DNA-Fueled Molecular Machine Enables Enzyme-Free Target Recycling Amplification for Electronic Detection of MicroRNA from Cancer Cells with Highly Minimized Background Noise

The variations in microRNA (miRNA) expression levels can be useful biomarkers for the diagnosis of different cancers. In this work, on the basis of a new miRNA-triggered molecular machine for enzyme-free target recycling signal amplification, the development of a simple electronic sensor for highly sensitive detection of miRNA-21 from human breast cancer cells is described. The three-stand DNA duplex probes are self-assembled on the gold electrode surface to fabricate the sensor. The miRNA-21 target binds to the terminal toehold region of the probes, displaces one of the short strands through toehold-mediated strand displacement reactions, and exposes the secondary toehold region for subsequent hybridization with the methylene blue (MB)-modified DNA fuel strand, which further displaces both the miRNA-21 target and the other short strand to activate the operation of the molecular machine. As a result, the miRNA-21 target is cyclically reused, and many MB-DNA fuel strands are attached to the sensor surface, leading to a significantly amplified current response for sensitive detection of miRNA-21 down to 1.4 fM. The developed sensor also shows high sequence discrimination capability and can be used to monitor miRNA-21 expression levels in cancer cells. Moreover, this sensor avoids the involvement of any enzymes for target recycling amplification and features with highly minimized background noise for miRNA detection, which makes this method hold great potential for convenient monitoring of different miRNA biomarkers for early diagnosis of various cancers.