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A kind of electrochemical sensor and its preparation and application

A sensor and electrochemical technology, applied in the field of electrochemistry, can solve problems such as time-consuming, low throughput, and unsuitable for precise quantitative analysis

Inactive Publication Date: 2016-08-24
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Northern blotting is the earliest technology used for the analysis of various miRNAs, and is considered the gold standard for miRNA research and analysis. Due to the complex detection steps, long time-consuming, low throughput, low sensitivity, and the need to detect a large number of samples, this method is not suitable. Profiling Everyday miRNAs
Microarray technology has high-throughput detection capabilities and can analyze a variety of miRNAs. However, miRNAs with short sequences and high sequence similarities have the possibility of cross-hybridization, which reduces the sensitivity and specificity of this method, so it is only suitable for General screening analysis, not suitable for precise quantitative analysis
The qRT-PCR method is widely used for the detection of miRNAs due to its high specificity and sensitivity, but the operation process requires reverse transcription steps and temperature cycles, and requires the design of primers and expensive instruments, which limits the use of this method

Method used

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  • A kind of electrochemical sensor and its preparation and application
  • A kind of electrochemical sensor and its preparation and application
  • A kind of electrochemical sensor and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Embodiment 1 prepares electrochemical sensor

[0102] 1. Materials and methods

[0103] 1.1 Materials

[0104] The sequences involved in the present invention are synthesized and purified at Shanghai Sangong, alkaline phosphatase-labeled streptavidin, 6-mercaptohexanol (MCH), bovine serum albumin (BSA), α-naphthyl phosphate ( α-NP) were purchased from Sigma-Aldrich, USA.

[0105] 1.2 Testing instruments

[0106] The CHI660D electrochemical workstation is a product of Shanghai Chenhua Instrument Company.

[0107] 1.3 Detection principle

[0108] Firstly, the capture probe for MicroRNA-21 is immobilized on the surface of the gold electrode by sulfhydryl groups to obtain the working electrode, and then the designed two stem-loops H1 and H2 and the target substance MicroRNA-21 to be detected are simultaneously added to the buffer for amplification After a period of time, the obtained amplified product was added dropwise to the working electrode, then alkaline phosphatase...

Embodiment 2

[0125] Characterization and inspection of the electrochemical sensor of embodiment 2

[0126] The electrochemical sensor obtained in embodiment 1 is characterized as follows:

[0127] 1. Electrochemical characterization of different modified electrodes

[0128] like figure 1 Shown is the square wave voltammetry response curve of different modified electrodes in 5mM potassium ferricyanide solution in each step:

[0129] a is a bare gold electrode;

[0130] b is the fixed capture probe;

[0131] c is the electrode sealed by MCH and BSA;

[0132] d is after adding the amplification product catalyzed by no target substance;

[0133] e is after adding the amplification product catalyzed by MicroRNA-21.

[0134] When the capture probe was immobilized on the surface of the bare gold electrode, the peak current decreased significantly ( figure 1 - Curve b), the result shows that the capture probe was successfully immobilized on the electrode. When MCH and BSA blocked the ele...

Embodiment 3

[0142] Embodiment 3 The optimization of electrochemical sensor and its service conditions

[0143] We also fixed several important conditions during the experiment, namely the number of base mismatches and H1 at 50nmol ml -1 The concentration of stem-loop H2, the temperature of the amplification system, and the reaction time of the amplification system were further optimized. For each condition, five points were selected from low concentration to high concentration to carry out a series of experiments.

[0144] In order to investigate the influence of base mismatch sites on electrochemical sensors, this experiment used stem-loops with different base mismatch sites (H1D1M2, H2D2M2, H2D3M2, H1D4M2), and then performed differential pulse voltammetry scanning. like image 3 , 4 It can be seen that the signal-to-noise ratio varies with different mismatched sites. When it is H2D2M2's turn, the signal-to-noise ratio reaches a maximum value of 33.6, which is higher than that of mis...

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Abstract

The invention provides an electrochemical sensor for detecting MicroRNA-21 and a detection system comprising the electrochemical sensor. The electrochemical detection electrode system comprises a working electrode, a reference electrode and a counter electrode, wherein the working electrode is obtained by fixing a capture probe aiming at MicroRNA-21 on the surface of a gold electrode. The MicroRNA-21 is detected by using the sensor or the detection system disclosed by the invention, the concentration of the MicroRNA-21 is in a linear relation in a range from 0.001nmol / ml to 25nmol / ml, the linear equation refers to Y=1.19999E<-5>+3.63774E<-6>X, the correlation coefficient is 0.9973, and the lowest detection limit is 0.6pmol / ml. Compared with the prior art, the sensor or the detection system is high in sensitivity, high in specificity, low in cost, easy and convenient to operate and short in detection period, is suitable for measuring the MicroRNA-21 in actual samples and is expected to become a sensor or a detection system with actual application values.

Description

technical field [0001] The invention relates to the field of electrochemistry, in particular to an electrochemical sensor and its preparation and application. Background technique [0002] Traditional miRNA detection methods mainly include Northern blotting, microarray hybridization, RT-PCR, etc., but each method has its own advantages and disadvantages, and there is still a lack of simple, rapid, highly sensitive, and highly specific detection methods. Northern blotting is the earliest technology used for the analysis of various miRNAs, and is considered the gold standard for miRNA research and analysis. Due to the complex detection steps, long time-consuming, low throughput, low sensitivity, and the need to detect a large number of samples, this method is not suitable. Analysis of daily miRNAs. Microarray technology has high-throughput detection capabilities and can analyze a variety of miRNAs. However, miRNAs with short sequences and high sequence similarities have the p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/327C12Q1/68
Inventor 丁世家颜玉蓉张晔袁泰先李胜强丁小娟杨建儒李新民袁睿吴茳铃
Owner CHONGQING MEDICAL UNIVERSITY
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