Preparation method and application of near-infrared fluorescent probe with AIE effect for hydrogen sulfide and hydrazine detection

Abstract

The invention discloses a near-infrared fluorescent probe with an aggregation-induced emission enhancement effect. The near-infrared fluorescent probe is used for dual detection of hydrogen sulfide and hydrazine. The fluorescent probe is synthesized by carrying out Knoevenagel condensation reaction on 4-hydroxy-3-(1, 4, 5-triphenyl-1H-imidazolyl) benzaldehyde (I) and 2-(2-methyl-4H-chromene-4-subunit) malononitrile (II) under the catalytic action of piperidine, and the chemical structural formula of the fluorescent probe is as shown in the formula (III). The fluorescent probe shows high selectivity and high sensitivity to both hydrogen sulfide and hydrazine, the detection limits are as low as 50.1 nM and 89.2 nM respectively, and the fluorescent probe can be used for various scenes such as food spoilage monitoring and environmental water sample detection, can be used for biological imaging, and shows important application potential in the aspect of detection of hydrazine and hydrogen sulfide in organisms.

Description

Technical Field

[0001] This invention belongs to the fields of organic compound synthesis, fluorescent probes and fine chemicals, and specifically relates to a near-infrared fluorescent probe with AIE effect for the detection of hydrogen sulfide and hydrazine. Background Technology

[0002] Hydrogen sulfide (H2S), as a key gaseous signaling molecule, plays a crucial role in physiological processes, pathological states, and food safety. It participates in cardiovascular regulation, nerve conduction, and inflammatory responses, and abnormal concentrations are associated with various diseases such as Alzheimer's disease and hypertension. Hydrazine (N2H4), with its unique chemical properties, is widely used in industries such as rocket propellants, pharmaceuticals, and agricultural chemicals. However, both compounds pose significant potential health risks: while hydrogen sulfide has important functions at physiological concentrations, it exhibits toxicity at high concentrations and is a marker of food spoilage; hydrazine, on the other hand, is a highly toxic substance, and even low-dose exposure can cause multi-organ damage. Furthermore, the widespread use of hydrazine increases its environmental pollution risk. Therefore, developing highly sensitive methods for accurate, real-time monitoring of hydrogen sulfide and hydrazine in biological systems and environmental samples is essential.

[0003] Traditional detection techniques for hydrogen sulfide and hydrazine, such as colorimetric, chromatographic, and electrochemical methods, often suffer from limitations such as complex operation, expensive instruments, cumbersome pretreatment, and potential invasiveness or damage, restricting their application in biological systems. Fluorescence imaging technology, with its real-time, non-invasive monitoring characteristics, combined with high sensitivity and good biocompatibility, offers an effective solution to these detection challenges. However, existing fluorescent probe materials often face the problem of aggregation-induced fluorescence quenching (ACQ), and developing a single probe capable of specifically distinguishing and dual-responding to hydrogen sulfide and hydrazine remains challenging. Combining aggregation-induced emission (AIE) characteristics with a ratiometric detection strategy can effectively overcome these limitations, providing a feasible path for constructing a high-performance dual-sensing platform.

[0004] Based on the above considerations, this invention prepares a novel near-infrared fluorescent probe with aggregation-induced emission (AIE) properties for the dual detection of hydrogen sulfide and hydrazine. This probe exhibits high selectivity and sensitivity for both analytes, with a low detection limit, and can be used in various scenarios such as food spoilage monitoring and environmental water sample detection. Furthermore, the probe utilizes ratiometric fluorescence signal output, effectively avoiding environmental interference, and its near-infrared emission characteristics and AIE properties demonstrate significant application potential for imaging analysis of hydrazine and hydrogen sulfide in vivo. Summary of the Invention

[0005] To address the shortcomings of existing methods for detecting hydrogen sulfide and hydrazine, this invention utilizes molecular design to prepare a near-infrared fluorescent probe with AIE effect that exhibits good selectivity, high sensitivity, strong anti-interference ability, and low detection limit for the dual detection of hydrogen sulfide and hydrazine.

