A chalcone-based fluorescent probe, its preparation method and application in detecting H2S

Abstract

The present invention discloses a chalcone-based fluorescent probe, its preparation method and application in detecting H2S, which relates to the technical field of hydrogen sulfide detection. The chalcone-based fluorescent probe of the present invention, the chemical formula of Cha-N3 is (E)-3-(4-azophenyl)-1-(2-hydroxy-4-methoxyphenyl)prop-2-en-1-one. This chalcone fluorescent probe has strong signal enhancement ability in the application of detecting hydrogen sulfide in aqueous environment. The fluorescence enhancement at the emission wavelength of 570 nm can reach 258 times, and qualitative analysis of hydrogen sulfide can be carried out by naked eyes. The detection limit is 122 nM, and it has the ability to monitor low-content hydrogen sulfide. At the same time, the monitoring of hydrogen sulfide in a variety of natural aqueous environments shows that it has strong anti-interference ability, and the fluorescence intensity of Cha-N3 shows a good linear relationship with the concentration of hydrogen sulfide.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogen sulfide detection, and more specifically, to a chalcone-based fluorescent probe, a preparation method thereof, and an application thereof in detecting H2S. Background Art

[0002] Effective hydrogen sulfide (H2S) is a colorless, malodorous and toxic gas, which exists in nature and various life production processes, such as waste gas emission, anaerobic digestion and petroleum production. Due to its good solubility, H2S is very easy to pollute the water system and even destroy biodiversity. Usually, it will react with water molecules to form acid rain or waste water containing toxic sulfides. On the other hand, H2S is a natural signal molecule in organisms. As the third largest signal molecule in the human body after CO and NO, it is an important participant in various physiological functions, including life activities such as vasodilation, nerve regulation, and apoptosis. However, abnormal levels of H2S in the human body can also cause various physiological diseases, including ischemic diseases, atherosclerosis, tumors, diabetes and hypertension. Therefore, monitoring the fluctuation of H2S concentration in natural water is an important indicator for water quality detection. Compared with traditional methods such as electrochemical analysis, chromatography and colorimetry, small molecule fluorescent probes have obvious advantages in terms of sensitivity, selectivity, in-situ imaging ability, etc. However, most of the fluorescent probes used for H2S detection have more or less problems such as complex synthesis routes, extremely small Stokes shifts, and limited applicability.

[0003] The prior art discloses a fluorescent probe (E)-3-(4-(2,4-dinitrophenoxy)styryl)quinolin-2(1H)-one for detecting hydrogen sulfide. The fluorescent probe uses 3-methylquinolin-2(1H)-one as a fluorophore and 2,4-dinitro ether bond as an identification site. Under the condition that acetonitrile is used as a solvent, there is no ultraviolet absorption peak at 550 nm. After adding hydrogen sulfide, the absorption peak at 380 nm decreases, and a new peak appears at 550 nm. When other anions are added, the ultraviolet absorption spectrum of the fluorescent probe does not change significantly. In the fluorescence spectrum, there is no fluorescence at the beginning. After adding HS - After that, the fluorescence intensity at 490 nm increases significantly. Under the irradiation of ultraviolet lamp at 365 nm, it is observed that fluorescence is lit up after adding HS - After that, while there is no change for other anions. In the presence of interference from other ions, after adding HS - After that, a new absorption peak still appears at 490 nm, and it is hardly interfered by other ions. This fluorescent probe has a certain detection specificity for hydrogen sulfide, but on the one hand, it can only perform qualitative detection and does not achieve quantitative detection. On the other hand, it does not improve the problem of extremely small Stokes shift of existing fluorescent probes. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of existing fluorescence probes for H2S detection, which have extremely small Stokes shifts, limited applicability, and inability to achieve qualitative and quantitative detection. A chalcone-based fluorescence probe is provided, whose structure can construct the fluorescence probe skeleton through multiple mechanisms such as fluorescence resonance energy transfer (FRET), excited state intramolecular proton transfer (ESIPT), or intramolecular charge transfer (ICT). It has the advantages of high sensitivity, good selectivity, large Stokes shift, low detection limit, and strong signal enhancement ability in the detection of hydrogen sulfide in an aqueous environment.

