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A method for constructing artificial cells based on Janus magnetic nanoparticles and DNA-directed immobilization technology

A magnetic nanoparticle, directional immobilization technology, applied to biochemical equipment and methods, and enzymes immobilized on or in inorganic carriers, can solve problems such as difficult separation, poor activity and reusability, and complicated preparation process, and achieve Easy separation, excellent biocompatibility, and simple preparation process

Active Publication Date: 2022-03-25
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • 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 complicated, it is difficult to separate from the reaction system, the activity and reusability are poor, and there are also great problems in activity.

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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Abstract

The invention discloses a method for constructing artificial cells based on Janus magnetic nanoparticles and DNA directional immobilization technology, which belongs to the field of artificial cell preparation. The present invention is divided into the following steps: first, prepare Janus magnetic nanoparticles modified by single-stranded DNA and glucose oxidase and horseradish peroxidase functionalized by complementary single-stranded DNA; then mix Janus particles with enzymes, and realize by DNA complementary hybridization Enzyme immobilization: co-incubating the immobilized double enzymes with tannic acid, encapsulating the immobilized enzyme through the self-polymerization of tannic acid, and constructing an artificial cell with a compartment structure. The artificial cell of the invention has a simple preparation process and mild conditions, high internal enzyme activity, easy separation from the reaction system, excellent stability, and can be used to simulate cascade reactions inside 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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Patent Type & Authority Patents(China)
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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