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Method for quickly modifying avidin on interface based on lipidosome

A technology of liposome and avidin, which is applied in the direction of immobilization on or in the inorganic carrier, can solve the problems of agglomeration of impurities on the glass surface, influence of single-molecule fluorescence observation, incomplete interface sealing, etc., to achieve a clean background, Uniform interface modification and time-saving effect

Inactive Publication Date: 2017-05-31
SHANGHAI NAT ENG RES CENT FORNANOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above method has been widely used in single-molecule research. However, this method is time-consuming and cumbersome, and because BSA and PEG are easy to agglomerate, it is easy to appear uneven modification and incomplete interface sealing during the modification process.
At the same time, when modifying with biotinylated polyethylene glycol, it is necessary to silanize the glass in advance, which will easily cause the aggregation of impurities on the glass surface and affect the observation of single-molecule fluorescence.

Method used

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  • Method for quickly modifying avidin on interface based on lipidosome
  • Method for quickly modifying avidin on interface based on lipidosome
  • Method for quickly modifying avidin on interface based on lipidosome

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Dissolve DOPC and biotinylated phosphatidylethanol in 5 mL of chloroform at a ratio of 100:1, mix well, add flowing nitrogen to dry, and then vacuum dry for 3 hr to remove residual organic solvent. Add PBS (pH=7.4) to 1 mL of the obtained phospholipids to hydrate the monolayer phospholipids to a final concentration of 5 mg / mL. The liposomes with biotin can be obtained by extruding 20 times with an extruder with a pore size filter membrane of 50 nm. The slides were ultrasonicated with lotion and ultrapure water for 10 minutes, then ultrasonicated with acetone and sodium hydroxide solution for 30 minutes, rinsed with ultrapure water, soaked in 1% hydrofluoric acid solution for about 30 seconds, and then cleaned with ultrapure water. Rinse with pure water and blow dry with nitrogen gas for later use. A fence was attached to the glass surface, and 100 μL of the prepared liposomes were added to the fence, followed by incubation at 45°C for 10 minutes. Subsequently, the exc...

Embodiment 2

[0028] Dissolve DOPC and biotinylated phosphatidylethanolamine in 5 mL of chloroform at a ratio of 100:1, mix well, add flowing nitrogen to dry, and then vacuum dry for 3 hr to remove residual organic solvent. Add 1 mL of PBS (pH = 7.4) to the resulting phospholipids to hydrate the monolayer of phospholipids to a final concentration of 5 mg / mL. The liposomes with biotin can be obtained by extruding 20 times with an extruder with a filter membrane with a pore size of 100 nm. The slides were ultrasonicated with lotion and ultrapure water for 10 minutes, then ultrasonicated with acetone and sodium hydroxide solution for 30 minutes, rinsed with ultrapure water, soaked in 1% hydrofluoric acid solution for about 30 seconds, and then cleaned with ultrapure water. Rinse with pure water and blow dry with nitrogen gas for later use. A fence was attached to the glass surface, and 100 μL of the prepared liposomes were added to the fence, followed by incubation at 45°C for 10 minutes. Su...

Embodiment 3

[0030] Dissolve DOPC and biotinylated phosphatidylethanolamine in 5 mL of chloroform at a ratio of 100:1, mix well, add flowing nitrogen to dry, and then vacuum dry for 3 hr to remove residual organic solvent. Add PBS (pH = 7.4) to the resulting phospholipids to hydrate the monolayer phospholipids to a final concentration of 5 mg / mL. The liposomes with biotin can be obtained by extruding 20 times with an extruder with a 200 nm pore size filter. The slides were ultrasonicated with lotion and ultrapure water for 10 minutes, then ultrasonicated with acetone and sodium hydroxide solution for 30 minutes, rinsed with ultrapure water, soaked in 1% hydrofluoric acid solution for about 30 seconds, and then cleaned with ultrapure water. Rinse with pure water and blow dry with nitrogen gas for later use. A fence was attached to the glass surface, and 100 μL of the prepared liposomes were added to the fence, followed by incubation at 45°C for 10 minutes. Subsequently, the excess liposom...

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Abstract

The invention relates to a method for quickly modifying avidin on an interface based on lipidosome. The method comprises the steps of mixing phospholipid and biotinylated phosphatidyl ethanol, adding chloroform for ultrasonic stirring, drying under nitrogen, and vacuum drying; adding a neutral solution, matching films with different bore diameters for a squeezer, and squeezing for multiple times so as to form lipidosomes with different sizes; after cleaning a glass slide, soaking in a hydrofluoric acid solution with the concentration being 1 percent for dozens of seconds, then washing with pure water, and drying under the nitrogen; putting the glass slide into a clean utensil, dropwise adding a certain amount of synthetic lipidosome, incubating for several minutes at the temperature higher than the phase-transition temperature, and using super-pure water for cleaning excessive lipidosome; later, adding the avidin for incubating for several minutes, using the super-pure water or solution to clean excessive avidin, and obtaining a glass interface with the avidin modified on the surface. The method is simple, convenient and quick in modification steps, uniform in modification, and complete in sealing, and since silanization reagents are not introduced during a modification process, the background interference is less. The prepared interface can meet the requirement on single molecule enzymology research.

Description

technical field [0001] The invention relates to a method for rapidly modifying the interface with avidin by using liposome as a medium. The invention belongs to the field of nano interface materials. Background technique [0002] The field of unimolecular enzymology, which takes a single enzyme molecule as the research object, is a hot spot and frontier of scientific research. Usually, single-molecule enzymology research uses a certain method to immobilize single-molecule enzymes, and uses single-molecule fluorescence microscopy to record real-time changes in the intensity of fluorescent signals generated by enzymes during the catalytic process, thereby reconstructing the catalytic process of single enzyme molecules unique dynamic process in . From a microscopic perspective, unimolecular enzymology reveals the dynamic fluctuations of individual enzyme molecules and substrates hidden under the overall average level, which deepens people's understanding of different reaction...

Claims

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Application Information

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IPC IPC(8): C12N11/14
CPCC12N11/14
Inventor 何丹农徐艳王萍金彩虹
Owner SHANGHAI NAT ENG RES CENT FORNANOTECH
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