A fluorescent probe for identifying hydrazine and its preparation method and application

Abstract

The present invention discloses a fluorescent probe for identifying hydrazine and a preparation method and application thereof, and relates to the technical field of hydrazine detection. The present invention designs and develops an "OFF-ON" type fluorescent probe MPD for detecting hydrazine through Knoevenagel condensation reaction. The probe can accurately identify hydrazine within 30 seconds, has many advantages such as good selectivity, high sensitivity, wide pH application range, and colorimetric-fluorescence dual-channel response. The prepared MPD-loaded TLC silica gel plate can quickly detect hydrazine vapor, and further successfully proves the excellent performance of MPD in detecting hydrazine in soil, water, hydroponic flowers, rice, water-grown bean sprouts and cells.

Description

Technical Field

[0001] The invention relates to the technical field of hydrazine detection, and in particular to a fluorescent probe for identifying hydrazine and a preparation method and application thereof. Background Art

[0002] Hydrazine (N2H4) is an important industrial raw material, widely used in medicine, pesticides, textile dyes and other fields; at the same time, hydrazine is polluting, and the widespread use of hydrazine inevitably causes serious pollution of water, soil, air and other ecosystems. Therefore, the rapid, simple and real-time quantitative detection of hydrazine is a technical problem that technicians in this field are more concerned about.

[0003] To date, researchers have done a lot of work to improve analytical methods for detecting and tracking low-concentration pollutants and developed many new feasible strategies. Traditional methods for detecting hydrazine include high-performance liquid chromatography, gas chromatography, electrochemical analysis, titration, etc. These methods have the advantages of accurate detection and low detection limit, but they also have the disadvantages of high cost, long time consumption, need for professional operation and fixed testing location, and are not conducive to real-time and long-term on-site monitoring. In recent years, fluorescent probes have attracted the attention of many researchers due to their advantages such as fast response, high sensitivity, non-invasiveness and ease of use.

[0004] So far, the fluorescent probes for detecting hydrazine that have been reported are divided into the following categories based on different recognition mechanisms, such as acetyl cleavage reaction, cyclization reaction, hydrazone formation, and selective ring-opening reaction promoted by hydrazine. However, most fluorescent probes have defects such as long reaction time, high detection limit, low signal-to-noise ratio, and fluorescence "ON-OFF", which seriously hinder their application in real-time detection and analysis and in the biological field. Secondly, the fluorescence signal of some probes is weak and cannot be detected visually.

[0005] Therefore, based on the above research, the inventors believe that how to design a fluorescent probe that has a rapid response to hydrazine, strong selectivity and anti-interference ability, and a low detection limit, and can realize visual detection of hydrazine, is a technical problem that technicians in this field urgently need to solve.

[0006] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0007] In view of the above technical problems, the embodiments of the present invention provide a fluorescent probe for identifying hydrazine and a preparation method and application thereof to solve the problems raised in the above background technology.

[0008] A fluorescent probe with the structural formula:

[0009] Among them, the above fluorescent probe is named MPD.

[0010] A method for preparing the fluorescent probe as described above comprises the following steps:

[0011] S1: dissolving compound a and cycloisopropyl malonate in dry ethanol to prepare solution A;

[0012] S2: Add glacial acetic acid dropwise to solution A and stir at room temperature until the reaction is complete to obtain solution B;

[0013] S3: filtering solution B to collect precipitates, and washing the precipitates with ethanol to obtain the fluorescent probe;

[0014] Wherein, the structural formula of compound a is:

[0015]

[0016] A use of the fluorescent probe as described above in hydrazine recognition.

[0017] Preferably, the fluorescent probe is used for detection and identification of hydrazine in soil, water, plants and food.

[0018] Preferably, the fluorescent probe is used for detecting and identifying exogenous hydrazine in cells for non-diagnostic purposes.

[0019] Preferably, the fluorescent probe is used by adding it to soil or water, or spraying it onto the surface of plants and food and then taking fluorescent photos.

