Hao Wu,Yaling Liu,Hongyong Wang,Jun Wu,Feifan Zhu,Pei Zou
Highly sensitive detection of DNA plays a crucial role in biomedical research and clinical diagnosis. Herein, we developed a simple, label-free, isothermal , and ultrasensitive colorimetric method for amplified detection of DNA on the basis of a new quadratic amplification strategy. With the presence of three ingeniously designed hairpin structures and Exonuclease III (Exo III), the target DNA can trigger two independent cycles of reactions: hairpin assembly reaction and Exo III cleavage reaction, which are designed to initiate target DNA recycling amplification and reporter DNA amplification, respectively. Therefore, the proposed method exhibits a high sensitivity toward target DNA with a detection limit of as low as 81 fM, and it can discriminate mismatched DNA from completely matched target DNA. Furthermore, this method could be used as a universal tool for the detection of various DNA sequences and might be further extended for the detection of aptamer-binding molecules.
The employment of DNAzyme probes for visual biodetections has received increasing interest recently due to the simple nature of this type of assay. However, achieving high sensitivity and detecting targets beyond nucleic acids remain two major challenges in DNAzyme-based visual detections. In this work, based on a new quadratic amplification strategy, we developed a sensitive and visual detection method for cytokines by using hairpin aptamer DNAzyme probes. The target cytokine, interferon γ (IFN-γ), associates with the aptamer sequences and unfolds the hairpin structure of the probes, leading to simultaneous recycling of the target IFN-γ (assisted by Bst-polymerase) and the DNA sequences (aided by λ exonuclease) to achieve quadratic amplification. This quadratic amplification results in the generation of numerous peroxidase-mimicking DNAzymes, which cause significantly intensified color change of the probe solution for highly sensitive detection of IFN-γ by the naked eye down to 50 pM. The proposed visual sensing method shows also high selectivity toward the target IFN-γ and can be performed in homogeneous solutions with using completely unmodified, synthetic aptamer DNAzyme probes. These distinct advantages of our developed assay protocol make it a potential platform for detecting various types of biomolecules with careful probe designs.
危险品化学品经营许可证（带存储）