Near-infrared fluorescent probe for detecting hydrazine as well as preparation method and application of near-infrared fluorescent probe

Abstract

The invention discloses a near-infrared fluorescent probe for detecting hydrazine as well as a preparation method and application of the near-infrared fluorescent probe, and belongs to the technical field of fluorescent probe detection and analysis of hydrazine. The near-infrared fluorescent probe for detecting hydrazine is (E)-2-bromo-4-(2-(3-(dicyanomethyl)-5, 5-dimethylcyclohex-1-ene-1-yl) vinyl) phenylacetate (HB), and the structural formula of the near-infrared fluorescent probe is shown in the specification. The invention also specifically discloses a preparation method of the near-infrared fluorescent probe for detecting hydrazine and application of the near-infrared fluorescent probe for detecting hydrazine in simultaneous rapid and high-selectivity identification of hydrazine and preparation of a hydrazine imaging detection reagent in an organism. The fluorescent probe prepared by the invention can realize colorimetric and fluorescence detection of hydrazine, is fast in response, low in detection limit and high in selectivity, and can be used for detection of hydrazine in an aqueous solution and imaging of hydrazine in a living body. When the probe is combined with different fluorescent equipment, the sample detection rate and the imaging effect of in-vivo hydrazine detection can be effectively improved.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probe detection and analysis of hydrazine (N2H4), and particularly relates to a near-infrared fluorescent probe for detecting hydrazine, a preparation method thereof, and imaging application thereof. Background Art

[0002] Hydrazine (N2H4), also known as hydrazine, is a simple, small molecule composed of two linked amino groups. Due to its high calorific value, it is widely used in the battery industry and as a motor fuel and rocket fuel. Hydrazine readily oxidizes under appropriate conditions, exhibiting strong reducing properties. It is widely used in the synthesis of herbicides, pesticides, natural products, and pharmaceuticals. The presence of hydrazine in the structures of drugs such as isoniazid, hydralazine, and carbidopa demonstrates its significant industrial value. However, hydrazine is also highly toxic, causing irreversible damage to humans and the environment. Long-term exposure to hydrazine compounds can irritate the respiratory system, cause dizziness and nausea, and produce hepatotoxicity and nephrotoxicity. Studies have also shown that hydrazine is a potential carcinogen, and the International Agency for Research on Cancer (IARC) recommends a lower threshold limit of 10 ppb (0.31 μM) for hydrazine. Hydrazine leakage during industrial use, discharge of hydrazine-containing wastewater, and endogenous hydrazine produced by drug metabolism in the human body all pose a potential risk. The development of technologies for hydrazine detection and tracing of hydrazine produced by drug metabolism in physiological environments is urgent. Traditional methods such as chromatography, mass spectrometry, and electrocatalysis are no longer sufficient for the detection of hydrazine in complex samples. Fluorescent probe technology, however, has gained significant attention for hydrazine detection due to its diverse structures, ease of modification, on-site detection, and non-destructive imaging. However, limitations such as slow response rates, complex synthesis, short emission wavelengths, small Stokes shifts, and difficulties in biological imaging continue to limit the widespread application of fluorescent probes. Summary of the Invention

[0003] The present invention aims to provide a near-infrared fluorescent probe for detecting hydrazine and a preparation method thereof. The fluorescent probe prepared by the method can be used for rapid and highly selective identification of hydrazine, and can realize the detection and biological imaging of hydrazine in organisms by combining various fluorescence detection technologies.

[0004] The present invention adopts the following technical scheme to achieve the above-mentioned purpose: a near-infrared fluorescent probe for detecting hydrazine, characterized in that: the fluorescent probe is (E)-2-bromo-4-(2-(3-(dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl acetate (HB), whose structural formula is

[0005]

[0006] The method for preparing a near-infrared fluorescent probe for detecting hydrazine of the present invention is characterized by the following specific preparation process: under nitrogen protection, using dichloromethane as a reaction solvent and (E)-2-(3-(3-bromo-4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile as a reaction raw material, sequentially adding triethylamine and acetyl chloride at -5 to 5°C for reaction, reacting at room temperature for 2 to 4 hours, removing the reaction solvent, and separating by column chromatography to obtain the target product, a near-infrared fluorescent probe for detecting hydrazine.

