Preparation method and application of near-infrared hydrazine fluorescent probe based on xanthene-malononitrile derivative

Abstract

The invention discloses a near-infrared hydrazine (N2H4) fluorescent probe based on a xanthene-malononitrile derivative and application of the near-infrared hydrazine (N2H4) fluorescent probe. The fluorescent probe is synthesized by carrying out Knoevenagel condensation reaction on 6-(diphenylamino)-2, 3-dihydro-1H-xanthene-4-formaldehyde and malononitrile, the chemical structural formula of the fluorescent probe is as shown in formula (I), and dicyanovinyl is used as a hydrazine recognition unit. The near-infrared fluorescent probe (I) disclosed by the invention has a specific recognition effect on hydrazine in a DMSO: PBS (v / v = 6: 4, and pH = 7.4) solution, the fluorescence emission intensity is remarkably quenched, the color is changed from purple to light yellow, the near-infrared fluorescent probe has high sensitivity and very strong anti-interference capability, and the detection limit can be as low as 2.5 mu M; the invention provides a fluorescent probe for simply and rapidly detecting hydrazine, and the fluorescent probe has a wide application prospect in the fields of environment and biology.

Description

Technical Field

[0001] The invention belongs to the technical field of organic synthesis, fluorescent probes and fine chemicals, and particularly relates to a near-infrared fluorescent probe for detecting hydrazine. Background Art

[0002] Hydrazine (N2H4) is a highly active alkaline reducing agent and an industrial raw material in the fields of chemical, pharmaceutical and agricultural industries, but it is highly toxic. Hydrazine has good solubility in water and high biological toxicity. It is easily absorbed by the human body through breathing or skin penetration, causing serious damage to the kidneys, liver and central nervous system, and even leading to gene mutations or cancer. Therefore, hydrazine has been listed as a potential human carcinogen, so it is of great significance to develop a simple and effective method for detecting hydrazine.

[0003] So far, there are many methods for detecting hydrazine, such as electrochemical method, spectral analysis, chromatography, titration, etc. However, these methods usually require special equipment, are time-consuming, and damage tissues or cells, and are not suitable for detecting N2H4 in vivo. In contrast, small molecule fluorescent probes have been widely used in many fields due to their high sensitivity and selectivity, excellent anti-interference and low cytotoxicity. Many small molecule fluorescent probes have been reported for the detection of hydrazine. However, most of these methods are expensive, require a lot of sample preparation, have a long detection time, and can only qualitatively detect N2H4; in addition, most hydrazine fluorescent probes have the characteristics of short emission wavelength and high background fluorescence, which are not suitable for practical application in complex systems such as environmental and biological samples. The emission wavelength of near-infrared fluorescent probes is 650-900nm, which can effectively avoid the interference of spontaneous fluorescence in biological systems and minimize the light damage to biological samples, thereby effectively overcoming the limitations of the application of fluorescent probes in biological samples. In addition, fluorescent probes with colorimetric signals are increasingly favored by people because they can show obvious color changes and provide intuitive changes.

[0004] Based on the above considerations, the present invention discloses a near-infrared fluorescent probe for detecting hydrazine, which has a specific recognition effect on hydrazine, is simple to synthesize, and has a high yield; after reacting with hydrazine, the fluorescence intensity is significantly quenched, the detection limit is low, and it has obvious color changes, and can be used for visual detection. Since the probe has the advantages of fast response speed, high selectivity and sensitivity, and little interference from other analytes, it has great application prospects in environmental detection. Summary of the invention

[0005] In view of the shortcomings of the existing hydrazine detection methods, the present invention prepares a near-infrared hydrazine fluorescent probe (I) of a xanthene-malononitrile derivative with good selectivity, high sensitivity, strong anti-interference ability and low detection limit through molecular design.

[0006] The present invention also provides a method for preparing the xanthene-malononitrile derivative hydrazine fluorescent probe.

[0007] The present invention also provides application of the xanthene-malononitrile derivative hydrazine fluorescent probe in the detection field.

[0008] Technical solution: In order to achieve the above-mentioned invention object, the technical solution of the present invention is: a near-infrared fluorescent probe for detecting hydrazine, whose chemical structure is shown in formula (I),

[0009]

[0010] The method for synthesizing the above-mentioned xanthene-malononitrile derivative near-infrared hydrazine fluorescent probe is characterized in that it is synthesized according to the following experimental steps:

[0011] With anhydrous ethanol as solvent and under the catalysis of piperidine, 6-(diphenylamino)-2,3-dihydro-1H-xanthene-4-carboxaldehyde and malononitrile react at 80°C for 6h to obtain the product.

