Cascade signal amplified assay of nucleic acids based on entropy-driven amplification strategy and Mg²⁺-dependent...

Nucleic acids-based biosensors are extremely important in modern life sciences and have been widely used for the detection of many biomarkers of disease and extensively applied in many fields, such as medical analysis, gene therapy, and pathogen determination. Therefore, it is necessary to develop some sensitive and selective methods for rapid detection of nucleic acids. In this work, an ultrasensitive and non-enzyme electrochemical biosensor has been developed for nucleic acids detection based on entropy-driven amplification (EDA) strategy and Mg²⁺-dependent DNAzyme cleavage method. In the presence of target DNA (T-DNA), the T-DNA could hybridize with the premade three-strand duplex (TD) through the toehold region to initiate the EDA process (Cycle I), leading to the generation of Mg²⁺-dependent DNAzyme served for Cycle II. The newly formed Mg²⁺-dependent DNAzyme could hybridize with the methylene blue (MB)-labeled hairpin DNA (MB-HP) on the gold electrode surface which induced the cleavage process of Mg²⁺, resulting in the recycle of Mg²⁺-dependent DNAzyme, accompanied by the release of MB-labeled DNA fragment from the gold electrode surface. Based on the proposed strategy, the developed electrochemical biosensor exhibited a wide linear relationship in the range from 5 fM to 1 nM with a limit of detection (LOD) of 2.7 fM (S/N = 3), which gave the developed electrochemical biosensor a great promising for the detection of nucleic acids in biomedical research and disease diagnosis.