A fluorescent probe based on cinnamic acid derivatives and a synthesis method and application thereof

Abstract

The application provides a fluorescent probe based on a cinnamic acid derivative as well as a synthesis method and application thereof, and relates to the technical field of synthesis of specific probes. The fluorescent probe is prepared by introducing benzyl bromide into the hydroxyl group of a modified structure compound FHA using p-hydroxycinnamic acid as raw material, and a novel bifunctional fluorescent probe capable of specifically detecting Al 3＋ and N2H4 is developed. The application overcomes the defects of the prior art, the probe can specifically detect Al 3+ and N2H4, and has the characteristics of low detection limit, short response time, wide pH application range and the like; the probe has the characteristics of good biocompatibility, small toxicity, good optical properties and the like, and can be applied to cell imaging experiments.

Description

Technical Field

[0001] This invention relates to the field of specific probe synthesis technology, specifically to an Al based on cinnamic acid derivatives. 3＋ Fluorescent probes for N2H4, their synthesis methods, and applications. Background Technology

[0002] Hydrazine hydrate (N₂H₄), an important chemical raw material, is widely used in rocket fuel, reducing agents, pesticides, and pharmaceuticals. However, its high toxicity, carcinogenicity, and environmental residues pose a serious threat to human health and ecological safety. Studies have shown that hydrazine hydrate can enter the body through skin contact, inhalation, or digestion; even low-concentration exposure can cause central nervous system damage and organ toxicity. Therefore, developing highly sensitive and selective hydrazine hydrate detection technologies is of great significance for environmental pollution monitoring, biomedical diagnosis, and industrial safety protection.

[0003] Aluminum is one of the most abundant metallic elements in the Earth's crust, but it is not an essential trace element for the human body. Excessive intake can lead to diseases such as Parkinson's disease, Alzheimer's disease, and osteoporosis.

[0004] Based on this design, an AI... 3＋ Probes that can specifically react with N2H4 are crucial. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a fluorescent probe based on cinnamic acid derivatives, its synthesis method, and its application. This probe can specifically detect Al. 3+ Both N2H4 and N2H4 have the characteristics of low detection limit, short response time and wide pH range; and the probes have good biocompatibility, low toxicity and good optical properties, and can be used in cell imaging experiments.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A fluorescent probe based on a cinnamic acid derivative, wherein the fluorescent probe is probe BFP, and the structural formula of probe BFP is shown below:

[0008] .

[0009] The synthesis method of the above-mentioned probe BFP includes the following steps:

[0010] S1. HPA was prepared by modifying p-hydroxycinnamic acid as a raw material:

[0011] p-hydroxycinnamic acid was placed in methanol, Pd / C catalyst was added, and after hydrogen gas was introduced, the mixture was stirred at room temperature for 5 h. After the reaction was complete, the mixture was filtered and the solvent was evaporated to obtain HPA.

[0012] S2. HPA was added to CHCl3 and reacted in 4M NaOH to obtain compound FHA:

[0013] 4M NaOH and HPA were added together to CHCl3 to obtain a mixture. The mixture was heated under reflux at 60°C for 6 h. Under acidic conditions, the reaction system was extracted with ethyl acetate after rotary evaporation. The organic phase was collected and dehydrated with MgSO4. The solvent was then recovered. The final product was eluted by silica gel column chromatography to obtain a white solid, which was compound FHA.

[0014] S3. The above compound FHA was mixed with benzyl bromide and K2CO3 and dissolved in acetonitrile. The mixture was heated to reflux and then concentrated under reduced pressure. The final product was eluted by silica gel column chromatography with gradient elution to obtain an orange solid as probe BFP.

[0015] The specific reaction process is as follows:

[0016] .

[0017] Preferably, in step S3, the mass ratio of compound FHA, benzyl bromide, and K2CO3 is 95-100:90-95:200-210.

[0018] Preferably, in step S3, the reflux temperature is 70°C, the reflux time is 5 hours, and the eluent used for gradient elution is a mixture of petroleum ether and ethyl acetate in a volume ratio of 50:1.

[0019] Preferably, the eluent used in the gradient elution in step S3 is a mixture of petroleum ether and ethyl acetate in a volume ratio of 50:1.

[0020] The aforementioned probe BFP is used for the specific detection of Al. 3＋ With N2H4.

