Pyrenyl fluorescent probe for high-sensitivity detection of hydrogen sulfide as well as preparation method and application of pyrenyl fluorescent probe

Abstract

The invention discloses a pyrenyl fluorescent probe for high-sensitivity detection of hydrogen sulfide as well as a preparation method and application of the pyrenyl fluorescent probe. Sodium hydrosulfide is used as a hydrogen sulfide donor. The probe can react with sodium hydrosulfide in a 50% DMSO aqueous solution, so that the probe has a fluorescence'opening 'effect. The probe molecule provided by the invention has the characteristics of high sensitivity, short response time (9min) and the like on detection of hydrogen sulfide, and has better stability under the condition of wide pH (2-9); the PYR-H2S probe solution is colorless and does not emit fluorescence, the solution becomes yellow and emits orange-yellow fluorescence after reacting with sodium hydrosulfide, a good colorimetric phenomenon is shown, hydrogen sulfide in a water sample can be quantitatively detected, and due to the characteristics of the PYR-H2S probe with the detection limit of 0.32 [mu] M, the PYR-H2S probe has important application value in the fields of environmental monitoring and the like.

Description

Technical Field

[0001] The invention belongs to the field of fluorescence detection, and specifically relates to a pyrene-based fluorescent probe for highly sensitive detection of hydrogen sulfide and a preparation method and application thereof. Background Art

[0002] Hydrogen sulfide (H2S), as the third endogenous gas signal molecule, has a strong odor similar to rotten eggs and can be found in fruits, vegetables and meats rich in organic sulfur. H2S can extend the quality assurance period of fruits and vegetables by increasing the activity of antioxidant enzymes and regulating physiological metabolic processes. However, during the process of food decay, H2S is produced excessively. It can be produced by sulfur-containing bacteria (such as Shewanella putrefaciens, Pseudomonas methylsulfonates and Citrobacter freundii) by converting sulfur amino acids (cysteine, methionine, etc.). Therefore, H2S may be a sign of food spoilage. In terms of food safety, it is important to use the level of H2S release as a freshness quality indicator in various foods, especially in raw foods, where excessive H2S concentrations can have a negative impact on food quality and may cause harmful damage to human health. In recent years, more and more studies have shown that H2S can mediate a variety of physiological and pathological processes such as redox state regulation, anti-inflammation, neurotransmitter transmission, cardiovascular function regulation, etc. However, abnormal levels of H2S often cause a series of health problems, such as Alzheimer's disease, Down syndrome, atherosclerosis, cirrhosis, etc. Therefore, monitoring H2S levels in food samples and complex biological systems has profound implications.

[0003] At present, there are many analytical techniques for detecting hydrogen sulfide, such as chemiluminescence, colorimetry, gas chromatography, electrochemical method and fluorescence method. However, most of these methods have limitations such as complex operation and time-consuming. Fluorescence spectroscopy has the advantages of high sensitivity, good specificity and fast response time. Fluorescence spectroscopy has certain application prospects for detecting hydrogen sulfide. Therefore, researchers have made considerable efforts in developing fluorescent probes for detecting these hydrogen sulfides. Although great progress has been made in the research on fluorescence detection of hydrogen sulfide, many reported probes still have problems such as cumbersome synthesis process, low selectivity and long response time. Therefore, it is very necessary to develop a simple, highly selective and fast-response fluorescent probe for the detection of hydrogen sulfide. Summary of the invention

[0004] The present invention aims to provide a method for preparing a highly sensitive pyrene-based fluorescent probe for detecting hydrogen sulfide in view of the deficiencies in the prior art.

[0005] The present invention provides a fluorescent probe with a simple synthesis route, mild reaction conditions and low cost; and also provides a fluorescent probe with high sensitivity, good selectivity and strong anti-interference ability, which can accurately identify hydrogen sulfide in a complex system. The structure is as follows:

[0006]

[0007] In the present invention, the two substituents are in adjacent positions, and the adjacent aldehyde groups can accelerate the removal of the recognition group, with a short response time (9 minutes), good water solubility, no need to add a surfactant, and a wide suitable pH range (2 to 9).

[0008]

[0009] The method for preparing the pyrene-based fluorescent probe for highly sensitive detection of hydrogen sulfide comprises the following steps:

[0010] 2-Hydroxypyrene formaldehyde and 1-fluoro-2,4-dinitrobenzene are uniformly mixed in a solvent and reacted. After the reaction, the solvent is spin-dried to obtain a solid crude product, and a pyrene-based fluorescent probe for highly sensitive detection of hydrogen sulfide is obtained by column chromatography analysis.

