A method for constructing artificial cells based on Janus magnetic nanoparticles and a DNA directional immobilization technology

A magnetic nanoparticle, directional immobilization technology, applied in biochemical equipment and methods, immobilized on or in inorganic carriers, enzymes, etc., can solve the problems of complex preparation process, poor activity and reusability, difficult separation, etc. The effect of simple preparation process, easy separation and simple method

Active Publication Date: 2020-04-10
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

Although these artificial cells have been proven to have good enzymatic activity and stability, the preparation process of such artificial cells is co

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Preparation of Janus magnetic nanoparticles (sspDNA@MJNPs) modified by single-stranded DNA

[0027]Add 300 μL of phosphate buffered saline to 0.5OD sspDNA, and vortex until the DNA is completely dissolved. 150 μL of DNA solution was added to Janus magnetic nanoparticles (MJNPs), and reacted for 3.5 hours at 37° C. on a shaker with a rotational speed of 400 rpm. After the product was washed twice with buffer A, 3 mL of bovine serum albumin solution (BSA, 5% wt) was added, and reacted for 30 min at 37° C. in a shaker with a rotation speed of 400 rpm. Click to close. After the reaction, the sspDNA@MJNPs were washed three times with buffered saline solution and stored at 4°C for future use.

Embodiment 2

[0028] Embodiment 2: the preparation of enzyme-single-stranded DNA conjugate complex (sscDNA-GOx and sscDNA-HRP)

[0029] Add 200 μL of phosphate buffer solution to 0.5OD sscDNA, vortex until the DNA is completely dissolved, then add 60 μL of TCEP aqueous solution (30 mM), and react for 1 h at 25°C on a shaker at 400 rpm. After the reaction, the mixture was filtered and washed with a 3K ultrafiltration centrifuge tube for 6 times, 22min each time, and the speed of the centrifuge was 8000xp to remove the DNA that did not participate in the reaction. Weigh 2 mg GOx (or HRP) and dissolve in 200 μL buffer solution, and vortex to mix evenly. Weigh 1 mg suflo-SMCC and dissolve it in 200 μL buffer solution by ultrasonic, add it into the GOx solution, and vortex to mix well. The mixture was placed in a shaker, and reacted for 1 h at a shaker temperature of 25° C. and a rotation speed of 400 rpm. After the reaction, the mixture was filtered and washed with a 10K ultrafiltration centr...

Embodiment 3

[0030] Example 3: Encapsulation and immobilization of enzymes with polytannic acid is used to prepare artificial cells.

[0031] (1) Preparation of single-stranded DNA-modified magnetic nanoparticles (sspDNA@MJNPs): same as Example 1 and Example 2.

[0032] (2) The single-stranded DNA modified magnetic nanoparticles prepared in Example 1, the enzyme-ssDNA conjugate complex prepared in Example 2, and 1 mL of phosphate buffer solution were placed in a shaker at 37° C. with a rotation speed of 400 rpm After reacting for 3 hours, the obtained DNA-directed immobilized enzyme (GOx-HRP@DNA@MJNPs) was obtained. Next, tannic acid (TA) was dissolved in 500 μL HEPES buffer solution (25 mM, pH=7.4), and GOx-HRP@DNA@MJNPs (200 μL, 1 mg mL -1 ) to form a suspension, mix the suspension for 4 hours, separate the product with a magnet, wash three times with a buffer solution, and obtain artificial cells.

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PUM

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Abstract

The invention discloses a method for constructing artificial cells based on Janus magnetic nanoparticles and a DNA directional immobilization technology, and belongs to the field of artificial cell preparation. The preparation method comprises the following steps: firstly, preparing single-stranded DNA modified Janus magnetic nanoparticles and complementary single-stranded DNA functionalized glucose oxidase and horseradish peroxidase; mixing the Janus particles with an enzyme, and realizing immobilization of the enzyme through DNA complementary hybridization; jointly incubating immobilized double enzymes and tannic acid, realizing encapsulation of the immobilized enzymes through self-polymerization of the tannic acid, and constructing the artificial cell with a compartment structure. The artificial cell prepared by the method is simple in preparation process, mild in condition, high in internal enzyme activity, easy to separate from a reaction system and excellent in stability, and canbe used for simulating cascade reaction in natural cells.

Description

technical field [0001] The invention belongs to the technical field of artificial cell preparation, and specifically relates to a method for simulating cell membranes by preparing Janus magnetic nanoparticles, double-stranded DNA complementation-mediated technology, immobilizing enzymes, and tannic acid self-polymerization encapsulating enzymes. Background technique [0002] Cells are the basic building blocks of life, and in-depth studies of various cellular activities (eg, biological cascades, metabolism, and reproduction) can effectively help us understand the origin of life. Although single living cells have proven to be powerful tools for studying cellular activities in vitro, their inherent complexity and fragility severely hamper their further applications. At present, researchers have prepared a variety of artificial cells. The purpose of this bottom-up approach in synthetic biology is to design compartmentalized microreactors to help us understand and manipulate wha...

Claims

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Application Information

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IPC IPC(8): C12N11/18C12N11/14
CPCC12N11/18C12N11/14C12N9/0006C12N9/0065C12Y101/03004C12Y111/01007
Inventor 杨屹沈昊苏萍宋佳一周梓昕贺雯婷
Owner BEIJING UNIV OF CHEM TECH
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