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Application of naphthyl derivatives used as targeted pH fluorescent probes for mitochondria

A technology of fluorescent probes and mitochondria, applied in the field of pH fluorescent probes, to achieve highly sensitive detection of pH changes, reduced interference of excitation light, and good cell membrane permeability

Inactive Publication Date: 2017-04-19
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the use of this derivative in pH fluorescent probes to detect intracellular mitochondrial pH changes and cell imaging has not been reported.

Method used

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  • Application of naphthyl derivatives used as targeted pH fluorescent probes for mitochondria
  • Application of naphthyl derivatives used as targeted pH fluorescent probes for mitochondria
  • Application of naphthyl derivatives used as targeted pH fluorescent probes for mitochondria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] With the probe HNEP in embodiment 1 + Use secondary water (conductivity 18.2) to prepare a stock solution with a concentration of 1 mM for storage. In the experiment, the probe was diluted to a final concentration of 10 μM with phosphate buffered saline (PBS) of different pH values, and the HNEP was recorded + In the PBS system, the ultraviolet absorption spectrum ( figure 1 ). As the pH value increased from 6.89 to 11.65, the absorption peak at 390nm decreased sequentially, the absorption peak at 445nm gradually increased, and an obvious isoabsorptive point appeared near 413nm. At the same time, the color of the solution changed from yellow to colorless ( figure 2 ).

Embodiment 2

[0022] The probe HNEP was also treated with different pH values ​​of phosphate buffer + Dilute to a final concentration of 10μM, fix the excitation wavelength at 317nm, record HNEP + Fluorescence emission spectrum changes with pH changes in PBS system ( image 3 ). As the pH value increases from 5.00 to 11.50, the fluorescence intensity at 586nm decreases gradually, which is suitable for the detection of pH changes in the alkaline environment (pH ~ 8.0) in mitochondria. Under the irradiation of ultraviolet light, the color of the solution changed from yellow to colorless ( Figure 4 ). According to HNEP + The Singmoidal fitting curve of the fluorescence intensity at 586nm as a function of pH ( Figure 5 ), calculate pK a The value is 8.8, and the pH response is linear in the range of 7.8-10.0. The linear regression equation is I=-9151.11×pH+94210.98, the correlation coefficient R 2 = 0.9948.

Embodiment 3

[0024] With the probe HNEP in embodiment 1 + The concentration was kept at 10 μM, and the effect of the probe on H in the presence of common ions and amino acids was investigated + selectivity. Such as Figure 6 As shown, in the PBS system, at pH 7.0, the probe has almost no response to the above substances, proving that HNEP + to H + Has high selectivity. Figure 6 The sequence and concentration of the substances in it are as follows: 1, blank; 2, F - (1mM); 3, Cl - (10mM); 4, Br - (1 mM); 5, I - (1mM); 6, SO 4 2- (1mM); 7,S 2 o 3 2- (1mM); 8,SO 3 2- (1mM); 9,HS - (1mM); 10, NO 3 - (1mM); 11, NO 2 - (1mM); 12,Ac - (1mM); 13, HCO 3 - (1mM); 14, ClO 4 - (1mM); 15,K + (140mM); 16,Cd 2+ (1mM); 17, Mg 2+ (1mM); 18, Li + (1mM); 19,Co 2+ (1mM); 20, Hg 2+ (1mM); 21, Ba 2+ (1mM); 22,Ni 2+ (1mM); 23,H 2 o 2 (1mM); 24,L-GSH(1mM); 25,Hcy(1mM); 26,Cys(1mM).

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Abstract

The invention discloses an application of naphthyl derivatives used as targeted pH fluorescent probes for mitochondria, and specifically discloses targeted marking of 1- methyl-4-[2-(6-hydroxyl-2-naphthalene)- vinyl]-pyridium (HNEP<+>) in the mitochondria of living cells, and an application thereof in pH detection. The derivatives are excited at a wavelength of 390nm, and achieve maximum fluorescence emission at a wavelength of 586nm. The fluorescence intensity at a wavelength of 586nm is gradually weakened with the rising of the pH value from 5.00 to 11.50 in a phosphate buffer system. The pKa value is 8.8, and the linear range of the pH value is 7.8-10.0 and suitable for detection for the mitochondria in an alkaline environment (with a pH value of 8.0). In addition, the probes have an ultra-large Stokes displacement (196nm), and are high in selectivity and water solubility, low in toxicity and beneficial to fluorescence imaging study in the cells. A colocalization experiment in the cells and a pH adjustment experiment for the mitochondria prove that the probes are capable of realizing specific targeted marking for the mitochondria, and capable of detecting the change of the pH value in the mitochondria with high sensitivity.

Description

technical field [0001] The present invention relates to a pH fluorescent probe, specifically 1-methyl-4-[2-(6-hydroxyl-2-naphthalene)-vinyl]-pyridine iodide salt (HNEP + ) as a mitochondrial-targeted pH fluorescent probe. Background technique [0002] Intracellular pH plays a key role in many cellular events, such as cell growth and apoptosis, endocytosis, receptor-mediated signal transduction, ion transport, and calcium regulation. There are respective acid-base balances between different subcellular structures of different prokaryotes and eukaryotic cells. Abnormal pH often leads to cellular dysfunction. Therefore, monitoring pH changes in living cells helps to better understand the physiological and pathological processes of cells. [0003] Mitochondria is a crucial organelle in eukaryotic cells. It is a sac-like structure wrapped by a double-layer membrane. The inner membrane contains an electron transport chain composed of four protein complexes. The energy released ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D213/30C09K11/06G01N21/64
CPCC07D213/30C09K11/06C09K2211/1029G01N21/6486
Inventor 樊丽林博葛金印董川双少敏
Owner SHANXI UNIV
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