[0007] It is further defined that the molar ratio of the (E)-2-(3-(3-bromo-4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile, triethylamine and acetyl chloride is 1:3:2.

[0008] The synthetic route in the preparation method of the near-infrared fluorescent probe for detecting hydrazine of the present invention is:

[0009]

[0010] The invention discloses an application of a near-infrared fluorescent probe for detecting hydrazine in the rapid and highly selective identification of hydrazine, characterized in that the fluorescent probe exhibits a light yellow color in a mixed solution of phosphate buffer and dimethyl sulfoxide in a volume ratio of 3:1 and exhibits no fluorescence under irradiation with a 365nm portable ultraviolet lamp; and after a solution containing hydrazine is added to the mixed system, the color of the mixed system changes to pink and exhibits bright red fluorescence under irradiation with a 365nm portable ultraviolet lamp.

[0011] The invention discloses an application of the near-infrared fluorescent probe for detecting hydrazine in the preparation of a hydrazine imaging detection reagent in a biological solution.

[0012] The invention relates to an application of the near-infrared fluorescent probe for detecting hydrazine in the preparation of a hydrazine imaging detection reagent in cells.

[0013] The invention discloses an application of the near-infrared fluorescent probe for detecting hydrazine in the preparation of a hydrazine imaging detection reagent produced by organism metabolism.

[0014] Compared with existing fluorescent probe technologies for detecting hydrazine, the fluorescent probe for detecting hydrazine prepared by the present invention has a simple preparation process and high yield, a large Stokes shift, and low self-absorption, which is beneficial for imaging of living organisms. The fluorescent probe prepared by the present invention can achieve rapid and highly selective detection of hydrazine in the near-infrared region. The fluorescent probe for detecting hydrazine prepared by the present invention can achieve both colorimetric and fluorescent detection of hydrazine, with a fast response, low detection limit, and high selectivity. When used in conjunction with a variety of fluorescent equipment, it can further achieve detection of hydrazine in living organisms and biological imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1It is the ultraviolet-visible absorption spectrum of the fluorescent probe HB prepared in Example 1 after adding hydrazine to the solution and the fluorescence spectrum at an excitation wavelength of 450 nm.

[0016] Figure 2 The UV spectra (a) and fluorescence spectra (b) of the fluorescent probe HB prepared in Example 1 after adding different equivalents of hydrazine to the solution are shown. The insets are photos of the solution observed with the naked eye and under 365nm handheld UV irradiation before and after adding hydrazine.

[0017] Figure 3 This is a linear fitting curve between the fluorescence intensity of the fluorescent probe HB prepared in Example 1 after reacting with different equivalents of hydrazine in solution and the concentration of the added hydrazine.

[0018] Figure 4 This is an image of the fluorescent probe HB prepared in Example 1 after reacting with different equivalents of hydrazine in a solution under a fluorescence imager.

[0019] Figure 5 The graph shows the change in fluorescence intensity of the solution at 675 nm over time after adding hydrazine of different concentrations to the solution of the fluorescent probe HB prepared in Example 1.

[0020] Figure 6 The fluorescence spectrum (a) of the fluorescent probe HB prepared in Example 1 reacting with different interfering ions and the fluorescence imaging diagram (b) under the fluorescence imaging instrument (0-blank; 1-Na + ;2-K + ;3-NH4 + ;4-Ag + ;5-Fe 3+ ;6-Cu 2+ ;7-Cl - ;8-Br - ;9-I - ;10-CH3COO - ;11-NO3 - ;12-SO4 2- ; 13-Methylamine; 14-Dimethylamine; 15-Aniline; 16-Phenylhydrazine; 17-Isoniazid; 18-Hydralazine; 19-N2H4).

