Hydrazine hydrate molecular fluorescent probe, preparation method and application thereof

Abstract

The present application relates to a kind of hydrazine hydrate molecular fluorescent probe and its preparation method and application, it is related to biochemical detection tool technical field.The present application provides a kind of hydrazine hydrate molecular fluorescent probe, its structural formula is as formula (1) :On this basis, the specific preparation method and application of hydrazine hydrate molecular fluorescent probe are further provided.The hydrazine hydrate molecular fluorescent probe provided by the present application is high in sensitivity, with good selectivity, low detection limit, and the hydrazine hydrate molecular fluorescent probe provided by the present application can be successfully applied to the monitoring of hydrazine hydrate in water environment and cell system.

Description

Technical Field

[0001] This invention relates to the field of biochemical detection tools. In particular, it relates to a hydrazine hydrate molecular fluorescent probe, its preparation method, and its applications. Background Technology

[0002] Hydrazine hydrate, an important fine chemical raw material, is widely used in agriculture, food, pharmaceuticals, textile dyeing, rocket propulsion, and missile systems. However, due to its high carcinogenicity, toxicity, and teratogenicity, its widespread use causes serious environmental pollution. Excessive hydrazine residues entering water, air, and soil can inhibit seed growth and cause leaf wilting. 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 concentration of hydrazine in drinking water is controlled below 0.625 μM (20 μg / L). The U.S. Environmental Protection Agency (US-EPA) has identified hydrazine hydrate as a potential carcinogen, with a minimum safe level of 0.312 μM (10 μg / L) for drinking water. Developing a simple, effective, and rapid tool for monitoring hydrazine hydrate levels in the environment and biological systems is of great significance for environmental protection and human health. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide a fluorescent probe for hydrazine hydrate molecules, its preparation method, and its application. This fluorescent probe can highly sensitively and specifically recognize hydrazine hydrate, causing the fluorescence color of the system to change from blue to green. This effectively solves the problems of requiring complex and expensive equipment and the long processing time in existing technologies.

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

[0006] The first objective of this invention is to provide a fluorescent probe for hydrated hydrazine molecules, the structural formula of which is shown in formula (1):

[0007]

[0008] The beneficial effects of this invention are: the fluorescent probe structure of this invention uses caffeoyl group as fluorescent group and aldehyde group as recognition unit. After reacting with hydrazine hydrate, it generates a phenylhydrazone structure, and the system shows obvious fluorescence enhancement. It can be applied to the specific monitoring of hydrazine hydrate in living cells and aquatic environments.

[0009] The second objective of this invention is to provide a method for preparing a fluorescent probe of hydrazine hydrate molecules, comprising the following steps:

[0010] (1) Add caffeic acid and N,N'-dicyclohexylcarbodiimide to tetrahydrofuran and stir, then add p-aminobenzaldehyde and stir to obtain residue;

[0011] (2) The residue was dissolved in ethyl acetate for washing, the organic layer was dried, and after filtration, the crude fluorescent probe product was obtained.

[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 this invention are: the hydrazine hydrate fluorescent probe provided by this invention has high sensitivity and good selectivity to hydrazine hydrate. 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 live cells.

[0014] This invention utilizes caffeic acid as a starting material to prepare a ratiometric hydrazine hydrate fluorescent probe. This compound can recognize hydrazine hydrate with high sensitivity and specificity, causing the fluorescence color of the system to change from blue to green. This probe can be used for trace detection of hydrazine hydrate in cells and in the aquatic environment. Therefore, this compound provides a new method for convenient and rapid detection of hydrazine hydrate in organisms and in the aquatic environment, and has good application prospects.

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

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

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

[0018] Further, step (2) is as follows: the residue is dissolved in ethyl acetate, washed at least 3 times with KHSO4, NaHCO3 and saturated brine respectively, the organic layer is dried with MgSO4 and filtered, the solvent is recovered, and the crude product is obtained.

