N2H4 ratiometric fluorescent probe based on cinnamic acid derivative as well as synthesis method and application of N2H4 ratiometric fluorescent probe

Abstract

The invention relates to the technical field of synthesis of specific probes, in particular to an N2H4 ratiometric fluorescent probe based on cinnamic acid derivatives as well as a synthesis method and application of the N2H4 ratiometric fluorescent probe. According to the fluorescent probe, p-hydroxycinnamic acid serves as a raw material, a tricyanofuran fluorophore is introduced into a probe framework to obtain the fluorescent probe CDP, C = C double bonds serve as recognition groups, and N2H4 can be specifically detected. Along with dropping of N2H4, the fluorescence intensity is gradually enhanced, the fluorescence color is changed from light orange to blue, and the detection process is not influenced by other interfering ions. Experiments prove that the probe CDP also has the characteristics of high sensitivity, short response time, wide pH application range and the like when being used for detecting N2H4. In addition, the probe CDP is low in toxicity and good in biocompatibility, and can be used for detecting N2H4 in cells and soil.

Description

Technical Field

[0001] This invention relates to the field of specific probe synthesis technology, specifically to an N2H4 ratiometric fluorescent probe based on cinnamic acid derivatives, its synthesis method, and its application. Background Technology

[0002] Hydrazine hydrate (N2H4), an important chemical raw material, is widely used in rocket fuel, reducing agents, pesticides, and pharmaceutical industries. 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, and even low-concentration exposure can lead to central nervous system damage and organ toxicity.

[0003] Therefore, developing highly sensitive and selective hydrazine hydrate detection technology is of great significance for environmental pollution monitoring, biomedical diagnosis, and industrial safety protection. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a ratiometric fluorescent probe for N2H4 based on cinnamic acid derivatives, its synthesis method, and its application, which specifically detects N2H4.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention proposes an N2H4 ratiometric fluorescent probe based on a cinnamic acid derivative, named CDP, whose structural formula is shown below: .

[0006] Secondly, this invention proposes a method for synthesizing an N2H4 ratiometric fluorescent probe based on a cinnamic acid derivative, characterized in that the synthesis method includes the following steps: Preparation of compound HPA: 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 4-6 h. After the reaction was complete, the mixture was filtered and the solvent was evaporated to obtain compound HPA. Preparation of compound FHA: Compound HPA and sodium hydroxide solution were added sequentially to chloroform to form a mixed system. The mixture was then refluxed under heating conditions. The reaction process was monitored by thin-layer chromatography. After the reaction was completed, the mixture was subjected to rotary evaporation under acidic conditions and extracted with ethyl acetate. The organic phase was collected, dehydrated, and the solvent was recovered by vacuum distillation. The final product was eluted by silica gel column chromatography to obtain a white solid compound FHA. Preparation of compound CFM: 3-hydroxy-3-methyl-2-butanone, malononitrile and sodium ethoxide were added sequentially to anhydrous ethanol to form a mixed system. The mixture was then refluxed under heating conditions. After the reaction was completed, the solvent was recovered by vacuum distillation. The crude product was eluted by silica gel column chromatography gradient to obtain the powdered compound CFM. Preparation of fluorescent probe CDP: Compounds FHA and CFM were dissolved in anhydrous ethanol, piperidine was added, and the mixture was refluxed under heating conditions. After the reaction was completed, the mixture was subjected to vacuum distillation, and the residue was purified by column chromatography to obtain solid fluorescent probe CDP.

[0007] Furthermore, the structural formula of the compound HPA is as follows:

[0008] The structural formula of the compound FHA is as follows:

[0009] The structural formula of the compound CFM is as follows: .

[0010] Furthermore, in the preparation of compound FHA, The molar ratio of the compound HPA to sodium hydroxide is 1:8-12; The reaction was refluxed at 50-70℃ for 5-8 hours. Acidity conditions were adjusted using 4M hydrochloric acid; The organic phase was dehydrated using magnesium sulfate, and the silica gel column chromatography used a mixture of petroleum ether, ethyl acetate, and glacial acetic acid with a volume ratio of 5-7:1:0.05-0.1 as the eluent.

