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Acid-sensitive double light emission probe, preparation method and uses thereof

An emission probe, acid-sensitive technology, applied in the field of probes, to achieve the effects of sensitive method, sensitive pH ratio determination, and simple synthesis

Inactive Publication Date: 2013-07-24
厦门生光生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, small molecule pH ratiometric fluorescent probes based on this dye have not been reported yet.

Method used

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  • Acid-sensitive double light emission probe, preparation method and uses thereof
  • Acid-sensitive double light emission probe, preparation method and uses thereof
  • Acid-sensitive double light emission probe, preparation method and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: the synthesis of Danyl-R6G

[0030] Add Rhodamine 6G (10 g) to 100 ml diethylenetriamine in a tube or bottle with a cap, and stir the reaction at 60°C until the color of Rhodamine 6G disappears. 500 ml of water was added to the reaction solution, and the mixture was extracted with dichloromethane. The organic phase was heated to remove the solvent, and the obtained solid was separated and purified by silica gel column to obtain a colorless solid (9 g). The solid was added to 100 ml of dichloromethane, followed by 3 g of dansyl chloride and 10 ml of triethylamine. The reaction was stirred at room temperature for 2 hours. The organic phase was heated to remove the solvent, and the obtained solid was separated and purified by silica gel column to obtain Danyl-R6G (5 g). Synthetic route see figure 1 .

Embodiment 2

[0031] Example 2: The ratiometric pH titration of Danyl-R6G under dual-wavelength light excitation

[0032] Danyl-R6G was prepared into a solution with a final concentration of 1 μg / ml in sodium phosphate buffer with different pH gradients, and the fluorescein excitation spectrum excited at 300 nm and the rhodamine 6G lactam excited at 532 nm corresponding to different pH were measured spectrum. see results image 3 and Figure 4 ;

[0033] image 3is the fluorescence emission spectrum of Dansyl-R6G in pH 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, and 3.5 buffer solution (the excitation wavelength of Dansyl-group is 300 nm, R6G-lactam group The excitation wavelength of the group is 532 nm). The abscissa is the fluorescence emission wavelength, and the ordinate is the fluorescence emission intensity; it can be seen from the figure that when the pH of the buffer varies from 5.5 to 3.5, the Danyl group emits the optimal fluorescence intensity at 485 nm, while R6G emits th...

Embodiment 3

[0035] Example 3: Localization of Danyl-R6G in L929 cells

[0036] L929 cells (from American Type Culture Collection) were cultured in a medium containing 0.1 mg / ml Danyl-R6G and Lyso Tracker green DND 26 for 2 hours, removed the culture medium and washed twice, replaced with new medium and continued to culture for half an hour . Followed by fluorescence confocal instrument (Zeisis 780) analysis, the results are shown in Figure 5A , 5B , 5C, 5D, where Figure 5A Fluorescence for LysoTracker Blue DND 26, Figure 5C is the fluorescence of the R6G-lactam group, Figure 5B is the fluorescence of the Dansyl group. Figure 5D The superimposed picture of the three shows that the distribution position of the three in the cell is the same. The scale length is 10 μm. It can be seen that the R6G signal ( Figure 5C ) can be clearly detected with Danyl-stained fractions ( Figure 5B ) similarly, suggesting that lysosomal pH regulates the ring opening of the R6G group of the pr...

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Abstract

The present invention relates to an acid-sensitive double light emission probe, a preparation method and uses thereof, wherein rhodamine 6G containing an intramolecular amide ring and dansyl chloride are linked to both ends of diethylenetriamine through amide linkages. The preparation method comprises that: rhodamine 6G is subjected to aminolysis in the presence of diethylenetriamine to obtain a colorless solid; and dansyl chloride reacts with the obtained colorless solid to obtain Danyl-R6G. The acid-sensitive dual light emission probe is provided for detecting the pH value of lysosome in cells. According to the present invention, different response performances of the rhodamine 6G derivative containing the intramolecular amide ring and the dansyl group to the pH value are adopted to achieve sensitive pH ratio determination; and synthesis of the probe is simple, and the method for detecting the pH value of lysosome by using the probe is sensitive, simple and rapid.

Description

technical field [0001] The invention relates to the field of probes, in particular to an acid-sensitive dual-light emission probe, a preparation method and an application thereof. Background technique [0002] The pH profile in eukaryotic cells is organelle-specific. For example, the cytoplasm is nearly neutral (pH 7.2) while the lysosome is acidic (pH 6.0-4.0). The acidic environment in the lysosome has an important impact on the processes of endocytosis, autophagy and apoptosis in eukaryotic cells. Changes in lysosome acidity can be observed in processes such as cell maturation and cancer cell proliferation, so detecting lysosome pH is of great significance for related scientific research. [0003] The single fluorescence intensity method based on a single luminophore has many shortcomings in practical applications, such as uneven distribution of probes in the sample, uneven sample thickness, etc., which make quantitative measurement difficult. In contrast, the ratiomet...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D491/107C09K11/06G01N21/64
Inventor 朱娅琴杨慧
Owner 厦门生光生物科技有限公司
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