A novel fluorescent probe for detecting hydrazine hydrate, its preparation method and application

Abstract

This invention discloses a novel fluorescent probe for the detection of hydrazine hydrate, its preparation method, and its application, belonging to the field of fluorescent probe technology. The novel fluorescent probe provided by this invention uses a hemicyanine dye derivative as the fluorophore; simultaneously, the thiophene ester in the novel fluorescent probe serves as a specific recognition group for N2H4. On the one hand, its electron-withdrawing effect can efficiently quench the fluorescence of the hemicyanine dye derivative, making the probe "fluorescently silent" when not in contact with the target analyte; on the other hand, its unique structure endows the probe with excellent selectivity and sensitivity, ensuring specific recognition of N2H4. Furthermore, this novel fluorescent probe exhibits advantages such as high accuracy, high sensitivity, immunity to interference from various amino acids, common cations and anions, and good specificity when used for the quantitative detection of N2H4. It also has good pH adaptability and water solubility, thus showing promising application prospects in the detection of hydrazine hydrate.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a novel fluorescent probe for detecting hydrazine hydrate, its preparation method, and its application. Background Technology

[0002] Hydrazine hydrate (N2H4·H2O) is a widely used but highly toxic industrial reducing agent. Although the human body cannot produce it, inhaling trace amounts of N2H4 from the environment can cause serious harm to organisms. Therefore, rapid and accurate detection methods are needed to quantify N2H4 in the environment in order to mitigate its environmental and health hazards.

[0003] Traditional methods for detecting N2H4 mainly include gas chromatography (GC), high-performance liquid chromatography (HPLC), electrochemical analysis (EA), and capillary electrophoresis (CE). These methods suffer from drawbacks such as long processing times, complex sample preparation, and damage to cell tissues. In contrast, fluorescence methods are gaining increasing popularity and attention due to their high sensitivity, good selectivity, simple operation, and easy observation of fluorescence signals. However, fluorescent probes for monitoring N2H4 in the environment and biological systems remain scarce, and fluorescent probes with good selectivity, biocompatibility, and high sensitivity are even rarer.

[0004] In view of this, it is necessary to develop a novel fluorescent probe for rapid and efficient monitoring of hydrazine hydrate in the environment and organisms. Summary of the Invention

[0005] The purpose of this invention is to provide a novel fluorescent probe for the detection of hydrazine hydrate, its preparation method, and its application. This addresses the problems of poor accuracy and specificity in existing methods for detecting N₂H₄.

[0006] In a first aspect, the present invention provides a novel fluorescent probe for detecting hydrazine hydrate, the novel fluorescent probe having the structure shown in formula (Ⅰ):

[0007] ; R1 is selected from methyl or ethyl; R2 is selected from diethylamine or nitrogen heterocycle.

[0008] In this invention, the inventors discovered that the hemicyanine dye derivative in the novel fluorescent probe, acting as a fluorophore, possesses core characteristics such as long-wavelength fluorescence emission, high fluorescence quantum yield, large molar extinction coefficient, and good biocompatibility, laying the foundation for detection performance. Simultaneously, the thiophene ester in the novel fluorescent probe, as a specific recognition group for N2H4, exhibits several advantages. First, its electron-withdrawing effect efficiently quenches the fluorescence of the hemicyanine dye derivative, resulting in a "fluorescently silent" state when the probe is not in contact with the target. Second, its unique structure endows the probe with excellent selectivity and sensitivity, ensuring specific recognition of N2H4. When the probe interacts with N2H4, N2H4 nucleophilically attacks the ester bond of the thiophene ester, triggering probe molecule hydrolysis and releasing a free fluorophore, accompanied by significant fluorescence enhancement, forming a typical "turn-on" detection mode. As a near-infrared fluorophore, the long absorption / emission wavelength characteristics of hemicyanine dye derivatives can effectively avoid background fluorescence interference and significantly improve the detection signal-to-noise ratio; at the same time, it enhances the tissue / sample penetration ability of the light signal, making it suitable for detection scenarios of deep tissues and complex matrix samples (such as turbid water and soil).

[0009] Furthermore, the introduction of positively charged structures, ester bonds, and tertiary amine groups into the probe molecule not only further optimizes biocompatibility but also significantly enhances the efficiency of cellular uptake of the probe, providing a guarantee for N2H4 monitoring in biological systems (cells, tissues). Through the synergistic design of fluorophore performance, recognition group specificity, and structural modification, this probe achieves multi-scenario, highly sensitive, and highly selective monitoring of N2H4 in environmental samples (water, soil) and biological systems (cells, living organisms), possessing broad application value.