[0021] Figure 7 The fluorescence spectrum (a) and fluorescence imaging (b) of the fluorescent probe HB prepared in Example 1 reacting with hydrazine and different interfering ions (0-blank; 1-Na + ;2-K + ;3-NH4 + ;4-Ag+ ;5-Fe 3+ ;6-Cu 2 + ;7-Cl - ;8-Br - ;9-I - ;10-CH3COO - ;11-NO3 - ;12-SO4 2- ; 13-Methylamine; 14-Dimethylamine; 15-Aniline; 16-Phenylhydrazine; 17-Isoniazid; 18-Hydralazine; 19-N2H4).

[0022] Figure 8 (a) is a cell imaging image of the fluorescent probe HB prepared in Example 1 reacting with hydrazine in HeLa cells; (b) is a fluorescence analysis image of HeLa cells after the fluorescent probe HB prepared in Example 1 reacted with different concentrations of hydrazine in a flow cytometer; (c) is an in vivo imaging image of the left and right legs injected with PBS and hydrazine respectively 30 minutes later, and then injected with the fluorescent probe HB respectively.

[0023] Figure 9 (a) is a fluorescence imaging image of the fluorescent probe HB prepared in Example 1 reacting with hydrazine in zebrafish; (b) is a layer scanning image of zebrafish at different depths; and (c) is a 3D reconstruction image achieved by the layer scanning image.

[0024] Figure 10 (a) is a cell image of the fluorescent probe HB prepared in Example 1 after being treated with different concentrations of isoniazid; (b) is an imaging image of the kidney and liver of a mouse injected with PBS and isoniazid through the tail vein one hour after the mouse was injected with the fluorescent probe HB through the tail vein. DETAILED DESCRIPTION

[0025] The above contents of the present invention are further described in detail below through examples, but this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above contents of the present invention fall within the scope of the present invention.

[0026] Example 1

[0027] Synthesis of Fluorescent Probe Compound (E)-2-Bromo-4-(2-(3-(dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenylacetate (HB)

[0028] The fluorescent probe compound (E)-2-bromo-4-(2-(3-(dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl acetate (HB) was prepared by the following synthesis route:

[0029]

[0030] Under nitrogen, (E)-2-(3-(3-bromo-4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile was dissolved in dichloromethane and cooled to 0°C in an ice-water bath. Triethylamine (3 equivalents) and acetyl chloride (2 equivalents) were then added sequentially to react. After complete addition, the mixture was allowed to return to room temperature and the reaction was continued for 2 hours. After removal of the solvent, column chromatography using 200-300 mesh silica gel was performed to obtain a yellow solid product in a 63% yield.

[0031] The yellow solid product prepared in this example has a hydrogen nuclear magnetic resonance spectrum: 1 H NMR (400MHz, CDCl3, 25℃, TMS): δ = 7.75 (d, J = 2.0Hz, 1H, Ar–H), 7.46 (dd, J = 8.4Hz, J = 2.0Hz, 1H, Ar–H), 7.16 (d, J = 8.4Hz, 1H, Ar–H), 6.94 (s, 2H, = CH), 6.86 (s, 1H, = CH), 2.61 (s, 2H, CH2), 2.45 (s, 2H, CH2), 2.38 (s, 3H, COCH3), 1.08 (s, 6H, 2 × CH3) ppm.

[0032] Its carbon NMR spectrum is: 13 C NMR (100MHz, CDCl3, 25℃, TMS) δ = 169.1, 168.4, 152.9, 148.9, 135.3, 134.2, 132.1, 130 .5,127.3,124.4,124.2,117.1,113.3,112.5,79.6,43.0,39.2,32.1,28.0,20.8ppm.

[0033] Its high resolution mass spectrum is: HRMS (ESI) m / z: calcd.for C 21 H 19 BrN2NaO2 + [M+H + ]:433.0523,found433.0519.