[0021] This invention provides a fluorescent probe based on cinnamic acid derivatives, its synthesis method, and its application. Compared with existing technologies, its advantages are as follows:

[0022] This invention introduces benzyl bromide at the hydroxyl position of the compound FHA to extend the probe's conjugated system and provide an oxygen-rich coordination environment for metal ions, thereby enhancing its coordination ability with metal ions and ultimately achieving the coordination of probe BFP with Al. 3＋The detection of N2H4 is achieved through the use of aldehyde groups in the BFP probe. Furthermore, the aldehyde group in the BFP probe's molecular structure is easily modified and can serve as a specific group for recognizing N2H4. When N2H4 is present, its C=O bond breaks, generating hydrazone compounds, which in turn alter the fluorescence signal of the BFP probe, thus enabling specific detection. The BFP probe solution itself exhibits a pale blue fluorescence. 3＋ The addition of [a specific ingredient] causes the fluorescence color of the system to turn yellow-green; however, when N2H4 is present, the fluorescence color of the solution turns sky blue. This means that the probe BFP can specifically detect Al. 3+ Both BFP and N2H4 exhibit low detection limits, short response times, and wide pH applicability. Furthermore, the probe BFP demonstrates good biocompatibility, low toxicity, and excellent optical properties, making it suitable for intracellular Al2O3 testing in HeLa cells. 3+ Real-time detection of N2H4. Attached Figure Description

[0023] Figure 1 (a) shows the fluorescence intensity of probe BFP in different solvents; (b) shows the fluorescence intensity of probe BFP at different DMSO / PBS values.

[0024] Figure 2 Add Al to probe BFP 3＋ Schematic diagram of UV absorption spectra before and after reacting with N2H4;

[0025] Figure 3 In (a), probe BFP (10 μM) was added to PBS buffer (v PBS / v DMSO = 3 / 7, 10 mM) with Al. 3＋ (a) Fluorescence spectra of probe BFP (10 μM) before and after the addition of N2H4 (100 μM) to PBS buffer (vPBS / v DMSO = 3 / 7, 10 mM) at an excitation wavelength of 350 nm;

[0026] Figure 4 In (a), different concentrations (0-100µM) of Al were added to the probe BFP (10µM). 3＋ (a) The fluorescence spectrum of the probe BFP at 495 nm and Al. 3＋ Linear relationship graph of concentration;

[0027] Figure 5 (a) shows the fluorescence spectra of probe BFP (10µM) after adding different concentrations (0-100µM) of N2H4; (b) shows the linear relationship between the fluorescence intensity of probe BFP at 445nm and the concentration of N2H4.

[0028] Figure 6Adding different interfering ions (analytes) to the probe BFP (10 µM) and then adding Al after adding different interfering ions. 3＋ The changes in fluorescence intensity after [the event], where the vertical axis represents values ​​1-15 respectively: Mg 2＋ Fe 3＋ Cu 2＋ Ca 2＋ Cr 3＋ Fe 2＋ Ba 2＋ Ag ＋ Hg ＋ Cd 2＋ Zn 2＋ Co 2＋ Mn 2＋ Ni 3＋ Al 3＋ ;

[0029] Figure 7 The fluorescence intensity changes are shown in the probe BFP (10 µM) after adding different interfering ions (analytes) and after adding N2H4. The vertical axis numbers 1-17 represent, in order, methylamine, ethylenediamine, urea, hydroxylamine, ethylamine, thiourea, Cys, Gly, Hcy, GSH, Glu, H2O2, and SCN. － PO4 3－ ,ClO － ·OH, N2H4;

[0030] Figure 8 (a) shows the addition of Al to the probe BFP. 3＋ (a) Fluorescence response time after Al; (b) is Al 3＋ The effect of pH on the fluorescence intensity of probe BFP (10 μM) in the presence of the virus;

[0031] Figure 9 (a) shows the fluorescence response time of probe BFP after the addition of N2H4; (b) shows the effect of pH on the fluorescence intensity of probe BFP (10 μM) in the presence of N2H4.

