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Red fluorescent probe, preparation and application of red fluorescent probe in cell imaging

A technology of red fluorescence and cell imaging, which is applied in the direction of fluorescence/phosphorescence, luminescent materials, and material analysis through optical means, can solve the problems of inability to truly feedback the fluorescent labeling of cell membranes, distortion, off-target, etc., and achieve low dark toxicity, Low-cost, easy-to-operate effect

Active Publication Date: 2021-11-30
ANHUI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the traditional membrane probes face off-target or distortion phenomenon, so they cannot truly feedback the fluorescent labeling of the cell membrane

Method used

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  • Red fluorescent probe, preparation and application of red fluorescent probe in cell imaging
  • Red fluorescent probe, preparation and application of red fluorescent probe in cell imaging
  • Red fluorescent probe, preparation and application of red fluorescent probe in cell imaging

Examples

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Embodiment 1

[0030] A method for preparing a red fluorescent probe for high-fidelity temporal-spatial imaging of cancer cell membranes, comprising the following steps

[0031] A, the preparation of intermediate M1

[0032] Mix 1,2-bis(2-iodoethoxy)ethane (1.0g, 2.7mmol, sigma-aldrich), 4-picoline (0.76g, 8.1mmol) and absolute ethanol, 65-67℃ The reaction was refluxed for 24 hours under the condition, and followed by TLC. After the reaction, silica gel column chromatography was performed to obtain a white oil M1 with a yield of 48.3%. 1 H NMR (400MHz, DMSO-d 6 ), δ (ppm): 8.93-8.92 (d, J = 6.48MHz, 4H), 8.06-8.05 (d, J = 6.32MHz, 4H), 4.78-4.76 (t, J = 4.76MHz, 4H), 3.88 -3.86(d,J=4.84MHz,4H),3.52(s,4H),2.66(s,6H). 13 C NMR (100MHz, DMSO-d 6 ), δ (ppm): 159.08, 144.01, 128.04, 69.46, 68.59, 59.43, 21.56; MS (ESI): calcd for: C 18 h 26 N 2 o 2 2+ [M / 2], 151.10; found, 151.0982.B, preparation of MO

[0033] Dissolve M1 (0.2g, 0.36mmol) and 7-(diethylamino)-2-oxo-2H-coumarin-3-carbal...

Embodiment 2

[0035] Colocalization map of the target product MO in HeLa cells

[0036] The excellent optical properties of probe MO prompted us to further explore its application at the cellular level. First, HeLa cells (BeNa culture collection) were selected to evaluate the cellular uptake capacity of MO, such as figure 2 As shown, MO was co-incubated with HeLa for 20 min and excited with 488nm excitation light. The imaging results showed that the target molecule MO could quickly and uniformly light up the cell membrane of HeLa cells (the cell membrane produced obvious red fluorescence, and there was almost no fluorescence in the nucleus). It basically coincides with the outline of bright field cells. In order to further explore the distribution of MO in cells, a commercial dye cell maskgreen (Thermo Fisher, Thermo Fisher) was used. We added cell mask green to the cells that had been incubated with MO and continued to incubate for 15 minutes. After the incubation, the cells were washed...

Embodiment 3

[0038] The uptake of MO by different cell lines and the cytotoxicity test chart

[0039] Considering that the surface of cancer cells usually has more negative charges than normal cells, the positively charged pyridinium salt probes prepared by the present invention theoretically have higher affinity for cancer cells. Based on this, we screened several common cell lines, including four cancer cells (HeLa cervical cancer cells, A549 lung cancer cells, Hep G2 liver cancer cells and MCF-7 breast cancer cells) and three normal cells (293T human embryonic kidney Cells, HL-7702 human hepatocytes and HK2 human renal tubular epithelial cells), among which, HeLa and HepG2 were purchased from BeNa, BeNa culture collection; other cells were from ATCC, American type culture collection. The staining of MO in different cell lines was studied. Interestingly, as expected, only cancer cells showed strong uptake of MO, and the cell membrane could be specifically lit up by the target product MO...

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Abstract

The invention provides a red fluorescent probe and application thereof in high-fidelity space-time imaging of a cancer cell membrane. The probe takes coumarin as an electron donating group (D), C=C is a pi bridge, a positive charge part of pyridinium is an electron withdrawing group (A), and a flexible ether oxygen chain is introduced to prepare an amphiphilic biscoumarin and bipyridinium target product MO with red emission. A cell developing experiment shows that the probe can uniformly brighten cell membranes of cancer cells, can be retained for 6 hours under a dark condition without generating a translocation condition, and realizes real-time high-fidelity imaging. Meanwhile, the developed probe has the characteristics of economical efficiency, good light stability, low cell dark toxicity, wide application range and the like, so that the red fluorescent probe can become potential cancer cell membrane dye as a membrane fluorescent marker.

Description

technical field [0001] The invention relates to a fluorescent imaging fluorescent probe and a preparation method thereof, in particular to a red fluorescent probe which is used for high-fidelity spatiotemporal imaging of cancer cell membranes. Background technique [0002] Biological imaging is an important research tool for characterizing the tissue structure of organisms and understanding their physiological functions. Usually, optical or electron microscopes are used to observe the microstructure of biological cells and tissues, and to analyze various physiological processes of biological cells. Compared with traditional detection methods, bioluminescent imaging has the advantages of high sensitivity, low trauma, high selectivity, and real-time imaging, making it a powerful tool for monitoring biomolecules in living systems. Fluorescence imaging technology has been widely used to monitor the pharmacokinetics of drug molecules because of its simple and flexible operation, ...

Claims

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Application Information

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IPC IPC(8): C07D405/14C09K11/06G01N21/64
CPCC07D405/14C09K11/06G01N21/6428G01N21/6486C09K2211/1029C09K2211/1088G01N2021/6439
Inventor 卜应翠周虹屏朱小姣余志鹏张杰汪联可
Owner ANHUI UNIVERSITY
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