[0011] Furthermore, in the preparation process of compound CFM, The molar ratio of 3-hydroxy-3-methyl-2-butanone, malononitrile, and sodium ethoxide is 2.7-3.3 mmol: 5.4-6.6 mmol: 0.54-0.66 mmol; The amount of anhydrous ethanol used is 6-8 mL; The reflux reaction temperature is 65-75℃, and the reaction time is 2-3h; the eluent used for gradient elution in silica gel column chromatography is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 280:1-320:1.

[0012] Furthermore, in the preparation process of the fluorescent probe CDP, The molar ratio of compound FHA to compound CFM is 0.9-1.1 mmol: 1.08-1.32 mmol; The amount of anhydrous ethanol used is 13-17 mL, and the amount of piperidine used is 25-31 μL; The reflux reaction temperature is 65-75℃, and the reflux time is 7-9h; The eluent used in the column chromatography purification is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 65:1-75:1.

[0013] Furthermore, in the preparation of compound FHA, The molar ratio of the compound HPA to sodium hydroxide is 1:10; The reaction was refluxed at 60°C for 6 hours. Acidity conditions were adjusted using 4M hydrochloric acid; The organic phase was dehydrated using magnesium sulfate, and silica gel column chromatography used a mixture of petroleum ether, ethyl acetate, and glacial acetic acid with a volume ratio of 6:1:1-0.07 as the eluent.

[0014] Furthermore, in the preparation process of compound CFM, The molar ratio of 3-hydroxy-3-methyl-2-butanone, malononitrile, and sodium ethoxide is 3 mmol: 6 mmol: 0.6 mmol; The amount of anhydrous ethanol used is 7 mL; The reflux reaction temperature is 70℃ and the reaction time is 2.5h; the eluent used in the silica gel column chromatography gradient elution is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 300:1.

[0015] Furthermore, in the preparation process of the fluorescent probe CDP, The molar ratio of compound FHA to compound CFM is 1 mmol: 1.2 mmol; The amount of anhydrous ethanol used is 15 mL, and the amount of piperidine used is 28 μL; The reflux reaction was carried out at a temperature of 70°C for 8 hours. The eluent used in the column chromatography purification is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 65:1-75:1.

[0016] Thirdly, this invention proposes the application of the N2H4 ratiometric fluorescent probe based on cinnamic acid derivatives in the specific detection of N2H4.

[0017] This invention provides a ratiometric fluorescent probe of N2H4 based on cinnamic acid derivatives, its synthesis method, and its application. Compared with existing technologies, its advantages are: This invention uses p-hydroxycinnamic acid as a raw material and introduces a tricyanofuran group into the 3-formyl-4-hydroxyphenylpropionic acid skeleton to maintain a long conjugated structure. The designed probe structure contains a furan ring, a cyano (-CN) group, and a malononitrile group as strong electron-withdrawing groups, which can regulate the electron distribution of the fluorophore and form a D-π-A structure to enhance the fluorescence signal. The carboxylic acid group (-COOH) can enhance the water solubility of the probe and can bind to biomolecules through hydrogen bonding or electrostatic interactions, improving the stability of the probe in the biological environment. The C=C double bond serves as a specific recognition site for N2H4. In the presence of N2H4, the C=C double bond breaks, causing the tricyanofuran structure to fall off, resulting in a change in the probe molecule structure, triggering a change in the fluorescence signal, and thus achieving the specific detection of N2H4 by the fluorescent probe CDP. This structure not only provides strong fluorescence and recognition properties but also endows the compound with good chemical stability, making it potentially applicable in multiple fields such as biosensors, environmental monitoring, and chemical analysis. As N2H4 was added, the fluorescence intensity gradually increased, and the fluorescence color changed from pale orange to blue. The detection process was unaffected by other interfering ions. Experiments showed that the fluorescent probe CDP exhibits high sensitivity, short response time, and a wide pH range for N2H4 detection. Furthermore, the fluorescent probe CDP has low toxicity and good biocompatibility, making it suitable for detecting N2H4 in cells and soil. Attached Figure Description

