Application of organic fluorescent dye in preparation of reagent or kit for observing change of mitochondrial membrane potential or bicolor imaging mitochondrial

Abstract

The invention discloses an application of an organic fluorescent dye in preparation of a reagent or a kit for observing mitochondrial membrane potential change or bicolor imaging mitochondria, and belongs to the field of organic fluorescent dyes. The fluorescent compound does not need to be washed, is used for bicolor observation of mitochondrial membrane potential, and can avoid influence of washing on the mitochondrial membrane potential and damage to cells. A new strategy is provided for detecting the mitochondrial membrane potential change.

Description

Technical Field

[0001] This invention relates to the application of an organic fluorescent dye in the preparation of reagents or kits for observing changes in mitochondrial membrane potential or for two-color imaging of mitochondria, and belongs to the field of organic fluorescent dyes. Background Technology

[0002] Mitochondria, enclosed by a double-membrane structure, are the main organelles in eukaryotic cells responsible for generating adenosine triphosphate (ATP), providing energy to the cell through aerobic respiration. As a key indicator for assessing mitochondrial function, mitochondrial membrane potential (MMP) plays a crucial role in evaluating cellular energy dynamics, particularly in the field of cell fate determination. Under physiological conditions, MMP maintains ATP synthesis and the stability of the proton gradient, ensuring fundamental cellular life activities. Therefore, the dynamic changes in MMP have become core biomarkers in cell biology and chemical biology research, and developing analytical methods that can visualize MMP in real time and in situ has become a key task in studying related physiological processes and pathological progression.

[0003] Currently, mitochondrial membrane potential is observed using the patch-clamp method, but the small size of mitochondria hinders observation. Fluorescence staining has become an important method for observing cellular life activities. However, currently developed organic fluorescent dyes generally suffer from two drawbacks: weak fluorescence and inability to effectively monitor dynamic changes in mitochondrial membrane potential. Furthermore, the washing step in routine cell staining processes can also have a significant impact on cell membrane potential. Therefore, developing a wash-free organic fluorescent dye capable of detecting intracellular mitochondrial membrane potential has significant scientific and practical value. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide an application of organic fluorescent dyes in the preparation of reagents or kits for observing changes in mitochondrial membrane potential or for two-color imaging of mitochondria.

[0005] Technical solution: The application of an organic fluorescent dye described in this invention in the preparation of reagents or kits for observing changes in mitochondrial membrane potential, wherein the structural formula of the organic fluorescent dye is:

[0006]

[0007] Furthermore, the kit also includes an incubation culture medium.

[0008] Furthermore, the incubation medium is a high-glucose culture medium containing 10% fetal bovine serum.

[0009] Furthermore, the concentration of the organic fluorescent dye is 5-15 μM.

[0010] The application of an organic fluorescent dye described in this invention in the preparation of dual-color imaging mitochondrial reagents or kits, wherein the structural formula of the organic fluorescent dye is:

[0011]

[0012] Furthermore, the kit also includes glycerin.

[0013] Furthermore, the concentration of the organic fluorescent dye is 5-15 μM.

[0014] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: This invention discloses for the first time the application of the organic fluorescent dye (E)-2-(2-(1H-indole-3)vinyl)-1-(2-hydroxyethyl)quinoline-1-iodide in two-color observation of mitochondrial membrane potential. This fluorescent compound requires no washing and can be used for two-color observation of changes in mitochondrial membrane potential, providing a new strategy for detecting changes in mitochondrial membrane potential. Attached Figure Description

[0015] Figure 1 This represents the crystal structure of QI.

[0016] Figure 2 The absorption and fluorescence spectra of QI (10 μM) in different solvents are shown.

[0017] Figure 3 The fluorescence spectra (A) and CIE chromaticity diagrams (B) of QI (10 μM) in Gly solution and solid are shown.

[0018] Figure 4 (A) is a monochromatic image of HeLa cells stained with QI (5 μM, 15 min) alone; (B) is a confocal fluorescence image of HeLa cells stained with both QI (5 μM, 15 min) and MTDR (0.2 μM, 15 min).

[0019] Figure 5 Confocal fluorescence images of HeLa cells stained with QI (5 μM, 15 min) for different times after CCCP treatment. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0021] The absorption spectroscopy instrument used in the following examples was a Hitachi U-2910 spectrophotometer; the fluorescence spectroscopy instrument was a Hitachi F-2700 spectrophotometer; and the cell imaging instrument was a Lecia confocal microscope.

