An Ultrasensitive Electrochemical Biosensor Integrated by Nicking Endonuclease-Assisted Primer Exchange Reaction Cascade Amplification and DNA Nanosphere-Mediated Electrochemical Signal-Enhanced System for MicroRNA Detection.

Specific and sensitive microRNAs (miRNAs) detection is essential to early cancer diagnosis. The development of these technologies including functional nuclease-mediated target amplification and DNA nanotechnology possesses tremendous potential for the high-performance detection of miRNAs in the accurate diagnosis of disease. In this study, we have established an ultrasensitive electrochemical biosensor by combining nicking endonuclease-assisted primer exchange reaction (PER) cascade amplification with a DNA nanosphere (DNS)-mediated electrochemical signal-enhanced system for the detection of miRNA-21 (miR-21). The cascade amplification is initiated by a nicking endonuclease that can cleave specific DNA substrates and highly amplify translation of the target to single-stranded DNA fragments (sDNA). Then, the PER cascade is powered by strand-displacing polymerase and generates a large amount of nascent single-stranded connector DNA (cDNA) via sDNA triggering of the dumbbell probe (DP), thus achieving the cascade amplification of the target. Finally, the DNS loaded with plenty of electroactive substances can be captured on the electrode via cDNA for further enhancing the electrochemical signal and highly sensitive detection of miR-21. The proposed electrochemical biosensor exhibits a wide detection range of 1 aM to 0.1 nM and a low detection limit of 0.58 aM. The excellent selectivity allows the biosensor to discriminate miR-21 from other miRNAs, even the one base-mismatched sequence. Moreover, the practicability of the biosensor is investigated by analyzing miR-21 in human serum and cancer cell lysate. Therefore, our proposed nicking endonuclease-assisted PER cascade amplification strategy provides a powerful platform for the early detection of miRNA-related disease and molecular diagnosis.