[0032] Figure 10 For probe BFP to N2H4 and Al 3＋ The response mechanism;

[0033] Figure 11 For probes BFP and BFP-N2H4 and BFP-Al 3＋ of 1 1H NMR comparison image; where 1 represents probe BFP. 1 HNMR spectrum; 2 is BFP-N2H4. 1 H NMR spectrum; 3 is BFP-Al 3＋of 1 H NMR spectrum;

[0034] Figure 12 For probes BFP and BFP-N2H4, BFP-Al 3＋ Optimized structure and frontier molecular orbital energy level distribution;

[0035] Figure 13 To quantitatively compare the optical signals used in detecting the effects of different concentrations of probe BFP (1-8 representing concentrations of 0.78 µM, 1.56 µM, 3.13 µM, 6.25 µM, 12.5 µM, 25 µM, 50 µM, and 100 µM, respectively) on the viability of HeLa cells using the CCK8 assay;

[0036] Figure 14 For probes BFP and Al 3＋ Confocal fluorescence images of HeLa cells after treatment;

[0037] Figure 15 Confocal fluorescence images of HeLa cells after treatment with probes BFP and N2H4. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] The instruments used in the following examples are:

[0040] Hitachi F-7100 fluorescence spectrophotometer records fluorescence spectra;

[0041] Ultraviolet absorption data were collected using a Hitachi UH5300 UV-Vis spectrophotometer;

[0042] Tested using a Bruker AV 500 MHz nuclear magnetic resonance spectrometer 1 H and 13 C NMR;

[0043] The Agilent 655A iFunnel Q-TOF instrument is used for recording high-resolution mass spectrometry (HRMS).

[0044] The Starter pH-Meter 2100 device is used to record pH measurements;

[0045] Compound purity was recorded using an Agilent 6460 LCMS spectrometer;

[0046] Fluorescent cell imaging was studied using a Leica TCS SP8 confocal laser scanning microscope. Example:

[0047] Synthesis of the fluorescent probe BFP:

[0048] (1) HPA was prepared by modifying p-hydroxycinnamic acid as a raw material;

[0049] p-Hydroxycinnamic acid (201 mg, 2 mmol) was placed in 10 mL of methanol. A catalyst, Pd / C (5% Pd, 0.1 g), was added, and the mixture was stirred at room temperature for 5 h after purging with hydrogen. After the reaction was complete, the mixture was filtered, and the solvent was evaporated using a rotary evaporator to obtain the expected product as a white solid, which was identified as compound HPA (195 mg), with a yield of 97%.

[0050] (2) HPA was added to CHCl3 and reacted in 4M NaOH to obtain compound FHA;

[0051] 4M NaOH (7.5 mL, 30 mmol) and compound HPA (500 mg, 3.01 mmol) were added to CHCl3 (10 mL) to obtain a mixture. The mixture was heated under reflux at 60 °C for 6 h. The reaction progress was monitored by thin-layer chromatography (TLC). Under acidic conditions (4M HCl), the reaction system was extracted with ethyl acetate after rotary evaporation. The collected organic phase was dehydrated with MgSO4, and the solvent was recovered by vacuum distillation. The final product was eluted by silica gel column chromatography (V... 石油醚 :v 乙酸乙酯 :v 冰乙酸 The ratio of 6:1:0.07 yielded a white solid, compound FHA (386 mg, yield 68.1%).

[0052] (3) Compound FHA (97 mg, 0.5 mmol), benzyl bromide (93.48 mg, 0.55 mmol), and K2CO3 (207.3 mg, 1.5 mmol) were dissolved in acetonitrile (10 mL), and the mixture was refluxed at 70 °C for 5 h. After terminating the reaction, the mixture was concentrated under reduced pressure, and the final product was subjected to gradient elution by silica gel column chromatography with a petroleum ether:ethyl acetate ratio of 50:1 to obtain the expected probe BFP as an orange solid (146 mg, yield 76%).

[0053] The specific synthesis route is as follows:

[0054]

[0055] And the probe BFP detection spectral data are as follows: 1 H NMR (500 MHz, CDCl3)δ12.03 (d, J = 6.2 Hz,1H), 10.52 (s, 1H), 7.68 (d, J = 2.4 Hz, 1H), 7.43 – 7.40 (m, 3H), 7.30 (dt,J = 7.4, 1.7 Hz, 3H), 6.95 (d, J = 8.5 Hz, 1H), 5.16 (s, 2H), 2.93 (t, J =7.6 Hz, 2H), 2.66 (t, J = 7.6 Hz, 2H). 13 C NMR (126 MHz, CDCl3)δ189.68, 172.43,159.71, 136.16, 135.97, 133.08, 128.75, 128.56, 128.29, 128.25, 127.85,127.30, 125.07, 113.26, 70.60, 35.71, 29.84. HRMS (ESI) calcd for C 17 H 16 O4[M+H] + 284.1143, found 285.1043.