[0010] In this invention, by further changing the types of substituents R1 and R2, not only can cyanine dye compounds with more different water solubility be designed and synthesized, but also cyanine dye compounds with more different optical properties can be designed and synthesized, thereby significantly enhancing the detection performance of novel fluorescent probes.

[0011] It is understandable that the types of substituents R1 and R2 can be conventionally selected according to actual needs, as long as they can enhance the detection performance of the novel fluorescent probe. For example, in this invention, R1 is preferably selected from methyl or ethyl groups; R2 is preferably selected from diethylamine or nitrogen-containing heterocycles.

[0012] In some embodiments, the nitrogen heterocycle is selected from the structures shown in formulas (A1)-(A3): , , .

[0013] Understandably, the type of nitrogen heterocycle can be conventionally selected according to actual needs, as long as it can enhance the detection performance of the novel fluorescent probe.

[0014] In a second aspect, the present invention provides a method for preparing a novel fluorescent probe as described above, comprising the step of esterifying a hemicyanine dye derivative with 2-acetylthiophene to obtain a novel fluorescent probe.

[0015] The preparation method provided by this invention is simple, and the raw materials used are cheap and readily available, making it easy to apply in large-scale industrial production.

[0016] In some embodiments, the preparation method includes the following steps: dissolving 2-acetylthiophene and diisopropylcarbodiimide in an organic solvent, adding 4-(dimethylamino)-pyridine-4-toluenesulfonate after a first stirring, lowering the temperature of the reaction system to 0-4°C after a second stirring, slowly adding a hemicyanine dye derivative, and then carrying out an esterification reaction under stirring; after the reaction is completed, the novel fluorescent probe is obtained by washing, drying, evaporating, and purifying.

[0017] In this invention, a novel fluorescent probe is obtained by esterification reaction using a hemicyanine dye derivative and 2-thiophene acetate as raw materials. In this novel fluorescent probe, the hemicyanine dye derivative acts as the fluorophore, and the thiophene ester acts as the recognition group. When the probe reacts with N₂H₄, N₂H₄ specifically recognizes the thiophene ester group and nucleophilically attacks the ester bond, causing the fluorescent probe to hydrolyze, releasing the fluorophore, and restoring the fluorescence signal. This change in fluorescence signal allows it to be used for the detection of hydrazine hydrate. Furthermore, this novel fluorescent probe exhibits good selectivity and high sensitivity, making it more suitable for the detection of trace amounts of N₂H₄ in the environment and biological samples. In addition, this novel fluorescent probe not only has excellent biocompatibility but also a long fluorescence emission wavelength, which is beneficial for bioimaging research.

[0018] In some embodiments, the molar ratio of 2-acetylthiophene, diisopropylcarbodiimide and 4-(dimethylamino)pyridine-4-toluenesulfonate is (0.5-1.5):(0.5-1.5):(0.5-1.5), preferably 1:1:1.

[0019] In this invention, by controlling the molar ratio of 2-acetylenol, diisopropylcarbodiimide, and 4-(dimethylamino)pyridine-4-toluenesulfonate within a specific range, the subsequent esterification reaction can be better carried out, resulting in a novel fluorescent probe with better performance.

[0020] In some embodiments, the mass-to-volume ratio of 2-acetylthiophene to the organic solvent is 36.5 mg:(20-40 mL), preferably 36.5 mg:30 mL; and the organic solvent includes dichloromethane.

[0021] In this invention, by controlling the mass-volume ratio of 2-acetyluphthiophene to organic solvent within a specific range, it is easier to carry out the subsequent esterification reaction and obtain a novel fluorescent probe with better performance.

[0022] It is understood that organic solvents can be conventionally selected based on actual usage needs, as long as they can efficiently dissolve the raw materials. For example, in this invention, dichloromethane is preferably included as the organic solvent.

[0023] In some embodiments, the first stirring includes stirring for 10-20 minutes at room temperature; and / or, the second stirring includes stirring for 4-6 hours at room temperature.

[0024] It is understood that the parameters of the first and second stirring can be adjusted according to actual usage needs, as long as the raw materials can be dissolved efficiently. For example, in this invention, the first stirring preferably includes stirring for 10-20 minutes at room temperature; the second stirring preferably includes stirring for 4-6 hours at room temperature.

[0025] In some embodiments, the molar ratio of hemicyanine dye derivative to 2-acetylthiophene is (0.05-0.15):(0.2-0.3), preferably 0.095:0.285.

