Dual-amplified strategy for ultrasensitive electrochemical biosensor based on click chemistry-mediated enzyme-assisted target recycling and functionalized fullerene nanoparticles in the detection of microRNA-141.

Rapid and efficient detection of tumor marker at the early stages is one of the crucial challenges in cancer diagnostics and therapy. In this study, an ultrasensitive electrochemical biosensor was fabricated by dual-amplified strategy for the detection of ultra-trace microRNA-141 (miRNA-141). Firstly, two split sequences contained G-quadruplex were connected by click chemistry-mediated nucleic acid strands self-assembly and the obtained complete G-quadruplex was complementary with miRNA-141 to formed DNA-RNA hybrid duplexes. Subsequently, the formed DNA-RNA hybrid duplexes were specifically recognized by duplex-specific nuclease (DSN), and the DNA part of the duplexes were cleaved and the miRNA-141 were released to trigger next cycle, which acquired a primal signal amplification by enzyme-assisted target recycling (EATR). Moreover, amino and thiol group multi-labeled functionalized fullerene nanoparticles (FC60) with a larger surface active sites and better biocompatibility, were designed rationally to modify the Au electrodes, which produced multiply-enhanced amplified signal. This dual-amplified sensing system exhibited a remarkable analytical performance for the detection of miRNA-141 in concentrations ranging from 0.1 pM to 100 nM and the detection limit of 7.78 fM was obtained. Compared with the biosensor with single amplification strategy such as EATR, this electrochemical biosensor based on dual-amplified strategy exhibited an excellent discrimination capability and higher analytical performance. Therefore, this electrochemical biosensor might hold a great potential for further applications in biomedical research and early clinical diagnosis.