0075] Depend on Figure 15 It can be seen that when HSO3 with a concentration of 200 μM is added to the TPA-CN working solution, - After solution, the fluorescence intensity ratio (I 458 / I 704 ) reaches its maximum value at about 90 seconds. This indicates that the probe can quickly respond to HSO3 - , which is crucial for the real-time detection of SO2 levels by the TPA-CN probe.

[0076] Test Example 6

[0077] TPA-CN probe and a certain concentration of HSO3 - and pH change relationship experiment

[0078] Experimental steps: 1.2 mg of the TPA-CN probe prepared in Example 1 was dissolved in DMSO to obtain a 1 mM probe stock solution, which was then diluted with PBS buffer (pH = 4, 5, 6, 7, 8, 9, and 10) to obtain a TPA-CN working solution; then, HSO3 at a concentration of 200 μM was added to the TPA-CN working solution. - The solution was added to a final concentration of 10 μM and reacted for 3 minutes to obtain a detection solution (containing 50% DMSO). The fluorescence spectra of the TPA-CN working solution and the detection solution were measured using a fluorescence spectrometer at two excitation wavelengths: a 5×5 nm slit width (380 nm) and a 10×10 nm slit width (500 nm), with emission wavelengths of 400 to 750 nm and 520 to 900 nm, respectively. The fluorescence intensity ratio (I 458 / I 704 ), the result is as follows Figure 16 shown.

[0079] Depend on Figure 16 It can be seen that in the absence of HSO3- Under different pH levels (4-9), the fluorescence intensity of the probe at 704nm and 458nm remained basically unchanged, which means that the TPA-CN probe is stable under physiological pH conditions. - The fluorescence intensity of the probe at 704nm and 458nm is significantly affected by pH value. - It is easier to deprotonate and increase nucleophilicity. At the same time, the value of the fluorescence intensity ratio (I 458 / I 704 ) further support the above results, indicating that the TPA-CN probe is suitable for monitoring SO2 in complex physiological environments.

[0080] Test Example 7

[0081] Selectivity experiments of TPA-CN probe

[0082] Experimental steps: 1.2 mg of the TPA-CN probe prepared in the example was dissolved in DMSO to obtain a 1 mM probe stock solution, which was then diluted with PBS buffer (pH = 7.4) to obtain a TPA-CN working solution; then, 200 μM of K + 、Na + Mg 2+ , Ca 2+ 、Fe 3+ 、Cl - , I - 、NO3 - 、CO3 2- 、AcO - 、NO2 - 、SO4 2- 、S2O3 2- 、SCN - , ClO - 、Hcy、Cys、GSH、SO3 2- and HSO3 - The solution was added to a final concentration of 10 μM and reacted for 3 min to obtain a detection solution (containing 50% DMSO). The fluorescence spectrum of the detection solution was measured using a fluorescence spectrometer at two excitation wavelengths: a 5×5 nm slit width (380 nm) and a 10×10 nm slit width (500 nm), with emission wavelengths of 400 to 750 nm and 520 to 900 nm, respectively. The fluorescence intensity ratio (I 458 / I 704 ), the result is as follows Figure 17 shown.

[0083] Depend on Figure 17 It can be seen that only HSO3- / SO3 2- Can lead to fluorescence intensity ratio (I 458 / I 704 ) was significantly enhanced, indicating that the TPA-CN probe was sensitive to HSO3 - / SO3 2- Has higher selectivity.

[0084] Test Example 8

[0085] Anti-interference experiment of TPA-CN probe

[0086] Experimental steps: 1.2 mg of the TPA-CN probe prepared in the example was dissolved in DMSO to obtain a 1 mM probe stock solution, which was then diluted with PBS buffer (pH = 7.4) to obtain a TPA-CN working solution; then, KCl was added to the TPA-CN working solution at a concentration of 200 μM. + 、Na + Mg 2+ , Ca 2+ 、Fe 3+ 、Cl - , I - 、NO3 - 、CO3 2- 、AcO - 、NO2 - 、SO4 2- 、S2O3 2- 、SCN - , ClO - 、Hcy、Cys、GSH、SO3 2- , then add 200μM HSO3 - The solution was added to a final concentration of 10 μM and reacted for 3 min to obtain a detection solution (containing 50% DMSO). The fluorescence spectrum of the detection solution was measured using a fluorescence spectrometer at two excitation wavelengths: a 5×5 nm slit width (380 nm) and a 10×10 nm slit width (500 nm), with emission wavelengths of 400 to 750 nm and 520 to 900 nm, respectively. The fluorescence intensity ratio (I 458 / I 704 ), the result is as follows Figure 18 shown.

[0087] Depend on Figure 18 It can be seen that even in the presence of potential interferents, I 458 / I 704 The above results indicate that the TPA-CN probe can specifically monitor SO2 in complex environments.