[0020] A TLC silica gel plate is loaded with the fluorescent probe as described above.

[0021] Preferably, the TLC silica gel plate is used to detect gaseous hydrazine.

[0022] Preferably, the method for using the TLC silica gel plate is: placing the TLC silica gel plate in air containing gaseous hydrazine, leaving it to stand for a period of time at room temperature, and then taking a fluorescent photograph of the plate.

[0023] Preferably, the method for preparing a TLC silica gel plate comprises the following steps: cutting the TLC silica gel plate into squares, soaking the plate in a solution containing a fluorescent probe, and then taking the plate out and drying it to obtain the TLC silica gel plate.

[0024] A fluorescent probe for identifying hydrazine, a preparation method and application thereof provided in an embodiment of the present invention have the following beneficial effects: the present invention designs and develops an "OFF-ON" type fluorescent probe MPD for detecting hydrazine through a Knoevenagel condensation reaction, the probe can accurately identify hydrazine within 30 seconds, has many advantages such as good selectivity, high sensitivity, a wide pH range of application, and a colorimetric-fluorescence dual-channel response. The prepared TLC silica gel plate loaded with MPD can quickly detect hydrazine vapor, and further successfully demonstrates the excellent performance of MPD in detecting hydrazine in soil, water, hydroponic flowers, rice, water-grown bean sprouts and cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The schematic diagram of the synthesis process of the fluorescent probe MPD and its mechanism of recognizing hydrazine;

[0026] Figure 2 is the H NMR spectrum of MPD ( 1 H NMR), solvent: DMSO-d6;

[0027] Figure 3 High resolution mass spectrometry (HRMS) for MPD, solvent: DMSO-d6;

[0028] Figure 4 Schematic diagram of adding different analytes to MPD (10 μM) in EtOH / Tris (v / v=5 / 5, pH=7.4); wherein,

[0029] Figure 4 a is the UV absorption spectrum; the inset is the sunlight change before and after identification;

[0030] Figure 4 b is the fluorescence spectrum; the inset shows the fluorescence changes before and after identification;

[0031] Figure 4 c Fluorescence response of MPD when hydrazine (140 μM) was added in the presence of different analytes (560 μM);

[0032] Among them, 1:Al 3+ 2:Fe 2+ 3:Hg + 4:Mn 2+ 5:Co 2+ 6:Zn 2+ 7:Cr 3+ 8:Mg 2+ , 9:Ca 2+ 、10:Cu 2+ 11:CH3COO - , 12:PO4 3- 、13:HPO4 2-、14:ClO - 、15:P2O7 4- 、16:SO4 2- 、17:S 2- , 18: HS - 、19:CN - 20:HSO3 - , 21:F - , 22:Br - 、23:Cl - 、24:S2O3 2- , 25: GSH, 26: Cys, 27: Hydroxylamine, 28: Ethylenediamine, 29: O-phenylenediamine, 30: Diethylamine, 31: Triethylamine, 32: Aniline, 33: Piperidine, 34: Histamine, 35: Lysine, 36: Glycine, 37: Pyridine, 38: Hydrazine;

[0033] Figure 4 d is the fluorescence response of MPD when different concentrations of hydrazine (0-140 μM) are added; the inset is the linear relationship between the hydrazine concentration (0-140 μM) and the fluorescence intensity of MPD at 520 nm;

[0034] Figure 4 e is the effect of different pH on the fluorescence intensity of MPD (10 μM) at 520 nm in EtOH / Tris (v / v=5 / 5) solution containing / not containing hydrazine;

[0035] Figure 4 f is the fluorescence response time trend of MPD (10 μM) recognizing hydrazine;

[0036] Figure 5 High resolution mass spectrometry (HRMS) of MPD+N2H4·H2O, solvent: DMSO-d6;

[0037] Figure 6 a is the proton spectrum control, MPD–FF, MPD+N2H4·H2O and MPD 1 H NMR spectrum, solution DMSO-d6;

[0038] Figure 6 b is the MPD transition diagram based on CAM-B3LYP / 6-31G(d);