[0018] Figure 1 The fluorescence intensity of the fluorescent probe CDP in different solvents; Figure 2 The fluorescence intensity of the fluorescent probe in different ratios of MSO / PBS; Figure 3 The UV-Vis spectra of the fluorescent probe CDP (10 μM) before and after adding N2H4 (100 μM) to PBS buffer (v PBS / v DMSO = 4 / 6, 10 mM); Figure 4 Fluorescence spectra of the fluorescent probe CDP (10 μM) before and after adding N2H4 (100 μM) to PBS buffer (v PBS / v DMSO = 4 / 6, 10 mM); Figure 5 Fluorescence spectra of the fluorescent probe CDP (10 µM) after adding different concentrations (0-100 µM) of N2H4; Figure 6 F for fluorescent probe CDP 454 / F 600 Linear relationship between fluorescence intensity ratio and N2H4 concentration; Figure 7The fluorescence intensity ratio changes of the fluorescent probe CDP (10µM) after adding different interfering ions and then adding N2H4 (1-16: methylamine, ethylenediamine, urea, hydroxylamine, ethylamine, thiourea, Cys, Gly, Hcy, GSH, Glu, H2O2, PO) 43－ ClO-, ·OH, N2H4); Figure 8 The fluorescence response time after adding N2H4 to the fluorescent probe CDP; Figure 9 The effect of pH on the fluorescence intensity ratio of the fluorescent probe CDP (10 μM) in the presence of N2H4; Figure 10 This is a diagram illustrating the response mechanism of the fluorescent probe CDP to N2H4. Figure 11 The image shows the 1H NMR comparison of the fluorescent probe CDP and the probe CDP-N2H4. Figure 12 This is a schematic diagram showing the optimized structure and front molecular orbital energy level distribution of the fluorescent probe CDP and probe CDP-N2H4. Figure 13 The results of quantitative comparison of optical signals for detecting the effect of different concentrations of fluorescent probe CDP (1-8: 0.78µM; 1.56µM; 3.13µM; 6.25µM; 12.5µM; 25µM; 50µM; 100µM) on the viability of HeLa cells using the CCK8 assay. Figure 14 Confocal fluorescence images of HeLa cells after treatment with fluorescent probes CDP and N2H4; Figure 15 Fluorescence imaging of mice at 0, 10, 30 min and 1 h after treatment with fluorescent probes CDP and N2H4; Figure 16 The fluorescence response of probe CDP to N2H4 in mountain soil, field soil and sandy soil. Detailed Implementation

[0019] 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.

[0020] All chemicals and solvents required for the experiments in the following examples, such as p-hydroxycinnamic acid, 3-hydroxy-3-methyl-2-butanone, malononitrile, sodium ethoxide, methanol, dichloromethane, anhydrous ethanol, ethyl acetate, piperidine, N,N-dimethyl sulfoxide (DMSO), hydrogen peroxide (H2O2), sodium thiocyanate (NaSCN), sodium phosphate (Na3PO4), sodium hypochlorite (NaClO), and hydrazine hydrate (N2H4), were purchased from two chemical suppliers (Titan and Aladdin) and were all commercially available analytical grade, ready for direct use without further processing.

[0021] The instruments used were as follows: a Hitachi F-7100 fluorescence spectrophotometer was used to record fluorescence spectra. A Hitachi UH5300 UV-Vis spectrophotometer was used to collect UV absorption data. A Bruker AV 500MHz nuclear magnetic resonance spectrometer was used for 1H and 13C NMR measurements. An Agilent 655A iFunnel Q-TOF spectrometer was used for high-resolution mass spectrometry (HRMS). A Starter pH-Meter 2100 instrument was used to record pH measurements. An Agilent 6460 LCMS spectrometer was used to record compound purity. A Leica TCS SP8 confocal laser scanning microscope was used to study fluorescence cell imaging. Example 1

[0022] The synthetic route for the fluorescent probe CDP is as follows: ; Preparation of compound HPA: p-hydroxycinnamic acid was placed in methanol, and catalyst Pd / C (Pd content 5%, 0.1 g) was added. 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 compound HPA. Synthesis of compound FHA: 4M NaOH (7.5 mL, 30 mmol) was added to a solution of compound HPA (500 mg, 3.01 mmol) in 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 (4 M HCl), the reaction system was extracted with ethyl acetate after rotary evaporation. The collected organic phase was dehydrated with MgSO2, 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 yields a white solid, FHA (386 mg, 68.1%).

