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Polyadenine-based DNA capture probe, biosensor and detection method thereof

A biosensor and capture probe technology, applied in the field of electrochemical detection, can solve the problems of cumbersome steps, restrict the development of electrochemical biosensors, and high synthesis cost, and achieve the effects of broad application prospects, favorable hybridization reaction, and easy operation.

Active Publication Date: 2019-07-12
SHANGHAI INST OF MEASUREMENT & TESTING TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above three methods all need to modify the DNA capture probe, the synthesis cost is high, and the steps are cumbersome
[0005] However, at present, it is difficult for those skilled in the art to know how to regulate the assembly density of DNA capture probes on the electrode surface of electrochemical biosensors, which directly restricts the further development of electrochemical biosensors. Therefore, it is urgent to obtain a new immobilization method for DNA capture probes And the electrochemical biosensor with excellent effect

Method used

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  • Polyadenine-based DNA capture probe, biosensor and detection method thereof
  • Polyadenine-based DNA capture probe, biosensor and detection method thereof
  • Polyadenine-based DNA capture probe, biosensor and detection method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0045] The construction of the DNA capture probe of embodiment 1 polyadenine

[0046] Design and synthesis of DNA capture probes containing different numbers (5, 10, 20, 30 and 40) of adenines (referred to as poly A5, poly A10, poly A20, poly A30 and poly A40) , the nucleotide sequences of which are respectively shown in SEQ ID No. 1-5 in the sequence listing, and the polyadenine DNA capture probes are obtained after artificial synthesis (Life Technology Biological Co., Ltd.).

[0047] Simultaneously, a single-stranded DNA with and only 5 adenines (namely A5, whose nucleotide sequence is shown in SEQ ID No. 6 in the sequence listing) was synthesized to serve as a control for subsequent experiments.

Embodiment 2

[0048] Example 2 Biosensors Containing Polyadenine-Based DNA Capture Probes

[0049] (1) Take the gold electrode (2mm diameter, Shanghai Chenhua Instrument Co., Ltd.) that has been physically polished and electrochemically cleaned with sulfuric acid, rinse the electrode surface thoroughly with ultrapure water Milli-Q, and then wash it with N 2 Blow dry to dispose of clean electrodes.

[0050] (2) Add 5 μL of 0.1 μM polyadenine DNA capture probe poly A30 obtained in Example 1 dropwise to the cleaned electrode obtained in step (1), react at 25° C., and assemble overnight to obtain an assembled electrode.

[0051] (3) Use 150 μL of 0.1 mM MCH (purchased from sigma) to seal the vacancy of the assembled electrode obtained in step (2) for 30 minutes, and the sealed electrode is a biosensor that can be used for detection.

[0052] (4) 1nM of the DNA to be tested (its nucleotide sequence is as shown in SEQ ID No.8 in the sequence listing) and 100nM signal probe (its nucleotide sequen...

Embodiment 3

[0056] Electrochemical detection of different assembly methods of embodiment 3

[0057] (1) Take the gold electrode (2mm diameter, Shanghai Chenhua Instrument Co., Ltd.) that has been physically polished and electrochemically cleaned with sulfuric acid, rinse the electrode surface thoroughly with ultrapure water Milli-Q, and then wash it with N 2 Blow dry to dispose of clean electrodes.

[0058] (2) Add 5 μL of 0.1 μM poly A30 obtained in Example 1 dropwise to the cleaned electrode obtained in step (1), react at 25° C., and assemble overnight to obtain an assembled electrode.

[0059] (3) Use 0.1 mM MCH and 0.1 μM A5 prepared in Example 1 to seal the gaps of the assembled electrodes obtained in step (2) for 30 minutes to obtain sealed electrodes A and B.

[0060] In addition, step (4) is directly performed after step (2) to obtain an electrode C assembled by adding poly A30 dropwise but not sealed by any substance.

[0061] All the other steps are identical to Example 2.

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Abstract

The invention discloses amulti-adenine based DNA capture probe, a biosensor and a detection method thereof. The nucleotide sequence of the DNA capture probe is shown as a sequence table SEQID No.1, SEQID No.2, SEQID No.3, SEQID No.4 or SEQID No.5. The mutual effect of adenines and a gold electrode to fix the multiple-consecutive-adenine DNA probe to the surface of the gold electrode, the electrochemical biosensor based on multi-adenine DNA is established for the first time, the effect same as a sulfydryl modified DNA based assembled gold electrode can be achieved, and the new era of novel electrochemical biosensors is opened. The multi-adenine based DNA capture probe can detect DNA in a high sensitivity and high specificity mode and is easy and convenient to operate and wide in application prospect, and the sensitivity can be up to 1 pM.

Description

technical field [0001] The invention belongs to the field of electrochemical detection, and in particular relates to a polyadenine-based DNA capture probe, a biosensor and a detection method thereof. Background technique [0002] The development of electrochemistry has a long history and is closely related to the development of cutting-edge science and technology and disciplines. Electrochemical biosensors have specific molecular recognition functions and have shown broad application prospects in disease gene diagnosis and anticancer drug screening. Cutting-edge research methods in the fields of science and medicine. [0003] The general steps for the electrochemical biosensor to measure DNA include: 1. Immobilization of the DNA capture probe, that is, immobilizing a single-stranded DNA (ssDNA) of a specific sequence on the surface of the electrode; 2. Hybridization, immobilizing the DNA capture probe The electrode is put into the measured solution containing the matched DN...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/327
CPCG01N27/3277
Inventor 李兰英闻艳丽刘刚王乐乐徐勤梁文
Owner SHANGHAI INST OF MEASUREMENT & TESTING TECH
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