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Electrochemical biosensor for detecting miRNA as well as preparation method and application of electrochemical biosensor

A biosensor, electrochemical technology, applied in biochemical equipment and methods, material electrochemical variables, scientific instruments, etc., can solve the problems of complicated instrument operation and expensive equipment, and achieve high sensitivity detection, low detection limit, and detection speed. quick effect

Active Publication Date: 2020-07-24
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims at the disadvantages of complex instrument operation, expensive equipment and professional operation in the existing detection method, and provides a detection method with high sensitivity, strong specificity and low cost Preparation method of electrochemical biosensor for detecting miRNA

Method used

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  • Electrochemical biosensor for detecting miRNA as well as preparation method and application of electrochemical biosensor
  • Electrochemical biosensor for detecting miRNA as well as preparation method and application of electrochemical biosensor
  • Electrochemical biosensor for detecting miRNA as well as preparation method and application of electrochemical biosensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Preparation of circular template and silver nanoclusters

[0063] (1) Mix 42 μL sterilized water, 6 μL linear template (100 μM), 6 μL ligation probe (100 μM) and 6 μL 10× T4 DNA ligase buffer, denature at 95°C for 5 min, then slowly Slowly cool down to room temperature to complete the hybridization, then add 3 μL T4 DNA ligase (60 U / μL) to the reaction system, and react at 16°C for 20 hours; after that, the reaction system is placed in a water bath at 65°C for 15 minutes, Inactivate T4 DNA ligase in the system.

[0064] (2) Add 1 μL of exonuclease Ⅰ (20 U / μL) and 2 μL of exonuclease Ⅲ (100 U / μL) to the above reaction system and react at 37°C for 2 h; then put the reaction system at 85°C Heated in a water bath for 10 min to obtain a circular template, which was stored at 4°C for future use.

[0065] (3) Mix 15 μL, 100 μM AgMBs nucleic acid sequence with 73 μL of 20 mM phosphate buffer solution (pH=7.0), and then add 6 μL, 1.5 mM AgNO3 solution into the system...

Embodiment 2

[0067] Example 2 Variation of electrochemical signal with S concentration

[0068] A preparation method of the electrochemical biosensor of the present invention, comprising the following steps:

[0069] a. The gold electrode is first polished in 0.3 and 0.05 µM alumina slurry until it becomes a mirror surface, and then rinsed repeatedly with PBS and secondary water;

[0070] b. Take 121 µL sterilized water, 44 µL 5×PBS buffer, 44 µL TCEP (10 µM), 11 µL S1 chain (2 µM) in a sterilized centrifuge tube;

[0071] c. Add 10 μL of the solution in step b to the surface of the electrode dropwise, incubate at 37 °C for 2 h, and wash.

[0072] So far, the modification process of the electrode has come to an end. The following describes the reaction in the homogeneous solution and the main steps in the homogeneous reaction:

[0073] a. Take 10 μL of 10×phi29 DNA polymerase reaction buffer, 10 μL of 10×RCA buffer, 5 μL of circular template (100 nM) and 2 μL of AgMBs (500 nM), 5 μL of S...

Embodiment 3

[0078] Example 3 Variation of electrochemical signal with concentration of cyclic template

[0079] A preparation method of the electrochemical biosensor of the present invention, comprising the following steps:

[0080] a. The gold electrode is first polished in 0.3 and 0.05 µM alumina slurry until it becomes a mirror surface, and then rinsed repeatedly with PBS and secondary water;

[0081] b. Take 121 µL sterilized water, 44 µL 5×PBS buffer, 44 µL TCEP (10 µM), 11 µL S1 chain (50 nM) in a sterilized centrifuge tube;

[0082] c. Add 10 μL of the solution in step b dropwise to the surface of the electrode, incubate at 37°C for 2 h, and wash.

[0083] So far, the modification process of the electrode has come to an end. The following describes the reaction in the homogeneous solution and the main steps in the homogeneous reaction:

[0084] a. Take 10 μL of 10×phi29 DNA polymerase reaction buffer, 10 μL of 10×RCA buffer, and 5 μL of circular template (concentrations are 0 nM,...

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Abstract

The invention relates to the technical field of biosensors, in particular to a biosensor for detecting miRNA through S specifically recognized by miRNA, conformation change of the S and rolling ring isothermal cycle amplification, and aims to solve the problems of low specificity and sensitivity and high cost of a method for detecting the miRNA in the prior art. According to the biosensor for detecting the miRNA, an electrode is sequentially modified with an S1 layer and an RCA product layer. The preparation method comprises the following steps: pretreating the electrode; modifying the S1 layer on the surface of the electrode; and modifying the RCA product layer on the surface of the electrode. The high-specificity detection on a target object is realized by utilizing the specific recognition of the miRNA; and three-step amplification of a target object signal is realized by using a DNAzyme and rolling ring isothermal amplification technology.

Description

technical field [0001] The invention relates to the technical field of electrochemical sensors, in particular to a biosensor for detecting miRNA based on DNAzyme, and also relates to a preparation method and application thereof. Background technique [0002] MicroRNAs (miRNAs) are a class of small endogenous non-coding RNAs (17-22 nucleotides), which play key roles in various biological processes such as gene expression, cell cycle and biological development. In the past 20 years, miRNAs have been widely expanded in the field of biology, and related basic studies have shown that miRNAs act as tumor suppressors or oncogenes, and changes in miRNAs can lead to the development and progression of many diseases, especially human cancers. These miRNAs may become a new branch of potential biomarkers in early diagnosis and clinical management of tumors. Therefore, accurate miRNA analysis has an important application prospect for further understanding the function of miRNA and reduci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12Q1/44G01N27/327G01N27/48
CPCC12Q1/6844C12Q1/44G01N27/3278G01N27/48C12Q2531/125C12Q2565/607C12Q2521/345C12Q2531/119C12Q2525/207C12Q2521/501
Inventor 黄加栋王业茹王玉刘素孙文玉张曼茹江龙
Owner UNIV OF JINAN
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