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Fluorescent probe for displaying two states of cell membrane potential by using fluorescence image and application thereof

A fluorescent probe and fluorescent image technology, applied in the field of fluorescent probes, to achieve the effect of eliminating tedious steps, high specificity and high selectivity

Active Publication Date: 2020-04-28
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In view of the deficiencies of the current methods for measuring cell membrane potential, the problem to be solved in the present invention is to provide a fluorescent probe that uses fluorescent images to display two states of cell membrane potential (normal state and near-zero state of cell membrane potential) and its application

Method used

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  • Fluorescent probe for displaying two states of cell membrane potential by using fluorescence image and application thereof
  • Fluorescent probe for displaying two states of cell membrane potential by using fluorescence image and application thereof
  • Fluorescent probe for displaying two states of cell membrane potential by using fluorescence image and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Preparation of the Fluorescent Probe (CP12) Using Fluorescent Images to Display Two States of Cell Membrane Potential in the Present Invention

[0030] Add 0.399g of N-dodecylcarbazole-3-carbaldehyde and 5ml of ethanol to a dry three-necked flask, protect it with nitrogen, heat up to 60°C, and dissolve it after heating for 30min. Add the compound 1-(2-hydroxyethyl )-4-picoline iodide salt 0.265g, add 5 drops of piperidine dropwise, heat up to reflux, stop heating overnight, evaporate the solvent after cooling, recrystallize with ethanol, dry to obtain 0.2g of yellow-green powder, the yield 51%, which is CP12.

[0031] Above-mentioned compound CP12 preparation reaction formula is as follows:

[0032]

[0033] 1 1H NMR (300MHz, DMSO-d6), δ (ppm): 8.83 (d, J = 6.9Hz, 2H), 8.60 (d, J = 0.9Hz, 1H), 8.19-8.26 (m, 4H), 7.90 ( d, J=9Hz, 1H), 7.74(d, J=8.7Hz, 1H), 7.67(d, J=8.4Hz, 1H), 7.49-7.58(m, 2H), 7.29(t, J=7.5Hz ,1H),5.27(t,J=5.4Hz,1H),4.54(t,J=4.5Hz,2H),4....

Embodiment 2

[0034] Embodiment 2 cancer cell (SiHa and HeLa) and normal cell (Fibroblast) culture

[0035] at 37°C, 5% CO 2 In a saturated humidity incubator, SiHa, HeLa and Fibroblast cell lines were grown adherently in H-DMEM medium containing 10% fetal bovine serum (containing 1% double antibody). After the cells grew to the logarithmic phase, the SiHa, HeLa, and Fibroblast cells were taken and cultured. The specific method is: ① Soak the coverslip in absolute ethanol for 30 minutes, dry it with an alcohol lamp, and put it into a disposable 35mm culture dish; ② Wash the cells that have grown to logarithmic phase in the cell dish three times with PBS, After digesting with 1mL 0.25% trypsin for 3-5 minutes, pour out the enzyme solution carefully, then add a small amount of fresh culture medium, pipette the cells to make a uniform cell suspension, after counting the cells, use the cell culture medium to adjust the final concentration of the cells 1×10 5 cells / ml, inoculated into a petri...

Embodiment 3

[0036] Example 3 Fluorescent probe CP12 stains living SiHa, HeLa and Fibroblast cells

[0037] Wash the cell slides prepared in Example 2 three times with PBS, stain live SiHa, HeLa and Fibroblast cells with 2 μM CP12 respectively, incubate at room temperature for 20 min, suck out the culture medium, and wash three times with PBS to wash away unbound cells. The excess staining solution, the cell growth side was covered on the glass slide, under the excitation of 473nm laser, the laser scanning confocal fluorescence microscope was used to observe the coloring part of the cell, the fluorescence distribution and the change of brightness, etc. It was found that the fluorescent probe CP12 can pass through The cell membrane enters the cell, and both the cell membrane and the cytoplasm are stained with bright green fluorescence.

[0038] see results figure 1 .

[0039] The results indicated that the membrane potential state of the living cells was normal. When the cells were in the...

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Abstract

The invention discloses a fluorescent probe for displaying two states (a normal state and a near-zero state) of cell membrane potential by utilizing a fluorescence image. The fluorescent probe is a compound of 4-[2-(9-dodecyl-9H-carbazole-3)vinyl]-1-(2-hydroxyethyl)-pyridine iodate, called CP12 for short. The invention also discloses an application of the fluorescent probe in detection of a normalstate and a near-zero state of cell membrane potential. When the potential of the cell membrane is in a normal state, the probe enables the cell membrane and cytoplasm to be dyed; when the potentialof the cell membrane is in a near-zero state, only the cell membrane is dyed by the probe. Moreover, the probe is not interfered by mitochondrial membrane potential when being used for measuring the cell membrane potential, and shows higher specificity. Compared with a currently used cell membrane potential probe product, the probe having the visual and rapid distinguishing mode has the advantagesthat calibration and standard curve drawing are not needed in the using process, time and labor are saved, and the probe has wide application prospects in the aspects of biological research and medical diagnosis.

Description

technical field [0001] The invention relates to a fluorescent probe for detecting cell membrane potential and its application, in particular to a fluorescent probe for displaying two states of cell membrane potential (normal state and near-zero state of cell membrane potential) by using fluorescent images and its application. Background technique [0002] The cell membrane potential is the potential difference between the inside and outside of the cell membrane when the cell is in a resting state. It is an important indicator reflecting cell activity and cell metabolism. The normal membrane potential of most cells is negative on the inside and positive on the outside, in the range of -50 to -100mV Inside. However, in some cell types, the membrane potential is significantly reduced. For example, U.V.Lassen found that the cell membrane potential of human erythrocytes was less than -14mV in 1967; S.Hodson reported that the cell membrane potential of rabbit corneal endothelial ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D401/06C09K11/06G01N21/64
CPCC07D401/06C09K11/06G01N21/6428C09K2211/1029
Inventor 何秀全张华淼于晓强刘志强
Owner SHANDONG UNIV
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