Bifunctional fluorescent probe for detecting hypochlorite ion and/or singlet oxygen in cell mitochondria, preparation method and application thereof

A hypochlorite and singlet oxygen technology, applied in the field of fluorescence detection, can solve the problems of data confusion, different distribution and metabolic curves, and inability to respond to hypochlorite ions and singlet oxygen, so as to prevent side reactions and synthetic methods Simple and effective in reducing impurities that are difficult to separate

Active Publication Date: 2022-04-26
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, many excellent hypochlorite ion fluorescent probes and singlet oxygen fluorescent probes have been constructed and applied to fluorescence imaging in living systems, but these fluorescent probes only focus on the selective detection of single Analyte (hypochlorite ion or singlet oxygen)
Although these two reactive oxygen species interact in many relevant biological issues, neither of them can respond to both hypochlorite ion and singlet oxygen
Although it is possible to use a fluorescent probe for hypochlorite ion and a fluorescent probe for singlet oxygen simultaneously in the applied study of such processes, the differences in the absorption, distribution and metabolism profiles of the two fluorescent probes lead to data confusion

Method used

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  • Bifunctional fluorescent probe for detecting hypochlorite ion and/or singlet oxygen in cell mitochondria, preparation method and application thereof
  • Bifunctional fluorescent probe for detecting hypochlorite ion and/or singlet oxygen in cell mitochondria, preparation method and application thereof
  • Bifunctional fluorescent probe for detecting hypochlorite ion and/or singlet oxygen in cell mitochondria, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Synthesis of fluorescent probes.

[0066] The preparation steps are summarized as:

[0067] Step 1, weigh 4-(diethylamino) salicylaldehyde and dissolve it in absolute ethanol, then add diethyl malonate, stir evenly, then add piperidine to obtain mixed solution A, mix the mixed solution AHeat to reflux and stir; after the reaction is completed, cool to room temperature, remove excess solvent, add an equal amount of acetic acid and hydrochloric acid in turn under ice-water bath conditions, and then continue to heat and reflux for reaction; after the reaction, pour the reacted mixture into In ice water, adjust the pH to 5 with sodium hydroxide solution, filter the precipitate under reduced pressure to obtain a filter cake, and recrystallize with absolute ethanol to obtain 7-(diethylamino)coumarin;

[0068] Step 2, at first prepare Wells reagent with equal amount of phosphorus oxychloride and DMF, then the 7-(diethylamino) coumarin that step 1 obtains is dissolved complete...

Embodiment 2

[0085] The fluorescent probe that embodiment 1 obtains is to hypochlorite ion (ClO - ) and singlet oxygen ( 1 o 2 ) detection selectivity.

[0086] Prepare the fluorescent probe molecule WG master solution with a concentration of 5 μM in methanol and set aside. Prepare a concentration of 1×10 with deionized water -3 M of H 2 o 2 , 1 o 2 , GSH, Cys, Mg 2+ , Fe 2+ , Fe 3+ , Zn 2+ solution, through the Fenton reaction to generate hydroxyl radicals (·OH). NO was generated using SNP (sodium(III) nitroferricyanide dihydrate). Use Na 2 MoO 4 / H 2 o 2 generated by the system 1 o 2 . When 300μM of various analyte solutions were added to the test solution containing 5μM fluorescent probe molecules (50mM phosphate buffer solution / methanol (1:4v / v, pH 7.4)), the probe WG (5μM) and Different analytes were incubated in the buffer solution for 30 minutes, and then the excitation wavelength was 480nm, and the fluorescence emission spectrum was measured to detect the respon...

Embodiment 3

[0091] Other common ions or molecules detect hypochlorite ion (ClO) to the fluorescent probe that embodiment 1 obtains - ) and singlet oxygen ( 1 o 2 ) interference experiment.

[0092] Prepare the test solution according to Example 2, first add 300 μM of other possible interfering substances to the 5 μM fluorescent probe molecule test solution, including blank, NO, OH, H 2 o 2 , 1 o 2 , GSH, Cys, Mg 2+ , Fe 2+ , Fe 3+ , Zn 2+ , and then added 300 μM hypochlorite ion (ClO - ). After mixing for 30 minutes, under the same conditions with excitation at 480nm, the fluorescence spectrum test was carried out to obtain the fluorescence spectrum of each group of solutions.

[0093] Prepare the test solution according to Example 2, first add 1.4mM of other possible interfering substances to the 5 μM fluorescent probe molecule test solution, including blank, NO, OH, H 2 o 2 , ClO - , GSH, Cys, Mg 2+ , Fe 2+ , Fe 3+ , Zn 2+ , and then add 1.4mM singlet oxygen ( 1 o 2 ...

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Abstract

The invention belongs to the technical field of fluorescence detection, and discloses a bifunctional fluorescent probe for detecting hypochlorite ions and / or singlet oxygen in the mitochondria of cells and its preparation method and application. By preparing 7-(diethylamino) After a series of steps such as coumarin and 7-(diethylamino)coumarin aldehyde, a bifunctional fluorescent probe for detecting hypochlorite ion and / or singlet oxygen in the mitochondria of cells is obtained. After the improvement of the synthesis method of the present invention, the occurrence of side reactions can be effectively prevented, and impurities that are difficult to separate during the reaction process can be greatly reduced, thereby obtaining a high-purity target product. The bifunctional fluorescent probe prepared by the present invention can not only detect chlorate ion or singlet oxygen alone, but also distinguish the levels of hypochlorite ion and singlet oxygen at the same time, and can also be used for endogenous hypochlorite ion and singlet oxygen in cells. and / or fluorescence imaging of singlet oxygen.

Description

technical field [0001] The invention relates to the technical field of fluorescence detection, in particular to a bifunctional fluorescent probe for detecting hypochlorite ions and / or singlet oxygen in mitochondria of cells, a preparation method and application thereof. Background technique [0002] Reactive oxygen species (ROS) are natural products of normal oxygen metabolism and participate in many important biological activities. Hypochlorite ion (ClO - ) as an important reactive oxygen species, plays an important role in host innate immunity and maintaining intracellular redox balance. On the other hand, singlet oxygen ( 1 o 2 ), as another important reactive oxygen species, plays an important role in both cell signal transduction and gene expression induction. Furthermore, photodynamic therapy (PDT) is a well-known technique for cancer treatment, and singlet oxygen also plays a crucial role in this process. ClO - and 1 o 2 Coexist in living systems, and they can...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07F9/655C09K11/06G01N21/64
CPCC07F9/65522C09K11/06G01N21/6428G01N21/6458C09K2211/1088C09K2211/1014
Inventor 龙凌亮刘卫国韩园园陈倩袁芳李璐璐
Owner JIANGSU UNIV
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