Electrochemical determination of microRNA-21 based on graphene, LNA integrated molecular beacon, AuNPs and biotin multifunctional bio bar codes and enzymatic assay system

Abstract

MicroRNAs (miRNAs), a kind of small, endogenous, noncoding RNAs (∼22 nucleotides), might play a crucial role in early cancer diagnose due to its abnormal expression in many solid tumors. As a result, label-free and PCR-amplification-free assay for miRNAs is of great significance. In this work, a highly sensitive biosensor for sequence specific miRNA-21 detection without miRNA-21 labeling and enrichment was constructed based on the substrate electrode of dendritic gold nanostructure (DenAu) and graphene nanosheets modified glassy carbon electrode. Sulfydryl functionalized locked nucleic acid (LNA) integrated hairpin molecule beacon (MB) probe was used as miRNA-21 capture probe. After hybridized with miRNA-21 and reported DNA loading in gold nanoparticles (AuNPs) and biotin multi-functionalized bio bar codes, streptavidin–HRP was brought to the electrode through the specific interaction with biotin to catalyze the chemical oxidation of hydroquinone by H2O2 to form benzoquinone. The electrochemical reduction signal of benzoquinone was utilized to monitor the miRNA-21 hybridization event. The effect of experimental variables on the amperometric response was investigated and optimized. Based on the specific confirmation of probe and signal amplification, the biosensor showed excellent selectivity and high sensitivity with low detection limit of 0.06pM. Successful attempts are made in miRNA-21 expression analysis of human hepatocarcinoma BEL-7402 cells and normal human hepatic L02 cells.