Fluorescent probe for detecting hydrogen sulfide as well as preparation method and bioimaging application thereof

Abstract

The preparation method of the fluorescent probe (S) comprises the following steps: adding absolute ethyl alcohol into p-phenylenediamine, refluxing and dissolving the solution (solution A) while heating at 80 DEG C, dissolving salicylaldehyde into a proper amount of absolute ethyl alcohol (solution B), dropwise adding the solution B into the solution A, and stirring to obtain a mixed solution; heating reflux is kept at 80 DEG C in the whole process, stirring is continuously carried out, and after reflux is carried out for 2 hours, filtering is carried out to obtain a faint yellow solid, namely the fluorescent probe (S). The fluorescent probe can specifically recognize hydrogen sulfide (H2S) under the interference of common ions, is strong in anti-interference capability and high in sensitivity, and can be used for real-time monitoring and fluorescence imaging of endogenous H2S of cells and mouse tumors.

Description

Technical Field

[0001] This invention relates to the field of bioanalytical detection technology, and more specifically, to a fluorescent probe for detecting hydrogen sulfide, a method for preparing the fluorescent probe, and the application of the fluorescent probe in bioimaging. Background Technology

[0002] Hydrogen sulfide (H2S) has traditionally been considered a toxic gas with a foul odor, but recent studies have confirmed it as a third endogenous gaseous neurotransmitter, playing a crucial regulatory role in physiological processes and pathological mechanisms in organisms (Zhang Peng, et al. Journal of Cardiovascular Pharmacology, 2021, 2). In mammals, H2S can be endogenously synthesized by three enzymes: cystathionine β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate thiotransferase (3-MST). Recent studies have shown that abnormal changes in H2S concentration in organisms are closely related to the occurrence and development of various diseases, including but not limited to neurodegenerative diseases, atherosclerosis, diabetes, and various cancers (Zhou Guijuan, et al. Science in China: Life Sciences, 2023, 10). Based on this, establishing a transient H2S detection method with both high specificity and high sensitivity to achieve accurate and rapid detection of H2S in biological samples has significant theoretical research value and practical application significance. Summary of the Invention

[0003] In view of the deficiencies of existing technologies, this invention provides a symmetrical Schiff base fluorescent probe, its preparation method, and its application. This fluorescent probe can detect H2S more efficiently and sensitively. This invention uses p-phenylenediamine as a raw material, which undergoes a condensation reaction with salicylaldehyde, attaching salicylaldehyde structures to both ends of p-phenylenediamine to form a symmetrical Schiff base fluorescent probe (hereinafter referred to as probe S). This probe can be used for the detection of exogenous H2S, and its structural formula is as follows:

[0004] The method for synthesizing the fluorescent probe (S) described in this invention specifically includes the following steps: p-phenylenediamine was placed in a round-bottom flask, anhydrous ethanol solution was added, and the mixture was heated under reflux at 80°C to obtain solution A. Prepare the appropriate amount of salicylaldehyde in a beaker, add an appropriate amount of anhydrous ethanol to dissolve the salicylaldehyde into a homogeneous solution, which is solution B. Add solution B dropwise to solution A, and keep the temperature at 80°C under reflux with continuous stirring. After refluxing for 2-4 hours, a pale yellow crude precipitate is obtained. The crude pale yellow precipitate was filtered under reduced pressure, and the filter cake was washed in anhydrous methanol and then filtered under reduced pressure again. This process was repeated three times to obtain a pure pale yellow solid, which is the symmetrical Schiff base fluorescent probe (S).

[0005] This invention provides a method for detecting endogenous or exogenous hydrogen sulfide using a fluorescent probe (S), the specific steps of which are as follows: The fluorescent probe (S) is prepared into a solution using DMF or DMSO, with a concentration of 5.0 × 10⁻⁶. -5 mol / L, as solution C; H₂S was prepared in deionized water to a concentration of 2.0 × 10⁻⁶ mol / L. -2 A solution containing mol / L was used as solution D. Solution D was mixed with a solution containing C in a certain proportion, and the mixture was detected by fluorescence spectroscopy to compare whether the original solution showed fluorescence.