[0088] Test Example 9

[0089] Experiment on the relationship between TPA-CN probe and viscosity change

[0090] Experimental steps: 1.2 mg of the TPA-CN probe prepared in the example was dissolved in a glycerol / methanol mixture containing different glycerol contents (0-95%) to obtain a detection solution with a concentration of 10 μM. The fluorescence spectrum of the detection solution was measured using a fluorescence spectrometer at two excitation wavelengths: a 5×5 nm slit width (380 nm) and a 10×10 nm slit width (500 nm), with emission wavelengths of 400-750 nm and 520-900 nm, respectively. The fluorescence intensity ratio (I 458 / I 704 ), the result is as follows Figure 19 shown.

[0091] Depend on Figure 19 It can be seen that as the proportion of glycerol in the mixture increases (0-80%), the fluorescence intensity of TPA-CN at 640nm gradually increases. The logarithm of the fluorescence intensity at 640nm and the logarithm of the viscosity at 1.43-399cp show a good linear relationship (R 2 Interestingly, when the glycerol ratio exceeds 80% (85% to 95%), the maximum fluorescence intensity of TPA-CN is red-shifted to 704 nm, which is related to the AIE effect of the probe.

[0092] Test Example 10

[0093] Fluorescence spectra of TPA-CN probe in different solvent systems

[0094] Experimental steps: 1.2 mg of the TPA-CN probe prepared in the example was dissolved in different solvents (DMSO, DMF, MeCN, MeOH, PBS, THF, DCM and Gly) to obtain a TPA-CN working solution with a concentration of 10 μM. The fluorescence spectrum of the TPA-CN working solution was measured using a fluorescence spectrometer at two excitation wavelengths: a 5×5 nm slit width (380 nm) and a 10×10 nm slit width (500 nm), recording emission wavelengths of 400-750 nm and 520-900 nm, respectively. The results are shown in Figure 2. Figure 20 shown.

[0095] Depend on Figure 20 As can be seen, significant fluorescence emission appears in low-polarity solvents, which is the main reason for the AIE property of the TPA-CN probe. However, in glycerol solvent, the fluorescence emission is significantly enhanced and red-shifted due to the restricted rotation of the TPA-CN probe caused by the increase in viscosity. These results demonstrate the potential of the TPA-CN probe to monitor viscosity changes in complex environments.

[0096] Test Example 11

[0097] TPA-CN probe cytotoxicity experiment

[0098] Experimental steps: HeLa cells were first seeded in a 96-well plate and cultured in DMEM medium. When the cell density reached about 80%, the old medium was replaced with fresh medium containing different concentrations of TPA-CN (0-20 μM) for 24 hours, then washed twice with PBS buffer and cultured in fresh medium containing MTT salt for another 4 hours. The medium was then discarded and 150 μL DMSO was added. The absorbance value of the 96-well plate at 450 nm was recorded on a microplate reader (BioTek Cytation5). The results are as follows: Figure 21 shown.

[0099] Depend on Figure 21 It can be seen that even after incubation with 20 μM TPA-CN, the viability of HeLa cells still remained above 90%, indicating that the TPA-CN probe has good biocompatibility and low cytotoxicity.

[0100] Test Example 12

[0101] Cellular uptake experiment of TPA-CN probe in HeLa cells

[0102] Experimental steps: HeLa cells were transplanted into 6-well plates (3×10 5 After overnight incubation with DMEM medium containing TPA-CN (10 μM) for different time periods (0, 1, 2, 3, and 4 hours), the cells were washed with PBS buffer and the fluorescence images were observed on an inverted fluorescence microscope (Olympus IX73). Figure 22 shown.

[0103] Depend on Figure 22 It can be seen that with the extension of incubation time, the red fluorescence gradually increased and reached a maximum value at about 3 hours.

[0104] Test Example 13

[0105] SO2 imaging experiment using TPA-CN probe

[0106] Experimental steps: After pre-treating HeLa cells with DMEM medium containing TPA-CN (10μM) for 3 hours, replace it with DMEM medium containing NaHSO3 (100, 200, 400μM) and incubate the cells for 30 minutes, and obtain fluorescence images under a laser confocal microscope. Similarly, first incubate the cells with DMEM medium containing Na2S2O3 (250μM or 500μM) and DMEM medium containing GSH (500μM or 1mM) for 1 hour, then add DMEM medium containing TPA-CN (10μM) and incubate for 3 hours. The changes in the red and blue fluorescence intensity in the cells were observed under a laser confocal microscope. The results are as follows: Figure 23 shown.

[0107] Depend on Figure 23 It can be seen that when the control group was exposed to TPA-CN (10 μM) alone, the cells in the red channel showed obvious red fluorescence, and the cells in the blue channel showed a small amount of blue fluorescence. - ) After culturing the cells for 30 minutes, they were incubated with TPA-CN and found that with the increase of HSO3 - As the concentration increases, the red fluorescence gradually decreases and the blue fluorescence gradually increases. These results show that the probe can effectively monitor the changes in endogenous SO2 concentration in living cells.

[0108] Test Example 14

[0109] Experimental study on the use of TPA-CN probe to detect endogenous SO2 derivatives in cells

[0110] Experimental steps: HeLa cells were transplanted into 6-well plates (3×10 5 The cells were cultured overnight in a 5% spherical flask, and sodium thiosulfate and glutathione were added for 1 hour, respectively. The cells were then incubated with DMEM medium containing TPA-CN (10 μM) for 3 hours. The changes in the red and blue fluorescence intensities in the cells were observed under a laser confocal microscope. Figure 24 shown.

