Hydrazine hydrate molecular fluorescent probe as well as preparation method and application thereof

Abstract

The invention relates to a hydrazine hydrate molecular fluorescent probe as well as a preparation method and application thereof, and relates to the technical field of biochemical detection tools. The invention provides a hydrazine hydrate molecular fluorescent probe with a structural formula as shown in a formula (1): # imgabs0 #, and further provides a specific preparation method and application of the hydrazine hydrate molecular fluorescent probe on the basis of the hydrazine hydrate molecular fluorescent probe. The hydrazine hydrate molecular fluorescent probe provided by the invention is high in sensitivity, good in selectivity and low in detection limit, and can be successfully applied to monitoring of hydrazine hydrate in a water environment and a cell system.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical detection tools, and in particular to a hydrazine hydrate molecular fluorescent probe and a preparation method and application thereof. Background Art

[0002] As an important fine chemical raw material, hydrazine hydrate is widely used in agriculture, food, pharmaceuticals, textile dye industry, rocket propulsion and missile systems. However, due to its high carcinogenicity, toxicity and teratogenicity, its widespread use will cause serious environmental pollution. Excessive hydrazine residues entering water, air and soil will inhibit seed growth and cause leaf withering. For humans, long-term exposure to hydrazine hydrate can cause skin irritation and serious damage to the central nervous system, kidneys, lungs and respiratory system.

[0003] In China, the hydrazine content in drinking water is controlled below 0.625 μM (20 μg / L). The United States Environmental Protection Agency (US-EPA) has identified hydrazine hydrate as a potential carcinogenic chemical, and the minimum value of hydrazine hydrate in drinking water is 0.312 μM (10 μg / L). Developing a simple, effective, and rapid tool for monitoring hydrazine hydrate content in the environment and biological systems is of great significance for environmental protection and human health. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a hydrazine hydrate molecular fluorescent probe and its preparation method and application, which can identify hydrazine hydrate with high sensitivity and specificity, and change the fluorescence color of the system from blue to green, effectively solving the problems of complex and expensive equipment detection and long time consumption in the prior art.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] The first object of the present invention is to provide a hydrazine hydrate molecular fluorescent probe, the structural formula of which is as follows:

[0007]

[0008] The beneficial effects of the present invention are as follows: in the fluorescent probe structure of the present invention, caffeoyl is used as a fluorescent group, and an aldehyde group is used as a recognition unit. After reacting with hydrazine hydrate, a phenylhydrazone structure is generated, and the system shows obvious fluorescence enhancement, and can be applied to the specific monitoring of hydrazine hydrate in living cells and water environments.

[0009] A second object of the present invention is to provide a method for preparing a hydrazine hydrate molecular fluorescent probe, comprising the following steps:

[0010] (1) adding caffeic acid and N,N'-dicyclohexylcarbodiimide to tetrahydrofuran and stirring, and then adding p-aminobenzaldehyde and stirring to obtain a residue;

[0011] (2) dissolving the residue in ethyl acetate for washing, drying the organic layer, and filtering to obtain a crude fluorescent probe product;

[0012] (3) After separating and purifying the crude fluorescent probe product, the solvent is recovered to obtain the fluorescent probe.

[0013] The beneficial effects of the present invention are as follows: the hydrazine hydrate fluorescent probe provided by the present invention has high sensitivity to hydrazine hydrate and good selectivity. Under physiological conditions and natural environments, the hydrazine hydrate fluorescent probe exhibits good fluorescence response to hydrazine hydrate and can be successfully applied to the monitoring of NO in living cells.

[0014] The present invention uses caffeic acid as a starting material to prepare a ratiometric hydrazine hydrate fluorescent probe, which can identify hydrazine hydrate with high sensitivity and specificity, and changes the fluorescence color of the system from blue to green. The probe can be used for trace detection of hydrazine hydrate in cells and in water environments, so the compound provides a new method for convenient and rapid detection of hydrazine hydrate in organisms and in water environments, and has good application prospects.

[0015] Furthermore, in step (1), the usage ratio of the caffeic acid, the N,N'-dicyclohexylcarbodiimide, the tetrahydrofuran, and the p-aminobenzaldehyde is (0.50-0.60) mmol: (0.5-0.7) mmol: (5-15) mL: (5-6) mmol.

[0016] Furthermore, in step (2), the ratio of the residue to the ethyl acetate is 1:20:100 mL.

[0017] Furthermore, step (1) is specifically as follows: caffeic acid and N,N'-dicyclohexylcarbodiimide are added to tetrahydrofuran and stirred at room temperature for 10-12 minutes, and then p-aminobenzaldehyde is added and stirred for 14-16 hours to obtain a residue.

[0018] Further, step (2) is specifically as follows: dissolving the residue in ethyl acetate, washing with KHSO4, NaHCO3 and saturated brine at least 3 times respectively, drying the organic layer with MgSO4 and filtering, recovering the solvent, and obtaining a crude product.

[0019] Furthermore, step (3) is specifically as follows: the crude product is separated by silica gel column chromatography, and the solvent is recovered to obtain a hydrazine hydrate molecular fluorescent probe.

[0020] Preferably, the volume ratio of ethyl acetate to petroleum ether during the chromatographic separation is 1:5.

[0021] The third object of the present invention is to provide an application of a hydrazine hydrate molecular fluorescent probe, wherein the hydrazine hydrate molecular fluorescent probe is used to detect hydrazine hydrate.

[0022] The beneficial effect of the present invention is to develop a simple, effective and rapid tool for monitoring the hydrazine hydrate content in the environment and biological systems, which is of great significance to environmental protection and human health.

[0023] Furthermore, the hydrazine hydrate molecular fluorescent probe is used to detect hydrazine hydrate in cells and in water environments.

[0024] Furthermore, the detection range of the hydrazine hydrate molecular fluorescent probe is 10 μM-100 μM, and the minimum detection limit is 0.106 μM. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The fluorescence spectra of the hydrazine hydrate molecular fluorescent probe in Example 1 of the present invention acting on hydrazine hydrate at different concentrations;

[0026] Figure 2 This is a diagram showing the fluorescence imaging result of HeLa cells after the hydrazine hydrate molecular fluorescent probe of Example 1 acts on hydrazine hydrate;

[0027] Figure 3 It is a schematic diagram of the results of treating the loaded probe paper with different concentrations of hydrazine hydrate under 365nm ultraviolet light;

[0028] Figure 4 Schematic diagram of the results of immersing different nitrogen-containing compound solutions with fluorescent probes. DETAILED DESCRIPTION

[0029] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0030] Example 1: Preparation of hydrazine hydrate molecular fluorescent probe

[0031] Preparation of hydrazine hydrate molecular fluorescent probe, the synthetic route is as follows:

[0032]

[0033] The specific synthesis steps are as follows:

[0034] (1) Caffeic acid (100 mg, 0.55 mmol) and N,N'-dicyclohexylcarbodiimide (123.7 mg, 0.6 mmol) were added to tetrahydrofuran (10 mL), stirred at room temperature for 10 min, and then p-aminobenzaldehyde (1197 mg, 5.55 mmol) was added and stirred for 15 h. The residue was filtered to obtain;

[0035] (2) The residue was dissolved in ethyl acetate (100 mL), washed with KHSO4 (1 mol / L), NaHCO3 (1 mol / L) and saturated brine, 3 times with 25 mL each time, the organic layer was dried with MgSO4, filtered, and the solvent was recovered to obtain a crude product of hydrazine hydrate molecular fluorescent probe;

[0036] (3) The crude product of hydrazine hydrate molecular fluorescent probe was separated by chromatography on a silica gel column (model 200-300 mesh, Qingdao Ocean) [eluent V (ethyl acetate): V (petroleum ether) = 1:5], and the solvent was recovered to obtain a yellow solid hydrazine hydrate molecular fluorescent probe (106.9 mg, 68.1%).

[0037] The characterization results of the hydrazine hydrate molecular fluorescent probe are as follows: 1 H NMR(500MHz,DMSO-d6)δ(ppm):10.50(s,1H),9.88(s,1H),9.38(s,3H),7.99-7.81(m,3H),7.47(d,J =15.5Hz,1H),7.03(d,J=2.1Hz,1H),6.94(dd,J=8.2,2.1Hz,1H),6.79(d,J=8.1Hz,1H),6.63(s,1H). 13 CNMR(125MHz,DMSO-d6)δ(ppm):189.94,168.10,163.51,157.07,154.04,1 48.25,146.09,142.07,126.58,121.40,116.26,116.14,114.11,113.3.HR MS(ESI),m / z:[C 16 H 13 NO4+H] + , theoretical value 284.0905; test value 284.0910.

[0038] Test Example 1:

[0039] The hydrazine hydrate molecular fluorescent probe prepared in Example 1 was dissolved in a DMF / H2O (V:V=1:1) buffer solution and prepared into 1×10 -5M solution; hydrazine hydrate was dissolved in distilled water to prepare solutions with concentrations of 0μM, 2.5μM, 5μM, 7.5μM, 10μM, 12.5μM, 15μM, 17.5μM, 20μM, 25μM, 30μM, 35μM, 40μM, 45μM, 50μM, 55μM, 60μM, 70μM, 80μM, 90μM, 100μM, 110μM, 120μM, 130μM, 140μM, 150μM, 160μM, 170μM, 180μM, 190, and 200μM.

[0040] The effect of the above-prepared concentration of hydrazine hydrate on the fluorescence emission spectrum of the hydrazine hydrate molecular fluorescent probe was detected by standard fluorescence spectrophotometry on a fluorescence spectrophotometer (Model F-7100; Hitachi). Figure 1 shown.

[0041] Depend on Figure 1 We can get:

[0042] The hydrazine hydrate molecular fluorescent probe has an obvious fluorescence emission peak at 450nm. As the concentration of hydrazine hydrate in the system increases, the fluorescence intensity at 484nm gradually increases, and the green fluorescence of the system gradually increases. This shows that the caffeic acid derivative L can be used as a hydrazine hydrate molecular fluorescent probe to detect hydrazine hydrate.

[0043] Test Example 2:

[0044] First, HeLa cells were placed in a 96-well plate containing 10% fetal bovine serum and 1% antibiotics, and incubated at 37°C and 5% CO2 for 24 hours. Subsequently, a cell imaging experiment was performed, and the fluorescent probe (HPA) prepared in Example 1 was formulated into a 10 μM fluorescent probe solution. After the HeLa cells were incubated with the fluorescent probe solution prepared in Example 1 for 30 minutes, the cultured HeLa cells were divided into 4 groups. The first group was not treated with hydrazine hydrate, and the second, third, and fourth groups were incubated with hydrazine hydrate (10 μM, 25 μM, and 50 μM) for 30 minutes, respectively. After incubation, the cells were rinsed three times with PBS, and the cell samples were imaged using a laser confocal fluorescence microscope (LeicaTCS SP8; Leica). The cell imaging is shown in FIG. Figure 2 shown.

[0045] Depend on Figure 2 We can get:

[0046] Hydrazine hydrate molecular fluorescent probe can detect hydrazine hydrate in cells

[0047] Test Example 3:

[0048] The fluorescent probe prepared in Example 1 was prepared into a 10 mol / L probe solution. A 2 cm × 2 cm circular filter paper was soaked in the probe solution for 2 min and then taken out. After natural air drying, the probe-loaded test paper was soaked in N2H4 solutions of different concentrations (5 mol / L, 10 mol / L, 20 mol / L, 30 mol / L, 40 mol / L) respectively. After 10 min, the soaked test paper was taken out and dried. The fluorescence color change was observed under ultraviolet light to obtain Figure 3 .

[0049] like Figure 3 As shown:

[0050] It can be seen that during application, as the concentration of N2H4 increases, the fluorescence color of the filter paper loaded with the compound gradually changes from blue to blue-green, which shows that the test paper loaded with the compound can be used to detect trace amounts of N2H4 in water samples, providing a new method for convenient and rapid detection of N2H4.

