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Two-photon fluorescent probe for detecting Cys as well as preparation method and application thereof

A two-photon fluorescence and probe technology, used in fluorescence/phosphorescence, chemical instruments and methods, luminescent materials, etc., can solve problems such as lack of deep penetration, avoid phototoxicity, improve fluorescence intensity, and have wide applications. Foreground effect

Inactive Publication Date: 2018-09-04
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Aiming at the lack of deep-penetrating two-photon Cys fluorescent probes in the prior art, the present invention provides a two-photon two-photon probe with less background interference, less scattering interference, high sensitivity and selectivity, and can avoid photobleaching and phototoxicity Cys fluorescent probe

Method used

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  • Two-photon fluorescent probe for detecting Cys as well as preparation method and application thereof
  • Two-photon fluorescent probe for detecting Cys as well as preparation method and application thereof
  • Two-photon fluorescent probe for detecting Cys as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Synthesis of embodiment 1 Co-Cys fluorescent probe

[0039] (1) In a 100 mL round bottom flask, add 1 mmol of 7-diethylamino-3-aminocoumarin (1), heat to 100°C in 20 mL of 1.5 mol / L hydrochloric acid aqueous solution, after the reaction is completed Suction filtration, the filter cake is 7-diethylamino-3-hydroxycoumarin (2). Wherein the hydrochloric acid aqueous solution is; The reaction time is 4 hours, productive rate: 91%; 1 H NMR (400 MHz, DMSO- d 6 ) δ9.51 (s, 1H), 7.29 (d, J =8,8 Hz, 1H), 7.02 (s, 1H), 6.65 (dd, J 1 =8.8Hz, J 2 =2.3 Hz, 1H), 6.51 (d, J =2.3 Hz, 1H), 3.38 (dd, J 1 =14.4Hz, J 2 =7.4 Hz, 5H),7.62 (t, J =7.0 Hz, 6H); 13 C NMR (126 MHz, DMSO) δ 159.50, 152.02, 148.10,137.47, 127.55, 117.53, 109.67, 108.86, 97.41, 44.33, 12.79; the product was directly carried on to the next step without purification:

[0040] ;

[0041] (2) Stir the compound 7-diethylamino-3-hydroxycoumarin (2) obtained in the previous step and acryloyl chloride (3) ...

Embodiment 2

[0045] Example 2 The selectivity of Co-Cys fluorescent probes to different molecules or ions

[0046] The Co-Cys fluorescent probe in Example 1 was prepared into a mother solution with a concentration of 1 mM.

[0047] The following substances: calcium chloride, magnesium chloride, sodium nitrate, sodium nitrite, sodium bisulfite, sodium sulfide, sodium sulfate, ferrous sulfate, potassium iodide, sodium bromide, sodium hypochlorite, hydrogen peroxide, tert-butyl hydroperoxide , sodium pyruvate, formaldehyde, acetaldehyde, chloral, glyoxal, Cys, Hcy and GSH were prepared in phosphate buffer (0.01 mM, pH=7.4) to prepare 5 mL of a stock solution with a concentration of 40 mM.

[0048] Take 22 test tubes, add 25 μL probe mother solution, 1 mL DMSO and the mother solution of each ion or molecule, and control with an equal amount of water instead of interfering substances; dilute to 5 mL with phosphate buffer (0.01 mM, pH=7.4) , so that the final concentration of interfering substa...

Embodiment 3

[0049] Example 3 Fluorescence intensity of Co-Cys under different concentrations of Cys

[0050] Prepare 10 mL of stock solution with a concentration of 100 mM Cys, and dilute with water to a total of 18 concentrations of 0-55 μM, using water as a control. Dilute the Co-Cys mother solution in Example 2 to 5 μM, add different concentrations of Cys respectively, and perform fluorescence detection (λex = 400 nm, λem = 496 nm) after reacting for 50 min to detect the fluorescence intensity in each system, and use the fluorescence intensity -Cys concentration curve, such as Figure 4 shown. It can be seen from the figure that with the increase of Cys concentration, the fluorescence intensity of the reaction system increases. When the Cys concentration is 2.5-50 μM, there is a good fluorescence intensity-concentration linear relationship; when the Cys concentration reaches 55 μM, the fluorescence intensity of the reaction system reaches saturation. state.

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Abstract

The invention provides a two-photon fluorescent probe for detecting Cys with the chemical name of 7-(diethylamino)-2-oxo-2H-chromene-3-acrylate, and the structural formula as shown in the description.The probe provided by the invention has small background interference, small scattering interference and high sensitivity and selectivity, can avoid photobleaching and phototoxicity, and can penetrate into tissue for deep imaging, and has broad application prospects in the field of biomolecule detection.

Description

technical field [0001] The invention relates to a two-photon fluorescent probe for detecting intracellular Cys (cysteine) and an application thereof, belonging to the field of biological thiol fluorescent probes. Background technique [0002] Intracellular thiols such as cysteine ​​(Cys), homocysteine ​​(Hcy) and glutathione (GSH) are substrates that play important roles in physiology, including redox homeostasis and cell growth. Deficiency of Cys can lead to a number of syndromes such as hair loss, lethargy, liver damage, muscle aches, skin damage and weakness. Therefore, it is promising and meaningful to develop the identification and detection of thiol biomolecules in biological samples. [0003] Although biothiols are closely related to many diseases, the metabolic mechanism and pathogenic mechanism of Cys in the body are still not very clear to medical workers. This is mainly due to the lack of simple and rapid methods for monitoring Cys in complex physiological envir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D311/16C09K11/06G01N21/64
Inventor 林伟英唐永和高世滢
Owner UNIV OF JINAN
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