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A fluorescent probe for specifically labeling lipid droplets in cells

A fluorescent probe and a specific technology, applied in the lipid droplet fluorescent probe and its application field, can solve the problems such as hindering further research on lipid droplets, difficult to achieve highly selective imaging of lipid droplets, unable to meet research needs, etc. The effect of dyeing ability, fast dyeing speed and good photostability

Inactive Publication Date: 2019-08-09
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with Nile Red, their selectivity has improved, but it still cannot meet the research needs, hindering further research on lipid droplets
It can be seen that it is difficult to achieve the purpose of imaging lipid droplets with high selectivity by simply relying on the lipophilicity of lipid droplets to design lipophilic lipid droplet probes.

Method used

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  • A fluorescent probe for specifically labeling lipid droplets in cells
  • A fluorescent probe for specifically labeling lipid droplets in cells
  • A fluorescent probe for specifically labeling lipid droplets in cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Synthesis of 4-(3'-bromopropylamino)-7-benzofurazan (1)

[0029] In a flask, 4-chloro-7-nitrophenylfurazan (2g, 10mmol) was dissolved in methanol, stirred for 15 minutes, then added with 3-bromopropylamine (2.19g, 10mmol), and reacted at room temperature for 8 hours. After the reaction, it was extracted with dichloromethane and washed with water. Dry with anhydrous sodium sulfate. Finally, a mixture of petroleum ether and ethyl acetate was used for column chromatography to obtain the final product.

[0030] 1 H NMR (300MHz, DMSO-d6): δ (ppm) 9.54 (t, J = 5.40Hz, 1H), 8.54 (d, J = 9.00Hz, 1H), 6.45 (d, J = 8.70Hz, 1H), 3.64(m,4H),2.23(m,2H).

[0031] Synthesis of 1-(3'-(7'-nitrobenzofurazan-4'-)aminopropyl)-4-picoline bromide (NPI)

[0032] The synthesized compound (1) (0.3g, 1mmol) was mixed with 4-picoline (240Ml, 1.5mmol), and heated to reflux with ethanol as a solvent. After 24 hours, the reaction was completed, cooled to room temperature, and the precipitate w...

Embodiment 2

[0034] Embodiment 2: the culture of HeLa cell

[0035] All HeLa cell lines were stored at 37°C, 5% CO 2 CO 2 cultured in an incubator. HeLa cells were cultured adherently in H-DMEM medium containing 10% fetal bovine serum and 1% double antibody.

[0036] When the cells grow to the logarithmic phase, culture the slices: ① Soak the coverslips in absolute ethanol for 30 minutes, dry them with an alcohol lamp, and place them in a disposable 35mm culture dish; ② Wash the cells in the 100mL cell bottle with PBS. Three times, digest with 1mL 0.25% trypsin for 3-5 minutes, pour out the medium carefully, add a small amount of fresh medium and pipette evenly, after counting the cells, leave cells with a suitable density, and add the medium to the required volume (The final concentration of control cells was 1×10 5 ), inoculated into a petri dish containing a coverslip, and placed in CO 2 Cultured in an incubator to allow the cells to grow on the sheet.

Embodiment 3

[0037] Example 3: Confocal fluorescence microscopy of lipid droplets in HeLa cells stained by NPI and Nile red

[0038] The attached cell slides were first incubated with nuclear dye Hoechst 33342 (5 μM) for 30 min, washed twice with PBS, and then stained with 4 μM NPI for 2 min or 5 μM Nile red for 20 min at room temperature. The cell slides were taken out, and the cell growth side was covered on the glass slide, and observed under a confocal fluorescence microscope. It was found that the lipid droplets in the cells were clearly colored by NPI, spherical in shape, and mainly distributed in the cytoplasm (not stained by NPI). Hoechst colored area). However, Nile red stains most intracellular structures in addition to dots. Therefore, the amphiphilic probe NPI of the present invention is a lipid droplet probe with ultrahigh selectivity, which can give a fluorescence picture of lipid droplets without background noise.

[0039] see results figure 1 . Confocal fluorescence ima...

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Abstract

The invention discloses a cell lipid droplet specific-labeling fluorescent probe. The fluorescent probe has a chemical name of 1-(3'-(7'-nitrobenzofuran-4'-)aminopropyl)-4-methylpyridine bromide and has a general chemical formula shown in the formula (I). The invention also discloses a use of the fluorescent probe in specific labeling or display of a lipid droplet form and distribution in living cells. An experiment result shows that the cell lipid droplet specific-labeling fluorescent probe has the characteristics of very high selectivity, background noise-free imaging, two-photon performances, very good light stability, fast dyeing ability, no use of washing and low cytotoxicity and has a great application prospect.

Description

technical field [0001] The invention relates to a lipid drop fluorescent probe and its application, in particular to a fluorescent probe for specifically marking lipid droplets in cells and its application. Background technique [0002] Lipid droplets have long been recognized as simple, inert spheres that store neutral lipids (triglycerides and cholesteryl esters) in cells. However, recent studies have shown that lipid droplets are not static, but highly dynamic organelles in cells, widely present in eukaryotic and prokaryotic cells. They play important roles in cells and participate in various activities in cells, such as regulating the metabolism of intracellular lipids, maintaining intracellular balance, and interacting with various organelles in cells. Moreover, the abnormality of lipid droplets can lead to many diseases, such as obesity, diabetes, atherosclerosis and so on. [0003] Lipid droplets have a large anhydrous neutral core, so some lipophilic probes have be...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D413/12C09K11/06G01N21/64
CPCC07D413/12C09K11/06C09K2211/1029C09K2211/1048G01N21/6486
Inventor 张尚立马剑锋马韫韬
Owner SHANDONG UNIV
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