[0022] Example 1 Synthesis of Fluorescent Compounds

[0023] (E)-1-(2-hydroxyethyl)-2-(2-(6-methoxynaphthalene-2)vinyl)quinoline-1-iodide (i.e., compound QI)

[0024] The reaction route is as follows:

[0025]

[0026] 1) Synthesis of benzothiazole iodide (compound 2)

[0027] Compound 1 (2-methylquinoline, 10 mmol, 1.43 g) and iodoethanol (1.76 mL, 10 mmol) were dissolved in 25 mL of anhydrous ethanol and stirred in a flask for 1 hour at room temperature. The mixture was then refluxed at 100 °C for 8 hours, cooled, filtered, and washed three times with anhydrous ethanol. After drying, a brown solid, compound 2, was obtained.

[0028] 2) Synthesis of compound QI

[0029] Compound 2 (1 mmol, 0.315 g) and compound 3 (1 mmol, 0.145 g) were dissolved in 25 mL of methanol and stirred in a flask for 1 h. Two drops of piperidine were then added. After stirring, the mixture was refluxed at 90 °C for 10 h. After cooling to room temperature, the mixture was washed with petroleum ether. The solution was purified by column chromatography using a CH₂Cl₂ / CH₃OH mixture (12:1–7:1, v / v) as the eluent. The resulting deep yellow solid was compound QI, and its crystal structure is shown in [reference needed]. Figure 1 .

[0030] 1 H NMR (400MHz, DMSO-d6) δ (ppm): 12.29 (s, 1H), 8.84 (d, J = 8.0Hz, 1H), 8.61 (q, J = 4.0Hz, 2H), 8.44 (d, J = 8.0Hz, 1H), 8.28 (t, J = 14.0Hz, 2H), 8.18 (t, J = 8 .0Hz,1H),8.07(t,J=8.0Hz,1H),7.89(q,J=9.4Hz,2H),7.56-7.81(m,1H) ,7.31(t,J=4.0Hz,2H),5.38(s,1H),),5.15(s,2H),4.14(t,J=4.0Hz,2H).

[0031] Example 2: Photophysical Property Testing Experiment

[0032] Test solutions containing 10 μM QI were prepared using DMF, DMSO, Gly, H2O, EtOH, MeCN, and 1,4-dioxane, respectively. The absorption spectra of these solutions were measured using a UV-Vis spectrophotometer, and their fluorescence emission spectra were measured using a fluorescence spectrometer. The results are shown in the figure. Figure 2 .

[0033] As can be seen from the figure, the fluorescent compound QI has an absorption peak at around 470 nm, and the absorption peak range is 350-600 nm (see figure). Figure 2 A). It exhibits a fluorescence peak in the 500-700 nm range (see...). Figure 2 B), but the fluorescence peak is most pronounced only in Gly solvent. The above results indicate that this fluorescent compound can be excited by light in the range of 350-600 nm, and its emission spectrum ranges from 500-700 nm.

[0034] A test solution containing 10 μM QI was prepared. The fluorescence emission spectra of the glycerol solution and the QI solid probe were measured using a fluorescence spectrometer. The CIE coordinate plots of the fluorescence spectra were then plotted. The results are shown in [Figure number missing]. Figure 3 .

[0035] like Figure 3 As shown in Figure A, the emission spectrum of the probe in glycerol ranges from 500 to 700 nm, with a fluorescence peak at 550 nm; the emission spectrum in the solid ranges from 550 to 700 nm, with a fluorescence peak at 600 nm. Furthermore, from... Figure 3 As shown in B, the probe changes from green to orange-red light. This indicates that the probe has two fluorescence peaks, making it suitable for two-color imaging.

[0036] Example 3: Experiment with probe staining of cells

[0037] Human cervical cancer cells (HeLa) originated from Thermo Fisher Scientific.

[0038] HeLa cells were cultured in a high-glucose culture medium (ThermoFisher) containing 10% fetal bovine serum and in a saturated humidity incubator at 37°C and 5% CO2. The culture medium was changed every 2-3 days and the cells were passaged.

[0039] Once the cells have grown to the logarithmic growth phase, mount them onto a slide for culture.

