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

Abstract

The present invention discloses a fluorescent probe for hydrazine detection, a preparation method and an application thereof, and belongs to the technical field of fluorescent probes for hydrazine (N2H4) detection. The fluorescent probe is 2-((10-butyl-10H-phenothiazine-3-yl)methylene)-1H-indene-1,3(2H)-dione, and its structural formula is #imgabs0# The present invention also specifically discloses a preparation method of the fluorescent probe for hydrazine imaging in an organism and its application in rapid and highly selective identification of hydrazine and preparation of imaging detection of hydrazine in cells and zebrafish. The present invention realizes colorimetric and fluorescence detection of hydrazine, and has fast response, low detection limit and high selectivity, and also realizes imaging detection of hydrazine in cells and zebrafish.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes for hydrazine (N2H4) detection, and specifically relates to a fluorescent probe for hydrazine detection and a preparation method and application thereof. Background Art

[0002] Hydrazine (N2H4) is an important industrial and laboratory raw material used in rocket fuel, imaging agents, antioxidants, dyes and the pharmaceutical industry. In addition, the synthesis of many pesticides and herbicides is inseparable from hydrazine. It can be said that hydrazine is closely related to human daily life. While hydrazine is widely used, it also produces a large amount of hydrazine waste, which harms the environment by penetrating the soil and polluting water bodies. It can also be ingested by the human body through the skin, eyes and inhalation, causing sensory irritation, shortness of breath, headache, coma, etc. Severe cases can also cause damage to the liver, kidneys and central nervous system of the human body. Hydrazine has therefore been regarded as a potential human carcinogen with a threshold limit of 10ppb (0.31μM). Traditional methods for detecting hydrazine include capillary electrophoresis, electrochemical analysis, chemiluminescence and chromatography. These detection methods cannot meet the current real-time detection needs due to their disadvantages such as expensive detection, time-consuming and complex operation. Currently, the widely used fluorescence spectroscopy technology has gained more attention due to its excellent sensitivity, specificity and biocompatibility. These fluorescent probes are designed and constructed based on the affinity properties of hydrazine. Although these fluorescent probes with functional groups have been constructed for the detection of hydrazine, they still have many defects, such as low water solubility, long reaction time, weak fluorescence signal, short emission wavelength, and narrow practical application range. Therefore, it is necessary to further explore the recognition mechanism to further improve the detection performance. Summary of the invention

[0003] The purpose of the present invention is to provide a fluorescent probe for hydrazine detection and a preparation method thereof. The fluorescent probe prepared by the method can be used for rapid and highly selective identification of hydrazine in organisms and for biological imaging.

[0004] The present invention adopts the following technical scheme to achieve the above-mentioned purpose: a fluorescent probe for hydrazine detection, characterized in that: the fluorescent probe is 2-((10-butyl-10H-phenothiazine-3-yl)methylene)-1H-indene-1,3(2H)-dione (DO), and its structural formula is

[0005]

[0006] The method for preparing a fluorescent probe for hydrazine detection of the present invention is characterized in that the specific process is: using ethanol as a reaction medium, using 10-butyl-10H-phenothiazine-3-carboxaldehyde and 1,3-indandione as reaction raw materials, using piperidine as a catalyst, reacting at 70-80°C under the protection of an inert gas, and after the reaction is complete, filtering to remove the solvent, and washing with ether to obtain the target product, which is a fluorescent probe for hydrazine detection.

[0007] It is further defined that the molar ratio of the 10-butyl-10H-phenothiazine-3-carboxaldehyde, 1,3-indandione and piperidine is 1:1:0.01-0.05.

[0008] The synthetic route in the preparation method of the fluorescent probe for hydrazine detection of the present invention is:

[0009]

[0010] The invention discloses an application of the fluorescent probe for hydrazine detection in the simultaneous rapid and highly selective identification of hydrazine, and the specific process is as follows: the fluorescent probe presents purple in a mixed solution of phosphate buffer and acetonitrile in a volume ratio of 4:1, and has no fluorescence under a 365nm portable ultraviolet lamp; after hydrazine is added to the mixed system, the color of the mixed system becomes colorless, and exhibits bright green fluorescence under a 365nm portable ultraviolet lamp.

[0011] The fluorescent probe for hydrazine detection of the present invention is used in preparing a hydrazine imaging detection reagent in cells or zebrafish.

[0012] Compared with the existing fluorescent probe technology for detecting hydrazine, the fluorescent probe for detecting hydrazine in an organism prepared by the present invention has a simple preparation process, can be obtained by only one-step reaction, has mild reaction conditions, is simple to operate, and does not require column chromatography. The fluorescent probe for detecting hydrazine in an organism prepared by the present invention can realize colorimetric and fluorescent detection of hydrazine, and has fast response, low detection limit and high selectivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a comparison chart of ultraviolet and fluorescence detection of the fluorescent probe DO prepared in Example 1 before and after the addition of the detection substance hydrazine.

