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Thermosensitive Fluorescent Compounds and Their Applications

一种荧光化合物、化合物的技术，应用在细胞检测领域，能够解决不均匀，只能看到一些小、细胞干扰、破坏等问题

Active Publication Date: 2018-06-15
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this temperature-sensitive fluorescent nanomaterial needs to be introduced into the cells by injection, causing interference and damage to the cells; and it can be seen from the reported fluorescent images that the distribution of the nanomaterials on the cells is very uneven, and only See some small bright spots[1], and the fluorescence intensity of temperature-sensitive fluorescent materials is not only related to temperature, but also related to its concentration distribution. Simply averaging the fluorescence intensity on the entire cell to reflect the temperature of the cell may have certain problems

Method used

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  • Thermosensitive Fluorescent Compounds and Their Applications
  • Thermosensitive Fluorescent Compounds and Their Applications
  • Thermosensitive Fluorescent Compounds and Their Applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0272] Example 1. Synthesis of Rh101AM and RhBAM

[0273] Rh101 (purchased from Santa Cruz), cesium fluoride, and bromoacetic acid were mixed and dissolved in ten times the amount of dimethylformamide (DMF) at a ratio of 1:2:1.2, and stirred at room temperature for 2 hours. Then, Rh101AM (compound represented by formula II) was obtained by separation and purification by preparative high-performance liquid chromatography.

[0274] The synthesis method of RhBAM is similar to that of Rh101AM:

[0275] RhB (purchased from Santa Cruz), cesium fluoride, and bromoacetic acid were mixed and dissolved in ten times the amount of dimethylformamide (DMF) at a ratio of 1:2:1.2, and stirred at room temperature for 2 hours. Then, RhBAM (compound represented by formula III) was obtained by separation and purification by preparative high-performance liquid chromatography.

Embodiment 2

[0276] Example 2. Synthesis of Rh101ME and RhBME

[0277] Mix Rh101 and thionyl chloride at a ratio of 1:5 and dissolve in ten times the amount of chloroform, heat to 60°C and stir for 10 minutes. Then the mixture was cooled to room temperature and then quenched with methanol, and then the solvent was removed by a rotary evaporator under negative pressure, and Rh101ME (compound represented by formula IV) was obtained by separation and purification by preparative high-performance liquid chromatography.

[0278] The synthesis method of RhBME is similar to that of Rh101ME:

[0279] Mix RhB and thionyl chloride at a ratio of 1:5 and dissolve in 10 times the amount of chloroform, heat to 60°C and stir for 10 minutes. Then the mixture was cooled to room temperature and then quenched with methanol, and then the solvent was removed by a rotary evaporator under negative pressure, and RhBME (compound represented by formula V) was obtained by separation and purification by preparative h...

Embodiment 3

[0280] Example 3. Using Rh101AM to measure cytoplasmic temperature distribution

[0281] Live cells were stained with Rh101AM and then imaged under a fluorescence microscope, and the fluorescence image was calculated using formula (1) to obtain an image of the distribution of intracellular temperature.

[0282] image 3 After staining HepG2 cells with 200nM Rh101AM in a cell culture incubator at 37°C for 60min, use EMCCD under a fluorescence microscope (BX61WI, Olympus Ltd., 40x mirror, numerical aperture NA 0.8, culture medium temperature 27.9°C for imaging) (Evolve 512, Photometrice Ltd.) Captured Stokes luminescence images. in image 3 (a) is a Stokes luminescence image formed by a monochromator (Optoscan monochromator, Cairn Research Ltd.) excited at a wavelength of 555nm (bandwidth 3nm) and collected at 573-613nm, image 3 (b) is the anti-Stokes luminescence image formed by the monochromator excited at a wavelength of 635nm (bandwidth 15nm) and received at 573-613nm, u...

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Abstract

New thermosensitive fluorescent compounds and their applications. The invention provides compounds represented by formula I, wherein R9 is an alkyl group of 1-3 carbon atoms substituted by a hydrocarbon group of 1-22 carbon atoms or an ester group of 2-3 carbon atoms, R5, R6, R7, R8 All are hydrocarbon groups or H, and R1, R2, R3, and R4 are all H or lower hydrocarbon groups; or, R9 is a hydrocarbon group of 2-22 carbon atoms or an ester group of 2-3 carbon atoms substituted with 1-3 carbon atoms The alkyl group of the atom is connected with R5 and R1, R6 and R2, R7 and R3, R8 and R4 to form a six-membered ring. The compound of the present invention has temperature-sensitive properties and can enter the cell, thereby obtaining an intracellular temperature distribution image with high spatial and temporal resolution; the compound of the present invention can also perform distribution calibration on the temperature-sensitive fluorescent compound. The invention also provides a method for measuring temperature distribution in living cells and a corresponding detection kit. This method meets the requirements of small size measurement and rapid measurement, thereby achieving high resolution in space and time.

Description

technical field [0001] The invention relates to the field of cell detection. In particular, the present invention relates to novel fluorescent dyes and the use of such novel fluorescent dyes to detect temperature distribution in living cells. Background technique [0002] During cellular activities such as metabolism, enzyme reaction, cell division, gene expression, etc., the temperature of the cell will change to a certain extent. These cellular activities are generally accompanied by the release of chemical energy in ATP, which generates heat and raises the temperature. In addition, when cells are stimulated by external drugs or signals, their metabolic activity will change rapidly, resulting in drastic fluctuations in intracellular temperature. However, due to the influence of heat exchange in the extracellular environment, these intracellular temperature changes are usually local and transient, so it is difficult to measure such intracellular temperature changes with t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D491/22C07D311/82C07D471/04C09K11/06C09B57/00G01K11/00
CPCC07D471/04C07D491/22C09K11/06C09K9/02C09K2211/1033C09K2211/1048C09K2211/1088G01K11/20C09B11/24C07D311/82C07D471/22C09K2211/1044
Inventor 康建胜谢涛嵘刘春凤
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI