An imino stilbene fluorescent probe for detecting hydrazine hydrate, a preparation method and application thereof

Abstract

The application belongs to the technical field of small-molecule organic fluorescent probes, and discloses an imino stilbene fluorescent probe for detecting hydrazine hydrate as well as a preparation method and application of the probe. The probe selects imino stilbene with strong power supply characteristics as a fluorophore, barbituric acid as an acceptor and N2H4 as a recognition part. The probe has excellent rapid response capability and high selectivity, and the fluorescence is quenched from pink after being combined with N2H4. Compared with other fluorescent probes, the application has AIE characteristics, and the fluorescence signal is more stable. Through development of a portable N2H4 recognition test strip, a powerful tool is provided for on-site rapid detection, and the application has broad application prospects. The fluorescent probe has high selectivity and rapid response, and brings significant benefits in the fields of biological imaging, drug screening, environmental monitoring and food safety. The application not only improves the accuracy and efficiency of research, but also helps to solve real-world problems such as environmental protection, drug development and food safety.

Description

Technical Field

[0001] This invention belongs to the field of small molecule organic fluorescent probe technology, specifically, it relates to an iminostilbene-derived fluorescent probe, its preparation method, and its application. Background Technology

[0002] Hydrazine hydrate (N₂H₄) is an important chemical substance with wide applications in chemical, pharmaceutical, agricultural, and fuel industries. However, due to its high water solubility and high toxicity, its potential environmental and health risks cannot be ignored. The U.S. Environmental Protection Agency (USEPA) has classified it as a possible carcinogen, setting a limit of 10 ppb for hydrazine. Therefore, monitoring during its production, transportation, and disposal is crucial for ensuring public safety and protecting human health. Many traditional analytical methods for hydrazine hydrate detection have been reported, such as electrochemical analysis, liquid chromatography, and voltammetry. However, these methods have significant limitations, including complex and time-consuming operation, and insufficient sensitivity and selectivity, especially when handling complex samples. In contrast, fluorescent probes have attracted considerable attention due to their ease of operation, high sensitivity, excellent selectivity, and real-time monitoring capabilities. This technology not only overcomes the limitations of traditional methods but also demonstrates great application potential in environmental analysis.

[0003] In fact, based on traditional fluorophores such as coumarin (Spectrochim. Acta Part A: Mol. Biomol. Spectrosc., 2019, 210: 381-386), naphthalimide (Spectrochim. Acta Part A: Mol. Biomol. Spectrosc., 2024, 305: 123450), and quinoline (Dyes Pigm., 2022, 206: 110618), many fluorescent probes have been reported for the detection of hydrazine hydrate. However, these probes suffer from traditional defects such as probe quenching (ACQ) due to aggregation, long response time, short emission wavelength, and small Stokes shift. These defects lead to fluorescent probe quenching and large errors in the detection process, limiting their application in environmental detection. Tang's discovery of aggregation-induced emission (AIE) can effectively solve the defects of ACQ (Chem. Commun., 2001(18): 1740-1741), enhance fluorescence intensity, and stabilize the fluorescence signal. Therefore, it is necessary to design an AIE-type fluorescent probe for detecting hydrazine hydrate with a long emission wavelength and a large Stokes shift. Summary of the Invention

[0004] This invention aims to solve the related technical problems of fluorescent probes in the detection of hydrazine hydrate, and provides an iminostilbene fluorescent probe for the detection of hydrazine hydrate, its preparation method and application. The probe uses iminostilbene as the fluorophore, barbituric acid as the acceptor and hydrazine hydrate recognition group, and forms a probe molecule with a D-π-A structure through benzene ring linkage. Its twisted structure helps to achieve AIE characteristics.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] According to a first aspect of the present invention, an iminostilbene fluorescent probe for detecting hydrazine hydrate is provided, having the following structural formula:

[0007]

[0008] Its chemical name is 5-(4-(5H-dibenzo[b,f]azapyro-5-yl)benzylidene)pyrimidin-2,4,6(1H,3H,5H)-trione, and its molecular formula is C2. 25 H 17 N3O3.