[0039] Figure 7 a is a schematic diagram of detecting hydrazine vapor using a TLC plate loaded with MPD;

[0040] Figure 7 b is the fluorescence change diagram of the MPD-loaded TLC plate when it was exposed to different concentrations of hydrazine vapor (0–200 μM) and interfering substances (50 mM);

[0041] Figure 8 a is a schematic diagram of the fluorescence changes of sand treated with hydrazine solution before and after spraying MPD;

[0042] Figure 8 b is a schematic diagram of the fluorescence changes after adding MPD (10 μM) to sandy soil solutions containing different concentrations of hydrazine;

[0043] Fig. 9 The fluorescence photo of MPD (10 μM) in the water sample of Xiaoling River and the comparison chart of fluorescence intensity and standard value;

[0044] Fig.10 a is a schematic diagram of the rose and chrysanthemum cultivation process;

[0045] Fig.10 b is a fluorescent photograph of the surface of flowers cultured with hydrazine solution after spraying MPD (5, 15, 30 min);

[0046] Fig.10 c is a fluorescent photo of rice and bean sprouts after different treatments;

[0047] Fig.11 Fluorescence photos of roses and chrysanthemums incubated with water, hydrazine solution, and water solution and then sprayed with MPD on the surface;

[0048] Fig.12 Schematic diagram of cell activity of MCF-7 cells in MPD solution (0, 5, 10, 15, 20 μM);

[0049] Fig.13 Bright field, green channel and superimposed field images of MCF-7 cells after incubation with hydrazine solution (0, 10, 50, 100, 150 μM) at 37°C for 30 min and then treated with MPD (10 μM); scale bar: 15 μm. DETAILED DESCRIPTION

[0050] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0051] In view of the above technical problems, the embodiments of the present invention provide a fluorescent probe for identifying hydrazine and a preparation method and application thereof to solve the problems raised in the above background technology.

[0052] Based on the current research status, we selected phenothiazine derivatives with methoxy groups bonded to the side chains as fluorophores, and introduced a strong electron-withdrawing group, cycloisopropyl malonate, to activate the adjacent C=C double bond, making the probe more susceptible to hydrazine attack, resulting in a nucleophilic addition reaction to generate a hydrazone product. The synthesis process of the probe MPD and its mechanism of recognizing hydrazine are as follows: Figure 1 shown.

[0053] It should be noted that the fluorescent probe designed by the present invention has a methoxy group bonded to the structure of phenothiazine. Studies have shown that the fluorescent probe with a methoxy group bonded to the side chain has better specificity than the fluorescent probe without a methoxy group, can specifically identify hydrazine, and can improve the response speed;

[0054] In addition, the hydroxyl-based "protection-deprotection" mechanism is one of the important strategies for designing fluorescent probes to identify hydrazine, and the methoxy group on the phenothiazine heterocycle can be converted into a hydroxyl group through a hydrolysis reaction with a high yield. If the current fluorescent probe sensing performance fails to meet the expected standards, we will further utilize the wide structural modifiability of phenothiazine derivatives to further design dual-recognition site fluorescent probes based on the "protection-deprotection" mechanism based on the current fluorescent probe structure.

[0055] 1 Experimental part

[0056] 1.1 Synthesis of MPD

[0057] Compound a (3.5mmol, 1.00g) and cycloisopropyl malonate (7mmol, 1.01g) were dissolved in 10mL dry ethanol, 0.5mL glacial acetic acid was added dropwise, and stirred at room temperature for 4h. The reaction progress was monitored by TLC. After the reaction was completed, the precipitate was filtered and washed with ethanol 3 times to obtain dark green solid MPD (0.99g, yield: 69.43%), Mp 236.7℃–237.1℃, QY=0.007% (in EtOH / Tris=5 / 5).

