A dual-responsive imino stilbene fluorescent probe, and a preparation method and application thereof

Abstract

This invention belongs to the field of small molecule organic fluorescent probe technology, and discloses a dual-response iminostilbene fluorescent probe, its preparation method, and its application. This probe is used to detect cyanide and hydrazine hydrate, selecting iminostilbene with strong electron-gathering properties as the fluorophore and dicyanovinyl group with strong electron-withdrawing ability as the CN group. ‑ And N2H4 recognition part; this probe has excellent selectivity and fast response capability, and is similar to CN ‑ Upon binding, the fluorescence color changes from yellow to orange-red, and upon binding with N2H4, fluorescence quenching occurs, exhibiting obvious AIE characteristics and a strong fluorescence signal. Compared to other dual-response fluorescent probes, this invention can rapidly achieve the fluorescence response to CN under the same solvent system and excitation wavelength. ‑ For the detection of N2H4, it is more convenient to use. Portable CN ‑ The development of N2H4 identification test strips provides a powerful tool for rapid on-site detection, and therefore has important application value in fields such as biomedicine, environmental monitoring, food safety, and materials science.

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] Cyanide (CN) - Cyanide and hydrazine (N₂H₄) are widely used in industrial processes such as electroplating, metallurgy, high-energy fuels, herbicides, and the synthesis of fibers and resins. However, their harmful effects on human health cannot be ignored compared to their wide range of applications. Currently reported traditional analytical methods for detecting cyanide and hydrazine often require expensive equipment, cumbersome sample preparation procedures, strict operational skills, and demanding experimental conditions, severely limiting their practical application. Compared to traditional analytical methods, fluorescent probes offer advantages such as ease of operation, rapid reaction speed, high selectivity, and strong sensitivity, providing an effective approach for the detection of cyanide and hydrazine.

[0003] Based on traditional fluorophores such as rhodamine (New J. Chem., 2018, 42(20): 17056-17061), BODIPY (Sens. Actuators B: Chem., 2017, 239: 1307-1317), coumarin (Dyes Pigm., 2023, 216: 111370), and naphthaleneimide (Analyst., 2023, 148(15): 3491-3497), various fluorophores have been developed for the individual detection of CN. - Fluorescent probes for N2H4 have been reported. However, the detection of CN using two different fluorescent probes is also problematic. - Detecting N2H4 and other analytes would be time-consuming and costly. In contrast, dual-response fluorescent probes detect two target analytes by outputting different fluorescence signals, overcoming the limitations of single-identification detection and possessing the potential to improve detection efficiency and reduce costs.

[0004] Currently regarding CN -There are relatively few reports on dual-response fluorescent probes for N2H4, and achieving dual-response detection of these two targets usually requires two different solvent systems, different excitation wavelengths, and long response times (Talanta, 2021, 221: 121606), which to some extent affects their practicality. In addition, these single-detection or dual-response fluorescent probes exhibit high quantum yields in dilute solutions, but weak fluorescence emission in aggregated states or high-concentration solutions due to the aggregation-induced quenching effect (ACQ) significantly limits their application in detection performance. The aggregation-induced emission effect (AIE) first proposed by Tang Benzhong's research group in 2001 fundamentally overcomes the ACQ effect (Chem. Commun., 2001(18): 1740-1741). Due to the large Stokes shift and good stability of AIE molecules, it has attracted widespread attention from researchers. Therefore, developing a probe with AIE characteristics for N2H4 under the same sensing medium and the same excitation wavelength is a key research objective. - Dual-response fluorescent probes that respond quickly to N2H4 are essential. Summary of the Invention

[0005] This invention aims to solve the technical problem of dual-response detection of two target compounds, cyanide and hydrazine hydrate, and provides a dual-response iminostilbene fluorescent probe, its preparation method, and its application. The dicyanovinyl group of this probe has strong charge-withdrawing ability, which helps to form strong ICT fluorescent materials and can react with CN. - It reacts with N2H4 in different reaction forms; molecules with a D-π-A structure can exhibit excellent fluorescence properties, and the twisted D-π-A structure of the molecule has been proven to be an effective design strategy for converting the ACQ system into the AIE system; iminostilbene with strong electron-gathering properties is selected as the fluorophore, and dicyanovinyl group with strong electron-withdrawing ability is selected as the CN. - In the N2H4 recognition part, phenyl groups were selected as π-bridges to construct AIE-type D-π-A probes.

