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A non-cleaning cell membrane red fluorescence imaging reagent and preparation method and use thereof

A red fluorescence and cell membrane technology, which is applied in the preparation of test samples, fluorescence/phosphorescence, and material analysis through optical means, can solve problems such as impracticability, achieve good cell membrane affinity, long imaging time, and improve imaging quality effect

Active Publication Date: 2020-03-31
SOUTHEAST UNIV
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  • Description
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  • Application Information

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Problems solved by technology

This is because most of the cell membrane imaging reagents need to go through cleaning operation, which is feasible in in vitro culture, but the cleaning operation cannot be implemented in vivo imaging observation, so the development of cell membrane imaging reagents that can be cleaned without cleaning is the key to realize in vivo imaging. The essential

Method used

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  • A non-cleaning cell membrane red fluorescence imaging reagent and preparation method and use thereof
  • A non-cleaning cell membrane red fluorescence imaging reagent and preparation method and use thereof
  • A non-cleaning cell membrane red fluorescence imaging reagent and preparation method and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0028] The synthesis process of the no-cleaning cell membrane red fluorescent probe is as follows:

[0029] Step 1. First, weigh 1 mg of N-hydroxysuccinimide ester-Cy5 (NHS-Cy5) dye molecule, and dissolve it in a phosphate buffer solution with pH=7.4. Then rapidly with 1.96mg of cholesterol-polyethylene glycol-amino (cholesterol-PEG2000-NH 2 ) molecules are mixed and reacted at room temperature for more than 4 hours;

[0030] Step 2. After the reaction is completed, the solution is purified in ultrapure water using a dialysis bag with a predetermined molecular weight cut-off, then freeze-dried, and stored frozen at -20° C. for future use.

[0031] The reagent obtained by the above method is denoted as cholesterol-polyethylene glycol-Cy5, and the molecular structural formula is shown in figure 1 .

Embodiment 2

[0033] The fluorescent molecule in Example 1 was replaced with NHS-Cy7, and other reaction parameters were the same as those in Example 1.

Embodiment 3

[0035] Observe the staining situation of the cholesterol-polyethylene glycol-Cy5 that embodiment 1 makes to human lung cancer cell (A549) plasma membrane, its method is as follows:

[0036] After A549 cells were cultured in 8-well plates for 24 hours, cholesterol-polyethylene glycol-Cy5 was dispersed in DMEM complete medium at a concentration of 2 μg / mL and added to 8-well plates (200 μL / well). CO 2 Observations were made after incubation in the environment for 10 minutes.

[0037] Confocal fluorescence microscope imaging observation: Cholesterol-polyethylene glycol-Cy5 emits red fluorescence under the excitation light of 638nm, see the results figure 2 . It can be seen from the figure that cholesterol-polyethylene glycol-Cy5 is very uniformly distributed on the cell membrane. Although it was not washed with PBS buffer before imaging, the imaging signal-to-noise ratio was still very high, and there was no red fluorescent interference in the background. Therefore, Choleste...

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Abstract

The invention discloses a no-clean cell membrane red fluorescence imaging reagent and a preparation method and application thereof. The imaging reagent is made from polyethylene glycol macromolecule with two terminals modified with cholesterol and indocyanine dye. The imaging reagent is simple to prepare, good in water solubility and biocompatibility, fast in imaging and free of clean. Furthermore, the reagent achieves in-situ imaging of the cell membranes of embryonic epidermal cells of zebra fish. The reagent plays an important role in cell membrane imaging at a cell and biological living level.

Description

technical field [0001] The invention relates to a no-cleaning cell membrane red fluorescent imaging reagent and its preparation method and application, belonging to the field of biotechnology. Background technique [0002] As a key boundary between cells and the environment, the cell membrane provides a relatively stable internal environment for cells, and also plays a vital role in the process of material transportation, energy conversion, and information transmission between cells and the external environment. Changes in the state of the cell membrane can reflect changes in the morphology and survival state of the cell itself. For example, cells that are about to undergo apoptosis often secrete many apoptotic vesicles on the cell membrane, and when cells are necrotic, large vesicles with a diameter of several microns will be produced on the cell membrane, accompanied by cell swelling and rounding. Cell membrane imaging technology is an important research method in the fie...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/28G01N1/30G01N21/64
CPCG01N1/2806G01N1/30G01N21/6428
Inventor 吴富根贾浩然祝雅璇
Owner SOUTHEAST UNIV
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