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Ferrocene-methylene blue double-labelled magnetic sphere nano-composite for detecting miRNA and preparation method of magnetic sphere nano-composite

A nanocomposite and methylene blue technology, applied in the field of electrochemical sensing, can solve the problems of inability to distinguish homologous miRNA sequences, low sensitivity, and cumbersome steps, and achieve easy magnetic separation, high detection sensitivity, and simple preparation.

Active Publication Date: 2017-10-24
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, Northern blotting is a semi-quantitative detection method with cumbersome steps, time-consuming and low sensitivity
PCR technology can perform highly sensitive and specific quantitative detection of miRNA, but the method is cumbersome and requires pretreatment of miRNA
Although the Microarray method can quickly and high-throughput detect a large number of miRNA sequences in a short period of time, it is expensive and cannot distinguish homologous miRNA sequences with small base differences.
"CN 105950711A" discloses a ferrocene-labeled magnetic nanocomposite, but the complex can only detect one miRNA sequence

Method used

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  • Ferrocene-methylene blue double-labelled magnetic sphere nano-composite for detecting miRNA and preparation method of magnetic sphere nano-composite
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  • Ferrocene-methylene blue double-labelled magnetic sphere nano-composite for detecting miRNA and preparation method of magnetic sphere nano-composite

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Effect test

Embodiment 1

[0037] Preparation method of ferrocene and methylene blue double-labeled magnetic sphere nanocomposite for detection of miRNA 1

[0038] (1) Preparation of magnetic sphere nano-gold composite

[0039] Mix and shake 8 mL of aminated gold nanoparticles with a diameter of 30-50 nm and 200 μL magnetic balls for 15 min, magnetically separate, remove the supernatant, and add 4.4 mM H 2 o 2 and 2.1mM HAuCl 4 4mL of the mixed solution was shaken for 12h, and the supernatant was removed to obtain the magnetic sphere nano-gold complex;

[0040] (2) Two kinds of hairpin DNA probes were immobilized on the surface of the magnetic sphere gold nanocomposite

[0041] Add 100 μL of a mixture of two hairpin DNA probes with a concentration of 1 μM to the magnetic sphere nano-gold complex, shake at room temperature for 3 h, and magnetically separate, use 50 μL of mercaptohexanol with a concentration of 0.1 mM for 0.5 h, magnetically separate, add 50 μL of bovine serum albumin solution with a ...

Embodiment 2

[0047] Preparation method of ferrocene and methylene blue double-labeled magnetic sphere nanocomposite for detection of miRNA 2

[0048] (1) Preparation of magnetic sphere nano-gold composite

[0049] Mix and shake 6mL of aminated gold nanoparticles with a diameter of 30-50nm and 150μL magnetic balls for 20min, magnetically separate, remove the supernatant, and add 2.2mM H to the magnetic balls. 2 o 2 and 1mM HAuCl 4 2 mL of the mixed solution, shaken for 10 h, removed the supernatant, and prepared the magnetic sphere nano-gold complex;

[0050] (2) Two kinds of hairpin DNA probes were immobilized on the surface of the magnetic sphere gold nanocomposite

[0051] Add 50 μL of a mixture of two hairpin DNA probes with a concentration of 0.5 μM to the magnetic sphere nano-gold complex, shake at room temperature for 4 hours, and magnetically separate, use 75 μL of mercaptohexanol with a concentration of 0.4 mM for 0.5 hours, and magnetically separate. Add 75 μL of bovine serum ...

Embodiment 3

[0057] Preparation method of ferrocene and methylene blue double-labeled magnetic sphere nanocomposite for detection of miRNA 3

[0058] (1) Preparation of magnetic sphere nano-gold composite

[0059] Mix and shake 10 mL of aminated gold nanoparticles with a diameter of 30-50 nm and 250 μL magnetic balls for 25 min, magnetically separate, remove the supernatant, and add 6.6 mM H to the magnetic balls. 2 o 2 and 3.1 mM HAuCl 4 6 mL of the mixed solution, shaken for 15 h, removed the supernatant, and prepared the magnetic sphere nano-gold complex;

[0060] (2) Two kinds of hairpin DNA probes were immobilized on the surface of the magnetic sphere gold nanocomposite

[0061] Add 200 μL of a mixture of two hairpin DNA probes with a concentration of 2 μM to the magnetic sphere nano-gold complex, shake at room temperature for 6 hours, magnetically separate, use 100 μL of mercaptohexanol with a concentration of 0.6 mM for 1 hour, magnetically separate, add 100 μL The bovine serum ...

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Abstract

The invention discloses a ferrocene-methylene blue double-labelled magnetic sphere nano-composite for detecting miRNA and a preparation method of the magnetic sphere nano-composite. The magnetic sphere nano-composite contains the following components: a magnetic sphere nanogold composite, hairpin DNA probes, ferrocene modified nanogold, methylene blue modified nanogold and miRNA, wherein miRNA is complementary with annular parts of the hairpin DNA probes. The preparation method comprises the steps of firstly preparing the magnetic sphere nanogold composite, fixing the two hairpin DNA probes to the surface of the magnetic sphere nanogold composite, carrying out miRNA hybridization, and fixing the ferrocene modified nanogold and the methylene blue modified nanogold. The magnetic sphere nano-composite provided by the invention has the advantages of simplicity in preparation, strong electrochemical responsiveness, high detection sensitivity and the like.

Description

technical field [0001] The invention belongs to the technical field of electrochemical sensing, and in particular relates to a ferrocene and methylene blue double-labeled magnetic sphere nanocomposite for detecting miRNA and a preparation method thereof. Background technique [0002] MicroRNA (miRNA) is an endogenous, non-coding single-stranded RNA containing 17-25 nucleotides, which plays a very important role in the regulation of gene expression. There are many forms of miRNA, the most primitive is pri-miRNA, which is about 300-1000 bases in length, and pri-miRNA forms pre-miRNA with a hairpin structure through the action of Drosha enzyme in the nucleus, and its length is about 70-1000 bases. 90 bases, the pre-miRNA is transported to the cytoplasm and digested by Dicer enzyme to become a mature miRNA with a length of about 17-25 nucleotides. miRNA is closely related to the occurrence and development of tumors, and its expression level can provide an important basis for cl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/26G01N27/327
CPCG01N27/26G01N27/3278
Inventor 王建秀王璟芮衣馨瑶卢知轩
Owner CENT SOUTH UNIV
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