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

[0007] According to a first aspect of the present invention, a dual-responsive iminostilbene fluorescent probe is provided, having the following structural formula:

[0008]

[0009] Its chemical name is 2-(4-(5H-dibenzo[b,f]azaporin-5-yl)benzylidene)malononitrile, and its molecular formula is C2. 24 H 15 N3.

[0010] According to a second aspect of the present invention, a method for preparing the above-mentioned dual-responsive iminostilbene fluorescent probe is provided, the synthetic route of which is as follows:

[0011]

[0012] The specific steps include:

[0013] (1) Synthesis of 4-(5-(5H-dibenzo[b,f]azaline))-benzaldehyde:

[0014] Iminostilbene, 4-iodobenzaldehyde, and K2CO3 were added to a reaction vessel, followed by the addition of toluene solution. N2 was then introduced for degassing to remove air from the system. Pd2(dba)3 and Xphos were immediately added to the system, and degassing continued. The mixture was refluxed under N2 protection. After the reaction was complete and cooled to room temperature, deionized water was added, and the mixture was extracted multiple times with dichloromethane. The organic phases were combined and dried over anhydrous MgSO4. The mixture was then concentrated under vacuum to obtain a crude product. Further purification of the crude product by column chromatography yielded a white solid product, 4-(5-(5H-dibenzo[b,f]azaline))-benzaldehyde.

[0015] (2) Synthesis of 2-(4-(5H-dibenzo[b,f]azaline-5-yl)benzylidene)malononitrile (ISB-PyMT):

[0016] The 4-(5-(5H-dibenzo[b,f]azaline))-benzaldehyde and malononitrile obtained in step (1) were placed in a reaction vessel, and then acetic acid and pyridine were added and stirred at room temperature. The reaction progress was monitored by TLC. After the reaction was completed, the reaction mixture was transferred to ice water, and a yellow precipitate was formed. The yellow precipitate was then filtered and the filter cake was washed several times with distilled water to obtain the crude product. The crude product was further purified by column chromatography to obtain a pure yellow solid, namely 2-(4-(5H-dibenzo[b,f]azaline-5-yl)benzylidene)malononitrile (ISB-PyMT).

[0017] Further, in step (1), the molar ratio of iminostilbene to 4-iodobenzaldehyde and K2CO3 is 1:(1.02~1.10):(2.5~3.5).

[0018] Furthermore, in step (1), the weight-to-volume ratio of iminostilbene to toluene is 1 g: (20-40) mL.

[0019] Further, in step (1), the molar ratio of iminostilbene to Pd2(dba)3 and Xphos is 1:(0.08-0.12):(0.32-0.48).

[0020] Furthermore, in step (1), the reflux time of the mixture reaction is 16–36 hours.

[0021] Furthermore, in step (1), the time for each degassing operation is 0.25 to 1 hour.

[0022] Furthermore, the weight-to-volume ratio of iminostilbene to deionized water and dichloromethane is 1.0:(20-40)mL:(20-40)mL.

[0023] 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 10 to 20:1.

[0024] Further, in step (2), the molar ratio of 4-(5-(5H-dibenzo[b,f]azaline))-benzaldehyde to malononitrile is 1:(1.2~2).

[0025] Further, in step (2), the weight-to-volume ratio of 4-(5-(5H-dibenzo[b,f]azaporin))-benzaldehyde to acetic acid and pyridine is 1 g: (0.33-0.68) mL: (8.5-13.6) mL.

[0026] Furthermore, in step (2), the stirring time after adding acetic acid and pyridine is 1 to 2 hours.

[0027] Further, in step (2), the weight-to-volume ratio of 4-(5-(5H-dibenzo[b,f]azaline))-benzaldehyde to ice water and distilled water is 1.0 g:(30-50 mL) mL:(25-35 mL).

[0028] 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 20:1.

[0029] According to a third aspect of the present invention, an application of the above-described dual-responsive iminostilbene fluorescent probe is provided for the preparation of portable CN... - And N2H4 identification test strip.

[0030] The ISB-PyMT fluorescent probe of this invention has high AIE characteristics, high selectivity, and fast response time, making it of great application value in fields such as biomedicine, environmental monitoring, food safety, and materials science. It not only improves the accuracy and efficiency of research, but also helps to solve many real-world problems.