[0006] The present invention also provides a method for preparing the above-mentioned fluorescent probe.

[0007] This invention also provides the application of the above-mentioned fluorescent probe in the field of detection.

[0008] Technical solution: To achieve the above-mentioned objectives, the technical solution of this invention is: a near-infrared fluorescent probe with AIE effect for the detection of hydrogen sulfide and hydrazine, the chemical structure of which is shown in formula (III).

[0009]

[0010] The above-mentioned method for synthesizing a near-infrared fluorescent probe with AIE effect for the detection of hydrogen sulfide and hydrazine is characterized in that it is synthesized according to the following experimental steps;

[0011] 4-Hydroxy-3-(1,4,5-triphenyl-1H-imidazolyl)benzaldehyde (I) and 2-(2-methyl-4H-chromene-4-ylidene)malonitrile (II) under piperidine catalysis undergo a Knoevenagel condensation reaction to give compound (III);

[0012] The specific synthetic reaction formulas for the near-infrared fluorescent probes with AIE effect for the detection of hydrogen sulfide and hydrazine are as follows:

[0013]

[0014] The experimental steps were completed using the following method:

[0015] 4-hydroxy-3-(1,4,5-triphenyl-1H-imidazolyl)benzaldehyde (I) was reacted with 2-(2-methyl-4H-chromene-4-ylidene)malonitrile (II) and piperidine in methanol and reacted at 70 °C for 2 h to give compound (III).

[0016] The beneficial effects of this invention are as follows: (1) The synthesis method of this fluorescent probe is simple, the reaction conditions are mild, and the purification and separation methods are convenient; (2) This fluorescent probe has good selectivity, strong anti-interference ability, and high sensitivity, and the detection limit can be as low as 50.1 nM (hydrogen sulfide) and 89.2 nM (hydrazine); (3) This fluorescent probe has aggregation-induced emission (AIE) characteristics and can still maintain a high fluorescence intensity in environments with high water content; (4) This fluorescent probe can be applied to the detection of hydrogen sulfide content after food (beef, pork and chicken) spoilage; (5) This fluorescent probe can be applied to the detection of hydrazine in actual water samples (deionized water, tap water and lake water), with high recovery rate and reliable results; (6) This fluorescent probe has good biocompatibility and can be used to detect the concentration changes of hydrogen sulfide and hydrazine in zebrafish, and has broad application prospects in analytical chemistry, environmental monitoring, food testing, biofluorescence imaging and other fields. Attached Figure Description

[0017] Figure 1 This is a fluorescence emission intensity diagram of fluorescent probe (III) (10 μM) after adding 5 equivalents of different analytes to a dimethyl sulfoxide:water (v / v = 1:1) solution.

[0018] Figure 2 This is a titration graph of hydrogen sulfide fluorescence emission intensity in a dimethyl sulfoxide:water (v / v = 1:1) solution of fluorescent probe (III) (10 μM). The vertical axis represents fluorescence emission intensity, and the horizontal axis represents emission wavelength and excitation wavelength λ. ex =450nm.

[0019] Figure 3 The ratio (F) of the fluorescence probe (III) with different concentrations of sodium hydrosulfide as the abscissa to the maximum fluorescence emission intensity at 549 nm and 650 nm is the value of the fluorescence intensity. 549 / F 650 The vertical axis represents the linear fit; the horizontal axis represents the concentration of sodium hydrosulfide added, in units of 10. -5 mol / L.

[0020] Figure 4 This is a titration graph of hydrazine fluorescence emission intensity of fluorescent probe (III) (10 μM) in a dimethyl sulfoxide:water (v / v = 1:1) solution. The vertical axis represents fluorescence emission intensity, and the horizontal axis represents emission wavelength and excitation wavelength λ. ex =450nm.

[0021] Figure 5 The ratio (F) of the fluorescent probe (III) with different concentrations of hydrazine as the x-axis to the maximum fluorescence emission intensity at 711 nm and 650 nm is given. 711 / F 650 The vertical axis represents the linear fit; the horizontal axis represents the concentration of hydrazine added, in units of 10⁻⁶.-5 mol / L.

