Amphiphilic lipid-droplet fluorescent probe with super-high selectivity and application thereof

A fluorescent probe and selective technology, applied in the lipid droplet fluorescent probe and its application field, can solve the problems of difficulty in imaging lipid droplets with high selectivity, hindering further research on lipid droplets, and failing to meet research needs, and achieve rapid Dyeing ability, good light stability, fast dyeing effect

Active Publication Date: 2017-06-30
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 diff

Method used

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  • Amphiphilic lipid-droplet fluorescent probe with super-high selectivity and application thereof
  • Amphiphilic lipid-droplet fluorescent probe with super-high selectivity and application thereof
  • Amphiphilic lipid-droplet fluorescent probe with super-high selectivity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Synthesis of 4-(3'-bromopropylamino)-7-benzofurazan (1)

[0033] 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.

[0034] 1H 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).

[0035] Synthesis of 1-(3'-(7'-nitrobenzofurazan-4'-)aminopropyl)-2,3,3-trimethylindole bromide (NII)

[0036] The synthesized compound (1) (0.3g, 1mmol) was mixed with 2,3,3-trimethylindole (240Ml, 1.5mmol), and heated to reflux with ethanol as a solvent. After 24 hours, the reaction was completed and cooled to room temperatur...

Embodiment 2

[0038] Embodiment 2: the cultivation of immortalized cancer cell HeLa

[0039] 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.

[0040] 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

[0041] Example 3: Confocal fluorescence microscopy experiments of lipid droplets in HeLa cells stained by NII and Nile red

[0042] 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 NII 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 stained by NII, spherical in shape, and mainly distributed in the cytoplasm (not stained by NII). Hoechst colored area). However, Nile red stains most intracellular structures in addition to dots. Therefore, the amphiphilic probe NII of the present invention is a lipid droplet probe with ultrahigh selectivity, which can give a fluorescence picture of lipid droplets without background noise.

[0043] see results figure 1 . Confocal fluo...

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PUM

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Abstract

The invention discloses an amphiphilic lipid-droplet fluorescent probe with super-high selectivity. The amphiphilic lipid-droplet fluorescent probe with super-high selectivity is characterized in that chemical name of the probe is 1-(3'-(7'-nitrobenzofurazan-4'-)aminopropyl)-2,3,3-trimethylindole bromine, NII for short; and the general chemical formula is as shown in the formula (I). The invention also discloses application of the probe in specifically marking or displaying the form and distribution of lipid droplets in living cells or tissues. Experiments confirm that the probe of the invention is a brand-new probe, has a wide range of application, has good single- and two-photon photostability, fast staining speed and low cytotoxicity, can specifically image lipid droplets in living cells and has a wide application prospect.

Description

technical field [0001] The present invention relates to a lipid droplet fluorescent probe and its application, in particular to a super-selective amphiphilic lipid droplet probe and its ability to specifically label or display lipid droplets in living cells or tissues Applications in Morphology and Distribution. 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, atherosclerosi...

Claims

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Application Information

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IPC IPC(8): C07D413/12C09K11/06G01N21/64
Inventor 于晓强刘志强郭丽方
Owner SHANDONG UNIV
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