[0031] This invention investigated ISB-PyMT with different water contents (f wThe spectral properties of ISB-PyMT in a DMSO / H2O mixed solution confirmed its AIE characteristics. In pure DMSO solution, the fluorescence intensity of ISB-PyMT at 548 nm was relatively weak, while with increasing f... w As f increases further, the fluorescence intensity gradually increases, when f w It reaches its maximum value when f = 90%. w As the fluorescence concentration increased from 0 to 90%, the maximum fluorescence emission peak redshifted from 484 nm to 548 nm. This change in fluorescence emission with increasing moisture content indicates that ISB-PyMT possesses AIE (Alternating Intensity Ionization) properties. The ISB-PyMT probe exhibits a high fluorescence quantum yield, with an absolute quantum yield of 26.89% in the solid state.

[0032] This invention studies the effect of ISB-PyMT probes on the addition of CN. - The basic spectra before and after N2H4 were analyzed to determine the response mechanism. This invention also examines the basic spectra of CN... - Both N2H4 and N2H4 were detected in a mixed solution of DMSO / H2O (9:1, v / v), with an excitation wavelength of 409 nm. In CN... - In the presence of [a specific substance], the absorption intensity of the absorption peak at 440 nm decreased significantly, the absorption band broadened, and the absorption peak red-shifted by 17 nm; the maximum fluorescence emission peak red-shifted by 20 nm to a wavelength of 568 nm, the fluorescence intensity decreased significantly, by approximately 2.3 times, and the fluorescence color changed from yellow to orange-red. - Upon binding to the acceptor, the electron-withdrawing ability is enhanced, promoting the intramolecular ICT process. In the presence of N₂H₄, the absorption peak at 430 nm disappears, while the absorbance at 210 nm and 310 nm increases; the fluorescence emission peak at 548 nm also disappears. This is mainly attributed to the conversion of the electron-withdrawing cyano group to the electron-donating amino group, a chemical reaction that inhibits the intramolecular ICT process, thereby triggering a change in the fluorescence signal. The ISB-PyMT probe can target CN₂H₄. - The two substances, N2H4 and N2H4, exhibit different fluorescence signals, which can be used to distinguish and detect them.

[0033] This invention performs ISB-PyMT on CN - The time-dependent response experiment with N2H4, when CN - When added to the ISB-PyMT solution, the fluorescence intensity stabilized within 9 minutes. However, after adding N₂H₄ to the ISB-PyMT solution, the fluorescence intensity required approximately 20 minutes to stabilize, indicating a significant difference in reaction rates between ISB-PyMT and the two analytes. ISB-PyMT and CN -CN reacts faster and stabilizes more quickly, while its reaction with N2H4 is relatively slower and requires a longer time to stabilize. This significant difference in stabilization time provides a qualitative way to distinguish CN. - The possibility of detecting N2H4. The above results indicate that this probe can be used to detect CN. - A rapid analytical probe for N2H4.

[0034] This invention aims to study the impact of ISB-PyMT on CN. - To mitigate the specificity of N2H4, the probe was treated with various interfering analytes. These interfering analytes included arginine, proline, glycine, isoniazid, and cations such as Li... + Zr 4+ Fe 2+ Zn 2+ NH4 + and anion SCN - ,Br - Cl - F - HCO3 - HPO4 2- CO3 2- SO4 2- I - H2PO4 - Only CN - The presence of N2H4 and other interfering analytes caused significant changes in fluorescence signal, while other interfering analytes caused smaller changes in fluorescence. In the presence of different interfering analytes, CN... - When the probe is treated with N2H4, the probe recognition is not significantly affected by competing species. These results indicate that ISB-PyMT is effective against CN-2H4. - Both N2H4 and N2H4 exhibit good selectivity and anti-interference properties.

[0035] This invention aims to enable rapid and convenient detection of CN using a probe. - In addition to N2H4, test strips containing ISB-PyMT were also prepared. It was observed that under 365 nm ultraviolet light, different concentrations of CN... - The presence of N2H4 will cause the test strip to exhibit different color changes. With CN... - As the concentration increases, the fluorescence color of the test strip gradually changes from yellow to blue. However, with increasing N2H4 concentration, the fluorescence gradually disappears. This test strip can detect CN at a concentration of approximately 20 μmol / L. - Both N2H4 and UV light showed significant color changes under UV light. These results indicate that the portable test strips are simple and convenient to operate, making them ideal for real-time on-site monitoring and possessing broad application potential.

[0036] The beneficial effects of this invention are:

[0037] (i) The dual-response iminostilbene fluorescent probe of the present invention has AIE characteristics, which can overcome the quenching effect (ACQ) caused by aggregation of dual-response fluorescent probes in aggregated state or high-concentration solution, enhance the signal intensity of the probe, and have good stability.