[0014] Figure 2 The UV (left) and fluorescence (right) spectra of the fluorescent probe DO prepared in Example 1 after adding 50 times the equivalent of hydrazine to the solution are shown in the insets, and the solution colors before and after adding hydrazine under sunlight and hand-held UV 365nm irradiation.

[0015] FIG3 is a fluorescence emission spectrum (A) of the fluorescent probe DO prepared in Example 1 reacting with different equivalents of hydrazine in solution and a linear fit between the fluorescence intensity at 515 nm and the added hydrazine concentration (B).

[0016] Figure 4 This is a curve showing the change of the fluorescence intensity of the solution at 515 nm over time after adding 20 equivalents of hydrazine to the solution of the fluorescent probe DO prepared in Example 1.

[0017] Figure 5 The fluorescence intensity bar graph of the fluorescent probe DO prepared in Example 1 in aqueous solution reacting with different interfering ions (blank, Na + , K + Mg 2+ 、Zn 2+ 、Ag + , Ca 2+ , Ba 2+ , Cu 2+ ,AcO - ,I - 、SO4 2- , Cl - 、NO3 - 、HPO4 2- Br - , PhOH, PhNH2, Cys, Hcy, GSH, N2H4).

[0018] Figure 6 These are photos of the fluorescent probe DO prepared in Example 1 before and after the reaction with hydrazine on the support and filter paper under sunlight and 365 nm portable ultraviolet lamp irradiation.

[0019] Figure 7 This is a cell imaging picture of the fluorescent probe DO prepared in Example 1 reacting with hydrazine in HeLa cells.

[0020] Figure 8 This is an imaging picture of the reaction of the fluorescent probe DO prepared in Example 1 with hydrazine in zebrafish. DETAILED DESCRIPTION

[0021] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0022] Example 1

[0023] Synthesis of Fluorescent Probe Compound 2-((10-Butyl-10H-phenothiazin-3-yl)methylene)-1H-indene-1,3(2H)-dione (DO)

[0024] Fluorescent probe compound The probe compound 2-((10-butyl-10H-phenothiazine-3-yl)methylene)-1H-indene-1,3(2H)-dione (DO) was reacted by the following synthetic route:

[0025]

[0026] Under nitrogen protection, 10-butyl-10H-phenothiazine-3-carboxaldehyde (400 mg, 1.4 mmol) and 1,3-indandione (206 mg, 1.4 mmol) were added to a single-necked bottle, and then 10 mL of ethanol and a catalytic amount of piperidine (0.028 mmol) were added. The reaction was carried out at 80 ° C for 8 hours. After the reaction was completed, it was cooled to room temperature and filtered to obtain a crude product. It was then washed with ether to obtain 470 mg of a brown solid product with a yield of 81%.

[0027] The brick-red solid product prepared in this example has a hydrogen nuclear magnetic resonance spectrum: 1 HNMR (400MHz, CDCl3, 25℃, TMS): δ = 8.33 (t, J = 2.4Hz, 1H, Ar–H), 8.30 (dd, J = 8.6Hz, J = 1.9Hz,1H,Ar–H),7.92-7.96(m,2H,Ar–H),7.71-7.77(m,2H,Ar–H),7.67(d,J=2.6Hz 1H,Ar-H),7.08-7.15(m,2H,Ar–H),6.94(t,J=7.4Hz,1H,Ar–H),6.84-6.87(m,2H,Ar–H),3.88(t,J =6.1Hz,2H,N-CH2),1.76-1.84(m,2H,CH2),1.43-1.50(m,2H,CH2),0.96(t,J=7.4Hz,3H,CH3)ppm.

[0028] Its carbon NMR spectrum is: 13 C NMR (400MHz, d6-DMSO, 25℃, TMS): δ = 190.3, 189.5, 149.8, 145.0, 142.9, 142.2, 139.8, 136.5, 136.2, 136.0, 13 2.6,128.5,127.8,127.8,126.7,124.3,123.3,123.2,122.8,122.7,117.1,115.8,47.2,28.7,19.8,14.1ppm.

[0029] Its high resolution mass spectrum is: HRMS (ESI) m / z calcd for C 26 H 20 NO2S + ,Exact Mass:412.1366,([M+H + ]),found412.1365.

