A novel dual-functional AIE fluorescent probe and its application in viscosity and ONOO - Application in detection

Abstract

The present invention discloses a novel dual-function AIE fluorescent probe and its application in viscosity and ONOO ‑ The bifunctional AIE fluorescent probe of the present invention can be used to detect ONOO by fluorescence spectroscopy. ‑ Identification and quantitative detection of ions and viscosity. ‑ The detection has excellent sensitivity, low detection limit (LOD = 6.64nM), strong anti-interference and high selectivity, and can respond to ONOO in a wider pH range (4-10). ‑ Furthermore, the probe can be used for intracellular imaging and can detect ONOO in two channels. ‑ and viscosity, can effectively eliminate the signal crosstalk during the detection process, in the detection of trace viscosity and ONOO ‑ It shows great potential in various aspects, demonstrating its potential application value in molecular biology.

Description

Technical Field

[0001] The present invention belongs to the technical field of ion detection, and specifically relates to a novel dual-function AIE fluorescent probe and its application in viscosity and ONOO - Application in detection. Background Art

[0002] Peroxynitrite (ONOO - ), is an endogenous reactive oxygen species (ROS) in biological systems, generated by the diffusion-controlled reaction of nitric oxide and superoxide anion radicals. ONOO - It plays a vital role in physiological and pathological processes and participates in biological reactions such as immunogenicity, cell signal transduction and cell apoptosis. Many physiological diseases are related to ONOO. - Abnormal accumulation of ONOO is associated with various diseases, such as inflammation, cardiovascular disease, neurodegenerative disease, stroke and cancer. Under physiological conditions, peroxynitrite is an extremely active substance with a very short half-life (20ms), so its steady-state concentration is very low (nanomolar level). This leads to ONOO - Since ONOO is difficult to measure in biological systems, a sensitive and reliable method is needed to detect ONOO. - Concentration for ONOO - The diagnosis and prevention of related diseases are of great significance.

[0003] Intracellular viscosity plays a key role in promoting the interaction between intracellular biomolecules and the transmission of chemical signals, as well as promoting the diffusion of metabolites in living cells. Abnormal viscosity can reflect the state of functional impairment and is closely related to many diseases, such as neurodegenerative diseases, diabetes, and cell malignancies. In particular, in vivo, due to the diffusion-limited reaction between the free radicals nitric oxide and superoxide radicals, changes in viscosity inhibit the endogenous ONOO - Therefore, it is crucial to develop specific and sensitive probes to detect intracellular viscosity in biological systems.

[0004] Organic small molecule fluorescent probe detection is a good detection method due to its advantages of simple operation, high sensitivity, direct observation, good selectivity and rapid detection. Although many fluorescent probes with different receptors have been successfully developed for ONOO - However, there are still some disadvantages such as small Stokes shift, relatively low sensitivity, interference from other reactive oxygen species, difficulty in preparation, and low yield. Therefore, in order to avoid the above defects, we are eager to develop a method for detecting ONOO -A new type of fluorescent probe. In contrast to common ACQ fluorescent dyes, aggregation-induced emission (AIE) fluorescent dyes have a rotatable structure or a twisted helical conformation, which has high emission due to restricted intramolecular motion. AIE fluorescent dyes usually have good photostability and large aggregates or solid-state Stokes shift, which can effectively avoid photobleaching problems and the influence of excitation light. Therefore, AIE fluorophores are very useful in detecting viscosity and ONOO in biomolecules. - It has good application prospects. Summary of the Invention

[0005] The present invention aims to provide a novel dual-functional AIE fluorescent probe and its application in viscosity and ONOO - The technical problem to be solved is to synthesize molecules that can monitor viscosity changes and identify ONOO - A new dual-functional AIE fluorescent probe.

[0006] The present invention is a novel dual-functional AIE fluorescent probe, the chemical formula of which is C 35 H 25 N3OS, the structural formula is as follows:

[0007]

[0008] The present invention's new dual-functional AIE fluorescent probe C 35 H 25 The preparation method of N3OS comprises the following steps:

[0009] With anhydrous ethanol as solvent, MA and benzothiazole-2-acetonitrile were added, and two drops of piperidine were added dropwise. The reaction was heated under reflux for 3 h. After cooling, a red solid was precipitated, which was washed three times with ethanol and dried to obtain the target product MAR.