[0038] (II) The dual-response iminostilbene fluorescent probe of the present invention uses iminostilbene, which has strong electron-donating ability, as a fluorophore, which helps to obtain probes with high fluorescence quantum yield and can obtain fluorescent probes with high signal intensity and high signal-to-noise ratio, thus promoting the development of iminostilbene in the field of fluorescent probes.

[0039] (III) The dual-response iminostilbene fluorescent probe of the present invention has good selectivity and fast response time, and can rapidly detect CN by outputting different fluorescence signals. - Compared to N2H4, it overcomes the limitations of single-identity detection and has the potential to improve detection efficiency and reduce costs. - With the N2H4 dual-response fluorescent probe, this invention can achieve the detection of CN under the same solvent system and the same excitation wavelength. - It makes the detection of N2H4 more convenient.

[0040] (iv) The dual-response iminostilbene fluorescent probe of the present invention can be used in portable CN... - The preparation of N2H4 identification test strips enables rapid detection of CN. - The presence of N2H4 makes its actual detection more convenient; the dual-response iminostilbene fluorescent probe of this invention has important application value in fields such as biomedicine, environmental monitoring, food safety, and materials science, improving the accuracy and efficiency of research. Attached Figure Description

[0041] Figure 1 The 2-(4-(5H-dibenzo[b,f]azaporin-5-yl)benzyl)malononitrile (ISB-PyMT) synthesized in Example 1 1 H NMR spectrum;

[0042] Figure 2 The fluorescence spectra (λ) of 2-(4-(5H-dibenzo[b,f]azaporin-5-yl)benzyl)malononitrile (ISB-PyMT) synthesized in Example 1 in mixed solutions with different water contents are shown. ex =409nm);

[0043] Figure 3 The image shows 2-(4-(5H-dibenzo[b,f]azaporin-5-yl)benzyl)malononitrile (ISB-PyMT) synthesized in Example 1, with the addition of CN... -Or the UV absorption and fluorescence spectra before and after N2H4 (λ) ex =409nm); where (a) is ISB-PyMT with added CN - Or the UV absorption spectra before and after the N2H4 reaction, (b) is the ISB-PyMT with CN added - Or fluorescence spectra before and after the N2H4 reaction;

[0044] Figure 4 The image shows 2-(4-(5H-dibenzo[b,f]azaporin-5-yl)benzyl)malononitrile (ISB-PyMT) synthesized in Example 1, with the addition of CN... - Time-dependent fluorescence change spectrum after N2H4 (λ) ex =409nm); where (a) is ISB-PyMT with added CN - (a) is a graph showing the change in fluorescence intensity over time after the addition of N2H4 to ISB-PyMT; (b) is a graph showing the change in fluorescence intensity over time after the addition of N2H4 to ISB-PyMT.

[0045] Figure 5 The figure shows the selectivity and anti-interference test results (λ) of 2-(4-(5H-dibenzo[b,f]azaporin-5-yl)benzyl)malononitrile (ISB-PyMT) synthesized in Example 1. ex =409nm); where (a) is the addition of CN to ISB-PyMT and different interfering analytes. - (a) Fluorescence changes before and after; (b) Fluorescence changes of ISB-PyMT and different interfering analytes before and after the addition of N2H4.

[0046] Figure 6 The image shows the 2-(4-(5H-dibenzo[b,f]azaporin-5-yl)benzyl)malononitrile (ISB-PyMT) portable CN synthesized in Example 1. - Photo of the N2H4 identification test strip. Detailed Implementation

[0047] 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.

[0048] Example 1: Molecular ISB-PyMT

[0049] (1) Synthesis of 4-(5-(5H-dibenzo[b,f]azaline))-benzaldehyde (1a):

[0050] Iminostilbene (3.87 g, 20.0 mmol), 4-iodobenzaldehyde (4.92 g, 21.2 mmol), and K₂CO₃ (6.91 g, 50.0 mmol) were added to a reaction vessel, followed by 80 mL of toluene solution. The mixture was degassed under N₂ for 0.25 h to remove all air from the system. Immediately afterwards, Pd₂(dba)₃ (1.46 g, 1.6 mmol) and Xphos (3.05 g, 6.4 mmol) were added, and degassed again for 0.25 h. The mixture was refluxed under N₂ protection for 16 h. After the reaction was complete and cooled to room temperature, 78 mL of deionized water was added, followed by extraction with 78 mL of dichloromethane. The combined organic phases were dried over anhydrous MgSO₄ and concentrated under vacuum to obtain the crude product. The crude product was further purified by column chromatography using petroleum ether:ethyl acetate (10:1, V / V) as the eluent to give a white solid product 1a in 48% yield.