[0058] 1 H NMR (400MHz, DMSO-d6) δ8.53(s,1H),8.12(s,1H),7.23(t,J=8.0Hz,8.4Hz,1H),7.15(t,J=8.8Hz,2H),7.02(t,J=7 .2Hz,7.6Hz,1H),6.63(s,1H),4.10(q,J=6.9Hz,2H),3.98(s,3H),3.34(s,3H),1.72(s,6H),1.35(t,J=6.8Hz,3H). 13C NMR(100MHz,DMSO-d6)δ163.89,162.49,160.92,152.33,148.65,141.85,130.68,122.6 4,114.97,112.75,109.49,104.30,98.82,57.08,42.51,27.28,12.91.High-resolution mass spectrum(HRMS,ESI - )m / z,C 22 H 22 NO6S - [M+H2O-H] - calcd:428.1173,found:428.1165( Figure 2-Figure 3 ).

[0059] 1.2 Preparation of MPD solution and analyte solution

[0060] Accurately weigh 4.11 mg of MPD solid and dilute it to 10 mL with DMSO in a volumetric flask to obtain a 1 mM MPD stock solution. 3+ 2:Fe 2+ 3:Hg + 4:Mn 2+ 5:Co 2+ 6:Zn 2+ 7:Cr 3+ 8:Mg 2+ , 9:Ca 2+ 、10:Cu 2+ 11:CH3COO - , 12:PO4 3- 、13:HPO4 2- 、14:ClO-、15:P2O7 4 -、16:SO4 2- 、17:S 2- , 18: HS - 、19:CN - 20:HSO3 - , 21:F - , 22:Br - 、23:Cl - 、24:S2O3 2

[0061] -, 25: GSH, 26: Cys, 27: Hydroxylamine, 28: Ethylenediamine, 29: o-phenylenediamine, 30: Diethylamine, 31: Triethylamine, 32: Aniline, 33: Piperidine, 34: Histamine, 35: Lysine, 36: Glycine, 37: Pyridine, 38: Hydrazine, 50mM). The prepared ion solution was stored in the refrigerator and diluted according to the required concentration when used. Test solution: EtOH / Tris (v / v=5 / 5, pH=7.4), test conditions: excitation slit 5.0nm, emission slit 5.0nm, test voltage 550V, excitation wavelength 380nm, emission wavelength 520nm.

[0062] 1.2 Preparation of MPD-loaded TLC silica gel plates

[0063] The TLC silica gel plate was cut into 2.5 cm × 2.5 cm squares, soaked in MPD stock solution, and dried for use. Then the MPD-loaded TLC plate was placed on top of 10 mL vials containing different concentrations of hydrazine (0, 15, 25, 50, 100, 150, 200 μM, 2 mL) and diethylamine, triethylamine, aniline, piperidine, histamine, and pyridine (560 μM, 2 mL) for 0.5 h. Photos were taken using a smartphone in a dark box UV analyzer (365 nm).

[0064] 1.4 Environmental monitoring

[0065] Soil testing: Sandy soil was collected and the residual hydrazine in the soil was tested by two methods within 24 hours. The detection ability of MPD for residual hydrazine in soil was evaluated by the above two detection methods.

[0066] Method 1: First, place a portion of sand in a culture dish and take fluorescence and daylight photos of it. Then, spray hydrazine solution (140 μM) into the soil to simulate soil contaminated by hydrazine. After stirring evenly, take fluorescence photos. Continue to spray MPD stock solution and take photos to record its fluorescence color changes.

[0067] Method 2: Soil extract experiment, 1g of soil was added to EtOH / Tris (3mL) solution, shaken thoroughly and then allowed to stand, and daylight and fluorescence photos were taken; then 1g of soil was weighed and mixed evenly with hydrazine solution (50mM, 4mL), and insoluble soil was filtered out to obtain a clear hydrazine test solution; different volumes of hydrazine test solution were added to the above EtOH / Tris system containing 1g of soil to prepare mixed solutions (3mL) with hydrazine concentrations (0, 15, 30, 60, 90, 110, 140μM), shaken evenly, and MPD stock solution (30μL) was added, and then fluorescence photos were taken in a dark box UV analyzer.