[0005] Another object of the present invention is to provide a preparation method of a chalcone-based fluorescence probe.

[0006] Another object of the present invention is to provide an application of a chalcone-based fluorescence probe in the qualitative detection of H2S in an aqueous environment.

[0007] Another object of the present invention is to provide an application of a chalcone-based fluorescence probe in the quantitative detection of H2S in an aqueous environment.

[0008] The above objects of the present invention are achieved by the following technical solutions:

[0009] A chalcone-based fluorescence probe, characterized in that the chemical formula of the chalcone-based fluorescence probe Cha-N3 is (E)-3-(4-azophenyl)-1-(2-hydroxy-4-methoxyphenyl)prop-2-en-1-one, and the structural formula is as follows:

[0010]

[0011] Among them, it should be noted that:

[0012] The main structure of the chalcone-based fluorescence probe of the present invention, chalcone, has an ESIPT effect, and the recognition group, the azide group, has an ICT effect. The synergistic effect of the two realizes the advantages of high sensitivity, good selectivity, large Stokes shift, low detection limit, and signal enhancement of the chalcone probe.

[0013] The chalcone molecule is a "turn-off-on" type fluorescence probe, and the mechanism of hydrogen sulfide detection is the excited state intramolecular proton transfer (ESIPT) coupled with the intramolecular charge transfer (ICT) mechanism.

[0014] The structure of chalcone-based fluorescent probes can construct the fluorescent probe framework through various mechanisms such as fluorescence resonance energy transfer (FRET), excited state intramolecular proton transfer (ESIPT), or intramolecular charge transfer (ICT). The detection limit for the detection of hydrogen sulfide in an aqueous environment is 122 nM, with high sensitivity. The fluorescence enhancement at the emission wavelength of 570 nm can reach 258 times, with strong signal enhancement ability. Moreover, the Stokes shift is as high as 140 nm, with a large Stokes shift. The responsive molecule emits orange-yellow fluorescence and has good anti-ion interference and selectivity against NaF, NaCl, KBr, KI, NaHSO3, Na2SO4, NaSCN, Na2CO3, NaHCO3, NaNO2, Na2SO4, CH3COONa, Na2S2O3, NaClO, H2O2, MgSO4, CaCl2, PhSNa, Cys, Hcy, GSH, etc.

[0015] The Stokes shift is the difference between the maximum emission wavelength of 570 nm and the maximum excitation wavelength of 430 nm, and its Stokes shift can be obtained based on the fluorescence data.

[0016] The present invention also specifically protects a preparation method of a chalcone-based fluorescent probe, which includes the following steps:

[0017] Mix 2-hydroxy-4-methoxyacetophenone, 4-azidobenzaldehyde, a base, and a solvent, and react for 4 - 12 hours under the protection of an inert gas. After the reaction ends, purify to obtain the fluorescent probe with a chalcone structure.

[0018] The molar ratio of 2-hydroxy-4-methoxyacetophenone to 4-azidobenzaldehyde is 1:0.8 - 1.2;

[0019] The molar ratio of 2-hydroxy-4-methoxyacetophenone to the base is 1:2 - 3.

[0020] It should be noted that:

[0021] The progress of the above reaction can be detected by thin layer chromatography (TLC). If the amount of the base is too low, 2-hydroxy-4-methoxyacetophenone cannot be fully activated.

[0022] The product purification can be carried out by column chromatography purification. After the reaction ends, the post-treatment steps include: filtering the reaction solution to obtain a filtrate, removing the solvent by vacuum rotary evaporation, and performing column chromatography separation and purification to obtain the chalcone fluorescent probe.

[0023] In the specific embodiment, the solvent can be any one of methanol, ethanol, acetone, dichloromethane, or ethyl acetate.

[0024] In the specific embodiments, the base can be any one of sodium hydroxide, potassium hydroxide, potassium carbonate, and triethylamine.

[0025] The present invention also specifically protects the application of a chalcone-based fluorescent probe in detecting sulfur-containing compounds, and the sulfur-containing compound is one of H2S, NaHS, and Na2S.

[0026] The present invention also specifically protects the application of a chalcone-based fluorescent probe in the qualitative detection of H2S in an aqueous environment.