[0019] Further, step (3) is 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] A third objective of this invention is to provide an application of a hydrazine hydrate molecular fluorescent probe for the detection of hydrazine hydrate.

[0022] The beneficial effects of this invention are: it develops a simple, effective, and rapid tool for monitoring the content of hydrazine hydrate in the environment and biological systems, which is of great significance for environmental protection and human health.

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

[0024] Furthermore, the detection range of the hydrazine hydrate molecular fluorescent probe is 10 μM-100 μM, and the limit of detection is 0.106 μM. Attached Figure Description

[0025] Figure 1 The fluorescence spectra of the hydrazine hydrate molecular fluorescent probe of Example 1 of the present invention interacting with hydrazine hydrate of different concentrations are shown.

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

[0027] Figure 3 This is a schematic diagram showing the results of treating the loaded probe test paper with different concentrations of hydrazine hydrate under a 365nm ultraviolet lamp.

[0028] Figure 4 This is a schematic diagram showing the results of soaking solutions of different nitrogen-containing compounds with a fluorescent probe. Detailed Implementation

[0029] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0030] Example 1: Preparation of a fluorescent probe for hydrazine hydrate molecules

[0031] The synthetic route for preparing the hydrazine hydrate molecular fluorescent probe is as follows:

[0032]

[0033] The specific synthesis steps are as follows:

[0034] (1) Add caffeic acid (100 mg, 0.55 mmol) and N,N'-dicyclohexylcarbodiimide (123.7 mg, 0.6 mmol) to tetrahydrofuran (10 mL) and stir at room temperature for 10 min. Then add p-aminobenzaldehyde (1197 mg, 5.55 mmol) and stir for 15 h. Filter to obtain residue.

[0035] (2) Dissolve the residue in ethyl acetate (100 mL), wash with KHSO4 (1 mol / L), NaHCO3 (1 mol / L) and saturated brine three times each with 25 mL each time, dry the organic layer with MgSO4, filter, and recover the solvent to obtain the crude product of hydrazine hydrate molecular fluorescent probe.

[0036] (3) The crude product of hydrazine hydrate molecular fluorescent probe was separated by silica gel column chromatography (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] Experimental 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 to prepare a 1×10⁻⁶ solution. -5The solution of M; hydrazine hydrate was dissolved in distilled water to prepare solutions with concentrations of 0 μM, 2.5 μM, 5 μM, 7.5 μM, 10 μM, 12.5 μM, 15 μM, 17.5 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM, and 200 μM.

[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 using a standard fluorescence spectrophotometer (model F-7100; Hitachi) via standard fluorescence spectrophotometric titration. Figure 1 As shown.

[0041] Depend on Figure 1 We can obtain:

[0042] The hydrazine hydrate molecular fluorescent probe exhibits a distinct fluorescence emission peak at 450 nm. As the concentration of hydrazine hydrate in the system increases, the fluorescence intensity at 484 nm gradually increases, and the green fluorescence of the system gradually intensifies. This indicates that the caffeic acid derivative L can be used as a hydrazine hydrate molecular fluorescent probe for the detection of hydrazine hydrate.

[0043] Experimental Example 2:

[0044] First, HeLa cells were placed in 96-well plates containing 10% fetal bovine serum and 1% antibiotics and incubated at 37°C and 5% CO2 for 24 hours. Subsequently, cell imaging experiments were performed. A 10 μM fluorescent probe solution was prepared using the fluorescent probe (HPA) prepared in Example 1. HeLa cells were incubated with the fluorescent probe solution prepared in Example 1 for 30 minutes. The cultured HeLa cells were then divided into four groups: Group 1 was not treated with hydrazine hydrate; Groups 2, 3, and 4 were incubated with hydrazine hydrate (10 μM, 25 μM, and 50 μM), respectively, for 30 minutes. After incubation, the cells were washed three times with PBS. Cell samples were imaged using a laser confocal fluorescence microscope (Leica TCS SP8). Cell imaging results are shown below. Figure 2 As shown.