[0030] This proves that the brick-red product is 2-((10-butyl-10H-phenothiazine-3-yl)methylene)-1H-indene-1,3(2H)-dione (DO), whose structural formula is

[0031]

[0032] Example 2

[0033] UV and fluorescence responses of fluorescent probe compound 2-((10-butyl-10H-phenothiazin-3-yl)methylene)-1H-indene-1,3(2H)-dione (DO) to hydrazine

[0034] The fluorescent probe DO prepared in Example 1 was dissolved in a mixed solution of 0.1 M pH 7.4 phosphate buffer and acetonitrile, wherein the volume ratio of the phosphate buffer to the acetonitrile was 4:1, and finally a test solution with a concentration of 10 μM fluorescent probe DO was obtained. Figure 1 The UV and fluorescence detection comparison diagram of the fluorescent probe DO prepared in Example 1 before and after the addition of the detection substance hydrazine is given. It can be seen from the figure that the UV and fluorescence spectra of the probe DO have undergone significant changes after contact with hydrazine. The UV fluorescence has decreased in the long-wave band, and the fluorescence has been significantly increased at 515nm. Figure 2 As shown, after adding 50 times the equivalent of hydrazine aqueous solution, the absorption of the probe solution at 510nm decreases rapidly, and the color of the reaction solution changes from purple to colorless. When excited at 392nm, the solution of the fluorescent probe DO has an obvious fluorescence opening process at 515nm, and the solution can be observed to have obvious green fluorescence under a portable ultraviolet lamp. Figure 3 is the fluorescence spectrum of the test solution of the fluorescent probe DO with a concentration of 10μM after adding 0-20 times the equivalent of hydrazine. When excited at 392nm, the fluorescence intensity at 515nm after adding solutions of different concentrations of hydrazine increases with the increase of hydrazine concentration ( Figure 3A ). The fluorescence intensity at 515 nm and the concentration of added hydrazine were linearly fitted, and the detection limit of the fluorescent probe DO for hydrazine was calculated to be 0.31 μM (S / N=3) ( Figure 3B ).like Figure 4 As shown, after adding 20 times the equivalent of hydrazine to the solution of the fluorescent probe DO and performing time tracking, it can be found that the fluorescence intensity of the probe solution at 515nm increases rapidly and reaches an equilibrium within 10 minutes, which shows that the response of the fluorescent probe DO to hydrazine is very sensitive and fast. Figure 1 , Figure 2 , Figure 3 and Figure 4 It is shown that the fluorescent probe compound 2-((10-butyl-10H-phenothiazine-3-yl)methylene)-1H-indene-1,3(2H)-dione (DO) prepared by the present invention can quickly realize the colorimetric and fluorescence detection of hydrazine.

[0035] Example 3

[0036] Anti-interference detection of fluorescent probe compound 2-((10-butyl-10H-phenothiazin-3-yl)methylene)-1H-indene-1,3(2H)-dione (DO)

[0037] To the test solution of the fluorescent probe DO with a concentration of 10 μM prepared in the above embodiment of the present invention, interfering cations (Na + , K + Mg 2+ 、Zn 2+ 、Ag + , Ca 2+ , Ba 2+ , Cu 2+ ), anion (AcO - ,I - 、SO4 2- , Cl - 、NO3 - 、HPO4 2- Br - ) and other active compounds (PhOH, PhNH2, Cys, Hcy, GSH) to a concentration of 500 μM. When the solution was excited at 392 nm, it was found that the fluorescent probe DO only had obvious fluorescence at 515 nm after reacting with hydrazine, but had no obvious changes with other ions or active compounds (such as Figure 5 As shown). In the presence of the above interfering ions, hydrazine (20 times equivalent) was added, and the above solution still reacted well with hydrazine, releasing obvious fluorescence at 515nm. The above data show that the fluorescent probe compound 2-((10-butyl-10H-phenothiazine-3-yl)methylene)-1H-indene-1,3(2H)-dione (DO) has a good selective detection effect on hydrazine and has good anti-interference ability.

[0038] Example 4

[0039] Application of fluorescent probe compound 2-((10-butyl-10H-phenothiazin-3-yl)methylene)-1H-indene-1,3(2H)-dione (DO) loaded on test paper for hydrazine detection

[0040] First, use a cotton swab dipped in DO solution (0.5 mM in acetonitrile) to write two letters DO on the filter paper, then spray a 30 μM hydrazine aqueous solution onto the filter paper, wait for it to dry, and then take pictures under sunlight and portable ultraviolet light (365 nm). Figure 6As shown in the figure, when not treated with hydrazine, the two letters DO on the filter paper appear purple and have no fluorescence under the 365nm irradiation of a portable ultraviolet lamp. After spraying hydrazine, the purple letters become colorless, and obvious green fluorescence can be observed under the irradiation of a portable ultraviolet lamp at 365nm. This shows that the simple test paper prepared with the fluorescent probe DO can detect hydrazine in the solution.

