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Electrochemical sensor based on stem-loop structure probe and preparation method thereof

A stem-loop structure, electrochemical technology, applied in the direction of material electrochemical variables, etc., can solve the problems of reaction condition interference, sensor accuracy, sensitivity and application range limitations, to achieve low-cost detection, improve sample utilization, and improve hybridization efficiency effect

Inactive Publication Date: 2017-01-18
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a routine quantitative analysis method, colorimetry is easily disturbed by reaction conditions, and the accuracy, sensitivity and application range of sensor measurement are largely limited.

Method used

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  • Electrochemical sensor based on stem-loop structure probe and preparation method thereof
  • Electrochemical sensor based on stem-loop structure probe and preparation method thereof
  • Electrochemical sensor based on stem-loop structure probe and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Reagents include:

[0051] 4 pieces of single-stranded DNA for self-assembly to form DNA probes, A (93bp, molecular weight 28496.0, 5'-end modified ferrocene ssDNA), B (55bp, molecular weight 17018.0, 5'-end modified sulfhydryl ssDNA), C (55bp , molecular weight 16898.0, 5'end modified sulfhydryl ssDNA), D (55bp, molecular weight 16877.0, 5'end modified thiol ssDNA). The four single-stranded DNAs that constitute the tetrahedral structure contain three structural domains, each of which is complementary to the corresponding domains of the other three single-stranded DNAs (17 base pairs). A forms a stem-loop structure at the 5' end, the stem structure is 6 base pairs, and the loop structure is 22 bases for the recognition of target molecules.

[0052] A (SEQ ID NO: 1):

[0053] 5'-Fc-TCCAGCGTTCAACATCAGTCTGATAAGCTAACGCTGTTACATTCCTAAGTCTGAAACATTACAGCTTGCTACACGAGAAGAGCCGCCATAGTA-3'

[0054] B (SEQ ID NO: 2):

[0055] 5'-HS-C6-TATCACCAGGCAGTTGACAGTGTAGCAAGCTGTAATAGATGCGAGG...

Embodiment 2-6

[0078] Reagents include:

[0079] The A chain and target microRNAs change according to the type of early screening cancer, see the table below for details.

[0080]

[0081]

[0082] The detection steps are the same as in Example 1.

Embodiment 7

[0084] Reagent is with embodiment 1.

[0085] Detection steps 1-4 are the same as in Example 1.

[0086] In the fifth step, the target microRNA: hsa-miR-21 was diluted with cell lysate to a concentration of 1 μM. The working electrode was placed in the reaction system to hybridize with the target microRNA for 15 minutes. After fast complementary pairing, the electrodes were taken out, rinsed with PBS buffer solution, and placed in PBS for differential pulse scanning. The electrochemical detection uses CHI electrochemical analyzer (CHI660) and adopts the traditional three-electrode system, the platinum plate electrode is the counter electrode, and the Ag / AgCl electrode is the reference electrode. The scanning voltage range is 0~0.6V, and the pulse period is 0.5s. Detect the relationship between the concentration of the target molecule hsa-miR-21 in the actual simulated sample and the detection signal, the results are as follows image 3 shown.

[0087] By detecting microRN...

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Abstract

The invention discloses an electrochemical sensor based on a stem-and-loop structured probe and a preparation method of the electrochemical sensor. Gold nanoparticles are deposited on the surface of a working electrode of the electrochemical sensor, and the surface of the working electrode is provided with the DNA probe, which has a stem-and-loop structure with a tetrahedral base and is modified with ferrocene, in a self-assembling manner. The invention further discloses a method for arranging the DNA probe having the stem-and-loop structure with the tetrahedral base on the surface of the working electrode of the electrochemical sensor in a self-assembling manner and a method for detecting microRNAs by utilizing the working electrode. According to the electrochemical sensor, the organic DNA nanostructure is combined with the inorganic gold nanoparticles, so that the surface density of the probe can be reasonably regulated and controlled, and the upright conformation of the probe can also be maintained. Thus, the electrochemical detection of a 'signal-on' system can be realized under a low surface density condition. The electrochemical sensor is simple and convenient to operate and ensures that the hybridization efficiency and the sample utilization rate are improved while the experiment cost is lowered. As a result, cancers in early stages can be simply and rapidly detected in a low-cost manner.

Description

technical field [0001] The invention relates to an electrochemical sensor, in particular to a "signal-on" electrochemical sensor based on a stem-loop structure probe and a preparation method thereof. Background technique [0002] Cancer is one of the most serious diseases threatening human health. According to statistics, cancer ranks second in the number of deaths due to diseases in the world, second only to cardiovascular diseases (Free Radical Biology and Medicine, 2004, 37, 287-303). The World Health Organization estimates based on cancer incidence, mortality, and population data that the total number of cancer deaths in the world is 6.9 million each year, and the new incidence is about 8.7 million. Cancer is caused by the abnormal mechanism of controlling cell growth and proliferation. It is characterized by unlimited proliferation and can be transferred to grow and reproduce throughout the body, destroying the structure and function of tissues and organs, and eventual...

Claims

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Application Information

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IPC IPC(8): G01N27/26
Inventor 刘硕鹏丁显廷李宗霖苏文琼
Owner SHANGHAI JIAO TONG UNIV
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