[0026] In this invention, controlling the molar ratio of hemicyanine dye derivative to 2-thiophene acetate within a specific range enables the esterification reaction to be complete, thereby obtaining a novel fluorescent probe with better performance.

[0027] In some implementations, the esterification reaction includes reacting at room temperature for 4-6 hours.

[0028] It is understood that the parameters of the esterification reaction can be routinely adjusted according to actual needs, as long as the esterification reaction is complete. For example, in this invention, the esterification reaction preferably includes reacting at room temperature for 4-6 hours.

[0029] In a third aspect, the present invention provides the application of the novel fluorescent probe as described above or the novel fluorescent probe prepared by any of the above preparation methods in the detection of hydrazine hydrate.

[0030] The novel fluorescent probe provided by this invention has long absorption / emission wavelength characteristics, which can effectively avoid background fluorescence interference and significantly improve the detection signal-to-noise ratio. At the same time, it enhances the tissue / sample penetration ability of the light signal, making it suitable for the detection of N2H4 in deep tissues and complex matrix samples (such as turbid water and soil). In addition, when used for the quantitative detection of N2H4, this novel fluorescent probe has the advantages of good accuracy, high sensitivity, no interference from various amino acids, common cations and anions, and good specificity. It also has good pH adaptability and water solubility. Therefore, it has good application prospects in the detection of hydrazine hydrate.

[0031] The beneficial effects of this invention are as follows: Unlike existing technologies, the hemicyanine dye derivative in the novel fluorescent probe provided by this invention serves as the fluorophore, possessing core characteristics such as long-wavelength fluorescence emission, high fluorescence quantum yield, large molar extinction coefficient, and good biocompatibility, laying the foundation for detection performance. Simultaneously, the thiophene ester in the novel fluorescent probe, as a specific recognition group for N2H4, exhibits electron-withdrawing effect that efficiently quenches the fluorescence of the hemicyanine dye derivative, resulting in a "fluorescently silent" state when the probe is not in contact with the target analyte. Furthermore, its unique structure endows the probe with excellent selectivity and sensitivity, ensuring specific recognition of N2H4. In addition, this novel fluorescent probe, when used for the quantitative detection of N2H4, offers advantages such as high accuracy, high sensitivity, immunity to interference from various amino acids, common cations and anions, and good specificity. It also possesses good pH adaptability and water solubility, thus showing promising application prospects in the detection of hydrazine hydrate. Attached Figure Description

[0032] Figure 1 The novel fluorescent probe CH-NO3 prepared in Example 1 of this invention 1 H NMR spectrum; Figure 2 The novel fluorescent probe CH-NO3 prepared in Example 1 of this invention 13 C NMR spectrum; Figure 3 The image shows the HRMS spectrum of the novel fluorescent probe CH-NO3 prepared in Example 1 of this invention. Figure 4 The UV absorption spectrum (A) and fluorescence emission spectrum (B) of the novel fluorescent probe CH-NO3 prepared in Example 1 of this invention before and after its reaction with N2H4 in PBS buffer solution are shown. Figure 5 The fluorescence emission spectra of the novel fluorescent probe CH-NO3 prepared in Example 1 of this invention before and after reaction with different concentrations of N2H4 in PBS buffer solution are shown. Figure 6This is a graph showing the fluorescence intensity results of the novel fluorescent probe CH-NO3 reacting with N2H4 in PBS buffer solutions at different pH values ​​in Example 2 of the present invention. Figure 7 The results of fluorescence intensity changes of the novel fluorescent probe CH-NO3 in Example 3 of this invention before and after reaction with various analytes in PBS buffer solution are shown; wherein: A1: control (no analyte added), A2: leucine, A3: glycine, A4: histidine, A5: Cys, A6: Hcy, A7: GSH, A8: alanine, A9: phenylalanine, A10: serine, A11: tryptophan, A12: valine, A13: kJ / mol. + A14: Na + A15: Ag + A16: Fe 3+ A17: Al 3+ A18: Cu 2+ A19: Hg 2+ A20: Zn 2+ A21: Mg 2+ A22: Cl A23: Br A24: I A25: NO 3 A26: AcO A27: SO4 2 A28: CO3 2- A29: N2H4. Detailed Implementation

[0033] The technical solutions of the present invention will be clearly and completely described below with reference to 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0034] Experimental methods not specified in the examples are generally performed under conventional conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, the general equipment, materials, reagents, etc. used are commercially available.

[0035] Example 1 Synthesis of a novel fluorescent probe CH-NO3 This embodiment provides a method for synthesizing a novel fluorescent probe CH-NO3, the synthesis reaction of which is shown below: .