[0056] Detection:

[0057] 1. Solvent effect:

[0058] Several different organic solvents (methanol (MeOH), ethanol (EtOH), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), tetrahydrofuran (THF), ethyl acetate (EAC), and acetonitrile (ACN)) were prepared by mixing with PBS buffer (v / v = 1 / 1), and then the probe BFP (500 μM) was added for fluorescence spectroscopy testing. Figure 1 As shown in (a), the probe BFP exhibited a significant fluorescence response in tetrahydrofuran. Simultaneously, it showed a moderate fluorescence signal in DMSO. Furthermore, the fluorescence response of the probe BFP (500 μM) under different water contents was analyzed by spectroscopic measurements. Figure 1As shown in (b), compared with different ratios of PBS / DMSO mixed solvents, the probe BFP exhibited the highest fluorescence intensity in the solvent system (vDMSO:vPBS=7:3). The fluorescence emission spectrum of the probe solution is shown. Therefore, in the next step of the fluorescence response investigation experiment, the solvent system (vDMSO:vPBS=7:3) was selected for optical property analysis.

[0059] 2. Solution preparation and spectral testing conditions:

[0060] After weighing the probe BFP, a probe BFP solution (1 mM) was prepared using a mixture of phosphate-buffered saline (PBS) buffer and dimethyl sulfoxide (DMSO) (vDMSO:vPBS=7:3, pH=7).

[0061] For optical response studies: the probe BFP solution (1 mM) was diluted to obtain a 10 μM test solution. In sensitivity studies, different concentrations of Al were added dropwise to the probe BFP solution. 3＋ And N2H4 (0-100μM), tested for different concentrations of Al 3＋ The fluorescence spectrum changes of the N2H4 probe BFP solution were observed. The minimum detectable concentration of the probe BFP was calculated using the limit of detection (LOD) formula. In the pH response experiment, probe solutions with different pH values ​​were prepared using PBS buffer at different pH values, and the fluorescence response under different pH conditions was analyzed. In the selectivity test and competition experiment, other analytes (Ca...) were compared. 2＋ Cr 3＋ Cu 2＋ Cd 2＋ Al 3＋ Ni 3＋ Co 2＋ Ba 2＋ Fe 3＋ Fe 2＋ Mg 2＋ Zn 2＋ Mn 2＋ Hg ＋ Ag ＋ Methylamine, ethylenediamine, urea, hydroxylamine, ethylamine, thiourea, Cys, Gly, Hcy, GSH, Glu, H2O2, SCN － PO4 3－ ,ClO － Both ·OH and ·OH are prepared in deionized water.

[0062] In the fluorescence spectroscopy test of this experiment, Al was detected. 3＋The excitation wavelength for detection was 380 nm, and the slit width was 10 nm / 10 nm; the excitation wavelength for detecting N2H4 was 350 nm, and the slit width was 10 nm / 5 nm.

[0063] (1) Analysis of the addition of Al by fluorescence spectroscopy and ultraviolet spectroscopy 3＋ Spectral changes of the probe solution before and after contact with N2H4:

[0064] Ultraviolet absorption spectrum such as Figure 2 As shown, the probe BFP (10 μM) exhibits a maximum absorption peak at 330 nm, which suggests the possible existence of a large conjugated system within the probe molecule. Adding 100 μM Al to the probe BFP solution... 3＋ Subsequently, the ultraviolet spectrum exhibits a redshift, and its maximum absorption peak decreases. This is due to the interaction between the probe and Al. 3＋ After coordination, the original conjugated system undergoes conformational distortion (such as reduced planarity), resulting in decreased conjugation and reduced UV absorption intensity. However, the addition of 100 μM N₂H₄ to the probe BFP solution caused a significant blue shift in the maximum UV absorption peak, appearing at 315 nm, with a noticeable decrease in peak value. This is because the aldehyde group in the probe structure reacts with N₂H₄, altering the probe structure and thus causing a significant blue shift in the UV absorption peak.