[0027] In the specific application, the specific operation of the qualitative detection can be referred to as follows: Add the sample to be tested into the solution containing the probe Cha-N3, mix well, and observe the color change under a 365 nm ultraviolet lamp. If the probe emits strong orange-yellow fluorescence, it contains hydrogen sulfide.

[0028] The present invention also specifically protects the application of a chalcone-based fluorescent probe in the quantitative detection of H2S in an aqueous environment.

[0029] In the specific application, the quantitative detection can refer to the following specific operation: Add the sample to be tested into the solution containing the probe Cha-N3, mix well, and perform fluorescence spectrophotometry detection at an excitation wavelength of 430 nm within the emission wavelength range of 450 nm to 700 nm. The linear equation between the fluorescence intensity y and the H2S concentration x is: y = 13.05662 * x + 125.80246.

[0030] The fitting degree of the above-mentioned linear equation between the quantitative detection fluorescence intensity y and the H2S concentration x is high, and R 2 = 0.99913.

[0031] When performing quantitative detection using the above method, it is preferred that the molar concentration range of H2S in the aqueous environment is 0 to 50 μM.

[0032] Compared with the prior art, the beneficial effects of the present invention are:

[0033] The chalcone fluorescent probe of the present invention has a strong signal enhancement ability in the application of detecting hydrogen sulfide in an aqueous environment. The fluorescence enhancement at the emission wavelength of 570 nm can reach 258 times. It can perform qualitative analysis of hydrogen sulfide by naked eyes, and the detection limit is 122 nM, having the ability to monitor low-content hydrogen sulfide. At the same time, the monitoring of hydrogen sulfide in various natural aqueous environments shows that it has a strong anti-interference ability, and the fluorescence intensity of Cha-N 3 shows a good linear relationship with the concentration of hydrogen sulfide. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The infrared spectrum of the probe Cha-N3 in Example 1.

[0035] Figure 2 1H NMR spectrum of probe Cha-N3 in Example 1.

[0036] Figure 3 13C NMR spectrum of probe Cha-N3 in Example 1.

[0037] Figure 4 High-resolution mass spectrum of probe Cha-N3 in Example 1.

[0038] Figure 5 Spectral properties of probe Cha-N3 in Example 1.

[0039] Figure 6 Response curve of fluorescence intensity of probe Cha-N3 in Example 1 to hydrogen sulfide concentration.

[0040] Figure 7 Linear relationship between fluorescence intensity of probe Cha-N3 in Example 1 and hydrogen sulfide concentration.

[0041] Figure 8 Changes in fluorescence intensity of probe Cha-N3 in Example 1 after adding various analytes.

[0042] Figure 9 Changes in fluorescence intensity of probe Cha-N3 in Example 1 after adding various interferents. Detailed implementation manners

[0043] The present invention will be further described below in conjunction with the detailed implementation manners, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the raw material reagents used in the embodiments of the present invention are raw material reagents purchased conventionally.

[0044] Example 1

[0045] A chalcone-based fluorescent probe, the chemical formula of the chalcone-based fluorescent probe Cha-N3 is (E)-3-(4-azidophenyl)-1-(2-hydroxy-4-methoxyphenyl)prop-2-en-1-one, and the structural formula is shown as follows:

[0046]

[0047] The preparation method of the chalcone-based fluorescent probe Cha-N3 in Example is as follows:

[0048] Add 2.94 g of 4-azidobenzaldehyde and 3.32 g of 2-hydroxy-4-methoxyacetophenone into a 250 mL reaction flask, add 50 mL of ethanol as a solvent, then add 2.0 g of sodium hydroxide, stir and react at room temperature for 12 hours, and separate and purify by column chromatography to obtain 2.12 g of probe Cha-N3 (eluent: ethyl acetate / petroleum ether = 1 / 20, v / v), and the yield is 36%.

[0049] The molar ratio of 2-hydroxy-4-methoxyacetophenone to 4-azidobenzaldehyde is 1:1.

[0050] The molar ratio of 2-hydroxy-4-methoxyacetophenone to the base is 1:3.