[0036] The specific synthesis method is as follows: 2-Acetylthiophene (36.5 mg, 0.285 mmol) and diisopropylcarbodiimide (DIPC, 36.0 mg, 0.285 mmol) were dissolved in 30 mL of anhydrous dichloromethane and stirred at room temperature for 15 minutes. Then, 4-(dimethylamino)-pyridine-4-toluenesulfonate (DPTS, 83.5 mg, 0.285 mmol) was added, and the reaction mixture was stirred for 5 hours. After cooling to 0 °C, the hemicyanine dye derivative CH-N (50 mg, 0.095 mmol) was slowly added, and the mixture was slowly restored to room temperature, followed by stirring for another 5 hours. After the reaction was complete, the mixture was washed three times with water, the organic layer was dried over Na2SO4, and evaporated under vacuum. The residue was purified by silica gel chromatography using CH2Cl2 / MeOH to obtain the novel fluorescent probe CH-NO3.

[0037] The structure of the novel fluorescent probe CH-NO3 prepared above was characterized.

[0038] Among them, the hydrogen spectrum (such as Figure 1 The results are as follows: the deuterated reagent is d 6-DMSO. 1 H NMR (400 MHz, d 6-DMSO,ppm): δ 8.15-8.14 (m, 1H), 8.07-8.06 (m, 1H), 7.81-7.80 (d, 1H), 7.72-7.70(d, 1H), 7.64-7.62 (d, 1H), 7.50-7.48 (m,3H), 7.35-7.33 (m, 1H), 7.13-7.11(d, 3H), 6.33-6.29 (d, 1H), 3.85-3.80 (m, 4H), 3.71 (s, 3H), 2.90-2.84 (m,2H),2.73-2.50 (m, 2H), 1.65 (s, 6H), 1.32-1.29 (m,6H).

[0039] Carbon spectrum (e.g.) Figure 2 The results are as follows: the deuterated reagent is d 6-DMSO. 13 C NMR (400 MHz, d6-DMSO, ppm): δ174.54, 167.73, 160.21, 152.60, 145.32, 143.22, 141.68, 138.04, 136.05,131.89, 129.59, 129.36, 128.51, 125.97, 122.83, 122.48, 120.75, 112.11,101.24, 49.57, 46.64, 41.79, 32.19, 28.20, 21.25, 14.37, 11.47.

[0040] Mass spectrometry (e.g.) Figure 3 The results are as follows: HRMS (ESI): calcd for C 32 H 35 N2O2S + ([M] + ) 511.2414, found 511.24208.

[0041] The above test results show that the novel fluorescent probe CH-NO3 in this invention was successfully synthesized.

[0042] Furthermore, the UV absorption and fluorescence emission spectra of the novel fluorescent probe CH-NO3 prepared above before and after its reaction with N2H4 in PBS buffer solution (pH 7.4) were tested, and the results are as follows: Figure 4 As shown.

[0043] from Figure 4 As can be seen, the novel fluorescent probe CH-NO3 has the largest UV absorption peak at 550 nm. After co-incubation with N2H4, the absorption peak at 550 nm decreases, while the absorption peak at 490 nm increases. Figure 4 A); Under excitation by 550 nm light, the fluorescence signal of the novel fluorescent probe CH-NO3 is weak, but after interaction with N2H4, the fluorescence signal at 640 nm gradually increases. Figure 4 (B) Clearly, the novel fluorescent probe CH-NO3 has the function of fluorescence-activated detection of N2H4. Furthermore, all the above tests were conducted in a pure water system, requiring no addition of any organic solvents for dissolution. Secondly, the fluorescence signal detection range is above 600 nm, and the long emission wavelength effectively reduces interference from the autofluorescence of organisms when detecting N2H4 in biological experiments (applications).

[0044] Furthermore, the fluorescence emission spectra of the novel fluorescent probe CH-NO3 prepared above were tested before and after interaction with different concentrations of N2H4 (0 μM, 40 μM, 80 μM, 120 μM, 160 μM, 200 μM, 240 μM) in PBS buffer solution (pH 7.4). The results are as follows. Figure 5 As shown.

[0045] from Figure 5 As can be seen, the fluorescence signal of the novel fluorescent probe CH-NO3 is weak under 550 nm light excitation, but the fluorescence signal at 640 nm increases with increasing N2H4 concentration. Therefore, this novel fluorescent probe CH-NO3 can be used for the detection of trace amounts of hydrazine hydrate (N2H4) in the environment and in organisms.