[0034] This proves that the yellow product is (E)-2-bromo-4-(2-(3-(dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl acetate (HB), whose structural formula is

[0035]

[0036] Example 2

[0037] UV and Fluorescence Responses of the Fluorescent Probe Compound (E)-2-Bromo-4-(2-(3-(dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenylacetate (HB) to Hydrazine

[0038] The fluorescent probe HB prepared in Example 1 was dissolved in a mixed solution of phosphate buffer and dimethyl sulfoxide in a volume ratio of 3:1 to obtain a test solution with a concentration of 10 μM fluorescent probe HB. Figure 1 The UV-visible absorption spectrum of the fluorescent probe HB prepared in Example 1 after adding the detection substance hydrazine and the fluorescence spectrum at an excitation wavelength of 450nm are shown. As can be seen from the figure, the maximum absorption peak of the fluorescent probe HB after adding hydrazine is located at 515nm, and the emission peak is located at 675nm. Therefore, the Stokes shift is 160nm, which lays the foundation for the good imaging effect of the fluorescent probe HB. Figure 2 As shown in (a), after adding different equivalents of hydrazine, as the concentration of hydrazine increases, the absorption of the fluorescent probe HB solution at 390nm decreases rapidly, while the absorption at 515nm gradually increases, and the color of the reaction solution changes from light yellow to pink. Figure 2 As shown in (b), when excited at 450 nm, the solution of the fluorescent probe HB has obvious fluorescence at 675 nm, and the fluorescence intensity increases with the increase of hydrazine equivalent. Obvious red fluorescence can be observed in the solution under a portable ultraviolet lamp. Figure 3 The curve is obtained by linear fitting of the fluorescence intensity at 675 nm and the concentration of added hydrazine after adding different equivalents of hydrazine to the test solution with a concentration of 10 μM fluorescent probe HB. The detection limit of the fluorescent probe HB for hydrazine is calculated to be 31 nM (S / N=3). Figure 4 The fluorescence probe HB reacted with different concentrations of hydrazine in a 96-well plate under a fluorescence imaging device. The fluorescence intensity increased with the increase of hydrazine concentration, which was consistent with the fluorescence spectrum test results. Figure 5 As shown in the figure, after adding 1, 3, and 5 times the equivalent of hydrazine to the solution of fluorescent probe HB and tracking the time, it can be found that the fluorescence intensity of the fluorescent probe solution at 675nm increases rapidly. When the hydrazine equivalent increases to 5 times the equivalent, the reaction reaches equilibrium within 12 minutes, which shows that the response of fluorescent probe HB to hydrazine is very sensitive and fast. Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 and Figure 5 The results show that the fluorescent probe compound (E)-2-bromo-4-(2-(3-(dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenylacetate (HB) prepared by the present invention can quickly realize colorimetric and fluorescent detection of hydrazine and is very suitable for biological imaging applications.

[0039] Example 3

[0040] Selective Detection of Fluorescent Probe Compound (E)-2-Bromo-4-(2-(3-(dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenylacetate (HB)

[0041] Various interfering ions (0-blank; 1-Na + ;2-K + ;3-NH4 + ;4-Ag + ;5-Fe 3+ ;6-Cu 2+ ;7-Cl - ;8-Br - ;9-I - ;10-CH3COO - ;11-NO3 - ;12-SO4 2- ; 13-Methylamine; 14-Dimethylamine; 15-Aniline; 16-Phenylhydrazine; 17-Isoniazid; 18-Hydralazine; 19-N2H4). Except for the concentration of hydrazine added at 50μM, the concentrations of other interfering substances were all 300μM. When the solution was excited at 450nm, it was found that the fluorescent probe HB only showed obvious fluorescence at 675nm after reacting with hydrazine, while there was no obvious change with other interfering substances (such as Figure 6 As shown in (a), when the test solution is placed in a 96-well plate under a fluorescence imaging device, only the sample with hydrazine added can be observed to have an obvious fluorescence response (as shown in Figure 6 If hydrazine and interfering ions are added to the fluorescent probe HB solution at the same time, it can be found that in addition to Cu 2+ The experimental group can cause fluorescence to weaken, and the presence of other interfering ions does not affect the detection of hydrazine by the fluorescent probe HB. 2+ The fluorescence attenuation is caused by Cu 2+It can complex hydrazine. Consistent responses were observed across the 96-well plate. These data demonstrate that the fluorescent probe compound (E)-2-bromo-4-(2-(3-(dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenylacetate (HB) exhibits excellent selectivity and anti-interference capabilities for hydrazine detection.