[0065] Al was added to the probe BFP solution (10 μM) respectively. 3＋ The fluorescence spectra of the probe BFP solution before and after the addition of N2H4 were analyzed. The BFP solution itself exhibited pale blue fluorescence, and the addition of Al... 3＋ Subsequently, the probe exhibited a distinct fluorescence emission peak at 495 nm. For example... Figure 3 As shown, the fluorescence color of the solution changed from pale blue to yellowish-green. When N₂H₄ was added dropwise to the probe BFP solution, the probe showed a distinct fluorescence emission peak at 445 nm, and the fluorescence color of the solution changed from pale blue to sky blue. These results indicate that the fluorescent probe BFP can be used in Al… 3＋ Dual-function detection of N2H4.

[0066] (2) Probe BFP to Al 3＋ And the sensitivity of N2H4:

[0067] Add 0-100 μM Al to the PBS / DMSO (3 / 7, v / v, pH = 7.4) mixture. 3＋ The titration fluorescence spectrum of the probe BFP solution was investigated using N2H4.

[0068] Specific results are as follows Figure 4 and Figure 5 As shown:

[0069] The fluorescence intensity of the probe BFP solution at 495 nm increased with Al 3＋ The concentration gradually increases with increasing concentration (0-100 μM). Figure 4 (a) Based on this emission spectrum, the fluorescence intensity of probe BFP at 495 nm was plotted against Al. 3＋ Concentration relationship graph ( Figure 4 (b) It was found that the fluorescence intensity of probe BFP at 495 nm affected Al 3＋ The concentration exhibits a good linear relationship (y = 8.1348x + 896.4242, R0). 2 = 0.9913). The probe BFP was obtained for Al. 3＋ The LOD is 21.6 nM.

[0070] Furthermore, with the continuous addition of N2H4 (0-100 μM), the fluorescence intensity of the probe BFP solution at 445 nm gradually increased. Figure 5 (a) The fluorescence intensity of probe BFP at 445 nm showed a good linear relationship with the N2H4 concentration. Figure 5 (b)) (y=15.1763x+563.9268, R 2 = 0.9964), the detection limit of probe BFP for N2H4 is 11.59 nM.

[0071] (3) Probe BFP to Al 3＋ And the selectivity and anti-interference properties of N2H4:

[0072] Select different ionic solutions (Mg 2＋ Fe 3＋ Cu 2＋ Ca 2＋ Cr 3＋ Fe 2＋ Ba 2＋ Ag ＋ Hg ＋ Cd 2＋ Zn 2＋ Co 2＋ Mn 2＋ Al 3＋ Ni 3＋ Methylamine, ethylenediamine, urea, hydroxylamine, ethylamine, thiourea, Cys, Gly, Hcy, GSH, Glu, H2O2, SCN － PO4 3－ ,ClO － (OH, N2H4) were tested as interfering analytes;

[0073] Specific results are as follows Figure 6-7As shown, no obvious fluorescence emission peaks were observed after adding other different ions to the probe BFP solution; only after adding Al... 3＋ A significant increase in fluorescence intensity only occurs after the addition of N2H4. Furthermore, the interference resistance of the probe BFP to other ions was investigated. Compared to adding only Al... 3＋ The fluorescence intensity of the solution containing N2H4 was compared, and it was found that the presence of other different ions did not significantly affect the fluorescence response. The results indicate that the probe BFP is effective against Al... 3＋ Both Al and N2H4 exhibit significant selectivity, while other metal ions did not affect Al. 3＋ It also has an impact on the detection process of N2H4, and has good anti-interference ability.

[0074] (4) Probe BFP to Al 3＋ And the time and pH response of N2H4:

[0075] like Figure 8 (a) and Figure 9 As shown in (a), Al is added 3＋ The fluorescence intensity of the system at 495 nm increased significantly with time, reaching a stable state after 150 s. However, after adding N₂H₄, the fluorescence intensity at 445 nm reached a stable state after 180 s. These results indicate that the probe BFP can effectively target Al₂H₃. 3＋ Rapid detection of N2H4.