[0111] Depend on Figure 24 As can be seen, the blue fluorescence of the group incubated with sodium thiosulfate and glutathione increased significantly, while the red fluorescence decreased significantly. This finding is consistent with the phenomenon observed when detecting exogenous SO2. These results show that the TPA-CN probe is suitable for monitoring endogenous and exogenous SO2 in living cells.

[0112] Test Example 15

[0113] Experimental study on the ability of TPA-CN probe to detect viscosity changes in living cells

[0114] Experimental steps: HeLa cells were transplanted into 6-well plates (3×10 5HeLa cells were incubated with different concentrations of nystatin (5 μM, 10 μM, and 20 μM) for 1 hour, and then incubated with DMEM medium containing TPA-CN (10 μM) for 3 hours. Fluorescence images were captured using an inverted fluorescence microscope (Olympus IX73) to monitor changes in cell viscosity. The results are shown in Figure 2. Figure 25 shown.

[0115] Depend on Figure 25 As shown, red fluorescence in cells was enhanced after incubation with nystatin. Compared with the unincubated group, the relative fluorescence intensity increased approximately 1.5-fold after incubation with nystatin (20 μM). Therefore, the TPA-CN probe has the potential to become a tool for monitoring the viscosity of living cells.

[0116] Test Case 16

[0117] Detection of SO2 in water and food using TPA-CN probe

[0118] Test solution: 10 μM TPA-CN solution

[0119] Samples: Tap water was used directly for measurement; beer and sugar solutions (100 mg / mL) were diluted 20-fold and 10-fold with the test solution, respectively, before measurement. Bean curd sticks (2 g) were soaked in deionized water (20 mL) overnight, centrifuged, and the supernatant was diluted 10-fold with the test solution before measurement.

[0120] Experimental steps: Add different concentrations (0, 10, 20 μM) of HSO3 to the above samples - , the TPA-CN fluorescence spectrum was measured, and the results are shown in Table 1.

[0121] Table 1. SO2 detection in water and food

[0122]

[0123] As shown in Table 1, the TPA-CN probe can detect SO2 derivatives in real samples with high recoveries (ranging from 91.81% to 105.52%) and low relative standard deviations (RSDs). These results demonstrate that the TPA-CN probe has the potential to be used for determining SO2 concentrations in real beverages and foods.

[0124] The above description is only a preferred embodiment of the present invention. Any slight modifications, equivalent changes and modifications made to the above embodiment according to the technical solution of the present invention are within the scope of the technical solution of the present invention.

Claims

1. A novel fluorescent probe based on triphenylamine, characterized in that: It has the following structural formula: 。 2. A method for preparing a novel fluorescent probe based on triphenylamine as claimed in claim 1, characterized in that: The following steps are involved: S1. 4-methoxyaniline and 1-bromo-4-iodobenzene are dissolved in solvent A, and then copper iodide, 1,10-phenanthroline, and potassium tert-butoxide are added to react to obtain compound 1; S2. Compound 1 obtained in S1, (5-formylthiophen-2-yl)boronic acid and anhydrous potassium carbonate are dissolved in solvent B, and then PdCl2(dppf) is added to react to obtain compound 2; S3. Compound 2 obtained in S2 and malononitrile are dissolved in solvent C, and then triethylamine is added to react to obtain a TPA-CN probe.

3. The method for preparing a novel fluorescent probe based on triphenylamine according to claim 2, wherein: The solvent A is toluene.

4. The method for preparing a novel fluorescent probe based on triphenylamine according to claim 2, wherein: The molar ratio of the 4-methoxyaniline, the 1-bromo-4-iodobenzene, the copper iodide, the 1,10-phenanthroline and the potassium tert-butoxide in S1 is 20:50:3:3:

60.

5. The method for preparing a novel fluorescent probe based on triphenylamine according to claim 2, wherein: The molar ratio of the compound 1, the (5-formylthiophene-2-yl)boric acid, the anhydrous potassium carbonate and the PdCl2(dppf) in S2 is: 1:3:10:0.

03.

6. The method for preparing a novel fluorescent probe based on triphenylamine according to claim 2, wherein: The solvent B in S2 is a mixture of toluene and methanol in a volume ratio of 1:

1.

7. The method for preparing a novel fluorescent probe based on triphenylamine according to claim 2, wherein: The molar ratio of the compound 2, the malononitrile and the triethylamine in S3 is 1:3:

1.

8. The method for preparing a novel fluorescent probe based on triphenylamine according to claim 2, wherein: The solvent C is dichloromethane.

9. A novel triphenylamine-based fluorescent probe as claimed in claim 1 for use in monitoring exogenous and endogenous SO2 and viscosity changes in living cells for non-disease diagnosis purposes.

10. A novel triphenylamine-based fluorescent probe as claimed in claim 1 for detecting SO2 in water and food.

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