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Preparation method of DNA walker-based coupled magnetic nano-composite, and product and application of nano-composite

A magnetic nano-composite technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of cumbersome operation, large sample requirements, limited biological and biomedical applications, etc., and achieve multiple signal amplification. , easy surface modification, easy magnetic separation effect

Active Publication Date: 2020-06-02
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the Northern blotting method is considered to be the gold standard method for miRNA detection, but it is not suitable for routine clinical diagnosis due to its cumbersome operation, time-consuming and laborious, relatively low sensitivity and large sample requirement (10 μg sample); PCR and gene chip detection methods have the disadvantages of low efficiency, time-consuming, low economy, expensive equipment and complicated operation, which limit their biological and biomedical applications. Therefore, it is of great significance to develop new detection strategies for miRNA
The use of electrochemical sensors to measure miRNA is mainly to detect miRNA through nanoparticle materials combined with DNA probes. Modified nanoparticle materials are of great significance for the detection of low concentrations of miRNA

Method used

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  • Preparation method of DNA walker-based coupled magnetic nano-composite, and product and application of nano-composite
  • Preparation method of DNA walker-based coupled magnetic nano-composite, and product and application of nano-composite
  • Preparation method of DNA walker-based coupled magnetic nano-composite, and product and application of nano-composite

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] In this example, the preparation of magnetic nanocomposites coupled with DNA walkers, the preparation flow chart is as follows figure 1 Shown: includes the following steps,

[0029] (1) Preparation of nano-gold: 1mL of mass concentration of 0.01g / mL of chloroauric acid and 99mL of secondary water are mixed, heated to solution boiling while stirring, then adding 2mL of mass concentration of sodium citrate of 0.02g / mL , continue heating for 15 min, and cool to room temperature to obtain a solution containing gold nanoparticles.

[0030] (2) Immobilization of DNA on the surface of gold nanoparticles: 50 μL of DNA with a concentration of 10 mM modified with a sulfhydryl group at one end and a biotin group at the other end (SH-T 30 -Biotin) was added to 1mL of the nano-gold solution prepared in step (1), reacted overnight, and then added a sodium chloride solution with a concentration of 3M every 2h, adding a total of 5 times, continued to react for 12h, and centrifuged wit...

Embodiment 2

[0034] Detection of miRNA-182 by DNA walker-coupled magnetic nanocomplexes

[0035] (1) Reaction of DNA walker-coupled magnetic nanocomplexes with miRNA-182 and enzyme cleavage: 50 μL concentrations of 0.001pM, 0.04pM, 0.08pM, 0.4pM, 0.8pM, 1.2pM, 1.5pM, 1.8pM, 2pM miRNA-182 solution was added to 20 μL DNA walker-coupled magnetic nanocomposites at a concentration of 5 mg / mL to react for 0.3-1 h, then magnetically separated and washed. Redissolve with the same volume of PBS, add 10μL, 10U / mL Nb.BbvCI endonuclease, cut reaction time is 30min, and then perform magnetic separation and washing to obtain multiple DNA walker-coupled magnetic nanocomposites to be tested thing.

[0036] (2) Pretreatment of the magnetic gold electrode: Polish the magnetic gold electrode with aluminum oxide with a particle size of 0.05 μm on a polishing cloth for 5 to 10 minutes, ultrasonically clean it with secondary water and absolute ethanol for 1 to 2 minutes respectively, and nitrogen blow dry.

...

Embodiment 3

[0040] In this example, the preparation of magnetic nanocomposites coupled with DNA walkers, the preparation flow chart is as follows figure 1 Shown: includes the following steps,

[0041] (1) Preparation of nano-gold: mix 1mL of chloroauric acid with a mass concentration of 0.005g / mL and 80mL of secondary water, heat until the solution boils while stirring, then add 1mL of sodium citrate with a mass concentration of 0.01g / mL , continue heating for 10 min, and cool to room temperature to obtain a solution containing gold nanoparticles.

[0042] (2) Immobilization of DNA on the surface of gold nanoparticles: 50 μL of DNA with a concentration of 15 mM modified with a sulfhydryl group at one end and a biotin group at the other end (SH-T 30 -Biotin) was added to 1.25mL of the nano-gold solution prepared in step (1), reacted for 6h, and then added a sodium chloride solution with a concentration of 3M every 2h, added 5 times in total, continued to react for 12h, and used a high-spe...

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Abstract

The invention discloses a preparation method of a DNA walker-based coupled magnetic nano-composite, and a product and application of the nano-composite. According to the invention, magnetic spheres easy to separate and enrich, gold nanoparticles with large specific surface area and DNA walkers are combined, the magnetic spheres can reduce the non-specific adsorption of interferents, the gold nanoparticles can increase the load capacity of DNA sequences, and the DNA walkers can gradually cut signal molecules under the action of enzymes to cause the changes of electrochemical signals. Compared with a traditional electrochemical detection method, the method avoids the complex steps of modifying the DNA sequences on the surfaces of electrodes, reducing the non-specific adsorption by using blocking agents, etc., has extremely high sensitivity, and can reach the lowest detection concentration of 1 fM for a target miRNA-182. The method of the invention has a wide detection range, ranging from0.001 to 2 pM. The content of miRNA-182 in a normal serum sample is about 0.2-1 pM, and the content of miRNA-182 in a glioma patient is about 1-2 pM. The method of the invention is feasible for the determination of miRNA-182 in the normal serum sample and the glioma patient serum sample.

Description

technical field [0001] The invention belongs to the technical field of biological materials and electrochemical biosensors, and in particular relates to a preparation method of a magnetic nanocomposite based on DNA walker coupling and its product and application. Background technique [0002] MicroRNA (miRNA) is an RNA regulatory factor widely present in eukaryotic cells, usually with a length of about 21 bases, and plays an important role in the regulation of development. Studies have found that the abnormal expression of miRNA is related to a variety of cancers. For example, miRNA-182 is closely related to glioma, which also makes miRNA a new biological marker for cancer diagnosis and provides a new treatment for human diseases. clinical information. However, due to its low content in cells, small size and easy degradation, it is very difficult to quantitatively detect miRNAs in cells. [0003] Traditional miRNA detection methods include: Northern blotting, real-time pol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6825C12Q1/6886
CPCC12Q1/6806C12Q1/6825C12Q1/6886C12Q2600/158C12Q2600/178C12Q2563/137C12Q2563/155C12Q2563/143C12Q2563/131C12Q2525/207C12Q2521/301C12Q2565/607Y02P20/55
Inventor 王建秀卢汉文高娟衣馨瑶
Owner CENT SOUTH UNIV
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