[0022] Figure 6 The ratio (F0) of the maximum fluorescence emission intensity at 549 nm to 650 nm after adding 5 equivalents of other competing analytes to a dimethyl sulfoxide:water (v / v = 1:1) solution containing fluorescent probe (III) (10 μM) and sodium hydrosulfide. 549 / F 650 Changes in the bar chart.

[0023] Figure 7 The ratio (F0) of the maximum fluorescence emission intensity at 711 nm to 650 nm after adding 5 equivalents of other competing analytes to a dimethyl sulfoxide:water (v / v = 1:1) solution containing fluorescent probe (III) (10 μM) and hydrazine is used. 711 / F 650 Changes in the bar chart.

[0024] Figure 8 This is a fluorescence emission spectrum of the fluorescent probe (III) (10 μM) in tetrahydrofuran / aqueous solutions at different ratios. Excitation source λ ex =450nm;

[0025] Figure 9 These are confocal fluorescence images of zebrafish under different treatment conditions using the fluorescent probe (III) (10 μM). Excitation source λ ex =450nm;

[0026] Figure 10 This is a graph showing the color changes of food spoilage detected by fluorescent probe (III) test strips under different treatment conditions.

[0027] Table 1 shows the detection results of hydrazine concentration in actual environmental water samples using the fluorescent probe (III). Detailed Implementation

[0028] The present invention will now be described in further detail with reference to embodiments and accompanying drawings.

[0029] Example 1: Preparation of fluorescent probe (III).

[0030] 4-hydroxy-3-(1,4,5-triphenyl-1H-imidazolyl)benzaldehyde (I) and 2-(2-methyl-4H-chromene-4-ylidene)malonitrile (II) were dissolved in methanol and reacted at 70 °C for 2 h. After the reaction was completed, the mixture was cooled to room temperature, the solvent was evaporated to dryness, and the mixture was purified by silica gel column chromatography (eluents were dichloromethane and n-hexane) to obtain an orange powder solid (III) in 57% yield. 1HNMR (600MHz, CDCl3): δ (ppm) 8.90 (m, 1H), 7.72 (m, 2H), 7.60-7.54 (m, 4H), 7.48 (d, J = 8.4Hz, 1H), 7.43 (t, J = 7.2Hz, 1H), 7.33- 7.29 (m, 9H), 7.22 (d, J = 3.6Hz, 1H), 7.20-7.16 (m, 2H), 7.10 (d, J = 8.4Hz, 1H), 6.96 (s, 1H), 6.58 (s, 1H), 5.64 (d, J = 15.6Hz, 1H). 13 C NMR (150MHz, CDCl3, ppm) δ161.056, 157.811, 153.258, 152.798, 152.284, 143.794, 138.282, 137.402, 13 5.321, 134.588, 134.504, 132.686, 131.568, 131.279, 131.028, 130.367, 129.852, 129.247, 128.896, 12 8.808, 128.653, 128.407, 127.312, 126.925, 126.053, 125.891, 125.817, 124.421, 123.782, 118.636, 11 8.506, 118.347, 117.882, 116.931, 116.032, 115.716, 113.699, 106.191, 105.498.ESI-HRMS: calculated for C 41 H 26 N4O2[M+H] + :607.2056; found:607.2111.

[0031] Example 2: Study on the selective recognition of different analytes by fluorescent probe (III).