[0009] According to a second aspect of the present invention, a method for preparing the above-mentioned iminostilbene fluorescent probe for detecting hydrazine hydrate is provided, the synthetic route of which is as follows:

[0010]

[0011] The specific steps include:

[0012] (1) Synthesis of 4-(5-(5H-dibenzo[b,f]azaline))-benzaldehyde:

[0013] Iminostilbene, 4-iodobenzaldehyde, and Cs₂CO₃ were added to a reaction vessel, followed by the addition of toluene solution. N₂ was then introduced for degassing to remove all air from the system. Pd(OAc)₂ and DPPF were immediately added to the system, and degassing continued. The mixture was refluxed in the presence of N₂. After the reaction was completed and cooled to room temperature, deionized water was added, and the mixture was extracted multiple times with ethyl acetate. The organic phases were combined and dried over anhydrous Na₂SO₄. The organic phase was concentrated to obtain a crude product, which was purified by column chromatography to obtain a white solid product, 4-(5-(5H-dibenzo[b,f]azaline))-benzaldehyde.

[0014] (2) Synthesis of 5-(4-(5H-dibenzo[b,f]azapyro-5-yl)benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (ISB-PyB):

[0015] Compound 4-(5-(5H-dibenzo[b,f]azaporin))-benzaldehyde and barbituric acid were placed in a reaction vessel, and anhydrous ethanol and piperidine were added to the system and the mixture was refluxed. After the reaction was cooled to room temperature, the solvent was evaporated and the crude product was purified by column chromatography to finally obtain a pink solid powder, which is 5-(4-(5H-dibenzo[b,f]azaporin-5-yl)benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (ISB-PyB).

[0016] Further, in step (1), the molar ratio of iminostilbene to 4-iodobenzaldehyde and Cs2CO3 is 1:(1.4-1.8):(2-3).

[0017] Furthermore, in step (1), the weight-to-volume ratio of iminostilbene to toluene is 1 g:(45-60) mL.

[0018] Further, in step (1), the molar ratio of iminostilbene to Pd(OAc)2 and DPPF is 1:(0.05~0.15):(0.10~0.30).

[0019] Furthermore, in step (1), the reaction reflux time of the mixture is 10 to 24 hours.

[0020] Furthermore, in step (1), the time for each degassing operation is 10 to 30 minutes.

[0021] Further, in step (1), the weight-to-volume ratio of iminostilbene to deionized water and ethyl acetate is 1.0 g: (30-50) mL: (15-25) mL.

[0022] Furthermore, in step (1), the eluent used in column chromatography purification is a mixed solution of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 12 to 25:1.

[0023] Further, in step (2), the molar ratio of 4-(5-(5H-dibenzo[b,f]azaline))-benzaldehyde to barbituric acid is 1:(1.0~1.5).

[0024] Further, in step (2), the weight-to-volume ratio of compound 4-(5-(5H-dibenzo[b,f]azaline))-benzaldehyde to anhydrous ethanol and pyridine is 1 g:(30-50) mL:(80-200) μL.

[0025] Furthermore, in step (2), the reflux time of the reaction after adding acetic acid and pyridine is 1-2 h.

[0026] Furthermore, in step (2), the eluent used in column chromatography purification is a mixed solution of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 5 to 10:1.

[0027] According to a third aspect of the present invention, an application of the above-described iminostilbene fluorescent probe for detecting hydrazine hydrate is provided for the preparation of a portable N2H4 identification test strip.

[0028] The fluorescent probes of this invention, possessing AIE (Adaptive Injection) properties, offer significant benefits in fields such as bioimaging, drug screening, environmental monitoring, and food safety due to their high selectivity and rapid response. These benefits not only improve the accuracy and efficiency of research but also help solve real-world problems such as environmental protection, drug development, and food safety.

[0029] This invention studies different water contents (f) w The fluorescence spectroscopic properties of ISB-PyB in a DMSO / H2O mixed solution confirmed its AIE characteristics. With f w With the increase of f, the fluorescence emission peak redshifts; when f w When the fluorescence intensity reaches 90%, a new fluorescence emission peak appears at 595 nm, and the fluorescence intensity reaches its maximum value. Furthermore, the fluorescence emission peak is consistent with f... w The fluorescence emission peak at 0% is redshifted by 77 nm. This change indicates that ISB-PyB possesses AIE characteristics. The ISB-PyB probe exhibits high fluorescence quantum yield, with an absolute quantum yield of 19.69% in the solid state.

[0030] This invention analyzes the fundamental spectra of the ISB-PyB probe before and after the addition of N₂H₄ to explore its response mechanism. In the presence of N₂H₄, the UV absorption peak at 448 nm and the fluorescence emission peak at 595 nm disappear. This is mainly attributed to a specific interaction between ISB-PyB and N₂H₄, which inhibits the intramolecular ICT process, thereby causing changes in the UV and fluorescence signals. Furthermore, the probe exhibits a large Stokes shift of 138 nm.