[0010] The synthetic route is as follows:

[0011]

[0012] The new dual-function AIE fluorescent probe of the present invention has the following characteristics: viscosity and ONOO - Application in detection.

[0013] The novel dual-functional AIE fluorescent probe is used to prepare detection reagents to detect viscosity changes and ONOO - Concentration detection.

[0014] Furthermore, the detection system is an aqueous medium, and fluorescence spectrum measurement is performed in the aqueous medium to detect ONOO by changes in fluorescence intensity. - and / or viscosity detection, and has strong anti-interference ability for multiple analytes.

[0015] The aqueous medium is a mixed solution of DMSO and water in a volume ratio of 4:1.

[0016] The bifunctional AIE novel fluorescent probe is used as a detection reagent for detecting ONOO - The detection limit is 6.64nM.

[0017] The bifunctional novel AIE fluorescent probe can realize the identification and quantitative detection of ONOO - Ion and / or viscosity by fluorescence spectroscopy, and can be used for preparing ONOO - Concentration and / or viscosity detection reagent, and has good anti-interference, high selectivity and sensitivity in the presence of other analytes; meanwhile, the probe can be used for intracellular imaging, and can detect ONOO - And viscosity in two channels, can effectively eliminate signal crosstalk in the detection process, and shows great potential in detecting trace viscosity and ONOO - , and shows its potential application value in molecular biology.

[0018] The beneficial effects of the present application are embodied in:

[0019] The bifunctional AIE novel fluorescent probe has multifunctionality, and can realize the identification and detection of viscosity and ONOO - Ion by fluorescence spectroscopy. The bifunctional AIE novel fluorescent probe can be used for quantitative detection of ONOO - Ion in an aqueous medium, and the detection limit is extremely low (6.64nM), indicating that the probe is quite sensitive to ONOO - Detection.

[0020] The bifunctional AIE novel fluorescent probe has high selectivity and good anti-interference ability for ONOO - Recognition, can respond to ONOO - In a wider pH range (4-10), and accompanied by obvious color change to realize naked eye recognition.

[0021] The bifunctional AIE novel fluorescent probe has good response to viscosity and ONOO - In biological imaging experiments, and the fluorescence wavelengths of the two channels are quite different, avoiding signal crosstalk, and realizing double-channel fluorescence detection. The above experimental results show that the probe has good application potential in environmental monitoring and biological bodies. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The nuclear magnetic resonance hydrogen spectrum of the bifunctional AIE novel fluorescent probe.

[0023] Figure 2 Figure a shows the fluorescence spectra of the new bifunctional AIE probe in different ratios of DMSO and water detection systems; Figure b shows the fluorescence intensity F of the probe 605nm Figure a shows the change with water content; Figure c shows the particle size distribution of the probe in a mixed solution of DMSO and water with a volume ratio of 4:1; Figure d shows the SEM image of the probe.

[0024] Figure 3 These are photos of the novel dual-functional AIE probe of the present invention under irradiation with 365nm ultraviolet light in detection systems with different ratios of DMSO and water.

[0025] Figure 4 Figure a shows the new dual-functional AIE fluorescent probe of the present invention at different viscosities (f gly = 0-100%) in methanol-glycerol solution; Figure b shows the fluorescence intensity F of the probe 600nm Follow f gly 's change graph.

[0026] Figure 5 Figure a is the UV-visible absorption spectrum of the novel bifunctional AIE fluorescent probe of the present invention when different analytes are added to a mixed solution of DMSO and water with a volume ratio of 4:1; Figure b is the fluorescence emission spectrum of the probe when different analytes are added to a mixed solution of DMSO and water with a volume ratio of 4:1 (λex = 323 nm).

[0027] Figure 6 Figure a shows the new dual-functional AIE fluorescent probe of the present invention in a mixed solution of DMSO and water with a volume ratio of 4:1, with the addition of 0-1.5 equiv of ONOO - Figure b shows the fluorescence intensity of the fluorescent probe at a fluorescence emission wavelength of 408 nm and the fluorescence emission spectrum of ONOO - Linear relationship with concentration.