[0068] Water sample testing: Water samples were collected from Xiaoling River in Jinzhou City to evaluate the ability of MPD to identify hydrazine in real water samples. The collected water samples from Xiaoling River in Jinzhou were filtered to remove the suspended matter visible to the naked eye, and the hydrazine solution (50mM) and Tris buffer solution were prepared with clarified Xiaoling River water. Different volumes of hydrazine solution (50mM) were added to the EtOH / Tris (4mL) system to prepare solutions with concentrations of (5, 10, 30, 60, 90, 120, 140μM), and then MPD stock solution (40μL) was added in sequence. The experiment was repeated three times. Fluorescence photos were taken in a dark box UV analyzer.

[0069] 1.5 Plant and food sample testing

[0070] Roses and chrysanthemums were purchased from the local market in Jinzhou and divided into four groups: tap water culture group, hydrazine solution (140 μM) culture group, surface spraying MPD stock solution and then cultured in water group, and first cultured with hydrazine solution for 3 hours and then sprayed with MPD solution on the surface group. The fluorescence changes were recorded with a smartphone in a dark box UV analyzer.

[0071] Bulk rice purchased from a local supermarket was divided into three groups and daylight photos were taken, namely, the group soaked in hydrazine solution (140 μM) for 1 hour, the group sprayed with MPD stock solution, and the group soaked in hydrazine solution (140 μM) for 1 hour and then sprayed with MPD stock solution. The fluorescence changes of the three groups of rice samples were observed in a dark box UV analyzer and photographed and recorded.

[0072] Soybean seeds purchased from the local market were soaked in water for 20 hours and then waited for germination. The sprouted soybeans were divided into three groups and daylight photos were taken, namely, the group soaked in hydrazine solution (140 μM) for 2 hours, the group sprayed with MPD stock solution, and the group soaked in hydrazine solution (140 μM) for 2 hours and then sprayed with MPD stock solution. The fluorescence changes of the bean sprout samples were observed in a dark box UV analyzer and photographed and recorded.

[0073] 1.6 Cell experiments

[0074] The cell counting kit (CCK-8) was used to detect the toxicity of MPD (0, 5, 10, 15, 20 μM) on MCF-7 cells. MCF-7 cells were incubated with N2H4 solution (0, 50, 100, 150 μM) for 30 min, then incubated with MPD (10 μM) for 30 min, and then washed with PBS solution three times before cell imaging.

[0075] 2 Results and discussion

[0076] 2.1 Spectral characteristics of MPD

[0077] Whether the probe can highly specifically recognize hydrazine is an important parameter to measure its performance. To this end, we studied the selectivity of MPD for hydrazine. First, we selected analytes (anions, cations, amino acids and other nitrogen-containing compounds) that may interfere with hydrazine detection and conducted UV-visible and fluorescence spectroscopy tests. Figure 4 As shown in a, MPD has an obvious absorption peak at 518nm. Only after adding hydrazine, the absorption peak blue-shifts to 360nm, and the color of the solution fades from purple to colorless ( Figure 4 In addition, the fluorescence spectrum ( Figure 4 b) It can be seen that only hydrazine can cause MPD to produce a new fluorescence emission peak at 520nm. Under 365nm ultraviolet light, the probe fluorescence can be observed to turn on and emit bright green fluorescence ( Figure 4 b inset), at the same time, the fluorescence quantum yield QY increased from 0.07% to 27.64%, while the MPD solution with other interfering ions did not produce any fluorescence signal at 520nm. The above results show that MPD has strong specificity and can realize dual-channel recognition of hydrazine by fluorescence and ultraviolet, with obvious fluorescence "turn-on" phenomenon and "naked eye" recognition effect.

[0078] Considering the complex situation in the actual environment, the presence of various analytes may interfere with the detection of hydrazine by MPD, so the anti-interference ability of MPD was further studied. Figure 4 As shown in Figure c, there is no significant difference in fluorescence intensity between the coexistence system of other analytes (560 μM), hydrazine (140 μM) and MPD and the MPD-N2H4 system. This shows that the specific recognition of hydrazine by MPD is not interfered by other analytes, which indicates that MPD has excellent selectivity and strong anti-interference ability for hydrazine.