[0031] The present invention conducted a time-dependent response experiment of ISB-PyB to N2H4. When N2H4 was added to the solution of ISB-PyB, the fluorescence intensity tended to stabilize within 2 min, which indicates that the ISB-PyB probe can be used as a rapid response probe for detecting N2H4.

[0032] To investigate the specificity of ISB-PyB for N2H4, this invention treated the probe with various interfering analytes. These interfering analytes included proline, glycine, isoniazid, and cations such as Li... + NH4 + and anion HCO3 - F- Cl - ,Br - I - SO4 2- SCN - HPO4 2- H2PO4 - Only N₂H₄ caused a significant change in the fluorescence signal. Furthermore, in the presence of different interfering analytes, probe recognition was not significantly affected by competing species when treated with N₂H₄. These results demonstrate that ISB-PyB exhibits good selectivity and anti-interference ability towards N₂H₄.

[0033] To enable faster and more convenient real-time detection of N2H4, this invention presents a test strip containing ISB-PyB. Under 365nm ultraviolet light, different concentrations of N2H4 result in different colors on the test strip. As the N2H4 concentration increases, the fluorescence color of the test strip gradually changes from white to blue until it is quenched. This portable test strip is not only simple and easy to use, but also suitable for on-site real-time monitoring, and has broad application prospects.

[0034] The beneficial effects of this invention are:

[0035] (i) This invention provides a fluorescent probe with AIE characteristics, which can overcome the quenching effect (ACQ) defect caused by fluorescent probe aggregation, stabilize the signal intensity of the probe, and facilitate detection.

[0036] (II) This invention uses iminostilbene as a fluorophore to obtain a probe with high fluorescence quantum yield, which can promote the development of iminostilbene in the field of fluorescent probes.

[0037] (III) The fluorescent probe for detecting hydrazine hydrate derived from iminostilbene of the present invention has the advantages of long emission wavelength, fast response time, good selectivity and large Stokes shift.

[0038] (iv) The fluorescent probe provided by the present invention can be used to prepare portable N2H4 identification test strips, which can detect N2H4 in real time and quickly; the fluorescent probe of the present invention has broad application prospects in fields such as bioimaging, drug screening, environmental monitoring and food safety. Attached Figure Description

[0039] Figure 1 The 5-(4-(5H-dibenzo[b,f]azaphen-5-yl)benzyl)pyrimidine-2,4,6(1H,3H,5H)-trione (ISB-PyB) synthesized in Example 1 1 H NMR spectrum;

[0040] Figure 2The fluorescence spectra (λ) of 5-(4-(5H-dibenzo[b,f]azaphen-5-yl)benzyl)pyrimidine-2,4,6(1H,3H,5H)-trione (ISB-PyB) synthesized in Example 1 in mixed solutions with different water contents are shown. ex =457nm);

[0041] Figure 3 The UV absorption and fluorescence spectra of 5-(4-(5H-dibenzo[b,f]azaphen-5-yl)benzyl)pyrimidin-2,4,6(1H,3H,5H)-trione (ISB-PyB) synthesized in Example 1 before and after the addition of N2H4 are shown in the image (λ). ex =457nm); where (a) is the UV absorption spectrum of ISB-PyB before and after the addition of N2H4; (b) is the fluorescence spectrum of ISB-PyB before and after the addition of N2H4;

[0042] Figure 4 The time-dependent fluorescence spectrum (λ) of 5-(4-(5H-dibenzo[b,f]azaphen-5-yl)benzyl)pyrimidine-2,4,6(1H,3H,5H)-trione (ISB-PyB) synthesized in Example 1 after the addition of N2H4. ex =457nm); where (c) is the fluorescence intensity of ISB-PyB after the addition of N2H4 over time; (d) is the fluorescence intensity of ISB-PyB at 567nm wavelength over time.

