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Difunctional fluorescent probe for detecting lipid droplets and/or protein aggregates as well as preparation method and application of difunctional fluorescent probe

A fluorescent probe and protein technology, applied in the field of fluorescence detection, can solve the problems of confusing probe data acquisition, unable to detect LDs and PAs at the same time, etc., and achieve the effects of reducing difficult-to-separate impurities, preventing side reactions, and simple synthesis methods.

Active Publication Date: 2021-11-05
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, currently available fluorescent probes can only be used to detect LDs or PAs
Although LDs and PAs play important interactions during aging, none of them can simultaneously detect LDs and PAs
Although fluorescent probes for LDs and fluorescent probes for PAs may be used together to obtain dual signals of LDs and PAs, due to the different uptake, distribution, and metabolic properties of the two probes in biological systems, the data acquisition of the two probes will be confusing

Method used

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  • Difunctional fluorescent probe for detecting lipid droplets and/or protein aggregates as well as preparation method and application of difunctional fluorescent probe
  • Difunctional fluorescent probe for detecting lipid droplets and/or protein aggregates as well as preparation method and application of difunctional fluorescent probe
  • Difunctional fluorescent probe for detecting lipid droplets and/or protein aggregates as well as preparation method and application of difunctional fluorescent probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] The synthesis of fluorescent probes, the preparation steps are summarized as follows:

[0066] Step 1. Weigh m-diethylaminophenol and dissolve it in a mixed solution of concentrated hydrochloric acid and water, then weigh sodium nitrite and dissolve it in water. Subsequently, the hydrochloric acid solution of m-diethylaminophenol was placed in an ice bath environment, and the sodium nitrite solution was added dropwise. After the dropwise addition was completed, the reaction was continued to stir in an ice bath. After the reaction was completed, the resulting solid was collected by filtration with a Buchner funnel, and then washed with saturated sodium acetate solution. Finally, the obtained solid matter was purified by recrystallization from acetone to obtain red crystals of 5-(diethylamino)-2-nitrosophenol.

[0067] Step 2. Dissolve 5-(diethylamino)-2-nitrosophenol in the flask with absolute ethanol, and then drop 85% hydrazine hydrate into the flask. Under the prot...

Embodiment 2

[0079] Fluorescent responses of the fluorescent probes obtained in Example 1 to various biomolecules.

[0080] With triolein, sarcosine, arginine, cysteine, homocysteine, DNA, RNA, glucose, vitamin C, 30% H 2 o 2 and OH as raw materials to prepare stock solutions of various biologically relevant substances. Test solutions were prepared by placing 25 μL of LW-1 stock solution and appropriate aliquots of each biologically relevant species stock solution into 5 mL volumetric flasks and then diluting to 5 mL with 50 mM potassium phosphate buffer (pH 7.4). The resulting solution was shaken and incubated at room temperature for 10 min before recording the fluorescence spectrum (λex = 540 nm and 600 nm). Test results such as figure 2 shown.

[0081] From figure 2 It can be found that only triolein can significantly enhance the red fluorescence of the fluorescent probe, while adding other biomolecules such as sarcosine, arginine, cysteine, homocysteine, DNA, RNA, Glucose, Vita...

Embodiment 3

[0085] Selectivity comparative test of fluorescent probe obtained in Example 1 and commercial lipid droplet probe Nile Red (Nile Red, NR).

[0086] Stock solutions of various biologically related substances were prepared from bovine serum albumin (BSA) and egg whites. Test solutions were prepared by placing 25 μLW-1 stock solution and appropriate aliquots of each biologically relevant species stock solution into 5 mL volumetric flasks and then diluting to 5 mL with 50 mM potassium phosphate buffer (pH 7.4). The resulting solution was shaken and incubated at room temperature for 10 min before recording the fluorescence spectrum (λex = 540 nm and 600 nm). Test results such as Figure 4 , Figure 5 shown.

[0087] From Figure 4 , Figure 5 From the results, it can be found that the probe LW-1 will not respond to BSA and egg white, but Nile red will have fluorescence enhancement. Therefore, compared with the commercial lipid droplet probe Nile Red, the specificity of the pr...

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Abstract

The invention belongs to the technical field of fluorescence detection, and discloses a difunctional fluorescent probe for detecting lipid droplets and / or protein aggregates as well as a preparation method and application of the difunctional fluorescent probe. The bifunctional fluorescent probe for detecting lipid droplets and / or protein aggregates is obtained through a series of steps such as preparation of 5-(diethylamino)-2-nitrosophenol and the like. After the synthesis method is improved, side reactions can be effectively prevented, and impurities difficult to separate in the reaction process are greatly reduced, so that a high-purity target product can be obtained. The difunctional fluorescent probe prepared by the method of the invention not only can be used for independently detecting the lipid droplets and the protein aggregates, but also can be used for simultaneously distinguishing the lipid droplets and the protein aggregates in cells, and also can be used for distinguishing and detecting fluorescence imaging of the lipid droplets and / or the protein aggregates in intestinal tissues of aged mice.

Description

technical field [0001] The invention relates to the technical field of fluorescence detection, in particular to a bifunctional fluorescent probe for detecting lipid droplets and / or protein aggregates, a preparation method and application thereof. Background technique [0002] The aging of the world's population is becoming more and more serious, and the health problems of the elderly have aroused great concern. Aging is a complex physiological process that often leads to structural and functional decline of tissues and organs, thereby increasing the risk of various diseases, such as cancer, neurodegenerative diseases, cardiovascular diseases, and diabetes. Over the past few decades, multiple mechanisms have been proposed for aging. More and more evidence shows that, in addition to epigenetic factors, cell damage caused by abnormal cell metabolism is also one of the causes of aging. However, the molecular mechanisms of aging are still not fully understood. Understanding th...

Claims

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Application Information

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IPC IPC(8): C07D265/38C09K11/06G01N21/64
CPCC07D265/38C09K11/06G01N21/6458C09K2211/1033
Inventor 龙凌亮袁芳刘卫国陈倩李璐璐陈晓东何丹
Owner JIANGSU UNIV
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