[0051] Test Example 4

[0052] The fluorescent probe prepared in Example 1 was prepared into a 10 mol / L probe solution, a 2 cm × 2 cm circular filter paper was placed in the probe solution and soaked for 2 min, taken out, and naturally air-dried. The probe-loaded test paper was immersed in different nitrogen-containing solutions (CH3NH2, C2H7N, C2H8N2, NH2OH, CO(NH2)2, CH4N2S, Cys, GSH, Hcy, Gly, Glu, N2H4) with a concentration of 10 mol / L. After 10 min, the immersed test paper was taken out, dried, and its fluorescence color change was observed under ultraviolet light to obtain Figure 4 ( Figure 4 The order of labels 1-12 is CH3NH2, C2H7N, C2H8N2, NH2OH, CO(NH2)2, CH4N2S, Cys, GSH, Hcy, Gly, Glu, N2H4).

[0053] like Figure 4 As shown:

[0054] It can be seen that when the filter paper loaded with the fluorescent probe prepared by the present invention is immersed in different nitrogen-containing compound solutions, the filter paper loaded with the compound is immersed in the N2H4 solution, and the fluorescence color of the test paper gradually changes from blue to blue-green, while the fluorescence color of the filter paper loaded with the compound does not change significantly when immersed in other solutions. This shows that the test paper loaded with the compound can be used for the specific detection of N2H4 in water samples.

[0055] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A hydrazine hydrate molecular fluorescent probe, characterized in that: Its structural formula is as follows (1):

2. A method for preparing a hydrazine hydrate molecular fluorescent probe according to claim 1, characterized in that: The preparation method comprises the following steps: (1) adding caffeic acid and N,N'-dicyclohexylcarbodiimide to tetrahydrofuran and stirring, and then adding p-aminobenzaldehyde and stirring to obtain a residue; (2) dissolving the residue in ethyl acetate for washing, drying the organic layer, and filtering to obtain a crude fluorescent probe product; (3) After separating and purifying the crude fluorescent probe product, the solvent is recovered to obtain the fluorescent probe.

3. The method for preparing a hydrazine hydrate molecular fluorescent probe according to claim 2, characterized in that: In step (1), the usage ratio of the caffeic acid, the N,N'-dicyclohexylcarbodiimide, the tetrahydrofuran and the p-aminobenzaldehyde is (0.50-0.60) mmol: (0.5-0.7) mmol: (5-15) mL: (5-6) mmol.

4. The method for preparing a hydrazine hydrate molecular fluorescent probe according to claim 2, characterized in that: The usage ratio of the residue to the ethyl acetate in step (2) is 1:20:100 mL.

5. The method for preparing a hydrazine hydrate molecular fluorescent probe according to claim 4, characterized in that: Step (1) is as follows: caffeic acid and N,N'-dicyclohexylcarbodiimide are added to tetrahydrofuran and stirred at room temperature for 10-12 minutes, and then p-aminobenzaldehyde is added and stirred for 14-16 hours to obtain a residue.

6. The method for preparing a hydrazine hydrate molecular fluorescent probe according to claim 2, characterized in that: Step (2) is as follows: the residue is dissolved in ethyl acetate, washed at least three times with KHSO4, NaHCO3 and saturated brine respectively, the organic layer is dried with MgSO4 and filtered to obtain a crude product.

7. The method for preparing a hydrazine hydrate molecular fluorescent probe according to claim 6, characterized in that: Step (3) is specifically as follows: the crude product is separated by silica gel column chromatography, and the solvent is recovered to obtain a hydrazine hydrate molecular fluorescent probe.

8. An application of a hydrazine hydrate molecular fluorescent probe, characterized in that: The hydrazine hydrate molecular fluorescent probe is used for detecting hydrazine hydrate.

9. The use of a hydrazine hydrate molecular fluorescent probe according to claim 8, characterized in that: The hydrazine hydrate molecular fluorescent probe is used for detecting hydrazine hydrate in cells and water environments.

10. The use of a hydrazine hydrate molecular fluorescent probe according to claim 9, characterized in that: The detection range of the hydrazine hydrate molecular fluorescent probe is 10 μM-100 μM, and the minimum detection limit is 0.106 μM.

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