PH sensitive probe molecule and application thereof

A CH2, electron-donating group technology, applied in the field of biomedicine, can solve the problems of affecting functional properties, changing physical properties, misleading information, etc., and achieves the effects of mild labeling conditions, high sensitivity, and simple operation

Active Publication Date: 2020-03-17
维思克思生物科技(武汉)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these dye-labeling methods have some shortcomings. First, because the dyes are mostly embedded in the membrane bilayer of extracellular vesicles in a non-covalent manner, this leads to the formation of dye aggregates or clumps of dyes in aqueous solutions similar to exosomes. , may bring misleading information to researchers in exosome uptake experiments; second, the structural modification of the labeled substance after dye labeling will change its physical properties and may also affect its functional properties; third, most The fluorescent signal of the dye is weak in an acidic environment, and cannot be well labeled in a buffer with a low pH

Method used

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  • PH sensitive probe molecule and application thereof
  • PH sensitive probe molecule and application thereof
  • PH sensitive probe molecule and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The preparation of embodiment 1 probe compound

[0037] Synthesis of dyes as figure 1 Shown:

[0038] (1) Synthesis of Compound 2: Compound 1,1,2-trimethyl-1H-benzoindole (3.13g, 15.0mmol) and 3-bromopropionic acid (2.43g, 16.0mmol) were mixed in 5.0mL In toluene, reflux at 100°C for 3 hours under nitrogen protection. After the reaction was monitored by TLC, the reaction solution was gradually cooled to room temperature, and the reaction solution was added dropwise to ether (150mL) solvent. A large number of powder particles were precipitated, and the powder was collected by filtration. Washing with cooled diethyl ether (50 mL) gave a magenta solid powder, compound 2 (4.11 g, yield 76%). HRMS(ESI,m / z) Calcd.for[C 18 h 20 BrNO 2 -Br - ], 282.1489; Found, 282.1613.

[0039] (2) Synthesis of Compound 3: Compound 2 (722.0mg, 2.0mmol) and 4-dianilinobenzaldehyde (546.0mg, 2.0mmol) were mixed in 5.0mL ethanol, refluxed at 80°C for 9h under nitrogen protection, and moni...

Embodiment 2

[0042] The labeling parameter determination of embodiment 2 dyes

[0043]Fluorescence spectroscopic detection of probes in the presence or absence of exosomes. The bladder cancer cell YTS-1 exosomes were used as the experimental object. The probe with a concentration of 50 μM was used for staining at room temperature in the dark for 30 min, and a blank control was set. The result is as Figure 4 Shown: When the probe is not bound to exosomes, the fluorescent signal is very weak, but when combined with exosomes, the fluorescent signal is significantly enhanced.

Embodiment 3

[0044] The screening of the optimum use concentration of embodiment 3 probe

[0045] Optimal concentration of probes for flow cytometry screening. Suspension cell SKM1 was used as the experimental object. Draw about 2.0×10 6 SKM1 cells were placed in a 15mL centrifuge tube, centrifuged at 5000rpm, fixed with 2% paraformaldehyde at room temperature for 15min, blocked with 1% BSA / PBS, resuspended in PBS, and evenly divided into 8 flow tubes, respectively using final concentrations of 0μM, 1μM, 2 μM, 5 μM, 10 μM, 50 μM, 100 μM probes were stained at room temperature in the dark for 30 min, neutralized with 100 mM glycine, washed once with PBS, and resuspended in 100 μL PBS for detection.

[0046] The result is as Figure 5 Shown: Compared with the negative control (probe concentration of 0 μM), the peak value of the cells labeled with 50 μM and 100 μM probes shifted greatly, so the probes with a concentration of 10 μM to 100 μM can better label the cells , 10 μM probes can al...

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Abstract

The invention discloses a pH sensitive probe molecule and an application thereof, and belongs to the technical field of biological medicines. The invention provides a detection method for marking extracellular vesicles based on covalent binding of N-hydroxysuccinimide (NHS) and amino to excite fluorophores. The probe compound has the characteristics of mild labeling conditions, stable product, noneed of additional chemical modification, simple operation, high sensitivity and the like, has strong fluorescence in an environment with the pH value of 2-6, solves the problem of accurate positioning of extracellular vesicles in in-vivo and in-vitro research, and has outstanding advantages for in-vivo tracing of the extracellular vesicles.

Description

technical field [0001] The invention relates to a pH-sensitive probe molecule and its application, belonging to the technical field of biomedicine. Background technique [0002] Extracellular vesicles are a subcellular component released spontaneously or under certain conditions by cells, and are closed spherical vesicles surrounded by lipid bilayers. Vesicles can be classified according to the biosynthesis or release pathway of extracellular vesicles: exosomes (exosomes) have a diameter of 30-150 nm and originate from the endocytic pathway; microparticles / microvesicles (microvesicles) have a diameter of about 100 nm -1000nm, released directly from the plasma membrane; apoptotic body (bleb) about 50nm-2μm in diameter, produced by apoptosis; tumor vesicles (tumorvesicles), about 1-10μm in diameter, released by tumor cells; and Various EV subpopulations. In recent years, extracellular vesicles, such as exosomes, have been the most popular research topic, and this study mostl...

Claims

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Application Information

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IPC IPC(8): C07D209/60C09K11/06G01N21/64
CPCC07D209/60C09K11/06C09K2211/1014C09K2211/1029G01N21/6428
Inventor 关锋周小满宋志辉张建健李想
Owner 维思克思生物科技(武汉)有限公司
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