A simple and effective biosensor for Hg2+ determination was investigated. The novel biosensor was prepared by the insertion approach that the moiety-labeled DNA inserted into a loosely packed cyclic-dithiothreitol (DTT) monolayer, improving the hybridization efficiency. Electrochemical impedance spectroscopy studies of two biosensors (single-hairpin and dual-hairpin structure DNA modified electrodes) used for Hg2+ detection indicated that the dual-hairpin modified electrode had a larger electron transfer resistance change (Delta R-ct). Consequently, the dual-hairpin structure was used as a signal amplifier for the preparation of a selective Hg2+ biosensor. This biosensor exhibited an excellent selectivity toward Hg2+ over Cd2+, Pd2+, Co2+ etc. Also, a linear relation was observed between the Delta R-ct and Hg2+ concentrations in a range from 0.1 nM to 5 mu M with a detection limit of 28 pM under optimum conditions. Moreover, the biosensor can be reused by using L-cysteine and successfully applied for detecting Hg2+ in real samples. (C) 2015 Elsevier B.V. All rights reserved.