[0041] Example 5

[0042] Application of fluorescent probe compound 2-((10-butyl-10H-phenothiazin-3-yl)methylene)-1H-indene-1,3(2H)-dione (DO) in cell imaging

[0043] Hela cells were cultured in DMEM high glucose medium supplemented with 100 U / mL penicillin-streptomycin. The cell culture medium was placed in a humidified incubator at 37°C with air containing 5% CO2. 4 The cells were seeded on a 14 mm glass coverslip at a density of 100 cells. Then, the fluorescent probe DO was added to the culture medium at a concentration of 10 μM for 1 hour, and then washed three times with phosphate buffered saline (PBS, pH = 7.4). Thereafter, hydrazine (20 μM or 40 μM) was added to the cells and incubated at 37°C for 30 minutes. The cells were rinsed three times with PBS, and then sealed with a sealing agent and imaged on a fluorescence microscope system.

[0044] Figure 7 The results of the fluorescent probe compound 2-((10-butyl-10H-phenothiazine-3-yl)methylene)-1H-indene-1,3(2H)-dione (DO) prepared by the present invention on hydrazine imaging are given. Figure 7 In the first row, it can be seen that the fluorescent probe DO has no obvious fluorescence in the green channel; in the second row, it can be found that the fluorescent probe DO can be observed in the green channel after being co-cultured with hydrazine for 30 minutes after adding 20 μM hydrazine. In the third row after the fluorescent probe DO is co-cultured with 40 μM hydrazine, brighter green fluorescence can be seen. Therefore, the fluorescent probe compound 2-((10-butyl-10H-phenothiazine-3-yl)methylene)-1H-indene-1,3(2H)-dione (DO) prepared by the present invention can well realize the imaging detection of hydrazine in cells.

[0045] Example 6

[0046] Imaging application of fluorescent probe compound 2-((10-butyl-10H-phenothiazin-3-yl)methylene)-1H-indene-1,3(2H)-dione (DO) in zebrafish

[0047] Three-day-old zebrafish were incubated in fresh E3 medium containing the fluorescent probe DO for 30 minutes, then washed three times with E3 medium, and hydrazine was added and incubated for another 30 minutes before imaging.

[0048] Figure 8 The results of the fluorescent probe compound 2-((10-butyl-10H-phenothiazine-3-yl)methylene)-1H-indene-1,3(2H)-dione (DO) prepared by the present invention on hydrazine imaging are given. Figure 8 In the first row, it can be seen that the zebrafish cultured with the fluorescent probe DO has no obvious fluorescence in the green channel; in the second row, after adding 20 μM hydrazine, it can be found that the fluorescent probe DO emits green fluorescence in the green channel after being co-cultured with hydrazine for 30 minutes. In the third row, after the fluorescent probe DO reacts with 40 μM hydrazine, brighter green fluorescence can be seen. Therefore, the fluorescent probe compound 2-((10-butyl-10H-phenothiazine-3-yl)methylene)-1H-indene-1,3(2H)-dione (DO) prepared by the present invention can well realize the imaging detection of hydrazine in the organism zebrafish.

[0049] As can be seen from the above, compared with the existing probe technology in the field of hydrazine detection, the fluorescent probe compound 2-((10-butyl-10H-phenothiazine-3-yl)methylene)-1H-indene-1,3(2H)-dione (DO) prepared by the present invention can well realize the recognition and detection of hydrazine, has the advantage of simple synthesis, and shows good selectivity and sensitivity for the detection of hydrazine, and can realize the imaging detection of hydrazine in cells and zebrafish.

[0050] The above embodiments describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. Application of a fluorescent probe in the preparation of a detection reagent for rapid and highly selective identification of hydrazine, characterized in that: The fluorescent probe is 2-((10-butyl-10H-phenothiazine-3-yl)methylene)-1H-indene-1,3(2H)-dione (DO), and its structural formula is The specific detection process is as follows: the fluorescent probe appears purple in a mixed solution of phosphate buffer and acetonitrile in a volume ratio of 4:1, and has no fluorescence under a 365nm portable ultraviolet lamp; after hydrazine is added to the mixed system, the color of the mixed system becomes colorless, and exhibits bright green fluorescence under a 365nm portable ultraviolet lamp.

2. The use according to claim 1, characterized in that: The fluorescent probe is used in preparing a reagent for imaging and detecting hydrazine in cells or zebrafish.

3. The use according to claim 1, characterized in that The specific preparation process of the fluorescent probe is: using ethanol as a reaction medium, using 10-butyl-10H-phenothiazine-3-carboxaldehyde and 1,3-indandione as reaction raw materials, using piperidine as a catalyst, reacting at 70-80° C. under the protection of an inert gas, and after the reaction is complete, filtering to remove the solvent, washing with ether to obtain the target product, which is a fluorescent probe for hydrazine detection.

4. The use according to claim 3, characterized in that: The molar ratio of the 10-butyl-10H-phenothiazine-3-carboxaldehyde, 1,3-indandione and piperidine is 1:1:0.01-0.

05.

5. The use according to claim 3, characterized in that The specific synthesis route of the fluorescent probe is:

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