A label-free electrochemical biosensor for microRNAs detection based on DNA nanomaterial by coupling with Y-shaped DNA structure and non-linear hybridization chain reaction

DNA nanomaterials have been widely used in bioassays due to their promising properties for sensitive and specific detection of biomolecules. Herein, a label-free electrochemical method was developed for quantitative detection of microRNAs by integrating Y-shaped DNA (Y-DNA) structures with non-linear hybridization chain reaction (non-linear HCR). The Y-DNA structures consisting of three sequences (Y1, Y2 and Y3) serve as stable and specific probes for recognizing target miRNAs. In the presence of target miRNA, competitive hybridization occurs between miRNA and Y-DNA, resulting in the release of Y3 and the disintegration of the Y-DNA structure. The triggers, which were blocked by Y3 previously, were exposed and initiated the non-linear HCR. Remarkable electrochemical signal changes were produced after the isothermal amplification reaction. Therefore, the proposed biosensor achieved sensitive detection of microRNAs (miRNAs). Under optimal conditions, the limit of detection (LOD) was reduced to 0.3334 fM and linear range was from 1 fM to 10 pM. The special design of Y-DNA helped the biosensor obtain the ability to distinguish between single base mutations. What's more, this biosensor was capable of detecting miRNAs in clinical serum samples. We hope that this developed biosensor would provide a potential application for DNA nanomaterials in the field of microRNAs detection and inspire more interests in the development of DNA nanomaterial biosensors.