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Fluorescent probe for detecting mitochondrial membrane potentials, and preparation method and application thereof

A technology of mitochondrial membrane potential and fluorescent probes, which is applied in the field of analytical chemistry, can solve the problems of single species, and achieve the effects of low biological toxicity, simple purification steps, and simple synthesis methods

Active Publication Date: 2020-02-11
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problem that the mitochondrial membrane potential probes in the prior art are of a single type and cannot realize the migration of subcellular organelles when the mitochondrial membrane potential changes, the present invention provides a mitochondrial membrane potential probe, which has good selectivity and high sensitivity. Has red emission, enabling subcellular organelle migration

Method used

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  • Fluorescent probe for detecting mitochondrial membrane potentials, and preparation method and application thereof
  • Fluorescent probe for detecting mitochondrial membrane potentials, and preparation method and application thereof
  • Fluorescent probe for detecting mitochondrial membrane potentials, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Synthesis of Fluorescent Probes

[0037] (1) Synthesis of 1,2-dimethyl-quinoline iodide salt (compound 1):

[0038] Add 10 mL of ethanol to the round bottom flask, then add 1.1 mL of 2-methylquinoline, add 0.5 mL of iodomethane, heat to 60 °C for 30 hours, after the reaction is completed, cool the reaction system to room temperature, and a solid precipitates , filtered and washed with ethanol to give 1,2-dimethyl-quinoline iodide salt (compound 2) with a yield of 92%. 1 H NMR (400 MHz, DMSO-d6) δ9.12 (d, J = 8.5 Hz, 1H), 8.60 (d, J = 9.0 Hz, 1H), 8.41 (dd, J = 8.2, 1.6Hz, 1H), 8.24 (ddd, J = 8.8, 7.0, 1.6 Hz, 1H), 8.14 (d, J = 8.5 Hz, 1H), 8.00 (t, J = 7.6 Hz, 1H), 4.45 (s, 3H), 3.09 (s , 3H).

[0039] (2) Synthesis of 4,4'-(piperazine-1,4-diyl)benzaldehyde (compound 2):

[0040] Piperazine (1.0 g, 11.62 mmol) was dissolved in H 2 O (18 mL) and 2-methoxyethanol (20 mL). The mixture was heated to reflux, then a solution of 4-fluorobenzaldehyde (0.63 mL, 5...

Embodiment 2

[0043] Example 2 Responses of fluorescent probes to different membrane potentials

[0044] Set the density to 3 x 10 5 cells / mL of HeLa cells were inoculated into sterile 35 mm imaging culture dishes in CO 2 Incubator (at 37°C, 5% CO 2 ) for more than 12 hours to allow the cells to adhere to the wall. Then prepare the DMSO solution of the probe HJI obtained in Example 1 with a concentration of 1 mM as the mother solution, add the mother solution to the cell culture dish so that the final concentration is 5 μM, continue to cultivate for 20 min, and then absorb the cell culture solution Go, wash the cells with PBS buffer 3 times, add 1 mL of fresh medium, and then add 10 μM CCCP (an oxidative phosphorylation uncoupler that can reduce the mitochondrial membrane potential) for imaging immediately, every 0.5 minutes, record different Imaging pictures under time.

[0045] In the cell imaging experiment, the excitation wavelength is 488 nm, and the detection band is the red ligh...

Embodiment 3

[0046] Example 3 Selectivity of fluorescent probes to different ions

[0047] Configure the DMSO mother solution of the fluorescent probe prepared in Example 1, the concentration is 5 mM, prepare different amino acids (Ile, Arg, Ser, Asn, Gln, Glu, His, Ala, Hcy, N-Ace, Val, GSH) and NaCl, KNO 3 、H 2 o 2 stock solution in PBS at a concentration of 100 mM. Then, 5 μL of the probe mother solution was added to 5 mL volumetric flasks, 10 μL of different analytes were added to each volumetric flask, and finally the volume was adjusted to 5 mL with PBS buffer solution. Fluorescence detection (excitation wavelength 500 nm) was then performed. Take the wavelength as the abscissa and the fluorescence intensity as the ordinate Figure 4 . It can be seen from the figure that the addition of different analytes has no effect on the fluorescence of the probe.

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Abstract

The invention belongs to the technical field of analytical chemistry, and provides a fluorescent probe for detecting mitochondrial membrane potentials as well as a preparation method and application thereof. The fluorescent probe for detecting the mitochondrial membrane potentials has a structural formula shown as: , and can be prepared by allowing reaction between a reaction product of 4-fluorobenzaldehyde and piperazine with 1,2-dimethyl-quinoline iodate. The fluorescent probe for detecting the mitochondrial membrane potentials provided by the invention has the characteristics of being low in biological toxicity, excellent in membrane permeability, simple in synthesis method and convenient in a purification step; and the fluorescent probe can be applied to cell imaging for detecting, labeling or displaying mitochondrial membrane potential changes.

Description

technical field [0001] The invention belongs to the technical field of analytical chemistry, and in particular relates to a fluorescent probe for detecting mitochondrial membrane potential and an application thereof. Background technique [0002] During the mitochondrial tricarboxylic acid cycle, the protons inside the mitochondria will be actively transported to the outside of the mitochondria, thus forming a mitochondrial membrane potential of -160mV~-180mV, which is negative on the inside and positive on the outside. The mitochondrial membrane potential powers the process of aerobic respiration, catalyzing its breakdown of highly stable compounds. In addition, the mitochondrial membrane potential is closely related to the state of the cell, and the level of the mitochondrial membrane potential can accurately reflect the healthy state of the cell. Therefore, real-time observation of changes in mitochondrial membrane potential has important physiological, pathological and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D215/12C09K11/06G01N21/64
CPCC07D215/12C09K11/06C09K2211/1029C09K2211/1044G01N21/6428G01N21/6486
Inventor 林伟英郭丁一田明刚孙洁
Owner UNIV OF JINAN
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