[0043] Figure 5 The selectivity and anti-interference experimental results (λ) of 5-(4-(5H-dibenzo[b,f]azapyro-5-yl)benzyl)pyrimidine-2,4,6(1H,3H,5H)-trione (ISB-PyB) synthesized in Example 1 are presented. ex =457nm);

[0044] Figure 6 Photograph of the portable N2H4 identification test strip 5-(4-(5H-dibenzo[b,f]azapyro-5-yl)benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (ISB-PyB) synthesized in Example 1. Detailed Implementation

[0045] To make the solutions and advantages of the present invention clearer, the present invention and its advantages will be described in detail below with reference to specific embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0046] Example 1: Molecular ISB-PyB

[0047] (1) Synthesis of 4-(5-(5H-dibenzo[b,f]azaline))-benzaldehyde:

[0048] Iminostilbene (1.93 g, 10.0 mmol), 4-iodobenzaldehyde (3.25 g, 14 mmol), and Cs₂CO₃ (6.52 g, 20 mmol) were added to a reaction vessel, followed by 87 mL of toluene solution. The mixture was then degassed with N₂ for 10 min to remove all air from the system. Immediately afterwards, Pd(OAc)₂ (0.11 g, 0.5 mmol) and DPPF (0.55 g, 1.0 mmol) were added to the system, and degassed for another 10 min. The mixture was refluxed under N₂ for 10 h. After the reaction was complete, the mixture was cooled to room temperature, and 58 mL of deionized water was added. The mixture was extracted with 29 mL of ethyl acetate, and the organic phases were combined and dried over anhydrous Na₂SO₄. The organic phase was concentrated to give the crude product. The crude product was purified by column chromatography using petroleum ether:ethyl acetate (12:1, V / V) as the eluent to give a white solid product 1a in 35% yield.

[0049] (2) Synthesis of 5-(4-(5H-dibenzo[b,f]azapyro-5-yl)benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (ISB-PyB):

[0050] Compound 4-(5-(5H-dibenzo[b,f]azaporin))-benzaldehyde (1a) (0.60 g, 2.0 mmol) and barbituric acid (0.26 g, 2.0 mmol) were placed in a reaction vessel, and 18 mL of anhydrous ethanol and 48 μL of piperidine were added to the system. The reaction was refluxed for 1 h. After the reaction was cooled to room temperature, the solvent was evaporated, and the crude product was purified by column chromatography using petroleum ether:ethyl acetate (5:1, V / V) as the eluent. The final product was a pink solid powder, ISB-PyB, in 75% yield.

[0051] The structure was characterized using nuclear magnetic resonance spectroscopy. The 1H NMR spectra were as follows: 1H NMR (500MHz, DMSO-d6) δ 11.15 (s, 1H), 11.01 (s, 1H), 8.19 (d, J = 8.8Hz, 2H), 8.09 (s, 1H), 7.63 (d, J = 5.8Hz, 6H), 7.51 (d, J = 7.2Hz, 2H), 7.01 (s, 2H), 6.23 (d, J = 8.8Hz, 2H). Figure 1 As shown.

[0052] Example 2: Molecular ISB-PyB

[0053] (1) Synthesis of 4-(5-(5H-dibenzo[b,f]azaline))-benzaldehyde:

[0054] Iminostilbene (1.93 g, 10.0 mmol), 4-iodobenzaldehyde (3.71 g, 16 mmol), and Cs₂CO₃ (8.15 g, 25 mmol) were added to a reaction vessel, followed by the addition of 102 mL of toluene solution. The mixture was then degassed by purging with N₂ for 20 min to remove all air from the system. Immediately afterwards, Pd(OAc)₂ (0.23 g, 1.0 mmol) and DPPF (1.11 g, 2.0 mmol) were added to the system, and the system was purged by purging for another 20 min. The mixture was refluxed under N₂ for 18 h. After the reaction was complete, the mixture was cooled to room temperature, and 77 mL of deionized water was added. The mixture was extracted with 39 mL of ethyl acetate, and the organic phases were combined and dried over anhydrous Na₂SO₄. The organic phase was concentrated to give the crude product. The crude product was purified by column chromatography using petroleum ether:ethyl acetate (18:1, V / V) as the eluent to give a white solid product 1a in 40% yield.

[0055] (2) Synthesis of 5-(4-(5H-dibenzo[b,f]azapyro-5-yl)benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (ISB-PyB):

[0056] Compound 4-(5-(5H-dibenzo[b,f]azaporin))-benzaldehyde (1a) (0.60 g, 2.0 mmol) and barbituric acid (0.32 g, 2.5 mmol) were placed in a reaction vessel, and 24 mL of anhydrous ethanol and 85 μL of piperidine were added to the system. The reaction was refluxed for 1.5 h. After the reaction was cooled to room temperature, the solvent was evaporated, and the crude product was purified by column chromatography using petroleum ether:ethyl acetate (8:1, V / V) as the eluent. The final product was a pink solid powder, ISB-PyB, in 73% yield.

