In the present work, an impedimetric nucleic acid biosensor has been designed for the purpose of detection of microRNA (miRNA). Ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate (IL))-modified chemically activated pencil graphite electrodes (PGEs) were used for the sensitive and selective detection of miRNA-34a. After covalent activation of the PGE surface using covalent agents (CAs), the ionic liquid (IL) was immobilized onto the surface of the chemically activated PGE by passive adsorption. The electrochemical and microscopic characterization of the IL/CA/PGEs was performed by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and scanning electron microscopy (SEM). DNA probe concentration, miRNA target concentration, and also the hybridization time and wet adsorption time were optimized by using the EIS technique. Then, the hybridization occurred between specific DNA probes and miRNA-34a was immobilized onto the surface of the IL/CA/PGEs. The impedimetric detection of miRNA-DNA hybrid was performed by EIS. The detection limit (DL) was calculated in a linear concentration range of 2–10 µg/mL miRNA-34a target, and it was found to be 0.772 µg/mL (109 nM) in phosphate buffer solution (PBS) and 0.826 µg/mL (117 nM) in diluted fetal bovine serum (FBS). The selectivity of impedimetric biosensor for miRNA-34a was also tested against to other non-complementary miRNA sequences both in buffer media, or diluted FBS.

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