[0032] Prepare a precise 10 μM solution of the fluorescent probe (III) in dimethyl sulfoxide and water (v / v = 1:1). Figure 1 As shown, five equivalents of different analytes, such as Ag, were added to the test solution containing the fluorescent probe (III). + Al 3+ Ba 2+ Ca 2+ Co 2+ Cr 3+ Cu 2+ Fe 2+ Fe 3+ Hg 2+ K +La 3+ Mg 2+ Mn 2+ Ni 2+ Na + Pb 2+ Zn 2+ Aniline, diethylamine, n-butylamine, pyridine, piperidine, 2-aminopyridine, dimethylamine, 4-dimethylaminopyridine, 4-methoxy-p-phenylene diamine, N,N-dimethylformamide, SO4 2- SO3 2- SCN - S2O3 - HSO3 - HSO4 - The fluorescence spectra of fluorescent probe (III) were compared with those of GSH, Cys, NaHS, and N2H4. The addition of sodium hydrosulfide resulted in a blue shift in the fluorescence spectrum of probe (III) from 650 nm to 549 nm with a significant increase in fluorescence intensity. The addition of hydrazine resulted in a red shift in the fluorescence spectrum of probe (III) from 650 nm to 711 nm with a significant increase in fluorescence intensity. The fluorescence intensity of the probe remained almost unchanged after the addition of other analytes. This indicates that, apart from hydrogen sulfide and hydrazine, the other analytes studied had little effect on the fluorescence spectrum of probe (III). These results suggest that fluorescent probe (III) has high selectivity for hydrogen sulfide and hydrazine, making it suitable for the specific detection of these two analytes.

[0033] Example 3: Fluorescence intensity change of fluorescent probe (III) with increasing hydrogen sulfide concentration.

[0034] The linear relationship between the fluorescence intensity of the fluorescent probe (HI) and the concentration of hydrogen sulfide was further investigated through titration experiments. Fluorescent titration of hydrogen sulfide was performed in a 10 μM solution of fluorescent probe (III) in dimethyl sulfoxide:water (v / v = 1:1). Figure 2 As shown, in the absence of hydrogen sulfide, under excitation at a wavelength of 450 nm, the maximum fluorescence emission peak of the fluorescent probe (III) is at 650 nm. However, with the increase of sodium hydrosulfide concentration (0 μM–13 μM), the fluorescence intensity of probe (III) at 650 nm gradually decreases, while the fluorescence intensity at 549 nm gradually increases until it reaches saturation. Figure 2 As shown. The ratio of the maximum fluorescence emission intensity at 549 nm to that at 650 nm (F) is selected. 549 / F 650 Using y = 0.38631x - 0.08039 as the x-axis, a linear regression equation was obtained by fitting the graph to the x-axis: y = 0.38631x - 0.08039. The results are as follows: Figure 3 As shown, the linear correlation coefficient R 2A value greater than 0.99 indicates a good linear relationship between the fluorescent probe (III) and hydrogen sulfide (e.g., ...). Figure 3 It has a minimum detection limit of 50.1 nM, exhibits good sensitivity, and can be used for quantitative analysis and detection of hydrogen sulfide within a certain concentration range.

[0035] Example 4: Fluorescence intensity change of fluorescent probe (III) with increasing hydrazine concentration.

[0036] The linear relationship between the fluorescence intensity of the fluorescent probe (III) and the concentration of hydrazine was further investigated through titration experiments. Fluorescent titration of hydrazine was performed in a 10 μM solution of fluorescent probe (III) in dimethyl sulfoxide:water (v / v = 1:1). Figure 4 As shown, in the absence of hydrazine, the maximum fluorescence emission peak of the fluorescent probe (IIII) under excitation at a wavelength of 450 nm is at 650 nm. However, with the increase of hydrazine concentration (0 μM to 26 μM), the fluorescence intensity of probe (III) at 650 nm gradually decreases, while the fluorescence intensity at 711 nm gradually increases until it reaches saturation. Figure 4 As shown. The ratio of the maximum fluorescence emission intensity at 711 nm to that at 650 nm (F) is selected. 711 / F 650 Using y as the ordinate and different concentrations of hydrazine as the abscissa, a linear regression equation was obtained by linear fitting: y = 0.18711x + 0.21759. The results are as follows: Figure 5 As shown, the linear correlation coefficient R 2 A value greater than 0.99 indicates a good linear relationship between the fluorescent probe (III) and hydrazine (e.g., ...). Figure 5 It has a detection limit of 89.2 nM, exhibits good sensitivity, and can be used for quantitative analysis and detection of hydrazine within a certain concentration range.

[0037] Example 5: Fluorescent probe (III) resistance to interference from different potential competing analytes when detecting hydrogen sulfide.