[0057] Example 3: Molecular ISB-PyB

[0058] (1) Synthesis of 4-(5-(5H-dibenzo[b,f]azaline))-benzaldehyde:

[0059] Iminostilbene (1.93 g, 10.0 mmol), 4-iodobenzaldehyde (4.18 g, 18 mmol), and Cs₂CO₃ (9.77 g, 30 mmol) were added to a reaction vessel, followed by 116 mL of toluene solution. The mixture was then degassed by purging with N₂ for 30 min to remove all air from the system. Immediately afterwards, Pd(OAc)₂ (0.34 g, 1.5 mmol) and DPPF (1.66 g, 3.0 mmol) were added to the system, and the system was purged by purging for another 30 min. The mixture was refluxed under N₂ for 24 h. After the reaction was complete, the mixture was cooled to room temperature, and 97 mL of deionized water was added. The mixture was extracted with 48 mL of ethyl acetate, and the organic phases were combined and dried over anhydrous Na₂SO₄. The organic phase was concentrated to give the crude product. The crude product was purified by column chromatography using petroleum ether:ethyl acetate (25:1, V / V) as the eluent to give a white solid product 1a in 45% yield.

[0060] (2) Synthesis of 5-(4-(5H-dibenzo[b,f]azapyro-5-yl)benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (ISB-PyB):

[0061] Compound 4-(5-(5H-dibenzo[b,f]azaporin))-benzaldehyde (1a) (0.60 g, 2.0 mmol) and barbituric acid (0.38 g, 3.0 mmol) were placed in a reaction vessel, and 30 mL of anhydrous ethanol and 120 μL of piperidine were added to the system. The reaction was refluxed for 2 h. After the reaction was cooled to room temperature, the solvent was evaporated, and the crude product was purified by column chromatography using petroleum ether:ethyl acetate (10:1, v / v) as the eluent. The final product was a pink solid powder, ISB-PyB, in 71% yield.

[0062] The target probes ISB-PyB synthesized in Examples 2 and 3 were characterized using the same methods as in Example 1. 1 The HNMR spectrum results are the same as in Example 1.

[0063] The probe ISB-PyB prepared in Example 1 above was tested as follows:

[0064] (I) AIE Nature Study

[0065] The study investigated ISB-PyB with different water contents (f) w The fluorescence spectral properties of the DMSO / H2O mixed solution confirmed its AIE characteristics. 100 μL of ISB-PyB stock solution (10...) was used. -3The solution (mol / L) was placed into ten 10 mL volumetric flasks. The following solutions were added to each flask: 1 mL DMSO + 9 mL H₂O, 2 mL DMSO + 8 mL H₂O, 3 mL DMSO + 7 mL H₂O, 4 mL DMSO + 6 mL H₂O, 5 mL DMSO + 5 mL H₂O, 6 mL DMSO + 4 mL H₂O, 7 mL DMSO + 3 mL H₂O, 8 mL DMSO + 2 mL H₂O, and 9 mL DMSO + 1 mL H₂O. The solution was then diluted to volume with 10 mL DMSO to prepare a solution with a water content f. w The test solutions with water contents of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, and 0% were tested by fluorescence spectroscopy to obtain fluorescence change spectra at different water contents. Figure 2 Fluorescence spectra of ISB-PyB mixed solutions with different water contents (λ) ex =457nm). ISB-PyB exhibits a fluorescence emission peak at 518nm in pure DMSO solution. With f w As the concentration increases from 0% to 80%, the fluorescence intensity gradually decreases, and the fluorescence emission peak gradually red-shifts. This is attributed to the change in solution polarity caused by the addition of water; this solvent effect leads to the red-shift of the emission peak. The increase in non-radiative relaxation pathways suppresses fluorescence emission, resulting in a decrease in fluorescence intensity. However, when f... w When the fluorescence intensity reaches 90%, a new fluorescence emission peak appears at 595 nm, and the fluorescence emission peak is similar to that at f. w The fluorescence emission peak at 0% is redshifted by 77 nm. This fluorescence change indicates that ISB-PyB possesses AIE characteristics.