[0079] In addition, we performed fluorescence spectroscopic titration experiments to evaluate the recognition sensitivity of MPD to hydrazine. Figure 4 As shown in Figure d, as the concentration of hydrazine solution increases, the fluorescence intensity of MPD at 520nm also increases gradually. When 140μM N2H4 is added, the fluorescence intensity reaches the maximum. After further addition of hydrazine, the fluorescence intensity of MPD remains basically unchanged, indicating that equilibrium has been reached. At the same time, there is a good linear relationship between the fluorescence intensity of MPD and the hydrazine concentration (0-140μM) (R 2 =0.99896, Figure 4 d inset). According to the equation LOD = 3S / K, the detection limit of MPD for hydrazine is calculated to be 12 nM, which is far below the maximum safety level (312 nM, 10 ppb) specified by the World Health Organization. The above results show that MPD has a high sensitivity to hydrazine and has the potential to detect trace amounts of hydrazine.

[0080] In order to evaluate the applicability of the probe in actual complex environments, the fluorescence responses of MPD and MPD-N2H4 under different pH conditions were investigated. Figure 4 As shown in Figure e, MPD exhibits a good response to hydrazine in the pH range of 6-12, which indicates that MPD can detect hydrazine in a wide pH range. In addition, the reaction rate of the probe with hydrazine is one of the important factors in evaluating the performance of the probe. Therefore, we further studied the time response of MPD to hydrazine. Figure 4 As shown in Figure f, the addition of hydrazine caused the fluorescence intensity of MPD to increase rapidly, reaching a maximum value at 30 s and remaining constant. This shows that MPD has the potential to quickly detect hydrazine in a practical environment.

[0081] 2.2 Sensing mechanism

[0082] Considering the nucleophilicity of hydrazine, we speculate that MPD reacts with hydrazine to form (Z)-10-ethyl-3-(hydrazinomethylene)-2-methoxy-10H-phenothiazine (MPD–FF) ( Figure 1 To confirm our hypothesis, we first added hydrazine hydrate (N2H4·H2O) to MPD to perform HEMS test. Figure 5 As shown, MPD+N2H4·H2O produced a new mass spectrum peak at m / z=318.2669, which corresponds to [M+H2O+H] + Subsequently, we react compound a with hydrazine hydrate and separate and purify the product. 1 H NMR confirmed that the product was MPD-FF ( Figure 6 a) Combine MPD–FF, MPD+N2H4·H2O and MPD 1 H NMR was used for comparison, such as Figure 6 As shown in a, after adding hydrazine hydrate, the proton signals of MPD at 8.6ppm (Ha) and 8.1ppm (Hb) shifted to 7.8ppm and 7.3ppm, respectively, which are completely consistent with the characteristic peaks (Ha', Hb') of MPD-FF. 1 The H NMR results are highly consistent. The above results fully prove that MPD reacts with hydrazine to form the product MPD-FF.

[0083] To further explore the sensing mechanism of MPD recognizing N2H4, we performed quantum chemical calculations using density functional theory (DFT) and time-dependent density functional theory (TD–DFT) methods. Figure 6As shown in b, for the probe MPD, the HOMO orbital is mainly localized in phenothiazine, the LUMO orbital is mainly localized in the isopropyl malonate group, and the S1 state is a charge transfer state. After photoexcitation, the PET process occurs with complete intramolecular charge transfer from the isopropyl malonate group to the phenothiazine, resulting in fluorescence quenching. The molecular orbital distribution of the product MPD-FF is delocalized on the conjugated system, and the S1 state is a localized excited state. After photoexcitation, electrons can directly fall back from the LUMO orbital to the HOMO orbital through radiation transition, thereby emitting fluorescence.