[0051] (2) Synthesis of 2-(4-(5H-dibenzo[b,f]azaline-5-yl)benzylidene)malononitrile (ISB-PyMT):

[0052] Compound 1a (0.59 g, 2.0 mmol) and malononitrile (0.16 g, 2.4 mmol) were placed in a reaction vessel, followed by the addition of 0.2 mL of acetic acid and 5 mL of pyridine. The mixture was stirred at room temperature for 1 h. The reaction was monitored by TLC. After the reaction was complete, the reaction mixture was transferred to 18 mL of ice water, resulting in the precipitation of a yellow precipitate. The mixture was then filtered, and the filter cake was washed with 15 mL of distilled water and dried to obtain the crude product. The crude product was further purified by column chromatography using petroleum ether:ethyl acetate (5:1, V / V) as the eluent to give a pure yellow solid, ISB-PyMT, in 81% yield.

[0053] Structural characterization was performed using nuclear magnetic resonance spectroscopy. 1 The H NMR spectroscopy results are as follows: 1 H NMR (400MHz, DMSO-d6) δ8.11(s,1H),7.73–7.66(m,2H),7.66–7.57(m,6H),7.51(ddd,J=7.0,5.7,2.4Hz,2H),7.00(s,2H),6.35–6.26(m,2H). like Figure 1 As shown.

[0054] Example 2: Molecular ISB-PyMT

[0055] (1) Synthesis of 4-(5-(5H-dibenzo[b,f]azaline))-benzaldehyde (1a):

[0056] Iminostilbene (3.87 g, 20.0 mmol), 4-iodobenzaldehyde (4.92 g, 21.2 mmol), and K₂CO₃ (8.29 g, 60.0 mmol) were added to a reaction vessel, followed by 120 mL of toluene solution. The mixture was degassed under N₂ for 0.45 h to remove all air from the system. Immediately afterwards, Pd₂(dba)₃ (1.83 g, 2 mmol) and Xphos (3.81 g, 8 mmol) were added, and degassed again for 0.45 h. The mixture was refluxed under N₂ protection for 26 h. After the reaction was complete and cooled to room temperature, 116 mL of deionized water was added, followed by extraction with 116 mL of dichloromethane. The combined organic phases were dried over anhydrous MgSO₄ and concentrated under vacuum to obtain the crude product. The crude product was further purified by column chromatography using petroleum ether:ethyl acetate (15:1, V / V) as the eluent to give a white solid product 1a in 50% yield.

[0057] (2) Synthesis of 2-(4-(5H-dibenzo[b,f]azaline-5-yl)benzylidene)malononitrile (ISB-PyMT):

[0058] Compound 1a (0.59 g, 2.0 mmol) and malononitrile (0.22 g, 3.2 mmol) were placed in a reaction vessel, followed by the addition of 0.3 mL acetic acid and 6.5 mL pyridine. The mixture was stirred at room temperature for 1.5 h. The reaction was monitored by TLC. After the reaction was complete, the reaction mixture was transferred to 24 mL of ice water, resulting in the precipitation of a yellow precipitate. The mixture was then filtered, and the filter cake was washed with 18 mL of distilled water and dried to obtain the crude product. The crude product was further purified by column chromatography using petroleum ether:ethyl acetate (10:1, v / v) as the eluent to give a pure yellow solid, ISB-PyMT, in 80% yield.

[0059] Example 3: Molecular ISB-PyMT

[0060] (1) Synthesis of 4-(5-(5H-dibenzo[b,f]azaline))-benzaldehyde (1a):

[0061] Iminostilbene (3.87 g, 20.0 mmol), 4-iodobenzaldehyde (5.10 g, 22 mmol), and K₂CO₃ (9.67 g, 70.0 mmol) were added to a reaction vessel, followed by 150 mL of toluene solution. The mixture was degassed with N₂ for 1 h to remove all air from the system. Immediately afterwards, Pd₂(dba)₃ (2.20 g, 2.4 mmol) and Xphos (4.58 g, 9.6 mmol) were added, and degassed for another 1 h. The mixture was refluxed under N₂ protection for 36 h. After the reaction was complete and cooled to room temperature, 155 mL of deionized water was added, followed by extraction with 155 mL of dichloromethane. The combined organic phases were dried over anhydrous MgSO₄ and concentrated under vacuum to obtain the crude product. The crude product was further purified by column chromatography using petroleum ether:ethyl acetate (20:1, V / V) as the eluent to give a white solid product 1a in 49% yield.