[0012] The near-infrared hydrazine fluorescent probe based on xanthene-malononitrile derivatives of the present invention has unique fluorescence responsiveness and significant color change to hydrazine in a mixed solution of dimethyl sulfoxide (DMSO):phosphate buffer solution (PBS) (v / v=6:4, pH=7.4).

[0013] Prepare a DMSO:PBS (v / v=6:4, pH=7.4) solution of the fluorescent probe (I), and add different analyte solutions containing quantitative amounts, such as Cd 2+ , Cu 2+ 、Al 3+ , Ca 2+ , Ba 2+ 、Ag + , Fe 2+ , Fe 3+ , Hg 2+ 、Zn 2+ , Pb 2+ 、Ni + 、Na + , Mn 2+ Mg 2 + ,La + , K + 、HSO3 - , aniline, diethylamine, triethylamine, n-butylamine, and N2H4 solutions were used to study the selective recognition of different analytes by testing their fluorescence emission spectra. Figure 1As shown, by examining the change in the intensity of its fluorescence emission spectrum, it is found that the fluorescent probe (I) of the present invention has a strong fluorescence response to hydrazine. When the fluorescent probe reacts with hydrazine, the fluorescence intensity is significantly quenched. In contrast, other analytes do not cause obvious changes in fluorescence intensity under the same conditions, indicating that the probe has high specific selectivity for N2H4. In addition, a certain amount of fluorescent probe (I) solution is taken, and N2H4 is gradually added; the fluorescence intensity of the fluorescent probe (I) at 657nm is gradually quenched as the hydrazine concentration increases. When the maximum fluorescence is completely quenched, the hydrazine concentration is continued to increase, and the fluorescence intensity remains almost unchanged, indicating that the detection of hydrazine has reached saturation. The results are as follows Figure 2 The maximum fluorescence emission intensity at 657 nm was selected as the ordinate and the hydrazine concentration was selected as the abscissa. Linear fitting was performed to obtain linear regression equations of two different ranges, y=821.49792-15.79839x and y=266.69803-2.63061x, respectively. The linear correlation coefficient R 2 All are greater than 0.99. The results are as follows Figure 3 The results show that the correlation is high, so the fluorescent probe can be used for quantitative analysis and detection of hydrazine in this range.

[0014] The near-infrared hydrazine fluorescent probe based on xanthene-malononitrile derivatives of the present invention has excellent anti-interference properties against different competing analytes when detecting hydrazine. Figure 4 As shown, after adding hydrazine, other analytes were then added respectively, and the degree of fluorescence quenching of the fluorescent probe (I) was almost unchanged and was not affected by other analytes. This confirms that the fluorescent probe (I) of the present invention has unique fluorescence selectivity and strong anti-interference ability for N2H4 in DMSO:PBS (v / v=6:4, pH=7.4) solution.

[0015] The near-infrared hydrazine fluorescent probe based on anthracene-malononitrile derivatives of the present invention has the characteristic of a short response time. In the presence of N2H4, the fluorescence of the fluorescent probe (I) can be quenched in a short time and quickly reach the minimum value. The results are as follows Figure 5 As shown, this faster-responding fluorescent probe has a relatively high sensitivity for the detection of hydrazine.

[0016] The near-infrared hydrazine fluorescent probe based on xanthene-malononitrile derivatives of the present invention has the characteristics of a wide range of pH value applications. Figure 6 As shown, within the pH range of 2-12, the probe still has a good effect on recognizing hydrazine, and a wider pH application range is helpful to improve the actual detection performance of the fluorescent probe.

[0017] The beneficial effects of the present invention are as follows: (1) The synthesis method of the fluorescent probe is simple, the reaction conditions are mild, and the purification and separation method is simple; (2) The fluorescent probe has good selectivity, strong anti-interference ability, high sensitivity, a detection limit as low as 2.5 μM, and can be used for visual detection. It has broad application prospects in the fields of analytical chemistry, environmental testing, food testing, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The concentration of fluorescent probe (I) is 1×10 -5 Fluorescence emission intensity diagram after adding 5 equivalents of different analytes to 1 mol / L DMSO:PBS (v / v=6:4, pH=7.4) solution.

[0019] Figure 2 The concentration of fluorescent probe (I) is 1×10 -5 Fluorescence emission intensity diagram of hydrazine titration in 1 mol / L DMSO:PBS (v / v=6:4, pH=7.4) solution, with the ordinate being the fluorescence emission intensity and the abscissa being the emission wavelength, and the excitation wavelength being 550 nm.

[0020] Figure 3 The linear fitting diagram of the fluorescent probe (I) is a graph with different concentrations of hydrazine as the horizontal axis and the maximum fluorescence emission intensity at 657nm as the vertical axis; the horizontal axis is the concentration of the added hydrazine, and the unit is 10 -5 mol / L.