[0023] Synthesis of compound CFM: 3-hydroxy-3-methyl-2-butanone (306 mg, 3 mmol), malononitrile (397 mg, 6 mmol), and sodium ethoxide (41 mg, 0.6 mmol) were sequentially added to anhydrous ethanol (7 mL), and the mixture was heated under reflux at 70 °C for 2.5 h. After the reaction was complete, the solvent was recovered by vacuum distillation. The crude product was then subjected to gradient elution by silica gel column chromatography. v 二氯甲烷 : v 甲醇 The ratio of 300:1 yielded a yellow-green powdery compound CFM (594 mg, yield 84.6%).

[0024] The nuclear magnetic resonance (NMR) spectrum and mass spectrometry (MS) data of compound CFM are as follows: 1 H NMR (500 MHz, DMSO- d 6) δ 3.51 (s, 6H), 2.50 (p, J = 1.8 Hz, 7H), 1.60 (s, 47H), 1.37 – 1.27 (m, 2H), 1.25 (d, J = 11.8 Hz, 2H), 1.17 (s, 3H), 0.77 (s, 2H). 13 C NMR (101 MHz, CDCl3) δ 182.41, 175.13, 110.99, 110.35,108.94, 104.85, 99.70, 77.36, 77.04, 76.72, 58.61, 29.70, 24.39, 14.18.HRMS(ESI) calcd for C 11 H9N3O [M+H] + 222.0638, found 223.0671. Synthesis of the fluorescent probe CDP: Compound FHA (194 mg, 1 mmol) and compound CFM (233 mg, 1.2 mmol) were dissolved in anhydrous ethanol (15 mL), piperidine (28 μL) was added, and the mixture was refluxed at 70 °C for 8 h, followed by vacuum distillation. The residue was purified by column chromatography. v 二氯甲烷 : v 甲醇 =70:1), the fluorescent probe CDP was obtained as an orange solid (286 mg, yield 67%).

[0025] The NMR and mass spectrometry data of the fluorescent probe CDP are as follows: 1 H NMR (500 MHz, DMSO) δ 12.12 (s, 1H), 10.69 (s, 1H), 8.15 (d, J = 16.4Hz, 1H), 7.72 (d, J = 2.2 Hz, 1H), 7.44 – 7.35 (m, 1H), 7.25 (dd, J = 8.4, 2.2Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 1.78 (s, 4H), 1.32 – 1.21 (m, 6H). 13 C NMR (126 MHz, MeOD) δ 176.82, 176.40, 157.11, 144.39, 134.11, 132.81, 129.02,121.26, 116.11, 114.60, 111.83, 111.34, 110.41, 98.44, 97.76, 48.05, 47.88,29.80, 24.95.HRMS (ESI) calcd for C 10 H 10 O4[M+H] + 398.1111, found 398.1119. Example 2

[0026] Spectral characteristics test of CDP: To investigate the solvent effect of the fluorescent probe CDP prepared in Example 1, several different organic solvents (methanol, ethanol, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), tetrahydrofuran, ethyl acetate, acetonitrile) were selected and reacted with PBS buffer solution ( v / v =1 / 1) was prepared into a solution (500 μM) and subjected to fluorescence spectroscopy. The results are as follows. Figure 1 As shown, the probe HMP exhibits a significant fluorescence response in methanol and ethanol. Simultaneously, it shows a moderate fluorescence signal in DMSO.

[0027] The effect of solvent water content on fluorescence performance was investigated by testing the changes in fluorescence spectra of CDP solutions (500 μM) in different ratios of DMSO / PBS mixed solvents (v / v = 1:9 to 7:3, pH = 7.4). Results Figure 2As shown in the figure, the experiment found that the fluorescence intensity of the probe reached its maximum value when vPBS / v DMSO = 4 / 6. Based on this, in the next fluorescence response investigation experiment, the optimal solvent system for the fluorescent probe CDP solution was determined to be a mixed solvent with a volume ratio of PBS to DMSO of 4 / 6. This detection system has both significant fluorescence characteristics and biocompatibility.

