Enzyme-Free and Triple Sensitivity Amplification for Electrochemical Detection of Exosomal microRNA

Exosomal miRNAs, particularly miRNA-21, are promising cancer biomarkers. Current enzyme-dependent detection methods face challenges, such as environmental limitations and high costs. In contrast, enzyme-independent sensors are highly desirable for on-site, miniaturized, and cost-effective miRNA detection. To address these limitations, we developed a nonenzymatic electrochemical sensor featuring a triple-signal amplification system for ultrasensitive detection of miRNA-21. This sensor utilizes cascade toehold-mediated strand displacement reactions to activate molecular machines triggered by target miRNA, generating biotinylated-and-thiol-modified double-stranded DNA for stable immobilization on a gold electrode. Preprepared biotinylated tetrahedron DNA (TDNA)-mediated hybridization chain reaction probes are then linked to the electrode via streptavidin–biotin binding. This amplification process allows for significant DNA duplex immobilization, with electroactive [Ru(NH3)6]3+ (RuHex) adsorbed onto them, producing a robust electrochemical signal. This approach enables accurate detection of miRNA-21 at concentrations as low as 0.43 fM, with a linear range from 1 fM to 1 nM. Clinical testing demonstrates its potential for cancer diagnostics.