Application of benzothiazole fluorescent compound in simultaneous observation of viscosity and pH value

Abstract

The invention discloses an application of a benzothiazole fluorescent compound in simultaneous observation of viscosity and pH value. The multipurpose fluorescent compound provided by the invention can be used for simultaneously detecting the viscosity and pH value change in a solution and cells. When the fluorescent probe is used as the fluorescent probe, compared with the existing single detection probe, one probe can be used for replacing two probes to solve the same problem, so that the cost can be reduced, and the detection operation is simpler and more convenient.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and in particular relates to the application of a benzothiazole fluorescent compound in the simultaneous observation of viscosity and pH value. Background Art

[0002] pH value is an important indicator in the field of chemical industry production. It reflects the physical quantity of the acidity and alkalinity of the solution and reflects the hydrogen ions (H + ) concentration. Measuring the pH of a solution can determine its chemical properties and better control and regulate chemical reactions. pH also reflects changes in the intracellular microenvironment and is closely related to biomolecular interactions, metabolite diffusion, and signal transduction. Therefore, pH is widely used in environmental protection, the food industry, biomedicine, and other fields.

[0003] Viscosity is also a crucial indicator in the chemical industry, reflecting the physical quantity of internal friction during fluid flow. Viscosity can also reflect changes in the intracellular microenvironment, being inextricably linked to biomolecular interactions, metabolite diffusion, and signal transduction. Therefore, monitoring changes in viscosity and pH is of great significance to chemistry and biology.

[0004] Currently, there are many methods for measuring viscosity and pH, including electrochemical methods, pH meters, pH test paper, capillary electrophoresis, high-performance liquid chromatography, and spectrophotometry. However, these methods require complex procedures and damage to the sample. Therefore, it is difficult to achieve non-invasive, highly sensitive, and real-time detection of pH and viscosity changes in vivo.

[0005] Organic fluorescent probes based on fluorescence imaging have become a powerful technology that can visualize viscosity and pH changes in situ, non-destructively, and independently. Currently, researchers have designed a variety of probes for detecting viscosity and pH, but there are few multi-purpose fluorescent probes that can simultaneously monitor changes in both viscosity and pH.

[0006] Therefore, it is of great significance to develop a multipurpose fluorescent probe that can simultaneously observe viscosity and pH value. Summary of the Invention

[0007] Purpose of the invention: The purpose of the present invention is to provide an application of a benzothiazole fluorescent compound in the simultaneous observation of viscosity and pH value.

[0008] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0009] The present invention provides an application of a benzothiazole fluorescent compound in viscosity detection for non-disease diagnosis and treatment purposes. The structural formula of the compound is:

[0010]

[0011] The fluorescent compound consists of two parts: thiazole salt and N, N-diethylbenzene.

[0012] The present invention also provides the use of the above-mentioned benzothiazole fluorescent compound in the simultaneous observation of viscosity and pH value for purposes other than disease diagnosis and treatment.

[0013] The fluorescent compound of the present invention can be used to observe changes in viscosity and pH value at the same time, and can be used as a multi-purpose fluorescent probe.

[0014] After the fluorescent compound stains the cells, changes in intracellular viscosity and pH value can be observed simultaneously.

[0015] Furthermore, the fluorescent compound can stain cells without washing the cells to observe changes in intracellular viscosity and pH.

[0016] In order to balance the toxicity of the probe to cells and the staining effect, the concentration of the fluorescent compound for staining cells is 1-15 μM.

[0017] The fluorescent compound of the present invention can also simultaneously detect changes in viscosity and pH value in a solution.

[0018] In order to balance the cost and the staining effect, the concentration of the fluorescent compound in the solution is 1-15 μM.

[0019] Furthermore, the concentration of the fluorescent compound is 10 μM.

[0020] The preparation method of the fluorescent compound is:

[0021] 2-Methylbenzothiazole reacts with iodoethanol to obtain compound 2; compound 2 and compound 3 are reacted with Knoevenagel to synthesize the fluorescent compound. The reaction scheme is:

[0022]

[0023] Since the pH and viscosity in cells are related to various diseases (cancer, etc.), the fluorescent compound of the present invention can be used to prepare pH-sensitive tumor diagnostic reagents, viscosity-sensitive tumor diagnostic reagents, and to prepare fluorescent imaging reagents or radioactive imaging reagents for tumors.