[0051] The structure of the prepared chalcone-based fluorescent probe Cha-N3 was characterized as follows:

[0052] Figure 1 The following is the infrared spectrum of the probe Cha-N3:

[0053] IR(KBr, cm -1 ) 3556.65, 3477.27, 3414.73, 2149.40, 1635.82, 1577.29, 1222.09, 825.56, 608.28.

[0054] Figure 2 The following is the 1H NMR spectrum of the probe Cha-N3:

[0055] 1 H NMR (500 MHz, DMSO-d 6 ) δ 13.49 (s, 1H), 8.30 (d, J = 9.1 Hz, 1H), 8.01 - 7.97 (m, 3H), 7.82 (d, J = 15.4 Hz, 1H), 7.21 (d, J = 8.6 Hz, 2H), 6.58 (d, J = 9.0 Hz, 1H), 6.52 (d, J = 2.5 Hz, 1H), 3.86 (s, 3H)

[0056] Figure 3 The following is the 13C NMR spectrum of the probe Cha-N3:

[0057] 13 C NMR (126 MHz, DMSO) δ 192.27, 166.52, 166.25, 143.69, 142.22, 133.19, 131.98, 131.42, 121.12, 120.12, 114.34, 107.93 101.40, 56.27.

[0058] Figure 4 The following is the high-resolution mass spectrum of the probe Cha-N3:

[0059] HRMS (ESI): m / z found 295.1179, calculated for [M] + : 295.0951.

[0060] Among them, the infrared peak at 2149.40 is the characteristic peak of the azide group, and the molecular ion peak obtained by mass spectrometry is consistent with that of the probe, which can prove the generation of the probe. Each group in the nuclear magnetic resonance corresponds to the probe product one by one. The characteristic peak of -OH is 13.49, and the characteristic peak of -OCH3 is 3.86.

[0061] Example 2

[0062] Spectral property test of the probe molecule Cha-N3

[0063] (1) Preparation process of the stock solution

[0064] Prepare a DMSO solution with a probe concentration of 1 mM in a 25 mL volumetric flask for later use. Prepare an aqueous solution with a Na2S concentration of 10 mM in a 25 mL volumetric flask for later use. The solvent system is a buffer system of DMSO / PBS = 4 / 1. Each time for sample measurement, take 5 mL of the mixed solvent and add 25 μL of the probe stock solution as the blank sample solution to be measured.

[0065] (2) Fluorescence spectrum test

[0066] When the probe Cha-N3 is excited at 380 nm, the blank sample solution to be measured has a weak fluorescence emission peak at 570 nm. Figure 5 This is the spectral property of the probe Cha-N3. When hydrogen sulfide is added, the fluorescence at 570 nm increases, and it increases with the increase of hydrogen sulfide concentration. See Figure 6 .

[0067] (3) Fluorescence titration experiment for detecting hydrogen sulfide

[0068] The linear fitting curve of fluorescence intensity and hydrogen sulfide concentration is y = 13.05662*x + 125.80246, (R 2 = 0.99913). The applicable hydrogen sulfide concentration range is 0 - 50 μM. See Figure 7 . The calculation formula for the lowest detection limit is (LOD = 3σ / k). The lowest detection concentration of Cha-N3 for hydrogen sulfide in the titration experiment is: 122 nM.

[0069] (4) Ion selectivity experiment

[0070] Prepare various analytes with a concentration of 10 mM, namely Na2S, NaF, NaCl, KBr, KI, NaHSO3, Na2SO4, NaSCN, Na2CO3, NaHCO3, NaNO2, Na2SO4, CH3COONa, Na2S2O3, NaClO, H2O2, MgSO4, CaCl2, PhSNa, Cys, Hcy, GSH, and let them react with the probe Cha-N3 respectively. Only hydrogen sulfide shows an obvious reaction with the probe, and the fluorescence intensity increases significantly at 570 nm. The experimental results show that Cha-N3 has the ability of specific selection for the detection of hydrogen sulfide. For details, see Figure 8 。