[0038] To verify the specific recognition of hydrogen sulfide by probe (III), we also investigated the resistance of probe (III) to interference from other possible analytes. For example... Figure 6 As shown, the addition of sodium hydrosulfide (10 μM) to the probe (III) solution first resulted in a significant decrease in fluorescence intensity at 650 nm and a significant increase in fluorescence intensity at 549 nm. Subsequently, the addition of various potential competitors (50 μM each) to the system did not cause significant changes in their fluorescence signals. This indicates that the presence of these competitors does not interfere with the specific recognition of hydrogen sulfide by probe (III), demonstrating the probe's good anti-interference ability. (Figure numbers 1-5: no addition, Ag) + Al 3+Ba 2+ Ca 2+ 6-10: Co 2+ Cr 3+ Cu 2+ Fe 2+ Fe 3+ 11-15: Hg 2+ K + La 3 + Mg 2+ Mn 2+ 16-20: Ni 2+ Na + Pb 2+ Zn 2+ 8O4 2- 21-25: SO3 2- SCN - S2O3 - HSO3 - HSO4 - 26-27: GSH, Cys).

[0039] Example 6: Fluorescent probe (III) resistance to interference from different potential competing analytes when detecting hydrazine

[0040] To verify the specific recognition of hydrazine by probe (III), we also investigated the resistance of probe (III) to interference from other possible analytes. For example... Figure 7 As shown, the addition of hydrazine (20 μM) to the probe (III) solution first resulted in a significant decrease in fluorescence intensity at 650 nm and a significant increase in fluorescence intensity at 711 nm. Subsequently, the addition of various potential competitors (100 μM each) to the system did not cause significant changes in their fluorescence signals. This indicates that the presence of these competitors does not interfere with the specific recognition of hydrazine by probe (III), demonstrating the probe's good anti-interference ability. (Figure numbers 1-5: no addition, Ag) + Al 3+ Ba 2+ Ca 2+ 6-10: Co 2+ Cr 3+ Cu 2+ Fe 2+ Fe 3+ 11-15: Hg 2+ K + La 3+ Mg 2+ Mn 2+ 16-20: Ni 2+ Na +Pb 2+ Zn 2+ 21-25: aniline, n-butylamine, pyridine, piperidine, 2-aminopyridine, 26-29: dimethylamine, 4-dimethylaminopyridine, 4-methoxy-p-phenylene diamine, N,N-dimethylformamide).

[0041] Example 7: Aggregation-Induced Enhancement Effect of Fluorescent Probe (III)

[0042] Different ratios of tetrahydrofuran / water solution containing 5 μM fluorescent probe (III) were used to measure its fluorescence emission spectra. The results are as follows: Figure 8 As shown, with the increase of the proportion of aqueous solution, the fluorescence intensity at 650 nm gradually increases and redshifts to 680 nm, indicating that probe (III) has aggregation-induced emission ability in tetrahydrofuran / aqueous solution.

[0043] Example 8: Confocal fluorescence imaging of fluorescent probe (III) (10 μM) in zebrafish under different hydrogen sulfide and hydrazine concentrations.

[0044] Zebrafish embryos were co-incubated with fluorescent probe (III) (10 μM) for 24 hours, and then exposed to solutions of different concentrations of sodium hydrosulfide and hydrazine. Figure 9 As shown, zebrafish maintained normal activity and morphology. With increasing sodium hydrosulfide concentration, the fluorescence of the red channel of fluorescent probe (III) decreased significantly, while the fluorescence of the green channel increased significantly; with increasing hydrazine concentration, the fluorescence of the red channel of fluorescent probe (III) increased, while the fluorescence of the green channel decreased, confirming that fluorescent probe (III) can detect changes in the concentration of hydrogen sulfide and hydrazine in live organisms (zebrafish).