[0024] Beneficial effects:

[0025] The multipurpose fluorescent compound described in this invention can simultaneously detect changes in viscosity and pH in solutions and cells. When used as a fluorescent probe, it can replace two existing separate detection probes to solve the same problem, reducing costs and simplifying detection procedures. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The absorption and fluorescence spectra of TN (10 μM) in different solvents.

[0027] Figure 2 Figure 3 is the fluorescence spectrum of TN (10 μM) in Gly-H2O solutions with different ratios. The arrows indicate that the fluorescence intensity increases with the increase of Gly concentration.

[0028] Figure 3 The absorption and fluorescence spectra of TN (10 μM) in solutions with different pH values.

[0029] Figure 4 These are confocal fluorescence images of normal and dexamethasone-treated HeLa cells stained with TN (5 μM, 15 min).

[0030] Figure 5 These are confocal fluorescence images of normal HeLa cells and HeLa cells treated with chloroquine solution stained with TN (5 μM, 15 min). DETAILED DESCRIPTION

[0031] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0032] The absorption spectrum was measured using a Hitachi U-2910 spectrophotometer, the fluorescence spectrum was measured using a Hitachi F-2700 spectrophotometer, and the cell imaging instrument was a Lecia confocal microscope.

[0033] In the following examples, the materials and reagents used were obtained from commercial sources unless otherwise specified.

[0034] Example 1 Synthesis of fluorescent compounds

[0035] (E)-2-(4-(dimethylamino)-2-hydroxyvinyl)-3-(2-hydroxyethyl)benzothiazole-3-iodide (TN)

[0036] The reaction route is as follows:

[0037]

[0038] 1) Synthesis of benzothiazole iodide (Compound 2)

[0039] Compound 1 (2-methylbenzothiazole, 10 mmol) and iodoethanol (1.72 mL, 10 mmol) were dissolved in 20 mL of anhydrous ethanol and stirred in a flask at room temperature for 1 hour. The mixture was then refluxed at 100°C for 8 hours, cooled, filtered, and washed three times with anhydrous EtOH. After drying, a white solid, compound 2 (mass: 3.23 g, yield: 90%), was obtained.

[0040] 2) Synthesis of compound TN

[0041] Compound 2 (1 mmol) and compound 3 (1 mmol) were dissolved in 20 mL of methanol and stirred in a flask for 1 hour. Five drops of piperidine were added. After stirring, the mixture was refluxed at 85°C for 8 hours. After cooling to room temperature, the mixture was washed with petroleum ether. Column chromatography using a CH2Cl2 / CH3OH mixture (10:1 to 6:1, v / v) as the eluent yielded compound TN (mass: 0.25 g, yield: 62%).

[0042] 1 H NMR (400MHz, DMSO-d6) δ (ppm): 10.76 (s, 1H), 8.20 (d, J = 8.0 Hz, 1H), 8.07 (d, J = 16. 0Hz,1H),8.04(d,J=12.0Hz,1H),7.80(d,J=8.0Hz,1H),7.71(t,J=8.0Hz,1H),7.6 2(t,J=6.0Hz,1H),7.51(t,J=16.0Hz,1H),6.45(d,J=8.0Hz,1H),6.19(s,1H),5.1 4(s,1H),4.77(t,J=4.0Hz,2H),3.87(s,1H),3.42-3.47(m,1H),1.14-1.23(m,6H).

[0043] 13 C NMR(400MHz,DMSO-d6)δ(ppm):180.14,143.01,142.54,139.82,136.16,13 2.13,128.20,120.49,114.21,113.36,105.76,51.35,34.73,33.70,26.97.

[0044] Example 2 Photophysical properties test experiment

[0045] Different solvents (see Figure 1 ) Prepare a test solution containing 10 μM TN, test its absorption spectrum with a UV-visible spectrophotometer, and test its fluorescence emission spectrum with a fluorescence spectrometer. The results are shown in Figure 1 .