[0011] More preferably, the molar ratio of 2-hydroxypyrene carboxaldehyde to 1-fluoro-2,4-dinitrobenzene is 1:0.5-1.5. The reaction is carried out at 15-35°C for 15-45 minutes. The silica gel used in the column chromatography analysis method is 200-300 mesh, and the eluent is: V 二氯甲烷 / V 石油醚 =5.5~6.5:1, and a pyrene-based fluorescent probe for highly sensitive detection of hydrogen sulfide was obtained.

[0012] Specific synthesis method: 2-hydroxypyrene carboxaldehyde and 1-fluoro-2,4-dinitrobenzene are stirred in DMF solvent and uniformly mixed at room temperature for 30 minutes. After the reaction, the DMF solvent is dried to obtain a yellow solid crude product, and the probe PYR-H2S is obtained by column chromatography; wherein, the molar ratio of 2-hydroxypyrene carboxaldehyde to 1-fluoro-2,4-dinitrobenzene is 1:1; the silica gel used in the column chromatography method is 200-300 mesh, and the eluent is: V 二氯甲烷 / V 石油醚 =6 / 1, a yellow product was obtained (yield 84%).

[0013] The reaction mechanism of the probe of the present invention is as follows:

[0014]

[0015] The mechanism of action of the fluorescent probe of the present invention is as follows: HS - The aldehyde group on PYR-H2S is attacked by a nucleophilic addition reaction, and the reaction may proceed through a six-membered transition state containing a thiol (-SH) moiety. The formation of this transition state is due to the special position of the aldehyde group in the ortho position of the pyrene ring skeleton and the participation of the thiol group. Through the above six-membered transition state, the pyrene ether bond is finally broken to form the corresponding product (PYR-H2S).

[0016] The pyrene-based fluorescent probe for detecting hydrogen sulfide is used in reagents for qualitative and quantitative detection of hydrogen sulfide.

[0017] The application specifically includes: dissolving the fluorescent probe PYR-H2S in DMSO solvent and configuring 1×10 - 3 mol / L probe stock solution. Sodium hydrosulfide was used as the hydrogen sulfide donor to prepare the NaHS stock solution (concentration of 1×10 - 2 mol / L)

[0018] The application specifically comprises: dissolving the fluorescent probe PYR-H2S mother solution in a DMSO aqueous solution to prepare a final concentration of the fluorescent probe PYR-H2S of 1×10 -5 mol / L solution, by adding different volumes of NaHS mother solution of the sample to be tested, after the reaction, the fluorescence change is observed at 340nm (fluorescence excitation) and 395nm (fluorescence emission) by fluorescence detection. The content of H2S is quantitatively calculated based on the linear correlation between the fluorescence intensity and the concentration of NaHS added to the sample to be tested. The PYR-H2S probe solution itself is colorless and does not fluoresce; after reacting with NaHS, the solution turns yellow and emits orange-yellow fluorescence, and the response time is 9min. Compared with the prior art, the present invention has the following advantages:

[0019] The probe molecule of the present invention has the characteristics of high sensitivity and short response time (9 minutes) for detecting hydrogen sulfide, and has good stability under a wide pH (2-9) condition; the PYR-H2S probe solution itself is colorless and does not emit fluorescence, but after reacting with sodium hydrosulfide, the solution turns yellow and emits orange-yellow fluorescence, showing a good colorimetric phenomenon, and can also quantitatively detect hydrogen sulfide in water samples, with a detection limit of 0.32 μM; at the same time, PYR-H2S can also quantitatively detect NaHS in water samples, and therefore has important application value in the fields of environmental monitoring.

[0020] The fluorescent probe of the present invention has the characteristics of simple synthesis route, rapid response and little interference from the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The hydrogen spectrum of the probe PYR-H2S in deuterated CDCl3 of the present invention, the abscissa is the chemical shift (ppm), and the ordinate is the intensity;

[0022] Figure 2 The carbon spectrum of the probe PYR-H2S in deuterated CDCl3 in the present invention, the abscissa is the chemical shift (ppm), and the ordinate is the intensity;

[0023] Figure 3For the mass spectrometer of the probe PYR-H2S in the invention, the ionization source is: ESI+;

[0024] Figure 4 The probe PYR-H2S (1×10 -5 mol / L), in 50% DMSO aqueous solution, at room temperature, compared with 12 common interfering compounds, and the UV selective spectrum recorded for 9 minutes;

[0025] Figure 5 The probe PYR-H2S (1×10 -5 mol / L), in 50% DMSO aqueous solution, at room temperature, respectively, compared with common interfering compounds, and the fluorescence spectra were recorded for 9 minutes; the excitation wavelength was 340nm, and the emission wavelength was 395nm;

[0026] Figure 6 The probe PYR-H2S (1×10 -5 mol / L), fluorescence titration spectra after reaction with different concentrations of NaHS in 50% DMSO aqueous solution at room temperature; excitation wavelength: 340nm, emission wavelength: 395nm;

[0027] Figure 7 The fluorescence intensity and linear relationship diagram of the probe PYR-H2S of the present invention after reacting with different concentrations of NaHS;

[0028] Figure 8 The mass spectrum of PYR-H2S-H2S in the present invention, the ionization source is: ESI-; DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with examples and drawings, but the present invention is not limited to the following embodiments.