[0066] (II) Analysis of the response mechanism of ISB-PyB probe to N2H4

[0067] Transfer 100 μL of ISB-PyB stock solution (10 -3 The concentration of N₂H₄ (mol / L) was placed in two separate 10 mL volumetric flasks. 200 μL of the N₂H₄ stock solution (10 mol / L) was then transferred to each flask. -2 The probe concentration (mol / L) was placed in one of the volumetric flasks and diluted to volume with 1 mL DMSO + 9 mL H2O to obtain a test solution with a probe concentration of 10 μmol / L. Subsequently, UV-Vis absorption spectroscopy and fluorescence spectroscopy were performed for scanning and testing. Figure 3The image shows the UV absorption and fluorescence spectra of ISB-PyB before and after the addition of N₂H₄. After the addition of N₂H₄, the band widths and absorbance of the absorption peaks at 296 nm and 306 nm of ISB-PyB increased, while the UV absorption peak at 448 nm and the fluorescence emission peak at 595 nm disappeared. These phenomena are mainly attributed to a specific interaction between ISB-PyB and N₂H₄, which inhibits the ICT process, leading to a significant change in the internal electron distribution of the molecule. This change in electron distribution causes changes in absorbance and fluorescence signal.

[0068] (III) Time Response Characteristics of ISB-PyB Probe to N2H4

[0069] To investigate the response rate of the ISB-PyB probe, the concentration of ISB-PyB in the test solution was maintained at 10 μmol / L, and the concentration of the analyte N2H4 was maintained at 100 μmol / L. The detailed preparation process is as follows: 100 μL of the ISB-PyB probe stock solution (10 μmol / L) was transferred... -3 mol / L), 100 μL of N2H4 stock solution (10 -2 The N2H4 test solution was prepared by placing a mol / L solution into a 10 mL volumetric flask and diluting it to volume with 1 mL DMSO and 9 mL H2O. The fluorescence spectrum of the system was then scanned over a period of time, and the fluorescence changes were recorded. When N2H4 was added to the ISB-PyMT solution, due to its excellent AIE properties, it rapidly aggregated, allowing its acceptor portion to bind quickly to N2H4. The fluorescence intensity stabilized within a short time (2 min), forming a stable fluorescence signal. This means that the probe can respond rapidly to N2H4.

[0070] (iv) Selectivity and anti-interference characteristics of ISB-PyB probe for N2H4

[0071] Preparation of selective system solutions for ISB-PyB: Transfer 100 μL of ISB-PyB probe stock solution (10 -3 mol / L) and different analyte stock solutions (10 -2 The probe concentration (10 μmol / L) was placed in a 10 mL volumetric flask and diluted to volume with 1 mL DMSO + 9 mL H₂O to maintain a probe concentration of 10 μmol / L. The concentration of each analyte was 100 μmol / L. These analytes included proline, glycine, isoniazid, and cations such as Li₂O₃. + NH4 + and anion HCO3 - F - Cl - ,Br - I - SO4 2-SCN - HPO4 2- H2PO4 - .

[0072] The preparation process of the anti-interference system for ISB-PyB probe detection of N2H4 is as follows: Take 100 μL of the probe stock solution (10... -3 mol / L), 100 μL of interfering analyte stock solution (10 -2 mol / L) and 100 μL N2H4 stock solution (10 -2 The sample was placed in a 10 mL volumetric flask and diluted to volume with 1 mL DMSO + 9 mL H2O to maintain the probe concentration in the test solution at 10 μmol / L, while the concentrations of various interfering substances and N2H4 were all 100 μmol / L.

[0073] After reacting at 27℃ for 5 min, the fluorescence spectra of each system were scanned to study the response of ISB-PyB in the presence of different interfering analytes and its anti-interference performance.

[0074] from Figure 5 It was observed that only N2H4 caused a significant change in the fluorescence signal. Furthermore, in the presence of different analytes, probe recognition was not significantly affected by competing species when treated with N2H4. These results demonstrate that ISB-PyB exhibits good selectivity and resistance to interference from N2H4, further expanding the potential applications of the probe in practical settings.