[0042] Example 4

[0043] Imaging of exogenous hydrazine in HeLa cells and mice using the fluorescent probe compound (E)-2-bromo-4-(2-(3-(dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenylacetate (HB)

[0044] HeLa cells were plated at 5 × 10 cells per coverslip. 4 Cells were seeded at a density of 10 μM on 14 mm glass coverslips. The fluorescent probe HB prepared above was then added to the culture medium at a concentration of 10 μM and incubated for 1 hour. The cells were then washed three times with phosphate-buffered saline (PBS, pH 7.4). Subsequently, different concentrations of hydrazine were added to the cells and incubated at 37°C for another 1 hour. The cells were rinsed three times with PBS and imaged under a laser confocal microscope. Figure 8 (a) shows the results of imaging hydrazine in cells using the fluorescent probe compound (E)-2-bromo-4-(2-(3-dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenylacetate (HB). The fluorescent probe HB has no obvious fluorescence in the red channel, but after adding hydrazine, it can be found that the fluorescent probe HB can be observed in the red channel. The fluorescence intensity increases with the increase of hydrazine equivalents. The fluorescence intensity of the probe HB before and after the reaction with hydrazine was detected by flow cytometry. It can be found that the fluorescence intensity also increases with the increase of the concentration of added hydrazine (such as Figure 8 (b)). This was followed by an in vivo imaging test of hydrazine in mice, as shown in Figure 8 As shown in (c), 50 μL of PBS and hydrazine (0.5 mM) were injected into the left and right legs of the mouse, respectively. 30 minutes later, 50 μL (50 μM) of fluorescent probe HB were injected into the same positions, and then imaging was performed under a mouse imager. It can be found that the left leg injected with PBS and fluorescent probe HB did not have an obvious fluorescent response, while the right leg injected with hydrazine and fluorescent probe HB showed an obvious fluorescent signal. Therefore, the fluorescent probe compound (E)-2-bromo-4-(2-(3-(dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenylacetate (HB) prepared by the present invention can well realize the imaging detection of exogenous hydrazine in HeLa cells and mice.

[0045] Example 5

[0046] Detection and Imaging of Hydrazine in Zebrafish Using the Fluorescent Probe Compound (E)-2-Bromo-4-(2-(3-(Dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenylacetate (HB)

[0047] Figure 9 (a) shows the results of the fluorescent probe compound (E)-2-bromo-4-(2-(3-(dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenylacetate (HB) for hydrazine detection imaging in zebrafish. Figure 9 In the first row of (a), it can be seen that the zebrafish cultured with the fluorescent probe HB has no obvious fluorescence in the red channel; while in the second row, a clear fluorescence signal can be observed in the zebrafish treated with the fluorescent probe HB and hydrazine. The distribution of fluorescence in the zebrafish after the reaction of the fluorescent probe HB and hydrazine can be clearly observed by scanning at a depth of 300μM (as shown in the figure). Figure 9 By recombining the deep scan images, we can observe the 3D stereoscopic imaging effect of zebrafish, providing an imaging tool for the study of hydrazine metabolism (such as Figure 9 Therefore, the fluorescent probe compound (E)-2-bromo-4-(2-(3-(dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenylacetate (HB) prepared by the present invention can well realize the detection and stereoscopic imaging of hydrazine in zebrafish.

[0048] Example 6

[0049] Imaging of endogenous hydrazine in cells and mice using the fluorescent probe compound (E)-2-bromo-4-(2-(3-(dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenylacetate (HB)

[0050] Endogenous hydrazine is produced by the metabolism of the tuberculosis treatment drug isoniazid (INH) in cells and mice.

