Ultrasensitive miRNA detection by AuNP-based 3D DNA walker and catalytic hairpin assembly (CHA) cascade amplification for early cancer diagnosis

By combining exquisitely designed hairpins with the catalytic hairpin assembly to form tripedal DNA walkers driven by enzyme, we constructed a 3D DNA walker with accordingly complementary hairpins attached on gold nanoparticles (AuNPs) and sensitive fluorescence sensing system for the sensitive detection of target miRNA-21. The presence of miR-21 triggers the CHA among three hairpins, which lead to the formation of the tripedal DNA walkers. For the walking trajectories, FAM-labeled hairpins were attached to the surface of AuNPs, the fluorescence of which was initially quenched due to its close proximity to AuNPs. After the binding/cleaving/moving process of tripedal DNA walkers with HP4 driven by Exonuclease III (Exo III), a number of single-stranded DNAs (ssDNAs) will be released with FAM fluorescence recovered. Benefiting from the DNA walker and CHA cascade amplification, the proposed sensing strategy showed remarkable improvement in sensitivity with the LOD of 46 aM. Also, the system was practically applied in human serum samples, demonstrating satisfactory outcome. Owing to the precise design of the system, this method exhibited excellent specificity to distinguish miR-21 from its single-, double-mismatched sequences and non-complementary sequences, showing great versatility and potential for the biological analysis and early disease diagnosis.