[0075] (iv) Preparation of portable N2H4 identification test strips

[0076] Portable N2H4 identification test strips were constructed using filter paper. The filter paper strips were immersed in a 10 μmol / L ISB-PyB solution in dichloromethane for 3 minutes, removed, and dried. Different concentrations of N2H4 solution were then sprayed onto the test strips. After complete air drying, images were captured under 365 nm ultraviolet light. The series of N2H4 concentrations were 5 μmol / L, 10 μmol / L, 15 μmol / L, 20 μmol / L, 25 μmol / L, 30 μmol / L, and 35 μmol / L. Figure 6 The N2H4 identification test strip prepared according to this invention is shown. It can be observed that under 365nm ultraviolet light, different concentrations of N2H4 cause different color changes in the test strip. As the N2H4 concentration increases, the fluorescence color of the test strip gradually changes from white to blue until it is quenched. This portable test strip is not only simple and easy to use, but also suitable for real-time on-site monitoring. The fluorescent probe of this invention has broad application prospects in fields such as bioimaging, drug screening, environmental monitoring, and food safety.

[0077] As described in the specification, those skilled in the art can modify the above examples. This invention is not limited to the above examples; any modifications, improvements, substitutions, or variations made within the concept and principles of this invention are within its protection scope. The specific terminology used in this specification is for ease of explanation and does not limit the invention.

Claims

1. An iminostilbene fluorescent probe ISB-PyB for detecting hydrazine hydrate, characterized in that, The compound ISB-PyB has the following structural formula: 。 2. A method for preparing an iminostilbene fluorescent probe for detecting hydrazine hydrate as described in claim 1, characterized in that, include: (1) Synthesis of compound 1a: Iminostilbene, 4-iodobenzaldehyde and Cs2CO3 were added to the reaction vessel, followed by the addition of toluene solution. N2 was then introduced to degas the system and remove all air from the system. Immediately afterwards, Pd(OAc)2 and DPPF were added to the system and degassing continued. The mixture was refluxed in the presence of N2. After the reaction was completed and cooled to room temperature, deionized water was added, and the mixture was extracted multiple times with ethyl acetate. The organic phases were combined and dried with anhydrous Na2SO4. The organic phase was concentrated to obtain the crude product. The crude product was purified by column chromatography to obtain a white solid product, which is compound 1a. Compound 1a has the following structural formula: ； (2) Synthesis of ISB-PyB: Compound 1a and barbituric acid were placed in a reaction vessel, and anhydrous ethanol and piperidine were added to the system and the mixture was refluxed. After the reaction was cooled to room temperature, the solvent was evaporated and the crude product was purified by column chromatography to finally obtain a pink solid powder, which is ISB-PyB.

3. The method for preparing the iminostilbene fluorescent probe for detecting hydrazine hydrate according to claim 2, characterized in that, In step (1), the molar ratio of iminostilbene to 4-iodobenzaldehyde and Cs2CO3 is 1 : (1.4~1.8) : (2~3); the weight-volume ratio of iminostilbene to toluene is 1 g : (45~60) mL.

4. The method for preparing the iminostilbene fluorescent probe for detecting hydrazine hydrate according to claim 2, characterized in that, In step (1), the molar ratio of iminostilbene to Pd(OAc)2 and DPPF is 1:(0.05~0.15):(0.10~0.30); the reaction reflux time of the mixture is 10~24 h.

5. The method for preparing the iminostilbene fluorescent probe for detecting hydrazine hydrate according to claim 2, characterized in that, In step (1), the time for each degassing operation is 10~30 min; the weight-volume ratio of iminostilbene to deionized water and ethyl acetate is 1.0 g: (30~50) mL: (15~25) mL.

6. The method for preparing the iminostilbene fluorescent probe for detecting hydrazine hydrate according to claim 2, characterized in that, In step (1), the eluent used in column chromatography purification is a mixed solution of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 12~25:

1.

7. The method for preparing the iminostilbene fluorescent probe for detecting hydrazine hydrate according to claim 2, characterized in that, In step (2), the molar ratio of compound 1a to barbituric acid is 1:(1.0~1.5).

8. The method for preparing the iminostilbene fluorescent probe for detecting hydrazine hydrate according to claim 2, characterized in that, In step (2), the weight-to-volume ratio of compound 1a to anhydrous ethanol and pyridine is 1 g:(30~50) mL:(80~200) μL, and the reflux time after adding acetic acid and pyridine is 1~2 h.

9. The method for preparing the iminostilbene fluorescent probe for detecting hydrazine hydrate according to claim 2, characterized in that, In step (2), the eluent used in column chromatography purification is a mixed solution of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 5~10:

1.

10. The application of the iminostilbene fluorescent probe for detecting hydrazine hydrate as described in claim 1, characterized in that, Used to prepare portable N2H4 identification test strips.

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