[0045] Depend on Figure 2 We can obtain:

[0046] A fluorescent probe for hydrazine hydrate can detect hydrazine hydrate in cells.

[0047] Experimental Example 3:

[0048] The fluorescent probe prepared in Example 1 was used to prepare a 10 mol / L probe solution. A 2 cm × 2 cm circular filter paper was immersed in the probe solution for 2 minutes, then removed and air-dried. The probe-loaded test paper was then immersed in N₂H₄ solutions of different concentrations (5 mol / L, 10 mol / L, 20 mol / L, 30 mol / L, and 40 mol / L) for 10 minutes. After 10 minutes, the immersed test paper was removed, dried, and its fluorescence color change was observed under a UV lamp. Figure 3 .

[0049] like Figure 3 As shown:

[0050] As can be seen, during application, the fluorescence color of the filter paper loaded with the compound gradually changes from blue to blue-green as the N2H4 concentration increases. This indicates 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 the convenient and rapid detection of N2H4.

[0051] Test Example 4

[0052] Prepare a 10 mol / L probe solution using the fluorescent probe prepared in Example 1. Immerse a 2 cm × 2 cm circular filter paper in the probe solution for 2 minutes, remove it, and air dry it naturally. Then, immerse the probe-loaded test paper in different nitrogen-containing solutions (CH3NH2, C2H7N, C2H8N2, NH2OH, CO(NH2)2, CH4N2S, Cys, GSH, Hcy, Gly, Glu, N2H4) at 10 mol / L concentrations for 10 minutes. Remove the immersed test paper, dry it, and observe the fluorescence color change under a UV lamp to obtain the desired results. Figure 4 ( Figure 4 The numbers 1-12 in the middle are CH3NH2, C2H7N, C2H8N2, NH2OH, CO(NH2)2, CH4N2S, Cys, GSH, Hcy, Gly, Glu, N2H4 in that order.

[0053] like Figure 4 As shown:

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

[0055] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A fluorescent probe for hydrazine hydrate molecules, characterized in that, Its structural formula is as shown in formula (1): 。 2. A method for preparing a hydrazine hydrate molecular fluorescent probe according to claim 1, characterized in that, The preparation method includes the following steps: (1) Add caffeic acid and N,N'-dicyclohexylcarbodiimide to tetrahydrofuran and stir, then add p-aminobenzaldehyde and stir to obtain residue; (2) The residue was dissolved in ethyl acetate for washing, the organic layer was dried, and after filtration, the crude fluorescent probe product was obtained; (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 ratio of the amount of caffeic acid, N,N'-dicyclohexylcarbodiimide, tetrahydrofuran, and 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, Step (1) is as follows: Add caffeic acid and N,N'-dicyclohexylcarbodiimide to tetrahydrofuran and stir at room temperature for 10-12 minutes. Then add p-aminobenzaldehyde and stir for 14-16 hours to obtain residue.

5. The method for preparing a hydrazine hydrate molecular fluorescent probe according to claim 2, characterized in that, Step (2) is as follows: Dissolve the residue in ethyl acetate, wash it at least 3 times with KHSO4, NaHCO3 and saturated brine respectively, dry the organic layer with MgSO4 and filter it to obtain the crude product.

6. The method for preparing a hydrazine hydrate molecular fluorescent probe according to claim 5, characterized in that, Step (3) is 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.

7. An application of a hydrazine hydrate molecular fluorescent probe, characterized in that, The hydrazine hydrate molecular fluorescent probe was used to detect hydrazine hydrate.

8. The application of the hydrazine hydrate molecular fluorescent probe according to claim 7, characterized in that, The hydrazine hydrate molecular fluorescent probe is used to detect hydrazine hydrate in the aquatic environment.

9. The application of the hydrazine hydrate molecular fluorescent probe according to claim 8, characterized in that, The detection range of the hydrazine hydrate molecular fluorescent probe is 10µM-100µM, and the limit of detection is 0.106µM.

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