Long-time multicolor fluorescence imaging reagent for cell membranes, and preparation method and purpose thereof

A fluorescence imaging, long-term technology, applied in fluorescence/phosphorescence, chemical instruments and methods, luminescent materials, etc., can solve the problems of cell shedding, intracellular uptake, unable to meet the needs of long-term cell membrane staining observation, etc. The effect of low toxicity, good safety and broad in vivo imaging application prospects

Active Publication Date: 2020-02-25
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently commonly used cell membrane fluorescent dyes (such as DiD family, FM family, and CellMask, etc.) are not ideal for imaging, and they face many problems such as easy endocytosis by cells, insufficient fluorescence stability, and incompatibility with immunofluorescence staining.
Secondly, the imaging effect of most commercially available cell membrane dyes cannot be maintained for a long time, and they are easily absorbed by cells or shed from cells, which cannot meet the needs of long-term cell membrane staining and observation.
[0004] In addition, current commercial membrane dyes are difficult to achieve membrane / wall imaging of bacterial and fungal cells
Finally, most of the existing cell membrane dyes can only realize the imaging of cultured cells in vitro, but cannot realize the cell membrane imaging of cells in vivo (such as zebrafish embryonic epidermal cells)

Method used

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  • Long-time multicolor fluorescence imaging reagent for cell membranes, and preparation method and purpose thereof
  • Long-time multicolor fluorescence imaging reagent for cell membranes, and preparation method and purpose thereof
  • Long-time multicolor fluorescence imaging reagent for cell membranes, and preparation method and purpose thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The synthesis process of the long-time multicolor fluorescence imaging reagent for cell membrane is as follows:

[0041] Step 1, at first weigh the N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol of 27mg, the PAMAM of 5mg (being N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol and PAMAM The molar ratio of substances is 32:1) and dissolved in dimethyl sulfoxide respectively, then mixed rapidly, and reacted at room temperature for more than 4h. After the reaction, the reaction mixture was dialyzed and purified in dimethyl sulfoxide dialysate for 24 h with a dialysis bag with a molecular weight cut-off of 3500, and then the dialysed liquid was replaced with ultrapure water for another 24 h of dialyzed purification. Finally, the purified imaging reagent precursor product was freeze-dried and stored at -20°C for future use;

[0042] Step 2. Weigh 20 mg of the imaging reagent precursor prepared in step 1, dissolve it in sodium carbonate / sodium ...

Embodiment 2

[0045] Similar to Example 1, except that 27 mg of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol in step 1 is replaced by 43.2 mg of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol, That is, the substance ratio of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol to PAMAM is 51.2:1. In step 2, 92.8 μg of TRITC molecules were replaced with 464 μg, that is, the ratio of imaging reagent precursor and TRITC molecules was 1:5 when mixed.

Embodiment 3

[0047] Similar to Example 1, except that 27 mg of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol in step 1 is replaced by 5.4 mg of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol, That is, the amount ratio of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol to PAMAM is 6.4:1. In step 2, 92.8 μg of TRITC molecules were replaced with 278.4 μg, that is, the ratio of imaging reagent precursor and TRITC molecules was 1:3.

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Abstract

The invention provides a long-time multicolor fluorescence imaging reagent for cell membranes, and a preparation method and a purpose thereof. The imaging reagent for the cell membranes is prepared bya two-step method with the steps of (1) grafting a certain number of polyethylene glycol-cholesterol molecules on the surfaces of PAMAM; and (2) grafting different fluorescent molecules on a productobtained in the first step to obtain the final membrane imaging reagent. The imaging agent has the advantages that the preparation is simple; the water dispersibility and the biocompatibility are good; the imaging speed is high; the application range is wide; and the imaging effect maintaining time is long. The types of fluorescent molecules in the imaging reagent are various, so that the cell membrane imaging at different colors can be realized. In addition, besides the excellent membrane imaging capability on various in vitro cultured mammal cells, the reagent can also be used for performingin-situ long-time imaging on the cell membranes / walls of bacteria and fungi and the cell membranes of epithelial cells of living zebrafish embryos.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a long-time multicolor fluorescent imaging reagent for cell membranes and its preparation method and usage. Background technique [0002] The cell membrane has important physiological functions. It not only maintains the intracellular environment of stable metabolism, but also regulates and selects substances to enter and exit the cell, ensuring the relative stability of the intracellular environment and enabling various biochemical reactions to run in an orderly manner. Functionally, the cell membrane is closely related to important life activities such as cell adhesion, migration, proliferation, endocytosis, apoptosis and signal transduction. Changes in the state of the cell membrane can reflect the process of various biochemical reactions in the cell and changes in cell activity to a certain extent, so the study of the structure and function of the cell membrane can ob...

Claims

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Application Information

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IPC IPC(8): G01N21/64C09K11/06C08G81/00
CPCC08G81/00C09K11/06G01N21/6428G01N21/6456G01N21/6486
Inventor 吴富根许可飞贾浩然
Owner SOUTHEAST UNIV
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