MicroRNA-21 detection based on molecular switching by amperometry

A sensitive and selective electrochemical method for microRNA-21 (miRNA-21) detection was developed. This detection tactic was based on graphene, gold nanoparticles (AuNPs), locked nucleic acid (LNA) incorporated hairpin DNA switching and enzymatic signal amplification. Graphene and AuNPs can improve the electrode conductivity and increase the electrode active surface. The LNA incorporated hairpin DNA probe was modified with biotin at its 5'-end and -SH at its 3'-end. After the probe hybridized with target miRNA-21, its loop-and-stem structure was unfolded to force the biotin away from the electrode surface. Through the specific interaction between biotin and streptavidin-horseradish peroxidase (streptavidin-HRP), miRNA-21 can be quantitatively realized by electrochemical detection of the enzymatic product of benzoquinone in the presence of hydrogen peroxide and hydroquinone. The determination conditions, such as probe concentration, probe immobilization time, hybridization time and applied potential, were optimized. This assay allowed the detection of miRNA-21 in the range of 1-5000 pM with a detection limit of 0.4 pM (S/N = 3). Based on the locked nucleic acid incorporated probe with hairpin structure, the sensor can well discriminated complementary and base mismatched sequences. Successful attempts were made in miRNA-21 expression analysis of human HeLa cells and normal human hepatic L02 cells.