[0040] ① Soak the coverslip in anhydrous ethanol for 30 minutes, dry it with an alcohol lamp, and then place it in a disposable 35mm petri dish for later use;

[0041] ② Wash the confluent cells in a 100mL cell culture flask three times with PBS, digest with 1mL of 0.25% trypsin (Gibco) for 5 minutes, carefully pour off the trypsin, add fresh culture medium, mix well by pipetting, and count the cells. Control the cell density by adding culture medium to achieve a final cell concentration of 1×10⁶ cells / mL. 5Each cell was seeded and then inoculated into a culture dish containing a coverslip. The dish was then incubated in a 5% CO2 incubator to allow the cells to grow in close contact with the culture dish. Once the HeLa cells had grown and completely covered the coverslip, they were used for cell experiments.

[0042] A 1 mM probe QI stock solution was prepared using DMSO. Viable HeLa cells were incubated in a culture medium containing 5 μM QI (high glucose medium containing 10% fetal bovine serum, Thermo Fisher) for 15 min (incubation conditions: 37℃, 5% CO2), followed by cell imaging. For colocalization experiments, after staining with 5 μM QI, 0.2 μM MTDR solution was added, and cells were stained for 15 min, then observed using a laser confocal microscope. The stained areas, fluorescence distribution and brightness changes, and colocalization information were recorded. Results are shown in [Figure number missing]. Figure 4 .

[0043] in, Figure 4 A is a confocal fluorescence image obtained using probe QI (5 μM, 15 min); Figure 4 Image B shows a confocal microscope image of viable HeLa cells stained with QI (5 μM, 15 min) and MTDR (0.2 μM, 15 min). The QI excitation wavelength was 488 nm, and the fluorescence collection wavelength was 530-630 nm. The MTDR excitation wavelength was 638 nm, and the collection range was 650-750 nm. Figure 4 As can be seen in A, the probe can image a structure similar to mitochondrial filaments in the red light channel; Figure 4 As can be seen in B, the two probes have a large overlap, with a co-localization coefficient of 88%, indicating that the probe stains mitochondria.

[0044] Example 4: Observation of changes in mitochondrial membrane potential using probe QI

[0045] The slide culture was the same as in Example 3. After slide culture, a 1 mM probe stock solution was prepared using DMSO. The cultured viable HeLa cells were incubated in culture medium containing 5 μM QI (high glucose culture medium containing 10% fetal bovine serum, Thermo Fisher) for 15 min (incubation conditions: 37°C, 5% CO2). Then, carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added for different treatment times, and the cells were observed using a laser confocal microscope. The stained areas, fluorescence distribution, and brightness changes in the cells were recorded. The results are shown in [Figure 1]. Figure 5 .

[0046] in, Figure 5 A shows confocal fluorescence images of normal HeLa cells stained with probe QI (5 μM, 15 min) and processed with CCCP at different times. Figure 5 B is Figure 5The fluorescence intensity ratio of the green and red channels of A. The excitation wavelength of QI in the green channel is 488 nm, the stimulated wavelength in the green channel is 500-550 nm, and the fluorescence collection wavelength is 550-650 nm. From... Figure 5 As shown in Figure A, with increasing processing time, the fluorescence intensity of the red channel gradually decreases, while the fluorescence intensity of the green channel gradually increases. This is due to the increasing ratio of fluorescence intensity between the red and green channels. Figure 5 B) further illustrates that the probe can observe changes in mitochondrial membrane potential.

Claims

1. The application of an organic fluorescent dye in the preparation of reagents or kits for observing changes in mitochondrial membrane potential, characterized in that, The structural formula of the organic fluorescent dye is:

2. The application according to claim 1, characterized in that, The kit also includes an incubation culture medium.

3. The application according to claim 2, characterized in that, The incubation medium is a high-glucose culture medium containing 10% fetal bovine serum.

4. The application according to claim 1, characterized in that, The concentration of the organic fluorescent dye is 5-15 μM.

5. The application of an organic fluorescent dye in the preparation of a two-color imaging mitochondrial reagent or kit, characterized in that, The structural formula of the organic fluorescent dye is:

6. The application according to claim 5, characterized in that, The kit also includes glycerol.

7. The application according to claim 5, characterized in that, The concentration of the organic fluorescent dye is 5-15 μM.

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