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Cell lipid droplet fluorescence imaging probe based on single benzene ring skeleton and application thereof

A fluorescence imaging and cell lipid technology, which is applied in the direction of fluorescence/phosphorescence, material analysis by optical means, material excitation analysis, etc., can solve the problems of crossover, low signal-to-noise ratio, low dyeing selectivity, etc., to avoid crossover, Good photostability and high dyeing selectivity

Active Publication Date: 2020-03-27
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, BODIPY and Nile Red (Materials, 2018, 11, 1768) are the most commonly used representative lipid droplet fluorescence imaging probes, but both probes have certain shortcomings
For example: the Stokes shift of BODIPY is relatively small, which will cause the cross-overlapping of excitation light and emission light; Nile Red dyeing selectivity is low, and when staining lipid droplets, it will also stain other hydrophobic structures in the cell, resulting in lower SNR

Method used

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  • Cell lipid droplet fluorescence imaging probe based on single benzene ring skeleton and application thereof
  • Cell lipid droplet fluorescence imaging probe based on single benzene ring skeleton and application thereof
  • Cell lipid droplet fluorescence imaging probe based on single benzene ring skeleton and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Synthesis of Dimethyl 2,5-Bis(cyclohexylamino)terephthalate (Ph-Red)

[0034] Add dimethyl 1,4-cyclohexanedione-2,5-dicarboxylate (228mg, 1.00mmol) into a two-necked flask, then add 10mL ethanol and 1mL acetic acid as solvent, add cyclohexylamine (0.35mL , 3.1 mmol), after which the reaction system was refluxed in air for 18 hours. After cooling to room temperature, the solid was filtered off and washed with ethanol to obtain 208 mg (0.535 mmol, 54%) of Ph-Red as a red powder.

[0035] 1 H NMR (400MHz, CDCl 3 ):δ7.37(s,2H),6.85(br,2H),3.95(s,6H),3.42(br,2H),2.09-2.06(m,4H),1.84-1.81(m,4H), 1.70-1.68(m,2H),1.52-1.32(m,10H). 13 C NMR (126MHz, CDCl 3 ): δ168.52, 140.05, 116.78, 114.95, 51.86, 51.03, 33.04, 26.02, 24.69.

[0036] figure 1 For the proton nuclear magnetic spectrum of embodiment 1 synthesis probe Ph-Red, figure 2 The C NMR spectra of the probe Ph-Red synthesized in Example 1 indicated that the target product Ph-Red was prepared.

[0037] We replace t...

Embodiment 2

[0042] Embodiment 2: the mensuration of the fluorescent probe Ph-Red absorption-emission spectrum prepared in embodiment 1

[0043] The probe Ph-Red synthesized in Example 1 was prepared into a solution at a concentration of 10 μM with 25 mL of toluene solvent. Use a UV-visible spectrophotometer to scan in the 300-700nm band to obtain the absorption spectrum, and use a fluorescence spectrometer to scan under the excitation of 490nm to obtain the fluorescence emission spectrum. Figure 9 The absorption-emission spectrum of the shown Ph-Red fluorescent probe in toluene solvent (the solid line on the left is the absorption spectrum, and the dotted line on the right is the emission spectrum), illustrating the absorption-emission peak of the fluorescent probe Ph-Red bit and its large Stokes shift.

Embodiment 3

[0044] Embodiment 3: the culture of HeLa cell

[0045] All percentages in this example are volume fractions.

[0046] HeLa cell lines were incubated at 37 °C with 5% CO 2 cultured in an incubator, and the culture medium was high-glucose DMEM containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixed solution). Among them, fetal bovine serum, double antibody and high-glucose DMEM were purchased directly from biological reagent companies.

[0047] When the cells grow to the logarithmic phase, we subculture the cells: absorb the culture medium in the cell culture flask, wash it with 2 mL of DMEM culture medium without fetal bovine serum, absorb the culture medium and wash it with 0.5 mL pancreatic Enzyme digestion for 2 minutes, after most of the cells detach, add 2mL of high-glucose DMEM culture medium containing 10% fetal bovine serum and 1% double antibody, pipette evenly, inoculate into new cell culture flasks and culture dishes, put into CO 2...

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Abstract

The invention discloses a cell lipid droplet fluorescence imaging probe based on a single benzene ring skeleton and application thereof, and belongs to the technical field of biological imaging. The structural formula of the fluorescence imaging probe is shown in the specification, R is methyl, ethyl, n-propyl, n-butyl, n-amyl, n-hexyl or cyclohexyl, and the Ph-Red effect of the fluorescence imaging probe taking R as cyclohexyl is the best. The invention also discloses an application of the fluorescence imaging probe in specific labeling of lipid droplets in cells or visualization of lipid droplet form and distribution in cells. Experiments prove that the probe Ph-Red disclosed by the invention is a lipid droplet fluorescent probe with the characteristics of ultrahigh dyeing selectivity, relatively high brightness, stability and cell membrane permeability, relatively low cytotoxicity and the like, and has a huge application prospect.

Description

technical field [0001] The invention belongs to the technical field of biological imaging, and in particular relates to a cell lipid droplet fluorescence imaging probe based on a single benzene ring skeleton and its application in specifically marking lipid droplets in cells or visualizing the shape and distribution of lipid droplets in cells. Background technique [0002] Lipid droplets are spherical organelles whose interior is composed of neutral lipids (triglycerides and cholesteryl esters) and which are covered with a phospholipid monolayer membrane immobilized by proteins. Lipid droplets are present in almost all organisms from prokaryotes to humans and play important roles in a variety of cellular activities including lipid storage, membrane transport, and protein storage. Lipid droplets are also closely related to many diseases, such as: neurodegenerative diseases, obesity, diabetes, atherosclerosis and cancer. [0003] To study the structure of lipid droplets and t...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6402G01N21/6486
Inventor 卢革宇王晨光周日刘晓敏闫旭刘方猛孙鹏
Owner JILIN UNIV
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