[0028] Figure 7 This is a bar graph showing the anti-interference properties of the novel dual-functional AIE fluorescent probe of the present invention when different analytes are added to a mixed solution of DMSO and water with a volume ratio of 4:1; The fluorescence intensity of the representative fluorescent probe when different analytes were added to a mixed solution of DMSO and water with a volume ratio of 4:1 (λex = 323 nm); Representative fluorescent probe compounds were added to a mixed solution of DMSO and water with a volume ratio of 4:1 and different analytes and ONOO - Fluorescence intensity of ions (λex = 323 nm).

[0029] Figure 8This is the fluorescence emission intensity spectrum of the novel bifunctional AIE fluorescent probe of the present invention under different pH conditions in a mixed solution of DMSO and water with a volume ratio of 4:1.

[0030] Figure 9 This is an image of the novel dual-functional AIE fluorescent probe of the present invention being used to detect viscosity changes in Hela cells.

[0031] Figure 10 The novel dual-functional AIE fluorescent probe of the present invention is used for ONOO in Hela cells - Fluorescence imaging of the detection. DETAILED DESCRIPTION

[0032] The present invention can be further illustrated by the following examples, but is not limited to the examples.

[0033] Example 1: Preparation and characterization of a novel bifunctional AIE fluorescent probe

[0034] MA (0.2 g, 0.53 mmol) and benzothiazole-2-acetonitrile (101.88 mg, 0.63 mmol) were dissolved in 10 mL of ethanol solution, and two drops of piperidine were added dropwise. The reaction was heated under reflux for 3 h. After cooling, a red solid precipitated, which was washed three times with ethanol and dried to obtain 207 mg of the target product MAR with a yield of 73%.

[0035] 1 H NMR (400MHz, DMSO-d6) δ8.21(s,1H),8.11(d,J=7.2Hz,1H),8.01–7.96(m,3H),7.64(d,J=8.6Hz,2H),7.59(d,J=8.8Hz ,2H),7.53(s,1H),7.41(d,J=8.0Hz,3H),7.24–7.20(m,5H),6.99(d,J=8.9Hz,2H),6.95(d,J=9.0Hz,2H),3.76(s,3H).

[0036] Example 2: AIE properties of a novel dual-functional AIE fluorescent probe

[0037] Accurately weigh a certain amount of the new dual-functional AIE fluorescent probe, use DMSO to prepare a probe stock solution with a concentration of 1 mM, and pipette 20 μL into 2.0 mL of a mixture of DMSO and water in different proportions, such as Figure 2 Figures 2a and 2b show that the MAR probe emits extremely weak and negligible fluorescence at low water content. As the water content increases from 30% to 100%, the fluorescence intensity at 605 nm increases significantly, reaching a maximum at 60% water content. These results demonstrate that the fluorescent probe exhibits typical AIE properties. Figure 3 The fluorescence probe solution in the sample tube with a water content of 40% showed orange-red light under a 365nm UV lamp. Dynamic light scattering (DLS) measurements showed that the average size of the probe particles in the solution was about 142nm ( Figure 2 c), SEM images show that the particle size is about 130nm ( Figure 2 d). Scanning electron microscopy and DLS revealed the conformation of the nanoaggregates.

[0038] Example 3: Fluorescence response of the novel bifunctional AIE fluorescent probe to viscosity

[0039] Accurately weigh a certain amount of the new dual-functional AIE fluorescent probe and prepare a 1mM probe stock solution with dimethyl sulfoxide. Pipette 20μL of the solution into 2.0mL of methanol-glycerol mixtures with different viscosities. After mixing, sonication, and standing, the solution is added to a fluorescence cuvette and the fluorescence spectrum of the probe is measured at an excitation wavelength of 468nm. The fluorescence spectra of the probe in methanol-glycerol mixtures with different viscosities are shown in Figure 2. Figure 4 Under 468nm excitation, weak fluorescence emission at 555nm was obtained in the methanol solution medium. As the proportion of glycerol increased, the solvent viscosity gradually increased, and the fluorescence emission of the probe increased accordingly. When the volume fraction of glycerol reached 100%, the fluorescence intensity reached its maximum.

