Electrochemical biosensor for detecting miRNA-122

A miRNA-122, biosensor technology, applied in the field of biosensors, can solve the problems of long time consumption, complicated instrument operation, etc., and achieve the effects of simple preparation method, good repeatability and mild reaction conditions

Inactive Publication Date: 2018-06-15
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Aiming at the disadvantages of complex instrument operation, long time-consuming and professional operators in the existing detection methods, a method based on exonuclease III-assisted cascading signal amplification with strong specificity, high sensitivity, low cost and fast detection speed is provided. Electrochemical biosensor for the detection of miRNA-122

Method used

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  • Electrochemical biosensor for detecting miRNA-122
  • Electrochemical biosensor for detecting miRNA-122
  • Electrochemical biosensor for detecting miRNA-122

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 The change of the current signal with the concentration of HAP1.

[0044] (1) The gold electrode was polished to a mirror surface in 0.3 µm and 0.05 µm alumina slurry in turn, and rinsed with PBS and sterile water;

[0045] (2) Add 10 μL of HAP2 (10 μM) dropwise to the pretreated electrode surface and incubate at 37 °C for 2 h; Incubate for 1 h;

[0046](3) Mix 12 μL sterilized water, 2 μL 10× NEBuffer, 2 μL HAP1 (final concentration 0.1 μM, 0.3 μM, 0.6 μM, 1 μM, 1.5 μM, 2 μM), 2 μL Exo Ⅲ (20 U / μL) and 2 μL miRNA-122 solution (1 pM) were added to the centrifuge tube, shaken for 30 s, and incubated in a 37 °C incubator for 2 h;

[0047] (4) Add the mixed solution in (3) dropwise onto the gold electrode modified with the HAP2-assisted probe, incubate at a constant temperature of 37 °C for 2 hours, and wash;

[0048] (5) Ag / AgCl was used as the reference electrode, the Pt electrode was used as the counter electrode, and the gold electrode obtained in step (4)...

Embodiment 2

[0050] Example 2 The change of the current signal with the concentration of HAP2.

[0051] (1) The gold electrode was polished to a mirror surface in 0.3 µm and 0.05 µm alumina slurry in turn, and rinsed with PBS and sterile water;

[0052] (2) Add 10 μL of HAP2 (1 μM, 3 μM, 6 μM, 10 μM, 15 μM, 20 μM) onto the pretreated electrode surface and incubate at 37 °C for 2 h; then add 10 μL Auxiliary probes were added dropwise to the electrode surface, and incubated at 37 °C for 1 h;

[0053] (3) Add 12 μL sterilized water, 2 μL 10× NEBuffer, 2 μL HAP1 (1 μM), 2 μL Exo III (20 U / μL) and 2 μL miRNA-122 solution (1 pM) into the centrifuge tube , shaken for 30 s, and incubated in a 37°C incubator for 2 h;

[0054] (4) Add the mixed solution in (3) dropwise onto the gold electrode modified with the HAP2-assisted probe, incubate at a constant temperature of 37 °C for 2 hours, and wash;

[0055] (5) Ag / AgCl was used as the reference electrode, the Pt electrode was used as the counter el...

Embodiment 3

[0057] Example 3 The change of current signal with the concentration of ExoIII.

[0058] (1) The gold electrode was polished to a mirror surface in 0.3 µm and 0.05 µm alumina slurry in turn, and rinsed with PBS and sterile water;

[0059] (2) Add 10 μL of HAP2 (10 μM) dropwise to the pretreated electrode surface and incubate at 37 °C for 2 h; Incubate for 1 h;

[0060] (3) Mix 12 μL sterile water, 2 μL 10× NEBuffer, 2 μL HAP1 (1 μM), 2 μL Exo III (5 U / μL, 10 U / μL, 15 U / μL, 20 U / μL, 25 U / μL, 30 U / μL) and 2 μL miRNA-122 solution (1 pM) were added to the centrifuge tube, shaken for 30 s, and incubated in a 37 °C incubator for 2 h;

[0061] (4) Add the mixed solution in (3) dropwise onto the gold electrode modified with the HAP2-assisted probe, incubate at a constant temperature of 37 °C for 2 hours, and wash;

[0062] (5) Ag / AgCl was used as the reference electrode, the Pt electrode was used as the counter electrode, and the gold electrode obtained in step (4) was used as the ...

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Abstract

The invention provides an electrochemical biosensor for detecting miRNA-122. The electrochemical biosensor comprises HAP1 (hairpin probe 1), ExoIII enzyme (exonuclease III), a gold electrode and a HAP2 (hairpin probe 2)-auxiliary probe modifying the surface of the gold electrode. The electrochemical biosensor has the advantages of good specificity, high sensitivity, mild reaction conditions, fastreaction speed, use of the simple gold electrode, small size, carrying easiness, reusability, simple preparation processes, stable performances, good electrode repeatability and feasibility of detection of miRNA-122 in human tissues and biosensor industrialization.

Description

technical field [0001] The invention belongs to the technical field of biosensors, in particular to an electrochemical biosensor for detecting miRNA-122 based on exonuclease III. Background technique [0002] MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs with regulatory functions found in eukaryotes, with a size of about 20-25 nucleotides. miRNA-122, its nucleic acid chain base sequence is 5'-UGGAGUGUGACAAUGGUGUUUG-3'. Studies have shown that miRNA-122 is transcribed from the HCR gene located on human chromosome 18, which can not only affect the growth and development of hepatocytes, but also play a role in the phenotype, differentiation metabolism and cell response of hepatocytes. It also plays an extremely important role in life activities. Many traditional methods such as Northern blot, microarray and polymerase chain reaction (PCR) have been used to identify and quantify miRNAs. These methods often have problems such as expensive instruments, long analy...

Claims

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Application Information

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IPC IPC(8): G01N27/48G01N27/327G01N33/543
CPCG01N27/3277G01N27/48G01N33/54366
Inventor 黄加栋裴倩倩王玉刘素
Owner UNIV OF JINAN
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