Novel fluorescent probe for simultaneously and quantitatively detecting Cys, Hcy and GSH in plasma as well as preparation method and application of novel fluorescent probe

A fluorescent probe and quantitative detection technology, applied in the field of fluorescence detection, can solve the problems of few reports on fluorescent probes, achieve the effects of reducing impurities that are difficult to separate, simple synthesis method, and preventing side reactions

Active Publication Date: 2021-12-10
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, there are few reports on fluorescent probes that can simultaneously recognize Cys and GSH and Cys / Hcy and GSH in different fluorescent channels
However, it remains a great challenge to develop fluorescent probes capable of simultaneously recognizing three biothiols

Method used

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  • Novel fluorescent probe for simultaneously and quantitatively detecting Cys, Hcy and GSH in plasma as well as preparation method and application of novel fluorescent probe
  • Novel fluorescent probe for simultaneously and quantitatively detecting Cys, Hcy and GSH in plasma as well as preparation method and application of novel fluorescent probe
  • Novel fluorescent probe for simultaneously and quantitatively detecting Cys, Hcy and GSH in plasma as well as preparation method and application of novel fluorescent probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The synthesis of fluorescent probes, the specific steps are as follows:

[0041] (1) Add 1.1 g of 4-(diethylamino) salicylaldehyde to a 50 mL flask, dissolve it with 13 mL of n-butanol, then add 0.6 mL of ethyl nitroacetate, stir well, then add 74 μL of piperidine, and then Add 0.26mL of acetic acid, heat the mixture under reflux at 102°C and stir for 11 hours; after the reaction, cool to room temperature, filter the precipitate with suction to obtain a filter cake, wash and purify the precipitate with n-butanol and petroleum ether, and dry in vacuo to obtain 7- (Diethylamino)coumarin-3-nitro orange solid 1.1g, yield 81%.

[0042] (2) Add 3mL of hydrochloric acid solution to a 50mL flask, then add 0.65g of stannous chloride in an ice-bath environment, and then add 0.21g of 7-(diethylamino)coumarin-3-nitro , stirred at room temperature, reacted for 1.5h, added sodium hydroxide to neutralize the pH to 7 under an ice bath, extracted and separated the mixed solution with d...

Embodiment 2

[0046] Fluorescent spectral responses of the fluorescent probes obtained in Example 1 to the fluorescent detection of Cys, Hcy and GSH.

[0047] Prepare a 5 μM fluorescent probe test solution with 20 mM potassium phosphate buffer / DMSO (4:1 v / v, pH 7.4 or 5.6), and set aside. The concentrations of Cys, Hcy and GSH solutions were prepared separately with deionized water to be 1×10 -2 M. With different concentrations of Cys, Hcy and GSH solutions added to the 5 μM fluorescent probe test solution, the excitation wavelengths were 350nm and 475nm respectively, and the fluorescence emission spectrum was tested. The measurement results are as follows: figure 2 shown.

[0048] from figure 2 It can be found from the results that when the pH is 7.4 and the excitation wavelength is 350nm, the fluorescence intensity at the emission wavelength of 508nm has a good linear relationship with Cys, Hcy and GSH in the concentration range of 0-60μM. The YF probe can detect the total concentra...

Embodiment 3

[0050] The selectivity of the fluorescent probes obtained in Example 1 to the fluorescence detection of Cys, Hcy and GSH.

[0051] Prepare test solution according to Example 2, prepare various analytes (Lys, Arg, Ser, Leu, Phe, Ala, Gly, Glu, Val, Gln, K with deionized water) + , Na + , Mg 2+ , Zn 2+ , H 2 o 2 ) The concentration of the solution is 1×10 -2 M. First add 12 equivalents of other possible interfering substances to the 5μM fluorescent probe molecular test solution, including blank, Lys, Arg, Ser, Leu, Phe, Ala, Gly, Glu, Val, Gln, K + , Na + , Mg 2+ , Zn 2+ , H 2 o 2 , and then add 12 equivalents of Cys, Hcy and GSH to these solutions respectively. After mixing for 10 minutes, under the same conditions, the excitation wavelengths of 350nm and 475nm were respectively used for fluorescence spectrum testing to obtain the fluorescence spectrum of each group of solutions.

[0052] from image 3 It can be found from the results that when the system is added...

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Abstract

The invention provides a novel fluorescent probe capable of simultaneously and quantitatively detecting Cys, Hcy and GSH in plasma as well as a preparation method and application of the novel fluorescent probe. The preparation method comprises the following steps: preparing 7-(diethylamino) coumarin-3-nitro group; preparing 7-(diethylamino) coumarin-3-amino group; preparation of the 7-(diethylamino) coumarin-3-hydroxyl group; adding 7-(diethylamino) coumarin-3-hydroxyl group and 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazole into an acetonitrile solution, then adding potassium carbonate, carrying out a stirring reaction at room temperature, and after the reaction is finished, removing a solvent and purifying to obtain the novel fluorescent probe capable of quantitatively distinguishing and detecting Cys, Hcy and GSH at the same time. The invention develops a novel fluorescent probe, and the novel fluorescent probe can quantitatively distinguish and detect the contents of Cys, Hcy and GSH in blood plasma at the same time under the conditions of different pH values and different fluorescent channels.

Description

technical field [0001] The invention relates to the technical field of fluorescence detection, in particular to a novel fluorescent probe for simultaneous quantitative detection and distinction of Cys, Hcy and GSH in plasma, a preparation method and application thereof. Background technique [0002] Cysteine ​​(Cys), homocysteine ​​(Hcy) and glutathione (GSH) are known to be the most abundant small molecule biothiols in the human body, and play roles in many human metabolism and homeostasis. play a key role in. Although the structures and activities of Cys, Hcy and GSH are very similar, they have different physiological and pathological roles. Among them, Cys is involved in enzyme catalysis, detoxification, protein synthesis and metabolism. Abnormal levels of Cys can cause a variety of diseases, such as growth retardation, skin damage, edema, lethargy, and liver damage; elevated levels of Hcy in plasma are considered to be the cause of cardiovascular disease, thrombosis, A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D413/12C09K11/06G01N21/64
CPCC07D413/12C09K11/06G01N21/6486C09K2211/1048C09K2211/1088
Inventor 龙凌亮袁芳刘卫国陈倩陈晓东李璐璐何丹
Owner JIANGSU UNIV
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