[0006] The preparation method of the symmetrical Schiff base fluorescent probe (S) described in this invention is simple, the raw material cost is low, it can be synthesized in one step, the purity can reach 99%, it can produce fluorescence effect for specific recognition of H2S, and it is more selective, more sensitive and has stronger anti-interference ability than ordinary unilateral fluorescent probes.

[0007] In terms of applications, the symmetrical Schiff base fluorescent probe (S) described in this invention can be widely used in the bioimaging detection of H2S, providing a novel technical solution for the rapid and accurate detection of H2S. Attached Figure Description

[0008] Figure 1 This is a curve showing the change in fluorescence value of the fluorescent probe (S) with H2S concentration; Figure 2 This is a standard curve of the fluorescence value of the fluorescent probe (S) changing with the concentration of H2S; Figure 3 This is the fluorescence emission spectrum of the fluorescent probe (S) selectively recognizing H2S in cations; Figure 4 This is the fluorescence emission spectrum of the fluorescent probe (S) selectively recognizing H2S in anions; Figure 5 This is the fluorescence emission spectrum of the fluorescent probe (S) selectively recognizing H2S in amino acids and bioactive molecules; Figure 6 This is fluorescence imaging of the fluorescent probe (S) in HeLa cells; Figure 7 This is real-time fluorescence imaging of a fluorescent probe (S) in mouse tumors (every 10 minutes). Detailed Implementation Plan

[0009] The present invention will now be analyzed in more detail with reference to the accompanying drawings and embodiments. Example 1

[0010] This invention provides a symmetrical Schiff base fluorescent probe (S), the structural formula of which is as follows:

[0011] p-Phenylenediamine (0.108 g, 1 mmol) was dissolved in 10 mL of anhydrous ethanol by heating. 2 mL of salicylaldehyde (0.244 g, 2 mmol) solution was added dropwise at 80 °C. The entire process was carried out under magnetic stirring and refluxed for 2 hours. The reaction progress was monitored by TLC until the reaction was complete. The reaction solution was then left overnight, which produced a large amount of pale yellow solid. The reaction solution was filtered under reduced pressure, and the filter cake was taken out and washed with anhydrous methanol. The above operation was repeated 3 times to obtain a pure pale yellow solid, which was the fluorescent probe (S) with a yield of 96%. 1 H NMR (600 MHz, DMSO -d 6 ): δ 9.04 (s, 2H), 7.68 -7.67 (m, 2H), 7.55 (s, 4H), 7.45 -7.42 (m, 2H), 7.01- 6.97 (m, 4H). 13 C NMR (151 MHz, DMSO) -d 6 ): δ 163.2, 160.3, 146.7, 133.4, 132.6, 122.6, 119.2, 116.6. MS(ESI): m / z 317.18 [M + H] + The reaction formula is as follows: Example 2

[0012] The symmetrical Schiff base fluorescent probe (S) described in this invention can specifically recognize H2S and produce a fluorescence effect, combined with Figure 1 As shown, the detection method involves preparing a solution of the fluorescent probe described above using DMF at a concentration of 5.0 × 10⁻⁶. -5 H₂S was prepared into a solution with a concentration of 5.0 × 10⁻⁶ mol / L using deionized water. -3 mol / L; using fluorescence spectroscopy, 2 mL of probe solution was placed in a glass cuvette, and 10 μL of quantitative H2S solution was added each time, for a total of 20 detections. The results showed that the fluorescence value of the fluorescent probe increased with the increase of H2S concentration. Example 3

[0013] The standard concentration curves of the probe (S) and H2S were plotted using fluorescence spectroscopy. Figure 2As shown, its minimum detection limit is 0.018 μM. Example 4