[0046] from Figure 1 It can be seen that the fluorescent compound TN has an absorption peak at 540nm, and the absorption peak range is 400-600nm ( Figure 1 A). It has a fluorescence peak in the range of 550-675nm ( Figure 1 B) This indicates that the fluorescent compound can be excited by light in the range of 400-600 nm, and its emission spectrum is in the range of 550-675 nm.

[0047] Example 3 Viscosity Test Experiment of Fluorescent Compound TN

[0048] Experimental methods:

[0049] (1) Take the organic fluorescent compound TN prepared in Example 1 and prepare a probe stock solution with a concentration of 1 mM using DMSO;

[0050] (2) Glycerol and water were mixed in different proportions (0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% glycerol by volume) to prepare mixed solutions with different viscosity values;

[0051] (3) adding the probe mother solution prepared in step (1) to the mixed solutions of different viscosity values ​​prepared in step (2) to prepare test solutions, so that the final concentration of the fluorescent probe TN in each solution is 10 μM;

[0052] (4) The above solution is tested for its fluorescence emission spectrum using a fluorescence spectrometer to obtain a corresponding curve. The emission spectrum wavelength is 500nm-655nm.

[0053] The fluorescence spectra of TN (10 μM) in Gly-H2O solutions with different ratios are shown in Figure 2 , the arrow indicates that the fluorescence intensity increases with the increase of Gly concentration. Figure 2 It can be seen that as the Gly ratio increases, the fluorescence intensity increases ( Figure 2 A), and its fluorescence intensity at 590 nm is proportional to the logarithm of viscosity ( Figure 2 B). This indicates that the probe has a significant response to viscosity.

[0054] Example 4 Fluorescence test experiment of fluorescent compound TN in solutions with different pH values

[0055] Experimental methods:

[0056] (1) Take the organic fluorescent compound TN prepared in Example 1 and prepare a probe stock solution with a concentration of 1 mM using DMSO;

[0057] (2) Prepare PBS solutions with different pH values ​​(pH = 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0);

[0058] (3) adding the probe stock solution prepared in step (1) to the solutions of different pH values ​​prepared in step (2) to prepare test solutions, so that the final concentration of the fluorescent probe TN in each solution is 10 μM;

[0059] (4) The above solution was tested for its absorption spectrum and fluorescence emission spectrum using a UV-visible spectrophotometer and a fluorescence spectrometer to obtain corresponding curves. TN has absorption in the range of 400-600 nm and a fluorescence emission spectrum range of 535-715 nm.

[0060] The absorption spectra of the above solutions were measured by UV spectrophotometer. The absorption spectra of TN (10 μM) in solutions with different pH values ​​are shown in Figure 2. Figure 3 A. Use fluorescence spectrometer to test its fluorescence emission spectrum, and fit its fluorescence spectrum to get the corresponding curve, see Figure 3 B.

[0061] As can be seen from the figure, as the pH value increases, the absorption peak changes from 530nm to 560nm, and the fluorescence intensity decreases with the increase of pH value. This shows that the probe is sensitive to pH value.

[0062] Example 5: Probe TN to observe changes in cell viscosity

[0063] HeLa cells were adherently cultured in high-glucose medium (ThermoFisher) containing 10% fetal bovine serum in a 37° C., 5% CO 2 saturated humidity incubator. The medium was replaced every 2-3 days, and subculture was performed.

[0064] When the cells grow to the logarithmic phase, culture them in sections:

[0065] (1) Soak the coverslip in anhydrous ethanol for 30 min, dry it with an alcohol burner, and place it in a disposable 35 mm Petri dish for later use;

[0066] (2) Wash the cells in the 100 mL cell flask three times with PBS, digest them with 1 mL of 0.25% trypsin for 5 minutes, carefully pour out the trypsin, add fresh culture medium, pipette evenly, and count the cells. The cell density is controlled by the amount of culture medium added to make the final cell concentration 1×10 cells per mL. 5 Then, inoculate the culture dish containing the cover glass and culture it in a 5% CO2 incubator to allow the cells to grow close to the culture dish. After the HeLa cells have grown and covered the cover glass, they can be used for cell experiments.

