Ratio-type electrochemical biosensor for eliminating LAMP background interference, construction method and application

A biosensor and construction method technology, applied in the field of ratiometric electrochemical biosensor construction, can solve problems such as difficulty in realization, achieve the effect of improving specificity and reducing false positive signals

Active Publication Date: 2020-04-24
SOUTHWEST UNIVERSITY
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Problems solved by technology

And this process is also extremely challenging, and it is difficult to achieve

Method used

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  • Ratio-type electrochemical biosensor for eliminating LAMP background interference, construction method and application
  • Ratio-type electrochemical biosensor for eliminating LAMP background interference, construction method and application
  • Ratio-type electrochemical biosensor for eliminating LAMP background interference, construction method and application

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Embodiment 1

[0048] 1. Sequence design:

[0049] Taking HIV-specific marker DNA as the target (hDNA) analysis model and as the external primer F3 of the LAMP reaction, on the NUPACK website ( www.nupack.org ) to design all relevant base sequences based on hDNA. As shown in Table 1.

[0050] Table 1. DNA base sequence used in the present invention

[0051]

[0052]

[0053] Among them, the Mg formed by the hybridization of S* and C* 2+ -The hairpin configuration and base sequence of DNAzyme, MB-H1 and Fc-H2 are as follows figure 1 shown.

[0054] 2. A ratiometric electrochemical biosensor for eliminating LAMP background interference, its construction method and application, the specific steps are as follows:

[0055] (1) LAMP reaction ( figure 2 ): The target hDNA (F3) reacts with the template DNA, B3, BIP, FIP, polymerase Bst and dNTPs in a closed-tube PCR instrument, and the amplification product solution contains a large amount of H + (LAMP(H + ))

[0056] (2) LAMP (H +...

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Abstract

The invention relates to a ratio-type electrochemical biosensor for eliminating LAMP background interference, a construction method and application. According to the method, a target object short-chain DNA is used as an LAMP amplification outer primer, the byproduct hydrogen ions induce i-motif conformation transformation, metal ions DNAzyme are assembled, cyclic shearing is carried out, and separation and purification of an LAMP complex system and products are achieved; two DNA hairpins marked with electroactive probes methylene blue and ferrocene respectively are triggered on the surface ofthe electrode for directional hybridization without any enzyme intervention, and when the two DNA hairpins are far away from and close to the surface of the electrode respectively, the current signalratio has high-sensitivity and high-specificity target concentration linear correlation. The electrode reaction of the established ratio-type sensor is more simplified, background interference and false positive signals are effectively reduced, the sensitivity is remarkably improved, and a new thought and a technical means are provided for electrochemical detection of DNA of various disease markers.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and electrochemical biosensor, and relates to a construction method and application of a ratiometric electrochemical biosensor for eliminating LAMP background interference. Background technique [0002] As an important and efficient nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP) can achieve 10 9 Replicate amplification. During the LAMP reaction, in addition to the main products of double-stranded DNA of different lengths, there are also a large amount of H + And pyrophosphoric acid, hydrolysis of pyrophosphoric acid further produces H + . Therefore, the pH of the LAMP reaction solution will vary depending on the target primer. [0003] Due to the advantages of mild reaction conditions, simple and fast operation, no need for special equipment, and high specificity, LAMP technology has been widely used in pathogen detection, clinical medicine, food ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/327G01N27/30
CPCG01N27/3276G01N27/3278G01N27/308
Inventor 许文菊
Owner SOUTHWEST UNIVERSITY
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