[0084] 3.3 Detection of gaseous hydrazine

[0085] Based on the excellent performance of MPD, we designed a TLC plate loaded with MPD and used it to detect hydrazine vapor. Figure 7 As shown in Figure a, the MPD-loaded TLC plate was placed on top of a vial containing different concentrations of hydrazine vapor source (0, 15, 25, 50, 100, 150, 200 μM) and other nitrogen-containing volatile substances (diethylamine, triethylamine, aniline, piperidine, histamine and pyridine, 50 mM), and the fluorescence changes of the TLC plate were observed under 365 nm ultraviolet light after being placed at room temperature for 0.5 h. As the concentration of hydrazine increases, the change process of the TLC plate's fluorescence signal from reddish brown to green can be clearly observed, while no obvious fluorescence signal changes were found for the TLC plate placed on top of the vial containing other interfering substances ( Figure 7 b) These results indicate that MPD-loaded TLC plates can rapidly and qualitatively detect hydrazine vapor.

[0086] 3.4 Determination of hydrazine in soil

[0087] The large-scale use of hydrazine and its improper handling will inevitably increase the possibility of soil contamination. Therefore, we studied the ability of MPD to detect hydrazine in sandy soil. Figure 8 As shown in a, there is no difference between uncontaminated soil and soil contaminated by hydrazine under fluorescence, but after MPD is sprayed on the surface of soil contaminated by hydrazine, it will emit strong green fluorescence under ultraviolet light. It can be seen that MPD can be used to directly detect soil contaminated by hydrazine and has an obvious fluorescence on signal for easy observation.

[0088] Considering that the inorganic colloids, organic colloids, and composite colloids in the soil have huge specific surface areas and carry a large amount of charge, the soil has a strong adsorption effect on organic and inorganic pollutants, which may cause MPD to be unable to effectively track hydrazine in the soil. In order to avoid the influence of soil adsorption on the detection effect, we tested the soil extract. Figure 8As shown in b, the MPD mixed system solution without hydrazine appears purple under ultraviolet light. This may be because the complex environment in the soil affects the original color of the MPD system solution. After the soil extract pretreated with different concentrations of hydrazine is added with MPD, the fluorescence color gradually changes from orange to green.

[0089] The experimental results of the above two methods show that MPD can directly perform qualitative detection of hydrazine in soil.

[0090] Application of 3.5MPD in actual water samples

[0091] In order to study the feasibility and accuracy of MPD detection of residual hydrazine in actual water samples, we selected water samples from the Xiaoling River in Jinzhou that may be contaminated by hydrazine. First, the fluorescence spectrum of the Xiaoling River water samples with hydrazine concentrations of (5, 10, 30, 60, 90, 120, 140 μM) was tested, and it was found that there was no significant difference between the measured values ​​of the fluorescence intensity of the Xiaoling River water samples and the predicted values ​​of the standard curve (Y = 54.4083X + 4.68803) we established ( Fig. 9 ). At the same time, we used the spike recovery method (Table 1) to measure the spike recovery rate of Xiaoling River water samples with hydrazine concentrations (5, 10, 15 μM) to be 98.14%–100.15%, with a small relative standard deviation. This shows that MPD has a strong anti-interference ability and is not affected by the complex environment in river water, and can quantitatively detect hydrazine in actual water samples.

[0092] Table 1 Detection of hydrazine in Xiaoling River water samples

[0093]

[0094] 3.6 Application of MPD in plants and food

[0095] In order to track and mark hydrazine in plant systems and food samples, we first used hydroponic flowers (roses and chrysanthemums) as experimental objects to simulate the accumulation of hydrazine in their bodies. After the flowers were cultured in the hydrazine solution for 3 hours, MPD ( Fig.10 a) Observe the fluorescence changes in a dark box UV analyzer to determine whether MPD can track hydrazine in plants. Fig.10 As shown in Figure b, as expected, the rose and chrysanthemum flowers pretreated with hydrazine can be observed to have obvious yellow-green fluorescence signals 5 minutes after spraying with MPD, and the fluorescence signals are significantly enhanced over time, while the flowers cultured with water, hydrazine solution, and sprayed with MPD only on the surface do not show any fluorescence signal changes within 60 minutes ( Fig.11 ). The above results indicate that MPD can track hydrazine accumulated in plants due to enrichment.

