MicroRNA biosensor coupling 3D DNA walking machine with catalytic hairpin assembly

A biosensor, 3-DDNA technology, applied in the field of biosensors, can solve the problems of reducing the colloidal stability, hindering DNA hybridization, and the connecting force is not strong enough.

Inactive Publication Date: 2019-12-20
CHONGQING MEDICAL UNIVERSITY
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Problems solved by technology

This connection force is not strong enough to avoid non-specific DNA adsorption, which seriously hinders DNA hybridization and reduces colloidal stability.
On the other hand, it needs to immobilize the affinity ligand and three nucleic acid complexes on the surface of AuNPs and optimize their ratio accordingly, resulting in poor reproducibility of DNA and AuNPs conjugated products.
[0004] The ratio of the corresponding optimized, resulting in poor reproducibility of DNA and AuNPs conjugated products

Method used

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  • MicroRNA biosensor coupling 3D DNA walking machine with catalytic hairpin assembly
  • MicroRNA biosensor coupling 3D DNA walking machine with catalytic hairpin assembly
  • MicroRNA biosensor coupling 3D DNA walking machine with catalytic hairpin assembly

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Embodiment Construction

[0043] To prepare a biosensor based on entropy-driven 3-D DNA walking machine coupled with catalytic hairpin self-assembly, follow the steps below:

[0044] (1) Preparation of 3-D DNA walking machine

[0045] The purified three nucleic acid strands LS, AS, S and TEmg buffer are annealed and then refolded to prepare the LS / AS / S triple nucleic acid complex (C3). The ratio of LS:AS:S is 1:1.5:1.5. The annealing process is to heat the solution in a PCR machine to 95°C for 5 minutes, and then slowly lower the temperature to 25°C at a rate of 0.1°C per second. Then, 50 μL of streptavidin-modified polystyrene microspheres (PS) were centrifuged at 12,000 rpm for 5 minutes, washed twice with binding buffer, and centrifuged to discard the supernatant. Resuspend in 50μL binding buffer and add 5μLμM C3. After incubating for 15 minutes in a 37°C incubator, wash with TEMg buffer 3 times, centrifuge at 12,000 rpm for 5 minutes to discard the supernatant. Finally, resuspend with 50μL of TEMg b...

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Abstract

In the patent, a new and entropy-driven three-dimensional DNA walking machine is provided and is combined with a catalytic hairpin assembly (CHA) reaction for successful detection of microRNA. The walking machine uses streptavidin wrapped polystyrene microspheres as a 3-D rail matrix to ensure good repeatability. A preparation method is as follows: miRNA-21 is used as a target, the target continuously walks on a DNA functionalized polystyrene microsphere rail through an entropy-driven strand displacement reaction, so that a DNA trinucleotide composite substrate on the polystyrene microspheresis decomposed, and circulation of the miRNA-21 is realized. In addition, release of a large number of helper strands from the DNA trinucleotide composite substrate can catalyze the CHA reaction with aconcomitant increase in obtained fluorescence signals. A linear range of the composite biosensor is between 50 pM and 20 nM with a detection limit as low as 41 pM. In addition, satisfactory results are obtained in both reproducibility tests (1.05%-4.22%) and recovery tests (99.5%-104.8%). The results show that the method has potential application value in aspects of disease diagnosis.

Description

Technical field [0001] The invention relates to a biosensor, in particular to a simple and entropy-driven 3-D DNA walking machine coupled with a catalyzed hairpin self-assembly reaction for microRNA detection biosensor Background technique [0002] DNA machines are molecular machines constructed from DNA. Due to the universality and predictability of Watson Crick's base pairing rules, it can be logically designed. Various DNA machines are constructed and synthesized through highly programmable methods, including DNA tweezers, DNA motors, DNA walkers and DNA robots. Among them, DNA walkers can be precisely controlled in micro-scale or nano-scale programmed oligonucleotide tracks, and have important application potential in biosensors, biocomputing, and drug delivery. Previous studies have reported that the DNA walking machine moves along a one-dimensional (1D) track, two-dimensional (2D) origami, or three-dimensional (3D) track. Among them, the 3D DNA walker has a more powerful ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6825C12Q1/682
CPCC12Q1/6825C12Q1/682
Inventor 谢国明杨廷燕方杰
Owner CHONGQING MEDICAL UNIVERSITY
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