[0021] Figure 4 The concentration of fluorescent probe (I) is 1×10 -5 Histogram of the fluorescence intensity change at the maximum fluorescence emission wavelength of 655 nm after adding 5 equivalents of other competing analytes to a DMSO:PBS (v / v=6:4, pH=7.4) solution containing 1 mol / L and an equivalent amount of hydrazine.

[0022] Figure 5 The concentration of fluorescent probe (I) is 1×10 -5 mol / L in DMSO:PBS (v / v=6:4, pH=7.4) solution without and with hydrazine.

[0023] Figure 6 The concentration of fluorescent probe (I) is 1×10 -5 mol / L before and after the addition of hydrazine, the fluorescence intensity changes with different pH values.

[0024] Figure 7 The concentration of fluorescent probe (I) is 1×10 -5 mol / L before and after the addition of hydrazine (100 μM). DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with implementation examples and accompanying drawings.

[0026] Embodiment 1

[0027] Preparation of Hydrazine Near-Infrared Fluorescent Probes Based on Xanthene-Malononitrile Derivatives (I)

[0028] With anhydrous ethanol as solvent, under the catalysis of piperidine, 6-(diphenylamino)-2,3-dihydro-1H-xanthene-4-carboxaldehyde and malononitrile were reacted at 80°C for 6 hours. After the reaction was completed, the mixture was cooled to room temperature, poured into deionized water (100 mL), and extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, and the filtrate was evaporated to dryness and separated and purified by silica gel column chromatography (eluents were ethyl acetate and dichloromethane) to obtain a black solid (I) with a yield of 66%. 1 H NMR (600MHz, CDCl3, ppm): 7.913 (s, 1H), 7.355 (t, J=7.2Hz, 4H), 7.194~7.151 (m, 6H), 7.050 (d, J=9Hz, 1H), 6.856 (s, 1H), 6.80 0 (dd, J=9Hz, J=2.4Hz, 1H), 6.740 (d, J=2.4Hz, 1H), 2.851 (t, J=6Hz, 2H), 2.585 (t, J=6Hz, 2H) 1.807 (t, J=6Hz, 2H); Esi-HRMS [C 29 H 21 N3O + ]: The theoretical value is 450.1685[M+Na + ], the actual measured value is: 450.1578.

[0029] Embodiment 2

[0030] Study on the Selective Recognition of Different Analytes by Hydrazine Fluorescent Probes Based on Xanthene-Malononitrile Derivatives (I)

[0031] A 10 μM solution of fluorescent probe (I) in DMSO:PBS (v / v=6:4, pH=7.4) was accurately prepared. Figure 1 As shown, 5 times of different analytes such as Cd were added to the test solution containing probe (I). 2+ , Cu 2+ 、Al 3+ , Ca 2+ , Ba 2+ 、Ag + , Fe 2+ , Fe 3+ , Hg 2+ 、Zn 2+, Pb 2+ 、Ni + 、Na + , Mn 2+ Mg 2+ ,La + , K + 、HSO3 - , aniline, diethylamine, triethylamine, n-butylamine, hydrazine (N2H4). Figure 1 It can be found that when only N2H4 is added, the fluorescence intensity of the fluorescence spectrum of probe (I) at 659nm is almost completely quenched, while after adding other analytes, the fluorescence emission intensity of probe (I) remains almost unchanged, indicating that except for hydrazine, the other analytes studied have almost no obvious effect on the fluorescence spectrum of probe (I). These results show that fluorescent probe (I) has high specific selectivity for hydrazine and can be used as a specific recognition fluorescent probe for detecting hydrazine.

[0032] Embodiment 3

[0033] Fluorescence intensity changes of xanthene-malononitrile derivative hydrazine fluorescent probe (I) with increasing hydrazine concentration

[0034] The linear relationship between the fluorescence intensity of the fluorescent probe (I) and the hydrazine concentration was further explored by titration experiments. Fluorescence titration of N2H4 was performed in a 10 μM fluorescent probe (I) DMSO:PBS (v / v=6:4, pH=7.4) solution. Figure 2 As shown in FIG. 1 , in the absence of hydrazine, under excitation at a wavelength of 550 nm, the fluorescent probe (I) emits strong fluorescence at 657 nm. However, as the concentration of hydrazine (0 to 130 μM) increases, the fluorescence intensity of the probe (I) at 657 nm is gradually quenched until it reaches saturation, as shown in FIG. Figure 2 The maximum fluorescence emission intensity at 657nm was selected as the ordinate, and the different concentrations of hydrazine were selected as the abscissa. Linear fitting was performed to obtain linear regression equations in two different concentration ranges, which were y=821.49792-15.79839x and y=266.69803-2.63061x. The results are shown in Figure 3 As shown, the linear correlation coefficient R 2 are greater than 0.99, and the fluorescent probe (I) and hydrazine show a good linear relationship (such as Figure 3 ), with a minimum detection limit of 2.5 μM, which has good sensitivity and can be used for quantitative analysis and detection of hydrazine within a certain concentration range.