[0040] Example 4: Bifunctional AIE novel fluorescent probe for ONOO - Specific response

[0041] Accurately weigh a certain amount of the new dual-functional AIE fluorescent probe, dissolve it and prepare it to a concentration of 1.0×10 -3 mol / L DMSO stock solution; dilute the stock solution to a concentration of 1.0×10 -5 mol / L of the test solution. Take 2mL of a solution with a concentration of 1.0×10 -5 mol / L of the test solution in a quartz cuvette (the thickness of the quartz cuvette is 1 cm), and then 10 μL of a 1.0×10 -2 mol / L of various analytes (Blank, ONOO - , ClO - ,H2O2,Hcy,Cys,GSH,CO3 2- ,HCO3 - ,HPO4 2- , H2PO4 - , NO 2- , SO3 2- , HSO3 - , ONOO - , Cl- , Br - , I - , K + , Fe 2+ , Fe 3+ ) aqueous solution, shake well and measure the UV-visible absorption spectrum of the solution. Figure 5 As shown in a, the UV-visible absorption spectrum of the probe solution has an obvious absorption peak at 463 nm. The addition of other analytes only causes a certain degree of absorption peak reduction, but the addition of ONOO - After the absorption peak of long wavelength almost disappeared, a new absorption peak appeared at 352 nm, indicating that under the conditions of UV-visible absorption spectrum, the probe has a strong affinity for ONOO - Ions have obvious recognition effect. Add ONOO - The color of the probe solution of ions changed from dark yellow to colorless and was visible to the naked eye, while the color of the probe solution added with other analytes did not change significantly.

[0042] Accurately weigh a certain amount of the new dual-functional AIE fluorescent probe, dissolve it and prepare it to a concentration of 1.0×10 -3 mol / L DMSO stock solution; dilute the stock solution to a concentration of 1.0×10 -5 mol / L of the test solution. Take 2mL of a solution with a concentration of 1.0×10 -5 mol / L of the test solution in a quartz cuvette (the thickness of the quartz cuvette is 1 cm), and then 10 μL of a 1.0×10 -2 mol / L of various analytes (Blank, ONOO - , ClO - ,H2O2,Hcy,Cys,GSH,CO3 2- ,HCO3 - ,HPO4 2- , H2PO4 - , NO 2- , SO3 2- , HSO3 - , ONOO - , Cl - , Br - , I - , K + , Fe 2+ , Fe 3+ ) aqueous solution, shake well and measure the fluorescence spectrum of the solution. Figure 5 As shown in b, it can be seen that this probe can specifically recognize ONOO - , and has almost no response to other ions. The results show that the fluorescent probe is sensitive to ONOO -Highly selective.

[0043] Example 5: Fluorescence intensity of the new dual-functional AIE fluorescent probe and ONOO - Correlation of concentration

[0044] Weigh the target product and dissolve it in DMSO to prepare a 10μM probe solution. Add the probe solution to each test tube and take ONOO - (0-1.5 equiv) was dripped into the test tube above. The fluorescence spectra of these test solutions were measured at 408 nm (e.g. Figure 6 As shown in Figure 2, the fluorescence intensity of the probe gradually increased with the increase of solution concentration. The fluorescence probe (10 μM) and ONOO - (0-1.5equiv) has a good linear relationship R 2 =0.991, which shows that the fluorescent probe can quantitatively detect ONOO in the fluorescence spectrum. - According to LOD = 3σ / k, the fluorescence probe is calculated to be ONOO in the fluorescence spectrum. - The detection limit is 6.64nM. Where σ is the standard deviation of blank measurements, k is the fluorescence intensity of ONOO - The slope of the concentration graph.

