Li Mei,Xu Xiong,Cai QingYou,Luo XuJian,Zhou ZhongGao,Xu GuoHai,Xie YongRong
A graphene oxide-based method has been developed for ultrasensitive and selective determination of microRNA-141 by means of rolling circle amplification (RCA) and exonuclease III (Exo III)-assisted recycling amplification. The method uses (a) a padlock probe with a hybrid sequence that is complementary to the target microRNA-141 at both the 5'- and the 3'-end, and (b) a long binding region of a signalling reporter strand. On addition of microRNA-141, it acts as the primer for triggering the RCA reaction following ligation. This results in the formation of a repeatedly concatenated sequence of the padlock probe. Subsequently, the RCA product hybridizes with the fluorescein-labelled signal strand to form the duplex DNA containing the blunt 3'-termini of signal strand. Addition of Exo III causes signal strand digestion and leads to RCA product recycling and liberation of fluorescein. Added graphene oxide does not absorb the fluorescein liberated because of the poor mutual interaction. Therefore, microRNA-141 can be quantified by measurement of the green fluorescence under excitation/emission wavelengths of 470/520 nm. The method has a 100 aM detection limit towards microRNA-141 and works in the wide range from 1 fM to 1 nM. It can discriminate even single-mismatched microRNA and shows good selectivity and sensitivity when applied to spiked human serum.
Schematic representation of a graphene oxide (GO)-based method for fluorometric determination of microRNA by using rolling circle amplification and exonuclease III (Exo III)-aided recycling amplification. With microRNA, the fluorescein-labelled signal strand becomes digested, and this genererates a fluorescent signal.
s 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.
危险品化学品经营许可证（带存储）