[0035] Embodiment 4

[0036] Anti-interference of xanthene-malononitrile derivative fluorescent probe (I) to different analytes in the detection of hydrazine.

[0037] In order to further investigate the specific recognition of probe (I) to hydrazine, the anti-interference of probe (I) to other possible analytes was also studied. After adding hydrazine (10 μM) to the solution of probe (I), the solution of probe (I) showed obvious fluorescence quenching at 655 nm. Then, 5 equivalents of other analytes were added, including: Cd 2+ , Cu 2+ 、Al 3+ , Ca 2+ , Ba 2 + 、Ag + , Fe 2+ , Fe 3+ , Hg 2+ 、Zn 2+ , Pb 2+ 、Ni + 、Na + , Mn 2+ Mg 2+ ,La + , K + 、HSO3 - , aniline, diethylamine, triethylamine, n-butylamine; such as Figure 4 As shown, the degree of fluorescence quenching of the solution containing hydrazine and other analytes did not change significantly, which indicates that the presence of other analytes does not interfere with the recognition and detection of hydrazine by probe (I), and probe (I) has good anti-interference ability for hydrazine recognition.

[0038] Embodiment 5

[0039] Fluorescence response time of hydrazine fluorescent probe (I) derived from xanthene-malononitrile after recognizing hydrazine

[0040] In order to determine the response time to hydrazine, the fluorescence intensity change of the fluorescent probe (I) with or without hydrazine in DMSO:PBS (v / v=6:4, pH=7.4) solution was tested. Figure 5 As shown; when hydrazine is not added, the fluorescence intensity is very high and the fluorescence intensity does not change with time; when hydrazine is added, the fluorescence intensity decreases significantly within 20 minutes, and the fluorescence is basically completely quenched within 30 minutes and tends to be stable, indicating that the fluorescent probe (I) has a relatively short response time.

[0041] Embodiment 6

[0042] Effect of different pH values ​​on the recognition of hydrazine by hydrazine fluorescent probes derived from xanthene-malononitrile (I)

[0043] In order to obtain the optimal pH range for detecting hydrazine, the changes in the fluorescence emission intensity of 10 μM fluorescent probe (I) in the presence and absence of hydrazine in the pH range of 2-12 were studied. Figure 5As shown, in DMSO:PBS (v / v=6:4, pH=7.4) without hydrazine, the fluorescence has stable characteristics in the pH range of 2-7, and the fluorescence emission intensity shows a trend of first increasing and then decreasing in the pH range of 8-12; while in the presence of hydrazine, the fluorescence quenching effect is kept stable in the pH range of 2-12, and the fluorescence intensity does not change significantly, indicating that the fluorescent probe (I) is applicable to a wide pH range, which helps to improve the actual detection performance of the fluorescent probe.

Claims

1. A near-infrared hydrazine fluorescent probe based on xanthene-malononitrile derivatives, characterized in that: Its chemical structure is shown in formula (I):

2. The method for synthesizing the near-infrared hydrazine fluorescent probe of xanthene-malononitrile derivative according to claim 1, characterized in that: It is synthesized according to the following experimental steps: Using anhydrous ethanol as solvent and under the catalysis of piperidine, 6-(diphenylamino)-2,3-dihydro-1H-xanthene-4-carboxaldehyde and malononitrile undergo Knoevenagel condensation reaction to obtain compound (I).

3. The method for preparing the near-infrared N2H4 fluorescent probe (I) of xanthene-malononitrile derivatives according to claim 2, characterized in that: The volume to amount ratio of anhydrous ethanol, piperidine, 6-(diphenylamino)-2,3-dihydro-1H-xanthene-4-carbaldehyde, and malononitrile is 15 mL: 1.5 mL: 1 mmol: 1.5 mmol.

4. Use of the near-infrared fluorescent probe of the xanthene-malononitrile derivative according to claim 1 in detecting hydrazine.

5. The use according to claim 4, characterized in that: The method can qualitatively and quantitatively detect N2H4 in a DMSO:PBS (v / v=6:4, pH=7.4) solution, wherein the detection is fluorescence detection; the fluorescent probe exhibits excellent detection performance for N2H4, with a detection limit as low as 2.5 μM; and can be applied to the detection of hydrazine molecules in environmental systems.

Images