[0045] Example 9: Detection of Food Spoilage Using Fluorescent Probe (III)

[0046] Three types of meat (pork, beef, and chicken) were selected as samples and sealed together with fluorescent probe (III) coated test strips, then stored at different temperatures. Figure 10 As shown, after being stored in a 2°C refrigerator for 24 hours, the meat sample test strips did not show significant color changes, indicating that the meat remained fresh. However, the meat sample test strips stored at room temperature for 24 hours showed a color change from yellow to red, indicating that a certain amount of H2S had been released and the meat was no longer fresh. These results demonstrate that the fluorescent probe (III) test strips, as a simple visualization tool, have application potential in the field of on-site monitoring of meat freshness.

[0047] Example 10: Application of Fluorescent Probe (III) in Actual Water Samples

[0048] Fluorescent probe (III) was applied to detect hydrazine concentrations in deionized water, tap water, and lake water samples. Hydrazine standard solutions of known concentrations were added to each water sample, and the concentration was determined by fluorescent probe (III) using fluorescence titration, with the recovery rate calculated. The results are shown in Table 1. Fluorescent probe (III) achieved good recoveries (98.44%–102.22%) for hydrazine in these actual water samples, and the relative standard deviations of the five parallel determinations were small, demonstrating the reliability and stability of this method for detecting hydrazine concentrations in real environmental samples.

[0049] Table 1

[0050]

Claims

1. A near-infrared fluorescent probe with AIE effect for the detection of hydrogen sulfide and hydrazine, characterized in that, Its chemical structure is shown in formula (III) below:

2. The method for synthesizing the near-infrared fluorescent probe with AIE effect for the detection of hydrogen sulfide and hydrazine as described in claim 1, characterized in that, It was synthesized according to the following experimental steps; 4-Hydroxy-3-(1,4,5-triphenyl-1H-imidazolyl)benzaldehyde (I) and 2-(2-methyl-4H-chromene-4-ylidene)malononitrile (II) undergo a Knoevenagel condensation reaction catalyzed by piperidine to give compound (III).

3. The method for preparing a fluorescent probe according to claim 2, characterized in that, The characteristic method includes the following steps: 4-hydroxy-3-(1,4,5-triphenyl-1H-imidazolyl)benzaldehyde (I) and 2-(2-methyl-4H-chromene-4-ylidene)malonitrile (II) and piperidine are dissolved in methanol and reacted at 70°C for 2 h to obtain compound (III).

4. The method for preparing a fluorescent probe according to claim 2, characterized in that, In the aforementioned step, the molar ratio of 4-hydroxy-3-(1,4,5-triphenyl-1H-imidazolyl)benzaldehyde (I) to 2-(2-methyl-4H-chromene-4-ylidene)malononitrile (II) is 1:1.

1.

5. The method for preparing a fluorescent probe according to claim 2, characterized in that, In the above steps, the reaction mixture is heated to 70°C and stirred for 2 hours to obtain compound (III).

6. The fluorescent probe according to claim 2 exhibits aggregation-induced emission (AIE) capability in tetrahydrofuran / aqueous solution.

7. The application of the fluorescent probe according to claim 6, characterized in that, The fluorescent probe enables the quantitative and qualitative detection of hydrogen sulfide and hydrazine in a 1:1 volume ratio mixture of dimethyl sulfoxide and water, and the detection is a fluorescence detection. The fluorescent probe exhibits excellent detectability for hydrogen sulfide and hydrazine, with detection limits as low as 50.1 nM (hydrogen sulfide) and 89.2 nM (hydrazine), respectively.

8. The application of the fluorescent probe according to claim 7, characterized in that... Used for the detection and analysis of hydrogen sulfide and hydrazine in environmental or biological systems.

9. The application of the fluorescent probe according to claim 8, characterized in that, The fluorescent probe can detect changes in the concentration of hydrogen sulfide and hydrazine in zebrafish. This application is not intended for the diagnosis or treatment of diseases.

10. The application of the fluorescent probe according to claim 8, characterized in that, The fluorescent probe can be used to detect the hydrogen sulfide content in different food samples after spoilage, including beef, pork, and chicken.

11. The application of the fluorescent probe according to claim 8, characterized in that, The fluorescent probe can be used to detect hydrazine content in water samples from different environments, including deionized water, tap water, and lake water.

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