[0051] Figure 10 (a) shows the imaging results of endogenous hydrazine in cells using the fluorescent probe compound (E)-2-bromo-4-(2-(3-(dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenylacetate (HB). Figure 10In (a), cells were treated with different concentrations of isoniazid for 8 hours, and then the fluorescent probe HB was added. It was found that obvious fluorescent signals could be observed in cells treated with different concentrations of isoniazid, and the fluorescence intensity increased with the increase in the concentration of isoniazid, indicating that the addition of isoniazid did produce endogenous hydrazine. Subsequently, the hydrazine produced by the metabolism of isoniazid in mice was detected. 100 μL of PBS and isoniazid were injected into the peritoneal cavity of mice, respectively. One hour later, 100 μL of fluorescent probe HB (1 mg / mL) was injected through the tail vein. Imaging was performed under a mouse imager 90 minutes later. Figure 10 As shown in (b), there was no obvious fluorescent signal in the abdominal cavity of the mice in the PBS group, but the INH group showed a significant fluorescence enhancement phenomenon, indicating that the fluorescent probe HB can be used for the detection and imaging of endogenous hydrazine in mice. The mice were dissected to obtain the kidneys and livers of the mice. As the most important organs for isoniazid metabolism, obvious fluorescent signals can be observed, indicating the production of hydrazine. Hydrazine is a toxic substance produced during the treatment of tuberculosis with isoniazid. The detection of hydrazine is conducive to the protection of the kidneys and liver and the adjustment of drug concentrations. Therefore, the fluorescent probe compound (E)-2-bromo-4-(2-(3-(dicyanomethyl)-5,5-dimethylcyclohex-1-ene-1-yl) vinyl)phenylacetate (HB) prepared by the present invention can well realize the imaging detection of hydrazine produced by isoniazid metabolism.

[0052] As can be seen from the above, compared with the existing fluorescent probe technology in the field of hydrazine detection, the fluorescent probe compound (E)-2-bromo-4-(2-(3-(dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenylacetate (HB) prepared by the present invention can well achieve rapid and highly selective detection of hydrazine, and has the advantage of simple synthesis. It also shows good selectivity and sensitivity for the detection of hydrazine, and can achieve imaging detection of endogenous and exogenous hydrazine in cells, zebrafish and mice, providing a visual imaging tool for environmental pollution detection and monitoring of drug metabolism processes. The combination with a variety of fluorescent imaging equipment greatly improves the detection efficiency of hydrazine-contaminated water samples and the effect of hydrazine detection imaging in living organisms.

[0053] 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. A near-infrared fluorescent probe for detecting hydrazine, characterized in that: The fluorescent probe is (E)-2-bromo-4-(2-(3-(dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl acetate (HB), and its structural formula is 2. A method for preparing a near-infrared fluorescent probe for detecting hydrazine according to claim 1, characterized in that The specific preparation process is as follows: under nitrogen protection, using dichloromethane as the reaction solvent and (E)-2-(3-(3-bromo-4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile as the reaction raw material, triethylamine and acetyl chloride are added in sequence at -5 to 5°C for reaction, and then the reaction is carried out at room temperature for 2 to 4 hours, and then the reaction solvent is removed. The target product is separated by column chromatography to obtain a near-infrared fluorescent probe for detecting hydrazine.

3. The method for preparing a near-infrared fluorescent probe for detecting hydrazine according to claim 2, wherein: The molar ratio of the (E)-2-(3-(3-bromo-4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile, triethylamine and acetyl chloride is 1:3:

2.

4. The method for preparing a near-infrared fluorescent probe for detecting hydrazine according to claim 2, wherein The synthetic route of the fluorescent probe is:

5. Use of the near-infrared fluorescent probe for detecting hydrazine according to claim 1 in rapid and highly selective identification of hydrazine.

6. The use according to claim 5, characterized in that The specific process is as follows: the fluorescent probe appears light yellow in a mixed solution of phosphate buffer and dimethyl sulfoxide in a volume ratio of 3:1, and has no fluorescence under 365nm portable ultraviolet lamp; after adding a solution containing hydrazine to the mixed system, the color of the mixed system turns pink and shows bright red fluorescence under 365nm portable ultraviolet lamp.

7. Use of the near-infrared fluorescent probe for detecting hydrazine according to claim 1 in preparing a hydrazine imaging detection reagent in a solution.

8. Use of the near-infrared fluorescent probe for detecting hydrazine according to claim 1 in preparing a reagent for imaging detection of hydrazine in cells.

9. Use of the near-infrared fluorescent probe for detecting hydrazine according to claim 1 in preparing a reagent for imaging detection of hydrazine produced by biological metabolism.

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