[0031] Example 1: Synthesis of pyrene-based fluorescent probe PYR-H2S

[0032] In a 50mL round-bottom flask, 2-hydroxypyrene carboxaldehyde and 1-fluoro-2,4-dinitrobenzene were uniformly mixed in DMF solvent (reacted at room temperature 25°C for 30 minutes). After the reaction, the solvent DMF was dried to obtain a yellow solid crude product, and the probe PYR-H2S was obtained by column chromatography; wherein, the molar ratio of 2-hydroxypyrene carboxaldehyde to 1-fluoro-2,4-dinitrobenzene was 1:1; the silica gel used in the column chromatography method was 200-300 mesh, and the eluent was: V 二氯甲烷 / V 石油醚 =6 / 1.

[0033] After synthesis, 1 H NMR, 13C NMR and mass spectrometry were used to characterize the probe structure: Figure 1 As shown, the obtained probe structure NMR hydrogen spectrum analysis is: 1 H NMR (CDCl3, 400MHz): δ10.51(s,1H), δ9.01(d,,1H), δ8.72(s,1H), δ8.31(t,2H), δ8.18(m,6H), δ6.58(d,1H). Such as Figure 2 As shown, the obtained probe structure NMR carbon spectrum analysis is: 13 CNMR (CDCl3, 125MHz) d (ppm): 188.74, 157.12, 145.88, 141.65, 131.83, 131.70, 130.49, 129.96, 129.19, 129.07, 128.97, 128.49, 127.43, 126.94, 126.68, 126.40, 124.95, 124.05, 122.47, 119.79, 116.83, 77.36, 77.04, 76.72, 31.45, 30.20. Figure 3 As shown, the mass spectrum of the obtained probe structure is: [M+H]+: 413.0772. All of these can confirm the correctness of the obtained probe structure.

[0034] Example 2: Selectivity of the pyrene-based fluorescent probe PYR-H2S for different interfering substances

[0035] The PYR-H2S fluorescent probe in Example 1 was configured as 1×10 -3 mol / L mother solution.

[0036] The following substances: homocysteine, cysteine, glutathione, glutamic acid, citric acid, sodium bisulfate, sodium carbonate, sodium bicarbonate, sodium nitrite, sodium fluoride, potassium bromide, potassium iodide were prepared into 1×10 -2 mol / L mother solution.

[0037] Take 12 test tubes and add 30 μL of the above-mentioned different interfering substance solutions to 30 μL of the probe solution respectively; for the control, replace the interfering substance with an equal amount of water: add 1470 μL of ultrapure water; then add 1470 μL of DMSO solvent to dilute to 3 mL. The final concentration of the fluorescent probe PYR-H2S is 1×10 -5 After the mol / L solutions were shaken well, UV and fluorescence detection (Ex = 340 nm) was performed. Figure 4 It can be seen that only NaHS can significantly red-shift the maximum UV absorption peak of PYR-H2S, and other interfering substances do not have a strong red-shift on its maximum UV absorption, indicating that the PYR-H2S probe has a specific response to NaHS. Figure 5 ;Depend on Figure 5 It can be seen that only NaHS can significantly enhance the fluorescence of PYR-H2S, and other interfering substances have almost no enhancing effect on its fluorescence intensity, indicating that the PYR-H2S probe has a specific response to hydrogen sulfide.

[0038] Example 3: Fluorescence intensity of the fluorescent probe PYR-H2S at different NaHS concentrations.

[0039] The concentration is 1×10 -2 mol / L NaHS stock solution, the final NaHS concentration was controlled by changing the volume added to the test tube (0μL to 30μL), forming 0 to 10 equivalents of NaHS. After adding different volumes of NaHS to react with the probe, fluorescence detection was performed at an excitation wavelength (Ex = 340nm) to detect the fluorescence intensity. A curve was drawn with the fluorescence intensity at 395nm as the ordinate and the concentration of NaHS as the abscissa, and a linear relationship was drawn ( Figure 7 ),from Figure 6 As can be seen from the figure, with the increase of NaHS concentration, the fluorescence intensity of the probe is also increasing (Ex = 340nm detection limit:

[0040] 3.2×10 -7 mol / L).