[0045] Example 6: Dual-responsive fluorescent probe for ONOO - Anti-interference of ion recognition

[0046] Weigh the target product and dissolve it in DMSO to prepare a 10 μM probe solution. Add the probe solution and 1 equivalent of each analyte (Blank, ONOO) to each test tube. - , ClO - ,H2O2,Hcy,Cys,GSH,CO3 2- ,HCO3 - ,HPO4 2- , H2PO4 - , NO 2- , SO3 2- , HSO3 - , ONOO - , Cl - , Br - , I - , K + , Fe 2+ , Fe 3+ ) solution, shake well, and measure its fluorescence emission spectrum at an excitation wavelength of λ = 396 nm. The results show that there is almost no effect on the fluorescence intensity of the fluorescent probe (such as Figure 7 ), which indicates that the fluorescent probe is sensitive to ONOO -Ion recognition has good anti-interference ability. Under 365nm ultraviolet light, the probe solution emits blue light, while the addition of other analytes to the probe solution does not change significantly.

[0047] Example 7: The novel dual-functional AIE fluorescent probe of the present invention responds to ONOO at different pH values - Impact

[0048] Weigh the target product and dissolve it in DMSO to prepare a 10μM probe solution. Add the probe solution and 1 equivalent of ONOO into the test tube. - solution, such as Figure 8 As shown in Figure 2, the fluorescent probe is very stable at pH 4.0–10.0 and has no effect on ONOO at pH 4.0–10.0. - Has good fluorescence response. Therefore, the fluorescent probe can respond to ONOO in a wider pH range. - and can be used as a turn-on fluorescence sensor to detect ONOO under physiological conditions. - .

[0049] Example 8: Detection of viscosity changes in Hela cells using a novel dual-function AIE fluorescent probe

[0050] After the well-grown Hela cells were treated with the fluorescent probe (10 μM) for 30 minutes, the cell imaging test was performed using a laser confocal microscope; after adding nystatin (20 μM) to the culture dish and culturing for 20 minutes, the fluorescent probe (10 μM) was added and incubated for 30 minutes, and the cell imaging test was performed. Figure 9 As shown, when HeLa cells were incubated with only the probe, the red channel showed no fluorescence. However, after pre-treating the HeLa cells with nystatin and then incubating them with the fluorescent probe, the red channel showed significant fluorescence. This fully demonstrates that the fluorescent probe can detect intracellular viscosity.

[0051] Example 9: Bifunctional AIE novel fluorescent probe for ONOO in Hela cells - Imaging applications

[0052] After the well-grown Hela cells were treated with the fluorescent probe (10 μM) for 30 min, the cells were imaged using a laser confocal microscope. - After culturing with ions (10 μM) for 10 min, cell imaging test was performed. Figure 10 As shown in the figure, when only fluorescent probe was added to Hela cells for incubation, there was no fluorescence in the blue channel. -After incubation again, it can be observed that the blue channel shows strong fluorescence. These results indicate that the fluorescent probe can be visualized by fluorescence imaging ONOO - .

Claims

1. A dual-function AIE fluorescent probe, characterized by: The chemical formula of the bifunctional AIE fluorescent probe is C 35 H 25 N3OS, the structural formula is as follows: 。 2. The method for preparing the bifunctional AIE fluorescent probe according to claim 1, comprising the following steps: With anhydrous ethanol as solvent, MA and benzothiazole-2-acetonitrile were added, piperidine was added dropwise, and the mixture was heated under reflux for 3 h. After cooling, a red solid was precipitated, which was washed with ethanol and dried to obtain the target product MAR. The synthetic route is as follows: 。 3. The dual-function AIE fluorescent probe according to claim 1 in terms of viscosity and / or ONOO - Application in detection, wherein the application is an application for non-disease diagnosis purposes.

4. The use according to claim 3, characterized in that: The detection system is an aqueous medium, in which fluorescence spectrum measurement is carried out, and ONOO is detected by the change of fluorescence intensity. - and / or viscosity testing.

5. The use according to claim 4, characterized in that: The aqueous medium is a mixed solution of DMSO and water in a volume ratio of 4:

1.

6. The use according to claim 5, characterized in that: The dual-functional AIE fluorescent probe is used as a detection reagent for ONOO - When ion detection was used, the detection limit was 6.64 nM.

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