[0028] Analysis in PBS / DMSO ( v / v =4 / 6) Fluorescence response before and after adding N2H4 to the mixed solution. The results are as follows. Figure 3 and Figure 4 As shown, the fluorescent probe CDP exhibits a distinct absorption peak at 455 nm. After the addition of 100 μM N₂H₄, the absorption peak at 455 nm gradually disappears. Figure 3 This phenomenon originates from the nucleophilic reaction between N₂H₄ and the α,β-unsaturated carbon-carbon double bond (C=C) in the probe structure, causing the C=C double bond to break and form a hydrazone derivative, resulting in a shortening of the conjugated system and an alteration of the electronic transition mode. Next, the fluorescence spectra before and after adding N₂H₄ to the fluorescent probe CDP solution (10 μM) were further analyzed. Figure 4 Before the addition of N₂H₄, the fluorescent probe CDP solution itself exhibited a pale orange fluorescence. However, after the addition of N₂H₄, the fluorescence intensity of the probe at 460 nm gradually increased, accompanied by a change in the solution's fluorescence color from pale orange to blue. These results indicate that the fluorescent probe CDP can be used for N₂H₄ detection. Example 3

[0029] Sensitivity test of fluorescent probe CDP to N2H4: To further investigate the sensitivity of the fluorescent probe CDP to N2H4, in v PBS / v DMSO The titration fluorescence spectrum of the fluorescent probe CDP solution was investigated by gradually adding 0-100 μM N2H4 to a CDP solution with a ratio of 4 / 6.

[0030] The results are as follows Figure 5 and Figure 6 As shown, with the addition of N2H4 (0-100 μM), the fluorescence intensity of the fluorescent probe CDP solution gradually increased at 454 nm, while the fluorescence intensity gradually decreased at 600 nm. Figure 5 F 454 / F 600 The fluorescence intensity gradually increased and showed a good linear relationship with the concentration of N2H4. Figure 6 y = 13.7489x + 769.4 R(²=0.995). The LOD of the fluorescent probe CDP for N2H4 was calculated to be 17.08 nM. These results indicate that the fluorescent probe CDP has high sensitivity to N2H4. Example 4

[0031] Selectivity and anti-interference test of fluorescent probe CDP against N2H4: To verify the selectivity of the fluorescent probe CDP for N2H4, the fluorescent probe CDP prepared in Example 1 was used in a solvent system of phosphate buffered saline (PBS, 10 mM, pH 7.0) and dimethyl sulfoxide (DMSO). v DMSO : v PBS A 1 mM CDP solution for the fluorescent probe was prepared using a solvent ratio of 6:4. In optical response studies, the solvent ( v DMSO : v PBS The fluorescent probe CDP solution (1 mM) was diluted to the desired concentration (10 μM) using a ratio of 6:4. In sensitivity testing, different concentrations of N2H4 (0-100 μM) were added to the fluorescent probe CDP test solution, and the changes in optical signal were recorded using a fluorescence spectrometer. The minimum detectable concentration of the fluorescent probe CDP was calculated based on the limit of detection (LOD) formula. To verify the probe's selective recognition ability for N2H4, a comparative system of various potential interfering substances was designed, including: methylamine, ethylenediamine, urea, hydroxylamine, ethylamine, thiourea, Cys, Gly, Hcy, GSH, Glu, H2O2, and PO4. 3－ ,ClO － All interfering solutions were prepared in deionized water, and the spectral response of the probes was tested either alone or in coexistence with N₂H₄. In this embodiment, the excitation wavelength was 350 nm and the slit width was 5 nm / 10 nm in the fluorescence spectroscopy test.

