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Single-molecule fluorescent probe capable of simultaneously imaging lipid droplets and endoplasmic reticulum in two colors and application of single-molecule fluorescent probe

A fluorescent probe, two-color imaging technology, applied in the field of fluorescent probes

Inactive Publication Date: 2021-01-22
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Searched for information on single-molecule imaging of lipid droplets and endoplasmic reticulum simultaneously in two colors using excited-state intramolecular p

Method used

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  • Single-molecule fluorescent probe capable of simultaneously imaging lipid droplets and endoplasmic reticulum in two colors and application of single-molecule fluorescent probe
  • Single-molecule fluorescent probe capable of simultaneously imaging lipid droplets and endoplasmic reticulum in two colors and application of single-molecule fluorescent probe
  • Single-molecule fluorescent probe capable of simultaneously imaging lipid droplets and endoplasmic reticulum in two colors and application of single-molecule fluorescent probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: the synthesis of probe EPC

[0032] A mixture consisting of 2'-hydroxyacetophenone (1 mL, 8.3 mmol) and 4-diethylaminobenzaldehyde (8.3 mmol) was added to the flask with ethanol (15 mL) as solvent. Then NaOH (66.4 mmol) dissolved in 2 mL of water was added to the flask and stirred at room temperature for 24 hours. To this mixture were then added NaOH (16.6 mmol), 2 mL of water and 3 mL of 30% H 2 o 2 , stirred at room temperature for 15 minutes, and then heated to 50° C. for 5 hours. After cooling to room temperature, the reaction system was neutralized to neutrality, then extracted with dichloromethane and washed with water three times. The residue was recrystallized to obtain a yellow powder product, namely 2-(4-(diethylamino)phenyl)-3-hydroxy-4H-chromen-4-one, referred to as EPC. The yield was 85%.

[0033] 1 H NMR (400MHz, DMSO-d 6 ):δ(ppm)9.08(s,1H),8.17-8.02(m,3H),7.80-7.64(m,2H),7.47-7.41(m,1H),6.96-6.69(m,2H),3.43 (q,J=7.2Hz,4H),1.14(t,J=7....

Embodiment 2

[0034] Example 2: Sensitivity test of probe EPC to changes in water content

[0035] Prepare a 2 μM EPC solution with 1,4-dioxane-water mixed solvent containing different volumes of water (0%, 2%, 4%, 6%, 8%, 10%), and then use ultraviolet light under 365nm excitation Naked-eye photographs obtained by light irradiation ( figure 1 Figure A); the fluorescence emission spectrum ( figure 1 Figure B), and get the corresponding CIE1931 coordinates ( figure 1 Figure C).

[0036] Depend on figure 1 As a result, it can be seen from the naked eye photos that EPC is in 1,4-dioxane-water mixed solvent containing different volumes of water (0%, 2%, 4%, 6%, 8%, 10%) Fluorescence of different colors was produced. With the increase of moisture, the fluorescence intensity of the T* state remained almost unchanged, but the fluorescence intensity of the N* state was significantly enhanced, and the fluorescence color also changed from orange-yellow to green. The corresponding CIE1931 coordi...

Embodiment 3

[0037] Embodiment 3: Preparation of live cell samples for testing

[0038] HeLa cells and SiHa cells were cultured in high glucose medium (H-DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. Mesenchymal stem cells (MSCs) were cultured in H-DMEM containing 10% FBS and 1% penicillin and streptomycin. At 37°C, 5% CO 2 Passage once every 2-3 days in an incubator with saturated humidity.

[0039] When the cells grow to the logarithmic phase, culture the slices: ① Soak the coverslips in absolute ethanol for 30 minutes, dry them with an alcohol lamp and place them in a disposable 35mm culture dish for later use; Wash the cells three times with PBS, digest with 1mL 0.25% trypsin for 3-5 minutes, pour out the trypsin carefully, add fresh culture medium and pipette evenly, and count the cells. The concentration is 1×10 per ml 5 , and then inoculated into the above-mentioned petri dish containing the coverslip in 5% CO 2 Cultivate in an incubator at 3...

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Abstract

The invention discloses a single-molecule double-target fluorescent probe capable of simultaneously imaging lipid droplets and endoplasmic reticulum in two colors and revealing the relationship between the lipid droplets and the endoplasmic reticulum, and the single-molecule double-target fluorescent probe is 2-(4-(diethylamino) phenyl)-3-hydroxy-4H-chromene-4-ketone, called EPC for short; the probe can realize real-time imaging of lipid droplets and endoplasmic reticulum in living cells and tissues at the same time, so that mature lipid droplets show orange-yellow fluorescence, and the endoplasmic reticulum shows green fluorescence. The probe realizes simultaneous distinguishing of lipid droplets and endoplasmic reticulum based on ESIPT reaction by utilizing fluorescence color change caused by tiny difference of water contents of the lipid droplets and the endoplasmic reticulum. The invention also discloses an application of the probe in in-situ or dynamic simultaneous visual monitoring of the relationship between lipid droplets and endoplasmic reticulum, and an application of the probe in sensitivity test of water content change. Compared with other existing probes, the probe disclosed by the invention has the characteristics of strong color development, strong light stability, accurate positioning and the like, and is expected to be deeply applied to distinguishing lipid droplets from endoplasmic reticulum and verifying the relationship between the lipid droplets and the endoplasmic reticulum.

Description

technical field [0001] The present invention relates to a fluorescent probe capable of simultaneous two-color imaging and its application, in particular to a method that utilizes excited-state intramolecular proton transfer between normal (N*) and tautomer (T*) states ( ESIPT) reaction single-molecule dual-target fluorescent probe capable of simultaneous two-color imaging of lipid droplets and endoplasmic reticulum and its application in dynamic monitoring of lipid droplets and endoplasmic reticulum and revealing their relationship. Background technique [0002] The organelles in the cell have a fine division of labor due to spatial regionalization and functional specialization. They cooperate with each other and closely contact each other to form an organelle interaction network to complete various physiological activities in the organism. Therefore, it is of great value to study the distribution and interaction of different organelles, especially closely related organelles...

Claims

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Application Information

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IPC IPC(8): C07D311/30C09K11/06G01N21/64
CPCC07D311/30C09K11/06G01N21/6428G01N21/6458C09K2211/1088C09K2211/1007
Inventor 郝秋华牛杰郭丽方于晓强
Owner SHANDONG UNIV
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