[0076] Furthermore, pH is a crucial factor in determining the suitability of novel fluorescent probes for biological applications. The effect of different pH values ​​on the BFP detection process was analyzed within the pH range of 2-13. Figure 8 (b) Figure 9 As shown in (b), when Al is added 3＋ Subsequently, the probe BFP exhibited strong fluorescence intensity within a pH range of 4-12. For N2H4, the BFP-N2H4 system also showed strong fluorescence intensity within a pH range of 3-12. These results indicate that the probe BFP can be used in complex biological systems involving Al... 3＋ Rapid detection of N2H4.

[0077] (5) Probe BFP to Al 3+ And the detection mechanism of N2H4:

[0078] use 1 1H NMR titration analysis was used to further determine the effect of probe BFP on Al. 3+ and the response mechanism of N2H4 ( Figure 10 ).like Figure 11 As shown, in the probe BFP 1In the 1H NMR spectrum, the Ha peak on the carboxyl group appeared at δ12.03, the chemical shift of Hb on the carbonyl carbon appeared at δ10.52, and the chemical shift of Hc on the ether bond oxygen appeared at δ4.26. After reacting with N2H4, the proton signal peak on the carboxyl group was significantly enhanced, the Hb on the carbonyl carbon shifted from δ10.52 to δ8.19, and a new proton signal peak appeared in the high-field region, namely Hd on the amino group. Therefore, the detection mechanism of the probe BFP molecule with N2H4 is concluded to be: the α,β-unsaturated ketone group (C=O) of the cinnamic acid derivative serves as the recognition site of N2H4. N2H4 directly undergoes nucleophilic addition to the carbonyl group to generate an open-chain hydrazone structure. In this pathway, the amino group of N2H4 attacks the carbonyl carbon to form the intermediate hydroxylamine, which is then dehydrated to generate a stable hydrazone bond (C=N-NH2). When Al 3＋ In its presence, the proton signal peak on the carboxyl group is slightly enhanced, the Hb peak on the carbonyl carbon shifts to a higher field (from δ10.52 to δ9.31), and the Hc chemical shift on the ether bond oxygen shifts to δ5.16. This is due to Al 3＋ The complexation with carbonyl oxygen and ether oxygen in the probe BFP molecule causes deprotonation, weakens the electron-withdrawing effect of the conjugated system, and triggers a change in the fluorescence signal of the probe BFP. The complexation ratio is 1:1.

[0079] (6) Probe BFP to Al 3＋ And the DFT calculation of N2H4:

[0080] DFT calculations were performed using Gaussian software to further verify the relationship between probe BFP and Al. 3＋ And the detection mechanism between N2H4.

[0081] like Figure 12 As shown, probe BFP and probe BFP-Al were obtained. 3＋ The optimized geometry of probe BFP-N2H4 was analyzed, and their lowest unoccupied molecular orbitals (LUMO) and highest occupied molecular orbitals (HOMO) were calculated. The LUMO electron clouds of probe BFP are mainly distributed on the benzaldehyde backbone, while its HOMO orbital energies are mainly distributed on the benzaldehyde and carboxyl groups. In contrast, the HOMO and LUMO electron clouds of probe BFP-N2H4 are mainly distributed on the 2-hydrazinomethylenephenol structure. Furthermore, for probe BFP-Al... 3＋ The molecule's HOMO orbital electron clouds are mainly distributed on the two benzene ring structures, while its LUMO orbital electron clouds are mainly distributed on the Al rings. 3+ The surrounding aggregation of the complex indicates that the complex BFP-Al 3＋ Charge transfer occurred within the molecule. Probe BFP, Probe BFP-Al 3＋Compared to the band gap of probe BFP-N2H4, the HOMO-LUMO band gap of probe BFP (4.55 eV) is significantly larger than that of BFP-N2H4 (3.76 eV), while BFP-Al 3＋ The smallest band gap value (3.59 eV) was observed. The results indicate that the probe BFP undergoes nucleophilic addition with the C=C bond of N2H4 to generate a hydrazone compound, and also reacts with Al... 3+ Complexation occurs, resulting in a significant increase in fluorescence intensity.