[0032] The results are as follows Figure 7 As shown, the horizontal axis 1-15 represent the addition of methylamine, ethylenediamine, urea, hydroxylamine, ethylamine, thiourea, Cys, Gly, Hcy, GSH, Glu, H2O2, and PO4 to the fluorescent probe CDP solution, respectively. 3－ ,ClO － F after ·OH 454 / F 600 The fluorescence intensity of the samples did not show obvious fluorescence emission peaks, but the addition of N2H4 (as shown in the bar graph corresponding to axis 16) induced an F emission peak. 454 / F 600The fluorescence intensity was significantly enhanced. Subsequently, the resistance of the fluorescent probe CDP to interference from other ions was investigated. Compared with the fluorescence intensity of the solution containing only N2H4, no significant change in fluorescence intensity was observed in the presence of other anions. The results indicate that the fluorescent probe CDP has significant selectivity for N2H4, and other ions did not affect the detection process of N2H4, demonstrating strong resistance to interference. Example 5

[0033] Time and pH response of fluorescent probe CDP to N2H4: To evaluate the practical application performance of the fluorescent probe CDP, its real-time response to N2H4 and its environmental pH adaptability were systematically investigated. The results are as follows: Figure 8 As shown, the fluorescent probe CDP solution (10 μM) was applied. v PBS / v DMSO After adding 100 μM N2H4 to (=4 / 6), F 454 / F 600 The fluorescence intensity increased rapidly within 35 seconds and remained stable. These results indicate that the fluorescent probe CDP can rapidly detect N2H4.

[0034] To further investigate the effect of pH on detection performance, the fluorescence intensity of the fluorescent probe CDP in buffer solutions of different pH values ​​(3-12) was measured in the presence of N2H4 (100 μM). Figure 9 As shown, the fluorescent probe CDP exhibits strong fluorescence intensity within a pH range of 5-12. Therefore, the fluorescent probe CDP can selectively recognize N2H4 within this pH range. These results indicate that the fluorescent probe CDP is suitable for real-time monitoring of N2H4 in physiological and slightly alkaline environments (such as cytoplasm and body fluids). Example 6

[0035] The detection mechanism of the fluorescent probe CDP for N2H4: To further determine the possible detection mechanism of the fluorescent probe CDP for N2H4 ( Figure 10 ), to conduct 1 1H NMR titration analysis. For example Figure 11 As shown, in the fluorescent probe CDP 1 In the 1H NMR spectrum, the H atoms on the two methyl groups d and H e The chemical shift appears δ At position 1.25, the chemical shift of the two methylene groups next to the carboxyl group appears δ At position 1.79. However, upon adding N₂H₄, the tricyanofuran structure falls off, at... δ H appears at position 1.25 dand H e The chemical shift also disappears, in δ A new peak appears at 4.21, namely the H on the amino group. c And originally in δ The chemical shifts of the two methylene groups at position 1.79 also moved to... δ 2.13, δ At position 2.83. Based on the above results, the possible detection mechanism of the fluorescent probe CDP for N2H4 is as follows: the C=C bond in the structure of the fluorescent probe CDP acts as a recognition site and reacts with N2H4 to generate hydrazone compounds, thereby leading to a change in the probe's fluorescence signal.

[0036] To further explore the potential detection mechanism between the fluorescent probe CDP and N2H4, DFT calculations were performed using Gaussian software to obtain the optimized geometric structures of the fluorescent probes CDP and CDP-N2H4. Based on these calculations, the lowest unoccupied molecular orbitals (LUMO) and highest occupied molecular orbitals (HOMO) of the fluorescent probes CDP and CDP-N2H4 were calculated. Figure 12 As shown, the HOMO and LUMO electron clouds of the fluorescent probe CDP molecule are mainly located on the structure excluding the carboxyl group and the two methyl groups. In contrast, the HOMO and LUMO electron clouds of the fluorescent probe CDP-N2H4 are mainly distributed on the 2-hydrazinomethylenephenol structure. The calculated HOMO-LUMO band gaps of the fluorescent probe CDP and CDP-N2H4 are 3.04 eV and 4.53 eV, respectively. It can be observed that the band gap of the fluorescent probe CDP (3.04 eV) is significantly smaller than that of the fluorescent probe CDP-N2H4 (4.53 eV). This is because after the addition of hydrazine hydrate, the C=C bond in the fluorescent probe CDP reacts with it as a recognition site to form a hydrazone compound, which alters the conjugated structure of the probe, increases the band gap, and causes a blue shift in the fluorescence emission peak. Example 7

[0037] An examination of the cell imaging effect of the fluorescent probe CDP on N2H4: The cytotoxicity of the fluorescent probe CDP against HeLa cells was investigated using CCK-8 assay. HeLa cells were cultured in MEM with 10% FBS. HeLa cells were then placed in 96-well plates and incubated for 24 h at 37°C, 5% CO2, and saturated humidity. Different concentrations of the fluorescent probe CDP (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. Negative and positive control groups were also established. 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 the inhibition rate was calculated. Results are shown below. Figure 13 As shown, when cells were treated with different concentrations of fluorescent probe CDP from 0.78 to 100 μM, the cell viability remained above 99%, indicating that the fluorescent probe CDP has extremely low toxicity in biological systems and can be applied to cell imaging experiments.