[0062] (2) Synthesis of 2-(4-(5H-dibenzo[b,f]azaline-5-yl)benzylidene)malononitrile (ISB-PyMT):

[0063] Compound 1a (0.59 g, 2.0 mmol) and malononitrile (0.26 g, 4 mmol) were placed in a reaction vessel, followed by the addition of 0.4 mL acetic acid and 8 mL pyridine. The mixture was stirred at room temperature for 2 h. The extent of the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was transferred to 30 mL of ice water, resulting in the precipitation of a yellow precipitate. The mixture was then filtered, and the filter cake was washed with 21 mL of distilled water and dried to obtain the crude product. The crude product was further purified by column chromatography using petroleum ether:ethyl acetate (20:1, v / v) as the eluent to give a pure yellow solid, ISB-PyMT, in 82% yield.

[0064] The target probes ISB-PyMT 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.

[0065] The probe ISB-PyMT prepared in Example 1 above was tested as follows:

[0066] (I) AIE Nature Study

[0067] The study investigated ISB-PyMT with different water contents (f) w The spectral properties of the DMSO / H2O mixed solution were analyzed to determine the AIE characteristics of the detection probe. 100 μL of ISB-PyMT 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: 10 mL DMSO, 9 mL DMSO + 1 mL H₂O, 8 mL DMSO + 2 mL H₂O, 7 mL DMSO + 3 mL H₂O, 6 mL DMSO + 4 mL H₂O, 5 mL DMSO + 5 mL H₂O, 4 mL DMSO + 6 mL H₂O, 3 mL DMSO + 7 mL H₂O, 2 mL DMSO + 8 mL H₂O, and 1 mL DMSO + 9 mL H₂O. The solutions were then diluted to volume to prepare solutions with a water content of f. w The test solutions with concentrations of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% were analyzed using fluorescence spectroscopy. Figure 2 The fluorescence spectra of ISB-PyMT in mixed solutions with different water contents (λ) ex =409nm). In pure DMSO solution, the fluorescence intensity of ISB-PyMT is relatively weak at 548nm, while with f w As f increases further, its fluorescence intensity gradually increases, when f w It reaches its maximum value when f = 90%. w As the fluorescence emission concentration increased from 0 to 90%, the maximum emission peak redshifted from 484 nm to 548 nm. This change in fluorescence emission with increasing moisture content indicates that ISB-PyMT exhibits AIE (Active Induction Emission) properties. Due to the distorted D-π-A structure of ISB-PyMT, the rotation of intramolecular CN single bonds in pure DMSO solution consumes excitation energy through a non-radiative pathway, thereby reducing fluorescence emission and causing the probe to exhibit weak fluorescence. However, as f... w With the increase of [amount], ISB-PyMT molecules cannot remain in a single-molecule state but instead aggregate into aggregates. This molecular aggregation behavior stimulates intramolecular motion-restricted processes (RIMS) in the system, thereby reducing non-radiative decay pathways and leading to a significant enhancement of fluorescence, exhibiting obvious AIE characteristics.

[0068] (II) ISB-PyMT probe for CN - Analysis of the response mechanism of N2H4

[0069] The test was conducted using a 10 μmol / L ISB-PyMT solution. The specific solution preparation is as follows: Transfer 100 μL of the ISB-PyMT stock solution (10 μmol / L...) -3 The concentrations (mol / L) were placed in three separate 10 mL volumetric flasks, and 100 μL of CN was added. - Mother liquor (10) -2 mol / L) and 200 μL of N2H4 stock solution (10 -2The solution was placed in two volumetric flasks and diluted to volume with 1 mL DMSO + 9 mL H2O to obtain the test solution. The solution was then scanned and tested using UV-Vis absorption spectroscopy and fluorescence spectroscopy. Figure 3 The image shows the addition of CN to ISB-PyMT. - Or, the UV absorption and fluorescence spectra before and after N2H4. ISB-PyMT in CN - In the presence of [specific component], the absorption peak red-shifted by approximately 17 nm, the absorption band broadened, and the absorption at 440 nm decreased significantly. ISB-PyMT and CN [specific component] - After binding, the maximum fluorescence emission peak redshifts by 20 nm to a wavelength of 568 nm, the fluorescence intensity decreases by approximately 2.3 times, and the fluorescence color changes from yellow to orange-red. These phenomena are mainly attributed to CN. - Upon binding to the acceptor, the electron-withdrawing ability is enhanced, promoting the intramolecular ICT process and leading to a shift in UV and fluorescence signals. However, after ISB-PyMT reacts with N₂H₄, the absorption peak at 430 nm disappears, the absorbance at 295 nm and 310 nm increases, and the fluorescence emission peak at 548 nm disappears. This is mainly attributed to the conversion of the electron-withdrawing cyano group to the electron-donating amino group. This chemical reaction inhibits the intramolecular ICT process, altering the intramolecular electron distribution and thus causing changes in absorbance and fluorescence signals.