[0041] Example 4: Detection of hydrogen sulfide in different water samples using pyrene-based fluorescent probe PYR-H2S

[0042] Four water samples were selected for hydrogen sulfide spike recovery experiments (including river water, tap water, beer, and wine). These water samples were used to prepare four NaHS mother solutions with concentrations of 0 / 20 / 40 / 60 μmol / L (NaHS was used as a hydrogen sulfide donor).

[0043] Add 30 μL of PYR-H2S mother solution to 4 test tubes, use 1470 μL of 4 water samples respectively, and then add 1470 μL of DMSO solvent to dilute to 3 mL. Perform fluorescence detection at Ex = 340 nm. Calculate the recovery rate and RSD (relative standard deviation) and draw Table 1. The recovery rates are all between 96.92% and 107.97%, and the RSDs are all less than 4.41. Table 1 shows the recovery rate of hydrogen sulfide in actual water samples using the probe PYR-H2S in the present invention.

[0044] Table 1: Hydrogen sulfide recovery data in actual water samples

[0045]

[0046] Example 5: Mechanism of detection of H2S by pyrene-based fluorescent probe PYR-H2S

[0047] HS on Hydrogen Sulfide - The aldehyde group on PYR-H2S is attacked by a nucleophilic addition reaction, and the reaction may proceed through a six-membered transition state containing a thiol (-SH) moiety. The formation of this transition state is due to the special position of the aldehyde group in the ortho position of the pyrene ring skeleton and the participation of the thiol group. Through the above six-membered transition state, the pyrene ether bond is finally broken to form the corresponding product (PYR-H2S-H2S). Figure 8 As shown, the mass spectrometry results of PYR-H2S-H2S have the same molecular formula as PYR-OH, which confirms the reaction mechanism.

Claims

1. A pyrene-based fluorescent probe for highly sensitive detection of hydrogen sulfide, characterized in that: It has the following structural formula:

2. The method for preparing the pyrene-based fluorescent probe for highly sensitive detection of hydrogen sulfide according to claim 1, characterized in that: The following steps are involved: 2-Hydroxypyrene formaldehyde and 1-fluoro-2,4-dinitrobenzene are uniformly mixed in a solvent and reacted. After the reaction, the solvent is spin-dried to obtain a solid crude product, and a pyrene-based fluorescent probe for highly sensitive detection of hydrogen sulfide is obtained by column chromatography analysis.

3. The method for preparing the pyrene-based fluorescent probe for highly sensitive detection of hydrogen sulfide according to claim 2, characterized in that: The molar ratio of the 2-hydroxypyrene formaldehyde to the 1-fluoro-2,4-dinitrobenzene is 1:0.5-1.

5.

4. The method for preparing the pyrene-based fluorescent probe for highly sensitive detection of hydrogen sulfide according to claim 2, characterized in that: React at 15-35°C for 15-45 minutes.

5. The method for preparing the pyrene-based fluorescent probe for highly sensitive detection of hydrogen sulfide according to claim 2, characterized in that: The silica gel used in the column chromatography analysis method is 200-300 mesh, and the eluent is: V 二氯甲烷 / V 石油醚 =5.5~6.5:1, and a pyrene-based fluorescent probe for highly sensitive detection of hydrogen sulfide was obtained.

6. The pyrene-based fluorescent probe for highly sensitive detection of hydrogen sulfide as claimed in claim 1 is useful for qualitative and quantitative detection of HS - Application in.

7. The use according to claim 6, characterized in that Specifically include: 1) dissolving the pyrene-based fluorescent probe PYR-H2S for highly sensitive detection of hydrogen sulfide in a solvent to prepare a probe solution, and using sodium hydrosulfide as a hydrogen sulfide donor to prepare a NaHS mother solution; 2) Adding different volumes of NaHS mother solution to the probe solution, after the reaction, using fluorescence detection at 340 nm for fluorescence excitation and 395 nm for fluorescence emission to observe the fluorescence change, and establishing a linear correlation standard curve between fluorescence intensity and added NaHS concentration; 3) Under 340nm fluorescence excitation, measure the fluorescence intensity of the sample solution at 395nm fluorescence emission to obtain the fluorescence intensity of the sample to be tested; 4) Compare the fluorescence intensity of the sample to be tested with the fluorescence intensity of step 1) and the linear correlation standard curve of the added NaHS concentration to quantitatively calculate the HS - The content.

8. The use according to claim 7, characterized in that The concentration of the probe solution is 0.5 to 1.5×10 - 5 mol / L.

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