[0046] Example 2: Effect of pH on the detection performance of a novel fluorescent probe for CH-NO3 The fluorescence intensity at 640 nm of the novel fluorescent probe CH-NO3 prepared in Example 1 was tested before and after the addition of N2H4 to PBS buffer solutions of different pH values ​​(pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.4, 8.0, 8.5, 9.0, 9.5, and 10.0). The results are as follows: Figure 6 As shown.

[0047] from Figure 6 As can be seen, under 550nm light excitation, the fluorescence intensity of the novel fluorescent probe CH-NO3 is almost unaffected by pH increase without the addition of N2H4; after the addition of N2H4, the fluorescence intensity is significantly enhanced in the pH range of 6.5-10. Therefore, the novel fluorescent probe CH-NO3 is suitable for the detection of hydrazine hydrate (N2H4) in most cases in the environment and in organisms.

[0048] Example 3: Specificity test of the novel fluorescent probe CH-NO3 The novel fluorescent probe CH-NO3 prepared in Example 1 was reacted with various amino acids, cations, and anions (leucine, glycine, histidine, Cys, Hcy, GSH, alanine, phenylalanine, serine, tryptophan, valine, and phosphodiesterase) in PBS buffer solution (pH 7.4). + Na + Ag + Fe 3+ Al 3+ Cu 2+ Hg 2+ Zn 2+ Mg 2+ Cl ,Br I NO 3 AcO SO4 2 CO3 2- After co-incubation with N2H4, the fluorescence intensity at 640 nm was measured, and the results are as follows: Figure 7 As shown.

[0049] from Figure 7 As can be seen, under 550nm light excitation, the fluorescence intensity of the novel fluorescent probe CH-NO3 hardly changed after 70 min with and without the addition of analyte. Only after the addition of N2H4 did the fluorescence intensity of the novel fluorescent probe CH-NO3 increase significantly. The results indicate that the novel fluorescent probe CH-NO3 has strong anti-interference ability and can be used for the detection of N2H4 in complex environments and biological systems.

[0050] In summary, the novel fluorescent probe provided by this invention has the advantages of high accuracy, high sensitivity, and good specificity when used for the quantitative detection of N2H4, and is not affected by various amino acids, common cations and anions. It also has good pH adaptability and water solubility. Therefore, it has good application prospects in the detection of hydrazine hydrate.

[0051] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0052] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A novel fluorescent probe for detecting hydrazine hydrate, characterized in that, The novel fluorescent probe has the structure shown in formula (Ⅰ): ； Wherein, R1 is selected from methyl or ethyl; R2 is selected from diethylamine and nitrogen heterocycles.

2. The novel fluorescent probe according to claim 1, characterized by, The nitrogen heterocycle is selected from the structures shown in formulas (A1)-(A3): 、 、 。 3. A method for preparing the novel fluorescent probe according to claim 1 or 2, characterized by, The method includes the step of esterifying a hemicyanine dye derivative with 2-acetylthiophene to obtain the novel fluorescent probe.

4. The method for preparing the novel fluorescent probe according to claim 3, characterized in that, Includes the following steps: 2-Acetylthiophene and diisopropylcarbodiimide were dissolved in an organic solvent. After a first stirring, 4-(dimethylamino)-pyridine-4-toluenesulfonate was added. After a second stirring, the temperature of the reaction system was lowered to 0-4℃, and a hemicyanine dye derivative was slowly added. Esterification reaction was carried out under stirring. After the reaction was completed, the novel fluorescent probe was obtained by washing, drying, evaporation, and purification.

5. The preparation method according to claim 4, characterized in that, The molar ratio of the 2-acetylthiophene, the diisopropylcarbodiimide, and the 4-(dimethylamino)-pyridine-4-toluenesulfonate is (0.5-1.5):(0.5-1.5):(0.5-1.5).

6. The preparation method according to claim 4, characterized in that, The mass-to-volume ratio of 2-acetylthiophene to the organic solvent is 36.5 mg:(20-40 mL), and the organic solvent includes dichloromethane.

7. The preparation method according to claim 4, characterized in that, The first stirring includes: stirring for 10-20 minutes at room temperature; and / or, The second stirring includes stirring for 4-6 hours at room temperature.

8. The preparation method according to claim 4, characterized in that, The molar ratio of the hemicyanine dye derivative to the 2-acetylthiophene is (0.05-0.15):(0.2-0.3).

9. The preparation method according to claim 4, characterized in that, The esterification reaction includes reacting at room temperature for 4-6 hours.

10. The application of the novel fluorescent probe as described in claim 1 or 2, or the novel fluorescent probe prepared by any one of claims 3-9, in the detection of hydrazine hydrate.

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