[0070] (III) ISB-PyMT probe for CN - and the time response characteristics of N2H4

[0071] To investigate the response rate of the probe ISB-PyMT, the concentration of ISB-PyMT in the test solution was maintained at 10 μmol / L, and the analyte CN was... - The concentrations of N2H4 were maintained at 100 μmol / L and 200 μmol / L, respectively. The specific preparation process is as follows: Transfer 100 μL of ISB-PyMT probe stock solution (10 -3 mol / L), 100 μL of CN - Mother liquor (10) -2 The concentration (mol / L) was placed in a 10 mL volumetric flask and diluted to volume with 1 mL DMSO + 9 mL H2O to obtain CN. - The test solution of the system. Transfer 100 μL of the ISB-PyMT probe stock solution (10... -3 mol / L), 200 μL of N2H4 stock solution (10 -2 The N2H4 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 spectra of both systems were then scanned over a period of time, and the changes in fluorescence intensity were observed and recorded. Figure 4The image shows the addition of CN to ISB-PyMT. - The time-dependent fluorescence change spectrum after N2H4, when CN - When added to the ISB-PyMT solution, the fluorescence intensity stabilized within 9 minutes, while the fluorescence intensity of ISB-PyMT bound to N2H4 required approximately 20 minutes to stabilize. ISB-PyMT and CN... - CN reacts faster and stabilizes more quickly, while its reaction with N2H4 is relatively slower and requires a longer time to stabilize. This significant difference in stabilization time provides a qualitative way to distinguish CN. - The possibility of N2H4. The above results indicate that the ISB-PyMT probe can rapidly target CN. - And N2H4 response.

[0072] (iv) ISB-PyMT probe for CN - Selectivity and anti-interference characteristics of N2H4

[0073] Preparation of selective ISB-PyMT system: Transfer 100 μL of ISB-PyMT probe stock solution (10 -3 mol / L) and different interfering analytes 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 and a concentration of each interfering analyte of 100 μmol / L. These interfering analytes included arginine, proline, glycine, isoniazid, and cations such as Li₂O₃. + Zr 4+ Fe 2+ Zn 2+ NH4 + and anion SCN - ,Br - Cl - F - HCO3 - HPO4 2- CO3 2- SO4 2- I - H2PO4 - After reacting at 27℃ for 25 min, the fluorescence spectra of each system were scanned.

[0074] ISB-PyMT probe detection of CN - The preparation process of the anti-interference system is as follows: Take 100 μL of probe stock solution (10 -3 mol / L), CN-mother liquor (10 -2mol / L) and mother liquor of interfering analytes (10 -2 The probe concentration (mol / 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 in the test solution. - The concentrations of all interfering substances were 100 μmol / L.

[0075] The preparation process of the anti-interference system for ISB-PyMT probe detection of N2H4 is as follows: Take 100 μL of the probe stock solution (10... -3 mol / L), 200 μL of interference analyte stock solution (10 -2 mol / L) and 200 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 200 μmol / L.

[0076] ISB-PyMT for CN - The responsive system was incubated at 27℃ for 10 min; while the ISB-PyMT system responding to N2H4 was incubated at 27℃ for 25 min. Subsequently, the fluorescence spectra of each system were scanned, and the fluorescence changes of each system were observed and recorded to study the probe response under the presence of different interfering analytes and to determine its anti-interference performance.

[0077] Figure 5 The results show the selectivity and anti-interference performance of ISB-PyMT. Only CN... - The presence of N2H4 and other interfering analytes caused significant changes in fluorescence signal, while other interfering analytes caused smaller changes in fluorescence. In the presence of different interfering analytes, CN... - When the probe is treated with N2H4, the probe recognition is not significantly affected by competing species. These results indicate that ISB-PyMT is effective against CN-2H4. - Both N2H4 and N2H4 exhibit good selectivity and anti-interference properties.