[0067] A probe stock solution with a concentration of 1 mM was prepared using DMSO. Active HeLa cells were incubated in a culture medium containing 5 μM TN (high-glucose culture medium containing 10% fetal bovine serum, ThermoFisher) (incubation conditions: 37°C, 5% CO2) for 15 minutes. A 2 mM dexamethasone solution (to enhance cell viscosity) was added and treated for 45 minutes. The cells were then observed using a laser confocal microscope. The control group was directly stained without any treatment. The staining sites, fluorescence distribution, and brightness changes in the cells were recorded. The results are shown in the table. Figure 4 .

[0068] in, Figure 4 A is a confocal microscopy image of active HeLa cells stained with the probe TN (5 μM, 15 min) and normal HeLa cells treated with 2 mM dexamethasone solution. Figure 4 B is Figure 4 The fluorescence intensity of A was measured. The excitation wavelength of TN in the green light channel was 561 nm, and the fluorescence was collected at a wavelength of 600-700 nm. The fluorescence intensity of the TN probe increased over time, confirming that the probe can monitor changes in intracellular viscosity.

[0069] Example 6 Observation of Cell pH Changes Using Probe TN

[0070] HeLa cells were adherently cultured in a high-glucose culture medium containing 10% fetal bovine serum in a 37°C, 5% CO2 saturated humidity incubator. The culture medium was replaced every 2-3 days, and subculture was performed.

[0071] When the cells grow to the logarithmic phase, culture them in sections:

[0072] ① Soak the coverslip in anhydrous ethanol for 30 minutes, dry it with an alcohol burner, and place it in a disposable 35mm Petri dish for later use;

[0073] ② Wash the cells in the 100 mL cell flask three times with PBS, digest them with 1 mL of 0.25% trypsin for 5 minutes, carefully pour out the trypsin, add fresh culture medium, pipette evenly, and count the cells. The cell density is controlled by the amount of culture medium added to make the final cell concentration of 1×10 cells per mL. 5 Then, inoculate the culture dish containing the cover glass and culture it in a 5% CO2 incubator to allow the cells to grow close to the culture dish. After the HeLa cells have grown and covered the cover glass, they can be used for cell experiments.

[0074] A 1mM probe stock solution was prepared in DMSO. Active HeLa cells were incubated in a culture medium containing 5μM TN for 15 minutes, and then treated with 10μM chloroquine solution for 2 hours. The cells were then observed using a laser confocal microscope. The control group was directly stained without any treatment. The staining site, fluorescence distribution and brightness changes, and colocalization information in the cells were recorded. The results are shown in Figure 2. Figure 5 .

[0075] in, Figure 5 A is a confocal microscopy image of active HeLa cells stained with probe TN (5 μM, 15 min) and treated with 10 μM chloroquine solution (to reduce the acidity of acidic substances in the cells) for 2 h. Figure 5 B shows the relative fluorescence intensity of active HeLa cells after 2 hours. The excitation wavelength of TN in the green channel was 488 nm, and the fluorescence was collected at wavelengths between 500 and 600 nm. The fluorescence intensity of the probe decreased over time, confirming that the probe can monitor changes in intracellular pH.

[0076] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to it in form and detail without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A use of a benzothiazole fluorescent compound in viscosity detection for non-disease diagnosis and treatment purposes, characterized in that: The structural formula of the compound is:

2. A use of a benzothiazole fluorescent compound in the simultaneous observation of viscosity and pH for non-disease diagnosis and treatment purposes, characterized in that: The structural formula of the compound is:

3. The use according to claim 1 or 2, characterized in that After the fluorescent compound stains the cells, changes in intracellular viscosity and pH value can be observed simultaneously.

4. The use according to claim 3, characterized in that The fluorescent compound stains cells at a concentration of 1-15 μM.

5. The use according to claim 1 or 2, characterized in that: The fluorescent compound can simultaneously detect changes in viscosity and pH value in a solution.

6. The use according to claim 5, characterized in that The concentration of the fluorescent compound in the solution is 1-15 μM.

7. The use according to claim 4 or 6, characterized in that The concentration of the fluorescent compound was 10 μM.

8. The use according to claim 1 or 2, characterized in that The fluorescent compound can be used to prepare a viscosity-sensitive tumor diagnostic reagent.

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