[0071] (5) Ion interference detection experiment

[0072] In the ion interference experiment, various interfering substances are added to the blank system to be measured, and then hydrogen sulfide is added for fluorescence emission spectrum testing. The interfering substances are NaF, NaCl, KBr, KI, NaHSO3, Na2SO4, NaSCN, Na2CO3, NaHCO3, NaNO2, Na2SO4, CH3COONa, Na2S2O3, NaClO, H2O2, MgSO4, CaCl2, PhSNa, Cys, Hcy, GSH. The experiment shows that the fluorescent probe Cha-N3 has a strong anti-interference ability against various interfering substances, and the presence of interfering substances does not affect the selective testing of the probe. For details, see Figure 9 。

[0073] Example 3

[0074] Experiment on the detection of hydrogen sulfide by the probe molecule Cha-N3 in different environmental water samples

[0075] Take four samples from the Pearl River water area in Guangzhou, Tianhe Lake water area, laboratory tap water, and Jingtian mineral water. After filtering to remove sediment, prepare them into PBS buffer solution with pH = 7.4, and prepare DMSO / PBS = 4 / 1 buffer system for standby. First, add 25 μL of fluorescent probe to 5 mL of buffer system, that is, the probe concentration is 5 μM. Then add hydrogen sulfide to different buffer systems with concentrations of 10 μM, 20 μM, and 30 μM respectively. After mixing evenly at room temperature for 30 minutes, measure their fluorescence spectra and calculate the recovery rate. The results are shown in Table 1 for details.

[0076] When the concentration of hydrogen sulfide is in the range of 0 - 50 μM, the fluorescence intensity of Cha-N3 shows a good linear relationship with the concentration of hydrogen sulfide, that is, this probe molecule can be used for the analysis and determination of low-concentration hydrogen sulfide in complex water environment system samples.

[0077] Table 1

[0078]

[0079] As can be seen from the recovery rate data in Table 1 above, the probe Cha-N3 of the present invention is not interfered by complex water environment systems and can be well applied to different water environments. The probe maintains good detection sensitivity and selectivity in complex water environment systems and can accurately quantify the content of H2S in the water environment.

[0080] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention and are not limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. Any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A chalcone-based fluorescent probe, characterized in that, The chemical formula of the chalcone fluorescent probe Cha-N3 is (E)-3-(4-azidophenyl)-1-(2-hydroxy-4-methoxyphenyl)prop-2-en-1-one, and its structural formula is as follows: 。 2. The preparation method of the chalcone fluorescent probe according to claim 1, characterized in that, It includes the following steps: Mix 2-hydroxy-4-methoxyacetophenone, 4-azidobenzaldehyde, a base, and a solvent, and react for 4 - 12 hours under the protection of an inert gas. After the reaction, purify to obtain the chalcone fluorescent probe. Among them, the molar ratio of 2-hydroxy-4-methoxyacetophenone to 4-azidobenzaldehyde is 1:0.8 - 1.2; The molar ratio of 2-hydroxy-4-methoxyacetophenone to the base is 1:2 - 3.

3. The preparation method according to claim 2, wherein, The solvent is any one of methanol, ethanol, dichloromethane, or ethyl acetate.

4. The preparation method according to claim 2, characterized in that, The base is any one of sodium hydroxide, potassium hydroxide, potassium carbonate, or triethylamine.

5. Use of the chalcone fluorescent probe according to claim 1 in detecting sulfur-containing compounds, characterized in that, The sulfur-containing compound is one of H2S, NaHS, or Na2S.

6. Application of the chalcone fluorescent probe described in claim 1 in the qualitative detection of H2S in an aqueous environment.

7. The application according to claim 6, characterized in that The specific detection operation is as follows: Add the sample to be tested into the solution containing the probe Cha-N3, mix evenly, and observe the color change under a 365 nm ultraviolet lamp. If the probe emits orange-yellow fluorescence, it contains hydrogen sulfide.

8. Application of the chalcone fluorescent probe described in claim 1 in the quantitative detection of H2S in an aqueous environment.

9. The application according to claim 8, wherein The specific operation is as follows: Add the sample to be tested into the solution containing the probe Cha-N3, mix evenly, and perform fluorescence spectrophotometry detection in the range of emission wavelength from 450 nm to 700 nm at an excitation wavelength of 430 nm. The linear equation of fluorescence intensity y and H2S concentration x is: y = 13.05662 * x + 125.80246.

10. The application according to claim 8, characterized in that The molar concentration range of H2S in the aqueous environment is 0 - 50 μM.

Images