[0078] (V) Portable CN - Preparation of N2H4 recognition test strips

[0079] Select filter paper to construct CN - Portable identification test strips for N2H4. The filter paper strips were immersed in a dichloromethane solution of ISB-PyMT (10 μmol / L) for 5 minutes, then removed and dried. Different concentrations of CN2H4 were then used for identification. - The N2H4 solution was sprayed onto the test strip, and images were captured under 365nm ultraviolet light after the solvent had completely evaporated. CN...- The series of N2H4 solutions were 20 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, 60 μmol / L, and 70 μmol / L. Figure 6 The test strip prepared according to the present invention is shown, and it can be observed that different concentrations of CN under 365nm ultraviolet light... - The presence of N2H4 solution will cause the test strip to exhibit different color changes. With CN... - As the concentration increases, the fluorescence color of the test strip gradually changes from yellow to blue. However, with increasing N2H4 concentration, the fluorescence gradually disappears. This test strip can detect CN at a concentration of approximately 20 μmol / L. - Both N2H4 and N2H4 show obvious color changes when exposed to ultraviolet light.

[0080] These results demonstrate that the portable test strip is simple and convenient to operate, making it ideal for real-time on-site monitoring and possessing broad application potential. This probe has significant application value in fields such as biomedicine, environmental monitoring, food safety, and materials science, improving the accuracy and efficiency of research.

[0081] 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. A dual-responsive imino stilbene fluorescent probe ISB-PyMT, characterized in that, Compound ISB-PyMT has the following structural formula: 。 2. A method for preparing the dual-responsive imino stilbene fluorescent probe according to claim 1, characterized in that, Comprising: (1) Synthesis of compound 1a: Imidodiyl, 4-iodobenzaldehyde and K2CO3 were added into a reaction vessel, and a toluene solution was added, N2 was introduced for degassing operation to exhaust the air in the system; then Pd2(dba)3 and Xphos were added into the system, and degassing was continued; the mixture was refluxed for 16-36 h under the protection of N2; after the reaction was completed and cooled to room temperature, deionized water was added, extracted with dichloromethane for several times, the organic phase was combined and dried with anhydrous MgSO4, vacuum concentration to obtain the crude product, which was further purified by column chromatography to obtain white solid product, which was compound 1a; Compound 1a has the following structural formula: ； (2) Synthesis of compound ISB-PyMT: Compound 1a obtained in step (1) and malononitrile were placed in a reaction vessel, then acetic acid and pyridine were added at room temperature for stirring, and the reaction progress was monitored by TLC; after the reaction was completed, the reaction mixture was transferred to ice water, and yellow precipitate was precipitated; then the yellow precipitate was filtered, and the filter cake was washed with distilled water for several times to obtain the crude product, which was further purified by column chromatography to obtain pure yellow solid, which was compound ISB-PyMT.

3. The method of claim 2, wherein the method is represented by the following scheme: ###0002### 2 3 In step (1), the molar ratio of imidodiyl, 4-iodobenzaldehyde and K2CO3 was 1: (1.02-1.10): (2.5-3.5); the weight-volume ratio of imidodiyl and toluene was 1 g: (20-40) mL.

4. The method for preparing the dual-response iminostilbene fluorescent probe according to claim 2, characterized in that, In step (1), the molar ratio of imidodiyl, Pd2(dba)3 and Xphos was 1: (0.08-0.12): (0.32-0.48); the refluxing time of the mixture was 16-36 h.

5. The method for preparing the dual-response iminostilbene fluorescent probe according to claim 2, characterized in that, In step (1), the degassing time was 0.25-1 h each time; the weight-volume ratio of imidodiyl, deionized water and dichloromethane was 1.0 g: (20-40) mL: (20-40) mL.

6. The method for preparing the dual-response iminostilbene fluorescent probe according to claim 2, characterized in that, In step (1), the eluent for column chromatography purification was a mixed solution of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate was 10-20:

1.

7. The method for preparing the dual-responsive iminostilbene fluorescent probe according to claim 2, characterized in that, In step (2), the molar ratio of compound 1a and malononitrile was 1: (1.2-2); the weight-volume ratio of compound 1a, acetic acid and pyridine was 1 g: (0.33-0.68) mL: (8.5-13.6) mL, and the stirring time after adding acetic acid and pyridine was 1-2 h.

8. The method for preparing the dual-response iminostilbene fluorescent probe according to claim 2, characterized in that, In step (2), the weight-volume ratio of compound 1a, ice water and distilled water was 1.0 g: (30-50) mL: (25-35) mL.

9. The method for preparing the dual-response iminostilbene fluorescent probe according to claim 2, characterized in that, In step (2), the eluent for column chromatography purification was a mixed solution of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate was 5-20:

1.

10. Use of the dual-responsive imino stilbene fluorescent probe according to claim 1, characterized in that, CN for making portable - and N2H4 identification test strips.

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