Enzyme-Free Electrochemical Biosensor Based on Localized DNA Cascade Displacement Reaction and Versatile DNA Nanosheets for Ultrasensitive Detection of Exosomal MicroRNA.

MicroRNA existing in exosomes (exo-miRNA) is a crucial and reliable biomarker for cancer screening and diagnosis. However, accurate detection of ultralow exo-miRNA amounts in real samples remains a challenge. Herein, a robust and ultrasensitive electrochemical biosensor was developed based on localized DNA cascade displacement reaction (L-DCDR) and versatile DNA nanosheets (DNSs) for enzyme-free analysis of exo-miRNA. The target activated L-DCDR repeatedly by consecutive toehold-mediated strand displacement, which released plentiful P strands to hybridize with capture probes immobilized on the electrode surface and DNS tags, generating an amplified electrochemical signal for the detection of exo-miRNA. The DNS could label-free load various electroactive molecules. The electrochemical biosensor revealed high sensitivity ranging from 0.1 fM to 1 nM with a limit of detection of 65 aM and good specificity. The constructed biosensor was demonstrated to be able to detect exo-miRNA derived from gastric cancer cell line (SGC-7901) and gastric cancer patients. In addition, the developed biosensor possessed several considerable advantages including simple substrate assembly, improved reaction rate, and high signal-to-noise ratio. Therefore, this strategy has great potential in bioanalysis and clinical diagnostics.