[0014] The fluorescent probe (S) described in this invention can specifically recognize H2S and produce a fluorescence effect, combined with Figure 3 As shown, the detection method involves preparing a solution of the fluorescent probe (S) described above using DMF, with a concentration of 5.0 × 10⁻⁶. -5 mol / L; various metal cations were prepared into solutions using deionized water (all metal cations were prepared from their corresponding chloride hydrates), namely Na + , K + Ni 2+ Cd 2+ Co 2+ Ca 2+ Mg 2+ ,Mn 2+ Cu 2+ , Cr 3+ The concentration was 2.0 × 10⁻⁶. -2 H₂S was prepared into a solution with a concentration of 2.0 × 10⁻⁶ mol / L using deionized water. -2 mol / L; using fluorescence spectroscopy, 2 mL of probe (S) solution was placed in a glass cuvette, and 20 equivalents of metal cations and H2S were added respectively. The results showed that under common cation interference, the fluorescent probe (S) could specifically recognize H2S without interference from cations. Example 5

[0015] The symmetrical Schiff base fluorescent probe (S) described in this invention can specifically recognize H2S and produce a fluorescence effect, combined with Figure 4 As shown, the detection method involves preparing a solution of the fluorescent probe (S) described above using DMF, with a concentration of 5.0 × 10⁻⁶. - 5 mol / L; solutions of various anions were prepared using deionized water (all anions were prepared from their corresponding sodium salt hydrates), namely (Cl... - ,Br - , I - H2SO4 - C2H2ClO2 - NO2 – NO3 – HSO4 - HSO3 - S2O3 2- The concentration was 2.0 × 10⁻⁶. - 2 H₂S was prepared into a solution with a concentration of 2.0 × 10⁻⁶ mol / L using deionized water. -2mol / L; Using fluorescence spectroscopy, 2 mL of the probe solution was taken in a glass cuvette, and 20 equivalents of anions and H2S were added respectively. The results showed that the fluorescent probe (S) could specifically recognize H2S without being interfered by the common anions. Example 6

[0016] The fluorescent probe (S) described in the present invention can specifically recognize H2S to produce a fluorescence effect. Combining Figure 5 as shown, the detection method is that the above-mentioned fluorescent probe (S) is formulated into a solution with DMF, and the concentration is 5.0×10 -5 mol / L; Various amino acids are formulated into solutions with deionized water, namely fumaric acid, lactic acid, citric acid, pyruvic acid, D-glucose, γ-aminobutyric acid, glycine, D-glutamic acid, D-alanine, and the concentration is 2.0×10 -2 mol / L; H2S is formulated into a solution with deionized water, and the concentration is 2.0×10 -2 mol / L; Using fluorescence spectroscopy, 2 mL of the probe solution was taken in a glass cuvette, and 20 equivalents of amino acids and H2S were added respectively. The results showed that the fluorescent probe could specifically recognize H2S without being interfered by the common amino acids. Example 7

[0017] Hela cells in the logarithmic growth phase with 80% fusion were treated with 10 μM of the probe (S) for 2 h, and the cells were rinsed three times with PBS solution to remove the excess probe, and then observed and photographed with a laser confocal microscope. Combining Figure 6 as shown, the probe (S) can recognize hydrogen sulfide in Hela cells and emit yellow-green fluorescence, while the blank group of Hela cells has no fluorescence. Example 8

[0018] The probe S (0.1 M) was injected onto the surface of mouse melanoma, and in vivo imaging was performed every 10 minutes. λex = 488 nm, λem = 520 nm. Combining Figure 7 as shown, it shows that (S) is applicable to real-time fluorescence imaging of mouse tumors.

Claims

1. A fluorescent probe for detecting hydrogen sulfide, its preparation method, and its application in bioimaging, relating to the application of this probe in the specific recognition of hydrogen sulfide molecules. The fluorescent probe has the following structural formula: 。 2. The application as described in claim 1, characterized in that, It can selectively recognize hydrogen sulfide in cationic aqueous solutions.

3. The application as described in claim 1, characterized in that, It can selectively recognize hydrogen sulfide in anionic aqueous solutions.

4. The application as described in claim 1, characterized in that, It can selectively recognize hydrogen sulfide in amino acid solutions.

5. The application as described in claim 1, characterized in that, It can selectively recognize hydrogen sulfide in bioactive molecules.

6. The application as described in claim 1, characterized in that, It can be used for fluorescence imaging of endogenous hydrogen sulfide in cancer cells.

7. The application as described in claim 1, characterized in that, It can be used for real-time fluorescence imaging of mouse tumors.

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