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Electrochemical analysis method for determining activity of T4 polynucleotide kinase based on magnetic nano material

A technology for polynucleotide and electrochemical analysis, applied in the field of electrochemical analysis based on magnetic nanomaterials to determine the activity of T4 polynucleotide kinase, to achieve the effect of improving selectivity, specificity and sensitivity

Active Publication Date: 2021-04-23
YUNNAN MINZU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is a lack of an electrochemical analysis method based on magnetic nanomaterials for the determination of T4 polynucleotide kinase activity that achieves highly sensitive, rapid and quantitative determination of T4 PNK activity

Method used

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  • Electrochemical analysis method for determining activity of T4 polynucleotide kinase based on magnetic nano material
  • Electrochemical analysis method for determining activity of T4 polynucleotide kinase based on magnetic nano material

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Embodiment 1

[0040] An electrochemical method for measuring T4 polynucleotide kinase activity based on magnetic nanomaterials of the present invention

[0041] The present invention is based on Fe 3 o 4 @TiO 2 The principle of the method for detecting T4 PNK activity by magnetic core-shell nanoparticles and AuNPs dual signal amplification technology is as follows: figure 1 shown. First prepare the Fe 3 o 4 @TiO 2 Magnetic core-shell nanoparticles, in the presence of ATP and T4PNK, nucleic acid strand S 1 Phosphorylated at the 5′ end, the phosphorylated end of S1 is passed with TiO 2 Specific reaction, modified to Fe 3 o 4 @TiO 2 Magnetic nanoparticle surfaces. After adding AuNPs-S 2 nanoparticles, AuNPs are modified to Fe due to the hybridization reaction between nucleic acid strands S1 and S2 3 o 4 @TiO 2 On the surface of magnetic nanoparticles, the formation of Fe 3 o 4 @TiO 2 -S1 / S2-AuNPs complex. Finally, the magnetic gold electrode (GME) was used as the working ele...

Embodiment 2

[0043] Oligonucleotide sequence design

[0044] The oligonucleotide sequence designed in the present invention was synthesized by China Shanghai Sangon Bioengineering Co., Ltd., purified and tested by HPLC, and freeze-dried. The oligonucleotide sequence designed by the present invention is as follows: the nucleic acid strand S1 has the nucleotide sequence shown in SEQ ID No.1; the nucleic acid strand S2 has the nucleotide sequence shown in SEQ ID No.2 ;

[0045] Nucleic acid strand S1: 5'-SH-CACTTGGTTGGTGTGGTTGG-3';

[0046] Nucleic acid strand S2: 5′-ACACCAACCAAGTGATGAT-3′; dissolve the oligonucleotide in ultrapure sterilized water and store at -18°C for future use.

Embodiment 3

[0048] Fe 3 o 4 @TiO 2 Preparation of Magnetic Core-Shell Nanoparticles

[0049] Weigh 0.615g FeCl 3 ·6H 2 Dissolve O in 20 mL of ethylene glycol, add 1.8 g of NaAc and 0.5 g of polyethylene glycol, stir the solution, transfer it to a 100 mL Teflon-lined stainless steel autoclave, react at 200 °C for 8 hours, and cool to room temperature , washed several times with ethanol, separated by magnetic force, and air-dried at room temperature to obtain Fe 3 o 4 magnetic nanoparticles. Dissolve 9.6mL glacial acetic acid and 6.8mL butyl titanate in 60mL ethanol, add 0.4g Fe 3 o 4 Magnetic nanoparticles were ultrasonically dispersed for 15 min, 2.4 g of polyethylene glycol and 3.6 g of urea were added, stirred electrically for 1 h, and then the mixture was transferred to a reaction vessel for crystallization at 180° C. for 8 h. After cooling to room temperature, the precipitate was filtered and washed several times with ethanol, and dried at 80 °C for 12 h to give Fe 3 o 4 @T...

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Abstract

The invention discloses an electrochemical analysis method for determining the activity of T4 polynucleotide kinase based on a magnetic nano material, the method comprises the following steps: preparing Fe3O4@TiO2 magnetic core-shell nanoparticles by a hydrothermal method, phosphorylating the 5'end of a nucleic acid chain S1 when ATP and T4 PNK exist, and modifying the surface of the Fe3O4@TiO2 magnetic nanoparticles with the end phosphorylated S1 by specific reaction with TiO2; adding a gold nanoparticle-nucleic acid chain S2 compound (AuNPs-S2), and forming a Fe3O4@TiO2-S1 / S2-AuNPs compound due to a hybridization reaction between the nucleic acid chain S1 and the nucleic acid chain S2; using hexamino ruthenium [Ru(NH3)63+] is used as an electroactivity indicator, and realizing high-sensitivity, rapid and quantitative determination of the activity of T4PNK through magnetic enrichment and electrochemical signal enhancement on the surface of GME.

Description

technical field [0001] The invention relates to the field of biochemical analysis or biochemical technology, in particular to an electrochemical analysis method for measuring T4 polynucleotide kinase activity based on magnetic nanometer materials. Background technique [0002] T4 polynucleotide kinase (T4 PNK) is a protein encoded by the pseT gene of bacteriophage. It has 5' kinase activity and can catalyze the phosphorylation of the 5' hydroxyl terminal of nucleic acid, and DNA recombination, Normal cellular activities such as replication and damage repair are closely related. In addition, T4 PNK is also an important molecular biology tool, and the discovery and application of T4 PNK has promoted the development of molecular biology to a certain extent. At present, T4 PNK has become an indispensable tool enzyme in genetic engineering and bioanalysis research, and is further used in the research of nucleic acid damage repair and enzyme inhibitors. Therefore, the determinat...

Claims

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Application Information

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IPC IPC(8): G01N27/327C12Q1/00C12Q1/48B82Y15/00B82Y40/00
Inventor 张艳丽陶锦彭庞鹏飞王红斌
Owner YUNNAN MINZU UNIV
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