[0082] 3. Cytotoxicity test:

[0083] The cytotoxicity of the probe BFP against HeLa cells was investigated using CCK-8. HeLa cells were cultured in MEM with 10% FBS. HeLa cells were then placed in 96-well plates and incubated for 24 h in a 37 ℃, 5% CO2, and saturated humidity incubator. Different concentrations of probe BFP (0.78 µM, 1.56 µM, 3.13 µM, 6.25 µM, 12.5 µM, 25 µM, 50 µM, 100 µM) were added to the culture medium, along with negative and positive control groups. After culturing HeLa cells for another 24 h, CCK-8 solution was added, and the cells were incubated for another 2 h, gently mixed with a shaker for 10 min. The optical density (OD) of each well was measured using a microplate reader, and cell viability was calculated.

[0084] The results are as follows Figure 13 As shown, when cells were treated with different concentrations of probe BFP from 0.78 to 100 μM, the cell viability remained above 95%, indicating that probe BFP has extremely low toxicity in biological systems and can be applied to cell imaging experiments.

[0085] 4. Cell imaging:

[0086] HeLa cells were first incubated for 24 h at 37°C and 5% CO2 in 96-well plates containing 10% FBS. Then, HeLa cells were co-cultured with 10 μM BFP probe for 30 min. Cells were washed three times with PBS, followed by incubation with 25 μM and 50 μM Al, respectively. 3＋ Incubate with N2H4 for 30 min. Cell imaging experiments were performed using laser confocal fluorescence microscopy. Before imaging, HeLa cells were washed three times with PBS to remove extracellular free probes BFP and Al. 3＋ With N2H4.

[0087] Specific results are as follows Figure 14As shown, no obvious fluorescence phenomenon was observed after HeLa cells were incubated with the probe BFP for 30 min. HeLa cells pretreated with the probe BFP were then incubated with different concentrations of Al... 3＋ After incubation for 30 min with (10 and 30 μM) solutions, HeLa cells showed obvious green fluorescence. Simultaneously, with Al... 3＋ As the concentration increases, the green fluorescence within the cells gradually intensifies. For example... Figure 15 As shown, as a control group, HeLa cells pretreated with the BFP probe were incubated with different concentrations of N2H4 (10 and 30 μM) for 30 min, and obvious blue fluorescence appeared in the HeLa cells. Meanwhile, the blue fluorescence gradually increased with increasing N2H4 concentration.

[0088] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fluorescent probe based on cinnamic acid derivatives, characterized in that: The fluorescent probe is probe BFP, and the structural formula of probe BFP is shown below: 。 2. A method for synthesizing a fluorescent probe as described in claim 1, characterized in that: The synthesis method includes the following steps: S1. HPA was prepared by modifying p-hydroxycinnamic acid as a raw material: p-hydroxycinnamic acid was placed in methanol, Pd / C catalyst was added, and after hydrogen gas was introduced, the mixture was stirred at room temperature for 5 h. After the reaction was complete, the mixture was filtered and the solvent was evaporated to obtain HPA. S2. HPA was added to CHCl3 and reacted in 4M NaOH to obtain compound FHA: 4M NaOH and HPA were added together to CHCl3 to obtain a mixture. The mixture was heated under reflux at 60°C for 6 h. Under acidic conditions, the reaction system was extracted with ethyl acetate after rotary evaporation. The organic phase was collected and dehydrated with MgSO4. The solvent was then recovered. The final product was eluted by silica gel column chromatography to obtain a white solid, which was compound FHA. S3. The above compound FHA was mixed with benzyl bromide and K2CO3 and dissolved in acetonitrile. The mixture was heated to reflux and then concentrated under reduced pressure. The final product was eluted by silica gel column chromatography with gradient elution to obtain an orange solid as probe BFP. The specific reaction process is as follows: 。 3. The synthesis method according to claim 2, characterized in that: In step S3, the mass ratio of compound FHA, benzyl bromide, and K2CO3 is 95-100:90-95:200-210.

4. The synthesis method according to claim 2, characterized in that: In step S3, the reflux temperature is 70°C and the reflux time is 5 hours. The eluent used for gradient elution is a mixture of petroleum ether and ethyl acetate in a volume ratio of 50:

1.

5. The synthesis method according to claim 2, characterized in that: The eluent used in the gradient elution in step S3 is a mixture of petroleum ether and ethyl acetate in a volume ratio of 50:

1.

6. A fluorescent probe as described in claim 1 for the specific detection of Al 3＋ Applications with N2H4.

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