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A two-photon pH ratiometric fluorescent probe for monitoring cell autophagy and its preparation method and application

A fluorescent probe and two-photon technology, applied in the field of two-photon ratio fluorescent probes, can solve problems such as unsatisfactory visualization effect and inability to monitor autophagy state, and achieve the effects of simple structure, good permeability and sensitive response.

Active Publication Date: 2020-10-02
ANHUI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these monitoring methods have their limitations, such as scanning electron microscopy and Western blot, they cannot monitor the autophagy state in living cells, and at the same time, the visualization of the monitoring results of these methods is not ideal. A simple and simple method for monitoring autophagy is necessary for the study of cell autophagy

Method used

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  • A two-photon pH ratiometric fluorescent probe for monitoring cell autophagy and its preparation method and application
  • A two-photon pH ratiometric fluorescent probe for monitoring cell autophagy and its preparation method and application
  • A two-photon pH ratiometric fluorescent probe for monitoring cell autophagy and its preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: the synthesis of fluorescent probe molecule Lyso-MPCB

[0030] Add 1g of compound A, 0.46g of o-phenylenediamine, 0.0082g of p-toluenesulfonic acid and 30mL of N,N-dimethylformamide into a three-necked flask, heat up to 120°C in an oil bath, and protect the reaction with argon for 12h After the reaction is finished, the liquid is spin-dried, extracted with dichloromethane and water, and the upper organic phase is collected and spin-dried to obtain the crude product; the crude product sample preparation is separated by column chromatography to obtain the target product and obtain the target product 0.7g (1.28mmoL), yield 60%. The structural formula of the compound A is:

[0031]

[0032] 1 H NMR (400MHz, DMSO-d 6)δ12.84(s,1H),9.04(s,1H),8.41(s,1H),8.33(d,J=8.6Hz,1H),7.84(d,J=8.7Hz,1H),7.73( d,J=8.5Hz,1H),7.65(d,J=8.5Hz,2H),7.55(t,J=11.5Hz,3H),7.20(dd,J=5.5,2.6Hz,2H),7.02( d,J=8.3Hz,2H),4.49(t,J=7.0Hz,2H),3.82(s,3H),3.53(s,4H),2.30(s,6H),1.89-1.79(m,...

Embodiment 2

[0033] Embodiment 2: Fluorescence test and two-photon test of fluorescent probe molecule

[0034] Dissolve the fluorescent probe Lyso-MPCB of the present invention in DMSO to prepare a 1mM mother solution, take 100 μL of the mother solution in a 10mL volumetric flask, and then use buffer solutions of different pH values ​​(phosphoric acid-acetic acid-boric acid solution system, by adding 1mM hydrochloric acid or sodium hydroxide solution (adjusted to different pH values) to dilute to 10 μM. The excitation wavelengths of the single-photon and two-photon fluorescent probes are 370nm and 760nm respectively, and the fluorescence spectrum changes in the wavelength range of 390-700nm are detected. And through the I corresponding to different pH values 475nm / I 410nm The pKa value (3.88) of the probe was calculated.

[0035] Fluorescent probe Lyso-MPCB is in the test solution adjusted back and forth with 1mM hydrochloric acid and sodium hydroxide solution, when pH=4.2 and pH=7.2, ...

Embodiment 3

[0037] Example 3: Cytotoxicity Test

[0038] The MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide) experiment was based on the reported articles to do some cytotoxicity tests. Add 0, 5, 10, 15, and 20 μM fluorescent probes to the same batch of cells, and the conditions are 37 ° C, 5% CO 2 Incubate in a cell incubator for 24 hours, according to the formula of cell viability: cell viability%=OD 570(样品) / OD 570(对照组) ×100, the cell survival rate can be calculated ( Figure 5 ). from Figure 5 We can see that when the concentration is 20 μM, the survival rate of the cells is about 90%, which shows that the fluorescent probe of the present invention has no toxic effect on the cells, so it can be used to detect the viscosity in the cells.

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Abstract

The invention discloses a two-photon pH (potential of hydrogen) ratio measurement fluorescence probe for monitoring cell autophagy, a preparation method of the fluorescence probe and an application. The structure of the two-photon pH (potential of hydrogen) ratio measurement fluorescence probe for monitoring cell autophagy is as shown in the specification. The fluorescence probe has specific fluorescence signal response to pH. By the aid of a cell co-localization test, the fluorescence probe can be specifically targeted to cell lysosomes, a cell toxicity test indicates that the fluorescence probe has almost no toxic and side effect on cells, a two-photon confocal fluorescence micro-imaging test indicates that the fluorescence probe has good penetrability for MCF-7 cells, pKa of the fluorescence probe is 3.88 through calculation, the fluorescence probe is suitable for monitoring change ranges of the pH of the cell lysosomes, and the cell autophagy process can be monitored in real time by detecting change of the pH of the cell lysosomes.

Description

technical field [0001] The invention relates to a two-photon ratiometric fluorescent probe, in particular to a two-photon pH ratiometric fluorescent probe for monitoring cell autophagy and its preparation method and application. Background technique [0002] Autophagy is a process in which cells engulf their own cytoplasmic proteins or organelles and invaginate them into vesicles when they are destructively stimulated, and then fuse with lysosomes to form autolysosomes, degrading the contents wrapped by them. Autophagy is the basis for the degradation and recycling of cell components, and it acts as a "scavenger" in cells. It is a normal way of natural metabolism of intracellular organelles and other structures. When tissue cells are damaged by various physical and chemical factors, autophagy The lysosomes increase in large quantities and play a protective role against cell damage. Traditional autophagy monitoring includes transmission electron microscopy (TEM), Western blo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D403/04C09K11/06G01N21/64
CPCC07D403/04C09K11/06C09K2211/1029C09K2211/1033C09K2211/1044G01N21/6486
Inventor 孟祥明候立玲宁鹏程浩然冯燕
Owner ANHUI UNIVERSITY
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