[0096] In addition, we investigated the detection effect of MPD on hydrazine in food samples. Fig.10 As shown in Figure c, only rice and bean sprouts treated with hydrazine showed obvious fluorescence signals under 365nm UV light after spraying MPD solution on their surface, while the hydrazine solution soaking group and the MPD stock solution spraying group did not show any changes. This shows that the probe MPD can be used to detect hydrazine in food samples.

[0097] 3.7 Imaging of hydrazine in living cells

[0098] In the cell experiment, MCF-7 cells were selected as the research object. First, the CCK-8 kit was used to perform a cytotoxicity test. The results showed that MPD had no significant toxicity to cells at low concentrations, and the cell survival rate was still over 80% when the concentration reached 20 μM ( Fig.12 In addition, cells incubated with MPD solution (10 μM) alone showed weak fluorescence signals in the green channel. However, when MCF-7 cells were first treated with hydrazine solutions of different concentrations (0, 10, 50, 100, 150 μM) for 30 min and then incubated with MPD solution for 30 min, it can be seen that the green fluorescence signal increased significantly with the increase in hydrazine concentration ( Fig.13 ). The above results show that MPD has good cell membrane permeability and can detect exogenous hydrazine in living cells.

[0099] In summary, we have successfully synthesized a new type of "OFF-ON" fluorescent probe MPD and used it for the rapid detection of hydrazine in various scenarios. MPD can achieve colorimetric and fluorescent dual-channel responses to hydrazine in the EtOH / Tris (v / v=5 / 5, pH=7.4) system, and the preparation of MPD-loaded TLC plates can quickly and qualitatively detect hydrazine vapor; in addition, MPD has been successfully used to label hydrazine enriched in hydroponic plants and foods, realizing the visual detection of hydrazine in plants and foods, and has also been successfully used for fluorescent imaging of hydrazine in living cells.

[0100] The embodiments described above are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the claims of the present invention.

Claims

1. A fluorescent probe, characterized in that The structural formula is: 。 2. A method for preparing a fluorescent probe as claimed in claim 1, characterized in that: The following steps are involved: S1: dissolving compound a and cycloisopropyl malonate in dry ethanol to prepare solution A; S2: Add glacial acetic acid dropwise to solution A and stir at room temperature until the reaction is complete to obtain solution B; S3: filtering solution B to collect precipitates, and washing the precipitates with ethanol to obtain the fluorescent probe; Wherein, the structural formula of compound a is: 。 3. Use of the fluorescent probe as claimed in claim 1 in preparing a hydrazine recognition reagent.

4. The use according to claim 3, characterized in that: The fluorescent probe is used for detecting and identifying hydrazine in soil, water, plants and food.

5. The use according to claim 3, characterized in that: The fluorescent probe is used for detecting and identifying non-diagnostic exogenous hydrazine in cells.

6. The use according to claim 4, characterized in that: The fluorescent probe is used by adding it to soil or water, or spraying it onto the surface of plants and food to take fluorescent photos.

7. A TLC silica gel plate, characterized in that The fluorescent probe as claimed in claim 1 is loaded thereon.

8. The TLC silica gel plate according to claim 7, characterized in that The TLC silica gel plate is used to detect gaseous hydrazine.

9. TLC silica gel plate according to claim 7, characterized in that, The method for using the TLC silica gel plate is as follows: placing the TLC silica gel plate in air containing gaseous hydrazine, leaving it to stand for a period of time at room temperature, and then taking a fluorescent photograph of the plate.

10. A method for preparing a TLC silica gel plate as claimed in claim 7, characterized in that: The method comprises the following steps: cutting a TLC silica gel plate into squares, soaking the plate in a solution containing a fluorescent probe, and then taking the plate out and drying it to obtain the TLC silica gel plate.

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