[0038] To further evaluate the ability of the fluorescent probe CDP to trace N2H4 in live cells, HeLa cells were treated with the fluorescent probe CDP for N2H4 bioimaging, such as... Figure 14 As shown, HeLa cells incubated with the fluorescent probe CDP for 30 min exhibited obvious yellow fluorescence. HeLa cells pretreated with the fluorescent probe CDP were incubated with different concentrations of N2H4 (15 μM and 30 μM) for 30 min, resulting in obvious blue fluorescence. Furthermore, the blue fluorescence gradually increased with increasing N2H4 concentration.

[0039] After clarifying the detection characteristics of the fluorescent probe CDP in HeLa cells, its ability to trace N2H4 in an inflammatory mouse model was further verified. Five- to six-week-old female BALB / c nude mice were used in the experiment. The in vivo fluorescence enrichment characteristics of the probe HMP were evaluated via tail vein injection combined with the VilberFusion imaging system. First, N2H4 was injected into the tumor of the mouse and allowed to diffuse for 30 minutes. Then, 100 μL of the fluorescent probe CDP solution (concentration 100 μM, solvent: physiological saline containing 5% DMSO and 1% Tween-80) was injected. After incubation for 0, 10, 30 min, 1 h, and 2 h, the mice were anesthetized and in vivo imaging was performed at an excitation wavelength of 649 nm and an emission wavelength of 670 nm. Figure 15 The results showed that the fluorescence intensity of the fluorescent probe CDP in the tumor area changed over time after injection, gradually increasing initially, reaching a peak at 30 min, and then decreasing at 1 h. This trend indicates that the fluorescent probe CDP can effectively track the dynamic changes of N2H4 in mice, with 30 min after injection being the optimal observation time. Example 8

[0040] Soil detection of N2H4 using the fluorescent probe CDP: The fluorescence response of the fluorescent probe CDP to N₂H₄ in three soil samples was evaluated. Soil samples were collected from farmland, mountainous areas, and a beach in a specific region. First, the soil samples were treated with N₂H₄ solution (10 mM, 1 mL) for 2 h. Then, the N₂H₄-treated soil samples were sprayed with the fluorescent probe CDP solution (1 mM, 1 mL). After 2 h, the fluorescence color change of the soil samples before and after the addition of the fluorescent probe CDP solution was measured under UV light. The soil samples were then extracted with DMSO solution, filtered, and a clear solution was obtained. Figure 16 As shown, soil samples treated with N2H4 emitted weak blue fluorescence in the presence of the fluorescent probe CDP, indicating that this method is insufficient to identify N2H4 pollutants. Therefore, DMSO solution was used to extract the soil samples, yielding a stable solution. The results show that the fluorescent probe CDP can be used for the detection of N2H4 in soil samples, and the fluorescence spectrum exhibits a distinct blue fluorescence.

