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MicroRNA biosensor for triggering 3-D double-leg DNA walker through exponential amplification reaction

A technology of biosensor and exponential amplification, applied in the direction of microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problem of limiting the walking efficiency of the total number of walkers

Pending Publication Date: 2021-01-22
CHONGQING MEDICAL UNIVERSITY
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Problems solved by technology

On the other hand, routine analysis of target molecules yields only one DNA walker, limiting the total number of walkers and their walking efficiency

Method used

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  • MicroRNA biosensor for triggering 3-D double-leg DNA walker through exponential amplification reaction
  • MicroRNA biosensor for triggering 3-D double-leg DNA walker through exponential amplification reaction
  • MicroRNA biosensor for triggering 3-D double-leg DNA walker through exponential amplification reaction

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Embodiment Construction

[0037] To prepare a biosensor based on a 3-D double-legged DNA walker triggered by an exponential amplification reaction, the steps are as follows:

[0038] (1) Isothermal Exponential Amplification Reaction (EXPAR) First, mix 100nM template, various concentrations of target microRNA, 1μL 10×CutSmart buffer and 1μL 10×NEBuffer 2, and heat in a water bath at 95°C for 5 minutes, then Slowly lower to room temperature. Then, 0.2 U of KF polymerase, 1 U of Ns.BsmAI, 250 μM dNTPs and DEPC-treated water were added to the mixture so that the final volume was 10 μL. Amplification reactions were performed at 37°C for 30 minutes. Finally, the reaction system was terminated by an enzyme inactivation treatment at 80° C. for 20 minutes. Store this solution at 4 °C for future use.

[0039] (2) Preparation of DNA walker probes 50 μL of streptavidin-coated microspheres were washed twice with 50 μL of binding buffer by centrifuging the microspheres at 12,000 rpm for 3 minutes, and then discar...

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Abstract

The invention relates to a DNA amplifier which is designed by using an incision enzyme-driven three-dimensional (3-D) DNA walker, wherein a double-leg DNA walker (BDW) can be continuously assembled ona fluorescently-labeled polystyrene microsphere track through an exponential amplification reaction (EXPAR) triggered by target miRNA (micro ribonucleic acid). According to comparative study, the situation that the walking speed of BDW is higher than that of a single-leg DNA walker (SDW) is proved firstly. In view of this, EXPAR is activated by the target miRNA by means of a polymerase and incision endonuclease to produce a large number of BDWs in a solution, so that the walker is activated in the presence of incision endonuclease, an enhanced fluorescence signal is produced in the supernatant. By taking microRNA 21 as a model target, the detection limit of the method under the optimal condition is as low as 5.2 fM, and compared with other DNA walkers, the method has higher sensitivity. The binding of the enzyme-assisted EXPAR in the solution to the enzymatic BDW on the particles can significantly improve signal amplification efficiency and improve detection sensitivity. Therefore, the method has huge potential for the application of biosensors related to BDW.

Description

technical field [0001] The invention relates to a biosensor, in particular to a 3-D dual-leg DNA walker triggered by an exponential amplification reaction for microRNA detection. Background technique [0002] DNA nanotechnology has made impressive progress in the field of analytical chemistry. Inspired by nature, DNA walkers, as an important branch of DNA nanotechnology, can mimic natural molecular walkers, such as dynein, myosin, and kinesin. For a typical DNA walker, it contains three basic components, namely walking chain, walking track and driving motor. During walking, DNA walkers can generate a large number of signaling molecules along tracks of different dimensions for signal amplification. However, both one-dimensional (1-D) tracks and two-dimensional (2-D) DNA origami have very limited persistence and walking space, which limits the execution capabilities of such DNA walking machines. To address the above issues, researchers have recently built 3-D tracks on the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6825
CPCC12Q1/6825C12Q2521/101C12Q2537/1376C12Q2521/301C12Q2525/207C12Q2563/107C12Q2565/607C12Q2563/149
Inventor 谢国明杨柳方杰
Owner CHONGQING MEDICAL UNIVERSITY
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