Ultra-sensitive detection of microRNAs using an electrochemical biosensor based on a PNA-DNA2 three-way junction nanostructure and dual cascade isothermal amplification.

BACKGROUND: MiRNA expression profiles may serve as valuable biomarkers for early cancer diagnosis. However, the detection of miRNA remains greatly challenging due to its shorter length and lower abundance in cells. Electrochemical biosensors based on DNA nanostructures have attracted significant attention due to their improved capture efficiency, high sensitivity, and ease of miniaturization, but the complex construction process, non-specific interactions among DNA probes, and their low stability continue to severely restrict their widespread application in clinical diagnostics. Therefore, developing robust and sensitive methods for miRNA analysis is still of great value.

RESULTS: We describe an ultra-sensitive electrochemical biosensor for miRNA by integrating the PNA-DNA2 three-way junction (3WJ) nanostructure with target-recycling catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) dual cascade isothermal amplification. The target miRNA triggers CHA amplification, resulting in the generation of substantial quantities of double-stranded DNA (dsDNA) products. These dsDNA are subsequently captured by PNA probes immobilized on the electrode surface, leading to the formation of a densely packed layer of PNA-DNA2 3WJ nanostructure. Subsequently, the single-stranded termini of the two branches extending from 3WJ function as promoters to initiate the HCR, resulting in the formation of a layer of intricately intertwined branched long dsDNA molecules, which could adsorb substantial quantities of [Ru(NH3)6]3+, thereby significantly amplifying the electrochemical signal. This electrochemical biosensor exhibits exceptional sensitivity towards miRNA-21, achieving a detection limit as low as 2.9 aM while effectively discriminating single base mutations.

SIGNIFICANCE: This represents the first electrochemical system for miRNA detection by integrating the PNA-DNA2 3WJ with CHA-HCR dual cascade isothermal amplification. The robust, specific and ultrasensitive feature makes cancer cell miRNA monitoring possible, suggesting its great application prospect as a promising sensing platform for monitoring various miRNA biomarkers in cancer diagnostics.