[0041] 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 ratiometric fluorescent probe for N2H4 based on cinnamic acid derivatives, characterized in that: The fluorescent probe is named CDP, and its structural formula is shown below: 。 2. A method for synthesizing an N2H4 ratiometric fluorescent probe based on cinnamic acid derivatives as described in claim 1, characterized in that: The synthesis method includes the following steps: Preparation of compound HPA: 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. After the reaction was complete, the mixture was filtered and the solvent was evaporated to obtain compound HPA. Preparation of compound FHA: Compound HPA and sodium hydroxide solution were added sequentially to chloroform to form a mixed system. The mixture was then refluxed under heating conditions. The reaction process was monitored by thin-layer chromatography. After the reaction was completed, the mixture was subjected to rotary evaporation under acidic conditions and extracted with ethyl acetate. The organic phase was collected, dehydrated, and the solvent was recovered by vacuum distillation. The final product was eluted by silica gel column chromatography to obtain a white solid compound FHA. Preparation of compound CFM: 3-hydroxy-3-methyl-2-butanone, malononitrile and sodium ethoxide were added sequentially to anhydrous ethanol to form a mixed system. The mixture was then refluxed under heating conditions. After the reaction was completed, the solvent was recovered by vacuum distillation. The crude product was eluted by silica gel column chromatography gradient to obtain the powdered compound CFM. Preparation of fluorescent probe CDP: Compounds FHA and CFM were dissolved in anhydrous ethanol, piperidine was added, and the mixture was refluxed under heating conditions. After the reaction was completed, the mixture was subjected to vacuum distillation, and the residue was purified by column chromatography to obtain solid fluorescent probe CDP.

3. The method for synthesizing the fluorescent probe according to claim 2, characterized in that: The structural formula of the compound HPA is as follows: The structural formula of the compound FHA is as follows: The structural formula of the compound CFM is as follows: 。 4. The method for synthesizing the fluorescent probe according to claim 2, characterized in that: In the preparation of compound FHA, The molar ratio of the compound HPA to sodium hydroxide is 1:8-12; The reaction was refluxed at 50-70℃ for 5-8 hours. Acidity conditions were adjusted using 4M hydrochloric acid; The organic phase was dehydrated using magnesium sulfate, and the silica gel column chromatography used a mixture of petroleum ether, ethyl acetate, and glacial acetic acid with a volume ratio of 5-7:1:0.05-0.1 as the eluent.

5. The method for synthesizing the fluorescent probe according to claim 2, characterized in that: In the preparation of compound CFM, The molar ratio of 3-hydroxy-3-methyl-2-butanone, malononitrile, and sodium ethoxide is 2.7-3.3 mmol: 5.4-6.6 mmol: 0.54-0.66 mmol; The amount of anhydrous ethanol used is 6-8 mL; The reflux reaction temperature is 65-75℃, and the reaction time is 2-3h; the eluent used for gradient elution in silica gel column chromatography is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 280:1-320:

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6. The method for synthesizing the fluorescent probe according to claim 3, characterized in that: In the preparation process of the fluorescent probe CDP, The molar ratio of compound FHA to compound CFM is 0.9-1.1 mmol: 1.08-1.32 mmol; The amount of anhydrous ethanol used is 13-17 mL, and the amount of piperidine used is 25-31 μL; The reflux reaction temperature is 65-75℃, and the reflux time is 7-9h; The eluent used in the column chromatography purification is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 65:1-75:

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7. The method for synthesizing the fluorescent probe according to claim 2, characterized in that: In the preparation of compound FHA, The molar ratio of the compound HPA to sodium hydroxide is 1:10; The reaction was refluxed at 60°C for 6 hours. Acidity conditions were adjusted using 4M hydrochloric acid; The organic phase was dehydrated using magnesium sulfate, and silica gel column chromatography used a mixture of petroleum ether, ethyl acetate, and glacial acetic acid with a volume ratio of 6:1:1-0.07 as the eluent.

8. The method for synthesizing the fluorescent probe according to claim 2, characterized in that: In the preparation of compound CFM, The molar ratio of 3-hydroxy-3-methyl-2-butanone, malononitrile, and sodium ethoxide is 3 mmol: 6 mmol: 0.6 mmol; The amount of anhydrous ethanol used is 7 mL; The reflux reaction temperature is 70℃ and the reaction time is 2.5h; the eluent used in the silica gel column chromatography gradient elution is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 300:

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9. The method for synthesizing the fluorescent probe according to claim 2, characterized in that: In the preparation process of the fluorescent probe CDP, The molar ratio of compound FHA to compound CFM is 1 mmol: 1.2 mmol; The amount of anhydrous ethanol used is 15 mL, and the amount of piperidine used is 28 μL; The reflux reaction was carried out at a temperature of 70°C for 8 hours. The eluent used in the column chromatography purification is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 65:1-75:

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10. The application of the fluorescent probe as described in claim 1 in the specific detection of N2H4.

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