Microelectrode biosensor of in-situ vivo detection plant miRNA and application of microelectrode biosensor

A biosensor and living body detection technology, applied in the direction of electrochemical variables of materials, etc., can solve the problems of large damage to plant samples and complicated processing process, and achieve the effect of easy to master and simple operation.

Inactive Publication Date: 2016-12-21
BEIJING RES CENT FOR INFORMATION TECH & AGRI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the defects of existing detection technology, the object of the present invention is to provide a kind of in situ detection method of plant miRNA based on biosensing technology, to overcome the in vitro sampling detection, complicated processing process and damage to plant samples in the prior art. Larger and other defects

Method used

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  • Microelectrode biosensor of in-situ vivo detection plant miRNA and application of microelectrode biosensor
  • Microelectrode biosensor of in-situ vivo detection plant miRNA and application of microelectrode biosensor
  • Microelectrode biosensor of in-situ vivo detection plant miRNA and application of microelectrode biosensor

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Embodiment 1

[0024] The preparation of embodiment 1 working electrode

[0025] (1) The microelectrode array is prepared by microelectromechanical processing technology (MEMS), including a reference electrode of Ag / AgCl, a platinum counter electrode, and a gold working electrode. The length of the microelectrode is 5 cm, and the length of the exposed conductive part is about 3-10 mm; The electrode was placed in a 0.5M dilute sulfuric acid solution for cyclic voltammetry scanning (-0.2 to 1.6V) to obtain a typical cyclic voltammetry spectrum to ensure that the electrode surface was clean.

[0026] (2) Gold nanoparticles with a diameter of 10 nm are deposited on the gold working electrode by electrodeposition to enhance the detection sensitivity of the sensor.

[0027] (3) The DNA probe sequence (5'-ggcatacaggggagccaggca-3') was designed according to the sequence of GmMIR160A, and the end was thiolated to be immobilized on the gold working electrode. Put 5 μL of 50 nM end-sulfhydrylated DNA ...

Embodiment 2

[0028] Embodiment 2 Application of microelectrode biosensor

[0029] (1) Prepare PBS (pH=7.0) standard solutions of 0, 0.01, 0.1, 0.5, 1, 5, and 10 μM miRNA respectively, using the microelectrode modified with probe DNA single strand prepared in Example 1 and different concentrations of miRNA standard solution reaction, detect the change of peak current value of MB before and after hybridization by pulse voltammetry, obtain a group of concentration and peak current relation curves, make the standard curve I (μ A) of miRNA=1.45log c (pM)+3.09( R=0.9857).

[0030] (2) Cultivation of plant samples: soybean seedlings hydroponically cultivated for 15 days were selected, a control group and a treatment group were set up, and 50 mM sodium chloride was used to treat them with salt stress for different periods of time. 30 repetitions were set for each treatment group.

[0031] (3) Insert the microelectrodes into the tender stems of soybean seedlings to be tested in different treatmen...

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Abstract

The invention relates to microelectrode biosensing technology, and specifically discloses a microelectrode biosensor for in-situ living body detection of plant miRNA and an application thereof. In the present invention, after the gold nanoparticles are electrodeposited on the working electrode, the DNA probes capable of recognizing the target miRNA end-mercaptolated are incubated in the methylene blue MB solution to form a DNA-methylene blue complex, and then the complex is drip-coated to the deposition The working electrode surface of the gold nanoparticles was obtained, and the working electrode modified with the DNA probe was obtained. The microelectrode biosensor containing the working electrode is used to monitor living plants, and the change of peak current value of MB before and after hybridization is detected by electrochemical pulse voltammetry, so as to achieve the purpose of quantitative detection of microRNA without causing essential damage to the tested sample. The obtained data result can reflect the content change of the target miRNA in the plant in real time and dynamically, and the practical application is easy to operate and easy to grasp.

Description

technical field [0001] The invention relates to microelectrode biosensing technology, in particular to a microelectrode biosensor for in-situ living body detection of plant miRNA and its application. Background technique [0002] Plant microRNA (miRNA) is an important class of small molecules with regulatory functions. It is a non-coding RNA with a length of about 20-25 nucleotides. The findings suggest that miRNAs are ubiquitous in higher plants. Many of the target genes of miRNA are transcription factors that play an important role in plants. Through the regulation of these target genes, miRNA plays a vital role in plant growth and development, hormone response, stress resistance and disease resistance. For example, miR1885 can be induced in Chinese cabbage infected by turnip mosaic virus, and miR1885 regulates disease resistance by regulating auxin receptor-nucleotide binding site-leucine rich region (TIR-NBS-LRR) Genes mediate the process of cabbage's resistance to vir...

Claims

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Application Information

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IPC IPC(8): G01N27/48
CPCG01N27/48
Inventor 李爱学王成胡叶王晓冬侯佩臣何璐璐周航高权陈泉赵勇
Owner BEIJING RES CENT FOR INFORMATION TECH & AGRI
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